Evaluating the innate immune response of the porcine uterus to vaccine adjuvants for the development of an intrauterine vaccine

Mucosal vaccination is a widely sought after method of vaccination for both its ease of delivery and because it can stimulate a strong local mucosal immune response. As the vast majority of pathogens enter the body through mucosal surfaces, a strong mucosal immune response has the potential to eliminate pathogens before them crossing the epithelial barrier, and can, in the case of the uterus, protect the developing fetuses from pathogens. For a mucosal vaccine composed of inactivated antigens to be effective, it must be formulated with potent adjuvants that will initiate a strong immune response in the uterus. This thesis aimed to evaluate the capacity of uterine epithelial cells to respond to vaccine adjuvants and to subsequently initiate and modulate the local uterine immune response of an intrauterine vaccine in sows. Because uterine epithelial cells (UECs) are the first point of contact for an intrauterine vaccine, we established an in vitro model using primary uterine epithelial cells to enable us to evaluate what receptors are present on these cells that could be targeted by the adjuvants in an intrauterine vaccine. Previous research showed that a triple adjuvant combination including polyI:C, a host defense peptide (HDP) and polyphosphazene (PCEP) administered as part of a mucosal vaccine induced strong humoral immunity. We tested these adjuvants as well as muramyl dipeptide and LPS alone and in combination with each other in the in vitro system. In vitro stimulations resulted with polyI:C alone and in combination with other potential adjuvants induced significant pro-inflammatory cytokines and chemokines, which was not further improved by the inclusion of HDP and PCEP. To determine if the observed response in vitro resulted in similar responses in vivo, the triple adjuvant combination was administered in vivo into the uterus of sows in combination with a standard semen dose. Twenty four hours post-breeding, epithelial cells had significantly increased CCL2 expression in response to the adjuvants combined with semen compared to the response generated to semen alone. Additionally animals bred with adjuvants included in the semen dose had a significant decrease in the proportion of γδ T cells and monocytes in the blood, however, there was no significant increase of these cells in the lumen indicating the γδ T cells were not being recruited into the uterine lumen. Following evaluation of infiltrated monocytes and macrophages in the tissue, no significant difference was observed in these cells levels compared to animals bred with semen alone. Lastly, returning to the in vitro UEC model, I took steps to quantify whether secretions from stimulated uterine epithelial cells impacted monocyte to DC differentiation or moDC activation. Secretions from triple adjuvant-stimulated uterine epithelial cells were not able to induce recruitment of monocytes nor did the secretions impact on the moDCs capacity to endocytose antigen, mature in response to stimulation or induce proliferation in T cells. The established primary UEC culture system established and characterized was observed to closely resemble the behaviour of UECs in vivo for studying the responses of adjuvants and could be further utilized for continuing the develop UECs role in the uterine immune response to stimulants and pathogens. Although uterine epithelial cells respond to vaccine adjuvants, the magnitude of this response is unable to significantly impact the immune response to vaccine adjuvants delivered by the intrauterine route in a semen dose

Dual infections of CD163 expressing NPTr epithelial cells with influenza A virus and PRRSV

In the pig, respiratory co-infections involving various pathogens are far more frequent than single infections. Amongst respiratory viruses, swine influenza type A virus (swIAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are frequently associated. Previously, we performed co-infections with swIAV and PRRSV in porcine alveolar macrophages (PAM) and precision cut lung slices (PCLS). With these two approaches it was practically impossible to have co-infections of the same cells as the main target cell of swIAV is the epithelial cell while the main target of PRRSV is the PAM. This constraint makes the study of interference between the two viruses difficult at the cellular level. In the current report, an epithelial cell line expressing, CD163, the main receptor of PRRSV was generated. This cell line receptive for both viruses was used to assess the interference between the two viruses. Results showed that swIAV as well as PRRSV, even if they interacted differently with the modified epithelial cells, were clearly interfering with each other regarding their replication when they infected a same cell with consequences within the cellular antiviral response. Our modified cell line, receptive to both viruses, can be used as a tool to assess interference between swIAV and PRRSV in a same cell as it probably happens in the porcine host

Molecular and Physiological Effects on the Small Intestine of Weaner Pigs Following Feeding with Deoxynivalenol-Contaminated Feed

We intended to assess how exposure of piglets to deoxynivalenol (DON)-contaminated feed impacted their growth, immune response and gut development. Piglets were fed traditional Phase I, Phase II and Phase III diets with the control group receiving 0.20–0.40 ppm DON (referred to as the Control group) and treatment group receiving much higher level of DON-contaminated wheat (3.30–3.80 ppm; referred to as DON-contaminated group). Feeding a DON-contaminated diet had no impact on average daily feed intake (ADFI) (p < 0.08) or average daily gain (ADG) (p > 0.10) but it did significantly reduce body weight over time relative to the control piglets (p < 0.05). Cytokine analysis after initial exposure to the DON-contaminated feed did not result in significant differences in serum interleukin (IL) IL1β, IL-8, IL-13, tumor necrosis factor (TNF)-α or interferon (IFN)-γ. After day 24, no obvious changes in jejunum or ileum gut morphology, histology or changes in gene expression for IL-1β, IL-6, IL-10, TNFα, or Toll-like receptor (TLR)-4 genes. IL-8 showed a trend towards increased expression in the ileum in DON-fed piglets. A significant increase in gene expression for claudin (CLDN) 7 gene expression and a trend towards increased CLDN 2-expression was observed in the ileum in piglets fed the highly DON-contaminated wheat. Because CLDN localization was not negatively affected, we believe that it is unlikely that gut permeability was affected. Exposure to DON-contaminated feed did not significantly impact weaner piglet performance or gut physiology

A DIO2 missense mutation and its impact on fetal response to PRRSV infection

Abstract Background Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection. Our objectives were to identify mutation(s) within the porcine DIO2 gene and to determine if they were associated with fetal outcomes after PRRSV-2 challenge. Sequencing of the DIO2, genotyping identified variants, and association of DIO2 genotypes with fetal phenotypes including DIO2 mRNA levels, viability, survival, viral loads, cortisol and thyroid hormone levels, and growth measurements were conducted. Results A missense variant (p.Asn91Ser) was identified in the parental populations from two independent PRRSV-2 challenge trials. This variant was further genotyped to determine association with fetal PRRS outcomes. DIO2 mRNA levels in fetal heart and kidney differed by the genotypes of Asn91Ser substitution with significantly greater DIO2 mRNA expression in heterozygotes compared with wild-type homozygotes (P < 0.001 for heart, P = 0.002 for kidney). While Asn91Ser did not significantly alter fetal viability and growth measurements, interaction effects of the variant with fetal sex or trial were identified for fetal viability or crown rump length, respectively. However, this mutation was not related to dysregulation of the hypothalamic-pituitary-adrenal and thyroid axis, indicated by no differences in circulating cortisol, T4, and T3 levels in fetuses of the opposing genotypes following PRRSV-2 infection. Conclusions The present study suggests that a complex relationship among DIO2 genotype, DIO2 expression, fetal sex, and fetal viability may exist during the course of fetal PRRSV infection. Our study also proposes the increase in cortisol levels, indicative of fetal stress response, may lead to fetal complications, such as fetal compromise, fetal death, or premature farrowing, during PRRSV infection