APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2010, p. 5140–5147
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 15
Determining UV Inactivation of Toxoplasma gondii Oocysts by Using
Cell Culture and a Mouse Bioassay?
Michael W. Ware,1§ Swinburne A. J. Augustine,1§ David O. Erisman,1Mary Jean See,3†
Larry Wymer,1Samuel L. Hayes,2J. P. Dubey,4and Eric N. Villegas1,3*
National Exposure Research Laboratory1and National Risk Management Research Laboratory,2U.S. Environmental Protection Agency,
Cincinnati, Ohio 45268; Department of Biological Sciences, McMicken College of Arts and Sciences, University of
Cincinnati, Cincinnati, Ohio 452203; and Animal Parasitic Disease Laboratory, Agricultural Research Service,
U.S. Department of Agriculture, Beltsville, Maryland 207054
Received 21 January 2010/Accepted 30 May 2010
The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water
disinfection. This study evaluated UV-irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro
T. gondii oocyst plaque (TOP) assay, and a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay.
The results from the animal bioassay show that 1- and 3-log10inactivation is achieved with 4 mJ/cm2UV and
10 mJ/cm2low-pressure UV, respectively. TOP assay results, but not RT-qPCR results, correlate well with
bioassay results. In conclusion, a 3-log10inactivation of T. gondii oocysts is achieved by 10-mJ/cm2low-pressure
UV, and the in vitro TOP assay is a promising alternative to the mouse bioassay.
Toxoplasma gondii is an obligate intracellular protozoan par-
asite that commonly infects humans (19). Its human prevalence
ranges worldwide by country from 10% to over 80% of the
population (4, 38). Infection with T. gondii is typically asymp-
tomatic in healthy individuals but results in a lifelong infection
that can reactivate, causing toxoplasmic encephalitis and death
if the individual becomes immunocompromised (49). In addi-
tion, if T. gondii is acquired during pregnancy, there is risk of
transplacental transmission to the fetus, causing fetal death,
abortion, malformation, or mental retardation. Recent studies
have reported that acquired acute toxoplasmosis can occur in
healthy individuals with symptoms of acute retinitis, prolonged
fever, elevated liver enzyme levels, and respiratory distress,
which resulted in one patient succumbing to acute disease (11,
12, 16). An outbreak of acquired acute T. gondii retinitis in
healthy individuals has also been reported (10). These recent
increases in the incidence of acute toxoplasmosis suggest the
emergence or reemergence of a T. gondii strain(s) that is more
pathogenic and poses greater human health risks than was
previously assessed (37).
Typically, T. gondii outbreaks are food borne and associated
with tissue cyst-contaminated meat from pigs, sheep, goats,
and poultry (58). However, improved animal husbandry and
hygiene management practices have significantly reduced the
prevalence of T. gondii cysts in meat (20, 58). Despite these
significant changes, the seroprevalence in humans remains rel-
atively high, suggesting that other sources of exposure to T.
gondii, such as oocysts in water or soil, are likely. Indeed,
worldwide, several waterborne outbreaks associated with pub-
lic water supplies have been documented (4, 5, 7, 17, 30). In the
1994 waterborne toxoplasmosis outbreak in Victoria, British
Columbia, the likely source of T. gondii was traced back to a
municipal water supplier that provided unfiltered chlorami-
nated drinking water to residents of the Greater Victoria re-
gion (7). The cases of toxoplasmosis reported coincided with
recent rainfall events but not with any breakdown in treatment
processes. Also, no increases in other pathogen levels were
reported. An epidemiological survey of wild felid species in the
surrounding area further revealed that cougars found living
near the watershed were seropositive for T. gondii antibodies,
with one cougar actively shedding T. gondii oocysts in its feces,
suggesting that the surface water sources were likely contam-
inated with T. gondii oocysts (2, 3, 7). Recent studies report
that toxoplasmosis in California sea otters can be attributed to
surface water runoff containing T. gondii oocysts that have
contaminated shellfish and other near-shore-dwelling marine
animals (35, 45–47). In addition, the oocyst can survive for a
long time period and is highly resistant to chemical disinfection
(18, 26, 27, 63). Taken together, all of these factors and studies
suggest that T. gondii oocysts may pose a significant risk to
drinking and recreational water quality worldwide.
Current drinking water regulations for protozoan pathogens
focus on Cryptosporidium oocysts and Giardia cysts. No specific
regulations, guidelines, or approved methods exist to monitor
or control T. gondii oocysts in drinking and recreational water
systems. Research efforts aimed at developing methods to
monitor for T. gondii oocysts in water are limited and have not
been used routinely to monitor source and recreational waters
(23, 24, 32, 53, 61, 64). Specific recommendations on disinfec-
tion treatment practices for publicly owned treatment works
(POTW) or drinking water utilities to inactivate waterborne T.
gondii oocysts also do not exist (1).
UV disinfection is a relatively old technology but has grown
in popularity and use in drinking water industries because it
forms few disinfection by-products (DBP) (31, 59). Cryptospo-
ridium is also highly resistant to chemical inactivation, but
* Corresponding author. Mailing address: Biohazard Assessment
Research Branch, Microbiological and Chemical Exposure Assess-
ment Research Division, National Exposure Research Laboratory,
U.S. Environmental Protection Agency, Cincinnati, OH 45268. Phone:
(513) 569-7017. Fax: (513) 569-7117. E-mail: email@example.com.
§ M.W.W. and S.A.J.A. contributed equally to the manuscript.
† Present address: Dynamac, Inc., Cincinnati, OH.
?Published ahead of print on 11 June 2010.
several studies show that Cryptosporidium parvum oocysts are
inactivated by UV radiation at fluences applicable to drinking
water treatment. These studies report that UV fluences of ?25
mJ/cm2achieve at least a 3-log10inactivation as determined by
either animal infectivity or cell culture (9, 13, 14, 51, 55).
Similar results are observed with Cryptosporidium hominis oo-
cysts (34). In addition, results from cell culture and animal
infectivity studies are not statistically different (51). The
USEPA Long-Term 2 Enhanced Surface Water Treatment
Rule (LT2) has defined that a 2-, 3-, and 4-log10inactivation of
C. parvum is achieved with UV doses of 5.8, 12, and 22 mJ/cm2,
respectively (59). To date, there are only two proceedings
documents demonstrating the potential for UV irradiation of
T. gondii oocysts (54, 56) and two published studies evaluating
UV irradiation and T. gondii oocysts (26, 62). The reports by
Sobsey and colleagues suggested that a 2-log10reduction of T.
gondii oocyst infectivity was achieved at 40 mJ/cm2(54, 56).
Dume `tre et al. reported a 4-log10inactivation after a UV dose
of 20 mJ/cm2(26), whereas Wainwright et al. reported that
some oocysts remained infectious to mice after UV doses of
?500 mJ/cm2(62). These results report a wide range of UV
exposures that are required to inactivate oocysts. To better
assess UV inactivation efficacies, a more comprehensive anal-
ysis of the effect of UV on T. gondii oocysts is warranted.
This study examines the effect of UV exposure on the via-
bility of T. gondii oocysts and determines the UV fluence re-
quired for a 3-log10inactivation using three analytical ap-
proaches established in our laboratory: (i) a SCID mouse
bioassay, (ii) a quantitative reverse transcriptase real-time
PCR (RT-qPCR) assay, and (iii) an in vitro T. gondii oocyst
plaque (TOP) assay.
MATERIALS AND METHODS
T. gondii oocysts. Partially purified, sporulated T. gondii (VEG strain) oocysts
were obtained from the feces of laboratory-infected cats as previously described
(22). The oocysts were further purified on a cesium chloride gradient as de-
scribed, except that the centrifugation was at 12,000 ? g (25, 57). The purified
oocysts were resuspended in 2% H2SO4and stored at 4°C. Prior to use, the
oocyst storage buffer was neutralized by addition of three-fifths of the original
volume (vol/vol) of 1 N NaOH. The oocysts were then washed with phosphate-
buffered saline (PBS). Oocysts used for mouse bioassays ranged in age from 1
month to 1 year, with no observed differences in infectivity, while oocysts that
were less than 3 months old were used for the TOP assay (18, 26, 27, 63).
Oocyst enumeration. Oocyst doses were enumerated by flow cytometry. All of
the studies evaluating the 4-, 40-, and 100-mJ/cm2-irradiated oocysts and their
respective controls were sorted with a FACS Vantage SE (Becton-Dickinson,
San Jose, CA) equipped with CloneCyt as previously described for C. parvum,
except that the sorting gate was set by autofluorescence isolating sporulated
oocysts (42, 48). The remaining studies were sorted with a FACS Aria (Becton-
Dickinson), and the oocysts were gated by forward- and side-scatter profiles, as
the autofluorescence could not be detected with the laser configuration. The sort
total was increased by 10% to normalize for the sporulated oocysts because 10%
of the total oocysts were unsporulated. For both flow cytometers, oocyst doses
were prepared from 5 to 25,000 oocysts per mouse in PBS by preparing a vial for
each dose group.
Mice. For each study, 20 to 85 male IcrTac:ICR-Prkdcscidmice aged 3 to 4
weeks were obtained from Taconic (Hudson, NY) and acclimated for at least 1
week prior to the start of experiments. They were housed in groups of 3 to 5
under barrier conditions, including sterile cages with 0.22-?m cage isolator
filters, sterile corn cob bedding, sterile water, and irradiated Pico Lab mouse diet
ad libitum. The cages were housed in an animal isolator (NuAire model 602-400;
Plymouth, MN). All animal and tissue manipulations were performed in an
animal cage station (NuAire model 650-600). All animal studies were approved
and overseen by the Cincinnati USEPA Institutional Animal Care and Use
UV inactivation. A low-pressure collimated beam apparatus was used to de-
liver UV fluences ranging from 4 mJ/cm2to 100 mJ/cm2as previously described
(29). The irradiance was measured using a factory-calibrated, U.S. NIST-trace-
able, model IL-1700 radiometer (International Light, Inc., Newburyport, MA) as
described previously (29). The UV fluences were calculated as described by
Bolton and Linden (6).
