Virulence factors associated with amoebic gill disease from Neoparamoeba perurans

Determining the virulence factors associated with pathogenic organisms is imperative in comprehending disease pathogenesis and ultimately paves the way for methods of treatment and disease management. Neoparamoeba perurans is an ectoparasitic amoeba responsible for the hyperplastic gill infection of marine cultured finfish referred to as amoebic gill disease (AGD). Neoparamoeba perurans is suspected to have a repertoire of putative virulence factors that enable the attachment and colonisation of the host, as well as resistance to host immune response. The primary aim of this study was to identify the virulence factors of N. perurans (both amoebic and bacterial) using a proteomic approach, and to increase understanding of the parasite’s mechanisms as an infectious agent in AGD of farmed fish. This thesis is divided into five chapters comprising: an introduction to AGD, N. perurans and virulence factors, three experimental chapters written as manuscripts for publication and finally, a general discussion that reviews the findings and future outcomes of this research. The first experimental chapter (chapter two) confirmed the reported loss of N. perurans virulence in long-term culture, as determined by an in vivo challenge trial. This work validated AGD infection in the virulent cohort and validated the lack of AGD infection in the attenuated cohort by gill scoring, histopathology, and qPCR of gill tissue. The microbiome of the virulent-1 and attenuated N. perurans were characterised by 16S rRNA Illumina MiSeq sequencing for informing the bacterial database protein search. Two-dimensional gel electrophoresis (2D gel) and LC-MS/MS analysis revealed that hydrophilic virulence proteins are differentially expressed between a virulent-1, virulent-2 and attenuated culture. Differentially expressed proteins were actin-associated proteins, lipoxygenase, an ABC membrane transporter and profilin. Overall, these proteins suggest that the virulent-2 culture of N. perurans maintains elevated levels of proteins involved in cytoskeletal, oxidative and immunomodulatory roles. The second experimental chapter (chapter three) assessed the extracellular secretions, or exoproteome, of a virulent-3 and attenuated parasite for virulence factors. Using label–free LC-MS/MS, proteins that were differentially expressed were found to be elevated in the attenuated parasite. Additionally, the extracellular secretions of the attenuated parasite had a greater cytotoxic effect on the salmonid RTgill W1 cell line compared with that of the virulent-3 isolate. This effect was believed to be associated with the increased growth of bacteria in the attenuated culture of N. perurans. Antibiotics were employed to reduce the presence of bacteria in the culture, which lessened the cytotoxicity observed in RTgill W1. Neoparamoeba perurans specific proteins were found in both exoproteomes that have serine protease activity which may facilitate AGD infection, but are not the main drivers of cytotoxicity. Additionally unknown and hypothetical proteins of N. perurans were identified that warrant further investigation. The third experimental chapter (chapter four) investigated the cell-surface and membrane proteome of a virulent-3 and attenuated N. perurans. Proteins involved in potential cell-mediated adhesion roles such as GAPDH and GTPase signalling proteins were found and may represent putative targets of treatment. Overall, the proteins identified reveal moonlighting roles for common proteins such as proteins involved in glycolysis that may aid host ligand attachment. The outcome of this work is expected to facilitate progress in the design of N. perurans targeted therapies for the treatment of AGD in farmed fish. Developing treatments that target and disrupt N. perurans will improve gill health in salmonids and will promote aquaculture sustainability and productivity.n

Mathematical modelling of the bovine estrus cycle and an investigation into silent heat and twinning /

ye

checking1

descriptionabstractn

Development of novel polymeric delivery systems for the controlled release of therapeutic agents to prevent the spread of Zoonotic diseases

