Thursday, May 31, 2012

CHRONIC WASTING DISEASE CWD PRION2012 Aerosol, Inhalation transmission, Scrapie, cats, species barrier, burial, and more

CHRONIC WASTING DISEASE CWD PRION2012 Aerosol, Inhalation transmission, Scrapie, cats, species barrier, burial, and more
PO-031: Aerosol transmission of chronic wasting disease to white-tailed deer

Nathaniel Denkers,1 Jeanette Hayes-Klug,1 Kelly Anderson,1 Sally Dahmes,2 David Osborn,3 Karl Miller,3 Robert Warren,3 Candace Mathiason,1 Edward Hoover1

1Colorado State University; Fort Collins, CO USA; 2WASCO Inc.; Monroe, GA USA; 3Warnell School of Forestry and Natural Resources, University of Georgia; Athens, GA USA

Purpose. A signature feature of chronic wasting disease (CWD) is its efficient lateral transmission in nature, almost surely by mucosal exposure. Our previous studies employing Tg(cerPrP) mice determined that CWD can be transmitted to a susceptible host by aerosol exposure, a route with relatively little investigation. The present study was designed to determine whether CWD is transmissible by aerosol to a native cervid host, white-tailed deer.

Materials and Methods. Nine white-tailed deer were exposed to two (2) aerosol doses of a 5% w/v CWD+ (n = 6) or CWD- (n = 3) brain homogenate, delivered via the nasal passages using a customized aerosol apparatus. At 3-month intervals post inoculation (mpi), tonsil and recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsies were collected and assayed for CWD infection by protein misfolding cyclic amplification (PMCA), western blotting (WB), and immunohistochemistry (IHC).

Results. At 3 mpi and 6 mpi, tonsil and RAMALT biopsies were collected from 5 of the 6 CWD + aerosol-exposed deer. Three of the 5 (60%) tested positive for CWD by PMCA but not IHC or western blot analysis at 3 mpi. By 6 mpi, 5 of 5 (100%) were tonsil and/or RAMALT biopsy positive by at least two of the three assays. Biopsies were collected from all CWD+ aerosol-exposed deer at 9 mpi, with 6 of 6 (100%) tonsil and/ or RAMALT positive by western blot or IHC. At 10 mpi 3 of the 6 prion-exposed deer have developed early clinical signs of CWD infection (hyperphagia, polydypsia, wide leg stance and head/neck dorsi-flexion). All sham-inoculated deer are showing no clinical signs and have remained CWD negative as assessed by all three assays. Interestingly, the prion dose delivered to the deer by aerosol-exposure is estimated to be 20-fold lower than the historical oral dose that has resulted in detectable CWD infection at 6 or 12 mpi.

Conclusions. This study documents the first aerosol transmission of CWD in deer. These results further infer that aerosolized prions facilitate CWD transmission with greater efficiency than does oral exposure to a larger prion dose. Thus exposure via the respiratory mucosa may be significant in the facile spread of CWD in deer and perhaps in prion transmission overall.

PO-073: Multiple routes of prion transepithelial transport in the nasal cavity following inhalation

Anthony Kincaid, Shawn Feilmann, Melissa Clouse, Albert Lorenzo, Jason Bartz Creighton University; Omaha, NE USA

Introduction. Inhalation of either prion-infected brain homogenate or aerosolized prions has been shown to cause disease, and in the case of inhalation of infected brain homogenate, the nasal route of infection has been shown to be 10–100 times more efficient than the oral route. The cell types involved in the in vivo transport of prions across the nasal cavity epithelium have not been determined. M cells in the follicular associated epithelium have been shown to mediate transcellular transport of prions in vitro and in the gut of experimentally infected mice. We tested the hypothesis that M-cell mediated transport was responsible for prion entry across nasal cavity epithelium following inhalation.

Materials and Methods. Hamsters were inoculated extranasally with 50 or 100ul of infected (n = 31) or mock-infected (n = 13) brain homogenate. Control animals were inoculated with buffer (n = 4) or were untreated (n = 5). Following survival periods ranging from 15 to 180 min, animals were perfused, skulls were decalcified and nasal cavities were embedded in paraffin. Tissue sections were cut and processed immunohistochemically for glial fibrillary acidic protein to identify brain homogenate, or for the disease-associated form of the prion protein. Tissue sections not further than 112 um apart through the entire extent of the nasal cavity were analyzed using light microscopy; photomicrographs were obtained wherever inoculum was observed on the surface of, within, or deep to the nasal mucosa for each animal.

