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Novel Cryptosporidium genotypes in sporadic cryptosporidiosis cases: first report of human infections with a cervine genotype. (Research).



In this study, we genotyped parasites from the fecal specimens of sporadic cryptosporidiosis Cryptosporidiosis Definition

Cryptosporidiosis refers to infection by the sporeforming protozoan known as Cryptosporidia. Protozoa are a group of parasites that infect the human intestine, and include the better known Giardia.
 cases in British Columbia from 1995 to 1999. Genotyping was conducted by polymerase chain amplification of the internal transcribed spacer ITS (for internal transcribed spacer) refers to a piece of non-functional RNA situated between structural ribosomal RNAs (rRNA) on a common precursor transcript. Read from 5' to 3', this polycistronic rRNA precursor transcript contains the 5' external transcribed sequence (5' ETS),  region, a hypervariable region hypervariable region

regions present on light and heavy chains of immunoglobulins where most of the variation in amino acid sequences occurs. These are also sites of antigen binding.
 in the 18S rRNA gene and the Cryptosporidium cryptosporidium (krĭp'tōspərĭd`ēəm), genus of protozoans having at least four species; they are waterborne parasites that cause the disease cryptosporidiosis.  oocyst oocyst /oo·cyst/ (-sist) the encysted or encapsulated ookinete in the wall of a mosquito's stomach; also, the analogous stage in the development of any sporozoan.

o·o·cyst
n.
 wall protein gene. Subsequent analysis was by restriction fragment length polymorphism restriction fragment length polymorphism
n. Abbr. RFLP
Intraspecies variations in the length of DNA fragments generated by the action of restriction enzymes and caused by mutations that alter the sites at which these enzymes act, changing
 and DNA sequencing. We identified two new Cryptosporidium genotypes in humans. One of these genotypes has been found recently in deer in New York New York, state, United States
New York, Middle Atlantic state of the United States. It is bordered by Vermont, Massachusetts, Connecticut, and the Atlantic Ocean (E), New Jersey and Pennsylvania (S), Lakes Erie and Ontario and the Canadian province of
 state. The other genotype has not been identified in humans or animals. These results have important implications for drinking water drinking water

supply of water available to animals for drinking supplied via nipples, in troughs, dams, ponds and larger natural water sources; an insufficient supply leads to dehydration; it can be the source of infection, e.g. leptospirosis, salmonellosis, or of poisoning, e.g.
 quality strategies, especially for communities that obtain drinking water supplies from surface sources located in forested regions with deer populations.

**********

In recent studies of cryptosporidiosis cases in North America, South America, Europe, and Australia, various polymorphic polymorphic - polymorphism  gene loci loci

[L.] plural of locus.

loci Plural of locus, see there
 were used to show that two major genotypes of Cryptosporidium parvum occur in humans (1-10). Genotype 1, or the human genotype of C. parvum, has been isolated almost exclusively from humans and associated mainly with anthroponotic (human-to-human) transmission cycles (1). Experiments to infect animals such as cattle and mice with the human genotype have been unsuccessful, and the only in vivo in vivo /in vi·vo/ (ve´vo) [L.] within the living body.

in vi·vo
adj.
Within a living organism.



in vivo adv.
 model that exists for this genotype is a gnotobiotic gno·to·bi·ot·ic
adj.
1. Of or relating to gnotobiology.

2. Free of germs or associated only with known or specified germs.



gnotobiotic

pertaining to a gnotobiote or to gnotobiotics.
 piglet Piglet

diffident little pig; tremulously courageous. [Children’s Lit.: Winnie-the-Pooh]

See : Timidity
 model (11). So far, the only animals reported to be infected with genotype 1 C. parvum are a monkey in the United States (5) and a dugong dugong: see sirenian.
dugong

Large marine mammal (Dugong dugon, the sole living member of the family Dugongidae) that lives in shallow coastal waters from the Red Sea and eastern Africa to the Philippines, New Guinea, and northern Australia.
 (Dugong dugon) in Australia (12). In contrast, genotype 2 or the calf genotype of C. parvum has been isolated from both human and bovine hosts, as well as other livestock and wild animals WILD ANIMALS. Animals in a state of nature; animals ferae naturae. Vide Animals; Ferae naturae.  such as sheep, goats, and deer. Genotype 2 has been associated with zoonotic Zoonotic
A disease which can be spread from animals to humans.

