Deletion variant near ZNF389 is associated with control of ovine lentivirus in multiple sheep flocks.

Ovine lentivirus (OvLV) is a macrophage-tropic lentivirus found in many countries that causes interstitial pneumonia, mastitis, arthritis and cachexia in sheep. There is no preventive vaccine and no cure, but breed differences suggest marker-assisted selective breeding might improve odds of infection and control of OvLV post-infection. Although variants in TMEM154 have consistent association with odds of infection, no variant in any gene has been associated with host control of OvLV post-infection in multiple animal sets. Proviral concentration is a live-animal diagnostic measure of OvLV control post-infection related to severity of OvLV-induced lesions. A recent genome-wide association study identified a region including four zinc finger genes associated with proviral concentration in one Rambouillet flock. To refine this region, we tested additional variants and identified a small insertion/deletion variant near ZNF389 that showed consistent association with proviral concentration in three animal sets (P < 0.05). These animal sets contained Rambouillet, Polypay and crossbred sheep from multiple locations and management conditions. Strikingly, one flock had exceptionally high prevalence (>87%, including yearlings) and mean proviral concentration (>950 copies/µg), possibly due to needle sharing. The best estimate of proviral concentration by genotype, obtained from all 1310 OvLV-positive animals tested, showed insertion homozygotes had less than half the proviral concentration of other genotypes (P < 0.0001). Future work will test additional breeds, management conditions and viral subtypes, and identify functional properties of the haplotype this deletion variant tracks. To our knowledge, this is the first genetic variant consistently associated with host control of OvLV post-infection in multiple sheep flocks.

Comparison of two PCR and one ELISA techniques for the detection of small ruminant lentiviruses (SRLVs) in milk of sheep and goats.

The aim of the present study was to compare the efficiency of two PCR techniques for the diagnosis of small ruminant lentiviruses (SRLVs). Detection of the proviral genome by PCR, though sensitive, is difficult due to the heterogeneity of the SRLV genomes. One of the PCR techniques amplifies a fragment in the pol gene (pol-PCR) and the other PCR targets the LTR region of the proviral genome (LTR-PCR). Milk from 194 sheep and 163 goats from farms in the Central Spain was analyzed by both techniques and compared to results obtained by ELISA. When compared to the serologic assay, the agreement of both PCR techniques was very low (0.024 and 0.020 in sheep, and 0.124 and 0.114 in goats). In view of these results, it may be concluded that the efficacy of PCR for the diagnosis of SRLVs is low and a combination of PCR and ELISA should be used for diagnosis.

Ovine progressive pneumonia virus capsid antigen as found in CD163- and CD172a-positive alveolar macrophages of persistently infected sheep.

In situ detection of ovine progressive pneumonia virus (OPPV) and the phenotypic identification of the cells that harbor OPPV have not been described for the OPPV-affected tissues, which include lung, mammary gland, synovial membranes of the carpal joint, and choroid plexus of the brain. In this study, the authors first developed a single enzyme-based automated immunohistochemical (IHC) analysis for detection of OPPV capsid antigen (CA) on OPPV-affected tissues, using 2 anti-CAEV CA monoclonal antibodies, 5A1 and 10A1, and 2 enzyme-based IHC systems. Out of 10 naturally and persistently OPPV-infected ewes, OPPV CA was detected in intercellular regions of the carpal synovial membrane of 1 ewe, in cells resembling alveolar macrophages and pulmonary interstitial macrophages in lung tissue of 3 ewes, and in mammary alveolar cells of 1 ewe. Furthermore, dual enzyme-based automated IHC analyses revealed that OPPV CA was predominantly detected in CD172a- or CD163-positive alveolar macrophages of the lungs and mammary gland. That anti-inflammatory (CD163) and downregulatory (CD172a) types of alveolar macrophage harbor OPPV CA leads to the possibility that during persistent infection with OPPV, the host alveolar macrophage might serve to limit inflammation while OPPV persists undetected by the host adaptive immune response in the lung and mammary gland.

