OCCURRENCE OF GIARDIA AND CRYPTOSPORIDIUM IN
THE KAU-PING RIVER AND ITS WATERSHED IN
SOUTHERN TAIWAN
BING-MU HSU
1, CHIHPIN HUANG
1*
*M, CHIH-LI LILIAN HSU
2,
YEONG-FUA HSU
3and J. H. YEH
41Institute of Environmental Engineering, National Chiao Tung University, Hsinchu, Taiwan;
2Department of Parasitology, Medical College National Cheng Kung University, Tainan, Taiwan;
3National Institute of Environmental Analysis, Environmental Protection Administration, Taipei,
Taiwan and4Bureau of Environmental Sanitation and Toxic Substances Control, Environmental
Protection Administration, Taipei, Taiwan
(First received July 1998; accepted in revised form October 1998)
AbstractÐGiardia and Cryptosporidium are important waterborne parasites. Thirteen water samples and 32 fecal specimens were collected from the Kau-Ping River and its watershed to test for Giardia cysts and Cryptosporidium oocysts. The detection methods are immuno¯uorescence assay and enzyme-linked immunosorbent assay for water samples and fecal specimens, respectively. Seven out of eight samples collected from raw water samples showed the presence of cysts, while six out of eight raw water samples contained oocysts. Cryptosporidium was present in 40% of the treated water, while Giar-dia occurred in all of them. Four out of 32 fecal specimens, collected from the hog farming region, tested positive for Giardia, and seven specimens tested positive for Cryptosporidium. Giardia was related only to Cryptosporidium but not to the others. # 1999 Elsevier Science Ltd. All rights reserved Key wordsÐGiardia, Cryptosporidium, water supply, Kau-Ping River
INTRODUCTION
Parasites Giardia and Cryptosporidium are common
pathogenic protozoa of the gastrointestinal tract
(Cook, 1995). Members of the genus Giardia infect
the proximal small intestine in humans and other
mammals, causing giardiasis with symptoms include
diarrhea, stomach cramps, nausea, and fatigue.
Many outbreaks of giardiasis have been reported in
the last few decades (Smith et al., 1995). Members
of the genus Cryptosporidium also cause
gastroen-teritis in humans and animals and are often
respon-sible for waterborne outbreaks. For instance, a
signi®cant outbreak in Milwaukee, WI, during 1993
was caused by Cryptosporidium, infecting 400,000
people (MacKenzie et al., 1994). The ®rst case of
giardiasis in Taiwan was discovered in 1975 on an
oshore island. Thirty two percent of the children
residing on the island were diagnosed with Giardia
in their stool specimens (Chung and Cross, 1975).
Another survey showed that over 50% of the avian
species
in
Taiwan
were
infected
with
Cryptosporidium spp. (Wang and Liew, 1990).
However, information pertaining to Giardia and
Cryptosporidium in the drinking water supply
sys-tems in Taiwan is very limited.
Majorities of Giardia cysts are oval in shape,
ran-ging from 8 to 14 mm in diameter. Cryptosporidium
oocysts, which range from 4 to 6 mm in diameter,
are characteristically spherical. These thick-walled
cysts and oocysts are extremely resistant to
com-monly used disinfectants such as chlorine (Korich
et al., 1990). They can remain viable for several
months in water between 4 and 108C (Medema et
al., 1997). The most commonly used laboratory
protocols for identifying cysts and oocysts in stool
specimens or environmental water samples are
immuno¯uorescent microscopic examination and
enzyme-linked immunosorbent assay (EIA) (Leng et
al., 1996).
The Kau-Ping River is the major raw water
source for the great Kaohsiung area of 2.5 million
people, approximately 12% of Taiwan's total
popu-lation. Another water source in this region is
groundwater. A survey on the Kau-Ping River
shows that the watershed in the upper region is
used for recreation. The middle and lower regions
are heavily polluted, due to inputs of domestic
sew-age, industrial and farm wastewaters. Although
conventional prechlorination, coagulation,
sedimen-tation, and ®ltration processes are employed by
# 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/99/$ - see front matter
PII: S0043-1354(98)00478-3
*Author to whom all correspondence should be addressed. [Tel: +886-35-726463; fax: +886-35-725958, e-mail: cphuang@green.ev.nctu.edu.tw].
local water treatment plants, the removal of
para-sites has never been evaluated.