The agricultural revolution and related activities have led to new connections between humans and farm animals, which are expected to expand due to the global trade of livestock and growth in the demand for animal protein for human consumption. Ireland is a globally recognised trading nation, providing high-quality meat, milk, and dairy products. In order to remain viable and grow over time, the Irish agricultural sector will need to focus on animal health to maximise productivity, mainly because parasitic infections represent a major production-limiting issue with the possibility of transmission within animal populations and from animal to human populations. Repidose® Ready Pulse bolus is the only orally administered anthelmintic intraruminal delivery device for grazing cattle in Ireland. Therefore, the aim of this research was to develop novel controlled-release solid dispersion formulations for the safe and efficient delivery of benzimidazole anthelmintics to cattle, achieved through processing the active ingredient with biodegradable polymers via hot-melt processing technologies. To accomplish this goal, five specific objectives were defined (i) to improve the poor water solubility of fenbendazole using a biocompatible, biodegradable, and hydrophilic polymer; (ii) to identify and analyse other polymers compatible with fenbendazole; (iii) to design and manufacture fenbendazole extended-release extruded formulations; (iv) to design and mould solid dispersion formulations for the extended-release of fenbendazole; and (v) to upgrade the solid dosage form with oxfendazole as is present in the commercial Repidose® bolus. The results achieved during this research revealed that (i) solid dispersions of fenbendazole and PEO are physically and chemically compatible, with higher percentages of PEO processed at lower screw speed being more effective in enhancing the drug dissolution properties; (ii) Kolliphor® P 407, PCL, and PLA are compatible with fenbendazole and have potential for further application in the development of solid dosage forms; (iii) melt-extruded matrices showed that the drug release rate from a PEO/PCL blend can be tailored by altering the ratio of PEO and PCL; (iv) moulded tablets of amorphous solid dispersions improved the dissolution properties of fenbendazole, with the PEO/PCL blend-based matrices showing an extended-release profile; and (v) hot-melt extrusion coupled with micro-injection moulding produced extended-release amorphous solid dispersion of oxfendazole, which is a novel pharmaceutical strategy with the potential to improve upon the Repidose® bolus. This research project successfully demonstrated the application of a semi-continuous manufacturing process for the production of plasticised solid dispersion tablets of oxfendazole as a potentially viable alternative to the current tablet formulation present in the Repidose® bolus, thus this technology could be viable on the market to provide effective parasite control.ye

Novel studies into the improvement of cattle fertility using multiple technologies that converges bioinformatics with machine learning.

This study aimed to evaluate the presence of a panel of 18 candidate lethal recessives in the Irish dairy, and beef cattle populations. Establishing the presence and frequency of such recessive alleles will assist the scientific, and animal breeding communities. The frequency of variants in Irish Holstein – Friesian dairy cattle are included in the dairy chapter. Furthermore, the beef cattle chapter assesses six different breeds Aberdeen Angus, Belgian Blue, Charolais, Hereford, Limousin and Simmental. Animal breeders have established that the optimal breeding goal is a more balanced and informed breeding approach; thus, the lethal recessives in the various population were assessed for any associated effects on 16 production traits in both the dairy and beef cattle prior to any breeding decisions made on-farm, for example culling, which may reduce the overall genetic diversity, increasing inbreeding depression and ultimately reducing population viability. A comparative genomics approach followed the above candidate lethal recessive association studies. This chapter identified variants in essential genes, using alternative mammalian species studies as supporting evidence. Variants were identified and prepared for genotyping approximately 350 K cattle, the results were analysed for potential candidates associated with embryo loss. Furthermore, variants within a 500 kb range of the candidates were selected and submitted to identify segregating variants that were possibly unknown to industry. Genetics and high throughput data are one aspect of improving cattle fertility. It is clear that in addition to genomic programmes, predictive technologies will be used in making forecasts in the agricultural sector, particularly machine learning and AI models. Such predictive technologies may ultimately incorporate genomic and environmental data into selection programmes; however, interdisciplinary studies are currently at the point of combining technologies. ReproDoc assesses cattle pregnancy status using mobile ultrasound technology and has amassed a database of animal-related details. This chapter prepared a private commercial database for predictive models to determine when individual cows are optimal for insemination. Customers would be the first to benefit from the success of such a technology, as the requirement for semen, and insemination attempts would decrease, and the need for herd replacements would be optimised, ultimately reducing the demand for replacements. Such information could provide farmers reassurance in allowing a cow to remain on the farm until the next breeding cycle, thus improving welfare and reducing wastage.ye

Novel investigations into the genomic architecture affecting the muscle growth and development of Irish beef and dairy cattle

Muscle growth and development in livestock is an important indicator of carcass yield and performance. Investigating the complex polygenic nature of carcass traits may yield additional knowledge and important insights into the role of genomic variation in muscle growth and development. This thesis describes the approaches undertaken to dissect the genomic variation and biological processes governing bovine muscle growth and development. Chapter two describes the estimation of the frequency and effects of genomic polymorphisms in Myostatin (MSTN) on milk, fertility and carcass performance in a population of dairy cows. The role of MSTN in muscle growth and development is long established however the aim here was to estimate both the beneficial and possible antagonistic effects on production traits of economic importance in the dairy industry. Similarly, the role μ-calpain (CAPN1) and Calpastatin (CAST) in post-mortem tenderisation is widely reported and in chapter three, the frequency and effects of genomic polymorphisms in CAPN1 and CAST on fertility and carcass performance were estimated in a population of beef cows. Chapter four describes the approaches taken to identify genomic polymorphisms with novel functional roles in bovine muscle growth and development and the disentanglement of their impact on gene function while also estimating the frequency of same polymorphisms in a large population of Irish cattle. Finally, the objective of chapter five was to identify novel associations between regions of the bovine genome with carcass performance using a GWAS in a population of beef cattle. Additionally, a functional genomics dataset in the form of gene expression data was integrated with GWAS results and subsequent gene ontology and pathway analysis contributed to an improved and enhanced understanding of the genomic variation underlying carcass performance. The project identified a number of gene polymorphisms, genomic regions and biological pathways significantly associated with carcass performance.ye