Mentioned in: Zoonosis
 (animal-to-human) transmission cycles.

Other genotypes of C. parvum are found in animals. including the dog, mouse, bear, pig, deer, and marsupial marsupial (märs`pēəl), member of the order Marsupialia, or pouched mammals.  genotypes of C. parvum, which have been differentiated by sequence polymorphisms in the small subunit ribosomal RNA ribosomal RNA
n.
See rRNA.


ribosomal RNA (rī´bōsō´m
 (13-15), the acetyl CoA acetyl CoA /ac·e·tyl CoA/ (as´e-til) (as´e-tel? ko-a´) acetyl coenzyme A.

acetyl CoA
abbr.
 synthetase synthetase /syn·the·tase/ (-the-tas) a term used in the names of some of the ligases, no longer favored because of its similarity to synthase and its emphasis on reaction products.

syn·the·tase
n.
 (15), and heat shock protein heat shock protein
n.
Any of a group of cellular proteins that are produced under conditions of heat stress and help to stabilize other cellular proteins exposed to high temperatures.
 70 (16), as well as the Cryptosporidium oocyst wall protein (COWP COWP Cowpens National Battlefield (US National Park Service)
CoWP Cobalt Tungsten Phosphide
) (17) genes, and named after the animals from which they were derived. Of these variant C. parvum genotypes, three human infections with the dog genotype have been reported--in an HIV HIV (Human Immunodeficiency Virus), either of two closely related retroviruses that invade T-helper lymphocytes and are responsible for AIDS. There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for the vast majority of AIDS in the United States.  patient (4), two Peruvian children (6), and a child in England (18). Aside from C. parvum, nine other Cryptosporidium species are recognized: C. felis (cat), C. muris (rodent), C. andersoni (cattle), C. wrairi (guinea pig guinea pig (gĭn`ē), domesticated form of the cavy, Cavia porcellus, a South American rodent. It is unrelated to the pig; the name may refer to its shrill squeal. ), C. baileyi (bird), C. meleagridis (bird), C. serpentis (reptile), C. surophilum (lizard), and C. nasorum (fish). Although previously thought to be host specific, these other Cryptosporidium species have been associated with a few reports of human infections. C. felis (4,18,19) and C. meleagridis (19) have been found in immunocompromised immunocompromised /im·mu·no·com·pro·mised/ (-kom´pro-mizd) having the immune response attenuated by administration of immunosuppressive drugs, by irradiation, by malnutrition, or by certain disease processes (e.g., cancer).  persons. In addition, C. felis (6,18), C. meleagridis (6), and possibly C. muris (20) infections have been reported in children.

In this study, we genotyped parasites from the fecal specimens of sporadic cryptosporidiosis cases in British Columbia (BC). Genotyping was conducted by polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is  (PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
) amplification of the internal transcribed spacer region, a hypervariable region in the 18S rRNA gene (4) and the COWP gene, (21). Subsequent analysis was by restriction fragment length polymorphism (RFLP RFLP
abbr.
restriction fragment length polymorphism



RFLP

restriction fragment length polymorphism.

RFLP 
) and DNA sequencing. We identified two new Cryptosporidium genotypes in humans. One of these genotypes has been found recently in deer in New York State (14). The other genotype has not been identified in humans or animals.

Materials and Methods

Cryptosporidiosis Cases and Community Information

C. parvum oocysts were isolated from patients in the Greater Vancouver and Fraser Valley Regional Districts of British Columbia over a 5-year period from 1995 to 1999. Demographic data on this geographic area have been described (2). Fecal specimens were collected from patients diagnosed with clinical symptoms consistent with cryptosporidiosis. Cryptosporidium oocysts were identified in stool specimens by standard concentration methods, acid-fast staining, and microscopy by the diagnostic parasitology Parasitology

The scientific study of parasites and of parasitism. Parasitism is a subdivision of symbiosis and is defined as an intimate association between an organism (parasite) and another, larger species of organism (host) upon which the parasite is
 laboratories to which the specimens were submitted. Oocyst-containing specimens were preserved in potassium dichromate solution (2.5% w/v) within 7 days of reception and stored at 4 [degrees] C. The study was conducted retrospectively on specimens that were coded without personal identifiers. Informed consent from subjects was obtained.