In this report we surveyed the prevalence of the
parasites and the microbes traditionally used as
water quality parameters from various locations in
the Kau-Ping River and their correlation to water
quality parameters. The risk analysis for human
infection of Giardia and Cryptosporidium from
drinking water supplies is also included in this
study.
MATERIALS AND METHODS
Sampling sites and sampling procedures
Samplings were carried out in three phases. In the ®rst phase, water samples were collected from upstream and downstream of the river running through the hog farming region. The second phase included the collection of ten samples (®ve raw water samples and ®ve treated water samples) from ®ve water treatment plants using the Kau-Ping River as a water source (Chiya-Shien, Pying-Ding, Cheng-Ching, Ueng-Gungyuan and Kau-Tan). The third phase was carried out to investigate the stool specimens of pasturing animals in the watershed for the presence of parasites. The hog farming region encompasses the middle region of the Kau-Ping River, where approximately 1.04 million pigs and other animals of less quantity are raised. These animals are potential reservoir hosts for the protozoa. Thirty two fecal speci-mens were randomly collected from farms located in the hog farming region.
Sampling method and procedure were adopted from the handout provided in the American Water Works Association training course (AWWA, 1994). They are the same method as speci®ed by the Information Collection Rule (ICR), using indirect ¯uorescent anti-body (IFA) procedures. 15±50 l of the raw water samples and 50±80 l of treated water samples were con-centrated through a 254 cm long and 1.0 mm nominal-pore-size polypropylene yarn-wound cartridge ®lter. The ®lter ®bers were eluted by 1±1.5 l of eluting solution (phosphate-buered saline, 1% Tween 80, 1% SDS) in a mechanical stomacher, and then the eluate was centri-fuged at 1050 g for 10 min in a 50-ml centrifuge tube in swinging-bucket rotor. Meanwhile, we recorded the packed pellet volume. After the supernatant was aspi-rated, the pellet was resuspended with an equal volume of 10% formalin and vortexed up to a total volume of 20-ml with an eluting solution in a 50-ml centrifuge tube. The mixture was underlaid with 30 ml of a per-coll±sucrose gradient (sp. gravity = 1.10) followed by centrifuging. The sample in the centrifuge tube was col-lected from the top 20 and 5 ml below interface, and then diluted with eluting solution to 50 ml. This 50 ml suspension was then centrifuged again and the top 45 ml was then aspirated. Finally, the last 5 ml suspen-sion was harvested for manifold ®ltering.
