New Cryptosporidium Genotypes in HIV-Infected Persons.Using DNA sequencing 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, we identified four distinct Cryptosporidium cryptosporidium (krĭp'tōspərĭd`ēəm), genus of protozoans having at least four species; they are waterborne parasites that cause the disease cryptosporidiosis. genotypes in HIV-infected patients: genotype 1 (human), [ILLEGIBLE TEXT] (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one [ILLEGIBLE TEXT] Cryptosporidium sp. isolate from a dog. This is the first identification of human [ILLEGIBLE TEXT] with the latter two genotypes. Protozoan protozoan (prō'təzō`ən), informal term for the unicellular heterotrophs of the kingdom Protista. Protozoans comprise a large, diverse assortment of microscopic or near-microscopic organisms that live as single cells or in simple apicomplexan parasites from the genus Cryptosporidium infect a wide hosts (1). The parasites are transmitted to humans through contaminated drinking contact with infected animals, and contact with infected persons (3). In the [ILLEGIBLE TEXT] 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. manifests itself as self-limited diarrhea, sometimes accompanied by abdominal cramps, fever, and vomiting. In the immunodeficient, however, [ILLEGIBLE TEXT] may be severe, chronic, and life-threatening (4). Cross-infection experiments, in which Cryptosporidium oocysts were obtained [ILLEGIBLE TEXT] one species and fed to animals of another species, have investigated the host [ILLEGIBLE TEXT] parasite (5). The differences observed in the host range of putative C. parvum [ILLEGIBLE TEXT] proposal to establish the Cryptosporidium isolate originating from guinea pigs, morphologically indistinguishable from C. parvum, as a new species--C, wrairi the basis of experimental infection (6). The possibility of many Cryptosporidium fostered the development of techniques suitable for typing isolates. Commonly [ILLEGIBLE TEXT] are isoenzyme isoenzyme /iso·en·zyme/ (-en´zim) isozyme. i·so·en·zyme n. See isozyme. i analysis (7), Western blotting (8,9), random amplified [ILLEGIBLE TEXT] analysis (10-12), 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 restriction fragment length [ILLEGIBLE TEXT] RFLP RFLP abbr. restriction fragment length polymorphism RFLP restriction fragment length polymorphism. RFLP ) analysis (13,14), and PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) followed by DNA sequencing (11,15-19). Early studies of the polymorphism of isolates classified as C. parvum found sign geographic variation among isolates (20) in the region coding for the small [ILLEGIBLE TEXT] RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic (SSU-rRNA), commonly used for taxonomic classification. Recently, it [ILLEGIBLE TEXT] (21) that one of the sequences used in this analysis (22) was erroneously [ILLEGIBLE TEXT] parvum sequence, while in fact it was C. muris. More recent work (e.g., Le Blan and GenBank entry AF040725) has shown that the SSU-rRNA region of the C. p zoonotic Zoonotic A disease which can be spread from animals to humans. Mentioned in: Zoonosis (bovine) genotype does not show heterogeneity and is practically [ILLEGIBLE TEXT] sequence submitted to GenBank in 1993 (accession number L 16996, [24,25]). Recently, consistent results of typing bovine and human C. parvum isolates led [ILLEGIBLE TEXT] recognition of two genotypes of C parvum. These two genotypes were [ILLEGIBLE TEXT] differentiated by sequencing the SSU-rRNA coding region (16), sequencing the Cryptosporidium thrombospondin-related adhesion protein (TRAP-C2) gene (17 RFLP analysis of the Cryptosporidium 