Characterization of an immunodominant epitope of small ruminant lentivirus (SRLV) nucleoprotein.

To extend and complete the epitope mapping of gag-encoded structural proteins, the immunodiagnostic potential of nucleoprotein (NP) of two different small ruminant lentivirus (SRLV) genotypes were antigenically characterized. Respective recombinant counterparts were generated and used in an enzyme-linked immunosorbent assay (ELISA) format to test a panel of sera from infected flocks. Results clearly indicate that a single linear epitope located within the C-terminal is partially cross-reactive among different SRLV genotypes and may complement multiple epitope ELISA for serological diagnosis of infection. However, in contrast to matrix and capsid antigen epitopes, which drive a genotype-specific immunoresponse, a moderate degree of variation was identified in NP independently of the genotype to which it belongs.

Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential.

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

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.

Dynamics of cell-associated viremia and antibody response during the early phase of lentivirus infection in sheep.

OBJECTIVE: To determine patterns of cell-associated viremia and antibody responses during the early phase of ovine lentivirus (OvLV) infection in sheep. ANIMALS: 18 neonatal lambs. PROCEDURES: 12 lambs were inoculated intratracheally with OvLV within 24 hours after birth; 6 lambs were inoculated with noninfected cell culture supernatant. Degree of cell-associated viremia was measured every other week for 16 weeks by use of a limited dilution assay. Antibody responses to OvLV transmembrane (TM) and p25 proteins were determined weekly by use of recombinant ELISA. Neutralizing antibody responses were measured before and 8 and 16 weeks after inoculation. RESULTS: Degree of cell-associated viremia peaked between 2 and 6 weeks after inoculation and then decreased. For inoculated lambs, mean anti-p25 titer peaked 5 weeks after inoculation then slowly declined, whereas mean anti-TM and neutralizing antibody titers increased steadily. Over time, mean degree of cell-associated viremia was negatively correlated with mean anti-TM titer. Maximum individual degree of cell-associated viremia was positively correlated with maximum individual anti-TM titer. CONCLUSIONS: Results suggest that after experimental inoculation, OvLV replicates actively for several weeks and that an increase in anti-TM titer coincides with a decrease in degree of cell-associated viremia. Although the role antibodies play in protecting against lentivirus infection remains uncertain, understanding the dynamics of the antibody response may have important implications for diagnosis of OvLV infection, and antibodies may prove to be valuable markers for prediction of infection and disease.

Risk factors for seroprevalence of ovine lentivirus in breeding ewe flocks in Nebraska, USA.

The prevalence of and risk factors for ovine lentivirus (OLV) infection in 1466 breeding ewes in nine US Meat Animal Research Center (MARC) flocks were determined using a recombinant transmembrane protein (PTM) enzyme-linked immunosorbent assay (ELISA) to detect serum anti-OLV antibodies and define infection. Based on multivariable logistic regression, confinement birth and rearing (odds ratio (OR) = 1.6), older weaning ages (OR = 1.1 week-1), and older age (OR = 1.3-2.5 year-1 beyond age 1 year) were significantly associated with higher OLV prevalence in ewes. Prevalence also varied significantly by flock, with Finnsheep and Texel ewes having the highest prevalences and Booroola Merino and Suffolk ewes having the lowest prevalences. These findings support the hypothesis that management control efforts should concentrate on events early in the life of sheep, as this period is associated with factors which can modulate the risk for OLV infection.

Effect of ewe ovine lentivirus infection on ewe and lamb productivity.