Results. Infected or uninfected brain homogenate was identified within the nasal cavities of animals at all time points and was seen crossing the nasal cavity epithelium within minutes of inoculation; the transepithelial transport of brain homogenate continued for up to 3 h after inoculation. Infected or uninfected brain homogenate was seen adhering to, or located within, M cells at all time points. However, larger volumes of infected or uninfected brain homogenate were identified crossing between cells of the olfactory and respiratory epithelia in multiple locations. In addition, infected or uninfected brain homogenate was identified within the lumen of lymphatic vessels in the lamina propria beneath the nasal mucosa at all time points.

Conclusion. Transepithelial transport of prions across nasal cavity mucosa begins within minutes of inhalation and can continue for up to 3 h. While M cells appear to transport prions across the follicular associated epithelium, larger amounts of prions are transported between the cells of the respiratory and olfactory epithelia, where they immediately enter the lymphatic vessels in the lamina propria. Thus, inhaled prions can be spread via lymph draining the nasal cavity and have access to somatic and autonomic nerves in the lamina propria of the nasal cavity. The increased efficiency of the nasal cavity route of infection compared with the oral route may be due to the rapid and prolonged transport of prions between cells of the respiratory and olfactory epithelia.

PO-033: Replication efficiency of soil-bound prions varies with soil type

Shannon Bartelt-Hunt,1 Samuel Saunders,1 Ronald Shikiya,2 Katie Langenfeld,2 Jason Bartz2 1University of Nebraska-Lincoln; Omaha, NE USA; 2Creighton University; Omaha, NE USA

Prion sorption to soil is thought to play an important role in the transmission of scrapie and chronic wasting disease (CWD) via the environment. Sorption of PrP to soil and soil minerals is influenced by the strain and species of PrPSc and by soil characteristics. However, the ability of soil-bound prions to convert PrPc to PrPSc under these wide-ranging conditions remains poorly understood. We developed a semiquantitative protein misfolding cyclic amplification (PMCA) protocol to evaluate replication efficiency of soil-bound prions. Binding of the hyper (HY) strain of transmissible mink encephalopathy (TME) (hamster) prions to a silty clay loam soil yielded a greater-than-1-log decrease in PMCA replication efficiency with a corresponding 1.3-log reduction in titer. The increased binding of PrPSc to soil over time corresponded with a decrease in PMCA replication efficiency. The PMCA efficiency of bound prions varied with soil type, where prions bound to clay and organic surfaces exhibited significantly lower replication efficiencies while prions bound to sand exhibited no apparent difference in replication efficiency compared to unbound controls. PMCA results from hamster and CWD agent-infected elk prions yielded similar findings. Given that PrPSc adsorption affinity varies with soil type, the overall balance between prion adsorption affinity and replication efficiency for the dominant soil types of an area may be a significant determinant in the environmental transmission of prion diseases.

PO-039: A comparison of scrapie and chronic wasting disease in white-tailed deer

Justin Greenlee, Jodi Smith, Eric Nicholson US Dept. Agriculture; Agricultural Research Service, National Animal Disease Center; Ames, IA USA

Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of these experiments was to determine susceptibility of white-tailed deer (WTD) to scrapie and to compare the resultant clinical signs, lesions, and molecular profiles of PrPSc to those of chronic wasting disease (CWD). We inoculated WTD intracranially (IC; n = 5) and by a natural route of exposure (concurrent oral and intranasal (IN); n = 5) with a US scrapie isolate.

All deer were inoculated with a 10% (wt/vol) brain homogenate from sheep with scrapie (1ml IC, 1 ml IN, 30 ml oral). All deer inoculated by the intracranial route had evidence of PrPSc accumulation. PrPSc was detected in lymphoid tissues as early as 7 months-post-inoculation (PI) and a single deer that was necropsied at 15.6 months had widespread distribution of PrPSc highlighting that PrPSc is widely distributed in the CNS and lymphoid tissues prior to the onset of clinical signs. IC inoculated deer necropsied after 20 months PI (3/5) had clinical signs, spongiform encephalopathy, and widespread distribution of PrPSc in neural and lymphoid tissues.