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 (COWP COWP Cowpens National Battlefield (US National Park Service) CoWP Cobalt Tungsten Phosphide ) gene (15). [ILLEGIBLE TEXT] anthroponotic genotype (genotype l) has been found only in infected humans, [ILLEGIBLE TEXT] zoonotic genotype (genotype 2) has been found both in infected humans and in [ILLEGIBLE TEXT] cows, lambs, goats, and horses. The published partial SSU-rRNA sequence of [ILLEGIBLE TEXT] genotype 1 (16) is identical to the Cryptosporidium SSU-rRNA sequence that we PCR-amplified 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. from a patient with AIDS (GenBank accession number L1 data from different laboratories for these two genotypes were reproducible calls [ILLEGIBLE TEXT] the recent conclusion of Tzipori and Griffiths (26) that "there are no clearly [ILLEGIBLE TEXT] characterized reference `strains' of Cryptosporidium." Other new Cryptosporidium genotypes have been identified in various animals (cats, and koalas) by sequencing the region coding for SSU-rRNA (16,27,28). [ILLEGIBLE TEXT] is thought to represent C. felis (28). When morphologic and other data are [ILLEGIBLE TEXT] new genotypes, they may be recognized as new Cryptosporidium species. We present results of typing Cryptosporidium isolates by direct sequencing the [ILLEGIBLE TEXT] of the SSU-rRNA amplified from fecal specimens with Cryptosporidium genus-s diagnostic PCR primers (25). Applying this method to a set of specimens from a longitudinal study longitudinal study a chronological study in epidemiology which attempts to establish a relationship between an antecedent cause and a subsequent effect. See also cohort study. on the risk for enteric enteric /en·ter·ic/ (en-ter´ik) within or pertaining to the small intestine. en·ter·ic adj. 1. Of, relating to, or within the intestine. 2. parasitosis par·a·si·to·sis n. pl. par·a·si·to·ses Infestation with parasites. parasitosis a disease caused by a parasitic infestation. See also helminthiasis. and chronic diarrhea in [ILLEGIBLE TEXT] patients with a low CD4+ count, we found the first human cases of infection by a newly identified zoonotic Cryptosporidium species possibly originating from a [ILLEGIBLE TEXT]. The Study All analyzed specimens were collected from January 1991 through September 19 assessing the impact of enteric parasite-associated diarrhea in persons infected [ILLEGIBLE TEXT]. Study participants answered comprehensive questionnaires concerning clinical a epidemiologic information and provided stool specimens monthly. All stool [ILLEGIBLE TEXT] examined for C. parvum by Kinyoun carbol-fuchsin modified acid-fast stain Acid-fast stain A special stain done to microscopically identify the bacteria that cause tuberculosis. Mentioned in: Sputum Culture acid-fast stain (30) immunofluorescence Immunofluorescence A technique that uses a fluorochrome to indicate the occurrence of a specific antigen-antibody reaction. The fluorochrome labels either an antigen or an antibody. (31). Stained slides were examined by observing 200 oil-[ILLEGIBLE TEXT] fields. C parvum was associated with 18 (5,1%) of the 354 acute episodes and 3 the chronic episodes of diarrhea reined by the participants. All specimens from were preserved in separate vials of 10% formalin formalin /for·ma·lin/ (for´mah-lin) formaldehyde solution. for·ma·lin n. An aqueous solution of formaldehyde that is 37 percent by weight. and polyvinyl alcohol (Para-[ILLEGIBLE TEXT] System; Meridian Diagnostics, Inc., Cincinnati, OH) and thus were not suitable [ILLEGIBLE TEXT] analysis. Some specimens were originally aliquoted and stored without [ILLEGIBLE TEXT] Of this set, we selected 18 available specimens for 