High levels of maternally derived antibodies do not significantly interfere with the development of humoral and cell-mediated responses to Porcine circovirus 2 after intradermal vaccination.

BACKGROUND: Vaccination of pigs against PCV2 is usually performed around weaning when animals still have maternally derived antibodies (MDA). The present study aimed to assess the possible interference of MDA in the development of the PCV2-specific immune response after vaccination of commercial weaners. For this purpose, a PRRS-negative 600-sow farrow-to-finish farm was selected. Half of the sows were vaccinated and revaccinated with Porcilis® PCV ID against PCV2 7 and 3 weeks before farrowing. After farrowing, piglets were tested by AlphaLisa to select 72 animals with high and low levels of MDA. Groups were further subdivided and vaccinated intradermally with Porcilis® PCV ID at 21 or 28 days of age. Unvaccinated controls were also included. Animals were followed afterward for 42 days to examine the development of PCV2-specific antibodies and interferon-γ secreting cells (IFN-γ SC). RESULTS: The average titres of antibodies of the groups vaccinated in the presence of low or high MDA levels were similar at 28 and 42 days post-vaccination while in the controls the titres declined throughout the observation period. Results of vaccinating at 21 or 28 days of age were equivalent with regard to antibody development. Regarding the IFN-γ SC, vaccinated animals produced significant frequencies of IFN-γ SC by day 28. Again, no differences were observed between the groups with high or low antibody levels. CONCLUSION: High levels of MDA did not interfere with the development of humoral and cell-mediated responses to Porcine circovirus 2 after intradermal vaccination at 21 or 28 days of age.

Introduction of a PRRSV-1 strain of increased virulence in a pig production structure in Spain: virus evolution and impact on production.

BACKGROUND: A strain of Porcine reproductive and respiratory syndrome virus (PRRSV), showing characteristics of enhanced virulence, affected a pyramidal production system from Spain with 7600 sows in 4 genetic nuclei and 13,000 sows in multipliers. Different PRRSV strains circulating in this production system from December 2020 to October 2021 were detected and sequenced. The spread of each isolate was examined and their impact on health and production in three of the affected farms was evaluated. RESULTS: The newly emerged PRRSV isolate with enhanced virulence entered the system before the onset of the study (January 2020) and afterwards four significantly different clades were detected during the study period in different farms, probably because of independent introduction events. The diversification of the enhanced virulence strain was higher for those clades (substitution rates up to 1.1% nucleotides/year) compared to other PRRSV strains present in the production system (up to 0.17%), suggesting a faster spread and adaptation. The impact of the infection in the first affected farm was dramatic, with an average abortion rate above 27% during 17 weeks before returning to the baseline production. Fertile sow mortality reached 6.5% for 39 weeks. In two farms infected later by other clades of this enhanced virulence strain, the impact was less acute; despite the fact that for parameters such as the proportion of stillbirths or mummies, more than 10 months were needed to recover pre-outbreak values. In the examined nurseries, mortalities reached peaks between 28 and 50% and several months were needed to return to normality. CONCLUSION: Introduction of a PRRSV strain of enhanced virulence in a production system where several farms were previously positive for other PRRSV strains, resulted in a fast spread such as would be observed in naïve farms. The productive and health impact was very high taking several months to return to normality.

Diversity of respiratory viruses present in nasal swabs under influenza suspicion in respiratory disease cases of weaned pigs.

Respiratory diseases in weaned pigs are a common problem, with a complex etiology involving both viruses and bacteria. In the present study, we investigated the presence of eleven viruses in nasal swabs, collected from nurseries (55 cases) under the suspicion of swine influenza A virus (swIAV) and submitted by swine veterinarians for diagnosis. The other ten viruses included in the study were influenza B (IBV) and D (IDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine respiratory coronavirus (PRCV), Porcine cytomegalovirus (PCMV), Porcine circovirus 2 (PCV2), 3 (PCV3) and 4 (PCV), Porcine parainfluenza 1 (PPIV1) and Swine orthopneumovirus (SOV). Twenty-six swIAV-positive cases and twenty-nine cases of swIAV-negative respiratory disease were primarily established. While IBV, IDV, PCV4 and PPIV1 were not found in any of the cases, PRCV, SOV, and PCMV were more likely to be found in swIAV-positive nurseries with respiratory disease (p < 0.05). Overall, PCV3, PRRSV, and PCMV were the most frequently detected agents at herd level. Taken individually, virus prevalence was: swIAV, 48.6%; PRCV, 48.0%; PRRSV, 31.6%; SOV, 33.8%; PCMV, 48.3%, PCV2, 36.0%; and PCV3, 33.0%. Moreover, low Ct values (<30) were common for all agents, except PCV2 and PCV3. When the correlation between pathogens was individually examined, the presence of PRRSV was negatively correlated with swIAV and PRCV, while was positively associated to PCMV (p < 0.05). Also, PRCV and SOV were positively correlated between them and negatively with PCMV. Besides, the analysis of suckling pig samples, collected in subclinically infected farrowing units under an influenza monitoring program, showed that circulation of PRCV, PCMV, SOV, and PCV3 started during the early weeks of life. Interestingly, in those subclinically infected units, none of the pathogens was found to be correlated to any other. Overall, our data may contribute to a better understanding of the complex etiology and epidemiology of respiratory diseases in weaners. This is the first report of SOV in Spain and shows, for the first time, the dynamics of this pathogen in swine farms.

