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Pneumonia is an important disease of bighorn sheep (BHS) that is primarily responsible for the drastic decline in numbers of these animals in North America. Members of the genus Mannheimia and Pasteurella have frequently been isolated from the pneumonic lungs of BHS. Antibodies to several respiratory viruses, including bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), and bovine herpesvirus 1 (BoHV-1), have been detected in herds of BHS. The availability of BHS fetal lung cell lines is likely to enhance the chances of isolation of these viruses. Here we report the development of such a cell line. This line is permissive for BPIV-3, BRSV, BVDV, and BoHV-1 infection, as revealed by an enzyme immunoassay of virus-infected cells with antibodies specific for each of these viruses. This cell line should be valuable for detecting these 4, and possibly other, respiratory viruses in BHS.
La pneumonie est une maladie importante chez les mouflons et est responsable en grande partie pour le déclin drastique de leur nombre en Amérique du Nord. Les membres du genre Mannheimia (anciennnement Pasteurella) ont fréquemment été isolés de poumons de mouflons avec des lésions de pneumonie. Des anticorps dirigés contre plusieurs virus respiratoires, incluant le virus parainfluenza 3 bovin (BPIV-3), le virus respiratoire syncytial bovin (BRSV), le virus de la diarrhée virale bovine (BVDV) et le herpèsvirus bovin de type 1 (BoHV-1), ont été détectés dans des troupeaux de mouflons. La disponibilité de lignées cellulaires pulmonaires fœtales de mouflon devrait probablement augmenter les chances d’isolement de ces virus. Nous rapportons ici le développement d’une telle lignée cellulaire. Cette lignée est permissive pour une infection par BPIV-3, BRSV, BVDV et BoHv-1, tel que démontré par une épreuve immunoenzymatique sur des cellules infectées par ces virus avec des anticorps spécifiques pour chaque virus. Cette lignée cellulaire devrait être utile pour détecter ces 4 virus, et probablement d’autres virus respiratoires chez les mouflons.
(Traduit par Docteur Serge Messier)
The North American population of bighorn sheep (BHS), Ovis canadensis, has declined from an estimated 2 million at the beginning of the 19th century to fewer than 70 000 today (1). The attributed reasons include loss of habitat, competition with domestic livestock for forage, and disease (1,2), an important type being respiratory disease (1). Several outbreaks of pneumonia during the past decade have reduced the annual population growth by 40% (3). Members of the genus Mannheimia and Pasteurella have frequently been isolated from the pneumonic lungs of BHS (1). M. haemolytica has long been identified as the secondary bacterial pathogen causing severe fibrinonecrotic pneumonia in cattle (4). In cattle, these bacterial infections do not cause pneumonia unless preceded by infection with BoHV-1 (5), BRSV (6), BVDV (6), or BPIV-3 (7). Antibodies to BPIV-3 (8) and BRSV (9), as well as BVDV and BoHV-1 (Mark Drew, Idaho Department of Fish and Game, Caldwell, Idaho: personal communication, 2008), have been detected in several herds of BHS. However, these viruses have not been routinely isolated from pneumonic BHS. The failure to isolate these viruses from the large number of BHS that have died from pneumonia so far could be due to the long delay before arrival of the carcasses or lung tissue at the diagnostic laboratory. This problem is difficult to circumvent because of the remoteness of the BHS habitats. However, the chances of isolation of these viruses from the pneumonic lungs of BHS are likely to be enhanced by the availability of BHS cell lines, particularly those of lung origin. Here we report the development of a BHS fetal lung cell line and its permissiveness for infection with respiratory viruses.
A 2nd-trimester fetus from a BHS ewe that was euthanized because of a compound fracture of the left femur was used as the source of fetal lung tissue. The tissue, aseptically removed from the fetus, was rinsed in calcium- and magnesium-free phosphate-buffered saline (PBS) (CMF-PBS: NaCl, 8.0 g; Na2HPO4·H2O, 2.16 g; KCl, 0.2 g; KH2PO4, 0.2 g/L; pH 7.2) supplemented with 20 μg/mL of gentamicin (Invitrogen, Carlsbad, California, USA) and placed in a large petri dish containing 300 mL of CMF-PBS. The lung tissue was chopped into small pieces. The tissue suspension was transferred to a beaker and allowed to settle for 10 min. The top 200 mL of PBS was poured off to get rid of debris and erythrocytes. The remaining 100 mL of minced cells and CMF-PBS was placed in a trypsinizing flask to which 200 mL of prewarmed (to 37°C) CMF-PBS and 100 mL of 1% trypsin (Invitrogen) was added. A stirring bar was placed in the flask, which was kept on a stir plate for 30 min in an incubator at 37°C for the trypsinizing process. The flask contents were then strained through sterile gauze over a beaker. The supernatant containing the cells was transferred into 50-mL centrifuge tubes and centrifuged for 10 min at 170 × g. The cell pellets were pooled, resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen), and centrifuged for 10 min at 100 × g. The cells were washed with DMEM until the supernatant was clear. After the last wash, the supernatant was decanted. The cells were counted and then suspended in DMEM containing 10% fetal bovine serum (Atlanta Biologicals, Norcross, Georgia, USA) and 20 × g/mL of gentamicin (complete growth medium) and dispensed into 75-cm2 tissue culture flasks. Each flask received approximately 0.5 mL of packed cells suspended in 100 mL of complete growth medium. The flasks were incubated in a humidified incubator at 37°C with 5% CO2. The growth medium was replenished every 24 h.