SCID mouse bioassay and necropsy. The mice were exposed to the oocysts by
vortexing the oocyst suspension and exposing each mouse to a 0.2-ml intraper-
itoneal (i.p.) injection with a 27-gauge needle. In a preliminary study, the mice
were exposed by cage to various numbers of T. gondii oocyst doses to establish
the baseline 50% infective dose (ID50). This curve was then used to evaluate the
log inactivation by UV by comparing the ID50of exposed oocysts to that of
control oocysts by using a modification of the approach taken by Korich et al.
(40). Each study also included a negative control group in which the mice were
exposed to 0.2 ml PBS. Mice were observed twice daily for signs of being
moribund, such as weight loss, ruffled fur, reduced activity, and shivering. Mor-
ibund mice were euthanized and then necropsied. In necropsy, the brains,
spleens, and lungs were aseptically removed, placed in individual cryovials, flash
frozen in liquid nitrogen, and stored at ?20°C. Mice which did not become
moribund were euthanized at 42 days postinfection and necropsied as described
Duplex qPCR tissue confirmation. Tissue samples were partially thawed and
divided by scalpel, and approximately 20% of the spleen and 10% of the lung and
brain from the center of the tissue were used to isolate genomic DNA. These
tissue portions were weighed, and the DNA was isolated using the Qiagen
QiaAmp DNA minikit (Qiagen, Valencia, CA) by following the manufacturer’s
protocol, except that RNase A was not used and the centrifugations of 6,000 ?
g were increased to 10,000 ? g. Approximately 200 mg of brain and lung tissue
and 100 mg of spleen tissue were processed for each sample.
The tissue samples were used to confirm the observed T. gondii infection data
using a duplex qPCR assay as previously described (44). All control results were
also confirmed by this assay. Sample amplification was performed in triplicate
with a duplex reaction using an Applied Biosystems 7900 real-time PCR detec-
tion system (Applied Biosystems, Foster City, CA). The primers, probes, and
GenBank accession numbers for the 35-fold B1 gene of T. gondii and the mouse
glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) are listed in Table 1
(39, 44). Each PCR assay consisted of 20 nM both probes, 20 nM GAPDH
primers, 200 nM B1 primers, 3.5 mM MgCl2, 200 ?M deoxynucleoside triphos-
phate (dNTP), 1? ROX reference dye (Invitrogen), and 2.5 U AmpliTaq gold
(Applied Biosystems). Amplification conditions were denaturation for 5 min at
TABLE 1. Real-time PCR primers for mouse GAPDH and the T. gondii B1 genes
Primer/probe name Sequence (5?33?)Nucleotide position
Mouse GAPDHGAPDH-ENV for
TCA TCT CCG CCC CTT CTG
TCG TGG TTC ACA CCC ATC AC
CGA TGC CCC CAT GTT
T. gondii B1b
CTA GTA TCG TGC GGC AAT GTG
GGC AGC GTC TCT TCC TCT TTT
CCA CCT CGC CTC TTG G
aGenBank accession numbers: GAPDH, NC_000072; and B1 gene, AF179871.
VOL. 76, 2010 INACTIVATION OF T. GONDII OOCYSTS BY UV DISINFECTION5141
95°C followed by 40 cycles of denaturation at 95°C for 30 s and annealing and
extension at 60°C for 1 min.
The cycle threshold (CT) values for each tissue sample were averaged by gene.
In those cases in which qPCRs failed to amplify within 40 cycles, the sample was
assigned a CTvalue of 40.1. This allowed undetermined values to be included in
the calculations while ensuring that the resulting average was a conservative
estimate of the CTvalue. Brain tissues were used for primary classifications, and
mice were considered positive for T. gondii if they became moribund and the
mean B1 CTwas ?34. Samples were considered negative for T. gondii if the
mouse was asymptomatic and the mean B1 CTwas ?38.7. Spleen and then lung
tissues were analyzed using the same criteria if the B1 CTwas not definitive or the
animal became moribund without detectable T. gondii DNA. The GAPDH re-
action was used both to serve as a PCR control and to confirm the presence of
mouse tissue. The qPCR results were not normalized, and total parasite burden
was not determined.