Immunolabeling of water and fecal samples
Indirect immuno¯uorescence assay (IFA) was used to detect Giardia cysts and Cryptosporidium oocysts in water samples. The concentrated water samples were labeled
with monoclonal antiserum (Hydro¯uor2Combo Giardia/
Cryptosporidium; Ensys, Inc., NC, U.S.A.) and examined with epi¯uorescent microscope at 200 , 400 or 1000 magni®cation (Olympus, Japan). Cysts and oocysts were identi®ed using following parameters: size, shape, surface feature, and staining reaction. The candidates exhibiting the right size and shape were further identi®ed by
epi¯uor-Table 1. The concentration of Giardia and Cryptosporidium and w ater quality parameters of eight sampling sites in the K au-Ping R iver Sampling site Sample type Sampling volume (l) Giardia (cysts/100 l) Cryptosporidium (oocysts/100 l)
Heterotrophic bacteria (CFU/1
m l) Total coliforms (CFU/100 m l) Fecal coliforms (CFU/100 m l) pH Temperature (8 C) Conductivity (m s) Turbidity (NTU) Chiya-Shien raw 25 651 <643 <5 <1 <1 7.05 20.5 437 80 Chiya-Shien treated 50 57 <56 < 5 < 1 < 1 7.10 19.7 436 0.40 Pying-Ding raw 50 397 4047 8900 280 31 7.30 25.7 845 20 Pying-Ding treated 80 40 159 <5 <1 <1 6.60 25.2 595 0.20 Cheng-Ching raw 50 1367 8972 5300 1200 10 7.70 25.4 526 17 Cheng-Ching treated 80 14 <41 < 5 2.0 <1 7.10 25.5 634 1.4 Ueng-Gungyuan raw 50 < 93 <237 5800 880 337 7.08 25.0 890 5.2 Ueng-Gungyuan treated 80 12 <35 < 5 < 1 < 1 7.20 22.6 770 0.50 Kau-Tan raw 50 1107 2540 1.2 10 5 1.0 10 5 3.4 10 4 7.24 22.1 644 11 Kau-Tan treated 80 49 72 <5 1.0 <1 7.15 24.1 546 0.30 Fwoguang-Shan raw 40 1398 2378 1.6 10 4 2.4 10 4 100 7.35 23.8 966 7.0 Guei-Yuan raw 15 3754 12,516 6.0 10 6 5.0 10 7 2.4 10 6 7.35 22.8 881 50 Shyi-Jichang raw 20 6978 7754 4.2 10 5 7.5 10 4 100 7.28 22.8 630 40
escent microscopy under a bright ®eld according to their internal morphological features. EIA was used to detect the presence of the organisms in fecal samples. ProSpecT
Giardia EZ Microplate Assay and ProSpecT
Cryptosporidium Microplate Assay were purchased from Alexon Inc. (Sunnyvale, CA, U.S.A.). Sample preparation and immuno-reaction procedures followed supplier's
pro-tocols. Readings of A450=0.05 were interpreted as positive
reactions and A450<0.05 as negative. The primary
anti-bodies used in the EIA test are speci®c to Giardia and Cryptosporidium. Scheer and Van Etta (1994) have proved that these antibodies would not cross-react with any other enteric parasites. To assess the sensitivity of the EIA, puri®ed oocysts were serially diluted 10-fold in PBS, and 200 ml aliquots from each dilution were placed in test wells.
Detection of water quality parameters
Water temperature and its pH were measured in situ using a portable pH meter (Radiometer Analytical SA, France). Turbidity was measured using a ratio turbidi-meter (HACH, Co., U.S.A.). Water samples were collected in sterilized 500 ml Schott bottles, which were kept in a cooler during transportation to the lab. The screening for microbiological parameters must be ®nished in eight hours after sampling. Heterotrophic bacteria were measured by the spread method. Total coliforms and fecal coliforms were measured by membrane ®ltration procedures described by the Standard Method for the Examination of Water and Wastewater (Methods 9222 B and D) (APHA, 1995).
Recovery eciency test
To determine the recovery eciency, cysts and oocysts were prepared and then calculated with a cali-brated hemacytometer. The tests were started with
9.37 105 cysts and 8.14 104 oocysts and the numbers
detected in 50 l of raw water and 80 l of treated water were calculated using IFA.
Statistical evaluation
The numbers of cysts and oocysts observed under the microscope were recorded and later normalized to number of counts per 100 l. These numbers were divided by the recovery eciency to obtain the ®nal counts. When the protozoa were not observed via epi¯uorescent microscope, we express the protozoa concentration in water samples as less than (<) their detection limits. The calculations of correlation coecients among turbidity, heterotrophic bac-teria densities, total coliforms densities, fecal coliforms densities, Giardia levels, and Cryptosporidium levels were conducted using the STATISTICA software (StatSoft, Inc., U.S.A.). In the statistical evaluation, data under the detectable levels were treated as zero. The mean and stan-dard deviation of the parasites and the parameters of water quality were calculated.
