Environmental inactivation of Cryptosporidium oocysts incatchment soils

C.M. Davies1,3, N. Altavilla1, M. Krogh2,3, C.M. Ferguson1,2,3, D.A. Deere3 and N.J. Ashbolt1,31Centre for Water and Waste Technology, School of Civil and Environmental Engineering, University of New South Wales, Sydney,

NSW, Australia, 2Sydney Catchment Authority, Penrith, NSW, Australia, and 3Cooperative Research Centre for Water Quality and

Treatment, Salisbury, SA, Australia

2004/0719: received 22 June 2004, revised 9 August 2004 and accepted 10 August 2004

ABSTRACT

C.M. DAVIES, N . ALTAVILLA , M. KROGH, C.M. FERGUSON, D.A . DEERE AND N.J . ASHBOLT. 2004.

Aims: To generate field-relevant inactivation rates for Cryptosporidium oocysts in soil that may serve as parameter

values in models to predict the terrestrial fate and transport of oocysts in catchments.

Methods and Results: The inactivation of Cryptosporidium oocysts in closed soil microcosms over time was

monitored using fluorescence in situ hybridization (FISH) as an estimate of oocyst ‘viability’. Inactivation rates for

Cryptosporidium in two soils were determined under a range of temperature, moisture and biotic status regimes.

Temperature and soil type emerged as significantly influential factors (P < 0Æ05) for Cryptosporidium inactivation. In

particular, temperatures as high as 35�C may result in enhanced inactivation.

Conclusions: When modelling the fate of Cryptosporidium oocysts in catchment soils, the use of inactivation rates

that are appropriate for the specific catchment climate and soil types is essential. FISH was considered cost-effective

and appropriate for determining oocyst inactivation rates in soil.

Significance and Impact of the Study: Previous models for predicting the fate of pathogens in catchments have

either made nonvalidated assumptions regarding inactivation of Cryptosporidium in the terrestrial environment or

have not considered it at all. Field-relevant inactivation data are presented, with significant implications for the

management of catchments in warm temperate and tropical environments.

Keywords: catchment, Cryptosporidium, inactivation, pathogens, soil.

INTRODUCTION

In many developed regions, the responsible authorities are

increasingly focusing attention on catchment management as

a means of reducing pathogen risks to drinking water

supplies. For instance, the United States Environmental

Protection Agency is seeking estimates of total maximum

daily loads for watershed pathogens (USEPA 2001). The

quantification of transport mechanisms and environmental

inactivation for key pathogens will enable models to be

constructed to predict source water quality and thus better

manage the factors that govern pathogen transport in

catchment environments (Ferguson et al. 2004).Previous work has investigated the transport of Cryptos-

poridium oocysts in terrestrial environments (Atwill et al.2002; Davies et al. 2004). However, one of the major

limitations for the development of catchment pathogen fate

models is the lack of accurate inactivation data that are

relevant to field conditions (Walker and Stedinger 1999).

The oocysts of the protozoan parasite Cryptosporidiumparvum are known to be environmentally robust but there

is little quantitative data that describes their inactivation

kinetics in the environment. Oocysts shed by infected

animals may remain enmeshed in faecal material for many

months before being dispersed by a combination of

mechanical, biological and hydrological means. During this

Correspondence to: Cheryl M. Davies, Centre for Water and Waste Technology,

School of Civil and Environmental Engineering, University of New South Wales,

Sydney, NSW 2052, Australia (e-mail: c.davies@unsw.edu.au).

ª 2004 The Society for Applied Microbiology

Journal of Applied Microbiology 2005, 98, 308–317 doi:10.1111/j.1365-2672.2004.02459.x

time, the combined effects of environmental factors on the

total number of oocysts, and more importantly on the

viability of the oocysts are unknown. Alternatively, they may

be released from the faecal matrix by the action of rainfall on

fresh or recent faecal deposits (Davies et al. 2004), the

release rate being higher when the water salinity is low

(Bradford and Schjiven 2002). Once dispersed from the

faecal matrix, inactivation may be dependant on the

physical, chemical and biological properties of the soil

milieu (Ferguson et al. 2003).Sentinel chambers have been employed in previous

studies to examine the inactivation of oocysts in various

aquatic and terrestrial environments, including soil (Jenkins

et al. 1999; Lim et al. 1999; Walker et al. 2001; Jenkins et al.2002; Udeh et al. 2003). To date there have been only two

reported studies that have investigated the inactivation of

Cryptosporidium oocysts in faecal material (Jenkins et al.1999; Olsen et al. 1999). Temperature has been identified as

the most influential factor (in the absence of sunlight) on

oocyst inactivation in soil (Jenkins et al. 2002). Soil texture,but not soil moisture, was also shown to be important to

survival (Jenkins et al. 2002). In the present study the effects

of soil type, temperature, moisture, and the presence of biota

on the inactivation rates of C. parvum oocysts were examined

in soils from the Sydney drinking water supply catchment.

The main objective was to generate field-relevant inactiva-

tion rates for Cryptosporidium oocysts in soil, which as part

of a larger project, would provide critical parameter

estimates in future models for predicting the fate and

transport of surface water pathogens in catchments.

