Materials and Methods

Swine and housing—Three hundred thirty-two 6- to 8-week-old pigs (principals) were obtained from a herd known to be free of PRRSV on the basis of 10 years of diagnostic testing. After arrival, pigs were confirmed to be uninfected by use of a commercial ELISA^a and RT-PCR assay.^b Pigs were individually identified with ear tags and randomly assigned to 5 groups (A to E) that were housed in separate rooms at the research farm of the University of Minnesota Swine Disease Eradication Center in west central Minnesota. Groups were designated as follows: group A, infected with wild-type PRRSV only (positive control pigs); group B, infected with PRRSV and administered 1 dose of MLV vaccine; group C, infected with PRRSV and administered 2 doses of MLV vaccine; group D, infected with PRRSV and administered 3 doses of MLV vaccine; and group E, sham inoculated only (negative control pigs; Table 1). Groups A, B, C, and D contained 80 pigs each, and group E contained 12 pigs. All pigs were vaccinated on arrival against H parasuis, Erysipelothrix rhusiopathiae,^c and Lawsonia intracellularis.^d In conjunction with the 332 principal pigs, 108 PRRSV-naïve age-matched sentinel pigs were introduced at designated periods following the initiation of the study. All protocols and procedures of pig management and care were reviewed and approved by the University of Minnesota Institutional Animal Care and Use Committee. Personnel practiced PRRSV-specific biosecurity protocols³⁵ across all study groups throughout the experiment.

Infection and vaccination—On day 0, all 320 pigs in groups A, B, C, and D were intranasally inoculated with 2 mL of cell culture fluid containing 10⁴ TCID₅₀/mL of PRRSV VR2332 (cell culture passage 3). At 7 DPI, pigs in groups B, C, and D were vaccinated IM with 2 mL of a homologous modified-live PRRSV vaccine.^e Groups C and D were then revaccinated 30 days later (37 DPI), and a final dose was administered to group D at 67 DPI. Pigs in group E were sham inoculated intranasally with 2 mL of sterile saline (0.9% NaCl) solution (Table 1). Modifiedlive PRRSV vaccine^e and PRRSV VR2332 GenBank accession numbers are AF066183 and PRU87392, respectively. To monitor the protocols of infection and vaccination, 12 pigs from groups A to D and 6 pigs in group E were randomly selected and serum samples were collected in sterile vacuum tubes^f via jugular venipuncture at 0, 7, 14, 37, 67, 97, and 127 DPI.

Assessment of transmission—To determine whether different protocols of vaccination affected virus shedding, groups of 10 individually tagged PRRSV-naïve pigs were introduced 30 days after the last vaccination into each treatment group (Table 1). All sentinel pigs were removed and euthanized, and samples were collected 30 days after introduction. Transmission was defined as either detection of PRRSV nucleic acid in serum or tissues by use of RT-PCR procedures in 1 or more sentinel pigs or detection of anti-PRRSV antibodies as determined by positive results of ELISA that was subsequently confirmed by positive results of indirect fluorescent antibody test³⁶ in 1 or more sentinel pigs.

Assessment of persistence—To evaluate whether different protocols of vaccination affected the proportion of PRRSV persistently infected pigs through time, 10 pigs/group were randomly selected and euthanized, and samples were collected 30 days following the last vaccination (Table 1). The study was terminated, and all remaining pigs were euthanized and sampled at 127 DPI. Serum and tissue samples were tested by use of RT-PCR assay for PRRSV nucleic acid. Tonsil, sternal lymph nodes, and superficial inguinal lymph nodes were collected in the processing plant, stored in separate plastic bags, and transported on ice to the laboratory for testing.