Calculation of ID50and statistical methodology. Dose-response curves and
associated ID50s were based on the logistic regression of the likelihood of
infection with a log10dose. Odds of infection were considered dependent on UV
irradiation level, taken as a categorical effect. Based on a lack of a statistically
significant (P ? 0.05) difference in their odds ratios, the data from UV irradia-
tions that were ?15 mJ/cm2were combined. Analysis of the results from the TOP
assays was based on a hierarchical Bayes model that assumed the plaque counts
to have a Poisson distribution, with the mean density being lognormally distrib-
uted from experiment to experiment and constant log reduction at 4 mJ/cm2UV
irradiation. SAS version 9.2 (SAS Institute, Cary, NC) and WinBUGS version 1.4
were used for analysis (43).
Total RNA extraction. RNA extraction was performed using a QIAgen
RNeasy microkit according to the manufacturer’s suggestions. Briefly, 3 ? 104to
1 ? 105oocysts were resuspended in 150 ?l of RLT buffer and subjected to five
freeze-thaw cycles in liquid nitrogen and a 55°C water bath. Proteinase K (30 ?l
of 2 mg/ml) and 270 ?l diethyl pyrocarbonate (DEPC) water were added to each
sample, vortexed, and incubated for 1 h at 55°C. Total oocyst lysates were then
further processed according to the manufacturer’s protocol. Briefly, RNA was
precipitated with absolute ethanol and 5 ?l (4 mg/ml) carrier RNA. The RNA
was washed with RNeasy wash solutions. The genomic DNA was removed by
adding DNase I (1 U) to each sample and incubating the sample for 15 min at
ambient temperature. Total RNA was then eluted with 35 ?l of RNase-free
water and stored at ?80°C until use.
RT-qPCR. RT-qPCR was performed to detect the ACT1 and SporoSAG
mRNA as previously described (60). Each sample analyzed contained total RNA
extracted from 1,000 oocysts as described above. The RT reaction mixture
contained 1 ?l of murine leukemia virus (MULV) RT (Promega, Madison, WI),
2.5 ?l 10? PCR buffer II (Applied Biosystems), 1.5 ?l 2.5 mM magnesium
chloride, 1 ?l 10 ?M reverse primer (Table 2), 2.5 ?l 25 ?M dNTPs (Promega),
and 0.75 ?l RNasin (Promega) in a total volume of 25 ?l. The RT reaction was
performed using a DMJ PTC-200 thermal cycler (MJ Research, Inc., Watertown,
MA) at 43°C for 1 h and then at 94°C for 5 min. Immediately following the RT
reaction, a qPCR was performed in DEPC-treated water containing 25 ?l RT
reaction mixture, 2.5 ?l 10? PCR buffer II, 5.5 ?l 25 mM magnesium chloride,
1 ?l ROX dye (Invitrogen), 1 ?l 10 ?M forward primer, 1 ?l 10 ?M probe, 0.5
?l AmpliTaq gold (Applied Biosystems) in a total volume of 50 ?l. The qPCR
was performed using ABI 7900HT (Applied Biosystems) consisting of 50°C for 2
min, 95°C for 10 min, and 40 cycles at 95°C for 15 s and then 60°C for 1 min. All
RT-qPCRs were done in triplicate. ACT1 and SporoSAG RT-qPCR primers and
probes were designed to specifically amplify the mRNA species and not genomic
DNA (Table 2). The ACT1 gene was selected to detect all forms of T. gondii and
the SporoSAG gene to detect infectious sporozoites. To verify that endogenous
DNA was destroyed, RT-qPCRs were performed without the addition of reverse
transcriptase in all experiments performed. No amplification occurred in these
Oocyst excystation. Oocyst excystation as reported by Villegas et al. was used
for this study (60). Briefly, oocysts at a concentration of 2 ? 106in 1 ml of 1?
PBS were added to 2-ml microcentrifuge screw cap conical tubes containing 0.5 g
of 0.5-mm glass beads (Biospec Products, Inc., Bartlesville, OK) and mechani-
cally disrupted using a mini-BeadBeater (BioSpec Products, Inc.) for 20 s at 250
rpm. Bovine bile (Sigma; 500 ?l, 10%) was then added to the sample and
incubated at 37°C for 1.5 h. The solution was then transferred to a new tube and
centrifuged at 21,000 ? g for 10 min at 4°C. The supernatant was then aspirated
and discarded, and the pellet was washed once with 1? PBS. The pellet was then
resuspended in 1 ml 10% Dulbecco modified Eagle medium (DMEM) (Gibco,
Gaithersburg, MD) in preparation for the TOP assay (see below).