MATERIALS AND METHODS

Preparation of soil microcosms

Surface soil (top 10 cm) collected from two drinking water

supply catchment locations (designated sites 6 and 11) was

air-dried and sieved using a 1200-lm soil sieve. Several

hundred portions of each of the sieved soils (0Æ5 g) were

weighed into 5 ml polyethylene vials. Approximately half of

the vials of soil from each site were sterilized by gamma-

irradiation at a dose of 90 kGy using a 60Co source.

Cryptosporidium oocysts were purified from fresh defatted

calf faeces by sucrose flotation (Upton 1997). Three batches

of oocysts were used for the entire experiment from separate

purifications of calf faeces collected from the same location.

An estimate of initial oocyst viability for each batch of

purified oocysts was undertaken by excystation using flow

cytometry as described by Vesey et al. (1997) and verified by

fluorescence in situ hybridization (FISH) (see below). The

genetic similarity of extracted DNA from each oocyst batch

was examined by PCR-PAGE using the method of Blasdall

et al. (2002).

Each vial containing 0Æ5 g of soil was inoculated with

0Æ1 ml of a suspension of C. parvum oocysts to achieve

approximate final number of oocysts per vial of 1 · 106. The

inoculum was distributed evenly throughout the soil during

inoculation by mixing. A number of control vials of each soil

type were left uninoculated, to be used for moisture

determinations. MilliQ water was added to each of these

vials in place of the inoculum to ensure that the moisture

content was similar to that in inoculated vials. Salt solutions

(ca 250 ml) containing 0Æ08 and 0Æ77 mol)1 NaCl were

placed into the bottom of sealable airtight jars (capacity ca2 l). According to the literature, these molal NaCl solutions

produce and maintain simulated soil matric potentials of

approximate field capacity and dry conditions respectively

(wilting point) (Walker et al. 2001), and were designated

‘wet’ and ‘dry’ in this study. In addition, to those vials

designated ‘wet’ a calculated volume of MilliQ water was

added to expedite the equilibration of the soil to the desired

moisture potential. Wire mesh discs were used to elevate the

vials above the level of the salt solution. The vials, with caps

loosened, were placed in the jars, which were incubated in

the dark at 4, 20 and 35�C. For microcosms incubated at 20

and 35�C, nonirradiated and gamma-irradiated soils were

inoculated. However, at 4�C, only nonirradiated soil micro-

cosms were prepared.

Enumeration of Cryptosporidium oocysts

The microcosms were sampled destructively by periodically

withdrawing five replicate-inoculated vials for each treat-

ment (soil type, moisture, temperature, biotic status) from

the sealed jars for the determination of Cryptosporidiumoocyst concentrations. In addition, duplicate uninoculated

vials were removed from each jar for percentage moisture

determination by drying in preweighed crucibles at 105�Cfor 48 h (APHA 1998).

The method used for the enumeration of Cryptosporidiumin soil was that reported for bovine faeces (Davies et al.2003). Briefly, each 0Æ5 g of inoculated soil was washed into

a separate 50-ml Falcon tube using 20 ml of 2 mmol)1

sodium pyrophosphate and vortexing. The soil slurry was

then vortexed for 2 min and allowed to stand for 30 min,

followed by centrifugation at 2500 g for 10 min. The pellet

was resuspended in MilliQ water and oocysts extracted

using immunomagnetic separation (IMS) (Dynabeads; Dy-

nal, Olso, Norway) followed by FISH to estimate viability,

and immunofluorescent antibody staining (see Davies et al.2003).

A recovery control was prepared for each soil type by

freshly inoculating 0Æ5 g of the appropriate soil with 100

ColorSeedTM C. parvum oocysts (BTF Decisive Microbio-

logy, North Ryde, NSW, Australia), and processing as

described above.

INACTIVATION OF CRYPTOSPORIDIUM IN SOIL 309

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x

Estimation of oocyst ‘viability’

The viability of the oocysts in each of the five replicates was

estimated using FISH (N. Altavilla and N.A. Ashbolt, in

prep.). Briefly, the oocyst suspension produced by IMS was

serially diluted in sterile MilliQ water and the oocysts were

permeabilized using the method of Deere et al. (1998) in

50% (v/v) ethanol for 10 min at 80�C. After cooling to

room temperature, the oocysts were loaded on to membrane

filters (0Æ8 lm pore size, 13 mm diameter; Millipore Aus-

tralia Pty Ltd, North Ryde, NSW, Australia) in Swinnex

filter housings by filtration. The filters were washed twice by

passing through 0Æ5 ml of PBS containing 1 mmol)1 vanadyl

ribonucleoside complex (VRC) (New England Biolabs Inc.,

Beverly, MA, USA) and once with 200 ll prewarmed

(42�C) hybridization buffer [0Æ9 mol)1 NaCl, 20 mmol)1

Tris-Cl, 0Æ5% (v/v) SDS]. The Texas Red-labelled CRY1

probe (Vesey et al. 1998) was placed on the surface of the

membrane filters at a concentration of 1 lmol)1 in 200 ll ofhybridization buffer. The filter housings were sealed at both

ends and incubated in the dark at 42�C for 2 h.