Clinical and virologic response to heterologous challenge—At 97 DPI, 10 randomly selected pigs from every group and 2 additional negative control pigs were transported to the isolation facilities of the College of Veterinary Medicine at the University of Minnesota. Each group was housed in 2 rooms (5 pigs/room) at a density of 1.2 m²/pig. Each isolation room had an independent ventilation system and a slurry pit to prevent cross-contamination of pathogens between rooms. All pigs in groups A, B, C, and D and 10 pigs from group E were intranasally inoculated with 2 mL of cell culture fluid containing PRRSV MN-184 isolate (10⁴ TCID₅₀/mL). Challenge control PRRSV-naïve pigs were labeled as E+. The group of 2 additional negative control pigs was sham inoculated intranasally with 2 mL of sterile saline solution and labeled as E–. The challenge virus, PRRSV MN-184 (GenBank accession No. AY656992), a highly virulent isolate from a farm in southern Minnesota affected with severe reproductive disease and high sow mortality rate in 2001,³⁷ was used at cell culture passage 2. The percentage divergence in the ORF 5 nucleotide sequence between MN-184 and VR2332 or the modified-live PRRSV was 15.5%.³⁷

Blood was collected from all 52 pigs at 0, 7, 14, and 21 DPI to evaluate viremia and antibody response. At 21 DPI, all pigs were euthanized and samples of tonsil, sternal lymph nodes, and superficial inguinal lymph nodes were collected in separate plastic bags. To evaluate the clinical response following heterologous challenge, rectal temperature, appetite, and mortality rate were measured at 0, 2, 4, 7, 10, 14, 17, and 21 DPI. Pigs were also weighed at 0 and 21 DPI, and ADG (g) per group was calculated. Rectal temperature (°C) was measured by the same person every sampling day between 8 and 9 AM. Every morning, the same operator measured the volume of feed still remaining from the previous day in each room and the percentage of reduction in feed intake was estimated (0% to 100%). Study personnel were unaware of group assignments.

Diagnostic testing—The PRRSV antibody response was evaluated by use of a commercial ELISA test.^a The presence of PRRSV nucleic acid in serum and tissues was determined by use of RT-PCR assay.^b Tissue samples collected at euthanasia were pooled by individual animal and 1 g was placed in 15 mL of lysis buffer^g in a sterile plastic tube.^h After homogenization with the appropriate equipment,ⁱ samples were centrifuged (700 X g for 15 minutes) to remove disrupted cells and debris. Total RNA was extracted and purified with a commercial kit^g according to the manufacturer's protocol from 200 μL of serum or 50 μL of the middle layer of homogenized tissue supernatant. The RNA was eluted in 50 μL of water, dried in a vacuum centrifuge,^j and rehydrated in 5 μL of water. Every sample was assayed in duplicate by use of 2 μL of the rehydrated sample in a 20-μL RT-PCR assay with primers and probe directed to the ORF 7 region of the North American PRRSV.³⁸ All reactions were conducted in a real-time PCR instrument.^k

Extracted RNA from selected tissue samples with positive PCR assay results was submitted to the University of Missouri, Columbia Veterinary Diagnostic Laboratory to sequence the PRRSV ORF 5 region by use of a procedure modified from a published protocol.³⁹ Amino acid sequence comparisons were used to differentiate wild-type from vaccine viruses.

Tissue RNA from pigs in the heterologous challenge phase of the study was tested by use of an isolate-specific PCR assay targeting a nucleotide sequence on the ORF 5 region of PRRSV MN-184. Total RNA was extracted as described. The PCR included the RT-PCR kit,^b forward primer (5a–TAACTTAACGATATGTGAGCTGAATG-GCAC–3a), reverse primer (5a–ACACAGTGATCAG-GCCGACC–3a), and probe (6FAM-CTGGCTGAACAA TCATTTTAGTTGGGCAGTGGAGACTTTCGTTATC-TAMRA). A standard curve was developed for the quantitative RT-PCR procedure by preparing 10-fold dilutions of PRRSV MN-184 stock starting at 10⁵ TCID₅₀/mL. Results were reported in number of RNA copies (ie, RNAc) per gram of tissue.

Statistical analysis—The proportions of PRRSV persistently infected pigs were compared among groups within the same DPI by use of the Fisher exact test. The number of PRRSV RNA copies per gram of tissue was log transformed to stabilize the variance prior to analysis. Rectal temperature (°C), ELISA S:P ratio mean, ADG (g) and log₁₀ RNAc per gram of tissue were compared among groups by use of 1-way ANOVA. All analyses were performed with standard software.^l A value of P <0.05 was considered significant.

