The inclusion of health-related traits, or functionally associated genetic markers, in pig breeding programs could contribute to produce more robust and disease resistant animals. The aim of the present work was to study the genetic determinism and genomic regions associated to global immunocompetence and health in a Duroc pig population. For this purpose, a set of 30 health-related traits covering immune (mainly innate), haematological, and stress parameters were measured in 432 healthy Duroc piglets aged 8 weeks. Moderate to high heritabilities were obtained for most traits and significant genetic correlations among them were observed. A genome wide association study pointed out 31 significantly associated SNPs at whole-genome level, located in six chromosomal regions on pig chromosomes SSC4, SSC6, SSC17 and SSCX, for IgG, γδ T-cells, C-reactive protein, lymphocytes phagocytic capacity, total number of lymphocytes, mean corpuscular volume and mean corpuscular haemoglobin. A total of 16 promising functionally-related candidate genes, including CRP, NFATC2, PRDX1, SLA, ST3GAL1, and VPS4A, have been proposed to explain the variation of immune and haematological traits. Our results enhance the knowledge of the genetic control of traits related with immunity and support the possibility of applying effective selection programs to improve immunocompetence in pigs.

Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions.

Swine influenza virus (SIVs) infections cause a significant economic impact to the pork industry. Moreover, pigs may act as mixing vessel favoring genome reassortment of diverse influenza viruses. Such an example is the pandemic H1N1 (pH1N1) virus that appeared in 2009, harboring a combination of gene segments from avian, pig and human lineages, which rapidly reached pandemic proportions. In order to confront and prevent these possible emergences as well as antigenic drift phenomena, vaccination remains of vital importance. The present work aimed to evaluate a new DNA influenza vaccine based on distinct conserved HA-peptides fused with flagellin and applied together with Diluvac Forte as adjuvant using a needle-free device (IntraDermal Application of Liquids, IDAL®). Two experimental pig studies were performed to test DNA-vaccine efficacy against SIVs in pigs. In the first experiment, SIV-seronegative pigs were vaccinated with VC4-flagellin DNA and intranasally challenged with a pH1N1. In the second study, VC4-flagellin DNA vaccine was employed in SIV-seropositive animals and challenged intranasally with an H3N2 SIV-isolate. Both experiments demonstrated a reduction in the viral shedding after challenge, suggesting vaccine efficacy against both the H1 and H3 influenza virus subtypes. In addition, the results proved that maternally derived antibodies (MDA) did not constitute an obstacle to the vaccine approach used. Moreover, elevated titers in antibodies both against H1 and H3 proteins in serum and in bronchoalveolar lavage fluids (BALFs) was detected in the vaccinated animals along with a markedly increased mucosal IgA response. Additionally, vaccinated animals developed stronger neutralizing antibodies in BALFs and higher inhibiting hemagglutination titers in sera against both the pH1N1 and H3N2 influenza viruses compared to unvaccinated, challenged-pigs. It is proposed that the described DNA-vaccine formulation could potentially be used as a multivalent vaccine against SIV infections.

Swine influenza viruses (SIVs), the causal agents of swine influenza, are not only important to control due to the economic losses in the swine industry, but also can be pandemic pathogens. Vaccination is one of the most relevant strategies to control and prevent influenza infection. Current human vaccines against influenza induce strain-specific immunity and annual update is required due to the virus antigenic shift phenomena. Previously, our group has reported the use of conserved hemagglutinin peptides (HA-peptides) derived from H1-influenza virus as a potential multivalent vaccine candidate. Immunization of swine with these HA-peptides elicited antibodies that recognized and neutralized heterologous influenza viruses in vitro and demonstrated strong hemagglutination-inhibiting activity. In the present work, we cloned one HA-peptide (named NG34) into a plasmid fused with cytotoxic T lymphocyte-associated antigen (CTLA4) which is a molecule that modifies T cell activation and with an adjuvant activity interfering with the adaptive immune response. The resulting plasmid, named pCMV-CTLA4-Ig-NG34, was administered twice to animals employing a needle-free delivery approach. Two studies were carried out to test the efficacy of pCMV-CTLA4-Ig-NG34 as a potential swine influenza vaccine, one in seronegative and another in seropositive pigs against SIV. The second one was aimed to evaluate whether pCMV-CTLA4-Ig-NG34 vaccination would overcome maternally derived antibodies (MDA). After immunization, all animals were intranasally challenged with an H3N2 influenza strain. A complete elimination or significant reduction in the viral shedding was observed within the first week after the challenge in the vaccinated animals from both studies. In addition, no challenged heterologous virus load was detected in the airways of vaccinated pigs. Overall, it is suggested that the pCMV-CTLA4-Ig-NG34 vaccine formulation could potentially be used as a multivalent vaccine against influenza viruses.