After incubation, the probe/hybridization solution was

removed by rinsing the membrane twice with 1 ml of PBS

containing 1 mMM VRC. Oocysts were stained with 80 llEasyStainTM (BTF Decisive Microbiology) containing

RNasin (Promega Corp., Annandale, NSW, Australia) at

40 units ml)1. After 15 min at room temperature, the

membranes were washed with 1 ml of EasyStainTM wash

buffer containing 1 mmol)1 VRC and mounted on micro-

scope slides.

Soil moisture determination

Moisture curves for the two soils were determined using the

evaporation method (Wendroth et al. 1993) for the higher

end of the moisture range (h ¼ 0Æ25–0Æ6) and by the

pressure plate method (Dane and Hopmans 2002) for

the lower end of the moisture range (h < 0Æ25). This enabledthe laboratory gravimetrically determined soil h-values to berelated to the moisture potential of the soil matrix.

Data analysis

The effects of the different environmental factors: soil type,

biotic status and moisture on concentrations of FISH-

positive Cryptosporidium oocysts were determined by ana-

lysis of variance (ANOVAANOVA) using the SAS Generalized Linear

Model procedure (Version 8.1; SAS Institute Inc., Cary,

NC, USA). The Student–Newman–Keuls Test was used to

test for significant differences between log10 means at the

a ¼ 0Æ05 level. The model used to determine inactivation

rates was log10 Nt/N0 ¼ )KT. Inactivation rates were

calculated using linear regression of log10 Nt against time,

where Nt was the concentration of viable oocysts at time t(least squares technique; SAS). The slope of the line of best

fit was equal to )K and the intercept was equal to log10 N0,

where N0 was the mean concentration at time zero. A

measure of the appropriateness of this approach was

derived by assessing the R2-value and significance of the

regression model and parameter values at the a ¼ 0Æ05level.

RESULTS

The initial viabilities (i.e. at T ¼ 0) of the three oocyst

batches as determined by excystation were 87Æ5, 80 and

93Æ3%, used for 4, 20 and 35�C microcosms, respectively,

and all >92% by FISH (minimum acceptable initial viability

was 80%; Anon. 1999). Examination of extracted DNA from

each of the oocyst batches using PCR indicated that they

were genetically identical to each other (not shown), and

based on morphology, considered to be C. parvum.Recoveries of 100 ColorSeedTM oocysts from soil were

determined throughout the experiment. However, a decision

was made not to adjust the data for the percentage recoveries

of ColorSeedTM based on the fact that the recoveries were

not significantly different (at a ¼ 0Æ05) for the two soil

types, and that the recovery of ColorSeedTM may not be

representative of the recovery of soil-aged oocysts. Mean

percentage recoveries were 41 ± 13% (n ¼ 16) and

39 ± 20% (n ¼ 15), for sites 6 and 11 soils respectively.

These recoveries were similar to those reported previously

for Cryptosporidium in soils (Davies et al. 2003).Total and viable (FISH-positive) concentrations of Cryp-

tosporidium oocysts over time, at 35, 20 and 4�C are shown in

Figs 1–3 respectively. In general, the total concentrations of

Cryptosporidium oocysts remained relatively constant over

time, whereas the concentrations of viable oocysts decreased

with time, except at 4�C where they appeared to remain

relatively constant.

Particle size analysis classified site 6 soil as a loam (49%

sand, 27% silt, 24% clay) and site 11 soil as a clay loam (7%

sand, 55% silt, 38% clay) (W. Hijnen and P. Stuyfzand,

pers. comm.). These two catchment soils were chosen for

the inactivation experiments because they were appreciably

different in texture and pH (pH 5Æ7 and 4Æ5 for sites 6 and

11 respectively).

Soil moisture in terms of the moisture characteristic h is

given in Table 1. In real terms, the lower and higher

moisture regimes designated ‘dry’ and ‘wet’, represented soil

moistures (h) of 0Æ05–0Æ2, and 0Æ2–0Æ6, respectively, depend-ing on temperature. ‘Dry’ conditions were close to soil

moisture potential at wilting point (less than )1500 kPa),

and ‘wet’ conditions approximated soil moisture potential

at field capacity ()10 kPa). The largest difference between

wet and dry soil moistures was seen at 35�C, and the

310 C.M. DAVIES ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x

smallest difference was seen at 4�C indicating the effect of

temperature on this technique as a means of maintaining

constant moisture conditions in microcosms.