Risk assessment
The exponential risk assessment model (Rose et al., 1991; Crabtree et al., 1996) was used to estimate the number of cases of illness resulting from the measured levels of organisms. The model gives a potential daily risk (P), assuming that a person drinks two liters of water per day. It is calculated using the following equation:
P 1 ÿ exp ÿrN , 1
where r is the coecient (0.0198 for Giardia and 0.004 for Cryptosporidium) and N is the number of cysts or oocysts per two liters of water. The model assumed that all cysts and oocysts were viable and infectious. The overestimate and the underestimate of the risks were canceled by each other.
Fig. 1. The watershed of the Kau-Ping River and sampling sites in this study. Abbreviations: C.S.: Chiya-Shien water plant; G.Y.: Guei-Yuan; F.S.: Fwoguang-Shan; S.J.: Shyi-Jichang; P.D.: Pying-Ding water plant; C.C.: Cheng-Ching water plant; U.G.: Ueng-Gungyuan water plant; K.T.: Kau-Tan water
RESULTS AND DISCUSSION
Distribution of cysts and oocysts in water samples
Thirteen water samples were collected from the
water treatment plants and hog farming region of
Kau-Ping River watershed. Sampling sites, sample
volumes, parasite concentrations and parameters of
water quality are listed in Table 1. During the
period of sampling, water temperatures of the river
ranged
from
19.7
to
25.78C
(mean2SD = 23.521.948C), the pH values
ran-ged from 6.60 to 7.70 (7.1920.26), and the
conduc-tivities ranged from 436 to 966 ms (677? 177 ms).
The location of sampling sites and the distribution
of protozoan parasites in this research are presented
in Fig. 1 and Table 1. It is evident that both
para-sites are widely distributed in the Kau-Ping River
watershed. Twelve out of thirteen water samples
showed the presence of cysts, while eight out of
thirteen water samples contained oocysts. All the
water samples taking from the watershed near the
hog
farms
contained
both
Giardia
and
Cryptosporidium. The percentages for Giardia- and
Cryptosporidium-positive were 80% and 60% in raw
water samples of water treatment plants, and 100%
and 40% in treated water samples, respectively.
Some literatures such as Ahmad et al. (1997) have
indicated that the lower rate of Giardia-positive in
raw water samples than in treated water may be
due to the interference of detection limit by
turbid-ity.
Relationship between protozoan parasites and water
quality parameters
Jennifer et al. (1994) conducted a blind survey of
sixteen commercial laboratories to evaluate the
recovery eciency for protozoa. The recovery
e-ciencies of Giardia and Cryptosporidium ranged
from 0.8 to 22.3% and from 1.3 to 5.5%,
respect-ively, and their average was 9.1% for Giardia and
2.8% for Cryptosporidium. In our research, the
recovery eciency of cysts ranged from 22.8 to
26.5%, and 8.93 to 8.94% for oocysts. The results
of recovery eciencies were higher than those of
Jennifer et al. LeChevallier et al. (1995) suggested
that careful handling in concentration and
clari®ca-tion may result in improvement in the recovery
e-ciency.
The mean concentration of Giardia cysts in raw
water samples was 1956 cysts/100 l (SD = 2320),
ranging from less than 93 to 6978 cysts; and was
4776
oocysts/100 l
for
Cryptosporidium
(SD = 4520), ranging from less than 237 to 12,515
Fig. 3. Relationship between densities of Giardia cysts and Cryptosporidium oocysts in raw water samples of the Kau-Ping river. Regression line: y = 2376.3 + 1.23x; r = 0.630, p = 0.094.
Fig. 2. The trends of protozoan parasites and water qual-ity parameters in raw water samples collected along the
oocysts (Table 1). Figure 2 is a 3D-diagram
show-ing the distribution of parasites and water quality
parameters in raw water samples collected from
upstream to downstream of the river watershed. All
of the data in the diagram were derived from log
transformation. The concentrations of Giardia and
Cryptosporidium along the river exhibited a similar
trend, while those of the heterotrophic bacteria,
total coliforms and fecal coliforms exhibited a
dierent pattern. Figure 2 also shows that the
sampling sites located downstream and near the
hog farming region contain more Giardia cysts and
Cryptosporidium oocysts than the upstream ones or
those using groundwater aected by the surface
water.
