Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) can be difficult to manage in commercial settings. A novel type I PRRSV vaccinal strain (94881) was evaluated for safety and efficacy/onset of immunity (OOI) in piglets. In 2 experiments, groups of piglets were vaccinated intramuscularly (IM) at approximately 14 d of age with a maximum-range commercial dose, an overdose, or a placebo in experiment 1 and either a minimum-range commercial dose or a placebo in experiment 2. The piglets in experiment 1 were evaluated for local and systemic reactions from days −2 through 14 after vaccination. The piglets in experiment 2 were challenged with a virulent heterologous type I PRRSV isolate 14 d after vaccination and observed once daily for general health from days −1 through 12 after vaccination and once daily for clinical signs associated with challenge from days 13 through 24 after vaccination. The average daily weight gain (ADWG) and the results of serologic and viremia testing were evaluated in experiments 1 and 2. Lung lesion scores and results of testing for PRRSV in lung tissue were evaluated in experiment 2. In experiment 1 the vaccine was shown to be safe, as there were no relevant differences between the vaccinated piglets and the piglets given a placebo. In experiment 2 the vaccine’s efficacy, with an OOI of 14 d after vaccination, was established, as the vaccinated and challenged piglets exhibited significantly lower lung lesion scores, viremia, viral load in lung tissue, and total clinical sign scores, along with a significantly greater ADWG, compared with the placebo-vaccinated and challenged piglets.
Résumé
La gestion du virus du syndrome reproducteur et respiratoire porcin (VSRRP) peut être difficile dans un environnement de production commerciale. Une nouvelle souche vaccinale du VSRRP de type 1 (94881) a été évaluée d’un point de vue sécurité et efficacité/début de l’immunité (DDI) chez des porcelets. Dans deux expériences, des groupes de porcelets ont été vaccinés par voie intramusculaire (IM) à l’âge d’environ 14 j avec une dose commerciale maximale, une surdose, ou un placebo dans l’expérience 1 et une dose commerciale minimale ou un placebo dans l’expérience 2. Les porcelets dans l’expérience 1 furent évalués pour des réactions locale et systémique à compter du jour −2 jusqu’au jour 14 post-vaccination. Les porcelets dans l’expérience 2 furent soumis à une infection défi avec un isolat virulent hétérologue de VSRRP de type 1 14 j après la vaccination et observés une fois par jour pour leur état de santé général du jour −1 jusqu’au jour 12 après la vaccination et une fois par jour pour des signes cliniques associés avec l’infection du jour 13 au jour 24 après l’infection. Le gain moyen quotidien (GMQ) et les résultats des analyses sérologiques et de virémie ont été évalués dans les expériences 1 et 2. Les pointages de lésions pulmonaires et les résultats de détection du VSRRP dans le tissu pulmonaire ont été évalués dans l’expérience 2. Dans l’expérience 1, le vaccin s’est montré sécuritaire étant donné qu’il n’y avait aucune différence significative entre les porcelets vaccinés et les porcelets ayant reçu un placebo. Dans l’expérience 2, l’efficacité du vaccin, avec un DDI de 14 j après la vaccination, a été établie, étant donné que les porcelets vaccinés, et soumis à une infection défi avaient des valeurs significativement moins élevées de pointage de lésions pulmonaires, de virémie, de charge virale dans le tissu pulmonaire, et des pointages de signes cliniques totaux, avec un GMQ significativement plus élevé, comparativement au porcs vaccinés avec un placebo et soumis à une infection défi.
(Traduit par Docteur Serge Messier)
Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic concern in the swine industry owing to losses resulting from decreased growth performance and increased mortality rates in weaned pigs (1–4). Vaccination with a modified live virus (MLV) vaccine is considered to be of value for decreasing these losses; however, the diversity of the virus tends to interfere with the protection provided by vaccines. Currently the virus has 2 main genotypes, type I (European) and type II (North American), which are genetically and antigenically different (1,3,5–11). Furthermore, 4 subtypes of type I PRRSV based on sequence analyses of open reading frame 5 (ORF5) have been identified (8). Although similarities exist between and within the type I strains, there is enough diversity that vaccines may not induce sufficient cross-protection against heterologous strains. Pileri et al (12) stated that the genetic diversity of PRRSV is such that all challenge situations in the field could be considered heterologous. Some studies have determined that the level of genetic similarity between the vaccine strain and the challenge strain was not necessarily an accurate predictor of vaccine efficacy (4,13), whereas others have concluded that the level of protection a vaccine provides against PRRSV infection may depend on the degree of relatedness between the vaccine and challenge strains (7,10).
It has been reported that MLV vaccines against PRRSV are at least partially effective at reducing symptoms and viremia, as well as virus shedding (14). Furthermore, MLV vaccines based on a single virus strain offer consistently good protection against homologous challenge; however, protection against heterologous challenge is neither as consistent nor as effective (2,6,14). In an attempt to broaden vaccine protection, a vaccine containing 5 type II strains of attenuated PRRSV was developed; this vaccine, however, was found to offer no more effective protection against heterologous challenge than a single-strain vaccine (15). Geldhof et al (5) summarized the difficulties facing development of an effective PRRSV vaccine by reporting that infection with different strains leads to diverse virologic and immunologic effects (i.e., various degrees of homologous and heterologous protection) and that the susceptibility of PRRSV strains to antibody neutralization likewise differs. Since new strains, including highly pathogenic ones (3,16), continue to be found, it is important to continue to search for a single-strain vaccine that is effective in the face of heterologous challenge. One important factor to consider in this search is the virulence of the challenge strain: it should reflect what is found currently in the field. In the past, clinical signs of PRRSV infection have been difficult to reproduce with a single-strain type I PRRSV challenge, through either virus administration or contact with infected pigs (1,4,7,12,17,18). In addition to efficacy concerns, the safety of PRRS MLV vaccines has been questioned. It has been reported that these vaccines may cause clinical signs of respiratory disease and decreased growth performance, as well as viremia, and that the virus may subsequently spread to other naïve animals when administered to piglets (2,5,9,14,19). Additionally, PRRS MLV vaccines have been found to initiate protection in a relatively delayed manner, at around 3 to 4 wk after administration (2,17,18).
