The PRRSV isolate (16244B) used in this experiment has been previously described (3
). Briefly, the virus was originally derived from the serum of a 7-day-old clinically affected pig from a breeding herd that was experiencing a severe outbreak of porcine reproductive and respiratory syndrome. The virus was propagated twice in the MARC-145 cell line (7
) at 37°C. The titer of the virus inoculum used in the study was determined by a microtiter assay of the same cell line.
Fifty 10- to 14-day-old segregated, early weaned, female pigs were obtained from a herd that was periodically tested for PRRSV antibodies by enzyme-linked immunosorbent assay (ELISA) (HerdChek PRRSV: IDEXX Laboratories, Westbrook, Maine) and known to be free from PRRSV infection. The pigs were held in isolation rooms, acclimated to their surroundings for 17 days, and then randomly assigned to one of five isolation rooms according to treatment group. The pigs in four of the rooms were designated principals, while the pigs in the fifth group were designated negative controls. Five days later, when the pigs were approximately 35 days of age, serum samples were collected from the pigs. The pigs designated principals were inoculated intranasally with 2 ml of inoculum containing approximately 106
50% tissue culture infective doses of PRRSV isolate 16244B, divided between both nostrils. The negative controls were not inoculated with virus. Three pigs in each room were randomly assigned to another research study (2
). They were treated the same as the other pigs in the rooms until 150 days p.i., at which time they were removed. Only information collected for the remaining 35 pigs is presented in this paper. The use of animals in this research project followed protocols approved by the Institutional Animal Care and Use Committee of the University of Nebraska—Lincoln and complied with all relevant federal guidelines.
Jugular or anterior vena cava blood samples were collected with blood collection tubes (Vacutainer; Becton Dickinson, Franklin Lakes, N.J.) on days 0, 7, 14, and 28 and then approximately monthly thereafter until day 225 p.i. After the blood samples were collected, the pigs were given an intravenous injection of an anesthetic cocktail that was made by reconstituting 250 mg of tiletamine and 250 mg of zolazepam (Telazol; Fort Dodge Animal Health, Fort Dodge, Iowa) with 12.5 ml of xylazine (100 mg/ml) and 12.5 ml of ketamine (100 mg/ml) (Ketaset; Fort Dodge Animal Health). The cocktail was administered at a dosage of 0.15 ml/10 lb of body weight. After the pigs were anesthetized, a 4.0-mm-diameter dermatology biopsy punch was used to harvest an approximately 4.0- by 8.0-mm section of tissue from each palatine tonsil. Immediately upon collection, one tonsil biopsy sample was placed in a sterile 1.5-ml Eppendorf tube on dry ice and stored at −70°C. The second biopsy sample, if collected on days 0 to 119 p.i., was placed in 0.5 ml of 10% buffered formalin. If collected on days 147 to 225 p.i., the second biopsy sample was placed on dry ice and stored at −70°C. On day 251 p.i., all inoculated pigs and one randomly selected control pig were euthanized. Blood, tonsil, bronchial lymph node, spleen, and lung samples were collected at necropsy. Portions of the tissue samples were placed in 10% buffered formalin. Other aliquots were frozen at −70°C. Blood samples were collected from the remaining six control pigs the next day (day 252 p.i.). Serum harvested from blood samples was aliquoted into 1.5-ml Eppendorf tubes. Serum samples were tested by using a commercial PRRSV ELISA (HerdChek PRRSV; IDEXX Laboratories). Serum was also stored at −70°C for later analysis.
Virus isolation on MARC-145 cells was conducted with serum and tonsil biopsy samples collected from the principals and a negative control pig randomly selected for a given collection day. Virus isolation was conducted with serum collected on days 7 to 84 p.i. and with tonsil homogenates prepared from biopsy samples or necropsy tissues collected on days 7 to 251 p.i. In each case, samples collected on the same day were assayed as a group. Tonsil homogenates were made by grinding the tissue with a pestle in an Eppendorf tube, adding 500 μl of minimal essential medium (MEM) with 50 μg of gentamicin per ml and vortexing with the pestle in the tube. The pestle was removed, an additional 500 μl of MEM was added, and the mixture was vortexed. The suspension was then passed through a 0.20-μm-pore-size filter and divided into two aliquots. One aliquot was used for virus isolation. The other aliquot of each tonsil homogenate from days 7 to 251 p.i. was reserved for analysis by nested reverse transcription (RT)-PCR.
Monolayers of 2- to 3-day-old MARC-145 cells in 12- or 24-well plates were used in virus isolation. The cells were inoculated with either 300 μl of serum or 150 μl of tonsil homogenate plus 150 μl of MEM with 50 μg of gentamicin per ml and incubated for 1 h at 37°C with 5% CO2. The media were removed by pipette and then replaced with 1 ml of MEM with 50 μg of gentamicin per ml. The cells were incubated for 7 days at 37°C with 5% CO2 and checked daily for signs of cytopathic effects (CPE). If CPE were observed or the 7 days had elapsed, the plates were frozen and thawed. Two hundred microliters of the supernatant was used to inoculate 2- to 3-day-old MARC-145 cells. The procedure for the first passage was duplicated. At the end of the 7-day incubation period or if CPE were seen sooner, the cells were fixed with acetone-methanol and stained with the PRRSV fluorescent monoclonal antibody conjugate SDOW17 (David Benfield, South Dakota State University, Brookings, S.Dak.).