Description of a New Clade within Subtype 1 of Betaarterivirus suid 1 Causing Severe Outbreaks in Spain.

This report describes 28 genome sequences from a new clade within subtype 1 of Betaarterivirus suid 1, formerly known as porcine reproductive and respiratory syndrome virus 1. All share a potential recombinant pattern, with a highly pathogenic Italian strain as the putative major parental sequence and three other possible parents.

Using commercial ELISAs to assess humoral response in sows repeatedly vaccinated with modified live porcine reproductive and respiratory syndrome virus.

BACKGROUND: Sows in breeding herds are often mass vaccinated against porcine reproductive and respiratory syndrome (PRRS) every few months using modified live vaccines (MLV). Field veterinarians repeatedly report that multiple vaccinated sows test negative in ELISA. Obviously, this creates uncertainty when assessing the compliance of vaccination and the status of sows. METHODS: In the present study, four commercial ELISAs were used to assess the serological PRRS status in gilts and sows of three farms that were PRRS MLV vaccinated every four months. Animals were tested before vaccination (BV) and postvaccination (PV). Total and neutralising antibodies and cell-mediated responses were also measured in animals that yielded negative results in all ELISAs. RESULTS: The proportion of seronegative animals BV varied depending on the farm and the ELISA used. When samples were analysed using only one ELISA, a substantial number of negative results obtained BV remained as negative afterwards. Five animals were negative BV and PV with all the examined ELISAs. Those animals also yielded negative results in all the other immunological assays. CONCLUSION: Our findings suggest that the use of ELISA for monitoring multiple PRRS MLV vaccinated sows is very limited due to the variability of the humoral responses and the moderate agreement between tests.

Prevalence of enteric pathogens in diarrheic and non-diarrheic samples from pig farms with neonatal diarrhea in the North East of Spain.

Diarrhea is one of the major causes of neonatal mortality in pigs. In the present study, 31 pig farms with outbreaks of neonatal diarrhea were investigated in Catalonia (NE Spain) from February 2017 until June 2018. Two hundred and fifteen diarrheic samples from 1 to 7 days old piglets were tested for a panel of enteric pathogens. In 19 of the studied farms additional fecal samples from apparently healthy pen-mates were collected and tested for the same panel of infectious agents. Samples were bacteriologically cultured and tested by PCR for E. coli virulence factors genes, C. perfringens types A and C toxins (Cpα, Cpß, Cpß2) and C. difficile toxins (TcdA, TcdB). Moreover, Rotavirus A (RVA), Rotavirus B (RVB), Rotavirus C (RVC), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) were also determined by RT-qPCR. More than one pathogen could be detected in all of the outbreaks. Nevertheless, RVA was the only agent that could be statistically correlated with the outcome of diarrhea. For the other viruses and bacteria analyzed significant differences between the diseased pigs and the controls were not found. In spite of this, the individual analysis of each of the studied farms indicated that other agents such as RVB, RVC, toxigenic C. difficile or pathogenic E. coli could play a relevant role in the outbreak of diarrhea. In conclusion, the large diversity of agent combinations and disease situations detected in neonatal diarrhea outbreaks of this study stand for a more personalized diagnosis and management advice at a farm level.

Full-genome characterization by deep sequencing of rotavirus A isolates from outbreaks of neonatal diarrhoea in pigs in Spain.

