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Porcine reproductive and respiratory syndrome virus (PRRSV) can be devastating to commercial breeding operations. The objective of this study was to evaluate a novel European PRRSV vaccinal strain for safety and efficacy in bred gilts. In 2 experiments, 110 gilts were vaccinated intramuscularly and the vaccine was evaluated for safety and efficacy. Gilts in Experiment 1 were evaluated for local and systemic reactions and gilts in both experiments were observed for clinical signs of disease through farrow. In both experiments, piglet clinical observations, piglet average daily weight gain (ADWG), gilt serology [determined by enzyme-linked immunosorbent assay (ELISA)], gilt and piglet viremia [determined by quantitative real-time polymerase chain reaction (qPCR)], as well as piglet lung lesion scores and PRRS virus in lung tissue (qPCR) were determined. The vaccine was shown to be safe as there were no significant differences among groups in either experiment. Efficacy was established in Experiment 2 as both vaccinated groups were associated with desirable significant differences in percentage of gilts with abnormal clinical findings; gilt viral load post-challenge [day 125, day of farrowing (DOF), and DOF + 13]; percentages of alive, healthy live, weak live, and mummified piglets per litter at farrowing and weaning; percentage of piglets per gilt that were positive for viremia; percentage of piglets per gilt with clinical disease; and piglet viral load on DOF. It was concluded that a vaccine formulated from the PRRSV modified live virus (MLV) strain 94881 is a safe and effective method of protection against the detrimental effects of virulent PRRSV infection in breeding female pigs.
Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) peut être dévastateur pour les opérations de reproduction commerciales. L’objectif de la présente étude était d’évaluer l’innocuité et l’efficacité d’une nouvelle souche vaccinale européenne du VSRRP chez des cochettes saillies. Lors de deux expériences, 110 cochettes ont été vaccinées par voie intramusculaire afin d’évaluer l’innocuité et l’efficacité du vaccin. Les cochettes de l’Expérience 1 ont été évaluées pour la présence de réactions locales et systémiques et les cochettes dans les deux expériences ont été observées pour vérifier la présence de signes cliniques de maladie jusqu’au moment de la mise-bas. Lors des deux expériences on nota les observations cliniques des porcelets, le gain de poids quotidien moyen des porcelets (GMQ), les titres sérologiques des truies [déterminés par épreuve immuno-enzymatique (ELISA)], la virémie chez les cochettes et les porcelets [déterminées par réaction d’amplification en chaine par la polymérase quantitative (qACP)], ainsi que par les pointages de lésions pulmonaires des porcelets et la quantité de virus SRRP dans le tissu pulmonaire (qACP). Le vaccin s’est avéré sécuritaire et il n’y avait pas de différence significative entre les groupes dans les deux expériences. L’efficacité a été établie lors de l’Expérience 2 alors que les deux groupes vaccinés ont été associés à des différences significatives souhaitées dans le pourcentage de cochettes avec des trouvailles cliniques anormales; dans la charge virale post-challenge par cochette [jour 125, jour de la mise-bas (JMB), et JMB + 13]; pourcentages de porcelets vivants, vivants en santé, vivants faibles, et momifiés par portée au moment de la mise-bas et au sevrage; pourcentages de porcelets par truie qui étaient positifs pour une virémie; pourcentage de porcelets par cochette avec une maladie clinique; et charge virale des porcelets au JMB. Il a été conclu qu’un vaccin formulé à partir de la souche 94881 du VSRRP vivant modifié est une méthode sécuritaire et efficace de protection contre les effets néfastes d’une infection par le VSRRP chez des porcs femelles de reproduction.
(Traduit par Docteur Serge Messier)
Porcine reproductive and respiratory syndrome virus (PRRSV) can be devastating to breeding herds due to losses resulting from reproductive failure, i.e., reduced fertility, abortions, and premature farrowing (1–8), as well as delayed return to estrus (2,4). Piglets born to affected dams suffer from increased weakness at birth, decreased growth rates, increased susceptibility to respiratory infections, and higher pre-weaning mortality (1,2,6,7,9). Vaccination before breeding is considered to be of value for decreasing these detrimental effects.
The 2 main types of commercially available vaccines, attenuated or modified live virus (MLV) and inactivated or killed virus (KV), confer various levels of protection. Several factors, including type of vaccine used (10), viral diversity (10,11), and timing (12,13), can directly affect the safety and efficacy of vaccinations. It is commonly accepted that inactivated vaccines are safe to administer to breeding animals (2,6,14) as they do not spread to other animals in the herd and do not induce reproductive failure in bred females (6) and are thus primarily recommended for immunizing animals used for reproduction.
Concerns have been raised about the safety of MLV vaccines due to the risk of vertical and horizontal transmission of vaccine virus (1,6,12,14–17) and the potential for reduced reproductive performance when administered during gestation (12). Despite safety concerns, MLV vaccines are commonly administered during gestation as they have been reported to provide more protection against infection than inactivated vaccines when faced with heterologous challenge (11,16,17). It has been reported that inactivated vaccines did not prevent viremia and/or clinical signs of PRRS (2,6,17,18). Vaccination with an MLV vaccine, however, has been shown to protect gilts from viremia and reduce congenital infection of piglets and pre- and post-natal death (2,6,16). In clinical trials, both attenuated (2,6,8,10,19) and inactivated (8,10,18) vaccines have been proven to provide effective protection against homologous challenge. Due to significant viral diversity, however, all challenge situations in the field could be considered heterologous (20).
Specifically, there are 2 main genotypes of PRRS virus, European (EU, Type 1) or North American (NA, Type 2) (3,8,15,19,21–25), which are further divided into multiple clusters (24). Since new strains continue to be found, including highly pathogenic strains (23), it is important that vaccines offer cross-protection against heterologous strains. In addition to the type of vaccine used, timing of vaccine administration is also important.