Aliquots of the tonsil homogenates prepared as inoculum for the virus isolation assay were analyzed by RT-PCR. Separate tonsil biopsy samples collected on days 147 to 225 p.i. and tonsillar tissue collected at necropsy were prepared specifically for RT-PCR analysis. In this case, the RNA extraction process was conducted directly on ground tonsillar tissue, not on tonsillar tissue mixed with MEM. The presence of viral RNA was also assessed by RT-PCR with serum samples collected on days 0 to 251. Samples that were collected on the same day were assayed as a group, with the exception of the serum samples collected from control pigs on day 252 p.i., which were analyzed with the samples collected on day 251 p.i.
Viral RNA was extracted by first adding 0.75 ml of Trizol (Gibco BRL, Gaithersburg, Md.) to 0.25 ml of serum, 0.25 ml of inoculum prepared for virus isolation from tonsil tissues, or directly to ground tonsillar tissue. A known PRRSV-positive serum sample and a PRRSV-negative fetal calf serum sample were included as positive and negative controls. After incubation at room temperature for 5 min, 0.2 ml of chloroform was added to each sample, which were shaken vigorously for 15 s, incubated for 15 min at room temperature, and then centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was removed and added to tubes containing 2 μl of glycogen. After 0.5 ml of isopropanol was added, the contents of the tubes were mixed and incubated at room temperature for 10 min. Following centrifugation at 10,000 × g for 10 min at 4°C, the isopropanol was drained, leaving a pellet that was then rinsed with 1 ml of 75% ethanol-25% diethyl pyrocarbonate water and allowed to dry. The RNA pellet was then dissolved in 20 μl of diethyl pyrocarbonate water. RT-PCR was performed by using an EZ rTth RNA PCR kit (Perkin-Elmer, Branchburg, N.J.) and was followed by nested PCR, both using primers which amplify open reading frame 6 of PRRSV. These primers included 14424 F (5′ AGGTGCTCTTGGCGTTCTCTATT 3′) and 14836 R (5′ GCTTTTCTGCCACCCAACACG 3′) for outer PCR and 14661 F (5′ CCTCCAGATGCCGTTTGTGCT 3′) and 14790 R (5′ TGCCGTTGACCGTAGTGGAGC 3′) for nested PCR.
Each reaction was performed in a final volume of 50 μl containing 4 mM Mn2+ (for the RT), a 0.3 mM concentration of each deoxynucleoside triphosphate, a 0.8 μM concentration of each primer, and 5 U of a recombinant Tth polymerase. The samples were then placed in a thermocycler for 30 min at 60°C and then for 3 min at 95°C, followed by 35 cycles consisting of 1 min of denaturation at 95°C, 1 min of primer annealing at 60°C, and 1 min of extension at 72°C. For the nested PCR, 2-μl aliquots of products from the previous RT-PCR were added to tubes containing 1 mM Mg2+, a 0.2 mM concentration of each deoxynucleoside triphosphate, a 0.8 μM concentration of each primer, and 5 U of Taq polymerase. Cycling parameters for the nested PCR were the same as those for the outer PCR. The samples were then electrophoresed on 2% Nu-Sieve 3:1 agarose (FMC BioProducts, Rockland, Maine).
The presence of infectious PRRSV in tissue samples collected at necropsy from the 28 principals and one control pig was assessed by bioassay. PRRSV-free sentinel pigs were inoculated with tissue preparations from the principals and the control pigs and then tested for evidence of infection. Samples of tonsil, lymph node, and lung tissue were used for pigs 1, 2, 16, 24, 26, 29, 34, and 40. The procedure was subsequently modified to also include samples of spleen to potentially improve the detection of virus in the remaining pigs. Approximately 8 g of each type of tissue was minced and mixed with 15 ml of MEM. Homogenates were placed in plastic bags and macerated (stomacher; Colworth), frozen at −70°C, thawed, and centrifuged at 1200 × g for 10 min. Eleven milliliters of supernatant was drawn into a syringe for inoculation of pigs. Serum samples (0.5 ml) collected from pigs 7, 29, and 40 on days 56, 251, and 225 p.i., respectively, were also used but without further processing. Eight 2- to 3-week-old pigs, obtained from a herd known to be PRRSV-free through periodic serologic testing, were used as sentinels. The pigs were housed in separate isolation rooms for the remainder of the experiment. For each round of bioassays, supernatant or serum from a single pig was injected into the peritoneal cavity of a sentinel pig. Serum samples were collected from sentinel pigs just prior to injection and again approximately 14 and 28 days later. Serum samples were assayed by PRRSV ELISA for the presence of anti-PRRSV antibodies. The eight recipient pigs remained seronegative and were used repeatedly as sentinels for all subsequent bioassays.