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Appl Environ Microbiol, April 1998, p. 1584-1586, Vol. 64, No. 4
Veterinary Medicine Teaching and Research
Center, School of Veterinary Medicine, University of California, Davis,
Tulare, California 93274,1 and
Department of Pathology, Microbiology, and Immunology, School
of Veterinary Medicine, University of California, Davis, Davis,
California 956162
Received 1 December 1997/Accepted 28 January 1998
We evaluated whether nucleic acid amplification with primers
specific for Cryptosporidium parvum followed by automated
DNA sequence analysis of the PCR amplicons could differentiate between California isolates of C. parvum obtained from livestock,
humans, and feral pigs. Almost complete sequence identity existed among the livestock isolates and between the livestock and human isolates. DNA sequences from feral pig isolates differed from those from livestock and humans by 1.0 to 1.2%. The reference sequence obtained by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688-694, 1991.)
differed from California isolates of C. parvum by 1.8 to
3.2%. These data suggest that DNA sequence analysis of the amplicon of
Laxer et al. does not allow for differentiation between various strains
of C. parvum or that our collection of isolates obtained
from various hosts from across California was limited to one strain of
C. parvum.
Cryptosporidium parvum, a
protozoan parasite, is an important etiologic agent of enterocolitis in
mammals. C. parvum appears to be infectious for 79 mammals,
including domestic and wildlife species (5). Transmission of
oocysts can occur by the direct fecal-oral route or through the
consumption of food or water contaminated with oocysts (5).
With respect to waterborne C. parvum, identifying the
mammalian source following a waterborne outbreak has proven to be very
difficult (7, 10, 15, 16). The standard approach has been to
collect fecal samples from mammalian populations of concern and to
determine the proportion with detectable levels of C. parvum
oocysts. Those mammalian populations found to be shedding C. parvum are presumed to have been the source of the waterborne
outbreak (7, 15, 16). The validity of this approach may be
strengthened by utilizing molecular fingerprinting to assess whether
C. parvum isolated from the human case(s) is similar to or
different from the isolate(s) obtained from the suspect source(s).
Desirable attributes for a DNA fingerprinting method would include the
ability to detect minute concentrations of oocysts, high specificity,
and the ability to function with fresh or archived fecal and water
samples. Isoenzyme typing, immunoblotting, restriction fragment length
polymorphism on whole genomes, and field inversion gel electrophoresis
typically require large numbers of oocysts and have not been able to
differentiate between animal isolates or could only distinguish between
animal and human isolates (2, 3, 11-13). Arbitrarily primed
PCR requires that contaminating DNA not be present in the sample. An
alternative method for differentiating C. parvum from
different mammalian sources would be nucleic acid amplification with
primers specific for C. parvum followed by automated DNA
sequence analysis of the amplicons. The present study was undertaken to
determine if such an approach could differentiate C. parvum
isolates obtained from different mammalian sources from throughout
California.
We utilized a previously developed set of PCR primers (8).
Although these primers were not known to target a specific gene (14), they have the test attributes we were seeking, e.g.,
sensitive for C. parvum and able to function with
formalin-fixed samples, and they have been shown not to amplify other
microorganisms commonly found in water and feces (6, 8, 9,
14). Additionally, we tested this primer pair against bovine
Neospora, Salmonella typhimurium,
Escherichia coli, Nematodirus battus,
Eimeria zurnii, Eimeria ellipsoidalis,
Cryptosporidium muris, Toxoplasma gondii, and
bovine Giardia duodenalis and found no cross-reaction.
Fecal samples were collected from dairy calves, beef calves, goats,
horses, and feral pigs from throughout California. Secondary treated
wastewater samples (presumably of human origin) were collected from one
plant. Samples were screened for C. parvum by a direct immunofluorescence assay (MERIFLUOR
Cryptosporidium/Giardia; Meridian Diagnostic, Inc.,
Cincinnati, Ohio). For each isolate, C. parvum oocysts were
purified by using a sucrose gradient (17) or low-speed centrifugation (4), followed by bleach sterilization. DNA
was extracted by incubating oocysts for 48 h in TES (10 mM Tris
HCl [pH 7.5], 1 mM EDTA, 10 mM NaCl) buffer containing 0.8% Sarkosyl (Sigma, St. Louis, Mo.) and 200 µg of proteinase K (Sigma) per ml.
