Methods in coccidiosis research: separation of oocysts from faeces

Factors which may be important in the large-scale extraction of coccidial oocysts from faeces ha.ve been investigated with Eimeria tenella. Age of bird, inoculum, feeding status at the time of inoculation, period of collection, feeding status during collection, collection medium, homogenization and sieving, flotation, washing, sporulation and further purification have all been considered. The aim has been to establish a method to produce the maximum number of oocysts of a required degree of purity and viability, with the expenditure of the minimum amount of physical effort, time, animals and chemicals. In our method, groups of chickens 3-4 weeks of age are inoculated with 5000 oocysts of E. tenella and food is supplied ad lib. Over the period 5-8 days after inoculation, faeces are collected in trays containing 2% (w/v) potassium dichromate solution, while food intake is restricted. The faecal material is homogenized, passed once through 40 and 100 mesh sieves, centrifuged and the oocysts recovered from the sediment by 3 flotations in saturated salt solution. Following washing, oocysts are sporulated by forced aeration at 30 degrees C and may be further purified by hypochlorite treatment, or passage in 5% Tween 80 solution through a glass bead column followed by sucrose density gradient centrifugation. Routine passages along these lines over a 5 year period have given a recovery of 46% of the oocysts excreted by over 7000 birds.