Cryptosporidium inactivation at the three different tem-

peratures could not be compared statistically by ANOVAANOVA

because, as a result of the large number of samples to be

processed at each sampling interval, microcosms at each

temperature had to be staggered and, therefore, sampled on

different occasions. In order to obtain sufficient data points

for inactivation rates to be estimated, microcosms had to be

sampled over a shorter time period at 35�C than at 4�C. Theeffects of time, soil type, biotic status and soil moisture were

therefore examined for each individual temperature. The

justification for not comparing data collected at different

temperatures is covered in the existing literature, which

provides ample evidence that inactivation of Cryptosporidium

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7·00

0 5 10 15 20 25 30 35 40 45Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7·00

0 5 10 15 20 25 30 35 40 45Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7·00

0 5 10 15 20 25 30 35 40 45Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7·00

0 5 10 15 20 25 30 35 40 45Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

(a)

(c) (d)

(b)

Fig. 1 Mean log10 concentrations of total and ‘viable’ Cryptosporidium oocysts at 35�C in (a) nonirradiated site 6 soil, (b) nonirradiated site 11

soil, (c) gamma-irradiated site 6 soil and (d) gamma-irradiated site 11 soil. (m) Total concentration in dry soil; (n) viable concentration in dry soil;

(j) total concentration in wet soil; (() viable concentration in wet soil. Error bars represent ± 1 S.D.S.D. of five replicates. Not adjusted for recovery

INACTIVATION OF CRYPTOSPORIDIUM IN SOIL 311

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x

oocysts occurs at a higher rate at higher temperatures

(Jenkins et al. 1999, 2002).Log10 transformation of the oocyst concentrations

improved the heterogeneity of the variances and the

normality of the residuals, as did square root transforma-

tion. However, only observations from analysis of the log10-

transformed data are included here. ANOVAANOVA was performed

on the log10 FISH-positive oocyst concentrations. The

moisture of the soil did not significantly effect the

concentration of these potentially viable Cryptosporidium

oocysts under any set of conditions, except in gamma-

irradiated soil at 20�C (P < 0Æ0001). At 35�C, ‘viable’

oocyst concentrations remained significantly higher in site

11 soil than in site 6 soil, and also in gamma-irradiated soil

compared with nonirradiated soil (P < 0Æ0001). However,

there was also a significant interaction between biotic status

and time at 35�C, which suggests that the significant

differences in ‘viable’ Cryptosporidium concentration for

different soil biotic status should be interpreted carefully.

At 4 and at 20�C, ‘viable’ oocyst concentrations also

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7·00

0 20 40 60 80 100 120 140 160 180Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7·00

0 20 40 60 80 100 120 140 160 180Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7·00

0 20 40 60 80 100 120 140 160 180Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7.00

0 20 40 60 80 100 120 140 160 180

Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

(a) (b)

(c) (d)

Fig. 2 Mean log10 concentrations of total and ‘viable’ Cryptosporidium oocysts at 20�C in (a) nonirradiated site 6 soil, (b) nonirradiated site 11 soil,

(c) gamma-irradiated site 6 soil and (d) gamma-irradiated site 11 soil. (m) Total concentration in dry soil; (n) viable concentration in dry soil;

(j) total concentration in wet soil; (() viable concentration in wet soil. Error bars represent ± 1 S.D.S.D. of five replicates. Not adjusted for recovery

312 C.M. DAVIES ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x

remained significantly higher in site 11 soil than in site 6

soil (P < 0Æ0001).Inactivation rates in terms of the log10 K-values derived

from each combination of temperature, moisture, biotic

status and soil type are given in Table 2. The R2-values for

the goodness-of-fit of the relationship log10 Nt/N0 ¼ )KTare also given. In general the R2-values were above 0Æ7 and

were significant at a ¼ 0Æ05, with the exception of those

derived from data collected at 4�C where the fit was poor.

However, this is not surprising given that there was little or

no inactivation of oocysts with time (over the 180 days

monitored), resulting in apparent positive K-values for site11 soil at 4�C.

Figure 4 summarizes the effects of the different factors on

Cryptosporidium oocyst inactivation rates for each combina-

tion of factors. The 95% confidence intervals for the mean

K-values are also given. It can be seen that the inactivation

rates are significantly different at the three different

temperatures with greatest inactivation occurring at 35�Cand least inactivation occurring at 4�C. There is a greater

difference between inactivation rates at 35 and 20�C, than at

20 and 4�C, particularly for site 6 soil. Inactivation of

oocysts in site 6 soil appears to be more rapid than in site 11

soil at 35 and 4�C. Most importantly, the moisture

characteristics of the soil and the biotic status appear to

have little effect on the inactivation rate.

Table 1 Soil moisture content for microcosms over time

Time (days)

Soil moisture characteristic (h) (m3 m)3)

Site 6 soil Site 11 soil

4�C 20�C 35�C 4�C 20�C 35�C

D W D W D W D W D W D W

12 0Æ05 0Æ24 – – – – 0Æ17 0Æ21 – – – –

13 – – 0Æ22 0Æ22 – – – – 0Æ31 0Æ31 – –

20 – – 0Æ05 0Æ13 – – – – 0Æ12 0Æ3 – –

26 – – – – 0Æ06 0Æ49 – – – – 0Æ15 0Æ641 – – – – 0Æ06 0Æ52 – – – – 0Æ15 0Æ5868 – – 0Æ05 0Æ14 – – – – 0Æ15 0Æ26 – –

76 0Æ05 0Æ2 – – – – 0Æ17 0Æ21 – – – –

103 – – 0Æ06 0Æ25 – – – – – – – –

144 – – 0Æ06 0Æ13 – – – – 0Æ16 0Æ25152 0Æ05 0Æ14 – – – – 0Æ14 0Æ27 – – – –

D, dry; W, wet.