The results of this study suggest that there are many similarities in the manifestation of CWD and scrapie in WTD after IC inoculation including early and widespread presence of PrPSc in lymphoid tissues, clinical signs of depression and weight loss progressing to wasting, and an incubation time of 21-23 months. Moreover, western blots (WB) done on brain material from the obex region have a molecular profile similar to CWD and distinct from tissues of the cerebrum or the scrapie inoculum. However, results of microscopic and IHC examination indicate that there are differences between the lesions expected in CWD and those that occur in deer with scrapie: amyloid plaques were not noted in any sections of brain examined from these deer and the pattern of immunoreactivity by IHC was diffuse rather than plaque-like.

After a natural route of exposure, 100% of WTD were susceptible to scrapie. Deer developed clinical signs of wasting and mental depression and were necropsied from 28 to 33 months PI. Tissues from these deer were positive for PrPSc by IHC and WB. Similar to IC inoculated deer, samples from these deer exhibited two different molecular profiles: samples from obex resembled CWD whereas those from cerebrum were similar to the original scrapie inoculum. On further examination by WB using a panel of antibodies, the tissues from deer with scrapie exhibit properties differing from tissues either from sheep with scrapie or WTD with CWD. Samples from WTD with CWD or sheep with scrapie are strongly immunoreactive when probed with mAb P4, however, samples from WTD with scrapie are only weakly immunoreactive. In contrast, when probed with mAb’s 6H4 or SAF 84, samples from sheep with scrapie and WTD with CWD are weakly immunoreactive and samples from WTD with scrapie are strongly positive. This work demonstrates that WTD are highly susceptible to sheep scrapie, but on first passage, scrapie in WTD is differentiable from CWD.

PO-041: Susceptibility of domestic cats to CWD infection

Amy Nalls, Jeanette Hayes-Klug, Kelly Anderson, Davis Seelig, Kevin Carnes, Susan Kraft, Edward Hoover, Candace Mathiason

Colorado State University; Fort Collins, CO USA

Domestic and non-domestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE); very likely due to consumption of bovine spongiform encephalopathy (BSE) contaminated meat. Because domestic and free-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease (CWD), we evaluated the susceptibility of domestic cats to CWD infection experimentally. Groups of n = 5 cats each were inoculated either intracerebrally (IC) or orally (PO) with CWD-infected deer brain homogenate.

Between 40 and 43 months two IC-inoculated cats developed slowly progressive symptoms including weight loss, anorexia, polydipsia, patterned motor behaviors, and ataxia”’ultimately mandating euthanasia. PrPCWD was detected in the brains of these animals by western blot, immunohistochemistry (IHC), and quaking-induced conversion (RT-QuIC) assays. No clinical signs of TSE were detected in the remaining primary passage cats at 86 months pi. Feline-adapted CWD (FelCWD) was sub-passaged into groups (n = 4 or 5) of cats by IC, PO, and IP/SQ routes.

All 5 IC inoculated cats developed symptoms of disease 20–24 months pi (approximately half the incubation period of primary passage). Additional symptoms in these animals included increasing aggressiveness and hyper responsiveness. FelCWD was demonstrated in the brains of all the affected cats by western blot and IHC. Currently, 3 of 4 IP/SQ, and 1 of 4 PO inoculated cats have developed abnormal behavior patterns consistent with the early stage of feline CWD. Magnetic resonance imaging (MRI) has been performed on 11 cats (6 clinically ill, 2 asymptomatic, and 3 age-matched negative controls). Abnormalities were detected in 4 of 6 clinically ill cats and included multifocal signal changes consistent with inflammation, ventricular size increases, more prominent sulci, and white matter tract cavitation.

These results demonstrate that CWD can be transmitted and adapted to the domestic cat, and raise the potential for cervid-to-feline transmission in nature.

