Differential Bioaccumulation of Mercury and Selenium in Stomach Contents and Tissues of Three Colorado, USA, Cutthroat Trout Populations.

Total mercury (THg) and selenium (TSe) levels were measured in stomach contents (SC) and twelve tissues of cutthroat trout (Oncorhynchus clarkii) occurring in three high-elevation lakes of Colorado, USA, inhabiting watersheds absent past and current mining activities. For 32 of 36 tissues, including muscle, mean THg wet weight (ww) concentrations were greater than in the diet (SC) for all sites, indicating biomagnification. Ranges of THg (µg/kg ww) for SC and stomach tissue (ST) were 1.23-73.54 and 14.55-61.35, respectively. Selenium concentrations in fish muscle were not greater than in the SC indicating a trophic transfer factor < 1.0. However, in several other tissues, mean Se dry weight (dw) levels were greater than in SC for all three lakes. Ranges of TSe for SC and ST were 166-7544 and 797-7523 (µg/kg dw), respectively. The muscle to egg/ovary ratio for Se averaged 2.30, 4.60, and 2.68 for the three populations. The variability of SC (planktonic vs. benthic) and differential distributions of THg and TSe in SC and organ-tissues generated questions focusing on the seasonal, physiological, and genetic drivers of these organometal(loid)s in subalpine trout.

Ovar-DRB1 haplotypes *2001 and *0301 are associated with sheep growth and ewe lifetime prolificacy.

BACKGROUND: The major histocompatibility complex (MHC) is an organized cluster of tightly linked vertebrate genes with immunological and non-immunological functions. While the important MHC gene DRB1 has been examined in regard to many sheep infectious disease traits, only one study, based on microsatellite markers, has previously examined DRB1 and sheep production traits. Furthermore, to our knowledge no studies have examined DRB1 relationship with lifetime ewe prolificacy traits. Therefore, we analyzed association between the presence of DRB1 SNP haplotypes with internationally recognized standard names and production traits including growth and lifetime prolificacy in 370 Rambouillet, Columbia, and Polypay sheep. RESULTS: The DRB1 *2001 haplotype was associated with increased weaning and mature weights, as well as average daily gain (Sidák P<0.05; corrected for the number of haplotypes tested). Interestingly, the *2001 haplotype also showed a trend toward association with increased total number of lifetime lambs born (Sidák P=0.084) and number of lambs born alive (Sidák P=0.084). In contrast, the DRB1 *0301 haplotype was associated with decreased mature weight (Sidák P=0.01). CONCLUSIONS: Since the *2001 haplotype was present in all three breeds, these results suggest there is at least one functional mutation in the region that influences growth and prolificacy traits that may be broadly present across several breeds. Furthermore, combined use of the similar *2001 and *0301 multi-marker haplotypes that nonetheless have opposing directions of production trait associations will enhance mutation discovery in this region. If undesirable alleles for underlying mutations can be identified, selective pressure against one or a small number of undesirable alleles may improve production with limited impact on MHC genetic diversity and infectious disease susceptibility.

A divergent Artiodactyl MYADM-like repeat is associated with erythrocyte traits and weight of lamb weaned in domestic sheep.

A genome-wide association study (GWAS) was performed to investigate seven red blood cell (RBC) phenotypes in over 500 domestic sheep (Ovis aries) from three breeds (Columbia, Polypay, and Rambouillet). A single nucleotide polymorphism (SNP) showed genome-wide significant association with increased mean corpuscular hemoglobin concentration (MCHC, P = 6.2×10(-14)) and genome-wide suggestive association with decreased mean corpuscular volume (MCV, P = 2.5×10(-6)). The ovine HapMap project found the same genomic region and the same peak SNP has been under extreme historical selective pressure, demonstrating the importance of this region for survival, reproduction, and/or artificially selected traits. We observed a large (>50 kb) variant haplotype sequence containing a full-length divergent artiodactyl MYADM-like repeat in strong linkage disequilibrium with the associated SNP. MYADM gene family members play roles in membrane organization and formation in myeloid cells. However, to our knowledge, no member of the MYADM gene family has been identified in development of morphologically variant RBCs. The specific RBC differences may be indicative of alterations in morphology. Additionally, erythrocytes with altered morphological structure often exhibit increased structural fragility, leading to increased RBC turnover and energy expenditure. The divergent artiodactyl MYADM-like repeat was also associated with increased ewe lifetime kilograms of lamb weaned (P = 2×10(-4)). This suggests selection for normal RBCs might increase lamb weights, although further validation is required before implementation in marker-assisted selection. These results provide clues to explain the strong selection on the artiodactyl MYADM-like repeat locus in sheep, and suggest MYADM family members may be important for RBC morphology in other mammals.