The present study was conducted to evaluate the safety and efficacy of a new European-derived PRRS MLV vaccine based on a novel type I strain (1) [European Collection of Cell Cultures accession numbers 11012501 (parent strain) and 11012502 (attenuated strain)] when administered to young piglets in the face of a disease-inducing heterologous PRRSV challenge.
Materials and methods
Animals
The protocols were reviewed and approved by the Contract Research Organization’s Institutional Animal Care and Use Committee before the study was started. Two experiments were conducted to establish safety (experiment 1) and efficacy/onset of immunity (OOI) (experiment 2) with 32 and 50, respectively, commercial mixed-breed castrated male and female piglets. The piglets in experiment 1 were blocked by weight and randomly assigned to group A (1A, n = 11), B (1B, n = 11), or C (1C, n = 10), except for the last block, which was assigned to either 1A or 1B. The piglets in experiment 2 were assigned to 1 of 3 treatment groups in the following manner: the piglets were blocked by weight, assigned a random number by means of the random number function in Microsoft Excel (Microsoft Corporation, Redmond, Washington, USA), and ranked in numerically ascending order by block; then the 2 lowest numbers of each block were assigned to group A (2A, n = 20), the next 2 numbers were assigned to group B (2B, n = 20), and the highest number was assigned to group C (2C, n = 10).
The piglets in experiment 2 were administered ceftiofur crystalline-free acid (Excede; Pharmacia & Upjohn Company, Division of Pfizer, New York, New York, USA) intramuscularly (IM) in the right ham, according to label directions, at the time of arrival. The piglets in groups 1A, 1B, and 2A received the test vaccine (Ingelvac PRRSFLEX EU; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) (1) at the maximum-range commercial dose, at an overdose, and at the minimum-range commercial dose, respectively, whereas the piglets in groups 1C, 2B, and 2C were administered a placebo. All the piglets were 14 ± 3 d of age at the time of treatment and were clinically healthy. The piglets in groups 1A, 1B, and 1C were monitored for local and systemic reactions to determine vaccine safety. The piglets in groups 2A and 2B were challenged with a heterologous strain of type I PRRSV, and those in group 2C were not challenged, serving as negative controls, to evaluate vaccine efficacy and OOI.
All the piglets were housed in multiple raised pens (5 or 6 pigs per pen) equipped with a nipple waterer, a feeder, and plastic slatted flooring. Each group was housed in separate but similar rooms for the duration of the experiments. The rooms were biosafety level 2-compliant, thermostat-controlled, and mechanically ventilated, with high-efficiency particulate air filtering, to ensure biosecurity. Additionally, appropriate measures were taken to prevent accidental cross-contamination from vaccinates and/or challenged animals. All the piglets were fed an age-appropriate commercially available ration medicated with tiamulin, 35 g/tonne, and chlortetracycline, 400 g/tonne (Lean Metrics Infant; Purina Mills, St. Louis, Missouri, USA). Feed and water were available ad libitum.
Serologic and viremia testing before vaccination
Blood was collected from all the piglets before vaccination. Briefly, venous blood was collected into serum separator tubes and allowed to clot at room temperature. Aliquots of serum were dispensed into appropriate tubes and held at either 2°C to 8°C or −70°C ± 10°C before testing. The samples held at 2°C to 8°C were tested for PRRSV antibodies at the Boehringer Ingelheim Vetmedica Health Management Center, Ames, Iowa, USA, with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (IDEXX HerdChek PRRS X3 ELISA; IDEXX Laboratories, Westbrook, Maine, USA). Results were reported as negative [sample to positive (S/P) ratio of < 0.4] or positive (S/P ratio ≥ 0.4). The samples held at −70°C ± 10°C were tested for PRRSV RNA by quantitative polymerase chain reaction (qPCR; bioScreen GmbH, Münster, Germany) as described by Revilla-Fernandez et al (20) with use of the 2× TaqMan Universal PCR Kit (Applied Biosystems, Foster City, California, USA) with AmpErase uracil N-glycosylase (Applied Biosystems), the EU6-MGB (CTGTGAGAAAGCCCGGAC) probe, and the primers EU6-343f-plus (GTRGAAAGTGCTGCAGGYCTCCA; sense) and EU6-462r-plus (CACGAGGCTCCGAAGYCCW; antisense). A PCR run was considered valid when the plasmid standard curve was linear, its R2 value was greater than or equal to 0.95, and the nontemplate control did not cross the threshold line. Test samples were evaluated for the presence or absence of a signal crossing the threshold, shown as the cycle threshold (Ct) value. For reporting purposes the samples were designated as positive or negative according to the point at which the PCR signal crossed the threshold in all replicates. Genomic equivalence (GE) was determined with use of the plasmid standard. The standard curve was generated and then used to calculate the initial concentration of an unknown sample by comparing its Ct value with the standard curve. The resulting initial concentration of the reaction (GE per reaction) was extrapolated to the amount of GE per milliliter.
Vaccination
All piglets in both experiments were administered the test vaccine or placebo at 14 ± 3 d of age. Those in groups 1A and 1B were vaccinated IM once with 1 mL of the vaccine (according to the manufacturer’s label instructions), at the maximum-range commercial dose (the maximum release dose of the vaccine at the time of production) and at an overdose (10 times the release dose), respectively. The piglets in group 1C served as negative controls and were administered 1 mL of sterile phosphate-buffered saline IM. The piglets in group 2A were vaccinated IM with 1 mL of the vaccine at the minimum-range commercial dose (minimum dose that provides protection). The piglets in groups 2B and 2C were injected IM with 1 mL of a lyophilized placebo product containing the inert vaccinal material without the PRRS MLV fraction.