The objective of this study was to evaluate the potential for safety and efficacy of a new PRRSV modified live virus (MLV) vaccine based on a novel strain [94881, US patent #8,765,142 B2, European Collection of Cell Cultures (ECACC) accession # ECACC 11012501 (parent strain) and ECACC 11012502 (attenuated strain)] (26) when administered to either bred gilts or to breeding-age gilts before conception and the subsequent heterologous challenge.
Protocols were reviewed and approved by the contract research organization’s Institutional Animal Care and Use Committee before study initiation. Two experiments were conducted to establish the safety (Experiment 1) and efficacy (Experiment 2) of the test vaccine in gilts. A total of 16 bred and 94 non-bred PRRSV-negative [enzyme-linked immunosorbent assay (ELISA) sample to positive (S/P) ratio of < 0.4], commercial mixed-breed, bred gilts were used for Experiment 1 and Experiment 2, respectively. For Experiment 1, a statistician randomly assigned gilts to either group 1A (n = 8) or 1B (n = 8) before day 0 and each group was then housed in 2 separate rooms. All gilts were 90 ± 3 d of gestation at the time of first vaccination and were clinically healthy. On days 0 and 14, gilts in group 1B were administered the test vaccine at approximately 10 times (10×) overdose, while gilts in group 1A received a placebo. Gilts in groups 1A and 1B were monitored for local and systemic reactions, including serology and viremia testing, to establish vaccine safety. All gilts in Experiment 1 subsequently farrowed on days 22 to 30 and piglets were monitored for number of piglets born live, dead (stillborn), weak, mummified, and crushed/mortality, as well as for viremia, average daily weight gain (ADWG), and lung pathology.
For Experiment 2, a statistician randomly assigned 94 healthy, non-bred, approximately 8-month-old, commercial mixed-breed gilts to 1 of 4 treatment groups (group 2A, n = 28; group 2B, n = 28; group 2C, n = 28; and group 2D, n = 10). Gilts in groups 2B and 2C were housed with their respective groups in 4 rooms (2 rooms per group) at the test facility for the duration of the trial, while gilts in groups 2A and 2D were housed together in a single room at an alternate facility. On day 0, approximately 28 d before breeding, gilts in groups 2A and 2D were administered a placebo product, while gilts in groups 2B and 2C were vaccinated with a low and high titer of the test vaccine, respectively. After vaccination, gilts were observed for local and systemic reactions. Gilts were tested for PRRSV serology and viremia, synchronized for breeding, artificially inseminated (AI), and evaluated for pregnancy by ultrasound. A total of 53 healthy bred gilts, [16 gilts each from groups 2A, 2B, and 2C and 5 gilts from group 2D (negative control group)] were randomly selected and subsequently enrolled into the challenge portion of the trial. Gilts in groups 2A to 2C were challenged with a heterologous [88.8% sequence homology at open reading frame (ORF) 5], Type 1 virulent strain of PRRSV and monitored for clinical signs of disease, including abortions and rectal temperatures. Gilts subsequently farrowed on days 134 to 147 and piglets were monitored for number of piglets born live, dead (stillborn), weak, mummified, and crushed/mortality, as well as for viremia and ADWG. Additionally, all piglets that were either born dead or died before DOF + 20 were necropsied and evaluated for lung pathology.
For the vaccination phase of Experiment 2, gilts in Groups 2B and 2C were housed by group in Biosafety Level 2 (BSL2) rooms at the test facility, while gilts in Groups 2A and 2D were housed together at an alternate facility until day 85 when the gilts were housed in rooms at the test facility. For Experiments 1 (entire trial) and 2 (beginning on day 85), bred gilts were housed in BSL2 rooms according to group and were placed in individual elevated crates. Crates were approximately 5 ft × 7 ft in size, were equipped with a nipple waterer, feeder, and plastic slatted flooring that was elevated above the floor, and did not allow for nose-to-nose contact. For biosecurity purposes, each BSL2 room was separately ventilated with both HEPA filters and mechanical ventilation, animal services staff were required to shower and don clean clothing before entering each room, and appropriate measures were taken to prevent accidental cross-contamination from vaccinates and/or challenged gilts to negative control gilts. All gilts were fed an age-appropriate, commercially available, non-medicated gestation or lactation ration (Heart of Iowa Cooperative, Roland, Iowa, USA) as appropriate for their condition. Food and water were available ad libitum.
Blood was collected by jugular venipuncture from gilts in Experiments 1 and 2 on days 0, 14, DOF, and DOF + 21 and on days 0, 7, 14, 21, 56, 84, 118, 125, 132, DOF, DOF + 7, DOF + 13, and DOF + 20, respectively. Venous whole blood was collected from piglets in Experiment 1 on the day of birth and DOF + 21 and from piglets in Experiment 2 on the day of birth, DOF + 7, DOF + 13, and DOF + 20, or when any piglet was found dead. Additionally, blood was collected from each mummified or stillborn piglet, but if this was not possible, thoracic or abdominal fluid was collected. Blood samples were processed for serum and fluids were aliquoted into appropriate tubes and held at either 2°C to 8°C or −70°C ± 10°C before testing. Samples held at 2°C to 8°C were tested for PRRSV antibodies at Boehringer Ingelheim Vetmedica Inc. Health Management Center, Ames, Iowa, USA (BIVI-Ames) using a commercially available ELISA kit (IDEXX Laboratories, Westbrook, Maine, USA). Results were reported as negative (ELISA S/P ratio of < 0.4) or positive (ELISA S/P ratio ≥ 0.4). Samples held at −70°C ± 10°C were tested for PRRSV ribonucleic acid (RNA) by quantitative real-time polymerase chain reaction (qPCR; bioScreen GmbH, Münster, Germany). Results were reported as genome equivalent/mL (log10 GE/mL).
