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The long-term objective of the study is to minimize the impact of swine rearing facilities on their watersheds by abrogating or reducing the number of oocysts of Cryptosporidium species leaving the facilities in their waste streams.<P> Specific objectives:<OL> <LI> Determine the viability of oocysts within swine lagoons and material leaving the lagoons for land application. <LI> Determine the effects of lagoon storage on the inactivation kinetics of oocysts placed within the lagoons in sentinel chambers. <LI>Determine the inactivation kinetics of oocysts that have been land applied to forage crops after their treatment within a swine lagoon.
Approach: To meet the first specific objective in the first year of the project we will sample once per month ten swine lagoons in the Southern Piedmont and Coastal Plain. Cryptosporidium oocysts will be extracted, enumerated, assayed for viability with a dye permeability protocol, and genetically typed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol and DNA sequence analysis. The genotyping will determine the frequency of oocysts pathogenic to humans.<P> The second specific objective will be met in the second year by extracting and purifying oocysts from swine feces obtained from one or two of the facilities that had the highest number of oocysts in its lagoon. The purified oocysts will be inoculated into lagoon effluent contained within a set of sentinel chamber which will be placed in two separate lagoons and removed at designated intervals in triplicate to be assayed for viability. Rates of oocyst inactivation in lagoons will be determined based on these studies.<P> In the third year the third specific objective will be met by placing purified oocysts in nylon filter bags (3 m-pore-size) which will then be placed in 10 m-pore-size nylon bags and in turn contained (and protected) in two stainless steel 80 US Standard mesh sieves sealed together. These oocysts will be submerged in a lagoon and aged for three months (Treatment 1) and six months (Treatment 2). The aged oocysts will be inoculated into sentinel chambers containing surface field soil that has been air-dried and sieved and brought to a soil moisture near field capacity. The inoculated chambers will be buried 2 cm in replicated 1.5 by 1.5 m tilled plots and irrigated with a suspension of the aged oocysts in lagoon effluent. At days 0, 20, 55, and 148 triplicate sentinel chambers will be removed, oocysts extracted and assayed for viability. Oocysts from the irrigated soil (in trioplicate) will be extracted and assayed for viability as well. Controls will consist of oocysts in distilled water and incubated at 4 C. Rates of oocyst inactivation will be determined and compared. Finally, oocysts from soils of actual spray fields will be extracted and assayed for viability.