Role of Bibersteinia trehalosi, respiratory syncytial virus, and parainfluenza-3 virus in bighorn sheep pneumonia.

Pneumonic bighorn sheep (BHS) have been found to be culture- and/or sero-positive for Bibersteinia trehalosi, respiratory syncytial virus (RSV), and parainfluenza-3 virus (PI-3). The objective of this study was to determine whether these pathogens can cause fatal pneumonia in BHS. In the first study, two groups of four BHS each were intra-tracheally administered with leukotoxin-positive (Group I) or leukotoxin-negative (Group II) B. trehalosi. All four animals in Group I developed severe pneumonia, and two of them died within 3 days. The other two animals showed severe pneumonic lesions on euthanasia and necropsy. Animals in Group II neither died nor showed gross pneumonic lesions on necropsy, suggesting that leukotoxin-positive, but not leukotoxin-negative, B. trehalosi can cause fatal pneumonia in BHS. In the second study, two other groups of four BHS (Groups III and IV) were intra-nasally administered with a mixture of RSV and PI-3. Four days later, RSV/PI-3-inoculated Group IV and another group of four BHS (Group V, positive control) were intra-nasally administered with Mannheimia haemolytica, the pathogen that consistently causes fatal pneumonia in BHS. All four animals in group III developed pneumonia, but did not die during the study period. However all four animals in Group IV, and three animals in Group V developed severe pneumonia and died within two days of M. haemolytica inoculation. The fourth animal in Group V showed severe pneumonic lesions on euthanasia and necropsy. These findings suggest that RSV/PI-3 can cause non-fatal pneumonia, but are not necessary predisposing agents for M. haemolytica-caused pneumonia of BHS.

Genome-wide association identifies multiple genomic regions associated with susceptibility to and control of ovine lentivirus.

BACKGROUND: Like human immunodeficiency virus (HIV), ovine lentivirus (OvLV) is macrophage-tropic and causes lifelong infection. OvLV infects one quarter of U.S. sheep and induces pneumonia and body condition wasting. There is no vaccine to prevent OvLV infection and no cost-effective treatment for infected animals. However, breed differences in prevalence and proviral concentration have indicated a genetic basis for susceptibility to OvLV. A recent study identified TMEM154 variants in OvLV susceptibility. The objective here was to identify additional loci associated with odds and/or control of OvLV infection. METHODOLOGY/PRINCIPAL FINDINGS: This genome-wide association study (GWAS) included 964 sheep from Rambouillet, Polypay, and Columbia breeds with serological status and proviral concentration phenotypes. Analytic models accounted for breed and age, as well as genotype. This approach identified TMEM154 (nominal P=9.2×10(-7); empirical P=0.13), provided 12 additional genomic regions associated with odds of infection, and provided 13 regions associated with control of infection (all nominal P<1 × 10(-5)). Rapid decline of linkage disequilibrium with distance suggested many regions included few genes each. Genes in regions associated with odds of infection included DPPA2/DPPA4 (empirical P=0.006), and SYTL3 (P=0.051). Genes in regions associated with control of infection included a zinc finger cluster (ZNF192, ZSCAN16, ZNF389, and ZNF165; P=0.001), C19orf42/TMEM38A (P=0.047), and DLGAP1 (P=0.092). CONCLUSIONS/SIGNIFICANCE: These associations provide targets for mutation discovery in sheep susceptibility to OvLV. Aside from TMEM154, these genes have not been associated previously with lentiviral infection in any species, to our knowledge. Further, data from other species suggest functional hypotheses for future testing of these genes in OvLV and other lentiviral infections. Specifically, SYTL3 binds and may regulate RAB27A, which is required for enveloped virus assembly of human cytomegalovirus. Zinc finger transcription factors have been associated with positive selection for repression of retroviral replication. DLGAP1 binds and may regulate DLG1, a known regulator of HIV infectivity.