TOP assay. Human foreskin fibroblasts (HFF) were purchased from the
American Type Culture Collection (ATCC; Manassas, VA; no. CRL-1634) and
grown in DMEM (Gibco) containing 10% fetal bovine serum (HyClone, Logan,
UT), 100 U/ml penicillin, 100 ?g/ml streptomycin, and 25 g/ml amphotericin B
(Gibco) at 37°C with 5% CO2. HFF cells were maintained as monolayers in
75-cm2cell culture flasks or seeded onto six-well tissue culture plates (Becton
Dickinson Labware, Franklin Lakes, NJ) and grown to confluence for TOP
assays. The cell monolayers were exposed to various dilutions (1 to 1 ? 106) of
excysted T. gondii oocysts and incubated at 37°C and in 5% CO2for 10 days. At
the end of the 10-day period, the cell monolayers were stained with crystal violet
for 30 min, washed, and analyzed microscopically by counting of plaques as
previously described (52, 60).
SCID mouse bioassay. The SCID mouse bioassay was de-
veloped and evaluated by determining the ID50for male SCID
mice via i.p. exposure. Figure 1 shows the effects on mice, with
times postexposure and at different oocyst doses, showing an
ID50between 10 and 50 oocysts. The date of the onset of
symptoms is related to the number of oocysts used to infect the
animal. Animals exposed to ?500 oocysts became moribund
approximately 2 weeks after exposure, while animals exposed
to ?500 oocysts became moribund approximately 3 weeks pos-
texposure. No animals developed signs of infection after day
33, even though the study continued for 42 days postinfection.
Results were confirmed by tissue analysis. A similar survival
curve was observed with SCID mice exposed by oral gavage
(44; data not shown).
FIG. 1. T. gondii oocyst infection by intraperitoneal exposure in
male SCID mice. Mouse survival by percentage of cohort after expo-
sure to T. gondii oocysts plotted against day of death postexposure by
oocyst dose. Uninfected (●) mice or mice exposed to 5 (ƒ), 10 (f), 50
(?), 500 (Œ), and 5,000 (E) oocysts were monitored for moribundity
for 42 days.
TABLE 2. Primer and probe sequences used for RT-qPCRa
ACT1TgACT1 forACA TCA AGG AGA AGC
TTT GCT ACA
TCA GCC GCC TTC ATT TCC
CGC CCT CGA CTT C
SporoSAGSporoSAG forGCG GAG ACA AGC GTT
AGC CTG TGG CTG CGC
CCT ATG CCA AAG AAC
5142 WARE ET AL.APPL. ENVIRON. MICROBIOL.
Effects of UV irradiation on T. gondii oocysts by SCID mouse
bioassay. The SCID mouse bioassay was used to estimate the
effects of UV irradiation on sporulated T. gondii oocysts as
shown in Table 3. Mice not exposed to oocysts were asymp-
tomatic. The control group exposed to untreated oocysts
showed that the ID50was ?50 oocysts because 83% of the mice
exposed to 50 oocysts and all of the mice exposed to ?500
oocysts became moribund. The 4-mJ/cm2group were asymp-
tomatic when exposed to ?50 oocysts, 48% became moribund
when exposed to 500 oocysts, and all mice exposed to ?5,000
oocysts became moribund. In contrast, the 10-mJ/cm2group of
mice were asymptomatic when dosed with ?5,000 oocysts, and
60% of the mice exposed to ?5,000 oocysts became moribund.
In oocysts exposed to ?15-mJ/cm2UV, all mice were asymp-
tomatic when exposed to ?5,000 oocysts, and 20 to 33% of the
mice exposed to ?5,000 oocysts became moribund. The data
presented in Table 3 were confirmed by the detection of T.
gondii B1 DNA by using a duplex qPCR assay.
Figure 2 shows the ID50dose-response curves developed
from the bioassay data, with the model shown by solid lines and
the dashed lines representing the 95% confidence intervals.
Table 4 presents the numerical data shown in Fig. 2 and the
log10inactivation achieved by the corresponding UV exposure.
The control group had an ID50of 24 oocysts (log10? 1.37)
(Fig. 2A and Table 4). The model derived from the 4-mJ/cm2
UV exposure group had an ID50of 346 oocysts (log10? 2.54),
achieving a 1.17-log10inactivation (Fig. 2B and Table 4). The
model from the 10-mJ/cm2UV exposure group had an ID50of
2.0 ? 104oocysts (log10? 4.31), with a 2.93-log10inactivation
(Fig. 2C and Table 4). All of the data from UV exposures of
?15 mJ/cm2were analyzed individually, but the models for all
of the groups were not statistically different and therefore com-
achieving a 3.58-log10inactivation (Fig. 2D and Table 4).
Duplex qPCR tissue confirmation. To confirm T. gondii-
induced pathology following infection, a duplex qPCR ampli-
fying the T. gondii B1 and mouse GAPDH genes was devel-
oped. Results from tissue analysis by this duplex qPCR are
shown in Table 5. Tissues from all animals were taken, and the
brains were used for primary classification of infection status.