Observations after vaccination
The piglets in experiment 1 were observed once daily for clinical signs of disease in terms of behavior (recumbency, shivering, lethargy, unconsciousness, or death), respiration (mild to severe coughing, sneezing, abdominal breathing, or rapid respiration), and digestion (vomiting, diarrhea, or change in appetite), as well as for other relevant clinical signs (e.g., hernia, thinness, lameness, and edema around the eyes) on days −2 and −1 and from days 1 to 14 after vaccination, as well as twice on day 0 (i.e., just before and at 4 h after vaccination). The piglets in experiment 2 were observed once daily for general health on days −1 to 12 after vaccination.
After vaccination, all injection sites in the piglets in experiment 1 were clearly identified with a circle by means of a black water-resistant marker and were examined for redness, swelling, heat, and pain on day 0 at 4 h after vaccination and once daily on days 1 to 14 after vaccination.
Challenge inoculation
On day 14 after vaccination the piglets in groups 2A and 2B were challenged with the virulent, low-passage type I PRRSV isolate 205817, at a median tissue culture infective dose of 1 × 104.71 per 3 mL. The challenge inoculum was derived from isolate 190136, originally obtained from the lung tissue of a newborn piglet on a farm showing typical reproductive signs of PRRSV (abortions in sows and weakness in newborn piglets) during an outbreak in Lower Saxony, Germany, in April 2004. The challenge isolate (205817) and the vaccine master seed isolate (94881) are heterologous members of type I PRRSV, subtype 1, isolated from geographically distinct regions in Germany and exhibiting less than 87% genetic identity within the complete genome (or 88% with the ORF5/ORF7 data) (21).
Observations after challenge
Clinical observations were conducted on the piglets in experiment 2 once daily from days 13 to 24 after vaccination (days −1 to 10 after challenge) by personnel that were blinded to the treatment-group assignment. Each piglet was visually examined in the pen before handling and was scored for clinical signs including coughing (none, soft or intermittent, harsh or severe and repetitive, or dead) and abnormalities of respiration (normal, panting/rapid respiration, dyspnea, or dead) and behavior (normal, mild to moderate lethargy, severely lethargic/recumbent, or dead).
Rectal temperature was determined with a self-calibrating digital thermometer in the piglets in experiment 1 on days −2 and −1 and from days 1 to 14 after vaccination, as well as twice on day 0 (just before and at 4 h after vaccination), and in the piglets in experiment 2 from days 13 to 24 after vaccination (days −1 to 10 after challenge). A normal physiological range was considered to be 38.7°C to 39.9°C, and fever was defined as an increase in temperature to 40.0°C or beyond.
In experiment 1 the individual body weight was measured in all the piglets on days −2, 0, and 14 after vaccination, and the average daily weight gain (ADWG) was determined from days 0 to 14 after vaccination. In experiment 2 the individual body weight was measured in all the piglets on days 0, 14, and 24 after vaccination, and the ADWG was calculated from days 0 to 14 and days 14 to 24 after vaccination.
Serologic and viremia testing for PRRS
Blood was collected via jugular venipuncture from the piglets in experiment 1 on days 7 and 14 after vaccination and from the piglets in experiment 2 on days 7, 14, 17, 21, and 24 after vaccination. Blood was processed for serum and used to determine the S/P ratio of antibodies against PRRSV by ELISA and PRRSV viremia (log10 GE/mL) by qPCR as described.
Postmortem examination and lung studies
All the piglets in experiment 1 were euthanized by sedation and then electrocution and underwent necropsy on day 14 after vaccination. Each injection site was palpated, incised, and evaluated for gross lesions. Additionally, the thoracic and abdominal cavities were exposed and examined for gross lesions. Two lung tissue samples containing lesions were collected from 1 piglet (in group 1A). One sample was placed in a Whirlpak (United States Plastic Corporation, Lima, Ohio, USA) and the other in a container with 10% formalin and submitted to the Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa, USA. The fresh sample was cultured for bacteria and the formalin-fixed sample examined histopathologically and by immunohistochemistry testing for PRRSV, porcine circovirus 2 (PCV-2), and Mycoplasma hyopneumoniae antigens.
All the piglets in experiment 2 were similarly euthanized and underwent necropsy on day 24 after vaccination (day 10 after challenge). The thoracic cavity was exposed, and the heart and lungs were removed. The lungs were examined for gross lesions, and the percentage of each lobe that was abnormal was recorded. Total lung lesion scores were determined 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 (i.e., 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 determined.
Lung samples were collected from all the piglets in experiment 2 for quantitation of PRRSV in the lung tissue. Samples in Whirlpaks were stored at −70°C ± 10°C until thawed and liquefied into a homogenate, subjected to RNA extraction procedures, and then tested for PRRSV RNA by qPCR as described. The results were reported as log10 GE/mL for left and right/intermediate lung samples.
Statistical analyses
The statistical analyses were conducted and data summaries prepared by Dr. Martin Vanselow (Biometrie & Statistik, Hannover, Germany) using SAS software release 8.2 or later (SAS Institute, Cary, North Carolina, USA).
For experiment 1, all data listings and summary statistics by treatment group, including mean, median, standard deviation, and/or frequency distribution, were generated for all primary variables, including local (injection-site) reactions, systemic (behavioral, respiratory, digestive, and other, as well as fever) reactions, and ADWG. Specifically, the proportions of piglets in each group with any abnormal clinical observation, with a specific clinical observation (behavioral, respiratory, digestive, or other score greater than 0), with an increase in rectal temperature greater than 1.5°C when compared with baseline (day 0), with any injection-site reaction score greater than 0, or with a specific injection-site reaction score greater than 0 for at least 1 d from 0 + 4 h to 14 d after vaccination were evaluated by Fisher’s exact test; the numbers of days per animal with any abnormal clinical observation and with any injection-site reaction from 0 + 4 h to 14 d after vaccination were evaluated by the Wilcoxon Mann–Whitney test; the mean daily rectal temperature for each group from 0 + 4 h to 14 d after vaccination and the initial mean body weight for each group on day 0 were evaluated by analysis of variance (ANOVA); and the mean ADWG for each group from 0 to 14 d after vaccination was evaluated by the t-test.