Results

Viremia and antibody response in monitor pigs—Porcine reproductive and respiratory syndrome virus nucleic acid was detected in serum at 7 DPI in 85% of monitor pigs from all inoculated groups (A to D). The proportion of viremic pigs decreased over time up to 67 DPI when all serum samples from monitor pigs in all groups yielded negative results of PCR assays. At 14 DPI, 90% of monitor pigs in all inoculated groups had positive results of ELISA (S: P ratio > 0.4). No significant differences in S:P ratio means were observed among inoculated groups during the first 97 DPI independently of the vaccination protocol; however, at 127 DPI, pigs in groups A and B had significantly (P = 0.008) higher S:P ratio mean values than pigs in groups C and D (Table 2). Negative control pigs (group E) had negative results of ELISA^b and PCR^a assay throughout the study.

Transmission to sentinel pigs—Transmission of PRRSV was detected at 37 to 67 DPI and 67 to 97 DPI in all groups in which sentinel pigs were introduced (Table 3). Between 97 and 127 DPI, sentinel pigs introduced into groups A (positive control pigs) and B became infected as determined by results of PCR assay or ELISA, whereas sentinel pigs in groups C and D remained uninfected.

Proportion of persistently infected pigs—The modified-live PRRSV vaccine did not reduce the proportion of persistently infected pigs at 37, 67, or 97 DPI (Table 3). However, at 127 DPI, the proportion of infected pigs was significantly reduced in vaccinated groups.

Twenty tissue samples that yielded positive results of PCR assay at 127 DPI from groups B, C, and D were submitted for nucleic acid sequencing of the ORF 5 region. Seven samples yielded sufficient product for sequencing. In every sample, the sequence corresponded more closely to that of VR2332 wild-type virus than to that of MLV vaccine.

Clinical and virologic response to MN-184 challenge—Porcine reproductive and respiratory syndrome virus RNA was not detected in serum of previously infected pigs (groups A, B, C, and D) at 7, 14, or 21 days after MN-184 inoculation. Only the challenge controls (group E+) had a detectable viremia: 10 of 10 pigs at 7 DPI, 7 of 10 at 14 DPI, and 1 of 10 at 21 DPI. The mean S:P ratios among previously infected groups (A to D) before MN-184 inoculation were not significantly (P = 0.321) different. Seven days after MN-184 administration, all challenge controls (E+) yielded negative ELISA results. In contrast, previously exposed pigs had increased ELISA S:P ratios by day 7, without significant (P = 0.934) difference across these groups. One week later, every pig in group E+ yielded ELISA positive results and the S:P ratio means were not significantly (P = 0.864) different among all inoculated groups (A to E+). Similar results were obtained 21 days after MN-184 inoculation (P = 0.965). Negative control pigs (E–) had negative results of ELISA and PCR assays during this phase of the study.

Isolate MN-184 was detected in tissue samples of 70% to 100% of the pigs from all challenged groups (A to E+) at 21 DPI. The proportion of infected pigs per group was not significantly affected by previous PRRSV exposure status. No significant (P = 0.366) difference among groups was detected in the mean number of PRRSV RNA copies per gram of tissue (Table 4).

Overall, pigs from groups that had been previously exposed to PRRSV had less severe clinical signs than newly infected pigs, 1 of which died 14 days after MN-184 challenge. It had fever (41.3°C), dyspnea, cyanosis of extremities, lethargy, and anorexia before death. Noncollapsed lungs and enlarged lymph nodes were the only remarkable lesions observed during necropsy. Interstitial pneumonia was detected via histologic examination, and PRRSV RNA was detected in lymph nodes and serum by use of RT-PCR assay. Although appetite was not affected in negative control pigs (E–) or groups A to D, a 5% to 15% mean reduction in feed intake was recorded in group E+ from 4 to 16 days after MN-184 inoculation. Pigs in group E+ also had significantly (P = 0.002) lower ADG than pigs in groups A, B, C, and D. Mean rectal temperatures in all groups were not significantly different before MN-184 inoculation (P = 0.071). Inoculated groups (A to E+) had significantly (P = 0.036) higher rectal temperatures than negative control pigs at 2 DPI; however, between 4 and 14 DPI, pigs in group E+ had significantly higher rectal temperature than previously infected (A to D) or negative control pigs (Table 4).