In samples collected from the Kau-Ping River
watershed,
the
mean
concentration
of
Cryptosporidium oocysts was 1.23 times that of
Giardia cysts with a correlation coecient of 0.630
(p = 0.094) (Fig. 3). This result was similar to the
®ndings of other investigators. For example,
LeChevallier et al. (1991) found higher oocyst
con-centrations in their samples and signi®cant
corre-lations between the concentrations of cysts and
oocysts (r = 0.59, p < 0.01). Rose et al. (1988) also
found a higher concentration of Cryptosporidium
oocysts and a signi®cant correlation between the
concentrations in a single watershed (r = 0.778,
p < 0.01, n = 39). The mean quality of the eight
raw water samples was 28.5 NTU (SD = 26) for
turbidity, 8.2 10
5CFU/ml (SD = 2.1 10
6) for
heterotrophic
bacteria,
6.3 10
6CFU/100 ml
(SD = 1.8 10
7) for total coliforms, and 3.0 10
5CFU/100 ml (SD = 8.5 10
5) for fecal coliforms.
The correlation between protozoan parasites and
water quality parameters in these samples was
ana-lyzed. The results indicated that neither cysts nor
oocysts were signi®cantly correlated with turbidity
levels (i.e., r = 0.300 at p = 0.471 and r = 0.154 at
p = 0.715) (Table 2). Highly positive correlations
were observed between Cryptosporidium
concen-trations and the levels of heterotrophic bacteria
(r = 0.713, p = 0.047), total coliforms (r = 0.692,
p = 0.057),
and
fecal
coliforms
(r = 0.690,
p = 0.058). It is also noted that no signi®cant
re-lationships were found between Giardia and
hetero-trophic bacteria, total coliforms or fecal coliforms.
Relationship between the removals of protozoa
para-sites and turbidity
All raw water samples had turbidity levels
greater than 5.0 NTU (with a mean of 28.5
NTU), while that of the treated water samples
showed a mean of 0.56 NTU (Table 1). The
removals of turbidity, cysts and oocyts in various
water treatment plants were presented in Table 3.
The mean removal percentages of all test samples
were calculated from the data in Table 3. They
are 92.61, 97.59, and 95.59% for cysts, oocysts,
and turbidity, respectively. Although the removal
percentage of turbidity did not correlate well
with either parasite, a positive correlation was
found between the removal percentages of both
parasites (r = 0.937 at p = 0.226).
Testing of animal fecal specimens
The data for the occurrences of Giardia and
Cryptosporidium in fecal specimens are shown in
Table 4. In the 32 collected samples, four were
positive for Giardia and seven positive for
Cryptosporidium. Both parasites were detected in
pig and cattle fecal specimens. Only one duck
and
one
sheep
were
tested
positive
for
Cryptosporidium. None of the parasites was
detected in any of goose or chicken fecal
speci-mens. Figures 4 and 5 are the EIA results of
the fecal samples. When the results of the
posi-tive controls were converted with IFA and
hemacytometer, samples within the detecting
range were estimated to contain 376 oocysts/
200 ml and 886 cysts/200 ml for Cryptosporidium
Table 4. Occurrence of Giardia and Cryptosporidium in the fecal specimens of hog farming region
Sample
category Number ofsamples positive samplesNumber of for Giardia Number of positive samples for Cryptosporidium Pigs 17 3 4 Cattle 5 1 1 Goose 1 0 0 Ducks 4 0 1 Sheep 2 0 1 Chickens 3 0 0 Total 32 4 7
Table 3. Removal percentages of Giardia, Cryptosporidium, and turbidity by water treatment plants
Water treatment plant Removal percentage of Giardia Removal percentage of Cryptosporidium Removal percentage of turbidity Chiya-Shien 91.28% ± 99.49% Pying-Ding 89.87% 96.07% 99.00% Cheng-Ching 98.98% >99.54%a 91.88% Ueng-Gungyuan <87.3%a ± 90.38% Kau-Tan 95.6% 97.17% 97.20%
aWhen no parasites were detected in samples, the limit of detection
method was recorded.