Gilts in both Experiment 1 and Experiment 2 were administered either the test vaccine or the placebo at approximately 90 ± 3 d of gestation and at 8 mo of age, respectively. In Experiment 1, gilts were vaccinated intramuscularly (IM) on the right side of the neck on day 0 and a repeat injection was administered on the left side of the neck on day 14. Gilts in group 1A were administered the placebo [phosphate-buffered saline (PBS)] and gilts in group 1B received a 10× overdose [approximately 1 × 108 50% Tissue Culture Infective Dose (TCID)50/dose] of the test vaccine (ReproCyc PRRS EU; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA). All gilts in Experiment 2 were vaccinated IM once on the right side of the neck. Specifically, gilts in group 2B (low titer) and 2C (high titer) were administered 2 mL of the test vaccine (ReproCyc PRRS EU; Boehringer Ingelheim Vetmedica) at the anticipated minimum immunizing dose (MID) of 1 × 102.43 (TCID)50/dose and at 1 × 103.90 TCID50/dose, respectively, while gilts in groups 2A and 2D received 2 mL of the placebo product (placebo matched product without PRRS 94881 MLV).
Gilts in Experiment 1 were observed once daily for clinical signs of disease including behavior (normal, recumbent, shivering, lethargic, or unconscious), respiration (normal, mild coughing, severe coughing, sneezing, abdominal breathing, or rapid respiration), digestion (normal, vomiting, diarrhea, and reduced or no appetite), and other (normal, hernia, thin, lame, edema around the eyes, etc.) on day −1 to their respective DOF + 21. Similarly, gilts in Experiment 2 were observed once daily for clinical signs of disease from day −1 to 21 and at least 3 times weekly from day 22 to 115.
For gilts in Experiment 1, all injection sites were examined for redness (none, slight, moderate, or severe), swelling (none, minimal, slight, moderate, or severe), heat, by feel, (normal, warm, or hot), and pain (absent or present during palpation) just before vaccination and at 1 and 4 h post-vaccination on day 0 and once daily from day 1 through day 14. All injection sites on the left side of the neck were monitored for signs of local reactions just before vaccination and at 1 and 4 h post-vaccination on day 14 and once daily from day 15 through day 28. Additionally, any gilt that exhibited an injection site reaction on the right and/or left side of the neck was observed until the reaction resolved or the end of the study 21 d post-farrow (DOF + 21).
Beginning on day 8 and continuing through day 21, all gilts in Experiment 2 were administered 6.8 mL of altrenogest (Matrix; Merck Animal Health, Whitehouse Station, New Jersey, USA) once daily on the feed for estrus synchronization purposes. On day 26 to 32, gilts were subsequently observed for estrus and were artificially inseminated a minimum of 2 times using semen obtained from a PRRS-negative boar farm upon determination of standing receptivity. Gilts were checked for pregnancy by ultrasound on day 84, approximately 55 ± 3 d after breeding.
No animals were removed from Experiment 1, while 41 gilts were excluded from Experiment 2 before challenge innoculation. Specifically, 5, 2, 3, and 1 gilt(s) from groups 2A, 2B, 2C, and 2D, respectively, did not display estrus after synchronization, were not bred, and were removed from the trial. After pregnancy was determined, 16 gilts (2 from group 2A, 9 from group 2B, 4 from group 2C, and 1 from group 2D) were removed from the trial by day 89 as a result of lameness, not being pregnant, or late breeding. Additionally, 5, 1, and 5 gilts from groups 2A, 2B, and 2C, respectively, were randomly selected for removal by day 104, so that there was a total of 16 bred gilts in each of the challenged groups. Similarly, 3 gilts from group 2D were randomly selected for removal, so that the negative control group consisted of 5 bred gilts.
On day 118, gilts in groups 2A, 2B, and 2C were challenged intranasally (IN) with 4 mL (2 mL per nare) and IM with 2 mL of a heterologous Type-1 PRRSV. Briefly, on the day of the challenge, the challenge virus was thawed and diluted with minimum essential medium (MEM) to a targeted titer of 1 × 106 TCID50/6 mL dose. The challenge virus was isolated from an 8-week-old pig on a German farm with lungs infected by Type-1 PRRS that had previously shown signs of respiratory distress. Postmortem findings confirmed interstitial pneumonia and lung lesions suggestive of PRRSV. The challenge isolate was directly propagated on MA104 cells and a culture stock containing only Type-I PRRSV was developed for future challenge studies. Pre- and post-challenge titers were carried out and the challenge material was determined to have a mean titer of 1 × 106.30 TCID50/6 mL dose.
Gilts in Experiment 2 were observed once daily for clinical signs of disease on study days 116 through DOF + 20. Gilts were visually examined in crates and scored for clinical signs including respiration (0 = normal, 1 = panting/rapid respiration, 2 = dyspnea, or 3 = dead), behavior (0 = normal, 1 = mild-to-moderate lethargy, 2 = severely lethargic or recumbent, or 3 = dead), and cough (0 = none, 1 = soft or intermittent, 2 = harsh or severe, repetitive, or 3 = dead). Observing personnel were blinded from treatment group assignment.
Rectal temperatures (°C) were collected from gilts in Experiment 1 once daily using a self-calibrating digital thermometer on days −1, 1 to 13, and 15 to 28. On vaccination days (days 0 and 14), rectal temperatures were recorded just before vaccination, as well as 4 h post-vaccination.
Gilts in Experiment 1 farrowed on day 22 to 30, while gilts in Experiment 2 farrowed on days 134 to 147. On the day of parturition, gilts in both experiments were administered oxytocin (Vet Tek, Blue Springs, Missouri, USA), according to label directions, to assist with farrowing as needed. On the day of birth, all live piglets in Experiments 1 and 2 were administered 1.0 mL of an iron injection (either Durvet, Blue Springs, Missouri, USA or Phoenix Scientific, St. Joseph, Missouri, USA) IM in the right ham to prevent iron deficiency anemia, as well as gentamicin (Sparhawk Laboratories, Lenexa, Kansas, USA), according to label directions, to prevent scours.