MHC class II DR allelic diversity in bighorn sheep.

We hypothesized that decreased diversity and/or unique polymorphisms in MHC class II alleles of bighorn sheep (BHS, Ovis canadensis) are responsible for lower titer of antibodies against Mannheimia haemolytica leukotoxin, in comparison to domestic sheep (DS, Ovis aries). To test this hypothesis, DRA and DRB transcripts from 24 captive BHS (Ovca-DRA and Ovca-DRB) were sequenced. Based on exon 2 (ß1 domain) sequences, eight different Ovca-DRB cDNA sequences were identified in BHS. Six of them were 100% identical to previously reported Ovca-DRB genomic DNA sequences. The new alleles DRB*23 and DRB*24, were closely related to two other Ovca-DRB exon 2 genomic DNA sequences. Nineteen out of 24 BHS (79%) Ovca-DRB exon 3 (ß2 domain) sequences were 100% identical to exon 3 sequence of DRB1 of DS (Ovar-DRB1). Ovca-DRA full length cDNA sequences exhibited >99% identity. Based upon exon 2 sequences, this BHS herd yielded higher Ovca-DRB allelic diversity than that reported in the previous study. Positively selected amino acid positions were identified in the peptide-binding groove of BHS and DS, but BHS showed more such sites. This highlights differing population histories, and may suggest differing needs for DR peptide-binding specificities. Presence of glutamine at position 52 (52Q) in some of the desert and captive BHS is predicted to alter the efficiency of DR dimerization, which may influence antigen presentation and T(h) cell activation. Functional assays with unique alleles should reveal whether the presentation of M. haemolytica leukotoxin peptides to T(h) cells by Ovca-DRB alleles is equivalent to that of Ovar-DRB1 alleles.

Reduced lentivirus susceptibility in sheep with TMEM154 mutations.

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100). In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43). Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.

Cell-surface expression of PrPC and the presence of scrapie prions in the blood of goats.

Although host-encoded prion protein (PrP(C)) expression in ovine PBMCs and prion infectivity in scrapie-infected sheep blood have been demonstrated, such studies have not been reported in goats. Therefore, this study characterized cell-surface expression of PrP(C) on PBMC subsets derived from normal goats and sheep, by flow cytometry, and determined prion infectivity in blood from a scrapie-infected goat using a transfusion bioassay in goat kids. Cell-surface PrP(C) expression was detected on all subsets of goat PBMCs. The highest PrP(C) cell-surface expression was found in CD2(+) T lymphocytes in goats. Transmission of infection was detected in all three recipients who received whole blood from a goat with classical scrapie. It was concluded that caprine PBMCs express PrP(C) similarly to sheep but with relative differences among PBMCs subsets, and that blood-borne infectious prions can be detected in scrapie-infected goats. Thus, similar to sheep, goat blood may be a suitable diagnostic target for the detection of scrapie infection.

Diagnostic assays used to control small ruminant lentiviruses.

The serologic diagnostic tests, such as the agar gel immunodiffusion assay and various types of enzyme-linked immunosorbent assays (ELISAs), have contributed to the reduction of small ruminant lentivirus (SRLV) infections worldwide. Because there are no treatments or efficacious vaccines, the serologic diagnostic tests have supported most of the eradication efforts by testing and removal or separation of adult animals that generate antibodies to SRLVs. With the advent of molecular diagnostics, standard and quantitative polymerase chain reaction (PCR)-based assays for the detection of provirus in peripheral blood cells are becoming more common and aid in the detection of infected goats and sheep before antibody detection by ELISA in some animals. Performance of the serologic and molecular diagnostic tests is dependent upon a number of factors, including the format of the assay, the percentage of identity between the viral nucleotide sequences in a flock or herd of a certain geographic region and the sequences used to generate SRLV test reagents, and the intrinsic pathogenesis or amount of provirus and SRLV antibody generated in a species or individual small ruminant. In addition, small ruminant genomics may help with establishing genetic markers of SRLV infection and disease, which could also aid eradication or reduction of SRLVs from herds and flocks throughout the world.

Non-maternal transmission is the major mode of ovine lentivirus transmission in a ewe flock: a molecular epidemiology study.