Since early 2017, in Spain there was an apparent increase in reports on rotavirus involvement in neonatal diarrhoea outbreaks, affecting also adult sows. In this study, 16 unrelated outbreaks of diarrhoea in suckling pigs and sows, where rotavirus A was the only pathogen detected, were investigated. Deep-sequencing was performed on total RNA from twenty-four positive faecal samples. Genotyping, phylogenetic and bayesian analyses showed that all isolates had a common ancestor of porcine, or human porcine-like, origin. The new strain was introduced in the population shortly before the onset of the outbreaks. Besides, a high diversification of the VP7 and VP4 genes occurred in a short time. Isolates presented a high number of amino acid changes in the neutralizing epitopes compared to vaccine sequences. The present report illustrates how a new rotavirus A strain may disseminate rapidly and the extremely high diversification that this pathogen may undergo in a short period.

Testing of umbilical cords by real time PCR is suitable for assessing vertical transmission of porcine reproductive and respiratory syndrome virus under field conditions.

The objective of this study was to test the suitability of umbilical cord (UC) sampling and ear vein swabbing (EVS) as alternatives to jugular vein bleeding (JVB) for the assessment of vertical transmission of porcine reproductive and respiratory syndrome virus (PRRSV). Twelve farms suspected to be PRRSV-positive unstable were selected and the three types of samples were obtained from 21 batches of newborn piglets (n=387). The proportions of positive results, viral loads and time spent to collect the samples were compared. UC yielded the highest detection rate, with 76 positives compared to 55 JVB- and 45 EV-positive results (P<0.05). Average Ct values were 26.6±8.5 for JVB, 30.8±6.4 for EV and 32.1±4.85 for UC (P<0.01). UC was the fastest collection method (mean 24s vs. 55s for EV and 72s for JVB; P<0.05). In this study, UC testing was a faster and more sensitive alternative to JVB or EV for the detection of PRRSV in newborn piglets.

Estimation of the transmission parameters for swine influenza and porcine reproductive and respiratory syndrome viruses in pigs from weaning to slaughter under natural conditions.

In the present study, the transmission parameters of swine influenza virus (SIV) and porcine reproductive and respiratory virus (PRRSV) have been calculated using the basic reproductive rate (R) parameter in two commercial pig farms (F1 and F2). In order to do this, a serological (PRRSV genotype 1 and SIV) and virological (SIV) follow-up of a batch of animals was carried out weekly from 3 weeks of age until the age of slaughter on each farm. Results of the analysis for SIV and PRRSV showed different transmission profiles depending on the farm, the pathogen, and time of transmission. In F1, transmission of both viruses was detected throughout the sampling. The Rt (R for a given period of time) value for SIV ranged from 1.5 [0.9-2.3] to 3.6 [2.3-4.9] from farrowing to the beginning of the fattening period, and the Rt value for PRRSV was 3.3 [2.9-4.3] to 3.5 [2.8-4.1] from farrowing until the slaughter age. These results indicated that both viruses were transmitted enzootically in that farm for these periods of time. A different transmission pattern with a higher incidence was also observed during the fattening period in F1 (after 15 weeks of age) for SIV, coinciding with the entrance of a new subtype. In this case, R value for SIV reached 3.3 [1.65-4.9]. On the other hand, in F2, SIV and PRRSV seemed to be restricted to the fattening period. R reached a value of 6.4 [4.1-8.8] for SIV and 7.1 [3.5-10.6] for PRRSV. These findings suggest a different origin of the virus, as well as a more epidemic circulation, especially for SIV, where most of the new cases were observed in a one week period. In conclusion, the present study offers a reliable estimation of the range of Rt values for SIV and genotype 1 PRRSV transmission under field conditions, suggesting that enzootic circulations of both viruses are similar in terms of transmission, probably higher for PRRSV, but also that transmission of SIV is more efficient (or epidemic) than transmission of a genotype 1 PRRSV isolate in naïve animals given the new cases observed in only in F2.

Phylogeny of Spanish swine influenza viruses isolated from respiratory disease outbreaks and evolution of swine influenza virus within an endemically infected farm.

In the present study, outbreaks of respiratory disease were investigated for the presence of swine influenza virus (SIV). In 14 cases the circulating SIV strains were isolated, fully sequenced and compared with other known SIVs. The viruses causing the outbreaks belonged to the H1N1 (including human pandemic H1N1), H3N2 and H1N2 subtypes. In 11/14 cases the phylogenetic analyses indicated the occurrence of probable reassortment events. In the second part of the study, the genetic evolution of H1N1 SIV was assessed in a longitudinal study in closed groups of pigs over six months. Sequencing of the 22 isolates indicated co-circulation of two different variants for the same virus, as well as the emergence of SIV reassortants at certain time-points. These results indicate that reassortment events in SIV are common, and point towards the need for a better understanding of the epidemiology of SIV, particularly in endemic farms.