DNA was extracted with phenol-chloroform-isoamyl alcohol (24:24:1), precipitated in 100% cold ethanol, dried, and stored at 4°C in Tris-EDTA buffer (pH 7.5). PCR amplifications were performed in a
Hot-Start tube with the GeneAmp Core Reagents (Perkin-Elmer, Foster
City, Calif.), 64 to 125 ng of DNA, and sterile water. DNA
concentrations were determined by the Nucleic Acid Test Instant Quantitation Kit (NBI, Plymouth, Minn.). Primer annealing temperature was 52°C. All PCR products were purified with the Qiaquick PCR purification kit (Qiagen, Inc., Valencia, Calif.). The products were
sequenced with the Taq FS Dye terminator mix (Perkin-Elmer), 20 ng of
template, and 10 pmol of primer. The sequence was generated on an ABI
377 automated DNA sequencer (Applied Biosystems, Alameda, Calif.).
Contiguous sequence was generated from the forward and reverse strands
with the Sequencher program (Genecodes, Ann Arbor, Mich.). Each isolate
was sequenced three times for the forward and reverse directions.
The DNA sequences obtained for the 12 C. parvum isolates had
various amounts of polymorphism when compared against each other (Fig.
1). Boldface and single-underlined
nucleotides indicate substitutions, black dots indicate deletions, and
double-underlined nucleotides on the Laxer sequence correspond to
diagnostic probe 127, a StyI restriction site, and
diagnostic probe 325 (4, 8). The degree of sequence
polymorphism (substitutions, deletions, insertions) ranged from 0.0 to
0.5% (Table 1) among the livestock isolates and between the livestock isolates and the secondary treated
wastewater isolate, presumably of human origin. The majority of
livestock hosts were located on watersheds which were hydrologically independent from each other and had no known common source of feed,
personnel, or other such factors which could serve as a means of
cross-transmission of C. parvum. The DNA sequences obtained from feral pig isolates differed from those of livestock and human C. parvum by 1.0 to 1.2%, at least twice the proportion
compared to the amount of polymorphism among livestock isolates (0.0 to 0.5%) and at least four times the proportion of polymorphism between livestock and human C. parvum (0.0 to 0.2%). These pigs
were trapped from a remote site that had no known cohabitating
livestock and minimal human contact (1). The DNA sequence
published by Laxer et al. (8) had the greatest amount of
polymorphism (2.0 to 3.2%) compared against all of the California
isolates. The Laxer sequence has been utilized by various researchers
to develop PCR-based diagnostic tests for C. parvum (4,
6, 9). Most of the polymorphism between the sequence of Laxer et
al. and California isolates did not occur at critical internal
diagnostic sites (probe or restriction enzyme), but all three feral
pigs had a substitution at probe 127 (A to T) and all 12 California
isolates had a deletion at probe 325 compared to the Laxer sequence.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
DNA Sequence Similarity between California
Isolates of Cryptosporidium parvum
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FIG. 1.
Sequence alignment of PCR amplicons from 12 isolates of
C. parvum from throughout California and the original
sequence obtained by Laxer et al. (8). Feral pigs 2 and 3 have a shorter sequence (201 bp) as a consequence of having to use
nested PCR (4) due to low oocyst concentrations in the two
fecal samples. Boldface and single-underlined nucleotides indicate
substitutions, black dots indicate deletions, and double-underlined
nucleotides on the sequence of Laxer et al. correspond to diagnostic
probe 127, a StyI restriction site, and diagnostic probe
325.
TABLE 1.
Levels of sequence similarity based on alignment of PCR
amplicons from 12 isolates of C. parvum from throughout
California and the original sequence obtained by Laxer et al.
(8)
Although the primers of Laxer et al. (8) have many of the test attributes that are desirable for amplifying a specific section of genomic DNA of C. parvum, there does not appear to be sufficient polymorphism within the amplicon to allow for reliable discrimination between C. parvum isolates obtained within California. Alternatively, the marked similarity between the DNA sequences obtained from each of the 12 isolates may indicate that infection with this protozoal parasite was limited to one strain of C. parvum.
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ACKNOWLEDGMENTS |
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We thank the California Veterinary Diagnostic Laboratory System in Tulare for providing oocysts and Salmonella typhimurium DNA, Steven A. Nadler for Nematodirus battus DNA, and Patricia A. Conrad for Toxoplasma gondii and bovine Neospora isolates.
This project was supported by the Center for Equine Health (formerly the Equine Research Laboratory) with funds by the Oak Tree Racing Association, the State of California satellite wagering fund, and contributions from private donors.
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FOOTNOTES |
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* Corresponding author. Mailing address: Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, 18830 Rd. 112, University of California, Davis, Tulare, CA 93274. Phone: (209) 688-1731. Fax: (209) 686-4231. E-mail: ratwill{at}vmtrc.ucdavis.edu.
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Appl Environ Microbiol, April 1998, p. 1584-1586, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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