Genomic DNA Extraction

Resuspended stool specimens were strained through cheesecloth cheese·cloth  
n.
A coarse, loosely woven cotton gauze, originally used for wrapping cheese.


cheesecloth
Noun

a light, loosely woven cotton cloth

Noun 1.
. Potassium dichromate was removed by washing the sedimented filtrate filtrate /fil·trate/ (fil´trat) a liquid or gas that has passed through a filter.

fil·trate
v.
To put or go through a filter.

n.
 3 times in distilled water. Lipids were then extracted by using ethyl acetate as described (2). Cryptosporidium oocysts were disrupted by repeated freezing in a dry ice-ethanol bath and thawing in a boiling water bath in a 20% w/v suspension of Chelex-100 (BioRad Laboratories, Hercules, CA) as described (2). The DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 extracts were stored at -20 [degrees] C.

PCR Amplification of C. parvum Oocyst DNA

Genomic DNA extracts from oocysts were centrifuged at 11,000 rpm (9,000 x g) for 20 minutes at 4 [degrees] C and the supernatants used as template DNA for PCR. The PCR reaction was carried out as described (2) by using the forward primer, cry7, and the reverse primer CP5.8R to amplify the entire internal transcribed spacer 1 (ITS1) region, resulting in a 600-bp product. The amplification procedure using the CPBDIAGF/CPBDIAGR primer pair described by Pieniazek et al. (4) was used to amplify the hypervariable region of the 18S rRNA gene, and the CRY-9/CRY-15 primer pair described by Patel et al. (21) was used for the COWP gene.

In addition, genomic DNA prepared from oocysts that had either been characterized in previous studies (1,2) or were isolated from well-defined sources were included as known genotype controls. Genotype 2 controls included one bovine isolate from a purified batch of Iowa strain oocysts that had been passaged in calves at the University of Arizona (body, education) University of Arizona - The University was founded in 1885 as a Land Grant institution with a three-fold mission of teaching, research and public service. ; two human isolates from 1996 Cranbrook and 1998 Chilliwack outbreak cases, where animals infected with cryptosporidiosis were found in the watershed area ([2] and Ong et al., unpub. data); and five other human isolates derived from sporadic cases in British Columbia that have been described in a previous study as C. parvum genotype 2 isolates (2). Genotype 1 controls included seven isolates from sporadic cases and one isolate from a 1996 Kelowna outbreak case (2), all identified previously as C. parvum genotype 1 isolates (2). Deionized water and a culture of a nonpathogenic strain of Escherichia coli Escherichia coli (ĕsh'ərĭk`ēə kō`lī), common bacterium that normally inhabits the intestinal tracts of humans and animals, but can cause infection in other parts of the body, especially the urinary tract.  were used as negative controls.

RFLP Analyses of PCR Products

PCR products were purified by using QIAquick spin columns (Qiagen, Mississauga, ON) according to the manufacturer's instructions before digestion with the restriction endonucleases Mse I (New England BioLabs New England Biolabs (NEB) produces and supplies reagents for the life science industry. NEB offers a large selection of recombinant and native enzymes for genomic research. It also offers products in the areas related to proteomics and drug discovery. , Mississauga, ON) for the ITS 1 locus and Rsa I (New England BioLabs) for the COWP gene. Two units of enzyme were added to a final volume of 20 [micro]L containing 15 [micro]L of PCR product and the appropriate dilution of the manufacturer's recommended buffer, and then incubated overnight at 37 [degrees] C. Restriction fragments were then separated on Metaphor FMC See fixed mobile convergence.  agarose agarose

more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments.
 gels (3% for Mse I digests of ITS1 products and 3.2% for Rsa I digests of COWP products) (Mandel Scientific, Guelph, ON) and stained with ethidium bromide; the patterns were visualized with a UV transilluminator. DNA band sizes were analyzed by using the ProRFLP program version 2.38 (DNA ProScan Inc., Nashville, TN).

DNA Sequencing and Analyses

PCR products from the variable 18S rRNA and COWP gene loci were cleaned by spin column purification using the QIAquick PCR Purification kits (Qiagen). Sequencing reactions were conducted in both directions, i.e., from the 5' and 3'-ends using the ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.


(Application Binary Interface) A specification for a specific hardware platform combined with the operating system.
 Prism BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and analyzed on an ABI 310 automated DNA analyzer (Applied Biosystems).