Farrowing data was recorded for all gilts in Experiments 1 and 2. On each gilt’s DOF (defined as the day the first piglet was delivered), all piglets were classified into 1 of 5 categories: mummy, stillborn, weak, healthy, or crushed/mortality. A live piglet at birth was defined as any piglet that was healthy, weak, or crushed/mortality. The number of surviving piglets was determined on the last day of the trial (DOF + 21 for Experiment 1 and DOF + 20 for Experiment 2).
All live piglets in both experiments were observed once daily for clinical signs beginning on DOF + 1 and continuing through to the end of the trial (DOF + 1 to DOF + 21 for Experiment 1 and DOF + 1 to DOF + 20 for Experiment 2). A daily total clinical observation score was determined as a summation of respiration, behavior, and cough scores, as previously described (post-challenge observations for gilts in Experiment 2).
Individual body weights (kg) of all piglets were collected on the day of birth. Body weights were also collected on DOF + 21 and DOF + 20 for piglets in Experiment 1 and Experiment 2, respectively, or on the day a piglet was found dead. Average daily weight gain (ADWG) was determined for all surviving piglets from DOF through DOF + 21 for Experiment 1 or DOF + 20 for Experiment 2.
All gilts in Experiment 1 were euthanized by sedation and electrocution on DOF + 21. If an injection site reaction (swelling) was still present, tissue samples were collected from the site and placed in a container with an appropriate amount of 10% formalin and submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL, Ames, Iowa, USA) for histopathologic examination (embedded in paraffin, sectioned, and stained with hematoxylin and eosin stain) by a board-certified veterinary pathologist who was blinded from animal treatments.
All surviving piglets in Experiment 1 were euthanized and necropsied on DOF + 21. The lungs were examined for gross lesions and the percent pathology for each lobe was recorded. Total lung lesion scores were determined for each pig using the European Union (EU) enzootic pneumonia formula. Specifically, total lung lesion scores were measured as a percentage of lung involvement calculated according to a weighting formula that accounts for the relative weight of each of the 7 lobes. The assessed percentage of lung lobe area with typical lesions was multiplied by the lobe factor (left apical = 0.05, left cardiac = 0.06, left diaphragmatic = 0.29, right apical = 0.11, right cardiac = 0.10, right diaphragmatic = 0.34, and intermediate = 0.05) and the total weighted lung lesion score was then determined. Similarly, lung pathology was recorded for all stillborn piglets (excluding mummies), as well as piglets that died or were euthanized before study completion.
For all surviving piglets in Experiment 1 and all piglets dead at delivery or that died or were euthanized before DOF + 21 (Experiment 1) or DOF + 20 (Experiment 2), 2 lung tissue samples were collected on DOF + 21 or the day of death. One sample was placed in a Whirl-Pak bag (US Plastic Corporation, Lima, Ohio, USA) and the other in a container with 10% formalin. Samples in Whirl-Pak bags were stored at −70°C ± 10°C and then shipped to bioScreen GmbH for viremia testing for PRRSV RNA by qPCR as described by Revilla-Fernandez et al (27). Specifically, the 2× TaqMan Universal PCR Kit (Applied Biosystems, Foster City, California, USA) with AmpErase UNG (Applied Biosystems), the EU6-MGB: CTGTGAGAAAGCCCGGAC probe, and the primers EU6-343f-plus: GTRGAAAGTGCTGCAGGYCTCCA (sense) and EU6-462r-plus: CACGAGGCTCCGAAGYCCW (antisense) were used. Results were reported as log10 GE/mL for left and right/intermediate lung samples. Formalin-fixed samples were stored at room temperature for approximately 1 wk before being submitted to ISU VDL for embedding in paraffin blocks and subsequent storage, again at room temperature, for possible future testing.
The statistical analyses and data summaries were done using SAS software, Version 8.2 (SAS Institute, Cary, North Carolina, USA). All data for both experiments were summarized descriptively (n = sample number, minimum, maximum, mean, median, interquartile range, or confidence interval) based on the type of variable. All data were analyzed assuming a completely random design structure and tests on differences were designed as 2-sided tests at α = 5%, with differences considered significant if P ≤ 0.05.
The main objective of Experiment 1 was to compare the vaccinated group (group 1B) with the non-vaccinated group (group 1A), with primary variables of gilt reproductive performance and survival rate of offspring at weaning (DOF + 21) and secondary, supportive parameters of systemic post-vaccination reactions, local injection site reactions, transplacental infection of piglets, and general health of offspring during the suckling period (clinical observations, ADWG, and lung lesions).
Specifically, gilt reproductive performance and the survival rate of offspring, as well as gilt rectal temperatures, mean duration of injection site reactions, transplacental infection of piglets, and live piglets plus mummies and stillborn piglets that were PRRSV-positive by qPCR, viremia in piglets per litter, and piglet lung lesions, were analyzed using the Wilcoxon-Mann-Whitney test. Clinical observations of gilts, proportion of gilts with an increase in rectal temperature > 1.5°C compared to baseline days, proportion of gilts with an injection site reaction for at least 1 d, viremia post-vaccination in gilts, post-vaccination serology of gilts, and proportion of piglets per litter with a positive clinical observation score for at least 1 d were evaluated using Fisher’s exact test. Finally, mean daily rectal temperature of gilts, mean piglet body weight per litter on DOF, and mean piglet ADWG per litter from DOF through DOF + 21 were compared using the analysis of variance (ANOVA) procedure.
The main objective of Experiment 2 was to compare the 2 vaccinated groups (groups 2B and 2C) to the unvaccinated challenge control group (group 2A). Animals removed from the trial were included in their respective parameter of analysis until removal. Primary variables included proportions of live piglets on day of farrowing (DOF) and proportions of live piglets at 20 d of age (DOF + 20), with supportive variables consisting of clinical observations post-vaccination and post-challenge, serology, viremia, and reproductive performance of the gilts, as well as viremia, clinical observations, and ADWG for the piglets.
