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Applied and Environmental Microbiology, September 2001, p. 3832-3836, Vol. 67, No. 9
Division of Microbiology, Hyogo Prefectural
Institute of Public Health,1
Department of Medical
Zoology,2 Department of
Microbiology,4 and Faculty of Health
Science,5 Kobe University School of
Medicine, Kobe, and National Institute of Infectious Diseases,
Tokyo,3 Japan
Received 11 January 2001/Accepted 3 June 2001
In Japan, only a few rivers have been inspected for
Cryptosporidium parvum contamination, and the methods
used had low sensitivity. In 1998 and 1999, we used a method with
higher sensitivity to examine all large rivers used as sources of water
supply in one prefecture (which we divided into four areas) in western
Japan for Cryptosporidium oocysts. One sample was
collected at each of 156 sites along 18 rivers, and samples were tested
for Cryptosporidium oocysts by immunomagnetic
separation. Samples were classified as being obtained on an island with
livestock and fishing industries, a densely populated urban area, a
western region including farming villages, or a still more rural
northern area with agriculture and fishing. Restriction fragment length
polymorphism analysis was used for identification of the C.
parvum found as the bovine or human type. C.
parvum was detected in at least one sample from 13 of the 18 rivers and in 47% (74 of 156) of the samples. One-third to all of the
samples from each area contained C. parvum oocysts. The
number of C. parvum oocysts per 20 liters of river water
varied in the same pattern as the number of cattle kept in the four
kinds of areas (as determined by the Mantel extension test). Oocysts isolated were of the bovine type; the C. parvum detected
in rivers probably came from cattle kept in that valley. As we had
expected, when tested with a more sensitive method, river water in
western Japan was found to be greatly contaminated with C.
parvum oocysts, as reported in other countries.
The protozoa Cryptosporidium
parvum and Giardia intestinalis (lamblia) are enteric
parasitic pathogens found in water systems (17). Their
oocysts and cysts, respectively, are environmentally robust, resisting
disinfectants. C. parvum is small enough to be difficult to
remove by filtration. It can cause outbreaks more readily than G. intestinalis because it produces many more oocysts in the host.
The infective dose of C. parvum is low. Okhuysen et al.
(19) found the 50% infective dose of the most virulent of
their three isolates to be nine oocysts for healthy adults. Calculation
on the basis of an epidemiological infection model showed that
infection may occur with even one oocyst (9).
Since the first report of an outbreak of C. parvum infection
caused by a contaminated water supply in 1985 (6),
epidemiological studies of such contamination have been done in many
countries, including the United States (20), Scotland
(27), Australia (32), and South Africa
(12). The methods used were diverse, so the results varied
widely. Nevertheless, C. parvum was detected in almost all
of the environmental waters tested (see reference 28 for a
review). Groundwater and lake water have been examined, but river water
used as the source of public water supplies has been surveyed more than
other water sources in such studies.
In a study of surface water in 17 states in the United States, Rose et
al. (23) reported finding oocysts in 93 (51%) of the 181 samples tested, and the mean number of oocysts detected was 4.3 per 10 liters. LeChevallier et al. (13) did a similar study in
one province of Canada and 14 states in the United States and found
oocysts in 74 (87%) of the 85 samples tested. In contrast, Roach et
al. (22), in a study in the Yukon, Canada, found
Cryptosporidium oocysts in only 3 (5%) of the 63 samples
tested. These differences in the percentage and intensity of
contamination of surface waters by Cryptosporidium oocysts
may be related to the season (23), rainfall
(7), or the presence of untreated feces of humans (16) or domestic animals such as cattle (26).
The methods used to detect C. parvum are complicated, and
results may be difficult to interpret because of low recovery at dilute
concentrations and other problems (5).
In Japan, only two studies of river water have appeared; none has been
done in western Japan or by the immunomagnetic separation assay based
on method 1622 of the U.S. Environmental Protection Agency (EPA)
(30). By immunomagnetic separation,
Cryptosporidium oocysts can be isolated from water. Recovery
is poor when the sample is contaminated with organic particles or
protozoa. The method has not been evaluated thoroughly for detection of
C. parvum oocysts in water in field studies. We recently
improved recovery in the field by adding three steps to this procedure.
Therefore, we used our modified method to establish the present state
of river contamination by Cryptosporidium oocysts in an
entire prefecture, and we examined the relationship between the
intensity of contamination and the numbers and varieties of domestic
animals kept in the valleys of the rivers in question. We also examined
whether Escherichia coli could be used as an index of
Cryptosporidium contamination.
Water sampling.
From July to November in 1998 or 1999, 18 rivers in Hyogo Prefecture in the Kansai area of western Japan were
sampled twice. The areas studied were classified as island, eastern,
western, and northern (Fig. 1). The
island area, in the southernmost part of the prefecture, was an island
in the Inland Sea, with flourishing livestock and fishing industries.