10 randomly chosen Cryptosporidium positive patients for this study. An aliquot aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share) of approximately 300 [micro]l of each stool specimen was suspended in 1 phosphate-buffered saline, pH 7.2, containing 0.01 M of EDTA EDTA: see chelating agents. (PBS-EDTA), a suspension was centrifuged at 14,000 x g, 4 [degrees] C for 5 minutes. The pellet from [ILLEGIBLE TEXT] centrifugation Centrifugation A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal was washed two more tunes under the same conditions. The pellet resuspended in 300 [micro]l of PBS-EDTA and used for DNA extraction, performed [ILLEGIBLE TEXT] FastPrep disrupter and the FastDNA kit (BIO 101, Inc., Vista, CA) (32). [ILLEGIBLE TEXT] stored at 4 [degrees] C until PCR amplification. Cryptosporidium genus-specific primers (CPBDIAGF and CPBDIAGR) were us the Cryptosporidium SSU-rRNA variable region (24,25). The conditions for PC for 15 minutes; 45 cycles of denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures. at 94 [degrees] C for 30 seconds, annealing annealing (ənēl`ĭng), process in which glass, metals, and other materials are treated to render them less brittle and more workable. at 65 seconds, extension at 72 [degrees] C for 1 minute, 30 seconds; followed by extension at 7 minutes; and finished with a hold step at 4 [degrees] C. PCR products were analyzed by electrophoresis on 2% SeaKem GTG (chat) gtg - Got to go. The user is about to stop chatting. agarose agarose more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments. (C FMC See fixed mobile convergence. Bioproducts, Rockland, ME), stained with ethidium bromide, and visualize transilluminator. The size of the diagnostic fragment amplified with these primer parvum genotype 1 (GenBank accession number L16997) was 438 bp, for C. par 2 (L16996) 435 bp, for C. baileyi (L19068) 428 bp, and for C. muris (L19069) 4 PCR products were purified by using the Wizard PCR Preps kit (Cat. No. A7170 Madison, WI). Sequencing reactions were done with the Perkin Elmer Big Dye [ILLEGIBLE TEXT] 4303149, PE Biosystems, Foster City, CA) and analyzed on the Perkin Elmer [ILLEGIBLE TEXT] automatic DNA sequencer. Sequences were assembled by using the program [ILLEGIBLE TEXT] (DNASTAR Inc., Madison, WI). Sequences were aligned with the program MA analyzed by programs from the PHYLIP PHYLIP Phylogeny Inference Package (genetics software) (phylogeny inference program) package Findings After the 18 fecal specimens were retrieved from storage and processed for PCR CPBDIAGF/CPBDIAGR diagnostic primer set, visualization of PCR products [ILLEGIBLE TEXT] gels found two different sizes of diagnostic bands. The size of the Cryptosporidium bands for patients 53, 75, 119, 124, 153,278, and 554 did not differ significantly of the standard band (approximately 435 bp) obtained with cloned SSU-rRNA [ILLEGIBLE TEXT] parvum genotype 2 (Figure 1, lane 1). On the other hand, the Cryptosporidium [ILLEGIBLE TEXT] bands in samples from patients 84, 184, and 485 were visibly larger, [ILLEGIBLE TEXT] (Figure 1, lane 3 shows data for patient 84). [Figure 1 ILLUSTRATION OMITTED] DNA sequence [ILLEGIBLE TEXT] types of sequences ([ILLEGIBLE TEXT] sequence of the [ILLEGIBLE TEXT] patient 53 was 435 bp 1 identical to the [ILLEGIBLE TEXT] fragment of the C. parvum 2 (GenBank accession L 16996) SSU-rRNA. [ILLEGIBLE TEXT] bands in patients 75, 12 and 554 were 438 bp [ILLEGIBLE TEXT] identical to the [ILLEGIBLE TEXT] fragment of the C. parv 1 SSU-rRNA (GenBan number L 16997). The [ILLEGIBLE TEXT] patient 119 was 429 bp diagnostic bands from 184, and 485 were 455 were identical to each [ILLEGIBLE