0·00

1·00

2·00

3·00

4·00

5·00

6·00

7.00

0 40 80 120 160Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 40 80 120 160Time (days)

Log 1

0 co

ncen

trat

ion

g–1 (

dry

wt)

(a) (b)

Fig. 3 Mean log10 concentrations of total and ‘viable’ Cryptosporidium oocysts at 4�C in (a) nonirradiated site 6 soil, (b) nonirradiated site 11 soil.

(m) Total concentration in dry soil; (n) viable concentration in dry soil; (j) total concentration in wet soil; (() viable concentration in wet soil.

Error bars represent ± 1 S.D.S.D. of five replicates. Not adjusted for recovery

INACTIVATION OF CRYPTOSPORIDIUM IN SOIL 313

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x

DISCUSSION

Previous studies have indicated that only a few oocysts from

a virulent strain are required to initiate Cryptosporidiuminfection in humans (Okhuysen et al. 1998) and that oocysts

may be released from animal faeces and transported overland

and into surface waters used as water supplies (Davies et al.2004). Thus, in order to assess the risk to surface water

supplies from animal faeces deposited on agricultural lands

it is important to be able to determine inactivation rates for

oocysts in various soil and faecal matrices. The inactivation

of Cryptosporidium oocysts in soil has been considered here

in the absence of sunlight, which was reported as being the

single most important factor affecting the inactivation of

bacteria in the environment (Chamberlain and Mitchell

1978). Nasser et al. (2003) also reported that oocyst

infectivity decreased significantly more rapidly in seawater

and in tap water in the presence of sunlight than in the dark.

The estimates of inactivation rates for Cryptosporidiumpresented in Table 2, therefore, are probably conservative,

as Cryptosporidium present on the surface of soil will

undoubtedly also be exposed to the microbicidal effects of

solar irradiation. However, it is assumed that the majority of

oocysts will be protected in the bulk of the soil matrix, albeit

in the top few centimetres of the bulk soil (Mawdsley et al.1996; McGechan 2002). To compliment the work carried

out in the present study, the inactivation of Cryptosporidiumoocysts in bovine faeces are the focus of further studies in

our laboratory.

In the absence of sunlight, temperature was the most

influential factor tested within the present study with regard

to Cryptosporidium oocyst inactivation. It is important,

therefore, when modelling the fate of pathogens in the

environment, that inactivation rates used are appropriate for

temperatures for the climate in question. Previous studies

have examined inactivation of Cryptosporidium at tempera-

tures of up to 30�C (Walker et al. 2001; Jenkins et al. 2002)but surface soil temperatures in some parts of the world

including Australia may exceed this during summer. Soil

type (texture) also significantly affected inactivation, and

similarly inactivation rates used in models must be appro-

priate for the soil types present. Soil moisture within the

range tested was not influential, an observation also noted by

Table 2 Log10 inactivation rates (K) for Cryptosporidium oocysts in soil

Temperature (�C) Biotic status Moisture regime

Site 6 soil Site 11 soil

K (95% CI) (day)1) R2 K (95% CI) (day)1) R2

35 NI Dry 0Æ0790 (0Æ0896, 0Æ0684) 0Æ92* 0Æ0425 (0Æ0533, 0Æ0317) 0Æ74*Wet 0Æ0818 (0Æ0928, 0Æ0708) 0Æ92* 0Æ0421 (0Æ0506, 0Æ0336) 0Æ82*

GI Dry 0Æ0599 (0Æ0703, 0Æ0494) 0Æ86* 0Æ0369 (0Æ0436, 0Æ0302) 0Æ86*Wet 0Æ0683 (0Æ0785, 0Æ0582) 0Æ89* 0Æ0249 (0Æ0309, 0Æ0189) 0Æ76*

20 NI Dry 0Æ0221 (0Æ0249, 0Æ0193) 0Æ90* 0Æ0135 (0Æ0167, 0Æ0102) 0Æ77*Wet 0Æ0213 (0Æ0242, 0Æ0185) 0Æ89* 0Æ0151 (0Æ0173, 0Æ0129) 0Æ89*

GI Dry 0Æ0221 (0Æ0255, 0Æ0186) 0Æ88* 0Æ0117 (0Æ0141, 0Æ0093) 0Æ83*Wet 0Æ0181 (0Æ0214, 0Æ0149) 0Æ85* 0Æ0095 (0Æ0109, 0Æ0081) 0Æ90*

4 NI Dry 0Æ0062 (0Æ0122, 0Æ0009) 0Æ17ns )0Æ0026� ()0Æ0024�, 0Æ0076) 0Æ05nsWet 0Æ0050 (0Æ0091, 0Æ0008) 0Æ22ns )0Æ0051� ()0Æ0021�, 0Æ0080) 0Æ36ns

GI Dry NA – NA –

Wet NA – NA –

CI, confidence interval; NI, nonirradiated, ns, not significant; GI, gamma-irradiated.