The eastern area was an urban area with a high population density. The
western area was a region mainly of small communities involved in
extensive farming. The northern area was a still more rural region with
much agricultural and fishing activity and with heavy snowfall in
winter. The number of rivers sampled and the number of sampling sites
were 4 and 10 in the island area, 5 and 87 in the eastern area, 4 and
40 in the western area, and 5 and 19 in the northern area,
respectively, and the total number of samples taken was 156 (Fig. 1).
One sample was taken from each sampling site.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.3832-3836.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Contamination of River Water by
Cryptosporidium parvum Oocysts in Western
Japan
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (21K):
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FIG. 1.
Areas surveyed. The inset shows Hyogo Prefecture on a
map of Japan excluding Okinawa. Samples of 20 liters were taken for
testing for C. parvum oocysts from four rivers (10 sampling sites) in the island area, from five rivers (87 sites) in the
eastern area, from four rivers (40 sites) in the western area, and from
five rivers (19 sites) in the northern area. In addition, three samples
of 100 liters for use in RFLP analysis were taken from a large river in
every area but the northern one.
Sample processing. We used a pressure device (model XX82 001 15; Millipore Corp., Bedford, Mass.) to filter the water samples through a disk-filter holder fitted with a nitrocellulose membrane (CF; pore size, 1.2 µm; Millipore) and retrieved the material caught on the surface of the membrane by use of ultrasound (Heat Systems Ultrasonics, Farmingdale, N.Y.) in the first step that we added. In the second step that we added, the material retrieved was suspended in 0.15 M phosphate-buffered saline (pH 7.6), and the mixture was sonicated for 1 min. A kit for immunomagnetic separation with Dynabeads coated with anti-Cryptosporidium antibodies (G-C Combo; Dynal A.S., Oslo, Norway) was used to treat the suspension. Preliminary experiments had shown that some captured oocysts remained attached to the beads, so we repeated this step (our third modification). The material that had bound to the antibodies was separated from them by treatment with 50 µl of 0.1 N HCl, the beads were held aside with a magnet, and the remaining suspension was removed from the tube with a pipette, placed on a slide, dried, and labeled with anti-Cryptosporidium monoclonal antibodies (Cellabs Pty. Ltd., Brookvale, New South Wales, Australia) conjugated with fluorescein so that oocysts could be identified (the first method [see below]). Water samples concentrated before transportation were first eluted into a buffer as described elsewhere (18) and then processed by immunomagnetic separation as described above.
Identification of oocysts from water by microscopy. Three methods were used for identification of oocysts from water, with the exception of the 100-liter samples mentioned in the next section, and when results were positive by all three methods, the oocysts were identified as being of C. parvum. In the first method, oocysts stained with the labeled monoclonal antibodies mentioned above were checked for having a diameter of 4 to 6 µm at magnifications of ×400 to ×1,000 with a 400- to 440-nm (blue-violet) filter. With the second method, microscopy was used for examination of the same stained oocysts for sporozoites at a magnification of ×1,000 with a Nomarski interference contrast filter. With the third method, fluorescent staining with 6-diamino-2-phenylindole (Sigma, St. Louis, Mo.) was used to look for nuclei in sporozoites detected at magnifications of ×400 to ×1,000 with a 330- to 385-nm (UV) filter.
Identification of oocysts by PCR. We used oocysts collected in capsule filters from 100-liter samples for analysis of RFLP by the method of Carraway et al. (4). Using primers that flanked a 515-bp region of an open reading frame in the C. parvum polythreonine locus, we amplified oocyst DNA from about 10 oocysts isolated from river water. The amplification product was digested with RsaI, and the pattern obtained by electrophoresis of the digest was compared with patterns of three strains of C. parvum of known origin and with the pattern of Cryptosporidium andersoni as well (15). For this comparison, we isolated one strain of C. parvum (diameter, 4.5 to 5.0 µm) from a calf and one strain of C. andersoni (5.5 to 7.5 µm) from a Guernsey cow. In addition, we used purified DNAs of C. parvum isolated from two patients with diarrhea. One strain had been identified as having the human profile by RFLP (only strains from humans have been found to have this genotype), and the other strain had been identified as having the bovine profile (isolates from human patients may have either the bovine or human profile). The National Institute of Infectious Diseases, Tokyo, Japan, provided both strains from patients.
Other.
