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Cryptosporidium pig genotype II in immunocompetent man.

To the Editor: Protozoan parasites from the genus Cryptosporidium have been described as a cause of diarrheal disease in immunodeficient and immunocompetent humans worldwide. Although C. hominis and C. parvum (cattle genotype) cause most infections, humans can be infected by several other Cryptosporidium species or genotypes: C. meleagridis; C. felis; C. canis; C. suis; C. muris; C. andersoni; C. hominis monkey genotype; C. parvum (mouse genotype); and Cryptosporidium rabbit genotype, deer genotype, skunk genotype, horse genotype, and chipmunk genotype I (1-4). Wild and domestic animals are sources of infection for humans (and other animals) and important contributors to contamination of food and drinking water; many nonhuman Cryptosporidium species or genotypes are detected in untreated water (5). We examined the diversity of Cryptosporidium spp. in immunocompetent persons in South Bohemia in the Czech Republic.

Diarrheal fecal samples (n = 457) from 203 anonymous immunocompetent patients [less than or equal to]69 years of age with suspected cryptosporidiosis (at least 2 samples/patient/3-day period) were obtained from local health departments and public hospitals in South Bohemia during 2005-2007. Samples were examined for Cryptosporidium oocysts by using aniline-carbol-methyl violet staining and light microscopy at x 1,000 magnification (6). The microscopically positive samples were confirmed by DNA sequencing of the small subunit (SSU) rRNA gene. Total DNA was extracted from 200-300 mg stool by using the QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions, after previous homogenization and disruption of oocysts with the Mini-BeadBeater (Biospec Products, Bartlesville, OK, USA). An [approximately equal to]830-bp fragment of the SSU rRNA gene was amplified by nested PCR according to Jiang et al. (7). Purified PCR products were sequenced in both directions on an ABI3130 sequencer analyzer (Applied Biosystems, Foster City, CA, USA) by using the secondary PCR primers and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were assembled by using Chromas Pro (www.technelysium.com.au/chromas.html) and aligned with reference sequences using ClustalX (ftp://ftp-igbmc.u-strasbg.fr/pub/ ClustalX). The BLAST server (www.ncbi.nlm.nih.gov/BLAST) was used for DNA database searches. The SSU rRNA gene partial sequences of the 7 patient isolates have been submitted to GenBank (Table).

Of the 203 patients, 7 (3.4%) (6 children and 1 adult) had positive results for Cryptosporidium spp. Moreover, all samples obtained from these persons during the 3-day period were Cryptosporidium spp. positive; partial sequences of the Cryptosporidium SSU rRNA gene were obtained from all positive samples identifying 3 different species or genotypes of Cryptosporidium. Five were C. parvum (bovine genotype), 1 was C. hominis, and 1 contained the Cryptosporidium pig genotype II (Table). Cryptosporidium pig genotype II was found in stool samples from a 29-year-old man who also was infected with Giardia intestinalis (assemblage A) (data not shown).

Only C. parvum (bovine genotype), C. hominis, and Cryptosporidium rabbit genotype have been implicated in waterborne outbreaks of cryptosporidiosis in humans. Further studies are needed to determine the potential of other cryptosporidia of animal origin. Recent genetic and biologic characterization studies have identified 2 distinct host-adapted cryptosporidia in pigs, C. suis and Cryptosporidium pig genotype II. Furthermore, both above-mentioned cryptosporidia have been identified in untreated water (8). Pigs could be sources of Cryptosporidium water and food pollution and a consequent risk to public health.

Although human infection with C. suis has been previously described (9), human infection with Cryptosporidium pig genotype II has been never reported. This genotype was found in diarrheal stool of 1 adult patient in this study. However, onset of diarrhea could have been caused by co-infection with G. intestinalis (assemblage A), which recently also has been described in pigs (10). Contact with infected animals and ingestion of contaminated food or water could be the source of both Cryptosporidium and Giardia infection in the Cryptosporidium pig genotype II-positive patient. The passage of oocysts can be excluded because of the number of oocysts detected in repeat samples (Table). Moreover, identification of the infection in an immunocompetent patient underlines the zoonotic potential of this pig genotype and possible presence of risk factors in rural areas with poor water treatment or inadequate biosecurity in pig units. Further evidence of the zoonotic potential of this Cryptosporidium genotype is needed to show its pathogenic potential in immunocompetent patients as a cause of gastroenteritis (in the absence of Giardia spp. and other established enteropathogens) and to demonstrate invasive tissue stages. The use of molecular techniques to identify Cryptosporidium spp. probably will show more zoonotic species or genotypes in humans.

This work was funded by the Grant Agency of the Czech Republic (project no. 523/07/P117) and by the Institute of Parasitology, Academy of Sciences of the Czech Republic (project no. Z60220518).

