Infectivity and neutralization of Cryptosporidium parvum sporozoites

Abstract

Cryptosporidiosis, a diarrheal disease of calves and humans caused by the coccidian parasite Cryptosporidium parvum, is terminated in hosts with normal immune systems. To assess the mechanisms of immunity in cryptosporidiosis, it is necessary to isolate and quantitate sporozoites, the infective stage of Cryptosporidium spp. Here we report the (i) separation of infective C. parvum oocysts from calf feces by ether extraction, sieving, and hypochlorite treatment; (ii) separation of viable C. parvum sporozoites from intact and excysted oocysts by anion-exchange chromatography; and (iii) quantitation of sporozoite infectivity in vivo by direct intraintestinal injection of isolated sporozoites in 7-day-old BALB/c mice. When isolated sporozoites were incubated with heat-inactivated immune bovine serum, 25 times the 50% infective dose for 7-day-old mice was completely neutralized. Sporozoites incubated with preimmune bovine serum were infectious for 7-day-old mice.

A simple and reliable method of producing in vitro infections of cryptosporidium parvum (apicomplexa)

“…We have used oocysts directly to infect cells previously [3,11], but have recently reduced the amount of time needed to manipulate the parasites. This method relies on previous observations that have shown C. parvum oocysts will readily excyst when the temperature is elevated to 37°C following treatment with 10% bleach [11,12]. Percent excystation of sodium hypochlorite-treated oocysts are nearly identical to that observed when bile salts are used [13], and all that is apparently needed for excystation is a reagent that affects the oocyst wall and suture [14]; non-pretreated oocysts excyst poorly [15,16].…”

Antibody fusions reduce onset of experimental Cryptosporidium parvum infection in calves

“…The Iowa C. parvum isolate (Heine et al, 1984), which was the source of oocysts for all experiments, has been maintained since 1988 by propagation in newborn Cryptosporidium-free Holstein bull calves (Riggs et al, 1989;Riggs and Perryman, 1987). Oocysts were isolated from calf feces by sucrose density gradient centrifugation and stored in 2.5% KCr 2 O 7 (4 • C) (Arrowood and Donaldson, 1996;Riggs and Perryman, 1987). For calf and mouse challenge studies, oocysts were used within 30 days of isolation and disinfected with 1% peracetic acid immediately prior to administration (Riggs et al, 1994).…”

Calf Clinical Model of Cryptosporidiosis for Efficacy Evaluation of Therapeutics

“…Decide on the C. parvum isolate to be used and obtain purified oocysts from a source production laboratory. We routinely use the Iowa C. parvum isolate [23,24] because it is well characterized and is maintained by monthly propagation in newborn Cryptosporidium-free Holstein bull calves in our laboratory [25,26] (Cryptosporidium Production Laboratory; https:// acbs.cals.arizona.edu/crypto). Other established laboratories and commercial sources exist for obtaining purified C. parvum oocysts in the numbers required for calf trials including Bunch Grass Farm (Deary, ID; bunchgrassfarm@gmail.com).…”

“…Immediately prior to administration to calves, disinfect oocysts with 0.5% peracetic acid (4 °C) to inactivate any possible contaminant bacterial or viral pathogens [23,27] (see Note 2). Peracetic acid is the preferred disinfectant as treatment of oocysts with diluted sodium hypochlorite (bleach) has been shown to promote spontaneous excystation at 37 °C [25,27]. Use of hypochlorite-disinfected oocysts could potentially result in excystation in the abomasum of experimental calves and sporozoite killing by acidic conditions, effectively reducing the intended infectious dose [25].…”

“…Peracetic acid is the preferred disinfectant as treatment of oocysts with diluted sodium hypochlorite (bleach) has been shown to promote spontaneous excystation at 37 °C [25,27]. Use of hypochlorite-disinfected oocysts could potentially result in excystation in the abomasum of experimental calves and sporozoite killing by acidic conditions, effectively reducing the intended infectious dose [25]. In contrast, peracetic acid disinfection does not promote spontaneous excystation.…”