Testing for E. coli was done by the method
used by public water departments in Japan (11). This
qualitative method is a combination of the defined-substrate method and
the standard method. In brief, a 50-ml water sample is examined for
coliforms by a method that uses
ortho-nitrophenyl-
-D-galactopyranoside
and 4-methylumbelliferyl--D-galactoside (Colilert; Idex Lab Inc., Westbrook, Maine). Culture is done at 44.5°C for 24 h in two tubes containing a broth for E. coli (Nissui, Tokyo, Japan), and bacteria that produce gas are
further cultured at 36°C for 20 h in a medium that uses
5-bromo-4-chloro-3-indolyl--D-glucuronide and
5-bromo-6-chloro-3-indolyl--D-galactopyranoside
(Nissui). Coliforms that react in indole-methyl
red-Voges-Proskauer-citrate tests are identified as being E. coli. We questioned residents of the area about the numbers of
their domestic animals and also used published statistics on the
numbers of domestic animals being kept (10).
2 test of proportions. A possible trend
for areas with larger numbers of livestock to have greater intensities
of C. parvum contamination, in terms of oocysts per 20 liters, was evaluated with the Mantel extension test. The
Kruskal-Wallis test was used to examine the differences in the
intensities of contamination in different sections of the rivers.
Differences with P values of <0.05 were defined as being
statistically significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Contamination of rivers and areas.
C. parvum
contaminated 13 (72%) of the 18 rivers tested, and E. coli
contaminated 9 (69%) of the 13 rivers tested (Table
1). C. parvum and E. coli were detected in all areas tested. The proportions of samples
with C. parvum were 10 of 10 (100%) in the island area, intermediate values in the eastern and western areas, and 7 of 19 (37%) in the northern area (Table 2;
P < 0.005). The proportions with E. coli
changed in the same pattern (P < 0.001).
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|
Relationship of contamination by oocysts to numbers of domestic
animals in the area.
Table 3 shows
the mean number of C. parvum oocysts found per 20 liters of
water, referred to as the intensity of contamination, and the numbers
of cattle, pigs, and chickens in each area. The mean concentration of
oocysts was largest (2.4 per 20 liters) in the island area and smallest
(1.4 per 20 liters) in the northern area. The intensity of
contamination by C. parvum and the number of cattle in the
different areas varied in the same pattern (P < 0.005).
|
Relationship of location along the river and intensity of
contamination.
The percentages of samples with C. parvum and the intensities of contamination in different sections
of the rivers are shown in Table 4. Of
the samples from the 156 sampling sites, 37% of the upstream samples,
46% of the midstream samples, and 54% of the downstream samples were
contaminated. The differences were not significant.
|
Identification of oocysts by PCR.
Results of electrophoresis
of PCR products are shown in Fig. 2. With
undigested DNAs from the three C. parvum strains of known origin and from the three large water samples, there was a single band
at about 510 bp, but C. andersoni was not amplified (Fig. 2A). After digestion, DNAs from five of the six strains of C. parvum had three main bands, at 270, 130, and 50 bp. However, the
C. parvum strain of the human type had two main bands, at about 400 and 50 bp. The oocysts that we detected in the water samples
were from C. parvum of the bovine type.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The results of two epidemiological studies of C. parvum in Japan have been published. In a study done by Hashimoto and Hirata (8) in central Japan, from a total of 13 sites along the Sagami River and its tributaries, water was sampled one to four times, with a large sample size (100 liters), and contamination was found at 9 (75%) of the 12 sites examined in 1996 and at 4 (67%) of the 6 sites examined in 1997. The rate of contamination that we found also was high, in agreement with their results, although they used an earlier method still used by the Ministry of Health and Welfare of Japan and we used a method more popular outside of Japan (method 1622 of the U.S. EPA).
Groups in Japan generally use a provisional method published in Japan in 1996 and based on an earlier EPA procedure, the information collection rule, with filtration usually of only 10 liters of water through a nitrocellulose membrane and dissolution of material caught on the filter in acetone. With this method, in a 1997 survey of sources of water supply that examined 277 sites in 94 rivers throughout Japan, the Ministry of Health and Welfare found Cryptosporidium oocysts in only 8 (2.9%) of the 277 sites tested (31). The different volumes of the samples (10 and 100 liters) used is the probable cause of the discrepant results in the two surveys mentioned above, rather than differences in the sites or seasons. The method that both surveys used, which is complicated, is termed provisional because there are doubts about recovery and sensitivity (21); reliability and validity also are questionable. We used immunomagnetic separation, an unrelated method, adding three modifications to improve recovery. Our results suggest again that the method would be useful in epidemiological studies as found earlier by Campbell and Smith (3) and Bukhari et al. (2).
