since 2000 , the uk cryptosporidium reference unit has maintained a national collection of cryptosporidium oocysts ( 8) .
over 16,000 cryptosporidium - positive human fecal samples have been submitted by primary diagnostic laboratories and characterized by the reference unit to identify the infecting species .
in addition to the expected c. hominis , c. parvum , and small number of c. meleagridis , c. felis , c. canis , and cervine genotype isolates , 3 other genotypes ( skunk , horse , and rabbit ) were identified in separate samples from individual patients after the onset of diarrhea in 2000 ( sample w971 ) , 2003 ( sample w6863 ) , and 2007 ( sample w16103 ) . a routinely collected minimum dataset
was submitted with each sample , and further exposure data were collected for each patient from the local consultant in communicable disease control . to prepare isolates for molecular characterization
, oocysts were concentrated by saturated salt flotation , disrupted by boiling for 1 hour and the dna purified by using a qiaamp dna mini kit ( qiagen , valencia , ca , usa ) as previously described ( 9 ) .
cambridge , uk ) at the small subunit ( ssu ) rrna ( 800-bp product ) ( 10 ) , cryptosporidium oocyst wall protein ( cowp ) ( 550-bp product ) ( 11 ) and heat shock protein ( hsp ) 70 ( 450-bp or 325-bp products ) ( 12 ) genes .
sequences were compared with genbank submissions by using the blast algorithm ( www.ncbi.nlm.nih.gov/education/blastinfo/blast_algorithm.html ) .   to confirm identification ,
phylogenetic analysis was conducted in treecon ( www.bioinformatics.psb.ugent.be/software/details/3 ) with other known cryptosporidium spp . and genotypes by using alignments generated in clustalx version 2.0 ( ftp://ftp.ebi.ac.uk/pub/software/clustalw2 ) and manually edited in bioedit version 7.0.9 ( www.mbio.ncsu.edu/bioedit/bioedit.html ) .
sequence analysis confirmed that w971 , w6863 , and w16103 were skunk , horse , and rabbit genotypes , respectively ( table ) .
isolate w971 was homologous with genotype w13 found in storm water , which , in turn , is the skunk genotype ( 6 ) .
initially , the blast search for isolate w6863 erroneously indicated c. parvum as the most probable identity at the ssu rrna gene , but this was due to the short length ( 484 bp ) of the only horse genotype sequence available ( ay273770 ) for comparison .
thus , c. parvum isolates that spanned our whole query sequence ( 787 bp ) were calculated to have greater identities by blast .
however , a detailed comparison between ay273770 and w6863 showed only 2-bp differences ( including 1 insertion in our sequence ) compared with 7-bp differences between w6863 and c. parvum .
w6863 was confirmed as a variant of the horse genotype by hsp70 gene sequence analysis and ssu rrna gene phylogenetic analysis ( figure ) . *
i d , identification ; ssu , small subunit ; hsp , heat shock protein ; cowp , cryptosporidium oocyst wall protein .
phylogenetic relationships between 3 unusual cryptosporidium genotypes and known cryptosporidium species / genotypes as inferred by a neighbor - joining analysis of the small subunit rrna gene .
evolutionary distances were calculated by the kimura 2-parameter model with eimeria tenella as an outgroup .
pcr - rflp analysis of the ssu rrna and cowp genes differentiated the skunk and horse genotypes from the most common human pathogens .
however , identifying the rabbit genotype by pcr - rflp at these loci was more problematic because of this genotype s close relationship with c. hominis .
the sequence and restriction pattern are identical at the cowp gene and , with only 4-bp substitutions ( 2 occurring in sspi cut - sites ) , the pattern is similar at the ssu rrna gene . increasing the resolution by running the agarose gel at an appropriate concentration and for as long as possible is important for the separation of the c. hominis diagnostic band ( 449 bp ) from the rabbit genotype ( 472 bp ) .
information on possible risk factors was collected for the 2 weeks before the onset of illness , but we can not be sure how these 3 persons became infected with the unusual genotypes .
the skunk genotype was found in a 25-year - old woman from a rural area of southwest england , who reported no foreign travel and no contact with animals .
she worked and swam regularly at an adult daycare center and had spent a week during the incubation period with clients at a holiday forest park in her region .
the horse genotype was found in a 30-year - old immunocompetent woman also from a rural area of southwest england , who reported swimming and foreign travel ( destination unknown ) but no contact with animals during the incubation period .
the rabbit genotype was found in a 48-year - old immunocompetent woman from a rural area of northwest england , who reported foreign travel to southern spain and contact with wild birds ( feeding ducks and geese ) but no contact with other animals .
previously , these 3 genotypes were known to cause infections only in wild or zoo animals ( 13,14 ) .
wild animals are known to be an important source of cryptosporidium oocysts in environmental samples and we have detected the rabbit genotype in surface waters and septic tank samples ( unpub .
data ) , but the source is unknown . since many isolates have yet to be found in humans and although little is actually known about them , they are assumed to be insignificant to public health ( 6,15 ) .
the importance of unusual genotypes in humans who seek treatment for diarrheal disease warrants further investigation .
 several cryptosporidium spp . are known to infect humans , but most cases of illness are caused by cryptosporidium hominis or c. parvum . during a long - term genotyping in the united kingdom , we identified 3 unusual cryptosporidium genotypes ( skunk , horse , and rabbit ) in human patients with diarrhea . 
The Study
Conclusions