*Significant at a ¼ 0Æ05.�Negative K-value indicates no inactivation.

–0·02

0

0·02

0·04

0·06

0·08

0.1

6NID

ry

6NIW

et

6GID

ry

6GIW

et

11N

IDry

11N

IWet

11G

IDry

11G

IWet

Factor combinations

K (

days

–1)

Fig. 4 Log10 inactivation rates (K) for Cryptosporidium exposed to

various combinations of soil type, moisture, biotic status and

temperature. (m) 4�C; (s) 20�C; (d) 35�C. Error bars are 95%confidence intervals for the mean K-values. 6, Site 6 soil; 11, site 11

soil; NI, nonirradiated; GI, gamma-irradiated; dry, wilting point; wet,

field capacity

314 C.M. DAVIES ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x

Jenkins et al. (2002) and Kato et al. (2004). However, in

contrast, Nasser et al. (2003) suggested that infectivity

(in HCT-8 cells) over 10 days at 30�C decreased by 90%

in a saturated loam soil compared with 99Æ99% in air-dried

soil.

Given that increases in either total or viable oocyst

concentrations with time are impossible, the observed

increase in site 11 soil at 4�C is most likely to be a result

of increased recovery of soil-aged oocysts by the technique

used and warrants further investigation.

The inactivation rates for Cryptosporidium oocysts in soil

in this study are similar to those reported by Jenkins et al.(2002) for similar soil types and conditions. For example, at

20�C in a silt clay loam soil, Jenkins et al. (2002) reportedan inactivation rate (K) of 0Æ0111 day)1. We report

inactivation rates (K) of 0Æ0135–0Æ0151 day)1 at 20�C in

clay loam soil (site 11 soil) depending on biotic status. In

the present study, the mean inactivation rates at 4�C in clay

loam soil ()0Æ0026 and )0Æ0051 day)1) suggest that there

was little inactivation of oocysts. However, if the 95%

confidence intervals for the means are taken into consid-

eration ()0Æ0024–0Æ0076 day)1), the inactivation rate over-

laps that of Jenkins et al. (2002) for similar conditions

(0Æ0030 day)1). It is difficult to make any further compar-

isons as the texture and probably the physicochemical

characteristics of the other soils in the two studies are

appreciably different. In addition, there are inherent

differences in the approaches used in different inactivation

studies, which may account for some differences in reported

inactivation rates. For instance Jenkins et al. (2002) used

the dye permeability technique to determine oocyst viabil-

ities, whereas in the present study FISH was used. The

sentinel chambers used by Jenkins et al. (2002) were placedin bulk soil, and because of their semipermeable nature

some exchange with the surrounding bulk soil was allowed.

Whereas, in the present study, closed microcosms contain-

ing a small amount of test soil were used. It has also been

suggested that during extraction/preparation of oocysts for

inactivation studies, the use of harsh chemicals that may

render the oocysts more sensitive to environmental factors

being tested should be avoided (Anon. 1999). However,

Slifko et al. (2000) reported that the use of defatting agents

such as diethyl ether and use of IMS (employing acid to

dissociate oocysts from beads) had no detrimental effects on

oocyst infectivity.

In a recent study, Kato et al. (2004) deployed sentinel

chambers containing soil spiked with C. parvum at field sites.

Oocyst viability was assessed using the dye exclusion

technique. No significant effect of soil moisture was found,

which supports the observations made in the present study.

Ambient temperature remained largely between 0 and 5�Cduring the experiment with occasional freezing. The

inactivation rates determined by Kato et al. (2004) were

generally more rapid (confidence interval 0Æ016–0Æ043 day)1)

than those presented for 4�C in the present study (confid-

ence interval 0Æ0008–0Æ0122 day)1). This may be due to

detrimental effect on oocyst viability of the repeated freeze–

thawing events that occurred during the field study. There is

a need for large-scale inactivation studies to be carried out to

verify that it is appropriate to extrapolate from data obtained

at the scale used in the present study and in the study of

Kato et al. (2004) to field conditions.

There has been much debate in recent years over the value

of oocyst viability data derived from the use of methods that

determine the ‘viabilities’/activities of oocysts rather than

their infectivities, for example, excystation, dye exclusion

and FISH. However, apparent oocyst viabilities measured

using FISH have shown modest agreement with the results

of cell culture infectivity assays, with the discrepancies

occurring mostly at low viabilities (Jenkins et al. 2003), andhigh agreement with excystation (Vesey et al. 1997). Sincethe publication of the comparative study of methods for

Cryptosporidium viability assessment by Jenkins et al. (2003),the FISH protocol (Deere et al. 1998; Vesey et al. 1998) hasbeen modified to include RNase pretreatment steps that

reduce the numbers of false-positive viable oocysts (Smith

et al. 2004). In addition, the modified FISH method used in

the present study included adaptation of the protocol for use

with membrane filters and the use of a combination of the

RNase inhibitors, RNasin and VRC to inactivate residual

RNase and stabilize the FISH signal thereby increasing the

allowable storage time of the slides (N. Altavilla and N.A.

