Limits...
Detection of CWD prions in urine and saliva of deer by transgenic mouse bioassay.

Haley NJ, Seelig DM, Zabel MD, Telling GC, Hoover EA - PLoS ONE (2009)

Bottom Line: The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role.In addition, PrP(CWD) was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA).These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

ABSTRACT
Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids (e.g. deer, elk, and moose). The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role. Here we report the presence of infectious prions in the urine and saliva of deer with chronic wasting disease (CWD). Prion infectivity was detected by bioassay of concentrated, dialyzed urine and saliva in transgenic mice expressing the cervid PrP gene (Tg[CerPrP] mice). In addition, PrP(CWD) was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA). The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrP(CWD) levels by traditional assays (western blot, ELISA) and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg(CerPrP) mice (373(+/-)3 days in 2 of 9 urine-inoculated mice and 342(+/-)109 days in 8 of 9 saliva-inoculated mice). These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.

Show MeSH

Related in: MedlinePlus

Serial PMCA amplification of PrPCWD in concentrated deer urine and in the brains of urine-inoculated mice.A) PrPCWD was detectable by serial PMCA (sPMCA) in control and urine inocula (lanes 1 and 2, respectively), while PrPCWD could not be identified in saliva and negative control inocula (lanes 3 and 4, respectively) after 3 rounds of amplification. B) Three rounds of sPMCA also amplified PrPCWD in the brains of CWD-infected mice, including positive-control inoculated mice and a single mouse inoculated with lyophilized urine (lanes 1 and 3, respectively). PrPCWD was not amplified in mice inoculated with negative control material (lanes 5 and 6) or in other mice inoculated with either urine (lane 2) or saliva (lane 4) from CWD+ deer. All flanking lanes represent undigested PrPC.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654070&req=5

pone-0004848-g004: Serial PMCA amplification of PrPCWD in concentrated deer urine and in the brains of urine-inoculated mice.A) PrPCWD was detectable by serial PMCA (sPMCA) in control and urine inocula (lanes 1 and 2, respectively), while PrPCWD could not be identified in saliva and negative control inocula (lanes 3 and 4, respectively) after 3 rounds of amplification. B) Three rounds of sPMCA also amplified PrPCWD in the brains of CWD-infected mice, including positive-control inoculated mice and a single mouse inoculated with lyophilized urine (lanes 1 and 3, respectively). PrPCWD was not amplified in mice inoculated with negative control material (lanes 5 and 6) or in other mice inoculated with either urine (lane 2) or saliva (lane 4) from CWD+ deer. All flanking lanes represent undigested PrPC.

Mentions: Concentrated samples used for mouse inoculation were assayed for PrPCWD by serial PMCA (sPMCA) over three rounds of amplification. In our experience, three rounds of amplification permits an approximate 4000-fold increase in sensitivity as compared to traditional western blotting detection, while avoiding both cross-contamination and generation of any spontaneously formed protease-resistant PrP, thereby maintaining 100% specificity [13]. In three independent experiments, PrPCWD was identified in lyophilized urine homogenate from CWD+ deer. (Figure 4A) PrPCWD was also found in both positive control inocula after the initial round of amplification, while PrPCWD was not detected in either saliva or negative control preparations.


Detection of CWD prions in urine and saliva of deer by transgenic mouse bioassay.

Haley NJ, Seelig DM, Zabel MD, Telling GC, Hoover EA - PLoS ONE (2009)

Serial PMCA amplification of PrPCWD in concentrated deer urine and in the brains of urine-inoculated mice.A) PrPCWD was detectable by serial PMCA (sPMCA) in control and urine inocula (lanes 1 and 2, respectively), while PrPCWD could not be identified in saliva and negative control inocula (lanes 3 and 4, respectively) after 3 rounds of amplification. B) Three rounds of sPMCA also amplified PrPCWD in the brains of CWD-infected mice, including positive-control inoculated mice and a single mouse inoculated with lyophilized urine (lanes 1 and 3, respectively). PrPCWD was not amplified in mice inoculated with negative control material (lanes 5 and 6) or in other mice inoculated with either urine (lane 2) or saliva (lane 4) from CWD+ deer. All flanking lanes represent undigested PrPC.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654070&req=5

pone-0004848-g004: Serial PMCA amplification of PrPCWD in concentrated deer urine and in the brains of urine-inoculated mice.A) PrPCWD was detectable by serial PMCA (sPMCA) in control and urine inocula (lanes 1 and 2, respectively), while PrPCWD could not be identified in saliva and negative control inocula (lanes 3 and 4, respectively) after 3 rounds of amplification. B) Three rounds of sPMCA also amplified PrPCWD in the brains of CWD-infected mice, including positive-control inoculated mice and a single mouse inoculated with lyophilized urine (lanes 1 and 3, respectively). PrPCWD was not amplified in mice inoculated with negative control material (lanes 5 and 6) or in other mice inoculated with either urine (lane 2) or saliva (lane 4) from CWD+ deer. All flanking lanes represent undigested PrPC.
Mentions: Concentrated samples used for mouse inoculation were assayed for PrPCWD by serial PMCA (sPMCA) over three rounds of amplification. In our experience, three rounds of amplification permits an approximate 4000-fold increase in sensitivity as compared to traditional western blotting detection, while avoiding both cross-contamination and generation of any spontaneously formed protease-resistant PrP, thereby maintaining 100% specificity [13]. In three independent experiments, PrPCWD was identified in lyophilized urine homogenate from CWD+ deer. (Figure 4A) PrPCWD was also found in both positive control inocula after the initial round of amplification, while PrPCWD was not detected in either saliva or negative control preparations.

Bottom Line: The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role.In addition, PrP(CWD) was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA).These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

ABSTRACT
Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids (e.g. deer, elk, and moose). The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role. Here we report the presence of infectious prions in the urine and saliva of deer with chronic wasting disease (CWD). Prion infectivity was detected by bioassay of concentrated, dialyzed urine and saliva in transgenic mice expressing the cervid PrP gene (Tg[CerPrP] mice). In addition, PrP(CWD) was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA). The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrP(CWD) levels by traditional assays (western blot, ELISA) and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg(CerPrP) mice (373(+/-)3 days in 2 of 9 urine-inoculated mice and 342(+/-)109 days in 8 of 9 saliva-inoculated mice). These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.

Show MeSH
Related in: MedlinePlus