The Molecular Characterisation by PCR RFLP and SDS-PAGE Western Blotting of Cryptosporidium parvum suspensions, treated by different cleaning procedures.
A total of 31 oocyst suspensions of Cryptosporidium parvum were examined which comprised 4 different ‘strains’ designated, Arizona, PHLS, Moredun and Iowa. The samples were received either as whole faeces, or as oocyst suspensions which had been treated with various cleaning procedures described as: sucrose; sucrose and caesium chloride; ether; and sodium chloride. Oocysts were stored in phosphate buffered saline or potassium dichromate.
Target DNA was extracted from all samples irrespective of the various cleaning treatments, and which resulted in the generation of amplicons for both the cryptosporidial COWP and TRAP C1 genes. Analysis of the restriction digest patterns showed that the C.parvum in all samples was of genotype 2
SDS/PAGE Western blotting analysis generated reproducible patterns with duplicate tests of each sample and patterns of between six and zero bands were obtained. Intra-strain analysis showed that differences were obtained from samples subjected to the various cleaning treatments and these resulted in the loss, but without change in apparent molecular weight, of bands within each profile. Identical results were obtained with whole faeces and ether treated samples for all four C.parvum strains. Compared to the results obtained with whole faeces, almost all samples stored in dichromate showed a loss of some or all of the bands: there was also a loss in intensity of the bands obtained from the dichromate preserved samples. Minor differences (loss or gain of one or two bands) were seen between some of the other treatments. Inter-strain comparisons showed that the Iowa and Arizona isolates gave identical banding patterns but were different from those obtained with the PHLS and Moredun isolates. The PHLS and Moredun strains gave distinct banding patterns. There were three bands common to all four strains examined (apparent molecular weights of 230, 90 and 80 KDa) and one band (260 KDa) common to all except the PHLS strain.
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