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

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(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: [e-mail]

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.