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The three cell culture sy terns routinely used in the study were commercially available continuous cells lines of RK- 1 3 and Vero cells, and primary equine foetal kidney cells ( EFK). Throughout the study, standard laboratory procedures were used for propagation and maintenance of cell cultures ( Freshney 1 994), and for isolation and propagation of viruses ( Lennette et aL. 1 988).

Media

The growth medium (GM) consisted of minimal essential medium with non-essential aminoacids ( MEM+n, S igma) supplemented with 1 0% v/v foetal bovine serum ( FBS) and 1 % v/v antibiotic olution ( PS K) ( Appendix E). Mai ntenance medium ( M M ) was the same as GM, except that it contained 2% v/v FBS. For preparation of primary cel ls, the GM was supplemented with 1 0% v/v lactalbumin hydrolysate (ELH, S igma). All media were prepared from powder according to the manufacturer' s instructions.

Subculturing of cells

The cells were routinely subcultured twice weekly. The monolayers were washed twice with warm phosphate buffered saline, pH 7.0 (PBS ) and cells were disassociated with

antibiotic-trypsin-versene solution (ATV) at 37 cc. After cells detached from the flask,

they were resuspended in an appropriate volume of G M . Usually, a split ratio of 1 : 2 was used for EFK cel ls, and a split ratio of 1 :4 to 1 : 8 for RK- 1 3 and Vero cells. For seeding 24-well plates, 2 ml of freshly diluted cell suspension was added to every wel l . Cell cultures were maintained at 37 cC in 5% CO2 atmosphere. For viru i olation, RK- 1 3 and Vero cel ls at passage 30-200, and EFK cells at a maximum passage five were used.

EHV- J/4 Serology ₆₉

Cryopreservation of cells

For freezing, cells were trypsinized, resuspended in 1 0 ml of GM, counted, pelleted by centrifugation at 300 g for 1 0 minutes, and resuspended in freezing medium (FM) at a concentration of 2 x 1 07 cells per m!. Freezing medium consisted of MEM+n with 20% v/v FBS and 1 0% v/v diemethyl sulfoxide (DMSO, S igma). The cell suspension was dispensed into 1 .7 ml cryo-vials (Nunc), and cooled slowly at 1 QC per minute in a 1 cC freezing container (Nalgene) at -70 QC, before v ials were placed in liquid nitrogen. In order to reconstitute the cells, one vial was thawed and transferred to a 1 75-cm3 or 80- cm' flask. An appropriate volume of warm GM was added slowly and cell s incubated at 37 °C in 5% CO2 atmosphere.

Preparation of primary equine kidney cells

The preparation of primary cell cultures were based on the methods described b y Freshney ( 1 994) . Enzymatic disaggregation in either cold or warm trypsin was used. The kidneys were removed aseptically from an equine foetus. The cortex was c leaned from unwanted tissue, chopped to fine pieces and washed three times in PBS . After the l ast wash the cortex pieces were transferred to a trypsinization fl ask and approximately 1 00 ml of 0.25% trypsin per 10 g of tissue was added. Tissue suspended in warm trypsin was stirred at 200 rpm at 3 7 °C for 2 hours. Every 1 5 minutes the pieces were allowed to settle, supernatant was collected and fresh trypsin added to the fl ask. The first supernatant was discarded, and all remaining supernatants were centrifuged at 500 g for 5 minutes. The cell pel let was resuspended in 1 0 m] of GM, and stored on ice. The chil led cell suspensions were pooled and seeded into 1 75-cm2 flasks at a concentration of 2 x 1 06 cells/ml.

The tissue suspended in cold trypsin was left undisturbed at 4 QC overnight. Then, the supernatant was removed, and the tissue was incubated in residual trypsin at 37 QC for approximately 20 to 30 minutes, after which time cells were resuspended i n approximately double the original trypsin volume of warm GM by gently pipetting up and down and seeded into flasks as described above. Irrespective of the method used, the cells were passaged once before they were frozen in liquid nitrogen.

Chapter 3 ₇₀

3.2.2 Collection of samples

Nasal swabs

Nasal swabs were collected using the Virocult transport system (Virocult, Medical Wire

& Equipment Co Australasia Pty Ltd), consi sting of a J 5-cm long swab and a transport tube w ith a foam pad saturated with 1 .2 ml of transport medium. After swabbing the nasal cavity of a horse, the swab was placed i n the transport tube and transported to the laboratory as quickly as possible. For samples collected from places more than 30 to 60 minutes drive away from the l aboratory, the swabs were transported on ice. For samples collected from closer locations, the swabs were usuall y transported at ambient temperatures. The manufacturer of the Virocult system assures survival of viruses at ambient temperatures for long periods of time: adenoviruses - 9 days, parainfluenza viruses - 3 days, herpesviruses - up to 1 2 days.

Blood samples

B lood for virus i solation was collected by venipunctu re using 1 0-ml heparinised vacutainer tubes (green caps) . One tube of blood for virus i solation from PBL was collected from every horse.

3.2.3 Processing of samples

Nasal swabs

Nasal swabs were processed directly on arrival at the l aboratory according to the manufacturer' s i nstructions. Approximately 2 ml of MEM+n was added to the transport

tube w ith the swab in situ. The tube was vortexed briefly and squeezed several times to

mix the contents. The liquid medium was w ithdrawn from the tube, passed through 0.22 f.lm filter, dispensed i nto 1 .7 ml Eppendorf tubes, and either used directly for

i nocul ation of cell cultu res, or frozen at -70 °C.

Blood

Peripheral blood leucocytes were separated from heparin ised blood as previously

described ( Gleeson & Coggins 1 985). The blood was allowed to settle for 1 0 to 20

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EHV- J/4 Serology 7 1

was collected, mixed with an equal volume of RBC lysing buffer (0.85% N H4CI, 0.0 1 7 M Tris, pH 7 .4) and incubated at RT for 5- 1 0 minutes. The cells were pelleted by centrifugation at 250 g for 1 0 minutes, washed once in PBS , pelleted again, and finally resuspended in 4 ml of warm PBS .

Primary inoculation and subculturing

For virus i solation, 200 /-11 of either PBL suspensIon or nasal swab filtrate was inoculated onto each of three different cell cultures, grown in 24-well plates. Thus, each sample was inocul ated into three wells containing three different cell cultures (EFK, RK- 1 3 and Vero). Three one-week passages were performed. Each time, cell cultures

were freeze-thawed and 200 !-1l of the cell l ysate was transferred to a corresponding well

in a new plate. The well s were inoculated either at the same time as the cel l s were seeded into plates, or on the foIJowing day, when monolayers were approximately 80% confluent.