For experiment 2, data were analyzed with the assumption of a randomized block design, and all tests on differences were designed as 2-sided at an α-value of 5%. Frequency tables of animals with at least 1 positive clinical finding between days 1 and 12 after vaccination, animals with at least 1 positive clinical finding from days 15 to 24 after vaccination (days 1 to 10 after challenge), and animals with positive ELISA results were generated, and differences between groups were compared by Fisher’s exact test. Differences in mean daily rectal temperature between treatment groups, as compared with baseline, were tested by ANOVA and t-tests, and least-squares (LS) means of groups and differences between LS means with 95% confidence intervals were calculated from the ANOVA. Comparisons between treatment groups for total lung lesion scores, maximum and mean clinical scores (for coughing and abnormalities of respiration and behavior, as well as for all 3 added together) per animal for days 15 to 24 after vaccination, the PRRSV qPCR (viremia) data (evaluated separately for each day), as well as the area under the curve (AUC) for individual responses between days 14 and 24 and between days 17 and 24 after vaccination were analyzed with the Wilcoxon Mann–Whitney test.
Results
After vaccination no piglets in groups 1A and 1B (single-dose and overdose vaccination, respectively) exhibited abnormal behavior, whereas 1 piglet in group 1C (placebo) was lethargic on days 8 and 9. There was no significant difference in behavior (P = 0.4762) between the vaccinated groups and the nonvaccinated group. Conversely, at least 1 piglet in each group exhibited abnormal respiration for a minimum of 1 d after vaccination: 1 piglet in group 1A, 2 piglets in group 1B, and 3 piglets in group 1C exhibited mild coughing; sneezing was noted for 3 piglets in group 1C; and severe coughing was noted for 1 piglet. There was no significant difference in respiration (P ≥ 0.1486) between the vaccinated groups and the nonvaccinated group. No piglets exhibited any abnormal digestive findings after vaccination. Several abnormal clinical findings not related to PRRSV vaccination (designated as “other”) were also noted: 1 piglet in group 1A was noted as thin on days 7 to 10 after vaccination (likely a result of decreased feed intake due to competition at the feeder) but was otherwise normal; 1 piglet in group 1B was found to have rectal prolapse on days 6 to 12; and 1 piglet in group 1B was noted as having a scrotal hernia on days 10 to 14. There was no significant difference in “other” clinical signs (P ≥ 0.4762) between the vaccinated groups and the nonvaccinated group. Likewise, no significant differences in clinical observations were noted between either of the vaccinated groups and the nonvaccinated group when all categories of clinical observation were combined (P ≥ 0.3615) or in the number of days piglets exhibited any clinical abnormalities (P = 0.2662 and 0.7726 for groups 1A and 1B, respectively) (Table I). No clinical abnormalities related to PRRSV were noted in any piglets in experiment 2 after vaccination and before challenge (i.e., on days −1 to 12 after vaccination); however, 1 piglet in group 2B had a lesion anterior to the right front leg beginning 9 d after vaccination.
Table I.
Number of days on which piglets exhibited any abnormal clinical signs after vaccination against porcine reproductive and respiratory syndrome virus (PRRSV)
| Group | Number of piglets | Number of days | P-valuea |
|---|---|---|---|
| 1A (maximum dose) | 11 | 4 | 0.2662 |
| 1B (overdose) | 11 | 7 | 0.7726 |
| 1C (placebo) | 10 | 8 | NA |
In comparison with group 1C.
NA — not applicable (no analysis conducted).
At the injection site, redness, heat, and pain were not noted in any piglet in experiment 1, but 1 piglet each in groups 1A (single dose) and 1B (overdose) exhibited minimal swelling (palpable only) for at least 1 d after vaccination; no piglets in group 1C (placebo) exhibited swelling after vaccination. These differences were not significant (P = 1.0000) (Table II).
Table II.
Number of days after vaccination on which any injection-site reaction was noted
| Group | Number of piglets | Number of days | P-valuea | ||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| Minimum | Maximum | Median | 95% CI | Mean | |||
| 1A (maximum dose) | 11 | 0 | 4 | 0 | 0 | 0.4 | 1.0000 |
| 1B (overdose) | 11 | 0 | 3 | 0 | 0 | 0.3 | 1.0000 |
| 1C | 10 | 0 | 0 | 0 | 0 | 0.0 | NA |
In comparison with group 1C.
CI — confidence interval; NA — not available.
After challenge, abnormal respiration was observed in 2 (10%) of the 20 vaccinates (group 2A) and 6 (30%) of the 20 challenge controls (group 2B); however, these proportions were not significantly different (P = 0.2351). Groups 2A and 2B had maximum abnormal respiration scores of 1 (panting/rapid respiration) and 2 (dyspnea), respectively, a difference that was not significant (P = 0.1872); both groups had a median maximum respiration score of 0. The mean respiration score for group 2A was lower than that for group 2B (0.02 versus 0.07; P = 0.1394). Similarly, although coughing was observed in more of the group 2B piglets than in the group 2A piglets [11 of 20 (55%) versus 6 of 20 (30%)], the difference was not significant (P = 0.2003). Groups 2A and 2B had maximum scores for coughing of 1 (soft or intermittent; median score of 0) and 2 (harsh or severe and repetitive; median score of 1), respectively; these differences were not significant (P = 0.1129). The mean coughing score was lower for group 2A than for group 2B (0.07 versus 0.17; P = 0.0535). Abnormal behavior was observed in significantly fewer piglets (P = 0.0012) in group 2A than in group 2B: 0 of 20 versus 9 of 20 (45%). Group 2A had a significantly lower (P = 0.0012) maximum behavior score than group 2B: 0 (normal) versus 1 (mild to moderate lethargy). The mean behavior score for group 2A was significantly lower (P = 0.0012) than that for group 2B: 0.00 versus 0.12. When combined, the percentages of piglets with total clinical scores greater than 0 were 30% (6 of 20) in group 2A and 65% (13 of 20) in group 2B and were not significantly different (P = 0.0562). Group 2A had a significantly lower maximum total score than group 2B: 1 versus 4 (P = 0.0072). The mean total scores for group 2A were significantly lower (P = 0.0103) than those for group 2B: 0.08 versus 0.35. No clinical signs were observed in the negative controls (group 2C) at any time after challenge, and the group had a score of 0 for each parameter.