Ashbolt, in prep.). Therefore, it may be assumed that as a

result of these modifications, the agreement of oocyst

viability data determined by FISH with oocyst infectivity

data may also have improved, although this remains to be

confirmed. For the purpose of generating inactivation rates

for use in predictive models, which should err on the

conservative side, the use of FISH was considered an

acceptable and cost-effective approach to assessing loss of

apparent oocyst viability under the influence of various

environmental factors.

One of the major limitations to modelling pathogen

export from land to surface waters at a level equivalent to

that carried out for sediment and nutrients, has been the

lack of accurate data that is relevant to field conditions.

Previous models for predicting the fate of pathogens in

catchments have either made nonvalidated assumptions

about inactivation rates (Walker and Stedinger 1999), or

have not considered inactivation at all (Fraser et al. 1998).The inactivation rates generated in the present study may

serve as suitable input functions to models for predicting

the fate and transport of surface water pathogens thereby

enabling better management of factors that govern the

attenuation and transport of pathogens in water supply

catchments.

INACTIVATION OF CRYPTOSPORIDIUM IN SOIL 315

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x

ACKNOWLEDGEMENTS

This work was funded by the American Water Works

Association Research Foundation (AwwaRF), the Cooper-

ative Research Centre for Water Quality and Treatment,

Sydney Catchment Authority, Melbourne Water Corpora-

tion, and the Water Services Association of Australia, as part

of AwwaRF project no. 2694. The authors would like to

thank Dr Peter Beatson and Christine Kaucner (UNSW),

Wim Hijnen and Pieter Stuyfzand (KIWA Water Research,

The Netherlands), and Dr Damien Field (University of

Sydney) for excellent technical assistance and advice.

REFERENCES

Anon. (1999) Towards a Standardised Experimental Design for Viability

and Inactivation Studies. Report on Workshop on Cryptosporidium

and Water. Tadley, Hampshire, UK: Tadley Court. 11–12 August

1999. http://www.dwi.gov.uk/pubs/tadley/pdf/tadleycr.pdf.

APHA (1998) Standard Methods for the Examination of Water and

Wastewater, 20th edn. Washington, DC: APHA.

Atwill, E.R., Hou, L., Karle, B.M., Harter, T., Tate, K.W. and

Dahlgren, R.A. (2002) Transport of Cryptosporidium parvum oocysts

through vegetated buffer strips and estimated filtration efficiency.

Applied and Environmental Microbiology 68, 5517–5527.

Blasdall, S.A., Ongerth, J.E. and Ashbolt, N.J. (2002) Sub-species

typing among bovine C. parvum isolates by PCR-PAGE using a

novel microsatellite + telomere primer scheme. Water Science and

Technology 2, 81–87.

Bradford, S.A. and Schjiven, J. (2002) Release of Cryptosporidium and

Giardia from dairy calf manure: impact of solution salinity.

Environmental Science and Technology 36, 3916–3923.

Chamberlain, C.E. and Mitchell, R. (1978) A decay model for enteric

bacteria in natural waters. In Water Pollution Microbiology, vol. 2. ed.

Mitchell, R. pp. 325–348. New York: Wiley.

Dane, J.H. and Hopmans, J.W. (2002) Pressure plate extractor. In

Methods of Soil Analysis, Part 4, 5th edn. ed. Topp, G.C. pp. 688–

690. Madison, WI: Soil Science Society of America.

Davies, C.M., Kaucner, C., Deere, D. and Ashbolt, N.J. (2003)

Recovery and enumeration of Cryptosporidium parvum in animal

faecal matrices. Applied and Environmental Microbiology 69, 2842–

2847.

Davies, C.M., Ferguson, C.M., Kaucner, C., Krogh, M., Altavilla, N.,

Deere, D.A. and Ashbolt, N.J. (2004) Dispersion and transport of

Cryptosporidium oocysts from fecal pats under simulated rainfall

events. Applied and Environmental Microbiology 70, 1151–1159.

Deere, D., Vesey, G., Milner, M., Ashbolt, N., Williams, K. and Veal,

D. (1998) Optimisation of fluorescent in-situ ribosomal RNA

labelling of Cryptosporidium parvum in suspension. Journal of Applied

Microbiology 85, 807–818.

Ferguson, C.M., de Roda Husman, A.M., Altavilla, N., Deere, D. and

Ashbolt, N.J. (2003) Fate and transport of surface water pathogens

in watersheds. Critical Reviews in Environmental Science and

Technology 33, 299–361.

Ferguson, C.M., Ashbolt, N.J. and Deere, D.A. (2004) Prioritization of

catchment management in the Sydney catchment – construction of a

pathogen budget. Water Science and Technology Water Supply 4,

35–38.

Fraser, R.H., Barten, P.K. and Pinney, D.A.K. (1998) Predicting

stream pathogen loading from livestock using a geographical

information system-based delivery model. Journal of Environmental

Quality 27, 935–945.

