2. Chronic Subclinical Transmissible Spongiform Encephalopathy Infections

3. Transmissible Spongiform Encephalopathies  Despite defining nosologic feature, < 1% of all human cases are a consequence of recognisable horizontal transmission events; ~ 85% “sporadic”; ~ 15% genetic.  Although infectivity very low compared to viruses/bacteria, epidemics have occurred given unique circumstances; e.g. natural scrapie -> recycled through cattle -> BSE epidemic-> vCJD  Incidence steady at 1-2 cases per million population/year, independent of country.  “Sporadic” cases thought to arise spontaneously as a consequence of failure of cellular clearance of misfolded PrPc, possibly related to somatic neuronal mutations or more likely decline in cellular protein “quality control”-> gradual accumulation of PrPres -> invariably and quite quickly leads to clinical disease.  Concept of “natural” reservoirs of infection serving as sources of point source or case-to- case transmission, probably through low-level, largely covert, contamination episodes -> ??helps to sustain low-level endemicity of the 1-2 cases per million/year; no clear epidemiological support despite detailed investigation of human CJD clusters, but very difficult to identify if host is asymptomatic at the time and for lengthy period thereafter and the contamination event not “dramatic”.  Interest in “natural” reservoirs and possible under/un-appreciated case-to-case transmissions, especially during provision of health care, rekindled by recent mouse models of chronic subclinical TSE infection; concerns supported by two large epidemiological case-control studies.

4. Probably first description of animal of model of chronic subclinical TSE infection Zlotnik 1965 Symptomatic scrapie in sheep; mice intragastricaly inoculated sheep brain; mice not develop scrapie after 13 months but brains showed vacuolation; mice brain/spleen pools IC and IP inoculated into further mice with all developing clinical scrapie. Current understanding would consider 13 months was not long enough an observation period for enteral inoculation route

5. Dickinson 1975 22A scrapie strain in C57BL and VM mice; inoculated log dilution series IC and IP; mice observed >600 days; mice IP at 10-1 developed clinical scrapie but higher dilutions not develop clinical disease and brains negative histol but spleens of 10-2 and 10-3 mice had low titres of infectivity on bioassay > 300 days post-IP inoculation Collis 1985 87V scrapie strain in IM mice; IP inoculation; 3/53 mice developed clinical disease after 600 days; however, spleens of these mice showed ubiquitous infectivity by bioassay from soon after inoculation which continued at similar levels until asymptomatic mice culled at 80 weeks. Implies low-level peripheral inoculation may never cause overt neurological disease but peripheral (especially lymphoreticular) organs are infectious over lengthy period of host’s life and in the incubation period may in fact exceed the life span of the host Also underscores that ID/LD 50 titres determined by clinical disease in recipients may be underestimated by 1000-fold.

6. Bueler 1994 RML strain in PrP+/+ and PrP+/- mice; IC inoculated; PrP+/- mice express half PrPc levels -> prolonged incubation period (~290 days cf ~140 days) and disease duration (~125 days cf ~13 days); however - PrPres detectable day 84, peaked ~168 days, plateaued thereafter, - vacuolation and astrogliosis evident from ~ 140 days, most prominent after ~290 days, plateaued thereafter, - titre ~8log10 at 20 weeks and not significantly increase thereafter.

7. Frigg 1999 RAG-2-/- and uMT immunodeficient mice; IP inoculated -> asymptomatic for 665 days with randomly selected mice culled during the time course; in 5/14 randomly selected mice brains positive for PrPres on western and/or transmitted on IC bioassay to Tga20 mice ; four mice further IC inoculated into Tga20, RAG-1-/-, and uMT mice. - titres (by time interval assay) frequently comparable to those seen in terminally sick mice. - only symptomatic mice had brain vacuolation. Immunodeficient mice (except SCID mice) all develop scrapie similar to wild-type mice after IC inoculation; suggests peripheral inoculation may be more relevant than immunodeficiency to creation of subclinical infection state.