For the piglets in experiment 1, all the mean rectal temperatures were within the normal physiological range, and no piglets exhibited an increase in rectal temperature of 1.5°C or more above baseline on any day after vaccination. In experiment 2, the mean and LS mean rectal temperatures for the piglets in group 2A (vaccinated and challenged) were 39.77°C on the day before challenge (day 13 after vaccination) and ranged from 39.69°C to 40.68°C after challenge (on days 1 and 2 after challenge, respectively), for the piglets in group 2B (nonvaccinated and challenged) they were 39.39°C on the day before challenge and ranged from 39.77°C to 40.61°C after challenge (on days 2 and 6, respectively), and for the piglets in group 2C (nonvaccinated and not challenged) they remained at 39.68°C or less throughout the study. The LS means were significantly lower (P < 0.0001) for the piglets in group 2B than for those in group 2A the day before challenge (39.39°C versus 39.77°C), the day of challenge (39.37°C versus 39.76°C), and 2 d after challenge (39.77°C versus 40.68°C). There were no other significant differences (P ≥ 0.0528) between the 2 groups on any day, as the rectal temperatures for both groups were at or above 40°C from days 4 (group 2B) and 5 (group 2A) through day 10 after challenge (Figure 1).
Figure 1.
Mean rectal temperatures of piglets after challenge with a heterologous strain of type I porcine reproductive and respiratory syndrome virus (PRRSV) (groups 2A and 2B) 14 d after administration of the test vaccine (Ingelvac PRRSFLEX EU; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) at the minimum-range commercial dose (group 2A) or a placebo (groups 2B and 2C). The asterisks indicate a significant difference (P < 0.0001) in the temperatures for group 2B compared with group 2A on days −1, 0, and 2 after challenge.
In experiment 1 there were no significant differences in mean body weight between the groups on days 0 and 14 after vaccination (P = 0.7582 and P ≥ 0.2273, respectively) and no significant differences (P = 0.1562 and 0.1628, respectively) in ADWG from 0 to 14 d after vaccination (Table III). In experiment 2 there were no significant differences in LS mean body weight between the piglets in groups 2A and 2B on days 0 (P = 0.8743) and 14 (P = 0.4297) after vaccination. However, on day 24 after vaccination (day 10 after challenge) the LS mean body weight of the piglets in group 2A (10.26 kg) was significantly higher (P = 0.0063) than that of group 2B (8.87 kg). Likewise, the difference in LS mean ADWG between groups 2A and 2B was not significant (P = 0.1889) from days 0 to 14 after vaccination; however, from days 14 to 24 (days 0 to 10 after challenge) the LS mean ADWG was significantly higher (P = 0.0003) for the piglets in group 2A compared with those in group 2B (Table IV). Therefore, the piglets vaccinated 14 d before challenge had an ADWG significantly higher than the piglets that were not vaccinated before challenge.
Table III.
Mean body weight and average daily weight gain (ADWG) on days 0 to 14 after vaccination in experiment 1
| Group | Weight (kg) | ADWG (kg/d) | P-valuea | ||
|---|---|---|---|---|---|
|
| |||||
| Day 0 | Day 14 | P-valuea | |||
| 1A | 4.5 | 8.1 | 0.7582 | 0.3 | 0.1562 |
| 1B | 4.3 | 8.0 | ≥ 0.2273 | 0.3 | 0.1628 |
| 1C | 4.4 | 8.6 | NA | 0.3 | NA |
In comparison with group 1C.
NA — not available.
Table IV.
Least-squares mean weight and ADWG on days 0 to 14 and 14 to 24 after vaccination and days 0 to 10 after challenge with a virulent heterologous type I PRRSV isolate 14 d after vaccination in experiment 2
| Weight (kg) | ADWG (kg/d) | |||||||
|---|---|---|---|---|---|---|---|---|
|
|
|
|||||||
| Group | Day 0 | Day 14a | Day 24b | P-valuec | On days 0 to 14 after vaccination | P-valued | On days 0 to 10 after challenge | P-valued |
| 2A (20 vaccinates) | 4.14 | 7.64 | 10.26 | 0.0063 | 0.25 | 0.1889 | 0.26 | 0.0003 |
| 2B (20 controls) | 4.17 | 7.39 | 8.87 | 0.23 | 0.15 | |||
Day 0 after challenge.
Day 10 after challenge.
For the difference between groups 2A and 2B on day 24 after vaccination. On days 0 and 14 after vaccination there were no significant differences between the 2 groups (P = 0.8743 and 0.4297, respectively).
For differences between groups 2A and 2B.
All the piglets in experiment 1 were PRRSV-seronegative on day 0 after vaccination. On day 14 all 11 piglets in group 1A and 10 (91%) of the 11 piglets in group 1B were seropositive, whereas the 10 piglets in group 1C remained seronegative. These results indicate that the single dose and overdose of vaccine were effective at causing an immune response. All the piglets in experiment 2 were PRRSV-seronegative on days 0 and 7 after vaccination. On day 14 the proportion of seropositive piglets was significantly higher (P < 0.0001) in group 2A than in group 2B: 17 of 20 (85%) versus 0 of 20. Three days after challenge (17 d after vaccination) the proportion of seropositive piglets in group 2A had increased to 19 of 20 (95%); the piglets in group 2B remained seronegative. On day 7 after challenge (day 21 after vaccination) 11 of 20 (55%) of the piglets in group 2B were seropositive, a significant difference (P ≤ 0.0012) from the 20 of 20 in group 2A. On day 10 after challenge (day 24 after vaccination), however, 20 (100%) and 19 (95%) of the piglets in groups 2A and 2B, respectively, were PRRSV-seropositive. All the piglets in group 2C remained seronegative throughout the experiment.