TEXT] Sequence similarity [ILLEGIBLE TEXT] GenBank database and phylogeny analysis of [ILLEGIBLE TEXT] types of sequences [ILLEGIBLE TEXT] while they were not [ILLEGIBLE TEXT] sequences in GenBank, within other [ILLEGIBLE TEXT] rRNA sequences from [ILLEGIBLE TEXT] Cryptosporidium ([ILLEGIBLE TEXT] Cryptosporidium [ILLEGIBLE TEXT] In addition, these [ILLEGIBLE TEXT] significantly different [ILLEGIBLE TEXT] reported partial SSU-r sequences (16,19,27,28 long diagnostic [ILLEGIBLE TEXT] 119 was identical to a [ILLEGIBLE TEXT] identified canine C. [ILLEGIBLE TEXT] SSU-rRNA sequence [ILLEGIBLE TEXT] accession number AF1 longest sequence (455 bp) from patients 84, 184, and 495 was identical in a 100-[ILLEGIBLE TEXT] an updated sequence (GenBank accession number AF097430) for C. felis (also [ILLEGIBLE TEXT] parvum genotype) (28). Alignment of these sequences is shown in Figure 2. In [ILLEGIBLE TEXT] hypervariable alignment region (from position 101 to 110), all genotypes display number of thymidines. Genotype 1 has the longest stretch, 11 thymidines, while Cryptosporidium sp. (canine genotype) has only one. The sequence for C. fells [ILLEGIBLE TEXT] insertions at alignment positions 45, 86, 111, 187, and 218, as well as one [ILLEGIBLE TEXT] thymidines at positions 197 and 198. Apart from the difference in the hypervaria Cryptosporidium sp. (canine genotype) has a deletion of two bases at positions 4 [Figure 2 ILLUSTRATION OMITTED] Results of genotyping the 18 specimens are in Table 2. For patients 53, 124, 153, 485, only a single specimen was available. For other patients (e.g., patient 554), [ILLEGIBLE TEXT] specimens collected during 12 months were available. The same Cryptosporidium persisted throughout a patient's infection.
Table 2. Persistence of Cryptosporidium genotypes in patients(a)
No. of Month of follow-up
Patient available
no. specimens 1 2 3 4 5 6
53 1 B
75 2 H - - H
84 4 F - F - F F
119 2 C - C
124 1 H
153 1 H
184 1 F
278 1 H
485 1 F
554 4 H - - - - -
No. of Month of follow-up
Patient available
no. specimens 7 8 9 10 11 12
53 1
75 2
84 4
119 2
124 1
153 1
184 1
278 1
485 1
554 4 - H - H - H
(a) B, C. parvum genotype 2 (zoonotic, bovine); C, Cryptosporidium sp. (zoonotic, canine); F, C. felis (zoonotic, feline); H, C. parvum genotype 1 (anthroponotic); -, specimen not available. Conclusions Using the sequence of a diagnostic fragment of SSU-rRNA, as well as two well-genotypes of C. parvum (anthroponotic genotype 1 and zoonotic genotype 2), we new Cryptosporidium genotypes. The first, in patients 84, 184, and 485, was [ILLEGIBLE TEXT] feline Cryptosporidium genotype (28), also described as C. felis. The second, in [ILLEGIBLE TEXT] from patient 119, represents the newly identified Cryptosporidium sp. found in a originating from a dog (GenBank accession number AF112576). New taxons may be established within the genus Cryptosporidium on the basis [ILLEGIBLE TEXT] host range together with molecular data, even though morphologic criteria are [ILLEGIBLE TEXT] lacking. We propose to use C. felis Iseki, 1979, instead of feline C. parvum [ILLEGIBLE TEXT] believe that the anthroponotic genotype 1 of C. parvum and the Cryptosporidium genotype) described here should be named as distinct species. Although the [ILLEGIBLE TEXT] protists is morphology-based, the taxonomy of bacteria is being reevaluated on [ILLEGIBLE TEXT] molecular data (35). There is no reason to use different approaches to these two [ILLEGIBLE TEXT] is it necessary to postulate that the basis for speciation speciation Formation of new and distinct species, whereby a single evolutionary line splits into two or more genetically independent ones. One of the fundamental