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.

  1. Rapid detection techniques
    1. 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.
    2. The automated microscopic screening for fluorescently labelled oocysts is impractical at present.
    3. 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.
    4. 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.
    5. 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.
  2. 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 indicated.

  3. Aging of Cryptosporidium oocysts under various environmental pressures
    1. Cryptosporidium oocysts are resistant to the majority of environmental pressures that they are likely to encounter, including sea water.
    2. 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 cryptosporidiosis.
    3. 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.
  4. Biochemical analysis of Cryptosporidium oocysts
    1. 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.
    2. 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:
      1. magnetisable particle technology,
      2. the verification of numbers,
      3. the usefulness of these techniques in waters of varying qualities.
      4. 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, non-viable.

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.