Investigation of Genetic
Variation within Cryptosporidium
Hominis for Epidemiological Purposes
DWI0851
March 2007
EXECUTIVE SUMMARY
Cryptosporidiosis is a diarrhoeal disease of humans and young animals
caused by the protozoan parasite Cryptosporidium.
Illness in neonatal animals is caused by Cryptosporidium parvum and
both this and a human-adapted species, Cryptosporidium hominis,
cause cryptosporidiosis in people. Each species causes approximately
half of the reported cases in England and Wales, but the distribution
by person, time and place (the epidemiology) differs, as do the risk
factors for infection. Further investigation of variants within C. parvum has
revealed more about the natural history and epidemiology of this
species, but genetic variation within C. hominis is poorly understood
and the distribution of variants unknown. In a previous study, we found
that 90% of C. hominis
isolates from apparently un-linked (sporadic) cases in England and
Wales were indistinguishable. A study of the population genetics of C. hominis in the
north east of Scotland showed isolates were genetically very similar,
indicating that the parasite population structure was almost clonal,
while in other countries more variation has been observed.
In this project, to investigate whether other typing methods for C. hominis isolates
could be useful for epidemiological purposes in England and Wales, more
genetic loci were studied and any relationship between C. hominis variants
and epidemiological factors was investigated. We identified sporadic
and outbreak cases from the national collection of Cryptosporidium
oocysts maintained at the UK Cryptosporidium
Reference Unit in Swansea. In the first instance, we read the DNA
sequence of part of the parasite genome by looking at a large part of
the GP60 gene. This is a highly variable gene, and many
“families” have been identified in both C. parvum and C. hominis. The
gene also contains variable numbers and forms of a repeating sequence
of nucleotides, microsatellite DNA, which can be used to identify
variation within these families. Cases in a case control study were
investigated for relationships between exposure and GP60 results.
Family Ib subtype A10G2 was the most common in sporadic cases,
representing over 90% all isolates. However, people with non-IbA10G2
isolates were statistically more likely to have returned from
non-European destinations than people with IbA10G2.
Isolates from two drinking waterborne outbreaks that occurred in the
Autumn of 2005 were compared with sporadic cases and exposure. In the
outbreak in north west Wales, C.
hominis isolates were exclusively IbA10G2. This was also
the predominant type locally in cases during the six years prior to the
outbreak. In the outbreak in south east England, IbAl0G2 also
predominated but two other C. hominis families were also present,
although there was no difference in the results of the epidemiological
analysis of the outbreak when these cases were excluded from the
analysis.
We also investigated the development of an alternative typing method
for C. hominis
and C. parvum
to DNA sequence analysis, which is costly and time consuming. We
investigated single strand conformation polymorphisms (SSCP) at two
genetic loci. Investigation of the ITS-2 region showed more variation
between sample runs than between samples. However, SSCP on the GP60
gene appeared to be much more reliable, and had the advantage of
providing direct comparison with the DNA sequence based analysis.
To conclude, this study:
- tells us that indigenous C. hominis in the
UK shows little genetic variation in the GP60 gene and
supports previous findings at other loci
- indicates little change in C. hominis over the
time investigated
- suggests wider global transmission may be subject to
host-related or social factors
- an international database of Cryptosporidium
variants with standardised nomenclature would
assist in interpretation of studies elsewhere
- Further method development is required for rapid methods
for epidemiological purposes.
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