The tissues from asymptomatic, unexposed controls were used
to determine the B1 gene CTvalue, ?38.7, for a T. gondii-
negative sample (mean minus 2 standard deviations [SD]). The
determined CTvalues observed in the unexposed controls may
be due to duplex qPCR. A mouse was considered positive for
T. gondii if the animal became moribund and the average B1
CTvalue was ?34, reflecting at least a 1.5 order of magnitude
change in the expression of T. gondii B1 DNA between nega-
tive and positive tissues.
FIG. 2. Dose-response results for SCID mice for unexposed (UV
? 0) and UV-exposed (4, 10, and ?15 mJ/cm2) T. gondii oocysts (A to
D, respectively). Open circles are animal infectivity results, and solid
lines are the dose-response model results developed from the animal
results with 95% confidence intervals. The dashed lines are the ID50s
determined by the dose-response model.
TABLE 3. SCID mouse bioassay results by T. gondii oocyst
and UV dose
No. of mice infected/no. of mice exposed to indicated no. of
oocysts per animala
a??, mouse/mice removed from study because tissue from moribund animal(s)
was negative for T. gondii B1 gene in all tissues (data not shown). ND, not done.
VOL. 76, 2010INACTIVATION OF T. GONDII OOCYSTS BY UV DISINFECTION 5143
Brain tissues were used to confirm the results for 366 out of
383 mice used in this study, and 10 were confirmed by spleen
and lung tissues. All tissues were positive for the mouse house-
keeping gene GAPDH, with the CTaveraging approximately 22
for the brain and 19 for the lung and spleen. There were no
significant differences in GAPDH CTvalues for the same tissue
type. In contrast to the GAPDH results, the B1 results were
significantly different for the moribund mice, with an average
B1 CTvalue of 28.8 ? 2.2 (SD) for brain tissues compared to
39.9 ? 0.3 and 39.7 ? 0.5 for the asymptomatic and unexposed
mice, respectively. Spleen and then lung tissue were analyzed if
the brain results could not be confirmed using the criteria
listed above or if there was a discrepancy between the bioassay
and qPCR results. Seven mice became moribund without de-
tectable T. gondii DNA. These animals were not included in
the analysis and are ?2% of the animals used in the study.
Analysis of UV irradiation on T. gondii oocysts by an RT-
qPCR assay. An RT-qPCR assay targeting the ACT1 or
SporoSAG gene was also employed as an alternative rapid
viability assay to the SCID mice bioassay described above. As
shown in Table 6, CTvalues for ACT1 and SporoSAG in un-
exposed live T. gondii oocysts were 28.12 ? 3.13 and 31.07 ?
4.45, respectively. Following UV exposure at 4, 40, and 100
mJ/cm2, CTvalues for ACT1 remained unchanged: 28.21 ?
3.43, 28.16 ? 1.99, and 28.46 ? 1.40, respectively. Similarly,
SporoSAG mRNA levels between unexposed and UV-treated
oocysts were not significantly different: 4 mJ/cm2? 30.98 ?
4.93, 40 mJ/cm2? 31.73 ? 3.38, and 100 mJ/cm2? 32.64 ?
Effects of UV irradiation on T. gondii oocysts by TOP assay.
A less-expensive alternative approach to the SCID mouse bio-
assay described above, the TOP assay was used as a faster
means of measuring effects of UV exposure on oocyst infec-
tivity. As shown in Table 7, a 1.8 (? 0.2)-log10reduction of
infectivity (95% interval range of 1.6 to 2.1) was observed with
oocysts exposed to 4 mJ/cm2UV irradiation compared to the
unexposed controls. Oocysts exposed to ?40 mJ/cm2UV irra-
diation resulted in at least a 3-log10reduction of infectivity. No
observable differences in excystation efficiencies were detected
between unexposed and UV-treated oocysts (data not shown).
The TOP assay revealed that at least 1- and 3-log10reductions
in infectivity can be achieved with 4 and ?40 mJ/cm2UV
The enhancement of disinfection practices utilized by the
water industry has been driven by concerns of DBP and chem-
ically resistant microorganisms, such as T. gondii and Crypto-
sporidium oocysts (26, 31, 40, 63). UV disinfection has been
widely adopted worldwide because it forms few DBPs, and
exposure to ?12 mJ/cm2achieves a 3-log10inactivation of
Cryptosporidium oocysts and Giardia cysts (9, 14, 15, 29, 41,
51). Information about the effects of UV on T. gondii oocysts
is limited. Sobsey and colleagues reported a 2-log10reduction
of T. gondii oocyst infectivity at 40 mJ/cm2(55, 56). These
studies, however, used a limited number of animals, and so, an
accurate dose-response model able to quantify inactivation
rates could not be developed. Wainwright et al. demonstrated
that oocysts were infectious to mice even after UV exposures
of ?500 mJ/cm2but did not report log inactivation (62). Du-
me `tre et al. reported an ID50of 6.2 oocysts and a 4-log10
inactivation by 40 mJ/cm2UV, the only UV dose evaluated by
their bioassay (26).
