Cryptosporidium Rabbit Genotype, a Newly Identified Human Pathogen

Most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or C. hominis, but pathogenicity of some unusual Cryptosporidium species/genotypes is uncertain (1). In July 2008, an outbreak caused by Cryptosporidium rabbit genotype was linked to consumption of tap water in Northamptonshire, England (2). On June 23 and 24, Cryptosporidium oocysts were detected by operational monitoring of treated water at a surface water treatment works. A precautionary boil-water notice was implemented on June 25.


Cryptosporidium sp.
Rabbit Genotype, a Newly Identifi ed Human Pathogen To the Editor: Most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or C. hominis, but pathogenicity of some unusual Cryptosporidium species/genotypes is uncertain (1). In July 2008, an outbreak caused by Cryptosporidium sp. rabbit genotype was linked to consumption of tap water in Northamptonshire, England (2). On June 23 and 24, Cryptosporidium oocysts were detected by operational monitoring of treated water at a surface water treatment works. A precautionary boil-water notice was implemented on June 25.
Enhanced surveillance for cases was established by the health protection team on June 25 in the affected area. Eight single-well immunofl uorescent microscopy slides, on which oocysts were detected by water company sampling of the distribution system, were sent to the UK Cryptosporidium Reference Unit, Swansea, for typing. Slides contained 49-259 oocysts. Coverslips were removed after softening the seal with nail polish remover. Fixed material was resuspended from the slides by thorough scraping of the entire well with a pipette tip twice with 50 μL lysis buffer AL (QIAGEN, Crawley, UK) and twice with 50 μL reverse osmosis water to a fi nal volume of 200 μL. Oocysts were disrupted in 3 dry ice/methanol freezethaw cycles, and DNA was extracted by using the QIAamp DNA Mini Kit (QIAGEN), which involved digestion with proteinase K in lysis buffer AL at 56°C for 30 min, purifi cation in a spin column, elution in 50 μL buffer AE, and storage at -20°C (3).
Cryptosporidium oocysts were also detected by direct immunofl uorescent antibody test (IFAT) (Crypto-Cel; TCS Biosciences, Buckingham, UK) in large bowel contents from a rabbit carcass removed by the water company from a tank at the water treatment works. Oocysts were separated from fecal debris by fl otation, resuspended in reverse osmosis water (4), and processed as above.
Cryptosporidium species were identifi ed by bidirectional sequencing of PCR products generated by nested PCR for the small subunit (SSU) rRNA gene (5) from 4 DNA aliquots of each sample. SSU rDNA sequences from 7 water samples, containing 49-197 oocysts, and the rabbit isolate were homologous with isolates from rabbits in the People's Republic of China (6) and the Czech Republic (7) (GenBank accession nos. AY120901 and AY273771, respectively) (online Appendix Table, available from www. cdc.gov/EID/content/15/5/829-appT. htm). One sample from 1,391 L of water contained 259 oocysts but was not amplifi ed. Other cryptosporidia were not identifi ed.
Human stool samples from 34 local laboratory-identifi ed cases of cryptosporidiosis in the affected area were sent to the UK Cryptosporidium Reference Unit for typing. To differentiate rabbit genotype from C. hominis (1), enhanced typing by SSU rRNA nested PCR-restriction fragment length polymorphism analysis with SspI and VspI (1,5) was used for all isolates submitted to the UK Cryptosporidium Reference Unit during July and August. Samples from 23 cases (22 primary and 1 secondary) with rabbit genotype profi les were identifi ed by visualization of 472-, 267-, and 109-bp bands generated by digestion with SspI (1). All case-patients lived in the area affected by the water supply incident and had onset dates consistent with exposure by drinking water consumption or by person-to-person spread. All 23 samples were homologous to AY120901 and AY273771 (online Appendix Table). Of the other 11 samples, 6 were not confi rmed by IFAT or PCR, 2 were C. hominis, 1 was C. parvum, and 2 were not typeable.

LETTERS
Sequences of the heat shock protein (HSP) 70 gene (8) and, to identify subtype family, the 60-kDa glycoprotein (gp60) gene (9) were determined for 7 water isolates and the rabbit and 9 outbreak case isolates. All HSP70 sequences were homologous with AY273775 from a rabbit in the Czech Republic (7) (online Appendix  Table). One water sample, the rabbit sample, and 8 human samples amplifi ed the gp60 gene. These sequences were homologous with each other, but distinct from those published for C. hominis (subtype family I), C. parvum (subtype family II), C. meleagridis (subtype family III), and C. fayeri (subtype family IV) (10). Each rabbit genotype isolate had 18 TCA (serine) tandem repeats in the gp60 microsatellite region. We propose subtype family Va, subtype A18 for these isolates. This subtype differs from the rabbit genotype previously identifi ed in a human in the United Kingdom (1) (subtype VaA22) (Gen-Bank accession no. EU437420) and from rabbits in the Czech Republic (subtype VbA19) and China (subtype VbA29). Sequences generated during this study have been deposited in GenBank under accession nos. FJ262724-FJ262734.
Six additional persons infected with Cryptosporidium sp. rabbit genotype were identifi ed by testing 394 stool samples that were routinely submitted for typing from diarrheic patients in July and August from throughout the UK. All persons had onset dates inconsistent with the affected period and were from other regions of the UK. This fi nding may indicate a low background level of rabbit genotype cases; however, prevalence is currently unknown.
The Cryptosporidium rabbit genotype has been identifi ed as the etiologic agent in an outbreak of diarrheal disease and should be considered a human pathogen. Further studies commissioned by the Drinking Water Inspectorate (England and Wales) and funded by the Department of Environment, Food and Rural Affairs UK are underway.

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