Jpn. J. Infect. Dis., 54, 2001

Laboratory and Epidemiology Communications

Sensitive Detection of Cf7PtOSPOridium Oocysts in Environmental

Water Samples by Reverse TranscrlPt10n‑PCR
Tatsuya Karasudani*, Satomi Aoki, Junko Takeuchi, Masaaki Okuyama, Mitsuaki Oseto,
Shigemi Matsuura, Tadao Asai and Hiroo Inouye
Ehime Prefectural Institute of Public Health and Environmental Science,

Sanban‑cho 8‑234, Matsuyama, Ehime 790‑0003
Communicated by Hiroo lnouye(Accepted July 24, 2001 )centrifugation. The concentrates were subjected to mRNA

Cryptosporidium lS Present in the environment as an oocyst
and causes outbreaks through contamination or the water

extraction and RT‑PCR ampliflCation in the manner describedby Kaucner et al･ (1)･ C･ paTum HNJI supplied by Dr･ Kuroki,

Pupply. With a reverse transcription‑PCR (RT‑PCR) amplify‑

lng heat shock protein 70 (hsp70) gene, we were able to detect

Kanagawa Prefectural lnstltute Of Public Health, was used as

two differenttypes of viable Cyyptosporidium oocysts froma positive control.

the environment.

The concentrated specimens were suspended in lnstaGenematrix (Bio‑Rad, Hercules, Calif., USA), and heated for 20
min at 450C for induction of hsp70 gene expression. Afterfreeze‑thaw, mRNA was extracted from the samples uslng
the Dynabeads mRNA DIRECT kit (Dynal, Oslo, Norway),

Water specimens were filtered under positive pressure
through a 3‑Elm polycarbonate filter (Nuclepore filter,Whatman, Maidstone, England). The trapped materials weredetached from the filter by sonication, and concentrated by

the hsp70 gene was RT‑PCRamplified. To monitor the sensi‑*Corresponding author: Fax: +8 1 ‑89‑947‑ I 262, E‑mail: karasudani‑tivity Of the RT‑PCR amplification, a poly A‑tailed RNA wastatsuya@pref. ehime. jp

included in the reaction as an intemal positive control (IPC122

Jpn. J. Infect. Dis., 54, 2001
RNA). The IPC RNA was a transcript fiom an artificial gene,at different locations (Table 2). lMS/FA detected two C.

which consisted of an SP伝 promoter and nt 27, 324‑27, 974 ofparvum‑like oocysts each from the No, 5 and No. 6 speci‑

the A‑phage genome, followed by I 5 Ts onthe complementarymens. In the RTIPCR assays performed twice f♭r each sample,strand. The artificial gene (688 bp in size) was first PCR‑

No. 5 was positive in one trial and No. 6 in both trials. The
RT‑PCR products were recovered from the gel, and theirsequences were detemined by the Gene Rapid (Amersham
Phamacia Biotech, Piscataway, NJ., USA) dye teminator

amplified, and then used for transcrlPtlOn Of IPC RNA by
the Competitive RNA Transcription kit (TAKARA, Kyoto)･
Primers used for RT‑PCR are listed in Table 1. SP6/^27324Fand T15/^27954R were the pnmers used for PCR ampliflCa‑

method･ One of the two Not 6 specimens was fわund to be ation of the 688‑bp template DNA, and入27324F and入27954R

mixture of at least two variants, as variations of two nucle‑

for RTIPCR amplification of IPC RNA. CPHSP2423 andotides were observed at 15 locations in the amplified 361‑nt

CPHSP2764 were used as prlmerS for amplification of the C.

fragment. As only one variant was cut by BsaJI and BsrBI,

parvum hsp 70 gene.

the sequence of the uncut fragment was determined in order

The effects of inclusion of lPC RNA and its PCR primers

to deduce the sequences of the two variants.

on the specific Cryptosporidium hsp70 gene amplificationln total, four sequences were obtained from this study. Thewas examined. As shown in Fig. 1, the presence of 4,000
copies oflPC RNA had no effect (compare lanes 4 and 5

phylogenetic tree was constructed from these sequenceswith lanes 7 and 8); addition of 10‑fold more copies, i.e.,(3). The sequences of Cl3 and C‑4 were identical to those c‑f
the C. parvum bovine genotype, and those of C‑1 and C‑2were identical to those of C. meleagridis (Fig. 2), i.e., speci‑
men No. 5 Contained C. meleagridis, and specimen No. 6
the C. parvum bovine genotype and C. meleagridis. C.

