Serum neurofilament light (Nf-L) concentrations.

(A) The incubation period for each animal was between 330 to 436 days post inoculation. Four lines are shown representing serum Nf-L concentrations for each animal used in the study. (B) To meaningfully compare changes in serum Nf-L concentrations between animals incubating scrapie, each sample timepoint was calculated as a fraction of that animal’s scrapie incubation period. Although, Nf-L elevations were detected at different chronological points between animals, elevations in serum Nf-L occurred at similar fractional times of the incubation period once that timeframe was normalized. All animals have a notable rise in Nf-L levels during the final 10% of the scrapie incubation period.</p

RT-QuIC detection of prion seeding activity in rectal biopsy tissue.

Each color/shape combination represents a different animal that was used in the study. Every sheep sample per timepoint had 4 technical replicates performed. Samples were considered RT-QuIC positive when 2/4 replicates reached threshold–a data point is present when a sample was positive for that timepoint. Negative samples appear at the baseline of the x-axis. The error bars indicate standard deviation of technical replicates.</p

Timepoint serum neurofilament concentrations from sheep inoculated with the scrapie agent.

Timepoint serum neurofilament concentrations from sheep inoculated with the scrapie agent.</p

Enzyme immunoassay results for brainstem and lymphoid tissue.

Enzyme immunoassay results for brainstem and lymphoid tissue.</p

Non-neurologic vs neurologic.

ObjectiveNeurofilament light chain (Nf-L) has been used to detect neuroaxonal damage in the brain caused by physical injury or disease. The purpose of this study was to determine if serum Nf-L could be used as a biomarker for pre-symptomatic detection of scrapie in sheep.MethodsFour sheep with prion protein genotype AVQQ were intranasally inoculated with the classical scrapie strain x124. Blood was collected every 4 weeks until 44 weeks post-inoculation, at which point weekly collection commenced. Serum was analyzed using single molecule array (Quanterix SR-X) to evaluate Nf-L concentrations.ResultsScrapie was confirmed in each sheep by testing homogenized brainstem at the level of the obex with a commercially available enzyme immunoassay. Increased serum Nf-L concentrations were identified above the determined cutoff during the last tenth of the respective incubation period for each sheep. Throughout the time course study, PrPSc accumulation was not detected antemortem by immunohistochemistry in rectal tissue at any timepoint for any sheep. RT-QuIC results were inconsistently positive throughout the timepoints tested for each sheep; however, each sheep had at least one timepoint detected positive. When assessing serum Nf-L utility using receiver operator characteristic curves against different clinical parameters, such as asymptomatic and symptomatic (pruritus or neurologic signs), results showed that Nf-L was most useful at being an indicator of disease only late in disease progression when neurologic signs were present.ConclusionSerum Nf-L concentrations in the cohort of sheep increased as disease progressed; however, serum Nf-L did not increase during the presymptomatic window. The levels increased substantially throughout the final 10% of the animals’ scrapie incubation period when other clinical signs were present. Serum Nf-L is not a reliable biomarker for pre-clinical detection of scrapie.</div

Determination of cutoff values for serum Nf-L that differentiated clinical stages of sheep scrapie using receiver operator characteristic (ROC) curves.

The appearance of clinical signs is displayed in a timeline overlay of the incubation period (not to scale). Arrows to the right of A-D illustrate the appearance of clinical signs over time. The comparisons, A-D, correspond to the ROC curves below. (A) The serum Nf-L cut-off for asymptomatic sheep compared to sheep with neurologic signs. (B) The serum Nf-L cut-off for asymptomatic compared to all symptomatic sheep. (C) The serum Nf-L cut-off for non-neurologic sheep compared to sheep with neurologic signs. (D) The serum Nf-L cut-off for asymptomatic sheep compared to only the sheep with pruritus; prior to when those sheep developed neurologic signs.</p

Number of ileal bacteria in healthy and diseased dogs.

Number of ileal bacteria in healthy and diseased dogs.</p

Dog cohort demographics.

<p>Canine cohort demographics with regards to age, gender, breed, and diet at the time of endoscopic biopsy.</p

Spatial distribution of the number of colonic bacteria based on FISH.

<p>Spatial distribution of the number of colonic bacteria based on FISH.</p

Probes used for fluorescence in situ hybridization.

<p>Probes used for in situ bacterial identification.</p