Table 2. The linear correlation among the concentrations of proto-zoa, turbidity and three microbiological parameters (r: correlation
coecient; p: statistical signi®cance level)
Parameters Giardia
cysts Cryptosporidiumoocysts Turbidity r = 0.300, p = 0.471 r = 0.154, p = 0.715 Heterotrophic bacteria r = 0.373, p = 0.362 r = 0.713, p = 0.047 Total coliforms r = 0.314, p = 0.449 r = 0.692, p = 0.057 Fecal coliforms r = 0.311, p = 0.453 r = 0.690, p = 0.058
and Giardia, respectively. False-positives might
have occurred due to the presence of the
anti-gens in stool rather than complete cysts and
oocysts. This was not considered while
interpret-ing the raw data.
The risks of giardiasis and cryptosporidiosis
infec-tions
The concentrations of cysts and oocysts observed
in the euents of the ®ve water treatment plants
ranged
from
12
to
57
cysts/100 l
(mean2SD = 34220 cysts/100 l) and from less
than 35 to 159 oocysts/100 l (46270 oocysts/100 l).
The detection of the protozoan parasites in treated
water indicates a potential risk for waterborne
dis-eases. The average risk of acquiring Giardia and
Cryptosporidium infections from drinking unboiled
tap water from these ®ve plants were estimated to
be 132 and 58 people per 10,000 people per day.
However, few people in Kaohsiung drink tap water
without further treatment. A survey conducted in
the Kaohsiung metropolitan area (Chuang, 1996)
showed that 64.6% of the entire population
con-sumed bottled waters containing distilled water,
R.O. water, or mineral water. The rest of the people
chose tap water as their main drinking water, which
they either boiled before drinking or treated with in
house water purifying device. This explains why
there is no outbreak of giardiasis and
cryptospori-diosis in this area.
Fig. 5. The A450 values of puri®ed oocysts, EIA controls and positive fecal specimens for detecting
Cryptosporidium oocysts by EIA. (No. 1 and 2: EIA negative and positive controls, No. 3±10: positive fecal specimens, No. 11±12:376 and 37.6 oocysts in 200 ml reaction volume.)
Fig. 4. The A450 values of puri®ed cysts, EIA controls and positive fecal specimens for detecting
Giardia cysts by EIA. (No. 1 and 2: EIA negative and positive controls, No. 3±6: positive fecal speci-mens, No. 7 and 8: 8860 and 886 cysts in 200 ml reaction volume.)
CONCLUSION
Giardia and Cryptosporidium are prevalent in the
Kau-Ping River which supplies the drinking water
for 2.5 million people. The waters from the hog
farming region and its downstream sampling sites
contain more parasites than the water from
upstream sampling sites. The concentrations of the
cysts and oocysts correlated well with each other.
The occurrence of Cryptosporidium also related to
those of heterotrophic bacteria, total coliforms and
fecal coliforms, which proves that the upstream
farming plays an important role in the
contami-nation of these parasites, which indicates the
im-portance of establishing regulations to prevent
domestic sewage, industrial and pastured
waste-water from directly reaching the river. Risk
assess-ments of the parasitic infection suggest that the tap
water in southern Taiwan is still not suitable for
drinking without boiling or point-of-use treatment.
AcknowledgementsÐThis work was funded by the Environmental Protection Administration, R.O.C. We are grateful to Dr. Meilin Yu for her assistance in statistical analysis, to Miss Guo-Ying Jiang for her experimental work, and to Dr. Jill Ruhsing Pan and Ms. Stephanie Jones for their editing.
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