Overlapping bidirectional The ability to move, transfer or transmit in both directions.  sequences were assembled by using the SeqManII (DNASTAR Inc., Madison WI) sequence analysis software. Consensus sequences obtained were aligned by using the ClustalX program (22), which was also used for calculating the phylogenetic tree by the neighbor-joining method with 1,000 replicates for bootstrap See boot.

(operating system, compiler) bootstrap - To load and initialise the operating system on a computer. Normally abbreviated to "boot". From the curious expression "to pull oneself up by one's bootstraps", one of the legendary feats of Baron von Munchhausen.
 values. Published 18S rRNA gene reference sequences included in the multiple sequence alignment A multiple sequence alignment (MSA) is a sequence alignment of three or more biological sequences, generally protein, DNA, or RNA. In general, the input set of query sequences are assumed to have an evolutionary relationship by which they share a lineage and are descended from a  are listed below with their corresponding accession numbers: AF093491 (23) and AF087575 (4) for C. parvum genotype 1 human isolates; AF112569 (13) for a C. parvum simian isolate; AF087576 (4) and AF093490 genotype 2 isolates from a human and a bovine source, respectively; AF087574 (4) and AF112576 (13) for C. parvum "dog" genotype isolates from a human and a canine source, respectively; AF115377 (13), AF247535 (24), and AF112571 (13) for C. parvum pig, bear, and mouse genotype isolates, respectively; AF297511 (14), AF297512 (14), and AF297515 (14) for C. parvum "deer" genotype isolates; AF297503 (14) for a C. parvum muskrat muskrat, North American aquatic rodent. The common muskrats, species of the genus Ondatra, are sometimes called by their Native American name, musquash.  isolate; AF087577 (4) and AF112575 (13) for C. fells from a human and a feline source, respectively; AF115378 (13), AF093498 (23), AF093496 (23), AF112574 (13), AF093495 (23), and AF093499 (23) for C. wrairi, C. muris, C. andersoni, C. meleagridis, C. baileyi, and C. serpentis. The phylogenetic tree was displayed visually by using TreeView (25). The C. muris, C. andersoni, and C. serpentis sequences were used in the outgroup, and the tree was rooted with this outgroup.

The 18S rRNA and COWP gene sequences of the 11 patient isolates listed in the table have been submitted to GenBank and assigned accession numbers AY030084 to AY030093 and AF411631 to AF411633. The BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/) was used for DNA databases searches.

Results

Cryptosporidium oocysts were isolated from fecal specimens of 150 sporadic cryptosporidiosis cases. Two characteristic restriction profiles were obtained for Mse I digests of the 600-bp ITS1 products (Figure 1). The first type of restriction profile (Figure 1, lanes 4 and 15) showed five major bands at approximately 270, 160, 90, 75, and 55 bp. The bovine isolate, patient isolates from the 1996 Cranbrook and the 1998 Chilliwack outbreaks, and 29 (19%) isolates from sporadic cases had this restriction profile. Based on results from previous molecular characterization of a number of these isolates (1,2), this restriction profile was considered to be the C. parvum genotype 2 restriction pattern. The second restriction pattern (Figure 1, lanes 5, 6, 14, and 16) with six major bands around 185, 150, 100, 60, 40, and 30 bp was obtained from isolates of 108 (72%) sporadic cases and the one patient from the 1996 Kelowna outbreak. This restriction profile was considered to be the C. parvum genotype 1 profile, based on results from previous molecular analyses on the other seven genotype 1 isolates that were included as human genotype controls (2).

[FIGURE 1 OMITTED]

Restriction profiles with varying patterns (Figure 1, lanes 7, 9 to 13 and 17) were obtained from 13 (9%) other human isolates. Of these, nine (6%) isolates had identical restriction profiles (Figure 1, lanes 7 and 9 to 12) with five major bands at 150, 85, 70, 45, and 35 bp and were designated cervine cer·vine  
adj.
Relating to, resembling, or characteristic of deer.



[Latin cerv
 genotype isolates. The other four isolates had unique restriction profiles and could be split into two groups of two isolates, based on the similarity of banding patterns. These were CS33 (Figure 1, lane 8) and MH222 (data not shown), which both had restriction fragments at 175 and 50 bp and additional variant bands at 65 and 70 bp, respectively. The remaining two isolates VF383 had bands at 315 and 105 bp (Figure 1, lane 13) and TK348 had bands at 325 and 85bp (Figure 1, lane 17), respectively.

