USE OF MAGNETIC PARTICLE TECHNOLOGY TO CONCENTRATE C. PARVUM OOCYSTS.
Report No DWI0375/14

Feb 1994

SUMMARY

I. OBJECTIVES

The objectives of this study were to show that a) tangential flow filtration and b) monoclonal antibody coated magnetic particles can be used to recover Cryptosporidium oocysts from water. In addition a sugar-lectin reaction was to be tested as a species-specific probe for C. parvum.

II. REASONS

The methods currently used for the recovery of Cryptosporidium oocysts from water samples are of low efficiency and time consuming. Methods to overcome these problems are required.

III. CONCLUSIONS

Immunomagnetic separation provided a successful method for separating oocysts from contaminating debris in environmental samples in order to assess their viability. Previously, assessing the viability of the low numbers of oocysts detected in water samp les was time consuming, painstaking work and, because of the small numbers of oocysts in water samples, recoveries for viability estimates were poor. Furthermore, contaminating debris could obscure the oocysts to the extent that it was impossible to determine the viability of the oocysts. The use of the magnetic separation technique described reduced this problem considerably. It enabled the concentration of the sample to a smaller final volume and allowed the viability of the whole sample to be assessed within a reasonable period of time. Previously only 10 to 20 % of the sample could be analysed in the same time taken to analyse the whole sample, following IMS. Although there remains room for technological improvement in this technique, an increased recovery of oocysts from water samples was effected in a shorter period of time than was necessary previously.

IV. RECOMMENDATIONS

Tangential flow filtration may provide an alternative means of concentrating oocysts from good quality water. If the problems associated with filter blocking and rupture can be overcome and if devices with larger pore sizes can be obtained, tangential flow filtration may provide an alternative means of concentrating oocysts from a variety of raw waters. Immunomagnetic separation can be used to recover oocysts from water samples. Lectins can be used to distinguish intact C. parvum from C. baileyi.

V. RESUME OF CONTENTS

Tangential flow filtration was compared with large scale centrifugation as a means of concentrating oocysts and found to give better recoveries when clean water seeded with oocysts was used. When the eluate from washing a filter seeded with oocysts was concentrated there was no significant difference in recoveries between the two methods. This was due to problems associated with blockage of the membrane by particulates released from the cut Cuno filter. Blockage of the filter, in turn, increased the time required for concentration of 4L of filter wash eluate and concentration by tangential flow filtration took longer than centrifugation. The membranes of the tangential flow devices often became blocked or ruptured.

Magnetic particles labelled with an anti-FITC monoclonal antibody were shown to recover between 50 and 100 % of oocysts labelled with a commercially available FITC conjugated monoclonal antibody.

A lectin specific to sugars on the surface of intact C. parvum oocysts was used to differentiate them from C. baileyi oocysts.

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