Conserved synthetic peptides from the hemagglutinin of influenza viruses induce broad humoral and T-cell responses in a pig model.

Outbreaks involving either H5N1 or H1N1 influenza viruses (IV) have recently become an increasing threat to cause potential pandemics. Pigs have an important role in this aspect. As reflected in the 2009 human H1N1 pandemia, they may act as a vehicle for mixing and generating new assortments of viruses potentially pathogenic to animals and humans. Lack of universal vaccines against the highly variable influenza virus forces scientists to continuously design vaccines à la carte, which is an expensive and risky practice overall when dealing with virulent strains. Therefore, we focused our efforts on developing a broadly protective influenza vaccine based on the Informational Spectrum Method (ISM). This theoretical prediction allows the selection of highly conserved peptide sequences from within the hemagglutinin subunit 1 protein (HA1) from either H5 or H1 viruses which are located in the flanking region of the HA binding site and with the potential to elicit broader immune responses than conventional vaccines. Confirming the theoretical predictions, immunization of conventional farm pigs with the synthetic peptides induced humoral responses in every single pig. The fact that the induced antibodies were able to recognize in vitro heterologous influenza viruses such as the pandemic H1N1 virus (pH1N1), two swine influenza field isolates (SwH1N1 and SwH3N2) and a H5N1 highly pathogenic avian virus, confirm the broad recognition of the antibodies induced. Unexpectedly, all pigs also showed T-cell responses that not only recognized the specific peptides, but also the pH1N1 virus. Finally, a partial effect on the kinetics of virus clearance was observed after the intranasal infection with the pH1N1 virus, setting forth the groundwork for the design of peptide-based vaccines against influenza viruses. Further insights into the understanding of the mechanisms involved in the protection afforded will be necessary to optimize future vaccine formulations.

Comparison of two commercial enzyme-linked immunosorbent assays for the diagnosis of Porcine reproductive and respiratory syndrome virus infection.

Early diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV) is critically important for control of the disease. Two new commercially available enzyme-linked immunosorbent assays (ELISAs) based on different methodologies have been developed. In the present report, the 2 ELISAs were compared using blood samples from experimentally and naturally infected pigs. One of the 2 ELISAs was shown to be more sensitive than the other. The higher sensitivity of one of the ELISAs could pose a problem in PRRS diagnosis in endemic farms, because it can detect maternally derived antibodies for a longer time, overlapping with the detection of antibodies developed after PRRSV infection. However, the ELISA with higher sensitivity could be suitable for early detection of PRRSV antibodies in individual pigs, especially in PRRS-free herds.

Swine influenza virus infection dynamics in two pig farms; results of a longitudinal assessment.

In order to assess the dynamics of influenza virus infection in pigs, serological and virological follow-ups were conducted in two whole batches of pigs from two different farms (F1 and F2), from 3 weeks of age until market age. Anti-swine influenza virus (SIV) antibodies (measured by ELISA and hemagglutination inhibition) and nasal virus shedding (measured by RRT-PCR and isolation in embryonated chicken eggs and MDCK cells) were carried out periodically. SIV isolates were subtyped and hemagglutinin and neuraminidase genes were partially sequenced and analyzed phylogenetically. In F1, four waves of viral circulation were detected, and globally, 62/121 pigs (51.2%) were positive by RRT-PCR at least once. All F1 isolates corresponded to H1N1 subtype although hemagglutination inhibition results also revealed the presence of antibodies against H3N2. The first viral wave took place in the presence of colostral-derived antibodies. Nine pigs were positive in two non-consecutive sampling weeks, with two of the animals being positive with the same isolate. Phylogenetic analyses showed that different H1N1 variants circulated in that farm. In F2, only one isolate, H1N2, was detected and all infections were concentrated in a very short period of time, as assumed for a classic influenza outbreak. These findings led us to propose that influenza virus infection in pigs might present different patterns, from an epidemic outbreak to an endemic form with different waves of infections with a lower incidence.

Experimental infection with H1N1 European swine influenza virus protects pigs from an infection with the 2009 pandemic H1N1 human influenza virus.

The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.