To identify the Cryptosporidium species and genotype of isolates with variant restriction profiles, sequencing of a polymorphic locus on the 18S rRNA gene was carried out. Eleven isolates were selected based on their ITS1 restriction patterns. These included one isolate (TK386) with a characteristic human genotype 1 restriction profile, three isolates (TK324, TK303, DE340) with patterns similar to the human genotype 1 restriction profile but with one restriction fragment shifted slightly in molecular size (e.g., Figure 1, lane 14), three cervine genotype isolates (MH205, TK320, DE302; Figure 1, lanes 7, 10, and 11), and four isolates (CS33, MH222, VF383, TK348) with unique variant profiles (Table). Comparison of the 18S rRNA gene sequences of these isolates with 22 other published reference sequences, derived from a variety of human and animal Cryptosporidium isolates by using multiple sequence alignment and phylogenetic phy·lo·ge·net·ic
adj.
1. Of or relating to phylogeny or phylogenetics.

2. Relating to or based on evolutionary development or history.
 analysis (Figure 2), showed that the 11 isolates fell into four main groups. The first group consisted of four isolates (TK386, TK324, TK303, and DE340) that had restriction profiles identical or similar to the characteristic human genotype 1 pattern and all human genotype 1 reference isolates. All human isolates in the genotype 1 group had the characteristic polyT repeat sequence reported previously (10,26) between positions nt 686 and 698. The second group consisted of three human isolates (TK320, DE302, and MH205) and two cervine genotype isolates (Figure 2). The sequences of these three human isolates in the hypervariable 18S rRNA region were identical to that of a genotype 3 deer isolate described by Perz and Le Blancq (14). The third group consisted of two isolates (VF383 and TK348) and a pig genotype isolate (Figure 2). Sequences between the two human isolates were variant from the pig genotype sequence at only two different nucleotide positions between nt 686 and 698. The fourth group consisted of two human isolates (MH222 and CS33) and a C. meleagridis isolate (Figure 2).

[FIGURE 2 OMITTED]

Twenty-five sporadic isolates were also characterized by using a second locus on the COWP gene. Rsa I digests of the 550-bp PCR products (Figure 3) also showed the same dimorphism dimorphism /di·mor·phism/ (di-mor´fizm) the quality of existing in two distinct forms.dimor´phicdimor´phous

sexual dimorphism 
1. physical or behavioral differences associated with sex.
 as the ITS1 locus with two predominant restriction patterns. Of these, 10 (40%) isolates had fragments at approximately 285, 125, 105, and 35 bp, which were characteristic for genotype 1 isolates (Figure 3, lanes 7 and 8). Another six (24%) isolates had the genotype 2 restriction profile with fragments at 410, 105, and 35 bp (Figure 3, Lanes 4 to 6). One isolate (CS33) had a variant restriction profile (Figure 3, Lane 10) with bands at approximately 370, 290, and 150 bp, which were similar in size to fragments reported for a C. meleagridis isolate (26). The 18S rRNA gene sequence of this isolate (CS33) was identical to that of C. meleagridis. The other isolate (MH205), had a restriction profile that was identical to those obtained from genotype 1 isolates (Figure 3, lane 9). This isolate had an identical ITS1 restriction profile with eight other sporadic human isolates and an 18S rRNA gene sequence that grouped with deer genotype 3 isolates from New York (Figure 2). The COWP gene sequences of MH205 and another cervine genotype isolate TK320 were determined to be identical and novel, sharing only 90% and 91% identity with the COWP gene sequences of the human (AF248741) and bovine (AF248743) alleles, respectively. BLAST analysis showed most alignment (92% identity) with a pig COWP gene sequence (AF266270) (17). However, the cervine allele allele (əlēl`): see genetics.
allele

Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome.
 had identical RsaI restriction sites to the human allele at nt 34, 228, 512, and 618, whereas the bovine allele lacked the site at nt 228. The RFLP patterns could not be determined for the remaining seven isolates as insufficient PCR product was obtained. Over half (51%) of the isolates in this study were derived from pediatric patients <10 years of age, which accounted for seven of the nine cervine genotype infections as well as the two C. meleagridis infections.