PO-055: Transgenetic modeling of the CWD species barrier to humans

Eri Saijo,1 Sehun Kim,2 Claudio Soto,3 Glenn Telling2 1University of Kentucky College of Medicine; Fort Collins, CO USA; 2Department of Microbiology, Immunology and Pathology; Colorado State University; Fort Collins, CO USA ; 3Department of Neurology; University of Texas Houston Medical School; Houston, TX USA

Our recent studies raise significant concerns about the zoonotic potential of CWD. First, we showed that tissues consumed by humans derived from deer or elk with CWD, including skeletal muscle and antler velvet, harbor infectious prions. In other studies, cervid PrPSc converted human PrPC after CWD prions were stabilized by successive passages in vitro or in vivo.

We also identified at least two distinct strains of CWD, referred to as CWD1 and CWD2, the host-range properties of which are currently undefined. Other studies showed that codon 129, and the corresponding 132 residue in elk, significantly influenced the transmission of BSE and CWD prions respectively in transgenic (Tg) mouse models.

We inoculated Tg mice expressing human PrP encoding either methionine (M) or valine (V) at codon 129 with deer or elk CWD prions that previously produced disease in Tg mice expressing deer PrP with characteristics typical of CWD1 and CWD2 strains, as well as CWD prions that had been passaged multiple times in Tg mice expressing deer PrP.

While most Tg mice remained free of signs of prion disease for >260 days, small numbers of inoculated mice developed multiple, progressive neurological signs, that were consistent with prion disease. However, examination of brain materials from diseased mice failed to confirm the presence of protease-resistant human PrP. These preliminary results are consistent with a significant species barrier in humans to these CWD strains, and indicate that the 129 polymorphism does not modulate susceptibility.

PO-056: PrPCWD profiling of white-tailed deer (Odocoileus virginianus) with different Prnp genotypes following experimental oral infection

Camilo Duque Velasquez,1 Allen Herbst,1 Chad Johnson,2 Judd Aiken,1 Debbie McKenzie1 1Centre for Prions and Protein Folding Diseases; University of Alberta; Edmonton, AB Canada; 2Department of Soil Science; University of Wisconsin; Madison, WI USA

Chronic wasting disease (CWD) affects captive and free-ranging cervid populations in North America and farmed cervids of South Korea. CWD and scrapie are the only prion diseases in which the transmission occurs horizontally. The cervid Prnp gene is polymorphic at various positions. The effect of these changes on infection is influenced by the sequence and structure compatibility between the host and the infectious source. Prion strains have been described virtually in every prion disease and strongly impact disease characteristics (clinical symptoms, neuropathological profiles, incubation periods, species tropism as well as biochemical and biophysical properties of the abnormally folded prion protein). Prion protein sequence differences can result, upon subsequent infection, in the generation of novel strains as documented in sheep scrapie. We have previously shown that Prnp polymorphisms influence susceptibility to CWD in free-ranging white-tailed deer. In CWD-positive deer populations, alleles Q95G96 (wt) were over-represented compared to the H95G96 and Q95S96 alleles. Experimental oral infection of white-tailed deer with known Prnp genotypes (with inoculum from CWDpositive wt/wt deer) confirmed the link between prion protein primary sequence and the progression of disease. Heterozygous interference occurred in varying degrees as evidenced by the difference in the extension of the incubation period as an effect of alleles Q95S96 and H95G96. Interestingly, the biochemical profiles of the wt/Q95S96 and wt/H95G96 isolates resemble the wt/wt, differing only in the amount of protease resistant PrPCWD present suggesting that the wt allele is preferentially converted. PrPCWD profiling revealed differences between deer with at least one wt allele and the H95G96/Q95S96 deer suggesting that this PrPCWD is unique compared to the PrPCWD from wt/wt animals.

PO-057: Host factors influence prion strain adaptation

Crystal Meyerett Reid, Mark Zabel Colorado State University; Fort Collins, CO USA

Background. Chronic Wasting disease (CWD) is one of many prion-mediated diseases known as transmissible spongiform encephalopathies (TSEs). There is ever-increasing biological and biochemical evidence that prion pathogenesis is caused by the conversion of the normal host protein (PrPC) into an abnormal disease causing conformation (PrPRES). How prions encipher heritable strain properties without nucleic acid remains unclear. Previously we have shown that host factors have contributed to the adaptation of an original deer CWD prion strain to transgenic mice.