No PRRSV RNA was detected in the serum of any piglets in experiment 2 on day 0. On days 7 and 14 after vaccination the piglets in group 2A had median values of 3.00 and 3.32 log10 GE/mL, respectively, whereas group 2B (nonvaccinated) remained free of PRRSV RNA; however, on day 17 (day 3 after challenge) the piglets in group 2B had significantly higher values (P < 0.0001) than those in group 2A: 8.18 versus 6.72 log10 GE/mL. There were no significant differences (P ≥ 0.0565) between the 2 groups on days 21 and 24 after vaccination (days 7 and 10 after challenge), the values for group 2A being 7.38 and 7.15 log10 GE/mL, respectively, and those for group 2B being 7.87 and 7.27 log10 GE/mL, respectively. However, on days 17 to 24 after vaccination (days 3 to 10 after challenge) the daily AUC for group 2A was significantly lower (P = 0.0039) than the AUC for group 2B: 49.52 GE/mL versus 54.35 GE/mL. No PRRSV RNA was detected in the serum of any piglet in group 2C (Figure 2).
Figure 2.
Amount of PRRSV RNA in the serum as tested by quantitative polymerase chain reaction. The genomic equivalence (GE) of the initial concentration of the reaction (GE per reaction) was extrapolated to the amount of GE per milliliter. The asterisks indicate significantly greater amounts (P < 0.0001) for the piglets in group 2A compared with those in groups 2B and 2C on days 7 and 14 after vaccination and significantly lower amounts (P < 0.0001) for the piglets in group 2A 2 d after challenge (day 17 after vaccination) compared with the nonvaccinated piglets in group 2B.
No injection-site abnormalities or gross lesions in the abdominal and thoracic cavities were noted at necropsy for any of the piglets in experiment 1, except for 1 piglet in group 1A that had exhibited mild coughing for 1 d and was found to have minor lung lesions. After sampling and testing, the lesions were determined to be a result of infection with Bordetella bronchiseptica. The piglet was negative for PRRSV, PCV-2, and M. hyopneumoniae in IHC testing. Interestingly, the 4 group 1C piglets that exhibited sneezing, mild coughing, or severe coughing did not have gross lesions at necropsy. Additionally, at the time of necropsy the weight of the group 1A piglet that had been observed as thin for 4 d was found to be within the range of the other piglets in the group, and no gross lesions were noted. Thus, the sneezing, coughing, and temporary thinness noted for the piglets were not associated with vaccination. In experiment 2, the mean total lung lesion score was significantly lower (P = 0.0002) for group 2A than for group 2B: 27.368% versus 54.841% (Table V).
Table V.
Total lung lesion scores (percentages of lung involvement) for the piglets in experiment 2 at necropsy on day 24 (day 10 after challenge)
| Groupa | Minimum | Maximum | Median | 95% CI | IQR | Mean | P-value | |
|---|---|---|---|---|---|---|---|---|
| 2A | 0.06 | 59.30 | 27.550 | 12.270 | 40.600 | 29.515 | 27.368 | 0.0002 |
| 2B | 13.86 | 91.60 | 55.200 | 47.300 | 66.500 | 21.850 | 54.841 | |
| 2C | 0.00 | 0.06 | 0.000 | 0.000 | 0.000 | 0.000 | 0.006 | NI |
The 20 group 2A piglets were vaccinated with the minimum-range commercial dose of the test vaccine, whereas the other 2 groups received a placebo. The 20 group 2B piglets were challenged with the same strain as group 2A, whereas the 10 group 2C piglets were not challenged.
IQR — interquartile range; NI — not included in the statistical analysis; CI — confidence interval.
Although PRRSV RNA was detected in the lung tissue of all piglets in groups 2A and 2B, the mean qPCR values were significantly lower (P = 0.0101) in group 2A than in group 2B at the time of necropsy (day 24 after vaccination; day 10 after challenge): 7.47 log10 GE/mL versus 7.88 log10 GE/mL.
Discussion
Vaccinating weaned piglets with a PRRS MLV vaccine is a beneficial practice for a variety of reasons, such as increased ADWG and decreased frequency of clinical signs in piglets exposed to PRRSV (1,5,21). The effectiveness of a PRRS MLV vaccine in protecting piglets from clinical signs is important because clinical disease generally leads to decreased health and growth. In addition to being effective, a PRRS MLV vaccine must be safe to administer (i.e., not cause local or systemic reactions and result in minimal viremia and virus shedding). Safety was evaluated in this study by monitoring for local and systemic reactions (including through clinical assessment) and calculating the ADWG; effectiveness was determined primarily from the total lung lesion scores, with supportive parameters including the results of clinical assessment, serologic study, viremia testing after vaccination, ADWG, and viral load in lung tissues. According to these criteria the data clearly demonstrated that vaccination with the novel type I PRRSV 94881-based MLV vaccine (Ingelvac PRRSFLEX EU) is a safe option for reducing the incidence of disease associated with PRRSV. Specifically, in experiment 1 young piglets (16 to 17 d of age) that had been vaccinated once with either the maximum-range commercial dose or an overdose of the test vaccine did not exhibit any relevant differences in local or systemic reactions when compared with nonvaccinated placebo recipients, and in experiment 2 no vaccinated piglets exhibited clinical abnormalities associated with vaccination. Likewise, in both experiments there were no significant differences between the vaccinated groups when compared with the nonvaccinated controls in regard to individual clinical signs, including behavior, respiration, digestion, and others, as well as in regard to total clinical signs after vaccination. However, the piglets in group 1B (those receiving a vaccine overdose) had significantly higher rectal temperatures than the piglets in group 1C (those receiving a placebo) on days 3, 5, 7, 9, and 11 after vaccination, and the piglets in group 2A (challenged vaccinates) had significantly higher rectal temperatures than those in group 2B (challenged piglets that had not been vaccinated) on days −1 and 0 after challenge; however, these differences were not biologically relevant. Thus, the significance of elevated rectal temperatures is ambiguous. Nevertheless, the serologic results from experiment 2 show that a single dose of the vaccine at the minimum commercial range induces immunity 14 d after vaccination when administered to very young piglets (14 ± 3 d of age). Moreover, when compared with the nonvaccinated piglets the vaccinated piglets in experiment 2 had significantly lower lung lesion scores, viremia, viremia over time (AUC), viral load in lung tissue, behavior scores, and total clinical scores, and they had a significantly higher ADWG after challenge (i.e., 14 to 24 d after vaccination).
Several previously reported studies examined the clinical effectiveness of MLV PRRS vaccines. Martelli et al (4) found that a single IM or intradermal (ID) dose of an MLV vaccine (based on the type I PRRSV vaccinal strain DV) administered to 5-week-old piglets offered approximately 70% protection against clinical signs of disease when the piglets were challenged with a heterologous type I Italian wild-type strain (05R1421) through natural exposure 45 d after vaccination. The challenge strain was shown to be virulent, as 100% of the controls had increased rectal temperatures, increased lethargy, and decreased appetite throughout the study compared with 90% of the vaccinated animals, which showed consistently less severe signs of disease. However, the frequency and severity of clinical signs in all the groups may have been complicated by the presence of other pathogens within the herd (i.e., PCV-2, M. hyopneumoniae, Pasteurella multocida, and Streptococcus sp.). Various reports have indicated that PRRSV may have an immunosuppressive effect, thus making pigs more susceptible to secondary infections (5,12,14). Additionally, the exposure levels of the field challenge will vary among herds and between individual pigs, such that it is difficult to determine the occurrence of a vaccinal effect or if some animals were less exposed to the challenge virus. Another study evaluated a single-strain MLV vaccine (based on the EU vaccinal strain ALL-183) (19) administered on 2 occasions, 3 wk apart, to 5-month-old gilts that were challenged with a heterologous Lelystad strain 28 d after initial vaccination (18). In this trial it was determined that the vaccine had no significant effect on clinical signs, rectal temperatures, and ADWG; however, the challenge strain was not considered virulent. On the other hand, there were significant differences between the vaccinated and nonvaccinated pigs in level of viremia and viral load in lungs and tonsils after heterologous challenge, as in the present study. In contrast, the vaccine did not induce a serologic response to PRRSV until 21 d after initial vaccination; the ELISA S/P ratio was negative (< 0.4) on day 14 after vaccination, whereas in the present study there was a positive strong serologic response, an S/P ratio ≥ 0.4, in 17 out of 20 piglets on day 14. Likewise, another study found that pigs vaccinated once at 7 wk of age with a commercially available attenuated vaccine based on the type I strain DV experienced a shortened duration of viremia compared with pigs vaccinated twice, at 5 and 9 wk of age, with a commercially available inactivated vaccine based on the type I strain P120 or with either of 2 experimental inactivated vaccines based on the type I field isolates 07 V063 and 08 V194 in the face of fever-inducing heterologous challenge with either 07 V063 or 08 V194 (resulting in temperatures > 39.5°C and ≤ 40.6°C, respectively) at 13 wk of age (5). Trus et al (11) reported a significant reduction in severity and duration of fever as well as in the AUC for viremia in 4- and 7-week-old pigs vaccinated with an MLV PRRS vaccine (type I strain DV) and challenged with a highly pathogenic PRRSV strain (Lena, isolated from a farm in the Republic of Belarus) 8 and 6 wk, respectively, after vaccination when compared with nonvaccinated controls. However, differences in gross lung lesions between the groups were not significant. This trial used older pigs that were challenged much later after vaccination, compared with the present study, in which the younger piglets were challenged 14 d after vaccination. Another study using an MLV PRRS vaccine based on the European DV strain found that vaccinated 4-week-old pigs exhibited significantly reduced viremia levels and shorter duration of viremia, compared with nonvaccinated controls, when challenged with a heterologous strain (3267, isolated from a Portuguese farm) by contact with inoculated pigs 37 d after vaccination (12). The challenge strain was not known to induce noteworthy clinical signs outside of mild respiratory symptoms, which is characteristic of type I PRRSV strains; however, PRRSV was detected by means of qPCR in the lung tissue of all vaccinated and nonvaccinated pigs that were subsequently euthanized (n = 5 and 4, respectively). In contrast, in the present study the vaccinated piglets had significantly lower scores for total clinical signs, reduced PRRS viral load in the lung tissue, and, most importantly, significant reductions in scores for PRRSV-specific lung lesions compared with the nonvaccinated piglets after challenge. Roca et al (22) found that a commercially available MLV vaccine based on type I PRRSV strain VP046 BIS administered to 4-week-old pigs offered partial protection against challenge with a highly virulent type II strain (HP-PRRS21) 6 wk after vaccination. In that trial the vaccinated piglets showed a significant reduction in fever on 2 d (days 11 and 12 after challenge), significantly increased weight gain, decreased lethargy and/or anorexia, and decreased viremia (which was detected only on day 7 after challenge); however, the vaccine did not significantly reduce the score for lung lesions. In comparison, in the present study the vaccinated piglets had significantly lower LS mean rectal temperatures 1 d after challenge, significantly increased ADWG in the postchallenge period, a significantly lower incidence of abnormal behavior (lethargy), and significantly reduced viremia (on day 3 after challenge and AUC on days 3 to 10 after challenge) and lung abnormalities compared with the nonvaccinates after challenge. Prieto et al (13) evaluated the efficacy of multiple doses of vaccine before challenge: 4-week-old pigs were vaccinated on 3 occasions, 21 d apart, with the type I strain DV, beginning at 28 d of age, and were challenged with the type I PRRSV strain 5710 (isolated in Spain) 4 wk after the final vaccination. There were no significant differences between the vaccinated and nonvaccinated pigs in clinical signs or virus titer in all tissues collected at necropsy. However, the clinical signs were less severe in the vaccinated pigs, and the virus was found less frequently in their tissues, compared with the nonvaccinated pigs. Another study using the MLV PRRS European DV strain determined (by virus titration in bronchoalveolar lavage fluids and serum by immunoperoxidase monolayer assay) that pigs vaccinated at 5 wk of age either IM or ID were fully protected from homologous wild-type challenge but only partially protected from heterologous challenge (with an Italian strain) at 49 d after vaccination, as evidenced by significantly lower mean virus titers in serum samples from the vaccinated pigs as compared with the nonvaccinated controls (4). Antibodies to PRRSV began developing around 7 d after vaccination; however, 100% seroconversion did not occur until 35 d after vaccination, whereas in the present study the pigs in groups 1A, 1B, and 2A were 100%, 91%, and 85% seropositive, respectively, on day 14 after vaccination. Likewise, the data from the present study show that the novel PRRSV vaccinal strain 94881 was effective against viremia after challenge, the vaccinates having significantly lower mean log10 GE/mL values on day 3 after challenge and a significantly lower AUC for 3 to 10 d after challenge compared with the controls.
Effectiveness of vaccination was shown in the above instances in a manner similar to that in the present study (i.e., through decreased clinical signs, viremia, and viral load in lung tissue, as well as increased ADWG). However, no other vaccine was shown to positively affect all these parameters in a single trial involving challenge with a heterologous European-derived, type I PRRSV that induced clinical signs and lung lesions. In contrast to the previously reported studies, the present study showed significant reductions in lung abnormalities in young pigs vaccinated with the PRRS 94881 MLV vaccine. Moreover, to our knowledge this is the first time a 14-d OOI has been reported in piglets vaccinated before 3 wk of age.
Most PRRS MLV vaccines are considered safe if there are no significant local and/or systemic reactions, as well as differences in clinical signs, after vaccination (5,7,9,13,17); however, little is known about the likelihood of European-derived PRRS MLV vaccinal strains to cause viremia as well as the shedding of vaccine virus and subsequent transmission to naïve pigs. One study aimed to alleviate this knowledge gap by evaluating the safety of 3 vaccines based on type I strains that are commercially available in Europe (9): 4-week-old pigs were vaccinated with 3 PRRS MLV vaccines based on the strains VP046 BIS, ALL-183, and DV, and unvaccinated sentinel pigs were introduced at day 3 after vaccination. As with the present study, the vaccines were all deemed clinically safe, as no significant differences were noted in clinical signs between vaccinated and nonvaccinated pigs, and vaccination did not negatively affect ADWG. In contrast, the authors found that the vaccinated groups had varied mild lung lesion scores on 1 or more necropsy days (i.e., 7, 14, and 21 d after vaccination) that decreased over time. Additionally, all the vaccine strains induced viremia (in 57.3% to 88% of the pigs), and PRRSV was found in lung tissues in all the groups (in 20% to 66.7%). These results are contrary to the data from the present study, as no pigs in experiment 1 had lung lesions associated with vaccination, and PRRSV was not isolated from lung tissues at the time of necropsy even after the pig had received an overdose of the vaccine. Although the vaccinated piglets in experiment 2 also exhibited viremia, the level was significantly less than in the nonvaccinated group after challenge. Moreover, the vaccinated groups in the safety trial (9) shed virus in oropharyngeal and nasal secretions as well as feces (4.89% to 6.67% of all samples tested positive); thus, the sentinel pigs for each group became viremic and subsequently shed virus (2.22% to 5.71% of all samples tested positive). Although the vaccinated pigs in the present study did exhibit viremia, there was no evidence that the vaccine virus infected their lungs even after an overdose. Additionally, good biosecurity practices ensured that all negative control animals remained negative for all parameters tested. Other studies have also reported the presence of vaccine virus in the blood after vaccination (4,9,13,17).
These studies, as well as the present data, support the knowledge that the variability of PRRSV continues to confound the development of a single-strain vaccine that may be effective against the virus in the field. Although it is difficult to compare these data in a side-by-side manner, similarities and trends do exist. Generally, vaccination with an MLV vaccine has been found to offer significant protection against certain aspects of PRRSV infection (viremia, clinical signs, decreased productivity, etc.). The present study offers evidence that a vaccine based on the novel type I PRRSV strain 94881 administered to very young piglets may offer similar, if not improved, protection against the negative clinical and economic effects of PRRSV infection.
In conclusion, a vaccine that is safe and effective in preventing the detrimental effects of type I PRRSV in a timely manner is an essential part of swine production practices. The results from this study support the clinical safety and efficacy of Ingelvac PRRSFLEX EU when administered to piglets approximately 14 d of age. When the vaccine was administered once at the maximum-range commercial dose or as an overdose, there were no significant differences in clinical signs attributable to the vaccine; only transient, minimal swelling was noted at the injection site of 1 piglet, and ADWG was not affected. Through the significant reduction in lung lesions, viremia, viral load in lung tissue, and clinical signs, as well as the improvement in ADWG, it has been shown that administration of a single IM dose of the novel PRRS 94881 MLV vaccine to piglets induced the response required to build protective immunity by 14 d after vaccination. Consequently, a vaccine formulated from the type I PRRS viral strain 94881 has been proven to be a safe and effective method of protection against the detrimental effects of virulent PRRSV infection in young piglets.
Acknowledgments
The authors thank Dr. Ryan Saltzman, Dr. Lyle Kesl, Dr. Stephan Pesch, Mr. Rex Smiley, Dr. Alicia Zimmerman, and Ms. Sarah Layton for technical assistance.
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