Development of a novel method for isolating and detecting Cryptosporidium parvum from lean and fat beef carcass surfaces
2004
Moriarty, E.M. | McEvoy, J.M. | Duffy, G. | Sheridan, J.J. | Blair, I.S. | McDowell, D.A.
This paper describes the first reported method for the recovery and detection of Cryptosporidium parvum oocysts from beef carcasses. C. parvum oocysts were mobilized from beef surfaces into phosphate buffer saline-Tween 80, and subsequently recovered from this suspending medium by membrane filtration. Oocysts were removed from the membrane, concentrated by centrifugation, labelling with an FITC conjugated monoclonal antibody and enumerated using fluorescence microscopy. The study compared 'pulsification', a novel method, with 'stomaching' an established method, as processes for resuspending the target microorganisms from beef. Both yielded similar levels of recovery of oocysts, but pulsification produced less suspended sample debris, allowing easier oocyst detection. Membrane filter pore size (0.45, 1.0 or 3.0 micrometer) did not significantly affect the number of oocysts detected (P>0.05). Centrifugation at 2500g for 15 min using a swing out no brake (SONB) during deceleration rotor gave higher recoveries than a fixed angle with braking (FAB) rotor during centrifugation (P<0.05). Using inocula containing 2000 oocyst cm-2, the optimized method (pulsification, filtration, SONB centrifugation and FITC labelling) allowed recovery and detection of 85.4% of inoculated oocysts from fat beef tissue and 128.4% from lean beef tissue. The developed method will be of value in establishing the incidence of C. parvum on beef and in future research on this parasite.
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