processes of evolution, speciation may occur in many ways. of Cryptosporidium [ILLEGIBLE TEXT] or that molecular data lend little support to separation of Cryptosporidium into [ILLEGIBLE TEXT] species (26). Further, we see no evidence for the unconventional conclusions of Griffiths that "... genetic markers in C. parvum can change upon passage to a [ILLEGIBLE TEXT] possibly through a selective mechanism favoring different populations" (26) nor Widmer (21) that "... genetic studies and experimental infections suggest that a [ILLEGIBLE TEXT] mechanism triggered by a change in the intestinal environment might be involve the genetic make-up of C. parvum populations." The finding of new Cryptosporidium genotypes in immunodeficient patients [ILLEGIBLE TEXT] unique susceptibility to infections by divergent Cryptosporidium species [ILLEGIBLE TEXT] companion animals or livestock. However, recent identification of C. fells in a [ILLEGIBLE TEXT] indicate a complex pattern of flow of different Cryptosporidium species in the [ILLEGIBLE TEXT] When more data on the distribution of these species become available, public he preventive measures will need to be reevaluated, and isolates may need to be [ILLEGIBLE TEXT] their natural history. Finally, it is clear that Kinyoun carbol-fuchsin modified [ILLEGIBLE TEXT] (30) and direct immunofluorescence (31) could detect all genotypes, but this may for diagnostic reagents that may be affected directly (PCR) or indirectly (Ab) by composition of the new Cryptosporidium isolates reported here. Thus, discovery Cryptosporidium genotypes in human cryptosporidiosis should cause existing [ILLEGIBLE TEXT] to be reevaluated. Table 1. Cryptosporidium genotypes for 10 selected patients in this study
Patient no. Band size(a) (bp) C. parvum genotype
53 435 Zoonotic (genotype 2)
75, 124, 153 438 Anthroponotic
278, 554 (genotype 1)
119 429 Cryptosporidium sp.
(canine genotype)
84, 184, 485 455 C. felis
(a) Size of the Cryptosporidium diagnostic band obtained with the CPBDIAGF/CPBDIAGR PCR primer pair Acknowledgment The authors thank Jane L. Carter for her expert help with running the DNA sequencer. References (1.) Fayer R, Speer CA, Dubey JP. The general biology of Cryptosporidium. In editor. Cryptosporidium and cryptosporidiosis. Boca Raton (FL): CRC (Cyclical Redundancy Checking) An error checking technique used to ensure the accuracy of transmitting digital data. The transmitted messages are divided into predetermined lengths which, used as dividends, are divided by a fixed divisor. Pre 41. (2.) MacKenzie WR, Schell WL, Blair KA, Addiss DG, Peterson DE, Hoxie N Massive outbreak of waterborne Cryptosporidium infection in Milwaukee, recurrence of illness and risk of secondary transmission. Clin Infect Dis 19 (3.) Navin TR. Cryptosporidiosis in humans: review of recent epidemiologic [ILLEGIBLE TEXT]t Epidemiol 1985;1:77-83. (4.) Arrowood MJ. Diagnosis. In: Fayer R, editor. 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Similarities and differences between DNA of [ILLEGIBLE TEXT] parvum and C. wrairi detected by the polymerase chain reaction. Folia [ILLEGIBLE TEXT] 1994;41:97-100. (19.) Carraway M, Tzipori S, Widmer G. Identification of genetic heterogeneity Cryptosporidium parvum ribosomal repeat. Appl Environ Microbiol 1996; (20.) Kilani RT, Wenman WM. Geographical variation in 18S rRNA gene [ILLEGIBLE TEXT] Cryptosporidium parvum. Int J Parasitol 1994;24:303-6. (21.) Widmer G. Genetic heterogeneity and PCR detection of Cryptosporidium Parasitol 1998;40:223-39. (22.) Cai J, Collins MD, McDonald V, Thompson DE. PCR cloning and [ILLEGIBLE TEXT] determination of the 18S rRNA genes and 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. 