together with published CりPtOSPOridium hsp 70 sequences

40,000 copies ofIPC RNA, tended to decrease the density ofthe specific band (lanes 1 and 2), but even under these condi‑
tions, the sensitivlty Was Sumcient fわr detectlng a Slngle

viable C. paTVum OOCySt.
Using the above RT‑PCR and immunomagnetic separation

meleagridis has been considered to be a parasite of turkeys,

and immunofluorescence assay (IMS/FA), W.e attempted tobut its infection to humans has recently been confirmed (4).

detect C77PtOSPOridium from river‑water speclmenS Obtained

It has been suggested that bird discharge may be a source ofTable 1. Primers for RT‑PCR
Primers
SP伝/入27324Ft

Targetuence

5 I‑CCGCTCCTCGCTCATAACAG‑3 I

5 I‑ccGCCTACCmCACGACTT‑3 'C pan,um

5 ′‑AAATGGTGAGCAATCCTCTG‑3 ′CPHSP2764‑i hsp70 gene

5 ∫‑CTTGCTGCTCmCCAGTAC‑3 ′CPliSP24233

Size (bp)5 '‑TTTTTTTTTTTTTTTCCGCCTACCTTTCACGACTT‑3 'IPC RNA

入27954Req

5 I‑AAmAGGTGACACTATAGAATACCGCTCCTCGCTCATAACAG‑3 '入DNA

T1 5/入27954R2

人27324FS

1 The 5'end of the pnmer was modified to contain the SP6 phage promotor sequence･
2 The 5′ end of the pnmer was modifiedwith 15 thymidine base to add a 3'tail of 15 adenosine base to the IPC.

3ref.2.

Table 2. Detection of viable Cryptosporidium oocysts in nonspikcd river samples
Sample No.

volume ( I )turbidity(NTU)

pellct ( LL I)

IMS/FAI RTIPCR2 (sequence)

1.27

40

20.94

30

35.44

50

41,4340

5not tested

20+ (C‑))

6not tested

15+(C‑23,C‑34) +(C‑4)

〜2･5 102･52 LrI

1000022

I numbers or detected oocysts･2 +, 361 bp fragment was detected; ‑, 650 bp fragment was detected but 361 bp fragment was not detected1
3 not fragmented by BsaJI.
4 not fragmented by BsrBI.

M

123

4

5

6

78IPC RNA 650 bp
C.payvum 361 bp

Fig･ L SensitivityofRT‑PCR for simultaneous detection of C. parvum and IPC RNA. Lane I ‑3 and 4‑6 represent RT‑PCR
reaction containing 40,000 Copy and 4,000 Copy lPC RNA, respectively. Lane I , 4 and 7, five oocysts; Lane 2, 5 and 8, oneoocyst; Lane 3 and 6, no oocyst. Lane M, DNA molecular weight marker (￠X174 HincII)123

Jpn. J. Infect. Dis., 54, 2001

Fig･ 2･ Phylogenetic relationship of RT‑PCR productswithinthe Crwtosporidium parsites by neighbor‑jolnlng analysIS.

Cywtosporidium contamination of the water (5). Our results

3. Sulaiman, I. M., Morgan, U. M., Thompson, R. C., Lal,demonstrate that RT‑PCR may be a useful tool for molecular

A･ A･ and Xiao,し. (2000): Phylogenetic relationships ofepidemiology of Cywtosporidium.

Cryptosporidium parasites based on the 701kilodaltonheat shock protein(HSP70) gene. Appl. Environ. Microbio1.,
66, 2385‑2391.

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Checkley, W, Cabrera,し., Gilman, 氏. H. and Lal, A. A.

detectlOn Of viable Giapdia cysts and Cyyptosporidium(2001): Identification of 5types of CfyPtOSPOridium

parvum oocysts in large‑volume water samples withparasites in children in Lima, Peru. J. Infect. Dis., 183,

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5. Sreter, T., Kovacs, G., da Silva, A. ∫., Pieniazek, N. ∫.,to detect and determine the infectivlty Of waterborne

Szell, Z., Dobos‑Kovacs, M., Marialigeti, K. and Varga,
I･ (2000): Morphologic, host specificity, and molecularcharacterization of a Hungarian Cryptosporidium
meleagridis isolate. Appl. Environ. Microbio1., 66, 735‑

Cywtosporidium parvum. Appl. Environ. Microbio1., 63,738.

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