8. Race 1998, 2001, and 2002. 263K hamster scrapie strain in C57BL mice; Hill 2000 Sc237 strain (=263K) in CD-1 mice; Hamster scrapie brain (at high titre) IC inoculated into mice; no clinical disease in any mice up to ~800 days; up to ~400-600 days mice brains negative for mouse PrPres, but after this period some mice contained murine PrPres in their brains (likelihood increased with the length of post-inoculation period); hamster PrPres never seen in mouse brains. If mouse brain negative for PrPres, only but very commonly transmitted to hamsters with IC inoculation (with incubation periods inversely related to length of observation period in mice). If mouse brain positive for PrPres, generally transmitted to mice but always to hamsters.

9. Thackray 2002 ME7 and RML scrapie strains in Tga20 mice; undertook IC inoculations with log dilution series. higher dilutions (10-6 and 10-7 in ME7; 10-8 and 10-9 in RML) associated with fluctuating/reversible neurological signs that did not progress to terminal disease; still classed as “subclinical”. length of “subclinical” mice survival not clearly stated but culled and found to have - abundant PrPres (more than in terminally sick mice) - high brain titres (by time interval assay in Tga20 mice): 100,000 ID 50/g brain in ME7; 1000-100,000 ID 50/g brain in RML. no brain vacuolation unless developed terminal scrapie.

10. Box #Final inoculum dilution Sick mice/total inoculated Incubation period range (days post-ICI) Mean incubation period (days) 202810-16/6151-175161.8 202910-26/6158-184170.6 203010-36/6158-193176.5 203110-45/5193-399*290.8 203210-52/6283 203310-60/6 203410-71/6240 203510-80/5 203610-90/3 * Two mice became ill much later and died at 391 and 399 days. Mean incubation period for the other three mice was 221.3 days.

11. Origin a l Box# Original Inoculum Dilution Mouse# fro m ori gin al Bo x Western blot For brain PrP res Histol for va cu ola tio n IHC for PrPres plaques Recipient or Indicator Tga/20 mice:Box# Successful transmission/ Total mice inoculated Positive western blot for PrPres of selected mice/total assessed Mean incubatio n period days (range) 203210-51Pos+++20854/4ND*62.3 (61-65) ““2neg0020862/3** 113.5 (107-120) ““3pos+/++++20874/41/167.5 (65-72) ““4pos++/++++/++20884/41/165.3 (63-68) 203310-61neg0020893/4***3/4133.3 (98-202) ““2neg0020903/4***3/4139.6 (123-154) ““3neg0020914/42/282.5 (79-86) ““4pos++/+++++2092A &B3/3ND63.7 (62-65) 203410-71neg0020930/5 ““2neg0020940/5 ““3neg0020950/5 ““4neg0020960/5 203510-81neg0020971/51/1142 ““2neg0020980/4 ““3neg0020990/5 ““4neg0021001/41/1129 203610-91neg00ND ““2neg00ND ““3neg00ND *ND=not determined. ** Single sick mouse culled 214 days post-inoculation with brain negative for PrPres on western immunoblot. ***There was a single remaining mouse, which was healthy at culling 218 days post-inoculation; brain negative for PrPres on western immunoblot

12. Conclusions “inefficient” inoculation – peripheral route, decreased PrPc expression, low titre innoculum, “cross-species” – associated with prolonged incubation periods, which can approximate or exceed life-span of host. During this prolonged incubation period, and possibly from relatively early, high titres of infectivity may exist in peripheral lymphoreticular organs (and perhaps lower titres in other organs and blood) as well as brain, allowing potential unrecognised transmission to occur. Great concerns regarding potentially large numbers of subclinical vCJD carriers. Titres underestimated by 2-3 logs if only clinically apparent transmissions used for calculations. Circumstantial support for relevance to human TSE: Kuru incubation periods now exceed 50 years in most recent victims. two large epidemiological case-control studies linking surgery to increased risk of “sporadic” CJD. Priorities: To minimise secondary transmission risk from subclinical infection in health care setting, screening biochemical tests will need to be highly sensitive (eg CDI or high performance western blots for PrPres) and approximate bioassay results seen in sensitive recipients like Tga20 mice.