Specifically, frequency tables were generated of gilts with at least 1 positive finding for clinical observations post-vaccination and post-challenge, for positive ELISA results, and for positive qPCR (qualitative analysis) of gilt viremia and differences between the challenge control and vaccine groups were tested using Fisher’s exact test. Gilt viremia data were also evaluated (quantitative analysis) separately for each day and positive versus negative (assigned values of 3.0 and 0.0 log10 GE/mL, respectively) were compared using the Wilcoxon-Mann-Whitney test. Differences in gilt reproductive performance between groups were also tested by the Wilcoxon-Mann-Whitney test.
Piglet viremia data were evaluated qualitatively (calculated percentages of positive piglets per litter used as single values for comparison of groups) and quantitatively (calculated median qPCR values per litter used as single values for comparison of groups) for separate comparisons for each day using the Wilcoxon-Mann-Whitney test. Individual piglet qPCR data were used for summary statistics and viral load in lung samples from dead piglets before DOF + 20 were evaluated descriptively only. Individual piglet ADWGs were calculated for between DOF and DOF + 20 and differences among treatment groups were analyzed using the ANOVA procedure and subsequent t-tests. Least squares means of groups and differences between LS means with 95% confidence intervals (CI) were derived from the ANOVA. The analysis for DOF + 20 and ADWG was repeated with weight at DOF as a covariate and weight data of piglets per gilt were summarized descriptively. The calculated percentages of piglets per litter with at least 1 positive finding for clinical observations on DOF + 1 to DOF + 20 were used as single values for comparisons among groups by the Wilcoxon-Mann-Whitney test and were analyzed assuming a completely random design structure.
In Experiment 1, no gilt mortalities occurred in either group 1A or 1B (placebo-vaccinated or repeat 10× overdose, respectively). From day 0 + 4 h to DOF + 21, 50% of gilts in group 1A and 75% of gilts in group 1B exhibited at least 1 abnormal clinical finding for a minimum of 1 d (difference between groups was not significant; P = 0.6084). Specifically, 4 gilts in group 1A and 6 gilts in group 1B exhibited abnormal clinical findings. Two gilts had reduced or no appetite for 2 d and 1 gilt was lethargic for 1 d; 1 gilt had reduced or no appetite for 2 d and was lethargic for 2 consecutive d; 1 gilt had rapid respiration, reduced or no appetite for 1 d each, and was lethargic on 2 consecutive days; and 1 gilt had a small abscess on the right side of the neck (noted before vaccination) for 28 d and other signs (no description other than farrowing) on 1 d. No analysis was conducted on gilts in Experiment 2 as no abnormalities were noted after vaccination.
In Experiment 1, 1 gilt from each group exhibited an injection site reaction for at least 1 d until day 14 (P = 1.0000), whereas no gilts in group 1A, but 7 out of 8 gilts in group 1B exhibited an injection site reaction of either redness, heat, and/or pain for at least 1 d from day 14 onwards (P = 0.0014). From day 0 + 1 h to day 14, gilts in groups 1A and 1B had median injection site reaction duration of 0.0 and 2.0 d, respectively. Swelling was measurable for only 1 gilt in group 1B (slight swelling on the right side on day 1 to day 4 and on day 17 and moderate swelling on the right side on days 18 to 23 and days 41 to 43), which was later determined to be an abscess. Swelling of the 1 gilt in group 1A (day 0 + 4 to day 1) and the 6 gilts in group 1B was found to be minimal.
In Experiment 2, percentages of total abnormal findings (a clinical score > 0 for respiration, behavior, and/or cough indicating clinical disease) for at least 1 d for gilts in groups 2A (placebo-vaccinated and challenged), 2B [low-titer (MID) vaccinated and challenged], 2C (high-titer vaccinated and challenged), and 2D (placebo-vaccinated and not challenged) were 25.0%, 25.0%, 38.0%, and 60.0% from day 116 (2 d before challenge) to DOF + 20, respectively. No significant differences in frequency of gilts positive for clinical disease were detected among vaccinated groups and the challenge control group during this time period (P ≥ 0.7043).
In Experiment 1, mean rectal temperature for gilts was within the normal limits throughout the study and ranged from 37.9°C to 38.8°C in group 1A and from 38.0°C to 39.2°C in group 1B (Figure 1).
No significant differences were found between groups 1A and 1B for median percentages of live piglets (healthy + weak + crushed/mortality) per litter at farrowing (89.0% and 91.7%, respectively; P = 1.0000). Median percentages of healthy live piglets per litter (83.8% and 89.3%, respectively; P = 0.9262) and median percentages of weak live, stillborn, mummified, and crushed/mortality piglets per litter were 0.0% for all parameters for both groups (P ≥ 0.8825). There was no significant difference between groups for median percentages of live piglets at the time of weaning (100.0% and 90.5% for groups 1A and 1B, respectively; P = 0.2103).
Groups 2B and 2C had significantly higher percentages of live and healthy live piglets per litter (P ≤ 0.0455) than group 2A. Group 2C had significantly lower percentages of weak live piglets per litter (P = 0.0024), groups 2B and 2C had significantly lower percentages of mummies per litter (P ≤ 0.0190), and there were no significant differences among groups for the percentages of stillborn or crushed/mortality piglets per litter (P ≥ 0.1965). Median percentages of live piglets per litter at weaning were significantly higher in groups 2B and 2C than in group 2A (P = 0.0203 and P = 0.0022, respectively). The number of piglets born in each category per group is summarized in Figure 2, while the number of piglets weaned per group is shown in Table I.
There was no significant difference between groups 1A and 1B for median percentages of piglets per litter that were positive for clinical disease for at least 1 d (8.7% and 10.6%, respectively; P = 0.9814). In Experiment 2, median percentages of piglets per litter that were positive for clinical disease for at least 1 d were significantly lower in groups 2B and 2C than in group 2A (25.0% and 25.0% versus 100.0%, respectively; P ≤ 0.0001).
