FINAL REPORT on the DoE SPONSORED RESEARCH ON CRYPTOSPORIDIUM SPP.
OOCYSTS UNDERTAKEN AT THE SCOTTISH PARASITE DIAGNOSTIC LABORATORY,
STOBHILL GENERAL HOSPITAL, GLASGOW G21 3UW.
Report No DWI0744
SUMMARY AND RECOMMENDATIONS.
- Rapid detection techniques
- Rapid concentration and detection using magnetisable particles is
capable of recovering over 75% of oocysts from seeded samples. The
efficiency of concentration of small numbers of oocysts from seeded
and environmental samples requires further investigation, as does
the role of the detergents, used in the standard method for the
isolation of oocysts from water, on the interaction between antibody
paratope and oocyst epitope. High affinity monoclonal antibodies
(McAbs) should overcome this latter problem.
- The automated microscopic screening for fluorescently labelled
oocysts is impractical at present.
- Automatic rapid detection, using a cooled slow-scan CCD, is
impractical at present only because of the need to design suitable
computer software. These impracticalities are seen as technological
difficulties rather than scientific impossibilities, and should be
relatively easy to overcome. In addition, it should be possible to
screen samples using a fluorescence activated cell sorter (FACS).
This is an expensive machine (c. £130,000). Collaboration with the
SPDL to develop these techniques is recommended.
- Enhanced chemiluminescent detection of Cryptosporidium oocysts is
capable of detecting between 5 - 10 oocysts in a sample and appears
to be reproducible. We do not feel able to recommend the enhanced
chemiluminescent detection of Cryptosporidium oocysts as a
preliminary screening procedure using the commercially available
McAbs as we are undecided which is the best way to incorporate this
technology into routine screening procedures. However, should a
threshold level for the number of oocysts in a water sample be set,
this technique could prove to be both useful and rapid. Consultation
between the SPDL and other interested parties could address these
points. The production of purified, high affinity McAbs raised
against defined epitopes expressed on Cryptosporidium oocysts would
improve this technique considerably.
- The capability of a cooled slow-scan CCD to detect
Cryptosporidium oocysts, indicates the enormous potential of these
instruments in a sensitive, reliable, rapid detection technique.
- Viability Assay
Inclusion/exclusion of the fluorescent vital dyes DAPI and PI
provides a method of assessing the viability of C.parvum oocysts
that is sensitive, reproducible, and "user friendly". For these
reasons, and because it can be applied to small numbers of oocysts
and individual oocysts, it is considered superior to the more
traditional method of in vitro excystation for assessing viability.
This fluorogenic viability assay is based on the principle that dead
oocysts, but not viable oocysts, will include PI which fluoresces
red [PI(+)], whereas viable oocysts will not only exclude PI
[PI(-)], but will also include DAPI which fluoresces sky-blue
[DAPI(+)/PI(-)]. Using the in vitro excystation protocol described
herein, oocysts which exclude both fluorescent dyes [i.e.
DAPI(-)/PI(-)] are not considered to be viable and they will not
excyst over 4 h in vitro. However, following a further stimulus (for
example, pH), DAPI(-)/PI(-) oocysts can be converted to
DAPI(+)/PI(-) oocysts. DAPI(+)/PI(-) oocysts are viable and are
capable of excystation in vitro. Thus both DAPI(+))/PI(-) and
DAPI(-)/PI(-) oocysts should be regarded as being potentially
infective. However, knowledge of the correlation between viability
and infectivity is minimal and further research in this area is
- Aging of Cryptosporidium oocysts under various environmental
- Cryptosporidium oocysts are resistant to the majority of
environmental pressures that they are likely to encounter, including
- While desiccation appears to be 100% lethal, a small proportion
will survive long periods of being frozen. Neither frozen food nor
ice should be considered without risk in the contraction of
- Water treatment processes, while being of importance in the
removal of oocysts from water, are unlikely to kill any oocysts at
the pH used. d) Faeces appear to confer some measure of protection
on oocysts. Rapid dispersal of faecal material is recommended to
maximise aging and vulnerability of oocysts.
- Biochemical analysis of Cryptosporidium oocysts
- The outer surface of Cryptosporidium oocysts expresses few
proteins and is predominantly carbohydrate in composition. A
polysaccharide containing GalNAC is hypothesised to be the major
constituent present in oocyst walls.
- Putative biochemical markers for viability have been identified
and require further research. Further work is necessary to draw
together the strings of this DoE funded research, especially in
areas such as:
- magnetisable particle technology,
- the verification of numbers,
- the usefulness of these techniques in waters of varying
- the relationship between viability as defined by fluorogenic dye
inclusion/exclusion and infectivity, and the use of such a viability
assay in identifying potential agents and processes which might be
capable of rendering viable oocysts, as defined by this assay,
Whereas it appears that the above techniques (a, b and c) can be
included into routine benchwork (and have been at the SPDL), further
collaboration between the SPDL and other interested parties in the
water industry is recommended. The incorporation of the viability
assay with a detection assay, either fluorogenic or luminescent; and
the use of FACS or CCD machines in the detection of oocysts should
be considered. The SPDL with its contacts in biotechnology, and its
in depth knowledge of the viability assay would be pleased to be
involved further in this research programme.
Copies of this report may be available as an Acrobat pdf download under the 'Pre 2000 Reports' heading on the DWI website.