Materials and Methods. We assessed strain differences using biological and biochemical assays and found that amplified cervid prions and serial-passaged cervid prions were significantly different than that of the original cervid strain. It is possible that prion mutation and adaptation can broaden the host range. Previous reports, however, indicate that there is a strict species barrier preventing CWD infection in wildtype mice.

Results. Here we show the generation of a mouse-adapted strain of CWD upon serial passage into transgenic cervidized mice and then subsequent passage into wildtype mice. All wildtype mice remained non-clinical upon first passage but became completely susceptible after second passage with similar incubation times to those of mice terminally ill from a mouse adapted scrapie strain. Inoculation of our mouse adapted CWD strain back into cervidized mice delayed progression to terminal disease.

Conclusion. We conclude that prion strain adaptation and mutation is highly dependent upon host factors and host encoded PrPC primary sequence. Upon serial passage the adapted prion strain shares more characteristics with prion strains from the new host rather than the original species.

OR-12: Chronic wasting disease transmission and pathogenesis in cervid and non-cervid Species

Edward A. Hoover, Candace K. Mathiason, Nicholas J. Haley, Timothy D. Kurt, Davis M. Seelig, Nathaniel D. Denkers, Amy V. Nalls, Mark D. Zabel, and Glenn C. Telling

Prion Research Program, Department of Microbiology, Immunology, and Pathology; Colorado State University; Fort Collins, CO USA

Since its recognition as a TSE in the late 1970s, chronic wasting disease (CWD) of cervids has been distinguished by its facile spread and is now recognized in 18 states, 2 Canadian provinces, and South Korea. The efficient horizontal spread of CWD reflects a prion/host relationship that facilitates efficient mucosal uptake, peripheral lymphoid amplification, and dissemination by exploiting excretory tissues and their products, helping to establish indirect/environmental and well as direct (e.g., salivary) transmission. Recent studies from our group also support the likelihood of early life mother to offspring and aerosol CWD prion transmission. Studies of cervid CWD exposure by natural routes indicate that incubation period for detection of overt infection, while still uncertain, may be much longer than originally thought.

Several non-cervid species can be infected by CWD experimentally (e.g., ferrets, voles, cats) with consequent species-specific disease phenotypes. The species-adapted prions so generated can be transmitted by mucosal, i.e., more natural, routes. Whether non-cervid species sympatric with deer/elk can be infected in nature, however, remains unknown. In vitro CWD prion amplification studies, in particular sPMCA, can foreshadow in vivo susceptibility and suggest the importance of the PrPC rigid loop region in species barrier permissiveness. Trans-species CWD amplification appears to broaden the host range/strain characteristics of the resultant prions. The origins of CWD remain unknown, however, the existence of multiple CWD prion strains/ quasi-species, the mechanisms of prion shedding/dissemination, and the relationship between sheep scrapie and CWD merit further investigation.

PO-060: Transmission of chronic wasting disease from mother to offspring

Candace Mathiason, Amy Nalls, Stephenie Fullaway, Kelly Anderson, Jeanette Hayes-Klug, Nicholas Haley, Edward Hoover

Colorado State University; Fort Collins, CO USA

To investigate the role mother to offspring transmission plays in chronic wasting disease (CWD) we have developed a cervid model employing the Reeve’s muntjac deer (Muntiacus reevesi). Eight muntjac doe were orally inoculated with CWD and tested PrPCWD lymphoid positive by 4 mo post infection. Twelve fawns were born to these eight CWD-infected doe, 3 were born viable, 6 were born non-viable, and 3 were harvested as fetuses (1 each from first, second or third trimester of pregnancy) from CWDinfected doe euthanized at end-stage disease. The viable fawns have been monitored for CWD infection by immunohistochemistry (IHC) performed on serial tonsil and rectal lymphoid tissue biopsies. One fawn that was IHC PrPCWD positive at 40 d of age is now, at 28 mo of age, showing early clinical signs associated with CWD infection. Moreover, CWD prions have been detected by sPMCA in placenta, brain, spleen and mesenteric lymphoid tissue harvested from 5 full-term non-viable fawns, and in fetal placenta and brain tissue harvested in utero from the second and third trimester fetuses. Additional tissues and pregnancy related fluids from doe and offspring are being analyzed for CWD prions. In summary, using the muntjac deer model we have demonstrated CWD clinical disease in an offspring born to a CWD-infected doe, and in utero transmission of CWD from mother to offspring. These studies provide basis to further investigate the mechanisms of maternal transfer of prions.