We used a previously described sensitive and specific ovine lentivirus (OLV) recombinant transmembrane (rTM) protein enzyme-linked immunosorbent assay (ELISA) to detect anti-OLV antibodies and define OLV infection in breeding ewes from nine US Meat Animal Research Center (MARC) flocks. We estimated the production impacts of dam rTM ELISA seropositivity on ewe and lamb productivity in the birth-to-weaning interval using production data from 1466 breeding ewes (of which 1242 actually lambed) and their 2452 lambs born in spring 1992 using several multiple linear and logistic regression models. By adjusting for lamb weaning age, gender, type of birth and rearing, birth difficulty, dam age, and flock, the component of ewe or lamb productivity related to ewe OLV infection alone was isolated. The rTM ELISA-negative ewes produced significantly more total weight of weaned lamb per ewe-lambing (3.84 kg) and per ewe ram-exposed (4.95 kg) compared to their OLV-positive flockmates. Negative ewes also weaned 0.11 more lambs per ewe-lambing and 0.09 more lambs per ewe ram-exposed, gave birth to 0.13 more lambs per ewe ram-exposed, and were more likely to lamb after breeding (odds ratio (OR) = 1.9) compared to equivalent OLV-positive ewes. Lambs reared by OLV-negative ewes weighed 0.15 kg more at birth, gained 8 g more per day through weaning, and weighed 0.59 kg more at 56-day weaning. Preweaning mortality was lower (OR = 0.8) among lambs born to OLV-negative compared to OLV-positive ewes, although this difference was not significant. Our results suggest that subclinical OLV infection has important detrimental effects on sheep production which occur in cumulative fashion from breeding through weaning and that OLV control efforts may be financially justified in some sheep flocks.

Nitric oxide production following in vitro stimulation of ovine pulmonary alveolar macrophages.

Production of inducible nitric oxide (NO) as measured by nitrite in supernatant from ovine pulmonary alveolar macrophage (PAM) cultures was assessed following stimulation of PAM with live cells and supernatants from Corynebacterium pseudotuberculosis and Pasteurella haemolytica cultures; purified bacterial lipopolysaccharide derived from both Escherichia coli and Pasteurella haemolytica alone and in combination with interferon-gamma or lymphocyte conditioned medium; or ovine lentivirus. PAM cultured ex vivo with no further stimulation for 24 h, 48 h or 72 h, produced low concentrations of NO that was not substantially increased following co-culture by the various additives. Assessment of NO production in PAM cultures containing P. haemolytica or supernatant from P. haemolytica cultures was complicated by production of high levels of nitrite in the bacterial cultures. Species differences in inducible NO production may affect the efficacy of clearance of bacterial infections and be responsible for inter-host differences in disease expression following infection by intracellular pathogens.

Recognition of ovine lentivirus gag gene products by serum from infected sheep.

In order to localize the immunodominant regions, 12 ovine lentivirus (OLV) gag-coding gene fragments were cloned and expressed in Escherichia coli and then tested in a Western blot (WB) assay against a panel of sera collected from US and Italian OLV-infected sheep. The most immunoreactive regions were mapped to the amino-terminal of p25 and carboxyl-terminal of p14. In addition, we found that the reactivity pattern between US and Italian sheep was very similar, suggesting the antigenic domain between US and Italian isolates in the gag gene structures could be conserved. Given the broad immunoreactivity of the amino-terminal of p25, this region could serve as an ideal diagnostic antigen for the serological identification of OLV-infected sheep.

Comparison of ovine lentivirus detection by conventional and recombinant serological methods.