Possible sources of contamination of rivers by C. parvum are raw or treated sewage from humans and feces of domesticated and wild animals (15, 25). C. parvum has a wide range of hosts, including 79 species of mammals. Atwill et al. (1) found that 5.4% of 221 wild pigs tested in California were shedding C. parvum oocysts. Hashimoto and Hirata (8) suggested that uncomposted feces from a large-scale pig-breeding facility on the upper reaches of the river studied might be contaminating the river. In the areas we studied, however, there were no such large-scale facilities, and the incidence of parasitism was low (there were no positive results for any of the 567 pigs tested); pigs were not a likely source of contamination in our study. Uga et al. (data not shown) found that 8.9% of 418 chicken tested were shedding oocysts of Cryptosporidium baileyi in our survey areas. Wild animals such as deer and boar live in the western and northern areas we surveyed, but in numbers too small to be a problem. In rural parts of the prefecture, human waste is gathered in underground domestic tanks emptied regularly by collection trucks. In those areas, raw sewage that accidentally overflows may end up in a river, but sewage is almost always properly treated and there is strict water quality control for treated sewage in Japan, so contamination probably does not arise in this way.
In small farms in Japan, cattle feces are not composted, and Cryptosporidium oocysts shed by these cattle may contaminate rivers. The island area, the smallest of the four, had the most cattle and the highest density. That the percentage of samples contaminated was highest in the island area and lowest in the northern area suggested that the percentage of contaminated samples was related to the number of cattle in that area, or in other words, that oocysts shed by cattle are contaminating the environment. Saeki et al. (24) detected C. parvum in 1 (0.2%) of 582 adult cattle in the same prefecture, and we (29) reported that 24 (80%) of 30 calves tested shed C. parvum oocysts during the first month of life. Our RFLP results showed that the genotype of the C. parvum oocysts retrieved from river water was that found in cattle. Humans can be infected with C. parvum of the genotype originally isolated from cattle and still called the bovine type; nevertheless, it is likely that cattle had shed a large majority of the oocysts that we found in river water.
In our study, the percentage of contaminated samples was higher downstream than upstream, and the intensity of contamination also was greater downstream. We estimated that about half of the farms with cattle were upstream, making it likely that contamination started upstream and later affected downstream areas. Our findings suggest that C. parvum oocysts do not decrease in number by settling out while moving downstream but that the intensity of contamination instead increases downstream, because of the confluence of contaminated tributary streams.
Studies of C. parvum in river water involve complex procedures and various kinds of equipment. Evaluation of the data obtained also requires skill. Accordingly, attempts have been made to find an index of contamination other than C. parvum oocysts, one for which testing is easier. LeChevallier and Norton (14) found that turbidity (which increases after a heavy rainfall) may be one index of contamination by C. parvum oocysts. In addition, protozoa in the water come from animal feces, so E. coli and coliform bacilli in general have been suggested for use as an index of protozoan contamination. However, such bacilli die more quickly in water than protozoa, preventing their detection by culture, and a satisfactory method for their use as an index of contamination has not been established. Rose et al. (23) found no correlation between the concentration of Cryptosporidium (or Giardia) and coliform bacilli in their samples of surface water, but LeChevallier et al. (13) reported a significant correlation between the numbers of Cryptosporidium oocysts and the numbers of total coliform bacilli in samples of raw water. We surveyed contamination by organisms other than C. parvum (G. intestinalis and Clostridium perfringens [data not shown], in addition to E. coli) and found that the percentage of samples contaminated by E. coli was closest to the percentage contaminated by C. parvum. Many samples were contaminated with both organisms, more so than for the two other combinations. In this survey, we used a qualitative method for detection of E. coli. In our previous quantitative study, we found no relation between the number of E. coli cells and the number of Cryptosporidium oocysts detected (data not shown). A similar observation was reported by Simmons et al. (25). If E. coli is an index for Cryptosporidium contamination, a simple qualitative test may be sufficient.
The results of our study showed that Cryptosporidium contamination is widespread in the rivers of Japan and that immunomagnetic separation is a useful method for detection of this protozoan. E. coli may be an index of Cryptosporidium contamination.
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ACKNOWLEDGMENTS |
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We thank the staffs of the Division of Public Health Sanitation, the health centers, and the water supply offices of Hyogo Prefecture for contributions to this work, including help in collection of water specimens. We also thank Caroline Latta of Osaka City University Medical School for reading the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Microbiology, Hyogo Prefectural Institute of Public Health, 2-1-29 Arata-cho, Hyogo-ku, Kobe 652-0032, Japan. Phone: 81 78 511 6784. Fax: 81 78 531 7080. E-mail: ono{at}iph.pref.hyogo.jp.
Present address: Department of Microbiology, Nepal Medical College,
Kathmandu, Nepal.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, September 2001, p. 3832-3836, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.3832-3836.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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[Abstract] [Full Text] -
Feng, Y., Alderisio, K. A., Yang, W., Blancero, L. A., Kuhne, W. G., Nadareski, C. A., Reid, M., Xiao, L.
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