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) at a concentration of 4.5 X 10 5 cells/well and incubated at 37 °C and 5% CO 2 . Experimental groups included 1) nontransduced CRFK control cells, 2) CRFK cells transduced with the control vector lentivirus (multiplicity of infection [MOI] of 10), 3

Open access
in American Journal of Veterinary Research

Abstract

Objective

To assess shedding of ovine lentivirus (OvLV) in semen of infected rams with or without epididymitis.

Design

Rams 1 and 2 were naturally infected with OvLV. Rams 3-6 were inoculated with OvLV strain 85/ 34. Ram 7 was inoculated with uninfected cell culture supernatatant (OvLV-negative control). 14 weeks after OvLV inoculation, rams 1-3, 6, and 7 were inoculated with Brucella ovis into the epididymis. Ram 4 was a natural case of B ovis epididymitis, and ram 5 was left non-inoculated (Bovis-negative control). Blood mononuclear cells (BMNC) and semen were collected between 0 and 44 weeks after OvLV inoculation.

Animals

Seven 2- to 3-year-old rams.

Procedure

Infective OvLV in BMNC and semen was determined by virus isolation and subsequent OvLV-DNA amplification by polymerase chain reaction (PCR). Bronchoalveolar lavage cells collected after death were used for DNA extraction and PCR amplification.

Results

OvLV was detected in the semen of rams 3 and 6, but only after Bovis inoculation. OvLV was isolated consistently from BMNC of rams 3 and 6, but only occasionally from rams 1, 2, 4, and 5. Leukocytospermia was evident in every ejaculate of all Bovis-infected rams after infection. Semiquantitative PCR determination of OvLV DNA from bronchoalveolar lavage cells revealed the highest OvLV DNA load in rams 3 and 6.

Conclusions

Leukocytospermia and a high virus load in infected animals are important factors that determine shedding of OvLV in semen.

Clinical Relevance

Dissemination of OvLV through contaminated semen could have important implications in the epidemiology and control of this infection. (Am J Vet Res 1996; 57:684–688)

Free access
in American Journal of Veterinary Research

Abstract

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 a 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. (Am J Vet Res 1998;59:563–568)

Free access
in American Journal of Veterinary Research

SUMMARY

A nested polymerase chain reaction (pcr) test was developed to examine infection with the bovine lentivirus, bovine immunodeficiency-like virus (biv), in cattle. Primers were designed to amplify 2 separate regions of the pol and env segments of the biv genome. Two calves were experimentally infected with an isolate derived from the original strain of biv, R29, or with a recent field isolate, FL491. Serial blood samples were collected and examined by virus isolation, protein immunoblot, and nested pcr. The nested pcr test detected biv infection by 3 days after inoculation, earlier than the other 2 methods, and continued to identify infected cattle 9 to 15.5 months after inoculation, even when results from virus isolation and serology became negative. Nested pcr also detected multiple-size env products in samples obtained later in the infection from the calf that received FL491, giving evidence that viral quasispecies were selected during in vivo replication of the virus. Results indicated that the nested pcr test is more sensitive than virus isolation or serology for the detection of biv infection in cattle.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To establish immortalized caprine fibroblastic cell lines permissive for replication of small ruminant lentiviruses.

Animals

Carpal synovial membrane explants collected aseptically from a surgically delivered fetus of a lentivirus-seronegative goat.

Procedure

Immortalization of goat embryonic fibroblasts was performed by DNA transfection with plasmids coding for simian virus 40 large T antigen. The generated cell lines were phenotypically characterized. Cytogenetics, growth pattern, and sensitivity to viral infection were studied.

Results

3 cell lines, designated TIGEF, mMTSV-54, and mMTSV-93, were generated. They had a more rapid doubling time than did fibroblasts from which they were derived, and retained morphologic and phenotypic fibroblastic characteristics. They were immortalized but not transformed because tumor formation was not promoted after their SC injection into athymic nude mice. The 3 cell lines were susceptible to caprine arthritisencephalitis virus and visna-maedi virus infections, and supported a complete virus replication cycle.

Conclusions

Cultured caprine fibroblastic cells were immortalized, using simian virus 40 large T antigen. The TIGEF, mMTSV-54, and mMTSV-93 immortalized cell lines were permissive to in vitro small ruminant lentivirus replication.