Transmission of ovine progressive pneumonia virus (OPPV), a lentivirus of sheep, occurs through both maternal and non-maternal means. Currently, the contribution of each route to the overall flock OPPV prevalence is poorly understood since previous serological epidemiologic studies lacked the ability to accurately track routes of transmission within an infected flock. In this study, the amount of maternal OPP transmission was assessed in a naturally infected ewe flock by applying molecular analyses to proviral sequences derived from peripheral blood leukocytes of OPP positive dam-daughter pairs (N=40). Both proviral envelope (env) and long terminal repeat (LTR) sequences, separately and combined, were utilized in the following 2 sequence analysis methods: phylogenetic analysis and pairwise distance calculations. True maternal transmission events were defined as agreement in 2 out of the 2 sequence analysis methods. Using this criterion, proviral env sequences resulted in a 14.3% maternal transmission frequency, and proviral LTR sequences resulted in a 10% maternal transmission frequency. Both proportions of maternal transmission varied significantly from equality (P<0.0001). This indicates that the remaining 85.7-90% of daughters are infected via non-maternal transmission. This is also the first study to calculate the OPP proviral rate of change for the env gene and LTR promoter. Accurately defining the routes of OPPV transmission provides critical epidemiological data supporting management intended to reduce flock transmission and viral dose.

Evaluation of a caprine arthritis-encephalitis virus/maedi-visna virus indirect enzyme-linked immunosorbent assay in the serological diagnosis of ovine progressive pneumonia virus in U.S. sheep.

A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect enzyme-linked immunosorbent assay (iELISA) was validated with samples from U.S. sheep and by the use of radioimmunoprecipitation as the standard for comparison. The sensitivity and the specificity were 86.0% (+ or - 5.8%) and 95.9% (+ or - 2.9%), respectively. The iELISA format and phylogenetic differences based on the MVV gag sequence contribute to the reduced sensitivity.

Peripheral ovine progressive pneumonia provirus levels correlate with and predict histological tissue lesion severity in naturally infected sheep.

Studies were undertaken to determine whether anti-ovine progressive pneumonia virus (OPPV) antibody responses in serum or OPP provirus levels in peripheral blood associate with the degree of histologically measured tissue lesions in naturally OPPV-infected sheep. Sections of formalin-fixed, paraffin-embedded, and hematoxylin- and eosin-stained lung, mammary gland, carpal synovial membrane, and brain tissues from 11 OPPV-infected ewes (mean age of 8.6 years) and 5 OPPV-uninfected ewes (mean age of 6 years) were evaluated for lesion severity. Ovine progressive pneumonia (OPP) provirus levels and anti-OPPV antibody titers in peripheral blood and serum samples, respectively, were measured upon euthanasia and 3 years prior to euthanasia. Both mean peripheral OPP provirus levels and mean serum anti-surface envelope glycoprotein (anti-SU) antibody titers at the time of euthanasia were significantly higher in ewes with moderate to severe histological lesions than in ewes with no to mild histological lesions. However, although mean peripheral blood OPP provirus levels at euthanasia and 3 years prior to euthanasia significantly correlated with the highest histological lesion score for any affected tissue (two-tailed P values, 0.03 and 0.02), mean serum anti-SU antibody titers, anti-capsid antibody titers, and anti-transmembrane 90 antibody titers at euthanasia did not show a significant correlation with the highest histological lesion score for any tissue (two-tailed P values, 0.32, 0.97, and 0.18, respectively). These data are the first to show that OPP provirus levels predict and correlate with the extent of OPPV-related histological lesions in various OPPV-affected tissues. These findings suggest that peripheral OPP provirus levels quantitatively contribute more to the development of histological lesions than the systemic anti-SU antibody host immune response.

Ovine progressive pneumonia provirus levels are unaffected by the prion 171R allele in an Idaho sheep flock.