The respiratory viruses used for inoculation of the BHS lung cells were BVDV NY-1 (Animal Plant and Health Inspection Service, Center for Veterinary Biologics, Ames, Iowa, USA), BRSV 236-652 (10), BPIV-3, and BoHV-1 (Veterinary Diagnostic Laboratory, University of Nebraska–Lincoln, Lincoln, Nebraska, USA). The enzyme immunoassay was performed as previously described (11). Briefly, BHS lung cells were seeded onto 12-well plates. When 60% confluent, the cells were inoculated with the viruses at a multiplicity of infection (ratio of infectious agents to infection targets) of 0.5. The cells were incubated at 37°C (BVDV, BPIV-3, and BoHV-1) or 33°C (BRSV) in 5% CO2. Cells inoculated with BPIV-3 or BRSV were incubated for 6 d, BVDV for 4 d, and BoHV-1 for 24 h. The cells were fixed with 20% acetone/PBS for 10 min and allowed to dry overnight at room temperature. The following monoclonal antibodies were used as the primary antibodies in the assay: 348, specific for the E2 glycoprotein of BVDV [Veterinary Medical Research and Development Inc. (VMRD), Pullman, Washington, USA]; 8G12, specific for the F protein of BRSV (12); F2, specific for glycoprotein C of BoHV-1 (VMRD); and 1B6, specific for a 69-kDa protein of BPIV-3 (VMRD). Biotinylated horse immunoglobulin antibodies to mouse antigen (Vector Laboratories, Burlingame, California, USA) were used as the secondary antibody. Streptavidin–horseradish peroxidase complex (Invitrogen) and 3-amino-9-ethyl-carbazole (Sigma Chemical Company, St. Louis, Missouri, USA) were used to complete the immunoperoxidase procedure.
The BHS lung cells were evaluated for mycoplasmal infection with use of the fluorescent dye 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Roche Diagnostics Corporation, Indianapolis, Indiana, USA). Briefly, the cells were seeded onto a Teflon-coated spot slide (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and incubated overnight at 37°C in a humidified chamber with 5% CO2 to allow cell attachment. After incubation the medium was discarded and the cells were washed once with DAPI–methanol (1 μg/mL). The cells were then covered with DAPI–methanol (1 μg/mL) and incubated at 37°C for 15 min. After incubation the DAPI–methanol solution was discarded, the cells were washed once with methanol, and a coverslip was mounted on the slide with the use of glycerol as a mounting medium. The slide was evaluated under a fluorescence microscope with a 340/380 excitation filter and an LP 430-nm barrier filter.
Monolayers of primary cells in the flasks attained more than 70% confluence within 7 d of harvesting and plating. The monolayers were trypsinized and the resultant cells passaged at a 1:4 dilution in 75-cm2 flasks. The cells in the flasks attained 100% confluence within 8 d. By the 3rd passage, 100% confluent monolayers were formed in 5 d. The cells from passage no. 8 were used in the enzyme immunoassay for the detection of infection with the respiratory viruses. The assay revealed that the fetal lung cells were permissive for infection with BPIV-3, BRSV, BVDV, and BoHV-1 (Figure 1). The DAPI staining revealed that the cells were free of Mycoplasma contamination and hence could be used for routine virus isolation. These cells have undergone 15 passages in our laboratory and thus could be referred to as a cell line (13).
The etiology of pneumonia in BHS has not been resolved completely, but M. haemolytica, Bibersteinia trehalosi, and P. multocida have frequently been isolated from the lungs of BHS that died of pneumonia (1). In bovine respiratory disease complex (BRDC), these organisms are widely accepted as the bacterial pathogens secondary to primary infection with 1 or more of the respiratory viruses, including BPIV-3, BRSV, BVDV, and BoHV-1 (4). Although these bacterial pathogens cause pneumonia in calves when deposited directly into the lungs by intratracheal injection or by means of a bronchoscope, they rarely cause disease if administered intranasally. However, intranasal administration of these bacteria does cause pneumonia if preceded by administration of respiratory viruses such as BoHV-1, BRSV, and BVDV (5–7,14,15). These findings indicate a primary role for these viruses in BRDC. The detection of antibodies specific for these viruses in several herds of BHS (8,9, and Mark Drew, Idaho Department of Fish and Game, Caldwell, Idaho: personal communication, 2008) suggests a role for these viruses in pneumonia in BHS as well. However, these viruses have not been routinely isolated from BHS. There are reports of isolation of BPIV-3 (16) and BRSV (17) from BHS; the cells used were bovine turbinate cells and domestic sheep fetal lung cells, respectively.
Only 3 BHS cell lines have been reported: the kidney and turbinate cell lines were permissive for BPIV-3 and BRSV in BHS (18), and the fetal tongue cell line was permissive for BoHV-1, BVDV, BPIV-3, and BRSV (19). There have been no previous reports of BHS fetal lung cell lines. The BHS fetal lung cell line developed in this study is easy to grow, is amenable to trypsinization, and easily reverts back to culture from frozen stocks. It is equally sensitive to infection with BPIV-3, BRSV, BVDV, and BoHV-1 and very likely would also be permissive for other respiratory viruses of BHS. This cell line, which is free of contamination with Mycoplasma and BVDV, should enhance the chances of isolation of respiratory viruses of BHS and other ruminants. It is freely available to anyone on request.
This work was supported by funds from the Foundation for North American Wild Sheep and its Eastern, Idaho, Oregon, and Washington chapters, as well as from the Rocky Mountain Bighorn Sheep Society.