PO-081: Chronic wasting disease in the cat— Similarities to feline spongiform encephalopathy (FSE)

Davis Seelig, Amy Nalls, Maryanne Flasik, Victoria Frank, Candace Mathiason, Edward Hoover Colorado State University; Fort Collins, CO USA

Background and Introduction. Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids with an as yet to be fully defined host range. Moreover, the risk that CWD poses to feline predators and scavangers, through crossspecies consumption and subsequent transmission, is unknown. Previous and ongoing studies in our laboratory evaluating the susceptibility of domestic cats (Felis catus) to CWD (Mathiason et. al., NeuroPrion 2011, Nalls et. al., NeuroPrion 2012) have documented the susceptibility of domestic cats to CWD following intracerebral (IC) inoculation. However, many of the pathologic features of feline-adapted CWD, including the neural and systemic patterns of PrPCWD accumulation and neuropathology, remain unknown.

The chief objectives of this work were:

(1) to design a sensitive, enhanced immunohistochemical (E-IHC) protocol for the detection of CWD prions (PrPCWD) in feline tissues;

(2) to document the systemic distribution of PrPCWD in CWD-infected cats through E-IHC;

(3) to utilize single and multiple-label immunostaining and laser scanning confocal microscopy (LSCM) to provide insights into the subcellular patterns of PrPCWD accumulation and neuropathologic features of CWD-infected cats; and

(4) to compare feline CWD to the other known feline TSE Materials and Methods. Periodate-lysine-paraformaldehyde (PLP)-fixed, paraffin-embedded (PLP-PE) from terminal, IC-inoculated (n = 9) and sham-inoculated (n = 2), 1st and 2nd passage, CWD-infected cats were examined by E-IHC for the presence of PrPCWD and its association with markers of cell phenotype and organelles.

Results. The most sensitive E-IHC technique for the detection of PrPCWD in feline tissues incorporated a combination of slide pretreatment with proteinase-K (PK) in concert with tyramide signal amplification (TSA). With this protocol, we identified PrPCWD deposits throughout the CNS, which, in the 1st passage cats was primarily restricted to the obex, but increased in distribution and severity upon 2nd passage to include a number of midbrain nuclei, cortical gray matter, the thalamus and hypothalamus, and the hippocampus. Peripheral PrPCWD deposits were detected only in the 2nd passage cats, and included the enteric nervous system, the Peyer’s patches, and the retropharyngeal and mesenteric lymph nodes. PrPCWD was not detected in the sham-inoculated cats.

Moreover, using multi-label analysis, intracellular PrPCWD aggregates were seen in association with neurofilament heavy chain (NFH)-positive neurons and GFAP-positive astrocytes. In addition, large aggregates of intracellular PrPCWD were identified within LAMP1-positive lysosomes.

Conclusions. Feline PrPCWD is present in CNS neurons, astrocytes and LAMP-1-positive lysosomes. The morphologic overlap between the PrPCWD deposits in feline CWD and BSE-origin feline spongiform encephalopathy (FSE), implicates the importance of the host as a key determinant in the development of prion neuropathology and suggest a signature for detection of potential spontaneous feline prion disease.

PO-099: Estimating the risk of CWD transmission to humans—An interim report of a comprehensive study in non-human primates

Ann-Christin Schmaedicke DPZ; Goettingen, Germany

Chronic Wasting Disease (CWD) is a transmissible prion disease that occurs primarily among North American cervid species. CWD has emerged as a prion disease in captive as well as freeranging cervids with rising incidence. Continuous consumption of cervid-derived products in conjunction with increasing CWD prevalence suggests a risk for human exposure to CWD prions.

Although surveillance data collected in the North American population did not provide epidemiological evidence for CWD transmissions to humans it remains unclear whether a biological risk for such transmissions exists or can be excluded. In order to gauge the potential transmissibility of CWD to humans, a comprehensive CWD risk assessment in cynomolgus macaques has been initiated. To test for the CWD interspecies transmission to humans, we used a primate species previously shown to mimic the BSE susceptibility of humans. In addition, this species is phylogenetically close to humans with a homologous amino acid sequence of the prion protein. When we assessed the genotype of macaque PrP, the homozygosity for methionine at codon 129 was confirmed in all animals.