For piglets in both Experiments 1 and 2, there were no significant differences in body weight among groups on DOF (P = 0.6766 and P ≥ 0.2972, respectively). Specifically, least squares mean body weights for piglets in groups 1A and 1B were 1.55 and 1.50 kg, respectively, and the mean body weight for piglets in groups 2A, 2B, 2C, and 2D were 1.34, 1.43, 1.40, and 1.39 kg, respectively. No significant difference in least squares means for ADWG was detected between groups 1A and 1B from DOF to DOF + 21 (222 g/d and 226 g/d, respectively; P = 0.8760). Conversely, piglets in groups 2B and 2C had significantly higher least squares mean ADWG than those in group 2A from DOF to DOF + 20 (194 g/d and 197 g/d versus 130 g/d, respectively; P ≤ 0.0028) when weight on DOF was used as a covariate for analysis (Figure 3).
No PRRSV RNA was detected in the serum of any gilt in either experiment on day 0. In Experiment 1, all gilts in group 1A remained negative for the duration of the experiment. After vaccination (day 0), 2 out of 8 (25%) of gilts in group 1B were positive for PRRSV RNA by qPCR on day 14 (difference not significant; P = 0.4667), whereas no gilts in either group were positive on DOF and DOF + 21. In Experiment 2, all gilts in both groups 2A and 2D remained negative until the day of challenge and for the duration of the experiment, except for 1 gilt in group 2D that was positive on DOF + 7 (tested negative at all other time points).
After vaccination (day 0), 50% of gilts in groups 2B and 2C were PRRSV RNA positive, whereas 36% were PRRSV RNA positive on day 7. From days 14 to 56, only 4% of gilts in group 2B remained positive, while up to 4% of gilts in group 2C were intermittently qPCR positive. All gilts were qPCR negative for PRRSV RNA on day 84 and day 118 (day of challenge). After the challenge, groups 2B and/or 2C had significantly lower percentages of positive gilts than group 2A on day 125 (7 days post challenge; DPC) (31.0% and 25.0% versus 100.0%, respectively; P ≤ 0.0001), day 132 (14 DPC) [19.0% (P = 0.0290) and 31.0% (P = 0.1556) versus 63.0, respectively], DOF (25.0% and 6.0% versus 94.0%, respectively; P < 0.0002), and DOF + 13 (0.0% and 6.0% versus 47.0%, respectively; P ≤ 0.0155). No significant differences in percentages of positive gilts were detected between the vaccinated groups and the challenge control group on DOF + 7 and DOF + 20 (P ≥ 0.1719) (Figure 4).
No significant differences between groups in Experiment 1 were found in median percentages of piglets that were positive for PRRSV RNA on the day of farrowing. Specifically, median percentages of zero of pre-colostral blood samples from live piglets per litter from groups 1A and 1B were qPCR positive for PRRSV RNA on DOF (P = 0.4615). When blood/fluid samples from piglets born dead were included in the analysis, however, median percentages of positive piglets were 0.0% (group 1A) and 16.7% (group 1B) (P = 0.0769). Likewise, no significant differences were noted between live piglets in groups 1A and 1B on DOF + 14 (0.0% and 34.6%, respectively; P = 0.0769) and DOF + 21 (0.0% and 37.5%, respectively; P = 0.0769).
Conversely, significant differences in median percentages of piglets positive for PRRSV RNA were found on multiple occasions when all piglets (alive or dead) born to gilts in Experiment 2 were evaluated. Specifically, groups 2B and 2C had significantly lower median percentages of positive piglets than group 2A (P = 0.0381 and P = 0.0018, respectively) on DOF. Similarly, groups 2B and 2C had significantly lower median percentages of positive piglets than group 2A on DOF + 7 (P ≤ 0.0293). On DOF + 13, however, only group 2B had a significantly lower median percentage of positive piglets than group 2A (P = 0.0216), while the difference between groups 2C and 2A was not significant (P = 0.0860). No significant differences were detected among groups for percentages of positive piglets on DOF + 20 (P ≥ 0.0614) (Table II).
All gilts in Experiment 1 were seronegative for PRRSV by ELISA testing on day 0. After vaccination, 75% of gilts in group 1B were shown to have seroconverted by day 14 and 100% by DOF. Gilts in group 1A remained seronegative throughout the study. In Experiment 2, all gilts were seronegative for PRRSV by ELISA testing on day 0 and day 7. All gilts in groups 2A and 2D remained seronegative up to and including the day of challenge (day 118) and until the conclusion of the trial (DOF + 20), respectively. Conversely, on day 14, the percentage of PRRSV ELISA positive gilts in group 2B was 18% and in group 2C was 21.0%, which increased to 65.0% in 2B and 60.0% in 2C on day 56. On the day of challenge (day 118), percentages of seropositive gilts in groups 2B (56.0%) and 2C (50.0%) were decreasing. On day 125, percentages of seropositive gilts in groups 2B and 2C were higher than in group 2A (88.0% and 100.0% versus 6.0%, respectively). On day 132, all remaining gilts in groups 2A, 2B, and 2C were PRRSV seropositive through to the conclusion of the trial (DOF + 20).
On DOF + 21 (Experiment 1), persistent swelling in the right neck region of 1 gilt in group 1B was evaluated. Necropsy and histopathologic examination revealed a well-encapsulated abscess approximately 3 cm in size. There were no other notable findings for any gilt in Experiment 1.
One piglet in group 2A died after blood collection and death was confirmed at necropsy to be due to a lacerated jugular vein and secondary blood loss. In Experiment 1, all piglets in group 1A that were necropsied on DOF + 21 had no lung pathology in any lobe, whereas 3 out of 68 piglets in group 1B had minor lung lobe pathology (total lung lesion scores of ≤ 1.85%; lesions described as very small areas of lung consolidation likely due to a past bacterial infection) and 1 piglet had 100% pathology in each lung lobe (total lung lesion score of 100%; lesions described as pneumonia and pleuritis likely due a bacterial infection). There were no significant differences among groups for median percentages of piglets per litter with lung lesion scores (P = 0.2000) and for median total lung lesion scores (P = 0.0531) (Table III).
