Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

To determine the suitability and estimate the sensitivity of an immunohistochemical (IHC) test for disease-associated prion protein (PrP(Sc)) in biopsy specimens of rectoanal mucosa-associated lymphoid tissue (RAMALT) for diagnosis of scrapie in sheep. 762 sheep at high risk for having scrapie and indemnified by the National Scrapie Eradication Program. The IHC test for PrP(Sc) was applied to 2 RAMALT and 2 third-eyelid biopsy specimens and a postmortem RAMALT specimen from each sheep. Results were compared with those of a reference test in which results for tissues from obex and retropharyngeal lymph nodes, tonsil, or both were considered in parallel. The reference test identified 139 sheep as having scrapie. Biopsy-related complications occurred in 3 sheep. Sensitivity of the IHC test in RAMALT ranged from 85.3% to 89.4%, depending on the anatomic location from which RAMALT was obtained. Results for the test applied to 1 RAMALT specimen were similar to results interpreted in parallel for 2 third-eyelid specimens (sensitivity, 87.0%). The proportion of inconclusive test results attributable to insufficient lymphoid follicles in biopsy specimens was lower when considering results for 2 RAMALT specimens in parallel (10.1%) than when considering results for 2 third-eyelid specimens in parallel (23.7%). Specimens of RAMALT that were inappropriately collected from an area caudal to the rectoanal interface yielded a high proportion of inconclusive results (33.3% to 50.0%). The IHC test for PrP(Sc) in RAMALT was an effective means of detecting subclinical scrapie in live, high-risk sheep.

Peanut agglutinin (PNA) and surface immunoglobulin (SIg) were investigated as markers for T and B lymphocytes in blood and lymphoid tissues of dogs of various ages. In the blood study, 4 age groups (n = 8 dogs/group) were used. The mean (+/- SD) percentages of PNA-positive (PNA +) cells were 68.4 +/- 8.6% (group 1, < 1 year old), 70.3 +/- 9.2% (group 2, 1 to 2 years old), 72.0 +/- 3.7% (group 3, 5 to 6 years old), and 63.8 +/- 10.1% (group 4, 10 to 11 years old). The mean percentages of SIg-positive (SIg+) cells in blood were 32.1 +/- 10.6% (group 1), 43.2 +/- 7.0% (group 2), 34.3 +/- 4.8% (group 3), and 35.0 +/- 6.8% (group 4). The mean total percentages of PNA+ and SIg+ cells were 100 +/-6% (group 1), 113.5 +/- 4.9% (group 2), 106.3 +/- 5.3% (group 3), and 98.9 +/- 9.2% (group 4). The proportions of PNA+ and SIg+ cells in dogs of group 2 were significantly (P < 0.05) different from those in dogs of the other groups. Serial changes in PNA+ and SIg+ cells were investigated in blood of 6- to 29-week-old pups (n = 8). A significant (P < 0.05) transient decrease in PNA+ cells and a corresponding increase in SIg+ cells was observed in pups between 14 and 17 weeks old. Lymphoid tissue specimens and blood samples were obtained from 2- to 6-month-old dogs (n = 11) and from 6- to 12-month-old dogs (n = 10). Percentages reflected the combined data from both groups because there were no significant differences between the 2 age groups. The mean percentages of PNA+ cells were: blood, 68.4 +/- 8.6%; thymus, 86.6 +/- 16.3%; spleen, 29.5 +/- 16.0%; lymph node, 48.5 +/- 16.0%; and bone marrow, 30.8 +/- 26.4%. The mean percentage of SIg+ cells were: blood, 32.1 +/- 10.6%; thymus, 3.1 +/- 5.5%; spleen, 69.3 +/- 10.3%; lymph node, 55.4 +/- 15.2%; and bone marrow, 65.4 +/- 22.4%. The procedureto identify T lymphocytes in blood and lymphoid tissue was easy to perform, was reproducible, and could be performed on as few as 10(6) cells. Percentages of T lymphocytes identified compared favorably with those of other studies.