A novel replicating agent isolated from the human intestinal tract having characteristics shared with Creutzfeldt-Jakob and related agents

Abstract

Summary.A novel replicating agent (IFDO) was isolated from ileal fluid. Growth occurred in vitro under aerobic and anaerobic conditions, and was faster at 37°C than at room temperature. The doubling time was 1543 min. Colonies were dark brown in colour and occurred beneath the surface of agar after conventional surface inoculation. Provisional data indicate that the agent may be a normal intestinal commensal. The agent was remarkably resistant to inactivation by steam at 134"C, formaldehyde and glutaraldehyde; it was relatively resistant to ionising radiation, and it was filterable through membranes with a nominal pore diameter of 10 nm. Such properties, with the exception of growth in cell-free medium, are shared by "unconventional agents" such as those of Creutzfeldt-Jakob disease and scrapie. Further comparison of the properties of the intestinal agent and of slow viruses revealed additional shared characteristics, including resistance to proteinase K and trypsin, and inactivation by guanidine thiocyanate, diethyl pyrocarbonate, phenol and sodium hydroxide. The agent differs from that of scrapie in being inactivated by ethidium bromide, zinc nitrate, EDTA, hydroxylamine in the presence Sarkosyl, and, under certain circumstances, by ribonuclease. Broth cultures of the agent contained particles possessing considerable size heterogeneity. The smaller filterable particles were generally more susceptible to inactivation, did not survive autoclaving, and were inactivated by papaya protease and lipase. It is possible that the replicating agent may be formed by crystallisation from constituents of the medium, and not by a biological process. This does not exclude the postulated relationship to slow viruses.

A Novel Way to Grow Hemozoin-Like Crystals In Vitro and Its Use to Screen for Hemozoin Inhibiting Antimalarial Compounds

“…As previously described [16], a “bacterial colony like” growth was observed following seeding of cultures in broth or on surface agar with “IFDO”. Structures resembling bacterial/fungal ‘colonies’ appeared on plates after 7 to 10 days of incubation (Figure 1) and there was a correlation between optical density of initial inocula and the number of colonies observed.…”

“…Moreover, precipitates can be broken into smaller size particles which can serve as nuclei for the generation of new aggregates. The nucleation phenomenon was described by Burdon et al as being inhibited by various physicochemical treatments in a way that compares to inhibition of prion propagation [16], [18] (Data S1).…”

“…With this perspective, we were interested to study a model described by Burdon et al . because it appears to share some similarities to prion agents since the, yet unidentified, “agent” forms self-propagating precipitates under well defined conditions [16], [18]. Moreover, precipitates can be broken into smaller size particles which can serve as nuclei for the generation of new aggregates.…”

“…Culture methods were adapted from the original protocol [16]. The broth medium was prepared by autoclaving 35.5 g of Mycoplasma Broth Base (Oxoid CM403) in 950 ml distilled water and 2 ml Tween 80.…”

“…Initially, we set out to find a novel and simple replacement assay for the currently used complex, time intensive and expensive in vivo and in vitro assays to evaluate the therapeutic and decontamination efficacy against transmissible misfolded proteins (prion diseases). We investigated a “replicating agent”, described in the 1980s, and termed ”Ileal Fluid Dependent Organism” (IFDO) which, interestingly, presented susceptibility to inactivation treatments comparable to prions: resistant to steam and aldehydes but inactivated by guanidine thiocyanate and sodium hydroxide [16], [17], [18], [19].…”