Study of the genetics of the porcine acute phase proteins

The principle aims of the work presented in this thesis were to further investigate the porcine acute phase proteins (APP) and determine the factors influencing the baseline concentrations of haptoglobin (Hp), C-reactive protein (CRP), Pig major acute phase protein (Pig-MAP) and transthyretin (TTR). The APP are used as markers of inflammation and sub-clinical disease and are considered potential biomarkers for pig health and welfare. They have also been identified as a possible means of breeding for disease resistance, however little is known about the genetics of the porcine APP. This study investigated associations between the APP genes and baseline concentrations and the heritability of those concentrations. An enzyme linked immunosorbent assay (ELISA) was developed for the measurement of porcine CRP, in-house methods were used for the determination of Hp and TTR and a commercial assay was used in the measurement of Pig-MAP. A population of pure line high health boars (n=~345) from 7 lines was used in the initial study each of which had an archived DNA sample and serum samples available for use. Baseline herd concentrations of the 4 APP were determined and correlations between Hp and CRP, Hp and Pig-Map and CRP and Pig-MAP were identified. Significant differences were found between the 7 breeding lines in CRP, Pig-MAP and TTR concentrations, indicating that selective breeding for performance traits may also have affected innate immune traits such as APP concentrations. Single nucleotide polymoiphisms (SNP) were identified in the 4 APP genes and 17 were genotyped across the boar population with line differences apparent in their allele frequencies for CRP, Pig-MAP and TTR. Statistical analysis showed that there were significant associations between 3 of the SNP located in the Hp gene and Hp baseline concentrations (p<0.01); all 3 SNP were also in high linkage disequilibrium. The association indicates that Hp is under genetic control and would also be better suited to use as a biomarker due to the lack of line effects in the boars. A heritability study was earned out utilising a mixed sex population of 297 animals (120 male, 177 female) comprising 7 breeding lines located on 2 farms. Initial analysis identified significant differences between male and females in Hp concentration, between pig lines in Hp, CRP and TTR and between the 2 farm units for CRP concentrations. This study showed that the baseline concentrations of the 4 APP could be affected by a variety of factors such as sex, commercial line and individual farm units and this must be taken into account if they are to be used as biomarkers. Heritability was estimated at 0.15, 0.13, 0.12 and 0.07 for Hp, CRP, Pig-MAP and TTR, respectively. All 4 APP show low heritability in serum concentration, this may prove problematic if they are utilised as a breeding trait. Overall, the findings from this study indicate that baseline concentrations of porcine Hp, CRP, Pig-MAP and TTR are influenced by various factors including sex, breeding line, and farm unit and this must be taken into account before they are utilised as biomarkers. They are traits of low heritability but the evidence suggests there may be a genetic component to their regulation thus requiring further investigation

Prion infectivity in the spleen of a <em>PRNP</em> heterozygous individual with subclinical variant Creutzfeldt-Jakob disease

Blood transfusion has been identified as a source of human-to-human transmission of variant Creutzfeldt–Jakob disease. Three cases of variant Creutzfeldt–Jakob disease have been identified following red cell transfusions from donors who subsequently developed variant Creutzfeldt–Jakob disease and an asymptomatic red cell transfusion recipient, who did not die of variant Creutzfeldt–Jakob disease, has been identified with prion protein deposition in the spleen and a lymph node, but not the brain. This individual was heterozygous (MV) at codon 129 of the prion protein gene (PRNP), whereas all previous definite and probable cases of variant Creutzfeldt–Jakob disease have been methionine homozygotes (MM). A critical question for public health is whether the prion protein deposition reported in peripheral tissues from this MV individual correlates with infectivity. Additionally it is important to establish whether the PRNP codon 129 genotype has influenced the transmission characteristics of the infectious agent. Brain and spleen from the MV blood recipient were inoculated into murine strains that have consistently demonstrated transmission of the variant Creutzfeldt–Jakob disease agent. Mice were assessed for clinical and pathological signs of disease and transmission data were compared with other transmission studies in variant Creutzfeldt–Jakob disease, including those on the spleen and brain of the donor to the index case. Transmission of variant Creutzfeldt–Jakob disease was observed from the MV blood recipient spleen, but not from the brain, whereas there was transmission from both spleen and brain tissues from the red blood cell donor. Longer incubation times were observed for the blood donor spleen inoculum compared with the blood donor brain inoculum, suggesting lower titres of infectivity in the spleen. The distribution of vacuolar pathology and abnormal prion protein in infected mice were similar following inoculation with both donor and recipient spleen homogenates, providing initial evidence of similar transmission properties after propagation in PRNP codon 129 MV and MM individuals. These studies demonstrate that spleen tissue from a PRNP MV genotype individual can propagate the variant Creutzfeldt–Jakob disease agent and that the infectious agent can be present in the spleen without CNS involvement

Insights into Mechanisms of Chronic Neurodegeneration

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177Chronic neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and prion diseases are characterised by the accumulation of abnormal conformers of a host encoded protein in the central nervous system. The process leading to neurodegeneration is still poorly defined and thus development of early intervention strategies is challenging. Unique amongst these diseases are Transmissible Spongiform Encephalopathies (TSEs) or prion diseases, which have the ability to transmit between individuals. The infectious nature of these diseases has permitted in vivo and in vitro modelling of the time course of the disease process in a highly reproducible manner, thus early events can be defined. Recent evidence has demonstrated that the cell-to-cell spread of protein aggregates by a “prion-like mechanism” is common among the protein misfolding diseases. Thus, the TSE models may provide insights into disease mechanisms and testable hypotheses for disease intervention, applicable to a number of these chronic neurodegenerative diseases.https://doi.org/10.3390/ijms1701008217pubpub

Identification of a Homology-Independent Linchpin Domain Controlling Mouse and Bank Vole Prion Protein Conversion

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion