DETECTION OF CRYPTOSPORIDIUM-PARVUM IN RAW-MILK BY PCR AND OLIGONUCLEOTIDE PROBE HYBRIDIZATION

Cryptosporidium spp, are potential contaminants of food. Suspected cas es of food-borne cryptosporidiosis are rarely confirmed because of the limited numbers of oocysts in the samples and the lack of sensitive d etection methods adaptable to food. PCR was investigated as a means of overcoming this problem. A PCR assay was designed for the specific am plification of a previously sequenced portion of an oocyst protein gen e fragment of Cryptosporidium parvum (N. C. Lally, G. D. Baird, S. J. McQuay. F. Wright, and J. J. Oliver, Mol. Biochem. Parasitol. 56:69-78 , 1992) and compared with the primer set of Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am, J. Trop. Med. Hyg. 45:688-694, 19 91), The PCR products were hybridized with digoxigenin-labeled interna l probes and detected by chemiluminescence to enhance sensitivity. The two sets of primers were compared with regard to their sensitivity an d specificity by using a variety of human and animal isolates of C. pa rvum and related parasites. Both assays enabled the detection of 1 to 10 oocysts in 20 mi of artificially contaminated ram milk. The assay b ased on the PCR set and probe of Laxer et al. detected DNAs from Eimer ia acervulina and Giardia intestinalis. The new assay has good specifi city for C. parvum bovine isolates and hence has a better potential fo r monitoring the prevalence of C. parvum in raw milk and other environ mental samples.