References

(1.) Feltus DC, Giddings CW, Schneck BL, Monson T, Warshauer D, McEvoy JM. Evidence supporting zoonotic transmission of Cryptosporidium spp. in Wisconsin. J Clin Microbiol. 2006;44:4303-8. DOI: 10.1128/JCM.01067-06

(2.) Nichols G. Epidemiology. In: Fayer R, Xiao L, editors. Cryptosporidium and cryptosporidiosis. Boca Raton (FL): CRC Press; 2007. p. 79-118.

(3.) Robinson G, Elwin K, Chalmers RM. Unusual Cryptosporidium genotypes in human cases of diarrhea. Emerg Infect Dis. 2008;14:1800-2. DOI: 10.3201/ eid1411.080239

(4.) Ajjampur SS, Gladstone BP, Selvapandian D, Muliyil JP, Ward H, Kang G. Molecular and spatial epidemiology of cryptosporidiosis in children in a semiurban community in South India. J Clin Microbiol. 2007;45:915-20. DOI: 10.1128/ JCM.01590-06

(5.) Xiao L, Fayer R, Ryan U, Upton SJ. Cryptosporidium taxonomy: recent advances and implications for public health. Clin Microbiol Rev. 2004;17:72-97. DOI: 10.1128/CMR.17.1.72-97.2004

(6.) Milaeek P, Vitovec J. Differential staining of cryptosporidia by aniline-carbol-methyl violet and tartrazine in smears from faeces and scraping of intestinal mucosa. Folia Parasitol (Praha). 1985;32:50.

(7.) Jiang J, Alderisio KA, Xiao L. Distribution of Cryptosporidium genotypes in storm event water samples from three watersheds in New York. Appl Environ Microbiol. 2005;71:4446-54. DOI: 10.1128/ AEM.71.8.4446-4454.2005

(8.) Ryan U, Read C, Hawkins P, Warnecke M, Swanson P, Griffith M, et al. Genotypes of Cryptosporidium from Sydney water catchment areas. J Appl Microbiol. 2005;98:1221-9. DOI: 10.1111/j.1365-2672.2005.02562.x

(9.) Xiao L, Bern C, Arrowood M, Sulaman I, Zhou L, Kawai V, et al. Identification of Cryptosporidium pig genotype in a human patient. J Infect Dis. 2002;185:1846-8. DOI: 10.1086/340841

(10.) Langkjaer RB, Vigre H, Enemark HL, Maddox-Hyttel C. Molecular and phylogenetic characterization of Cryptosporidium and Giardia from pig and cattle in Denmark. Parasitology. 2007;134:339-50. DOI: 10.1017/S0031182006001533

DOI: 10.3201/eid1506.07621

Martin Kvac, Dana Kvetonova, Bohumil Sak, and Oleg Ditrich

Address for correspondence: Bohumil Sak, Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branisovska 31, 370 05 Eeske Budijovice, Czech Republic; email: casio@paru.cas.cz

Author affiliations: Academy of Sciences of the Czech Republic, Eeske Budijovice, Czech Republic (M. Kvae, D. Kvitooova, B. Sak, O. Ditrich); and University of South Bohemia, Eeske Budijovice (M. Kvae)
Table. Cryptosporidium genotypes identified by using sequencing of
partial sequences of the small subunit rRNA gene in the stool samples
of immunocompetent humans, Czech Republic

 Examination Cryptosporidium
Patient no. Age, y/sex year species/genotype

H15 9/M 2005 C. parvum ([dagger])
H23 10/M 2005 C. hominis
H98 10/F 2005 C. parvum ([dagger])
H101 11/M 2006 C. parvum ([dagger])
H132 8/M 2006 C. parvum ([dagger])
H158 11/M 2007 C. parvum ([dagger])
H199 29/M 2007 Cryptosporidium pig
 genotype II

 Infection intensity *

 GenBank
Patient no. Sample 1 Sample 2 accession no.

H15 56 78 EU331237
H23 77 121 EU331242
H98 43 25 EU331238
H101 11 5 EU331239
H132 150 62 EU331240
H158 26 85 EU331241
H199 38 27 EU331243
 ([double dagger]) ([double dagger])

* Numbers of oocysts per 30 fields at x1,000 magnification, unless
otherwise indicated.

([dagger]) Bovine genotype.

([double dagger]) Numbers of oocysts per whole slide at x1,000
magnification.
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Title Annotation:LETTERS
Author:Kvac, Martin; Kvetonova, Dana; Sak, Bohumil; Ditrich, Oleg
Publication:Emerging Infectious Diseases
Article Type:Report
Geographic Code:4EXCZ
Date:Jun 1, 2009
Words:1334
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