ARS | Publication request: Detection of Cryptosporidium Parvum in Polluted Stream Water

Submitted to: World Association for the Advancement of Veterinary Parasitology
Publication Type: Abstract
Publication Acceptance Date: June 1, 2003
Publication Date: August 11, 2003
Citation: Higgins, J.A., Belt, K., Hohn, C.A., Shelton, D.R. 2003. Detection of Cryptosporidium parvum in polluted stream water [Abstract]. World Association for the Advancement of Veterinary Parasitology. p. 116.

Technical Abstract: Currently, most surveys of water samples for the presence of C. parvum involve the use of capsule filtration, immunomagnetic separation (IMS), and IFA microscopy; for example, the "USEPA Method 1623". While published reports conducted using low-turbidity water indicate recoveries with this method can approach 70%, it is expensive and time-consuming. We are interested in screening stream water samples for the presence of C. parvum oocysts on a weekly, long-term (ie., one-year) basis, and thus prefer a less expensive method that offers sensitivity equivalent to that of capsule filtration. Accordingly, we adopted a method recently described by the L. Xiao and A. Lal laboratory at the CDC, in which 125 -200 ml samples of water are processed using simple centrifugation and IMS. Recovered oocysts are subjected to RNA extraction and RT-PCR is performed using several different sets of published primers targeting the C. parvum hsp70, viral symbiont, and 18S rRNA genes. Our selection of these primers was determined by a series of experiments using discrete quantities of oocysts, enumerated using micromanipulation (kindly provided by G. Sturbaum) and FACS (purchased from the Wisconsin State Laboratory). These purified oocysts were lysed and subjected to RT-PCR; the detection limits for various primer combinations ranged from 1 to 10 oocysts per RT-PCR. Since March 2003, weekly analyses have been done on water samples obtained from Gwynns Falls / Gwynns Run, a highly polluted stream located in metropolitan Baltimore, MD. This stream has a history of high coliform counts and turbidity readings and receives water from both rural and urban sources. Preliminary results indicate that our method can indeed detect oocysts in this stream, and sequencing of amplicons has indicated that they are C. paruvm. Data on the frequency of positive samples, and the species / genotype characteristics of the detected oocysts, will be presented.