Infectivity of Cryptosporidium parvum oocysts stored in water at environmental temperatures

Oocysts of Cryptosporidium parvum obtained from calves were cleaned of fecal debris by density gradient centrifugation and suspended in deionized water in microcentrifuge tubes. The tubes were placed in circulating water baths at temperatures of -10, -5, 0, 5, 10, 15, 20, 25, 30, or 35 C, and 2 tubes were removed from each water bath 1, 2, 4, 8, 12, 16, 20, and 24 wk later. Oocysts from 1 tube were administered at the rate of 1.5 x 105 oocysts per mouse to 2 litters of neonatal BALB/c mice and were considered infective when developmental stages were found in histologic sections of mouse gut and/or a positive polymerase chain reaction (PCR) was obtained for C. parvum DNA in mouse ileum. The second tube was held at -70 C until tubes from all time periods were available, then oocysts within the tubes were assayed for amylopectin concentration. Oocysts held at -10 C were infectious up to 1 wk of storage, and those held at -5 C were infectious up to 8 wk of storage, as determined by PCR but not histology. Oocysts held at 0, 5, 10, 15, and 20 C were still infectious after 24 wk of storage. By microscopic examination of mouse tissue, oocysts held at 20 C infected only 1 of 10 mice after 24 wk of storage, and the number of developmental stages began declining after 4 wk of storage; those held at 25 and 30 C each produced infections up to 12 wk after storage in 1 of 10 mice with reduced numbers of developmental stages beginning 4 wk after storage. Those held at 35 C produced light infections in 2 of 10 mice only up to 1 wk of storage. Amylopectin concentration decreased with increasing length of storage time or temperature. These findings provide a guide for estimating the potential duration of oocyst infectivity within a wide range of environmental temperatures and demonstrate the relationship between amylopectin concentration and infectivity.