Fewer PrPc myeloid-based cells in sheep with the prion-resistant genotype.

Numerous sheep with the prion gene (PRNP) that encodes for 171QQ develop scrapie, a neurodegenerative prion disease of sheep. Relatively few cases of scrapie-affected sheep with PRNP genetics that encode for 171RR, however, have been found. Using flow cytometric analysis, statistically fewer PrPc-positive microglia and monocytes were observed from sheep with 171RR PRNP genetics than from sheep with 171QQ PRNP genetics (P<0.05). One possibility for the lack of PrP(Sc) accumulation in brains and lymph nodes of scrapie-exposed sheep with 171RR PRNP genetics is because of fewer PrPc-positive myeloid-derived cells available for conversion of PrPc to PrP(Sc).

Identification and phylogenetic analysis of 15 MHC class II DRB1 beta1 expressed alleles in a ewe-lamb flock.

Ovar-DRB1 is part of the major histocompatibility complex (MHC) class II of sheep and functions by presenting extracellular-derived peptides to the immune system. Although there are over 100 different Ovar-DRB1 DNA sequences reported in GenBank, only two Ovar-DRB1 mRNA sequences have been reported. As a first step in understanding MHC Class II function as it relates to disease progression in sheep, Ovar-DRB1 transcripts encoding the peptide-binding site or the first domain (beta1) of Ovar-DRB1 in a 32-ewe-lamb flock were identified and characterized by using reverse transcriptase-polymerase chain reaction, cloning, sequencing, and phylogenetic analysis. Fourteen new Ovar-DRB1 beta1 cDNA sequences out of a total of 15 Ovar-DRB1 beta1 cDNA sequences in a ewe-lamb flock of 32 sheep were identified. One Ovar-DRB1 beta1 cDNA sequence was 100% identical to M93432, one of the two Ovar-DRB1 mRNA sequences reported in GenBank. Twelve out of 15 Ovar-DRB1 beta1 cDNA sequences were 100% identical to the corresponding previously reported Ovar-DRB1 genomic DNA sequences, indicating that these Ovar-DRB1 genomic DNA sequences are also transcribed. One of three of the remaining Ovar-DRB1 beta1 cDNA sequences, DRB1*07012, had a synonymous substitution resulting in an identical deduced amino acid sequence to DRB1*0701. Two of the remaining three Ovar-DRB1 beta1 cDNA sequences had nucleotide differences and subsequent deduced amino acid sequence differences when compared to known Ovar-DRB1 beta1 genomic DNA sequences, and therefore, DRB1*0206 and DRB1*0353 represent new Ovar-DRB1 beta1 expressed alleles. Phylogenetic analysis of the 15 Ovar-DRB1 beta1 cDNA sequences revealed that DRB1*0206 had a strong phylogenetic relationship to DRB1*0203, and DRB1*0353 had a strong phylogenetic relationship to DRB1*0303.

A real-time PCR assay for measuring alcelaphine herpesvirus-1 DNA.

Alcelaphine herpesvirus-1 (AlHV-1) is a rhadinovirus that causes malignant catarrhal fever in certain ruminant species and is an important pathogen in Africa and other areas where carrier species and clinically susceptible ruminants intermingle. In this study, a real-time PCR for AlHV-1 DNA was developed and compared to an established nested PCR. The nested PCR amplifies a region of the AlHV-1 gene coding for a transactivator protein (ORF 50), while the real-time PCR assay targets the AlHV-1 gene coding for a tegument protein (ORF 3). The real-time PCR assay reproducibly detected 10 copies of target DNA. In a dilution series of the target DNA there was linearity of the assay across 8 orders of magnitude (10(1)-10(9) copies). The nested PCR was more sensitive (approximately with 1 log) than the real-time PCR. The assay specifically amplified samples containing only AlHV-1, but not other common herpesviruses of cattle. In conclusion, we have developed a rapid, relatively sensitive, and reliable real-time PCR assay specific for AlHV-1. Similar to the real-time PCR for Ovine herpesvirus-2, this assay should prove useful for differential diagnostics of clinical MCF and for research to better define the epidemiology of AlHV-1 in wildebeest as well as in animals with wildebeest-associated MCF.

Surface envelope glycoprotein is B-lymphocyte immunodominant in sheep naturally infected with ovine progressive pneumonia virus.

The B-lymphocyte-immunodominant antigen involved in naturally ovine progressive pneumonia virus (OPPV)-infected mature sheep remains unknown. Therefore, the amount of antibody in sera from 10 naturally OPPV-infected sheep was evaluated by immunoprecipitation (IP) of the major viral proteins in [(35)S]methionine/cysteine-labeled OPPV (whole virus) lysate. Using an excess of OPPV proteins in whole-virus lysate, 8 out of 10 sheep had the highest serum antibody IP endpoint titers to the gp135 surface envelope glycoprotein (SU). Also, 2 out of 10 sheep had equivalent serum antibody IP endpoint titers to the transmembrane glycoprotein oligomer (TM90) and SU. Since these data indicate that SU is the immunodominant protein in most mature sheep persistently infected with OPPV, SU-specific diagnostic serological assays can be utilized for OPPV diagnosis.

Seven new ovine progressive pneumonia virus (OPPV) field isolates from Dubois Idaho sheep comprise part of OPPV clade II based on surface envelope glycoprotein (SU) sequences.