[FIGURE 3 OMITTED]

Discussion

This study describes the discovery of the first zoonotic infections in humans with a novel cervine Cryptosporidium parvum genotype. Perz and LeBlancq (14) described this genotype recently after characterizing 111 Cryptosporidium isolates from wildlife in New York state. Those researchers did not detect human infections with this genotype in cryptosporidiosis cases in New York City New York City: see New York, city.
New York City

City (pop., 2000: 8,008,278), southeastern New York, at the mouth of the Hudson River. The largest city in the U.S.
. Other molecular epidemiologic studies in England (8,18) of 1,705 cases also did not identify cervine genotype infections, although rare zoonotic infections in humans with the dog genotype of C. parvum as well as other Cryptosporidium species such as C. felis and C. meleagridis were found (27). It is possible that cervine genotype infections in humans were not identified because the novel deer genotype had not been reported at the time of the study. As well, the PCR/RFLP profile of the COWP gene from the cervine genotype isolate was identical to that obtained from human genotype 1 isolates. Sequencing of the COWP gene from two cervine genotype isolates confirmed that the human and cervine alleles had identical RsaI restriction sites. Therefore, RFLP analysis using this endonuclease endonuclease /en·do·nu·cle·ase/ (-noo´kle-as) any nuclease specifically catalyzing the hydrolysis of interior bonds of ribonucleotide or deoxyribonucleotide chains.  could not differentiate between isolates with these two genotypes.

Xiao et al. (28) have also found this novel cervine genotype in storm water samples collected from a stream in the watershed area of New York State that contributes to the New York City water supply. The transmission of cryptosporidiosis from wildlife to humans in British Columbia is not surprising as many communities are supplied with unfiltered Please wikify (format) this article or section as suggested in the Guide to layout and the Manual of Style.
Remove this template after wikifying. This article has been tagged since
 drinking water drawn from surface sources where Cryptosporidium spp. oocysts have been detected (29). Many of these watersheds are situated in remote forested areas, where wildlife such as deer are present in abundance. Deer with cryptosporidiosis infections have been identified in these watersheds (Ong et al., unpub. data). Therefore, to have as many as 6% of sporadic cases infected with this novel deer genotype is not an unexpected finding.

The ITS1 and 18S rRNA genes are reportedly multicopy genes with four copies of the Type A and one copy of the Type B rDNA units per haploid haploid /hap·loid/ (hap´loid)
1. having half the number of chromosomes characteristically found in the somatic (diploid) cells of an organism; typical of the gametes of a species whose union restores the diploid number.
 genome (30). The sequence divergence found between Type A and Type B units in the ITS1 region was a concern to us initially, as we first characterized the isolates using this locus before this report. However, Morgan et al. (31), who conducted a similar PCR-RFLP PCR-RFLP Polymerase Chain Reaction–Restriction Fragment Length Polymorphism  analysis of the ITS1 region, found that the restriction profiles were specific for different C. parvum genotypes. This study also indicated that intraorganism variation caused by the difference between Type A and Type B rDNA units may not be such a problem. Using primers to amplify the Type B unit, Morgan et al. (31) found that the Type A unit was amplified preferentially for human genotype isolates. To confirm that the observed variation in the ITS 1 RLFP patterns was not due to heterogeneous products amplified from different copies of rDNA, further characterization of a select number of sporadic isolates was performed with the 18S rRNA as well as the COWP genes. Results from these additional analyses showed that isolates with distinctly different ITS 1 RFLP patterns had different COWP RFLP patterns as well as 18S and COWP gene sequences. Therefore, ITS1 RFLP was useful for generating characteristic fingerprints that could distinguish between different C. parvum genotypes and Cryptosporidium species.

Using this method of genotyping, we were able to detect two new genotypes of C. parvum that had not been reported previously. Nine isolates (including three, MH205, TK320, and DE302, which had been characterized at the 18S locus; and two, MH205 and TK320, which had been characterized at the COWP locus) had the cervine genotype. Two other isolates (VF383 and TK348) had novel genotypes that were most closely related to a pig genotype isolate from Switzerland (13). These results have important implications for drinking water quality strategies, especially for communities that obtain drinking water supplies from surface sources located in forested regions with deer populations.
Table. Analysis of Cryptosporidium isolates collected from 1995 to
1999 from sporadic cases in British Columbia, Canada