Critical studies on the novel development and implementation of vaporized hydrogen peroxide (VH202) for terminal sterilization of medical devices encompassing use of flow cytometry for the real-time commensurate monitoring of microbial inactivation

Aims and Rationale: Sterilization microbiology relies on determining the linearity of microbial survivor curves for calculating decimal reduction times (D-values) that is used to inform sterility assurance levels (SAL), critical to efficacious sterilization of medical device for patient care application. Medical devices play an important role in the provision of healthcare, with a global market estimated at $432bn and expected to surpass$650bn by 2028. Terminal sterilization of medical devices is currently provided by established methods including Gamma, E-beam and X-ray radiation with Ethylene Oxide gas sterilization continuing to be the method of choice due to wide ranging material compatibility. Given the hazards associated with EO gas, alternative methods are actively being pursued by the global medical device industry. Furthermore, there is an increasing opportunity to develop sustainable sterilization microbiology processes necessary to meet emerging needs including evolution of device features containing heat sensitive polymers, inclusion of sensor technology and biologics. This constitutes the first, and timely study to (i) establish and alternative method using vaporized hydrogen peroxide (VH2O2) at an industrial sterilization scale and (ii) report on the linearity of microbial inactivation plots for treated Geobacillus stearothermophilus and Bacillus atrophaeus spores to demonstrate the appropriateness of process validation as a means of confidently determining SAL.ye

Estimation of the frequency and effects of casual mutations on fertility and production traits in Irish dairy cattle

Fertility is a major driver for profitability and sustainability of livestock enterprises. Identifying and estimating the effects of known lethal recessive genetic mutations and genes of major effect on production traits in cattle populations provides additional information to the industry for potential incorporation into breeding programs. Such information may support breeders to make more informed decisions through the identification of carrier animals and the evaluation of potential strategic matings in cases where carrier animals may be of otherwise high genetic merit. This project aims to estimate the frequency and effects of a panel of DNA polymorphisms (n=18) in Irish Holstein Friesian cattle, some of which are validated as causative mutations responsible for lethal recessive disorders (CVM, BLAD, DUMPS and Brachyspina), and some of which have been observed to have major effects on production and functional traits in previous research studies (STAT1, STAT3, STAT5). Genotypes on 21,707 Holstein Friesian dairy cattle were obtained from the ICBF, as were phenotypic data on milk, fertility, carcass and health traits (n=16). Phenotypes, expressed as predicted transmitting abilities (PTAs) were prepared for inclusion in the analysis by removal of parental contributions through a deregression process. Haplotypes were predicted using PHASE for all SNPs with positions on the same chromosome. Subsequently, associations between each SNP/haplotype and PTA were analysed in ASReml using a weighted mixed animal model. Several associations between the genes of major effect and production and functional traits were evident and consistent with previous reports of such associations, for example SNPs within the DGAT and Casein genes were associated with milk composition traits as expected, however, they were also associated with fertility and carcass traits. SNPs within the STAT genes, of which there has not been extensive previous studies on in cattle populations, were associated with both production and functional traits in the population studied. Additionally, a candidate novel lethal recessive mutation in LFNG has been identified. The results from this project will be evaluated by our industry partners, the ICBF, responsible for national genomically assisted breeding programs in Irish cattl

Optimal transport and incentive design in multi-agent economic control /

This paper develops a principled framework for incentive design in multi-agent economic systems using tools from optimal transport (OT) theory and decentralized control. We consider a class of stochastic multi-agent environments in which each agent selects actions to minimize individual cost functions that depend on both private preferences and aggregate outcomes. To promote socially desirable allocations, we introduce an OT-based mechanism design approach, where incentives are computed as gradients of a Lagrangian dual formulation over probability measures. Our main results establish: (i) a KKT-type characterization of incentive compatibility in Wasserstein space, (ii) monotonicity and fairness of equilibrium allocations under convex coupling, (iii) structural convexity of cost functionals over coupled agent dynamics, (iv) convergence of iterative market updates to optimal allocations, and (v) efficiency guarantees under decentralized feedback. We demonstrate that fairness and incentive alignment emerge naturally as solutions to constrained OT problems, allowing for scalable, interpretable, and robust economic control policies. This formulation provides a unifying perspective on decentralized optimization, mechanism design, and ergodic fairness in economic networks, and opens new directions for data-driven social planning under uncertainty.ye