Selective breeding of sheep for arginine (R) at prion gene (PRNP) codon 171 confers resistance to classical scrapie. However, other effects of 171R selection are uncertain. Ovine progressive pneumonia/Maedi-Visna virus (OPPV) may infect up to 66% of a flock thus any affect of 171R selection on OPPV susceptibility or disease progression could have major impact on the sheep industry. Hypotheses that the PRNP 171R allele is 1) associated with the presence of OPPV provirus and 2) associated with higher provirus levels were tested in an Idaho ewe flock. OPPV provirus was found in 226 of 358 ewes by quantitative PCR. The frequency of ewes with detectable provirus did not differ significantly among the 171QQ, 171QR, and 171RR genotypes (p > 0.05). Also, OPPV provirus levels in infected ewes were not significantly different among codon 171 genotypes (p > 0.05). These results show that, in the flock examined, the presence of OPPV provirus and provirus levels are not related to the PRNP 171R allele. Therefore, a genetic approach to scrapie control is not expected to increase or decrease the number of OPPV infected sheep or the progression of disease. This study provides further support to the adoption of PRNP 171R selection as a scrapie control measure.

Minimum intravenous infectious dose of ovine progressive pneumonia virus (OPPV).

The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6month-old lambs. Twelve groups of two 6month-old lambs were inoculated intravenously (i.v.) with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titers ranging from 10(7.6) TCID(50)/lamb down to 10(-3.4) TCID(50)/lamb and were monitored for seroconversion using the OPPV agar gel immunodiffusion assay (AGID). Fifteen of the 16 lambs given equal or greater than 10(0.6) TCID(50) seroconverted, and virus could be isolated from peripheral blood leukocytes in 13 out of the 15 of these lambs. None of the eight lambs receiving less than 10(0.6) TCID(50) seroconverted during the 12months. The results of this study indicated that 10(0.6) or 4 TCID(50)/lamb given i.v. was capable of establishing infection.

Ovine progressive pneumonia provirus levels associate with breed and Ovar-DRB1.

Previous studies initiated defining the role of host genetics in influencing the outcome of exposure to ovine progressive pneumonia virus. However, specific genes influencing host control of virus replication and disease progression have not been identified. This study, using 383 ewes of the Columbia, Polypay, and Rambouillet breeds, tested the hypothesis that host control of OPPV as measured by provirus levels in the peripheral blood associates with certain breeds and MHC class II Ovis aries (Ovar)-DRB1 expressed alleles. Rambouillet ewes were less likely to have measurable provirus levels as compared to Columbia ewes at ages 5 and 6 (P value < 0.02), and they exhibited lower provirus levels when compared to both Columbia and Polypay ewes of the same ages (P value < 0.05). The presence of DRB1*0403- or DRB1*07012-expressed alleles were significantly associated (P value = 0.019 and 0.0002, respectively) with lower OPP provirus levels but only were only found in 11% of the ewe flock. Analysis of each segregating amino acid in the beta1 domain of DR beta-chain revealed that amino acids Y31, T32, N37, T51, Q60, or N74 significantly associated (P value range = 0.0003-0.018) with lower OPP provirus levels, whereas amino acids H32, A38, or I67 associated (P value range = 0.013-0.043) with higher OPP provirus levels. These results suggest that Ovar-DRB1 contributes as one host genetic factor that controls OPP provirus levels, but does not fully account for the breed-specific OPP proviral differences.

Small-ruminant lentivirus enhances PrPSc accumulation in cultured sheep microglial cells.

Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrP(C) [C superscript stands for cellular]) to disease-associated prion protein (PrP(Sc) [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrP(Sc), sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrP(Sc) accumulation over time. The accumulated PrP(Sc) demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrP(Sc) demonstrated an approximately twofold increase in PrP(Sc) accumulation compared to that of primary microglia infected with PrP(Sc) alone. The results demonstrate the in vitro utility of PrP(Sc)-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.

Genomic analysis of Ovis aries (Ovar) MHC class IIa loci.

Determining the genomic organization of the Ovis aries (Ovar) major histocompatibility complex class IIa region is essential for future functional studies related to antigen presentation. In this study, a bacterial artificial chromosome (BAC) library of genomic DNA from peripheral blood leukocytes (PBL) of a Rambouillet ram was constructed, and BAC clone consisting of the major histocompatibility complex (MHC) class II DQB2, DQA2, DQB1, DQA1, and DRB1 loci was identified and completely sequenced. The BAC clone consists of 160,889 bp of finished sequence with the loci arranged in the following order: DQB2, DQA2, DQB1, DQA1, and DRB1 with 14.3, 25, 6.6, and 40.9 Kb spanning between the loci, respectively. All five of these loci were transcribed in the animal used to generate the MHC class II BAC clone. Repeat or retrotransposable elements along with MHC class II cis promoter elements consisting of S, X, and Y boxes were identified in the sequence. In addition, 16 non-coding conserved sequences amongst primates, carnivores, and ruminants were identified (p < 0.001). These conserved sequences include binding sites for transcription factors with known roles in immune cells, and they provide a basis for further functional investigation of the genes in this region. This is the first ruminant finished sequence of the DQB2-DRB1 region, and this sequence information will aid in whole genome and transcriptome analyses of MHC class II.

