A novel inactivated vaccine against Lawsonia intracellularis induces rapid induction of humoral immunity, reduction of bacterial shedding and provides robust gut barrier function.

Porcine proliferative ileitis is a major economic burden for the swine industry, affecting growing pigs and young adult pigs. In this study, the protective efficacy of an inactivated, injectable whole-cell bacteria vaccine against L. intracellularis - Porcilis® Ileitis was evaluated under field conditions. Eighty-five, three-week-old pigs on a commercial farrow-to-finish farm were vaccinated by the intramuscular route, either with a dose of injectable vaccine, or with saline. A subset of vaccinates and control pigs were necropsied at 21 days post-challenge. Incidence and severity of ileitis were evaluated by gross and microscopic observation of ileal tissues. Colonization of the gut after challenge was examined by L. intracellularis-specific immunohistochemistry, and qPCR of ileal scrapings. Integrity of the intestinal barrier was evaluated to quantify a range of intestinal markers including secreted mucin and intestinal alkaline phosphatase, and innate immune markers including Caspase-3 and Calprotectin. A second subset of pigs was monitored for fecal shedding of L. intracellularis, until resolution of shedding. Our investigation indicated that Porcilis Ileitis provided robust protection against ileitis, reduced bacterial shedding 15-fold (p < .05) and preserved normal gut barrier function in the face of an experimental challenge with virulent L. intracellularis.

An intronic polymorphism in the porcine IRF7 gene is associated with better health and immunity of the host during Sarcocystis infection, and affects interferon signalling.

Interferon regulatory factor 7 (IRF7), as a key regulator of type I interferon response, plays an important role during innate response against viral infection. Although well conserved across species, the structure of IRF7 and its function during parasite infection are not well documented in farm animals, such as the pig. To bridge this gap, we have determined the porcine IRF7 gene structure and identified two intronic single nucleotide polymorphisms (SNPs), SNP g.748G>C and SNP g.761A>G, in commercial pig breeds. The distribution of SNP g.761A>G in multiple breeds suggested that it was in Hardy-Weinberg equilibrium and allowed us to map it at the top of SSC2. We found that during Sarcocystis miescheriana infection, the G allele was associated with high lymphocyte levels (P < 0.02), reduced drop in platelet levels (P < 0.002) and IgG1-Th2-dominated response (P < 0.05). This suggests that the G allele was associated with better health and immunity of the host during Sarcocystis infection. Furthermore, we have also provided suggestive evidence that the G allele of SNPc.761A>G enhances the transactivation activity of IRF7, possibly by improving IRF7 transcript splicing of intron-3. These findings would suggest that IRF7, as a transcriptional regulator, is involved in the defence mechanism against a larger spectrum of pathogens, and in more host species, than initially anticipated.

Dynamic differential regulation of innate immune transcripts during the infection of alveolar macrophages by the porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, is the etiologic agent of an infectious disease of that name, characterized by respiratory disorders, abortion in pregnant sows and high mortality in piglets, resulting in significant economic losses in the pig industry worldwide. In order to identify whether genetic differences in PRRSV response may exist in pigs, alveolar macrophages were used to assess the progression of the type-I interferon (IFN) transcript response in porcine alveolar macrophages infected by PRRSV. Our results suggest that a dynamic differential regulation of the type-I IFN and chemokine transcripts may operate during the first hours of infection with and entry of the virus in alveolar macrophages, and provide a compelling mechanism for the establishment of PRRSV replication in susceptible cells.

Regulation of gibberellin 20-oxidase and gibberellin 3beta-hydroxylase transcript accumulation during De-etiolation of pea seedlings.

Gibberellin (GA) 20-oxidase (GA 20-ox) and GA 3beta-hydroxylase (GA 3beta-hy) are enzymes that catalyze the late steps in the formation of active GAs, and are potential control points in the regulation of GA biosynthesis by light. We have investigated the photoregulation of the GA 20-ox and GA 3beta-hy transcript levels in pea (Pisum sativum L.). The GA 20-ox transcript level was higher in light-grown seedlings than in etiolated seedlings, whereas GA 3beta-hy mRNA accumulation was higher in etiolated seedlings. However, transfer of etiolated seedlings to light led to a 5-fold increase in the expression of both transcripts 4 h after transfer. GA 20-ox mRNA accumulation is regulated by both phytochromes A and B. Transfer to light also resulted in a 6-fold decrease in GA(1) levels within 2 h. These results suggest that the light-induced drop in GA(1) level is not achieved through regulation of GA 20-ox and GA 3beta-hy mRNA accumulation. The application of exogenous GA(1) to apical buds of etiolated seedlings prior to light treatments inhibited the light-induced accumulation of both GA 20-ox and GA 3beta-hy mRNA, suggesting that negative feedback regulation is an important mechanism in the regulation of GA 20-ox and GA 3beta-hy mRNA accumulation during de-etiolation of pea seedlings.

The first step of gibberellin biosynthesis in pumpkin is catalyzed by at least two copalyl diphosphate synthases encoded by differentially regulated genes.

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression.

The LS locus of pea encodes the gibberellin biosynthesis enzyme ent-kaurene synthase A.

Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent-kaurene synthase A (KSA) and ent-kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA-responsive ls-1 mutant of pea suggested that GA levels are reduced in a developmental- and organ-specific manner due to reduced GA biosynthesis. Analysis of cellfree enzyme preparations from WT and ls-1 embryos at contact point reveals that ls-1 reduces the activity of KSA but not KSB. To characterize the ls-1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls-1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls-1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent-kaurene in vitro. Based on the sequence of RT-PCR products, three different truncated KSA proteins are predicted to exist in ls-1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls-1 allele is not null and is able to encode at least a partially functional KSA since a more severe is allele has been identified. The ls-1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.