The zoonotic potential of CWD is evaluated by challenging groups of animals via different inoculation routes. General transmissibility of CWD to macaques is tested by intracerebral (i.c.) inoculation of brain homogenate from CWD-infected white-tailed deer (WTD) and elk. This was either done by direct injection of 10 mg CWDWTD to two animals (> 880 dpi) or by surgical implantation of CWD-WTD or CWD-elk contaminated steel wires in two animals (> 750 dpi) or three animals (> 400 dpi), respectively.

To address the risk of hunters while field dressing carcasses, we inoculated two animals with CWD-WTD by dermal scarification (> 920 dpi). To simulate human consumption of CWDinfected food products, we orally challenged three animals with 10 g CWD-WTD brain (> 820 dpi) and three animals with 3 kg CWD muscle tissue from different cervid species (> 950 dpi) by repeated feeding. Mock-inoculated macaques are co-housed in the same cage allowing exposure to saliva and faeces of CWDinoculated macaques.

To date, all inoculated animals remain asymptomatic. Cerebrospinal fluid and blood from all animals were and are going to be collected bimonthly, processed and stored in a repository. Analysis of these samples using ultrasensitive prion detection methods has been initiated. Oral challenge of 50 mg BSE to cynomolgus macaques can lead to fatal disease only after more than 5 y of incubation time (1952 dpi).

Thus, to estimate whether CWD could be transmitted to macaques or not, we assume that observation of challenged animals would be necessary for at least 8–10 y.

PO-248: TSE infectivity survives burial for five years with little reduction in titer

Allister Smith, Robert Somerville, Karen Fernie The Roslin Institute and R(D)SVS; University of Edinburgh; Edinburgh, UK

BSE infected animals, BSE-contaminated materials and other sources of TSE (prion) infection, such as carcasses from scrapie infected sheep, CWD infected deer and cadavers of individuals infected with CJD may all end up in the environment through burial or other methods of disposal. They may continue to act as a reservoir of TSE infectivity if cattle or other susceptible animals were to be exposed to these sources in the future. In order to address these concerns, we performed two large scale demonstration experiments under field conditions which were designed to mimic some of the ways by which TSE infected materials may have been disposed of. The project examined the fate of TSE infectivity over a period of five years in two scenarios; when the infectivity was contained within bovine heads and when the infectivity was buried without any containment. Two soil types were compared: a sandy loam and a clay loam. We used the 301V TSE strain which was derived by serial passage of BSE in VM mice.

TSE infectivity was recovered from all the heads exhumed annually for five years from both types of soil, with little reduction in the amount of infectivity throughout the period of the experiment. Small amounts of infectivity were found in the soil immediately surrounding the heads, but not in samples remote from them. Similarly there was no evidence of significant lateral movement of infectivity from the buried bolus. However large amounts of TSE infectivity were recovered at the site of burial of both boluses. There was limited vertical upward movement of infectivity from the bolus buried in clay soil and downward movement from the bolus buried in sandy soil.

Now that these experiments are completed we conclude that TSE infectivity is likely to survive burial for long periods of time with minimal loss of infectivity and restricted movement from the site of burial. These experiments emphasize that the environment is a viable reservoir for retaining large quantities of TSE infectivity, and reinforce the importance of risk assessment when disposing of this type of infectious material.

see more here ;

Epidemiology Update March 23, 2006

As of today, 13 locations and 32 movements of cattle have been examined with 27 of those being substantially completed. Additional investigations of locations and herds will continue. In addition, state and federal officials have confirmed that a black bull calf was born in 2005 to the index animal (the red cow). The calf was taken by the owner to a local stockyard in July 2005 where the calf died. The calf was appropriately disposed of in a local landfill and did not enter the human or animal food chain.

> The calf was appropriately disposed of in a local

> landfill and did not enter the human or animal food chain.

Wednesday, May 30, 2012

PO-248: TSE infectivity survives burial for five years with little reduction in titer



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