We report an ID50of 24 oocysts, and 1- and 3-log10reduc-
tions are achieved with UV fluences of 4 and 10 mJ/cm2,
respectively, by mouse bioassay. The differences in the ID50
and maximal log inactivation values observed between these
studies may be explained by several factors, including the an-
imal model, confirmation method, oocyst strain, and study size.
This study used SCID mice, which are completely susceptible
to T. gondii infection, making this model potentially more
sensitive for determining oocyst infectivity compared to immu-
nocompetent mouse models (36). This sensitivity leads to a
TABLE 4. Mouse bioassay numerical dose-response model and
determined log inactivation
10 2.0 ? 104
9.0 ? 104
aNA, not applicable.
TABLE 5. Summary of tissue resultsc
Brain B1 mean CT? SD
GAPDH mean CT? SD
39.7 ? 0.5
22.1 ? 0.9
39.9 ? 0.3
22.1 ? 1.5
28.8 ? 2.2
21.9 ? 1.2
Spleen B1 mean CT? SD
GAPDH mean CT? SD
40.1 ? 0.1
19.9 ? 1.3
30.9 ? 3.1
19.0 ? 2.1
Lung B1 mean CT
GAPDH mean CT
aTissue results are averages from triplicate duplex qPCRs. Undetermined
results were given a value of 40.1. Moribund mice were positive for T. gondii if
they had a mean B1 CTvalue of ?34. Asymptomatic mice were negative if they
had a mean B1 CTvalue of ?38.7. Primary determination was by brain tissue
and, if required, by spleen and then by lung.
bSeven moribund mice did not have T. gondii and were not included in the
analysis (data not shown).
cNA, no tissues analyzed.
TABLE 6. RT-qPCR results to determine T. gondii oocyst viability
following UV exposure
Unexposed 28.12 ? 3.13
28.21 ? 3.43
28.16 ? 1.99
28.46 ? 1.40
31.07 ? 4.45
30.98 ? 4.93
31.73 ? 3.38
32.64 ? 2.14
aAverage CTvalues from three independent experiments ? standard devia-
tion. Triplicate RT-qPCRs were performed for each condition in each of the
three independent experiments.
5144WARE ET AL.APPL. ENVIRON. MICROBIOL.
more conservative estimate of the risk of infection. SCID mice
become moribund with advanced toxoplasmosis, allowing for a
simple preliminary determination of oocyst infectivity com-
pared to asymptomatic immunocompetent models, which may
require further analysis, such as serology, to reveal results.
Wainwright et al. used a different strain of T. gondii (Type X)
which may be more resistant to UV. The different oocyst strains
used may not account for the discrepancies since studies using
different Cryptosporidium oocyst strains and species showed all to
have similar sensitivities to UV (14, 34).
Oocyst infectivity appears to have a nonlinear response to
UV fluences above 10 mJ/cm2, and based on the dose-response
model shown in Fig. 2, a 4-log10inactivation could be achieved
within error of the model. More importantly, a 3-log10in-
activation of T. gondii oocysts is easily achieved by a UV
fluence of ?10 mJ/cm2as reported here and by Dume `tre et
al. (26). Another possible explanation for this nonlinear
effect is the unique ability of T. gondii oocyst and sporocyst
walls to autofluoresce upon exposure to UV (42). These walls
may be protecting the sporozoites from UV radiation by con-
verting UV energy into its autofluorescent properties. It should
be noted that ionizing and nonionizing irradiation exposure
affects parasite intracellular replication, with a minimal effect
on the invasion process (21, 28).
The gold standard for determining T. gondii oocyst viability
is through animal infectivity, which is expensive and labor-
intensive and requires at least 6 weeks to allow for infection to
develop in the SCID mice used in this study. This study deter-
mines the mouse infectivity status primarily by animal health
followed by a novel confirmation by a duplex qPCR assay
instead of serology or immunohistochemistry. The B1 qPCR
assay used to detect T. gondii in infected tissues was adopted
from an assay developed by Kompalic-Cristo et al. (39). This
approach is very reliable and less labor-intensive than serology
and immunohistochemistry and resulted in a dual detection of
both mouse tissue and the presence or absence of T. gondii.
No attempt was made to quantify the parasite load by qPCR
since the primary purpose of this assay is to confirm T.
gondii-induced pathology and morbidity in SCID mice. The
duplex qPCR assay was also able to determine that seven
moribund mice were negative for T. gondii. We believe that
these immunocompromised SCID mice had an undetermined
secondary infection. These results emphasize that mouse sur-
vival data must be confirmed by tissue analysis.
