Publications

The ability to control an immune response for the benefit and production efficiency of animals is the objective of immunomodulation in food-producing animals; substances that exert this control are called immunomodulators. A Spanish product (Inmunicin MAYMO(R)), based on food plant phytosterols, is being commercialized as complementary feed. The main component of this product is Beta-sitosterol (BSS). BSS and its glycoside (BSSG) have been shown to exhibit anti-inflammatory, anti-neoplasic, anti-pyretic and immune-modulating activity demonstrated by in vitro and in vivo experiments. The objective of the present study was to characterize the effect of BSS on the pig immune system using in vitro cell cultures first and to elucidate whether BSS possesses any in vivo activity in fattener pigs after vaccination with porcine reproductive and respiratory syndrome virus (PRRSV) modified life vaccine (MLV). Firstly, our in vitro results showed that BSS increased viable peripheral blood mononuclear cell (PBMC) numbers and it activated swine dendritic cells (DCs) in culture. Secondly, pigs treated with phytosterols prior to vaccination with PRRSV-MLV vaccine exhibited some changes in immunological parameters at different times post-vaccination, such as the proliferation ability of PBMC after phytohemaglutinin stimulation and increased apolipoprotein A1 plasma concentration which may contribute to enhance PRRSV vaccine response. In conclusion, the data in this report show that BSS can be considered an immunomodulator in pigs.

Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and beta(2)m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-beta(2)m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MOP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-beta(2)m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1 kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins. SLA-I monomeric proteins of 41.6 kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an a-helical structure, and the average alpha-helix, beta-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro

The human IL-1 family contains eleven genes encoded at three separate loci. Nine, including IL-36 receptor antagonist (IL-36RN), also known as IL-1F5, are present at a single locus on chromosome 2, whereas IL-18 and IL-33 lie on chromosomes 11 and 9 respectively. There are currently only three known orthologues in the chicken - IL-1 beta, IL-18 and IL-1RN - which are encoded on chromosomes 22, 24 and unplaced, respectively. A novel chicken IL-1 family sequence representing IL-36RN (IL-1F5) was initially identified from an expressed sequence tag (EST) library by its similarity to both chicken IL-1 RN and chicken IL-1 beta. Following isolation of the cDNA from the liver of an uninfected bird, a number of unique sequence features were identified. The predicted protein has a longer NH2-terminus than the human protein; however, as in mammals, this region contains neither a prodomain nor a signal peptide. A putative nuclear export sequence is also apparent, yet a similar motif is absent in mammalian IL-36RN. Although chIL-36RN exhibits low homology with its mammalian orthologues, it encodes a predicted beta-trefoil structure whose beta-strands are conserved with those of the mouse sequence. Unlike in mammals, chIL-36RN expression was constitutive in all tissues and cell subsets examined. In response to viral infection, expression was significantly downregulated in a line of birds which are susceptible to the virus. Chicken IL-36RN, like chIL-1RN, is not encoded at the chIL-1 beta locus, further emphasising the genomic fragmentation of the large IL-1 gene cluster found in mammals. This suggests differential evolution of this cytokine family since the divergence of birds and mammals from a common ancestor, and underlines the difficulty of determining the full repertoire of chIL-1 family members.

Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser (R) C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-gamma responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-gamma response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination.