In Situ Hybridization of PRRSV-1 Combined with Digital Image Analysis in Lung Tissues of Pigs Challenged with PRRSV-1.

Betaarterivirus suid 1 and 2 are the causative agents of porcine reproductive and respiratory syndrome (PRRS), which is one of the most significant diseases of the swine industry, causing significant economic losses in the main pig producing countries. Here, we report the development of a novel, RNA-based in situ hybridization technique (RNAscope) to detect PRRS virus (PRRSV) RNA in lung tissues of experimentally infected animals. The technique was applied to lung tissues of 20 piglets, which had been inoculated with a wild-type, highly pathogenic PRRSV-1 strain. To determine the RNAscope's applicability as a semi-quantitative method, we analysed the association between the proportion of the virus-infected cells measured with an image analysis software (QuPath) and the outcome of the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) tests performed in parallel. The results of the quantitative approach of these two molecular biological methods show significant association (pseudo R2 = 0.3894, p = 0.004). This is the first time RNAscope assay has been implemented for the detection of PRRSV-1 in experimental animals.

Detection and genotyping of Porcine circovirus 2 (PCV-2) and detection of Porcine circovirus 3 (PCV-3) in sera from fattening pigs of different European countries.

PCV-2 is considered one of the most economically important viral agents in swine worldwide. Recently, PCV-3 has been found in pigs affected by different disorders and in healthy animals. The objective of this epidemiological work was to describe the frequency of detection of PCV-2 and PCV-3 in pig farms of 9 European countries. Moreover, a second aim was to assess the most frequent PCV-2 genotypes found in the studied farms. Sera from 5 to 10 pigs per farm were collected from 2 to 11 farms per studied country. A total of 624 sera of fattening pigs (10-25 week old) from 64 farms from Spain (n = 11), Belgium (n = 10), France (n = 8), Germany (n = 8), Italy (n = 7), Denmark (n = 8), the Netherlands (n = 5), Ireland (n = 5) and Sweden (n = 2) were analysed by conventional PCR. In addition, one or two PCV-2-positive samples per farm were genotyped by sequencing the ORF2 gene. PCV-3 PCR-positive samples with relatively low Ct values were also sequenced and phylogenetically analysed. PCV-2 DNA was detected in pig sera from all European tested countries, but Sweden. A total of 132 out of 624 (21%) sera were positive for PCV-2 PCR, corresponding to 30 out of the 64 (47%) tested farms. PCV-3 DNA was detected in 52 out of 624 (8%) sera, corresponding also to 30 out of the 64 (47%) studied farms from all tested countries. A total of 48 PCV-2 PCR-positive samples were successfully sequenced and genotyped, being PCV-2d the most frequently genotype found (n = 28), followed by PCV-2b (n = 11) and PCV-2a (n = 9). These results pointed out PCV-2d as the most prevalent genotype currently in Europe. The PCV-3 phylogenetic analysis showed high identity (>98%) among sequences from all the analysed countries. The relatively low co-infection (3%), likely suggest an independent circulation patterns of PCV-2 and PCV-3.

Initial vaccination and revaccination with Type I PRRS 94881 MLV reduces viral load and infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) causes respiratory distress in pigs, reproductive failure in breeding-age gilts and sows, and can have devastating economic consequences in domestic herds. Several PRRS vaccines are available commercially. This study compared the effectiveness of single-vaccination and revaccination schedules using the PRRS 94881 Type I modified live virus (MLV) vaccine ReproCyc® PRRS EU with no vaccination (challenge control) in protecting against a PRRS virus (PRRSV) challenge in non-pregnant gilts. RESULTS: Data were available from 48 gilts across three groups: a challenge control group (n = 16), which received no vaccination; a revaccination group (n = 16), which received ReproCyc® PRRS EU on Days 0 and 56; and a single vaccination group (n = 16), which received ReproCyc® PRRS EU on Day 56. All gilts were PRRSV RNA-negative (based on reverse transcription and quantitative polymerase chain reaction [RT-qPCR]) and PRRSV seronegative (based on enzyme-linked immunosorbent assay [ELISA]) at Day 0. All gilts were challenged with PRRSV strain 190136 on Day 91.Viral RNA loads in both vaccination groups were significantly reduced compared with the challenge control group on Days 98 (P < 0.0001) and 101 (P < 0.0001), indicating that vaccinated gilts were better able to respond to challenge than unvaccinated gilts. At all timepoints following challenge, mean viral RNA load and the percentage of PRRSV RNA-positive gilts were numerically higher in the single-vaccination group than in the revaccination group; these differences were statistically significant on Day 101 (P = 0.0434). Furthermore, viremia levels after challenge were significantly lower in the revaccination group than in the single-vaccination group based on median area under the curve (AUC) values for viral RNA load from Day 91 to Day 112, suggesting that revaccinated gilts had better protection from viral infection than gilts who received a single vaccination. Protection from viremia did not correlate with the proportion of seropositive gilts on Day 91. In the single-vaccination group, 94% of pigs were seropositive on Day 91 compared with 56% in the revaccination group. Vaccination was well tolerated and no safety concerns were identified. CONCLUSIONS: Both single-vaccination and revaccination with ReproCyc® PRRS EU were effective in reducing PRRSV viremia post-challenge. These findings have important implications for herd management as both the single-vaccination and revaccination schedules protect against PRRSV challenge, with revaccination appearing to provide better protection from viremia than single vaccination.

Long duration of immunity against a type 1 heterologous PRRS virus challenge in pigs immunised with a novel PRRS MLV vaccine: a randomised controlled study.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is widespread in commercial pig farms worldwide, and has a significant cost to the swine industry. Herd owners need a vaccine that will confer long-lasting immunity to prevent PRRSV infection and transmission. The studies described here evaluated duration of immunity conferred by a European-derived PRRS (isolate 94,881) modified live virus (MLV) vaccine, Ingelvac PRRSFLEX® EU, at 20, 24, and 26 weeks post-vaccination. Primary endpoints were the assessment of gross and histological lung lesions and viral RNA load in lung tissue 10 days following heterologous PRRSV challenge. Secondary endpoints included clinical observations, average daily weight gain (ADWG) and viral RNA load in serum 10 days post-challenge. Three blinded, vaccination-challenge efficacy studies were performed using separate cohorts of pigs (n = 56 per study). Pigs received either Ingelvac PRRSFLEX® EU (Group 1) or placebo (Groups 2 and 3). Groups 1 and 2 were subsequently challenged with heterologous European PRRSV isolate 205,817 at 20, 24 or 26 weeks post-vaccination. RESULTS: Mean gross lung lesion scores were significantly lower in Group 1 than in Group 2 at 24 and 26 weeks (p <  0.0001), but not at 20 weeks (p = 0.299). Significantly lower mean histological lung lesion scores were observed in Group 1 versus Group 2 at 20 (p = 0.0065), 24 (p <  0.0001) and 26 weeks (p <  0.0001). Mean viral RNA load in lung tissue was significantly lower in Group 1 than in Group 2 (p <  0.0001) at 20 (p <  0.0001), 24 (p <  0.0001) and 26 weeks (p <  0.0001). Cumulative viral RNA loads in serum during days 1-10 post-challenge were significantly lower in Group 1 than in Group 2 (p <  0.0001) in all studies. A significant increase in ADWG was observed in Group 1 compared with Group 2 at 20 weeks (p = 0.0027) and 24 weeks (p = 0.0004), but not at 26 weeks (p = 0.1041). There were no significant differences in clinical signs post-challenge in any study. CONCLUSION: These results suggest that Ingelvac PRRSFLEX® EU confers long-term immunity to European heterologous PRRSV, which is maintained up to 26 weeks after vaccination, corresponding to the expected lifespan of commercial pigs.

Production results from piglets vaccinated in a field study in Spain with a Type 1 Porcine Respiratory and Reproductive virus modified live vaccine.

BACKGROUND: PRRS is a viral disease of pigs and sows that is one of the most costly to the pig industry worldwide. The disease can be controlled by focusing on different aspects. One of them is the vaccination of piglets, which is more controversial and difficult to manage than the vaccination of sows. However, pig producers could consider a piglet vaccination strategy if it reduces the negative clinical disease and improves zootechnical performance, decreases the probability to be infected and/or reduces the spread of the virus once the vaccinated piglet is infected. The efficacy of a novel PRRS modified live vaccine (Ingelvac PRRSFLEX® EU) was studied in a blinded, side-by-side placebo controlled field study of piglet vaccination including piglets weaned for three consecutive weeks (week groups 1, 2 and 3). RESULTS: This study established that PRRS piglet vaccination resulted in significantly better weight gain, seen as early as 4 weeks after vaccination, in naturally challenged pigs. Vaccine efficacy was supported by statistically significant increases in Average Daily Weight Gain (ADWG) among week group 3 vaccinated pigs from vaccination to the end of the study and statistically significant increases in bodyweight and ADWG from inclusion to 10 weeks of age in week group 2 vaccinated piglets. However, no differences were noted in week group 1 presumably because more than 30 % of the vaccinated pigs were viremic at the time of vaccination. Furthermore, the proportion of pigs showing any abnormal clinical sign at least once at any of the examination time points was lower in vaccinated pigs than in control pigs. Based on the viremia results (qPCR), early onset of PRRS was detected in this herd. Viremia occurred at the time of vaccination in week group 1 and shortly after vaccination in week groups 2 and 3. Peak wild type PRRSV infection was assumed at 4 weeks post vaccination in all groups based on the number of PRRS positive pigs in the control groups. CONCLUSION: This study establishes that vaccination of piglets with Ingelvac PRRSFLEX® EU at 4 weeks of age improves weight gain and reduces the appearance of clinical sings during the growing period, even when the piglets are infected shortly after vaccination.