Discussion

The purpose of the study reported here was to understand the dynamics of endemic PRRSV infection in pig populations following the application of a modified-live PRRSV vaccine as an initial means to determine whether vaccination could be a potential tool in control or eradication programs or both. After inoculation of pigs with PRRSV VR2332, the infection and seroconversion pattern was consistent with previous observations.^37,40 Various protocols of repeated vaccination with MLV did not affect the ELISA s:p ratio dynamic from 14 to 97 DPI; however, at 127 DPI, the 2 groups that received multiple vaccinations (C and D) had significantly lower S:P ratios than the positive control (A) and 1-dose group (B). These results are in accordance with previous reports^41,42 that the commercial ELISA test detected an antibody response to initial infection but not to single or repeated homologous inoculations. In the present study, repeated vaccination reduced the variation in ELISA results, as reflected in the SE of the S:P ratio mean.

Another observation was that repeated vaccination (2 or 3 doses) reduced the duration of PRRSV transmission to sentinels, perhaps because vaccinated groups had a significantly lower proportion of persistently infected pigs at 127 DPI. Despite the significant effect of the vaccination on viral persistence, the originally inoculated wild-type virus was not eliminated from the pigs in 127 days.

Although the reduction in persistence and duration of transmission by vaccination of infected populations may offer relevant information to improve PRRSV-control strategies such as herd closure and acclimation of gilts, results of the present study need to be interpreted with caution for a number of reasons. A limitation of the study was that the wild-type virus in the population was homologous to the commercially available MLV vaccine, which is not common. Because this experiment was part of a long-term study, the first logical step was to analyze the use of mass vaccination under homologous conditions to determine whether vaccination provided any benefit under optimum conditions. Detection of actively shedding pigs at 127 DPI was unexpected, and therefore, a longer trial may have provided more information. However, given the duration of shedding reported in the literature¹¹ and the standard acclimation period used by the industry,^16–18 it was expected that any evidence of vaccination effect on transmission or persistence would have been observed within 127 days. It is possible that performing mass vaccination on a population with low infection prevalence would reveal a larger benefit of mass vaccination than in the present experiment. Most of the prior reports of success of vaccination in controlling PRRSV resulted from a study that combined this strategy with herd closure.^31–34 Constant introduction of sentinels in the study, which was necessary to assess transmission over time, may have allowed for reinfection of principal pigs. However, reinfection of pigs with a homologous PRRSV has not been reported.

The final phase of the study attempted to recreate the common situation of external PRRSV reintroduction to endemically infected swine herds to gain knowledge about the clinical and virologic responses against a highly virulent and heterologous challenge. After MN-184 challenge, only the challenge controls had viremia, whereas previously exposed pigs generated a rapid serologic response without evidence of viremia. The MN-184 virus was found in the tissues of nearly all pigs from all groups regardless of the previous PRRSV infection status. A low level of transient viremia may have occurred before the first sampling at 7 DPI because an antibody response and tissue infection were observed. Consistent with partial protection against infection, fewer clinical signs and enhanced growth performance were observed in all groups that were previously exposed to PRRSV.

Under the conditions of this study, the use of a modified-live PRRSV vaccine reduced the proportion of persistently infected pigs and the duration of shedding of wild-type virus in a population of pigs infected with a homologous PRRSV isolate. Previous exposure to PRRSV significantly improved the clinical response against a highly virulent heterologous challenge, but vaccination did not eliminate the wild-type virus from lymphoid tissues or prevent heterologous infection. These results offer a detailed and controlled evaluation of the benefits and risks of the use of PRRSV mass vaccination under controlled field conditions. Although some field experiences have been reported, to the authors' knowledge, large-scale controlled experiments evaluating the effect of therapeutic vaccination on persistence, transmission, and clinical response against a heterologous PRRSV challenge have not been published. Although previous reports^31–34 provide valuable information about the potential applications of therapeutic vaccination by interpreting field results, the present experiment included positive and negative control groups; intense diagnosis monitoring via modern diagnosis tools; and strict control of a number of factors such as infection time, feeding, genetics, health status, and management to analyze the results with scientific criteria. Further studies are needed to assess the effect of mass vaccination on PRRSV-infected populations with heterologous isolates under field conditions to further define the role of MLV vaccines in control-eradication programs.