Jenkins, M.B., Walker, M.J., Bowman, D.D., Anthony, L.C. and

Ghiorse, W.C. (1999) Use of a sentinel system for field measure-

ments of Cryptosporidium parvum oocyst inactivation in soil and

animal waste. Applied and Environmental Microbiology 65, 1998–2005.

Jenkins, M., Bowman, D.D., Fogarty, E.A. and Ghiorse, W.C. (2002)

Cryptosporidium parvum oocyst inactivation in three soil types at

various temperatures and water potentials. Soil Biology and

Biochemistry 34, 1101–1109.

Jenkins, M., Trout, J.M., Higgins, J., Dorsch, M., Veal, D. and Fayer,

R. (2003) Comparison of tests for viable and infectious Cryptospor-

idium parvum oocysts. Parasitology Research 89, 1–5.

Kato, S., Jenkins, M., Fogarty, E. and Bowman, D. (2004) Cryptos-

poridium parvum oocyst inactivation in field soil and its relation to

soil characteristics: analyses using the geographic information

systems. Science of the Total Environment 321, 47–58.

Lim, Y.A.L., Ahmad, R.A., Osman, A. and Zulkeflie, Z. (1999)

Survival of Cryptosporidium parvum oocysts in river and soil

environments. Tropical Biomedicine 16, 7–15.

Mawdsley, J.L., Brooks, A.E. and Merry, R.J. (1996) Movement of the

protozoan pathogen Cryptosporidium parvum through three contrast-

ing soil types. Biology and Fertilisation of Soils 21, 30–36.

McGechan, M.B. (2002) Transport of particulate and colloid-sorbed

contaminants through soil, Part 2: trapping processes and soil pore

geometry. Biosystems Engineering 83, 387–395.

Nasser, A.M., Teuto, E., Tenenbaum, L. and Netzan, Y. (2003) Die-off

of Cryptosporidium spp. in Tap Water, in Seawater and in Soil:

Comparison Between Infectivity and Viability. Presented at the IWA

Health Related Water Microbiology Symposium, 14–17 September

2003. Cape Town, South Africa. London: IWA.

Okhuysen, P.C., Chappell, C.L., Sterling, C.R., Jakubowski, W. and

Dupont, H.L. (1998) Susceptibility and serologic response of

healthy adults to reinfection with Cryptosporidium parvum. Infection

and Immunology 66, 441–443.

Olsen, M.E., Goh, J., Phillips, M., Guselle, N. and McAllister, T.A.

(1999) Giardia cyst and Cryptosporidium oocyst survival in water,

soil, and cattle feces. Journal of Environmental Quality 28, 1991–

1996.

Slifko, T.R., Coulliette, A., Huffman, D.E. and Rose, J.B. (2000)

Impact of purification procedures on the viability and infectivity of

Cryptosporidium parvum oocysts. Water Science and Technology 41,

23–29.

Smith, J.J., Gunasekera, T.S., Barardi, C.R.M., Veal, D. and Vesey,

G. (2004) Determination of Cryptosporidium parvum oocyst viability

by fluorescence in situ hybridization using a ribosomal RNA-directed

probe. Journal of Applied Microbiology 96, 409–417.

Udeh, P.J., John, G. and Veenstra, J.N. (2003) Field inactivation of

oocysts exposed to agricultural land. Water, Air and Soil Pollution

142, 211–228.

Upton, S.J. (1997) In vitro cultivation. In Cryptosporidium and

Cryptosporidiosis ed. Fayer, R. pp. 181–207. Washington, DC:

CRC Press.

316 C.M. DAVIES ET AL.

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x

USEPA (2001) Protocol for Developing Pathogen TMDLs. EPA 841-R-

00–002. Washington, DC: Office of Water, United States Environ-

mental Protection Agency.

Vesey, G., Griffiths, K.R., Gauci, M.R., Deere, D., Williams, K.L.

and Veal, D.A. (1997) Simple and rapid measurement of Cryptos-

poridium excystation using flow cytometry. International Journal for

Parasitology 27, 1353–1359.

Vesey, G., Ashbolt, N., Fricker, E.J., Deere, D., Williams, K.L., Veal,

D.A. and Dorsch, M. (1998) The use of a ribosomal RNA targeted

oligonucleotide probe for fluorescent labelling of viable Cryptospor-

idium parvum oocysts. Journal of Applied Microbiology 85, 429–440.

Walker, F.R. and Stedinger, J.R. (1999) Fate and transport model

of Cryptosporidium. Journal of Environmental Engineering 125, 325–

333.

Walker, M., Leddy, K. and Hagar, E. (2001) Effects of combined

water potential and temperature stresses on Cryptosporidium

parvum oocysts. Applied and Environmental Microbiology 67, 5526–

5529.

Wendroth, O., Ehlers, W., Hopmans, J.W., Kage, H.J.H. and Wosten,

J.H.M. (1993) Reevaluation of the evaporation method for deter-

mining hydraulic functions in unsaturated soils. Soil Science Society

of America Journal 57, 1436–1443.

INACTIVATION OF CRYPTOSPORIDIUM IN SOIL 317

ª 2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 308–317, doi:10.1111/j.1365-2672.2004.02459.x