Seven new ovine progressive pneumonia virus (OPPV) field isolates were derived from colostrum and milk of 10 naturally OPPV-infected sheep from the US Sheep Experiment Station in Dubois, Idaho, USA. Sixteen sequences of the surface envelope glycoprotein (SU) from these seven Dubois OPPV field isolates and SU sequence from OPPV WLC1 were obtained, aligned with published SRLV SU sequences, and analyzed using phylogenetic analysis using parsimony (PAUP). Percent nucleotide identity in SU was greater than 95.8% among clones from individual Dubois OPPVs and ranged from 85.5 to 93.8% between different Dubois OPPV clones. SU sequences from Dubois OPPVs and WLC1 OPPV had significantly higher percent nucleotide identity to SU sequences from the North American OPPVs (85/34 and S93) than caprine-arthritis encephalitis virus (CAEVs) or MVVs. PAUP analysis also showed that SU sequences from the Dubois OPPVs and OPPV WLC1 grouped with other North American OPPVs (85/34 and S93) with a bootstrap value of 100 and formed one OPPV clade II group. In addition, Dubois and WLC1 SU amino acid sequences had significantly higher identity to SU sequences from North American OPPVs than CAEV or MVV. These data indicate that the seven new Dubois OPPV field isolates along with WLC1 OPPV are part of the OPPV clade II and are distinct from CAEVs and MVVs.

A solid-phase assay for identification of modulators of prion protein interactions.

The progression of the transmissible spongiform encephalopathies (TSEs) is characterized in part by accumulation of a proteinase K-resistant form of the prion protein, which has been converted from the endogenous, proteinase K-sensitive form. This conversion reaction provides a target for possible anti-TSE strategies. We have adapted a cell-free conversion reaction to a high-throughput, solid-phase format that can be used to screen possible therapeutic compounds for inhibitory activity or to illuminate inhibition and conversion mechanisms. The solid-phase assay was compatible with reactions performed under a variety of conditions. Using this assay, we report that phthalocyanine tetrasulfonate, a known modulator of conversion, inhibited conversion by interfering with binding between the protease-sensitive and the protease-resistant forms of the prion protein. A biotinylated form of the protease-sensitive prion protein was successfully converted to the protease-resistant isoform in the solid-phase assay, indicating that biotinylation provides a nonisotopic labeling strategy for large-scale screens.

Detection of serum antibodies to ovine progressive pneumonia virus in sheep by using a caprine arthritis-encephalitis virus competitive-inhibition enzyme-linked immunosorbent assay.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.

Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: diagnostic tool for successful eradication.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was evaluated for the detection of serum antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) in goats. This assay utilized 96-well microtiter plates containing CAEV-63 SU captured by monoclonal antibody (MAb) F7-299 and measured the competitive displacement of horseradish peroxidase-conjugated MAb GPB 74A binding by undiluted goat sera (F. Ozyörük, W. P. Cheevers, G. A. Hullinger, T. C. McGuire, M. Hutton, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 8:44-51, 2001). Two hundred serum samples from goats in the United States were used to determine the sensitivity and specificity of cELISA based on the immunoprecipitation (IP) of [(35)S]methionine-labeled viral antigens as a standard of comparison. A positive cELISA was defined as >33.2% inhibition of MAb 74A binding based on 2 standard deviations above the mean percent inhibition of 140 IP-negative serum samples. At this cutoff value, there were 0 of 60 false-negative sera (100% sensitivity) and 5 of 140 false-positive sera (96.4% specificity). Additional studies utilized IP-monitored cELISA to establish a CAEV-free herd of 1,640 dairy goats.

CD21-positive follicular dendritic cells: A possible source of PrPSc in lymph node macrophages of scrapie-infected sheep.

Natural sheep scrapie is a prion disease characterized by the accumulation of PrP(Sc) in brain and lymphoid tissues. Previous studies suggested that lymph node macrophages and follicular dendritic cells (FDC) accumulate PrP(Sc). In this study, lymph nodes were analyzed for the presence of PrP(Sc) and macrophage or FDC markers using dual immunohistochemistry. A monoclonal antibody (mAb) to the C-terminus of PrP reacted with CD172a+ macrophages and CD21+ FDC processes in secondary follicles. However, a PrP N-terminus-specific mAb reacted with CD21+ FDC processes but not CD172a+ macrophages in secondary follicles. Neither the PrP N-terminus nor C-terminus-specific mAb reacted with CD172a+ macrophages in the medulla. These results indicate that lymph node follicular macrophages acquire PrP(Sc) by phagocytosis of CD21+ FDC processes. The results also suggest that follicular macrophages have proteases that process full-length PrP(Sc) to N-terminally truncated PrP(Sc).

PrP(Sc) is not detected in peripheral blood leukocytes of scrapie-infected sheep: determining the limit of sensitivity by immunohistochemistry.

Peripheral blood leukocytes (PBLs) from scrapie-infected sheep were evaluated for the presence of PrP(Sc) by using dissociated retropharyngeal lymph node (DRLN) cells and immunohistochemistry (IHC). PrP(Sc)-positive cells were detected in 2.05% +/- 0.28% of 3 x 10(6) DRLN cells, but were not detected in 3 x 10(6) PBLs from scrapie-infected sheep. Titration of DRLN cells mixed with PBLs showed that IHC detects a minimum of 0.00205% or 60 PrP(Sc)-positive cells in 3 x 10(6) PBLs.