Selected isolates      Genotype      RLFP loci    Genes sequenced

TK386                   Human           ITS1            18S
TK303                   Human           ITS1            18S
TK324                   Human           ITS1         18S, COWP
DE340                   Human           ITS1            18S
MH205                  Cervine       ITS1, COWP      18S, COWP
TK320                  Cervine          ITS1         18S, COWP
DE302                  Cervine          ITS1            18S
MH222               C. meleagridis      ITS1            18S
CS33                C. meleagridis   ITS1, COWP         18S
VF383                Other novel        ITS1            18S
TK348                Other novel        ITS1            18S

RFLP = restriction fragment length polymorphism; ITS1 = internal
transcribed spacer 1; COWP = Cryptosporidium oocyst wall protein.


Acknowledgments

We thank Claudia Sutherland, Saima Kassam, Merrilee Hughes, and Tracy Kirkham for stool specimen processing; Tara Bonham Bonham can refer to:
  • Bonhams, a British auction house
  • Dr. Bonham's Case, a legal case decided in 1610 concerning the supremacy of the common law in England
  • Bonham, Texas, USA
  • Bonham (band), heavy metal band formed by Jason Bonham
People:
 and Linda Simpson for patient stool specimens; Reuben Chen for the 18S gene sequence of CS33 and COWP gene sequence of MH205; Ian Wilson for the COWP gene sequence of TK320; and Sean Byrne and Dr. Swee Han Goh for advice on DNA sequencing and polymerase chain reaction.

Funding for this project was provided by grants #153 (97-2) and #163 (98-2) from the British Columbia Health Research Foundation.

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1. CSL - Computer Structure Language. A computer hardware description language, written in BCPL.
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(25.) Page RDM RDM Ring Deutscher Makler (German Realty Association)
RDM Red Mage (Final Fantasy, gaming)
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FEMS Federation of European Materials Societies
FEMS Fabrication Engineering Management System
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(29.) Ong C, Moorehead W, Ross A, Isaac-Renton J. Studies of Giardia Giardia /Gi·ar·dia/ (je-ahr´de-ah) a genus of flagellate protozoa parasitic in the intestinal tract of humans and other animals, which may cause giardiasis; G. lam´blia (G. intestina´lis) is the species found in humans.  spp. and Cryptosporidium spp. in two adjacent watersheds. Appl Environ Microbiol 1996;62:2798-805.

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(31.) Morgan UM, Deplazes P, Forbes DA, Spano F, Hertzberg H, Sargent KD, et al. Sequence and PCR-RFLP analysis of the internal transcribed spacers of the rDNA repeat unit in isolates of Cryptosporidium from different hosts. Parasitology 1999;118 (Pt 1):49-58.

Corinne S. L. Ong, * ([dagger]) Diane L. Eisler, * Alireza Alikhani, * Vicki W. K. Fung, * Joan Tomblin, ([double dagger]) William R. Bowie, * Judith L. Isaac-Renton * ([dagger])

* University of British Columbia Locations
Vancouver
The Vancouver campus is located at Point Grey, a twenty-minute drive from downtown Vancouver. It is near several beaches and has views of the North Shore mountains. The 7.
, Vancouver, British Columbia; ([dagger]) BC Centre for Disease Control, Vancouver, British Columbia; ([double dagger]) BC Biomedical bi·o·med·i·cal
adj.
1. Of or relating to biomedicine.

2. Of, relating to, or involving biological, medical, and physical sciences.
 Laboratories Ltd., Surrey, British Columbia Surrey is a Canadian city in the province of British Columbia that lies within the Metro Vancouver district, and geographically at the centre of the larger region known as the Lower Mainland of BC. It is the province's second-largest city by population after the city of Vancouver. , Canada.

Dr. Ong is an assistant professor at the University of British Columbia and a senior scientist at the British Columbia Centre for Disease Control. Her research interests are in pathogenic protozoa.

Address for correspondence: Corinne S.L. Ong, Pathology & Laboratory Medicine, BC Centre for Disease Control Laboratory Services, 655 West 12th Avenue, Vancouver, British Columbia, Canada, V5Z 4R4; fax: 604-660-6073; e-mail: cong@interchange.ubc.ca
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Author:Isaac-Renton, Judith L.
Publication:Emerging Infectious Diseases
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Geographic Code:1CBRI
Date:Mar 1, 2002
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