There are ethical concerns associated with animal research;
thus, a scientifically acceptable alternative is needed. In vitro
cell culture results have been correlated with animal infectivity
results for Cryptosporidium disinfection studies (51); however,
to date there are only limited data for T. gondii oocysts. Du-
me `tre et al. evaluated the UV inactivation of T. gondii by both
in vitro and animal infectivity assays, but the relationship be-
tween the two assays was not rigorously evaluated, in that only
two groups, unexposed and exposed to 40 mJ/cm2, were eval-
uated. Nevertheless, they report that both assays achieved a
4-log10inactivation after exposure to 40 mJ/cm2UV. In addi-
tion, they determined that UV fluences of 7.9, 11.8, 15.3, and
17.5 mJ/cm2were required to achieve 1-, 2-, 3-, and 4-log10
reductions, respectively, only by the in vitro assay (26). Our
study also compares a mouse bioassay and TOP assay and
demonstrates 1- to 2-log10inactivation at 4 mJ/cm2by bioassay
and TOP assay and ?3-log10inactivation at ?40 mJ/cm2UV
by both assays. Taken together, cell culture assays appear to
be a promising alternative to animal bioassays; however, a
more complete evaluation is needed to determine their abil-
ity to assess disinfection efficacies of other inactivation
Cell culture obtains data in a week to 10 days, and this time
period would not be rapid enough to assay risk reduction in the
case of a water utility contamination event. A potential alter-
native is RT-qPCR, by which results are obtained in a few
hours. Our studies demonstrate that RT-qPCR results after
UV exposure do not correlate with the TOP assay or bioassay
results. Jenkins et al. compared fluorescence in situ hybridiza-
tion (FISH), cell culture, and animal bioassay to assess the
sensitivity of these methods in detecting the viability and in-
fectivity of C. parvum oocysts in water and found that even
when mRNA levels were not detected or minimally detected by
RT-PCR, the oocysts were able to effectively infect mice and
cell cultures (33). Major unknown factors in the use of RT-
PCR analysis of T. gondii oocyst inactivation following UV
light exposure are the rate and conditions of decay of the T.
gondii ACT1 and SporoSAG mRNA following inactivation.
The mRNA decay may be a significant contributing factor to
the low sensitivity observed when using this method and should
be further elucidated. Although the SporoSAG and ACT1 RT-
qPCR assay was not effective at predicting T. gondii viability
following UV exposure, it may still provide a rapid and sensi-
tive alternative assay for determining total levels of T. gondii
oocysts present in the environment. In addition, an RT-qPCR
technique could be combined with the TOP assay to more
TABLE 7. TOP assay results of UV-irradiated T. gondii oocysts
No. of plaques with indicated no. of oocystsa
0.33 ? 0.557
21.33 ? 4.73
0.33 ? 0.56
171.17 ? 6.51
1.67 ? 0.58
421.67 ? 3.52
180.33 ? 4.51
0.33 ? 0.56
22.33 ? 4.51
0.67 ? 0.57
167.67 ? 13.61
3 ? 1.00
416.67 ? 4.04
175.00 ? 9.85
aData represent mean number of plaques from triplicate wells ? standard deviation. *, too numerous to count.
VOL. 76, 2010INACTIVATION OF T. GONDII OOCYSTS BY UV DISINFECTION 5145
accurately assess oocyst viability as well as reduce the time in
culture required to obtain a result; similar methods have been
described for C. parvum (50). Another promising alternative to
an integrated cell culture/RT-qPCR assay is the use of pro-
pidium monoazide in conjunction with a molecular assay such
as PCR, as recently applied to assess viable Cryptosporidium
This study more fully elucidates the effectiveness of UV
irradiation against T. gondii oocysts and shows that 3-log10
inactivation is fully achievable at a 10-mJ/cm2UV dose, which
is applicable to the water industry. Low-pressure UV appears
to be effective against T. gondii oocysts under ideal conditions;
however, environmental samples have not been evaluated, and
research is needed to better understand the true susceptibility
of this pathogen in drinking water and wastewater systems. In
addition, while the RT-qPCR approach did not appear to be a
reliable indicator of infectivity, the TOP assay showed promise
as a sensitive alternative to established mice bioassays or the
SCID mouse model presented here.
We acknowledge the animal care and technical assistance of Sharon
Detmer, Paula McCain, Diana Miller, and Katrina Pratt. We also
acknowledge Eugene Rice and Frank W. Schaefer III for their critical
reviews of the manuscript.
The United States Environmental Protection Agency through its
Office of Research and Development funded and collaborated in the
research described here under interagency agreement number DW-
12-92289801-0 to USDA and contract number EP-D-06-100 to the
McConnell Group. It has been subjected to agency review and ap-
proved for publication.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Author contributions were as follows. E.N.V., S.A.J.A., and M.W.W.
conceived and designed the experiments. M.W.W., S.A.J.A., D.O.E.,
M.J.S., S.L.H., and E.N.V. performed the experiments. M.W.W.,
S.A.J.A., L.W., and E.N.V. analyzed the data. J.P.D. contributed re-
agents/materials/analysis tools. E.N.V., M.W.W., and S.A.J.A. wrote
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