Development and validation of an ovine progressive pneumonia virus quantitative PCR.

Ovine progressive pneumonia virus (OPPV) infects at least one sheep in 81% of U.S. sheep flocks, as determined by serology, and can cause viral mastitis, arthritis, dyspnea, and cachexia. Diagnostic tests that quantify OPPV proviral load in peripheral blood leukocytes (PBL) provide an additional method for identification of infected sheep and may help to further understanding of the pathogenesis of OPPV-induced disease. In this study, we compared a new OPPV real-time quantitative PCR (qPCR) assay specific for the transmembrane region of the envelope gene (tm) with a competitive inhibition enzyme-linked immunosorbent assay (cELISA) using 396 PBL samples and sera from Idaho sheep. The OPPV qPCR had a positive concordance of 96.2% +/- 2.3% and a negative concordance of 97.7% +/- 2.5% compared to the cELISA, with a kappa value of 0.93, indicating excellent agreement between the two tests. In addition, the presence of tm in the three OPPV qPCR-positive and cELISA-negative sheep and in 15 sheep with different OPPV proviral loads was confirmed by cloning and sequencing. These data indicate that the OPPV qPCR may be used as a supplemental diagnostic tool for OPPV infection and for measurement of viral load in PBLs of infected sheep.

Evidence of proviral clearance following postpartum transmission of an ovine lentivirus.

Lentiviral transmission by transfer of infected colostrum and/or milk is considered to be highly efficient. In this study, postpartum transmission of ovine progressive pneumonia virus (OPPV) from 10 naturally infected ewes to their 23 lambs was followed from the perinatal period throughout a four-year period. The lambs were allowed to suckle from their dam from birth through 32 weeks of age. Virus was tracked by virus isolation, quantitative PCR (qPCR), and anti-OPPV antibody responses as measured by cELISA. Cell-associated OPPV was isolated from colostrum/milk cells in 7 out of 10 ewes and provirus envelope (env) loads ranged 8 to 10(5) copies/mug DNA in colostrum/milk cells from the 10 ewes using qPCR. Provirus env loads were also detected in the peripheral circulation of 21 lambs at 8 weeks and two lambs at 22 weeks. The qPCR product at 8 weeks was confirmed as the transmembrane (tm) gene of OPPV by cloning and sequencing. Both cELISA titers ranging from 325 to 3125 and cross-neutralizing antibody titers ranging from 6 to 162 to seven different OPPV strains were found in the colostrum of the 10 ewes. Furthermore, cELISA titers in serum from lambs remained detectable through 32 weeks following the clearance of provirus at 24 weeks. After 32 weeks, both provirus and anti-OPPV antibody responses have subsequently remained undetectable through 4 years of age. These data suggest the clearance of cell-associated lentiviruses from lamb circulation after passive transfer of antibody via colostrum.

Ovine progressive pneumonia virus capsid is B-cell immunodominant using Western blot analysis: a comparison of sensitivity between Western blot analysis and immunoprecipitation.

A Western blot assay was developed and analyzed against the comparable standard, immunoprecipitation of (35)[S]-methionine/cysteine-labeled ovine progressive pneumonia virus (OPPV) proteins, for its ability to detect anti-OPPV antibodies using endpoint titers. Western blot analysis is 12-fold more sensitive in detecting endpoint anti-capsid antibody titers than IP, and the capsid is the B-cell immunodominant OPPV protein when utilizing Western blot analysis. Since the surface envelope glycoprotein is the B-cell immunodominant OPPV protein when utilizing immunoprecipitation, this suggests immunoprecipitation and Western blot analysis measure different types of antibody that are more specific for conformational and linear OPPV protein epitopes, respectively.