There was no significant difference between groups 1A and 1B in median percentages of piglets per litter that were positive for PRRSV RNA in the lung tissues by qPCR (0.0% and 10.0%, respectively; P = 0.0769). In Experiment 2, of the piglets that were either born dead, died, or were euthanized before DOF + 20, the mean lung qPCR results were 4.676, 4.092, 3.547, and 0.000 log10 GE/mL for groups 2A, 2B, 2C, and 2D, respectively (Table IV) (no statistical analysis conducted).
Vaccinating gilts and sows with a PRRS MLV vaccine is beneficial for several reasons, including increased farrowing and weaning rates and decreased number of premature farrowings (1,4,7,8). Vaccination of dams also has positive effects on the piglets, including increased ADWG and decreased clinical signs (12). The effectiveness of a PRRS MLV vaccine in improving reproductive performance is vital because loss from piglet deaths, for whatever reason (mummified, stillborn, and/or disease-related), results in fewer piglets weaned and therefore dramatically affects the economic productivity of the herd. A PRRS MLV vaccine must be safe to administer, however, in that it does not cause reproductive failure, local or systemic reactions, and negatively affect piglet survivability and growth performance.
In Experiment 1, primary criteria for evaluating safety included reproductive performance at farrowing and survival rate at weaning, with supportive parameters of local and/or systemic reactions to vaccination and transplacental infection, as well as general health of piglets (clinical observations and growth performance) during the suckling period.
In Experiment 2, effectiveness was primarily determined by evaluating reproductive performance at farrowing (number of live-born piglets) and the number of piglets at weaning. This was supported by evaluating gilts for clinical assessments post-vaccination and post-challenge, PRRSV serology, and viremia, as well as evaluation for total number of piglets, healthy live piglets, weak live piglets, mummies, stillborn piglets, and crushed/mortality piglets born per litter. Piglets were assessed for viremia, clinical observations, and ADWG. According to these criteria, the present data clearly demonstrated that vaccination of gilts with the novel PRRSV strain 94881 MLV vaccine (ReproCyc PRRS EU; Boehringer Ingelheim Vetmedica) is a safe option for preventing reproductive losses associated with the PRRS virus.
Specifically, PRRS-naive gilts that were given a repeat 10× overdose did not exhibit any relevant differences in median percentages of live piglets born or live piglets at weaning compared to non-vaccinated placebo controls. During the suckling period, there were no significant differences between the vaccinated and non-vaccinated groups for systemic reactions to vaccination (post-vaccination observations), gilt viremia, and general health of piglets (clinical observations, least squares mean body weight at birth, and ADWG). There were no significant differences between groups for injection site observations after the initial 10× overdose and no biologically relevant differences in rectal temperatures after vaccination (did not exceed 40.0°C).
In Experiment 2, the vaccine also met the necessary requirements for efficacy as significantly higher percentages of live pigs were born and weaned in the vaccinated group than in the non-vaccinated challenge control group. There were significant beneficial differences in 1 or both vaccinated groups compared to the challenge control group for percentages of healthy live piglets, weak live piglets, and mummified piglets born per litter. Results of gilt post-vaccination clinical observation were in alignment with the safety study, as there were no abnormal assessments for any group from day 1 through day 21 and no significant difference among groups for abnormal clinical assessments for at least 1 d from day 1 through day 113.
Similarly, there were no significant differences in gilt clinical observations post-challenge between the low-titer and high-titer vaccinated groups compared to the placebo-vaccinated and challenged group. Interestingly enough, however, a higher percentage of gilts in the negative control group (placebo-vaccinated and not challenged) exhibited a clinical observation for at least 1 d from day 116 through DOF + 20. Vaccinated gilts were also shown to have seroconverted as early as 14 d post-vaccination. Vaccinated gilts experienced a brief period of viremia after treatment was administered, as evidenced by positive gilts on day 7. Differences in viremia between vaccinated groups (groups 2B and 2C) were not significant from days 14 through 56, however, and all gilts were negative on day 84 and day 118.
Conversely, there were significantly lower percentages of qPCR-positive gilts in the vaccinated groups than in the challenge control group after the challenge [on days 125, 132 (low dose only), DOF, and DOF + 13]. Piglets born to vaccinated gilts also exhibited favorable results compared to piglets born to challenge control gilts, as 1 or both vaccinated groups had significantly lower percentages of piglets per litter that were positive for abnormal clinical findings for at least 1 d from DOF + 1 through DOF + 20, significantly higher ADWG, and significantly lower percentages of piglets per litter that were viremic on DOF, DOF + 7, and DOF + 13. These results clearly indicate that even a 10× overdose of the vaccine was safe to administer during gestation and effectively prevented the detrimental effects of PRRSV infection in both gilts and piglets born to vaccinated gilts.
While a number of studies have been published describing the safety of NA-type PRRS modified live virus (MLV) vaccines in bred females, less information is available about the safety of EU-type MLV vaccines. One trial examined the safety of a single dose of a commercially available EU-type MLV PRRSV vaccine in gilts and sows that were in either early or late pregnancy and in lactating sows (7). In the trial, safety was established according to the lack of significant differences in local or systemic reactions between vaccinated and non-vaccinated animals. Additionally, fluid and/or tissue samples collected from aborted piglets were negative for PRRSV RNA. These results are similar to the present safety evaluation data, as vaccinates exhibited neither significantly increased incidence of local or systemic reactions after the first vaccination and there were no significant differences between groups for piglet viremia on DOF, even though a 10× overdose of the vaccine had been administered.
