Susceptibility of two bighorn sheep cell cultures to selected common ruminant viruses.

Abstract

Respiratory disease is a major factor in mortality in bighorn sheep populations in the western United States and Canada. The most common microorganisms isolated from the lungs of bighorn sheep that have died from respiratory disease are Pasteurella sp.9,12,13,16 Pasteurellae are opportunistic pathogens commonly found in the respiratory tract of both wild and domestic ruminants.8,20 These organisms often require other predisposing factors to establish disease in their host. There is evidence that viral infections predispose domestic livestock to develop pneumonic pasteurellosis;6,17,22 however, the role of Pasteurella in respiratory disease of bighorn sheep is not well understood. Antibody titers to respiratory syncytial virus (RSV), parainfluenza 3 (PI3) virus, infectious bovine rhinotracheitis virus, and bovine viral diarrhea virus (BVDV) have been found in sera from healthy and clinically ill bighorn sheep.4,19 PI3 virus was isolated from a captive herd of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in Wyoming14 and later from free-ranging bighorns in Colorado.18 More recently, another respiratory virus, RSV, was isolated from a captive bighorn herd.5 Most laboratory cell lines currently used for virus isolation are of domestic animal origin. Viruses adapted to bighorn sheep may be more readily propagated in bighorn sheep cell cultures than in tissues from other animal species. The objectives of this study were to establish and evaluate cells of bighorn sheep origin for use in virus isolation. When 1 of 5 Rocky Mountain bighorn ewes captured for a study of lamb mortality9 died at 5 months gestation, the lamb was surgically removed for the production of tissue cultures. Kidneys were removed aseptically and placed in Hanks’ balanced salt solution. Cell suspensions were prepared from kidney cortical tissue by standard techniques.11 The prepared bighorn sheep kidney (BHSK) cells were suspended in growth medium (GM: minimal essential medium [MEM]a with 10% fetal bovine serum [FBS]b), seeded in 75cm2 tissue culture flasks, and incubated at 37 C in 5% CO2. The GM was exchanged frequently until confluent monolayers were established. Confluent monolayers were split 1:2 and passaged twice before freezing in GM with 10% dimethyl sulfoxidec in liquid nitrogen. Additionally, tissue fragments were removed from nasal turbinate bone and cartilage of the fetus. Tissue dissaggregation and cell collection, propagation, and preservation were carried out as above to produce turbinate cell cultures (BHST). The BHST and BHSK cells were passaged repeatedly to determine the number of viable passages. Coverslip cultures of both BHST and BHSK cells were examined for the presence of adventitious BVDV by direct fluorescence microscopy using fluorescein-conjugated antiserum to BVDVd and