Clinical Relevance

Because lentivirus detection, as well as detailed studies of host-lentivirus interactions, are hampered by differences in viral susceptibility of each primary culture, permanent cell lines are essential tools for such analysis. (Am J Vet Res 1997;58:579–584)

Free access
in American Journal of Veterinary Research

SUMMARY

Static lung compliance, static lung volumes, and transfer factor for carbon monoxide were measured in 12 anesthetized adult Texel ewes seropositive for maedi-visna virus (MVV) and in 11 breed-, sex-, and age-matched seronegative controls. Median static lung compliance in MVV-infected sheep (1.24 L⋅kPa−1; range, 0.27 to 2.20 L⋅kPa−1) was not significantly different from that in controls (1.58 L⋅kPa−1 range, 0.82 to 2.08 L⋅kPa−1). Median body weight of MVV-infected sheep (56 kg; range, 40 to 75 kg) was significantly (P < 0.05) less than that of controls (65 kg; range, 53 to 87 kg). Median effective alveolar lung volume in MVV-infected sheep (3.36 L; range, 1.44 to 4.52 L) was significantly (P < 0.01) less than that in controls (4.12 L; range, 3.75 to 4.90 L). Median effective end expiratory lung volume in MVV-infected sheep (1.20 L; range, 0.56 to 1.99 L) was significantly (P < 0.001) less than that of controls (1.98 L; range: 1.76 to 2.78 L). Median lung volumes expressed per unit of body weight did not differ significantly between the groups. Median single-breath transfer factor for carbon monoxide in MVV-infected sheep (7.89 mmol⋅min−1⋅kPa−1; range, 3.45 to 12.74 mmol⋅min−1⋅kPa−1) was significantly (P < 0.001) less than that in controls (14.10 mmol⋅min−1⋅kPa−1; range, 10.02 to 18.30 mmol⋅min−1⋅kPa−1). Median transfer factor expressed per liter of alveolar volume in MVV-infected sheep (2.44 mmol⋅min−1⋅kPa−1⋅L−1; range, 1.28 to 3.72 mmol⋅min−1⋅kPa−1⋅L−1) was significantly (P < 0.05) less than that in controls (3.22 mmol⋅min−1⋅kPa−1⋅L−l; range, 2.47 to 3.74 mmol⋅min−1⋅kPa−1⋅L−1). These findings indicate that static lung volumes and transfer factor for carbon monoxide are significantly decreased in adult sheep naturally infected with MVV.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether monocyte-derived macrophages from Mouflon-domestic sheep hybrids (Ovis musimon X Ovis spp) were susceptible to productive infection with caprine arthritis-encephalitis virus (CAEV) in vitro and whether experimental inoculation of Mouflon-domestic sheep hybrids with a molecularly cloned CAEV would result in persistent infection.

Animals—5 Mouflon hybrids.

Procedure—Macrophage monolayers were inoculated with virus in vitro. Three animals were inoculated with virus intratracheally.

Results—Productive replication of CAEV was demonstrated in monocyte-derived macrophages following in vitro and in vivo inoculation. Titer of infectious cytopathic CAEV produced by macrophages from the Mouflon hybrids was similar to titers produced by macrophages from an infected goat or by synovial membrane cells. Isolation of virus from monocytederived macrophages and use of a semiquantitative polymerase chain reaction assay to amplify a portion of the viral genome demonstrated persistent virus replication in all 3 inoculated animals. Two weeks after inoculation of sheep, approximately 1 of 5,000 monocytes was harboring the virus.

Conclusions and Clinical Relevance—Results indicate that Mouflon-domestic sheep hybrids are susceptible to infection with isolates of CAEV that cause infection in domestic small ruminants. (Am J Vet Res 2000;61:456–461)

Full access
in American Journal of Veterinary Research

Ovine progressive pneumonia is a grossly underdiagnosed and frequently fatal disease of sheep that causes devastating economic losses. 1–6 The causative agent of OPP is a retrovirus of the genus Lentivirus also known as Maedi-Visna virus that

Full access
in Journal of the American Veterinary Medical Association

that equine bronchial epithelial cells (EBECs) express ACE2 and would be susceptible to transduction with a lentivirus construct expressing the spike protein of SARS-CoV-2 that binds to ACE2. Our objectives were to characterize the expression of ACE2 by

Open access
in American Journal of Veterinary Research