A number of studies have examined the effects of the use of EU-type MLV vaccines in endemic situations. The effects of an EU-type MLV PRRS vaccine were evaluated in lactating sows and pregnant gilts and sows on 3 Polish farms with histories of chronic PRRSV infection (7). The vaccine was found to be beneficial as live piglets born and piglets weaned increased significantly compared to non-vaccinated animals. When the vaccine was administered to the remainder of the breeding females, regardless of pregnancy status, there were significant favorable differences in farrowing rate, live-born piglets, stillborns, weaned pigs, and abortions compared to pre-vaccination data for the same animals.
In a study conducted on an endemically PRRSV-infected farrow-to-finish farm in Greece, a group of gilts and sows were administered a single dose of an EU-type MLV vaccine at approximately 6 mo of age and 10 d after farrowing, respectively, while an additional group of gilts and sows were left untreated (1). The vaccinated group had significantly improved farrowing rates, higher numbers of live-born piglets, fewer dead or mummified piglets, and more weaned pigs (average of 0.7 piglets per litter) than the non-vaccinated group.
Another study described significantly improved farrowing rates, including higher numbers of live-born and fewer numbers of mummified and stillborn piglets per litter, when pigs on PRRSV-positive farms in Thailand were treated with either an EU-type or NA-type PRRSV MLV vaccine (4). Unfortunately, the data were reported as a generalization of vaccinates versus non-vaccinates (no groups based on timing of vaccination or type of PRRSV vaccination received). The authors did note, however, that vaccination improved fertility rate in gilts by a greater percentage (7.3%) than in sows (6.7%, 3.8%, 2.0%, and 0.4% in parity numbers 1, 2 to 3, 4 to 5, and ≥ 6, respectively).
While results similar to the present data [significantly improved reproductive performance at farrowing (more live and healthy live piglets born and fewer weak live piglets and mummies born) and more live pigs at weaning (average of 3.65 piglets per litter)] have been reported, these studies contrast directly with Experiment 2 as they did not involve an administered challenge of known virulence at a time of high susceptibility, the serological status of each animal before vaccination was not known, and the PRRSV strains present at the trial locations were not identified.
Only 1 study was found that examined the efficacy of 2 EU-type MLV PRRSV vaccines in gilts that also involved an administered challenge of PRRS virus. Using gilts vaccinated (IM) once at 24 d before AI and subsequently challenged with a heterologous strain, Scortti et al evaluated the effects of 2 different EU-type MLV PRRSV vaccines on gilts and their progeny (8). While no adverse reactions to either vaccine were noted, all vaccinated gilts except 1 developed vaccine-induced viremia. Neither vaccine provided complete protection against challenge-induced viremia as the virus was isolated in the serum of approximately 40% of all vaccinated gilts. Conversely, reproductive performance significantly improved in both groups of vaccinated gilts, with more live piglets and fewer stillborn and mummified piglets born, and decreased incidence of transplacental infection of piglets after the challenge. Additionally, ADWG and piglet mortality rates were not significantly affected by congenital infection. While the reproductive results in this study are similar to the data presented here, only 50.0% of vaccinated gilts in group 2B and 36.0% of vaccinated gilts in group 2C became viremic as a result of vaccination. The majority of vaccinated gilts (69.0% of group 2B and 75.0% of 2C) were protected from challenge-induced viremia and, while congenital infection was noted for piglets born to vaccinated gilts, piglet growth and survival rates were significantly improved by vaccination.
The data presented here has shown that this novel vaccine strain provides gilts with far better protection against PRRSV during pregnancy than inactivated vaccines. Inactivated EU-type vaccines have been shown to be protective in the face of a homologous PRRSV challenge, significantly increasing percentages of live and healthy piglets born (18) and decreasing duration of viremia and occurrence of congenital infection, reducing percentage of mummified fetuses, and improving fetal survival (16). When a heterologous challenge was used, however, an inactivated EU-type vaccine failed to significantly protect gilts from clinical signs and viremia and decreased reproductive performance and transplacental infection (17).
These cited studies support the fact that the heterogeneity of the PRRS virus continues to make it difficult to develop a single-strain vaccine virus that is effective against the various strains of PRRSV in the field. It is difficult to directly compare the safety and efficacy of EU-type PRRS MLV vaccines in breeding females, due to the limited nature of available data. Vaccination with a PRRS MLV vaccine, however, has generally been found to positively affect reproductive performance when used in PRRSV endemic situations. The present study provides evidence that a vaccine based on the novel PRRSV MLV strain 94881 is a safe and effective way to protect against the negative clinical and economic effects of PRRS viral infection when administered to gilts before breeding.
When the novel PRRS 94881 MLV vaccine, ReproCyc PRRS EU-type, was administered as a repeat 10× overdose (approximately 1 × 108 TCID50/6.0 mL) to bred gilts at approximately 90 d of gestation, there were no relevant differences for piglets in live-born, healthy live-born, general health during the suckling period, ADWG, live piglets at weaning, lung pathology, and viral load in lung tissue, and for gilts, in systemic reactions to vaccination and viremia. When a single dose of the test vaccine at various titer formulations was administered to breeding-age gilts 26 to 32 d before breeding, vaccinated gilts had more live born, healthy live-born, and weaned piglets per litter. The piglets had higher ADWG, fewer weak live and mummified piglets per litter, fewer piglets with abnormal clinical signs, fewer incidences of piglet viremia, no statistically relevant abnormal clinical assessments post-vaccination, positive serological responses as early as 14 d post-vaccination, and lower incidence of viremia following challenge.
In conclusion, A vaccine formulated from the PRRSV MLV strain 94881 has therefore been proven to be a safe and effective method of protection against the detrimental effects of virulent PRRSV infection in breeding female pigs.
This work was funded by Boehringer Ingelheim Vetmedica. The authors thank Dr. Ryan Saltzman, Dr. Lyle Kesl, Dr. Stephan Pesch, Dr. Martin Vanselow, Mr. Rex Smiley, and Ms. Sarah Layton for technical assistance.