Glaesserella parasuis is the etiological agent of Glässer’s disease, a common pathology in the pork industry with higher prevalence in the postweaning period. Vaccination is one of the strategies to control this disease. Here, we investigated the effect that sow vaccination against virulent strains of G. parasuis had in the nasal microbiota of their offspring. Nasal swabs from fifteen days-old piglets from vaccinated (vs-P, n = 11) and unvaccinated sows (cs-P, n = 11) were obtained and DNA was extracted for 16S amplicon sequencing. Microbiota composition was different, with lower diversity in vs-P, and a strong clustering of the groups in beta diversity analysis. Among the 1509 sequences associated to either study group, all the sequences classified as G. parasuis (10 ASVs) had lower relative abundance in the vs-P group. A list of 32 inferred metabolic pathways were statistically different between groups. A distinctive structure of the two microbial networks was detected, with modules in the cs-P not conserved in the vs-P network. In conclusion, vaccination of the sows had a large effect in the microbiota composition of their offspring that went beyond the effect on the targeted pathogen. The mechanisms underneath these changes may include alteration of the microbiota network due to the elimination of the targeted pathogen and/or immunological changes.

Haemophilus parasuis is part of the microbiota of the upper respiratory tract in swine. However, virulent strains can cause a systemic disease known as Glässer’s disease. Several virulence factors have been described in H. parasuis including the virulence-associated trimeric autotransporters (VtaAs). VtaA2 is up-regulated during infection and is only found in virulent strains. In order to determine its biological function, the vtaA2 gene was cloned with its native promotor region in pACYC184, and the transformed Escherichia coli was used to perform functional in vitro assays. VtaA2 was found to have a role in attachment to plastic, mucin, BSA, fibronectin and collagen. As other VtaAs from H. parasuis, the passenger domain of VtaA2 contains collagen domains. In order to examine the contribution of the collagen repeats to VtaA2 function, a recombinant vtaA2 without the central collagen domains was obtained and named vtaA2OL. VtaA2OL showed similar capacity than VtaA2 to adhere to plastic, mucin, BSA, fibronectin and plasma but a reduced capacity to adhere to collagen, suggesting that the collagen domains of VtaA2 are involved in collagen attachment. No function in cell adhesion and invasion to epithelial alveolar cell line A549 or unspecific binding to primary alveolar macrophages was found. Likewise VtaA2 had no role in serum or phagocytosis resistance. We propose that VtaA2 mediates adherence to the host by binding to the mucin, found in the upper respiratory tract mucus, and to the extracellular matrix proteins, present in the connective tissue of systemic sites, such as the serosa.

The aim of this study was to characterise bacteria in the genus Bergeyella isolated from the nasal passages of healthy piglets. Nasal swabs from 3 to 4 week-old piglets from eight commercial domestic pig farms and one wild boar farm were cultured under aerobic conditions. Twenty-nine Bergeyella spp. isolates were identified by partial 16S rRNA gene sequencing and 11 genotypes were discriminated by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Bergeyella zoohelcum and Bergeyella porcorum were identified within the 11 genotypes. Bergeyella spp. isolates exhibited resistance to serum complement and phagocytosis, poor capacity to form biofilms and were able to adhere to epithelial cells. Maneval staining was consistent with the presence of a capsule. Multiple drug resistance (resistance to three or more classes of antimicrobial agents) was present in 9/11 genotypes, including one genotype isolated from wild boar with no history of antimicrobial use. In conclusion, Bergeyella spp. isolates from the nasal cavities of piglets showed some in vitro features indicative of a potential for virulence. Further studies are necessary to identify the role of Bergeyella spp. in disease and within the nasal microbiota of pigs.

Glaesserella (Haemophilus) parasuis, an early colonizer of the nasal cavity in piglets, is a highly heterogeneous species, comprising both commensal and virulent strains. Virulent G. parasuis strains can cause fibrinous polyserositis called Glässer’s disease. Colostrum is a source of passive immunity for young piglets. When vaccinating sows, protective antibodies are transferred to their offspring through the colostrum. Here, sow vaccination was performed with a protein fragment, F4, from the outer membrane trimeric autotransporters VtaAs exclusively found in virulent G. parasuis. Piglets were allowed to suckle for 3 weeks, following which a challenge with two virulent strains of G. parasuis was performed. A group of nonvaccinated sows and their piglets were included as a control. Antibodies against F4 were confirmed using ELISA in the vaccinated sows and their offspring before the G. parasuis challenge. Compared to the control group, F4-vaccination also resulted in an increased level of serum TGF-β both in vaccinated sows and in their offspring at early time points of life. After the challenge, a lower body temperature and a higher weight were observed in the group of piglets from vaccinated sows. One piglet from the non-vaccinated group succumbed to the infection, but no other significant differences in clinical signs were noticed. At necropsy, performed 2 weeks after the virulent challenge, the level of surfactant protein D (SP-D) in bronchoalveolar lavage was higher in the piglets from vaccinated sows. Vaccination did not inhibit the nasal colonization of the piglets by the challenge strains.