Recombinant (r) transmembrane protein (TM), major capsid protein P25, and matrix protein P16 of ovine lentivirus (OLV) were used as solid phase antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies against OLV in sheep sera. Sensitivity, specificity, and agreement of these three recombinant assays were compared with each other and with two currently available conventional OLV serological assays, the agar gel immunodiffusion (AGID) test and a whole-virus (WV) ELISA. Field sera from a total of 412 Midwestern United States sheep were tested and compared by the five OLV detection methods, including visibly healthy sheep selected for public sale (Group A, n = 171), samples from a breeding flock of Finnsheep and Finn-cross ewes (Group B, n = 184) and moribund sheep with clinical signs associated with OLV (Group C, n = 57). The rTM ELISA was the most sensitive OLV detection assay, both overall and within each group. Sera from 48.1% (198/412) of field samples were rTM ELISA positive. By contrast, positive rates for the rP25, rP16, and WV ELISAs and AGID test were 34.2%, 32.3%, 36.9%, and 26.9%, respectively. The rTM ELISA reactivity was 36.8% for Group A sera, 50.0% for Group B sera, and 75.4% for Group C sera. Among the 21 Group C sheep possessing OLV lung lesions at necropsy, 20 (95.2%) were rTM ELISA positive. The greatest test agreement occurred between the rP25 and the rP16 ELISAs. The data suggest that the recombinant TM immunoassay is the most accurate and sensitive of the five methods evaluated for the detection of serum anti-OLV antibodies in sheep, both at the subclinical infection and overt clinical disease stages.

Oligopeptide-based enzyme immunoassay for ovine lentivirus antibody detection.

Ovine progressive pneumonia virus (OPPV) is a lentivirus which causes a progressive disease in sheep. Immunodominant epitopes have been identified in the envelope gp40 glycoprotein. Synthetic peptides representing these regions are able to detect the presence of OPPV antibodies in 96% of infected sheep.

Maternal factors associated with prenatal transmission of ovine lentivirus.

Prenatal transmission of ovine lentivirus (OvLV) was studied in 85 eyes and their offspring. The animals were from a flock with endemic OvLV infection and 49 (58%) had serum antibodies to OvLV. Blood was collected from all lambs before they nursed. Using the polymerase chain reaction (PCR), OvLV DNA was detected in peripheral blood mononuclear cells of 13 (11%) of 117 lambs, including two sets of twins. Mothers with OvLV-infected lambs (n = 11) were younger (mean, 2.5 years) and had fewer pregnancies (mean, 2.4) than seropositive ewes (3.2 years and 3.2 pregnancies; P < .05). Of mothers with OvLV-positive lambs, 4 had plasma antigenemia (mean, 31.3 +/- 2.1 ng/mL OvLV) in conjunction with indeterminate antiviral antibody responses by immunoblotting. These results suggest that maternal factors (age and parity) and host-virus interactions (antiviral antibody and antigenemia) are important risk factors in prenatal transmission of OvLV.

A comparison of whole virus and recombinant transmembrane ELISA and immunodiffusion for detection of ovine lentivirus antibodies in Italian sheep flocks.

Sera from two sheep experimentally infected with ovine lentivirus (OLV) and from 186 sheep selected from flocks with known high or low prevalence of infection or on the basis of virological or histopathological examination were simultaneously tested by whole virus (WV) ELISA, recombinant transmembrane (r-TM) ELISA and AGID assay. Antigens for both the WV ELISA and AGID were prepared from an Italian field isolate; recombinant antigen was derived from the N'-terminal region of the transmembrane envelope protein of strain K1514. The WV ELISA detected the highest number of seropositives, followed by the r-TM ELISA and AGID test. The sensitivity and specificity of the r-TM ELISA relative to the WV ELISA were 0.66 and 0.95, respectively. Immunoblot analysis of 14 WV ELISA-positive and r-TM ELISA-negative sera showed that the major core protein was immunodominant on WV antigen. It is concluded that the r-TM ELISA was more sensitive than the AGID test but less sensitive that the WV ELISA, particularly for detecting antibodies in the early stages of infection.

Evaluation of agar gel immunodiffusion serology using caprine and ovine lentiviral antigens for detection of antibody to caprine arthritis-encephalitis virus.

The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test was 0.56. The AGID test with either antigen was 100% specific. The lower sensitivity of the OPPV AGID test in detecting caprine antibody to CAEV indicates that OPPV antigen is of limited value for use in CAEV diagnosis and control programs.