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Atlanta: Centers for Disease Control; 1982. Publication no. (CDC See Control Data, century date change and Back Orifice. CDC - Control Data Corporation ) (31.) Garcia LS, Shum AC, Bruckner DA. Evaluation of a new monoclonal [ILLEGIBLE TEXT] combination reagent for the direct fluorescent detection 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. cysts a Cryptosporidium oocysts in human fecal specimens. J Clin Microbiol 199 (32.) da Silva AJ, Bornay-Llinares FJ, Moura INS INS abbr. 1. Immigration and Naturalization Service 2. International News Service Noun 1. INS , Slemenda SB, Tuttle JL, [ILLEGIBLE TEXT] Fast and reliable extraction of protozoan parasite DNA from fecal [ILLEGIBLE TEXT] Diagnosis. In press 1999. (33.) Schuler GD, Altschul SF, Lipman DJ. A workbench for multiple [ILLEGIBLE TEXT] and analysis. Proteins 1991 ;9:180-90. (34.) Felsenstein J. PHYLIP--phylogeny inference package. Cladistics cladistics (klədĭs`tĭks) or phylogenetic systematics (fī'lōjənĕt`ĭk) 1989;5: (35.) Roth A, Fischer M, Hamid ME, Michalke S, Ludwig W, Mauch H. [ILLEGIBLE TEXT] phylogenetically phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history: a phylogenetic classification of species. related slowly growing mycobacteria Mycobacteria that form colonies clearly visible to the naked eye in more than 7 days on subculture are termed slow growers. List of slowly growing Mycobacteria Nonchromogenic Rough
(36.) Bornay-Llinares F J, da Silva AJ, Moura IN, Myjak P, Pietkiewicz H, [ILLEGIBLE TEXT] Lozowska W, et al. Identification of Cryptosporidium felis in a cow by [ILLEGIBLE TEXT] molecular methods. Appl Environ Microbiol 1999;65:1455-8. Comments/Responses Have a comment on this article? Please use this form to reply. We're always happy to hear your views. [ILLUSTRATION OMITTED] Home | Top of Page | Current Issue | Expedited | Upcoming Issue | Past Issue | EID EID Emerging Infectious Diseases (journal) EID Electronic Identification EID Endpoint Identifier EID Employee Identification EID Ecological Interface Design EID Earned Income Disregard EID Education and Information Division Search | Contact Us CDC Home | Search | Health Topics A-Z This page last reviewed July 1, 1999 Emerging Infectious Diseases Journal National Center for Infectious Diseases Centers for Disease Control and Prevention Centers for Disease Control and Prevention (CDC), agency of the U.S. Public Health Service since 1973, with headquarters in Atlanta; it was established in 1946 as the Communicable Disease Center. URL URL in full Uniform Resource Locator Address of a resource on the Internet. The resource can be any type of file stored on a server, such as a Web page, a text file, a graphics file, or an application program. : http://www.cdc.gov/ncidod/eid/vol5no3/pieniazek.htm Norman J. Pieniazek,(*) Fernando J. Bornay-Llinares,(*) Susan B. Slemenda,(*) da Silva,(*) Iaci N. S. Moura,(*) Michael J. Arrowood,(*) Oleg Ditrich,([dagger]) and [ILLEGIBLE TEXT] Addiss(*) Centers for Disease Control and Prevention, Atlanta, Georgia, USA; and ([dagger]) [ILLEGIBLE TEXT] Parasitology AS CR, Ceske Budejovice, Czech Republic Dr. Pieniazek is a microbiologist with the National Center for Infectious Diseases, CDC. His include molecular reference diagnosis of parasitic diseases and molecular systematics systematics: see classification. . Address for correspondence: Norman J. Pieniazek, Division of Parasitic Diseases, Centers for and Prevention, 4770 Buford Highway NE, Mail Stop F13, Atlanta, GA, 30341-3724, USA; fax: e-mail: nxp3@cdc.gov. |
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