Relatively little is known about Salmonella Virchow as a pathogen. Its prevalence varies from country to country, however it is constantly associated with invasive disease, particularly in children and the immuno-compromised. The main sources of S. Virchow are humans and poultry. The aims of this study were to determine the genetic relatedness of S. Virchow isolates from difference sources in England, to characterise its infection biology using in vitro and in vivo based models, to establish the immune response produced by poultry in response to infection with the serovar and to begin to determine the level of protection and cross-protection that could be achieved against the serovar and S. Typhimurium. The genetic relatedness of the S. Virchow isolates was determined using molecular typing techniques including MLST and PFGE. The isolates were screened for the presence of 12 virulence genes that have been associated with adhesion, invasion and persistence. Human and avian cell lines and in vivo poultry infection experiments were used to characterise S. Virchow’s invasiveness, persistence and ability to elicit an immune response, compared to a well characterised S. Typhimurium isolate. Immune responses were evaluated by immunohistochemistry, RT-PCR and ELISA, to establish aspects of the innate, cellular and humoral response, as well as cytokine and chemokine expression. An in vivo poultry infection experiment was performed to gain an indication of the level of protection and cross-protection offered by primary infection with S. Virchow against secondary infection. Bacteriology, ELISA and western blot methods were used analyse this. Overall, S. Virchow appears to be a relatively clonal serovar, regardless of the source and the results indicate this is widespread and not solely in the UK. All of the isolates possessed the 12 virulence genes, which could contribute to its virulence in some hosts. S. Virchow was particularly persistent and inflammatory in the human Caco2 cells, which is consistent with the increased virulence previously reported in humans. The in vitro HD11 assay and the in vivo poultry infection experiments were consistent in showing S. Virchow colonises the chicken intestine to high levels, causes transient systemic infection and stimulates a moderate inflammatory response, very similar to S. Typhimurium infection. S. Virchow infection stimulated all aspects of the chicken immune system, characteristic of a broad-range serovar. Initial results from the in vivo protection experiment showed primary infection with S. Virchow does offer some protection against systemic invasion, although adequate protection against caecal colonisation was not found. However, 2 proteins were identified that strongly reacted and cross-reacted with sera from infected chickens, providing optimism that a vaccine to protect against S. Virchow colonisation could be developed with further research.

Canine infection from pathogenic Leptospira serovars remains an issue within the UK, despite the availability of a canine vaccine. Canine leptospirosis cases are non-reportable and data regarding current levels for both suspected and confirmed cases is limited. A questionnaire based survey was undertaken to determine the number of canine leptospirosis cases within UK practices over a 12 month period. Average canine vaccination coverage across responding practices was determined as 60%, with 1669 vaccines administered per practice on average within the responding practices. No significant difference was witnessed between doses administered in either mixed or dedicated small animal practices (1692.40 and 1653.38 respectively), demonstrating that vaccination habits vary between individual clinicians rather than practice type. Diagnosing leptospirosis remains an issue, particularly relating to vague clinical signs during early infection. Survey results emphasised the priority that clinicians base on initially vague signs, with leptospirosis being typically considered once icteric signs present, where mortality rates are greater and treatment is less effective. Despite well documented associations linking leptospirosis and rodents, a current uncertainty remains regarding serovars maintained within the UK. In an attempt to clarify the situation, 283 wild rodents were sampled from rural (n=7) and urban sites (n=8). Infection was identified within 23 (8.13%) samples belonging to wood mice (n=16/152), bank voles (n=5/47) and field voles (n=2/10). Initial Leptospira identification using direct sequencing of PCR amplicons showed a single infecting pathogenic species (Leptospira interrogans). Serology data was obtained for 71 rodents using the microscopic agglutination test (MAT). Positive samples from pooled antigen testing (n=7/71; 9.86%) were further tested using four individual antigens. Data further confirmed a single infecting species (L. interrogans) and serogroup (Australis). Interestingly, we did not detect Leptospira within the portion of 67 rat kidney samples investigated. Stained kidney sections (n=11) showed limited association between inflammation and leptospire presence, indicating that rodents may shed the bacteria asymptomatically. Multi-locus sequence typing (MLST) schemes have been successfully applied to identify the sequence types (STs) of pathogenic Leptospira strains. The PCR positive rodent samples were tested using MLST (n=23). All samples with a full profile (n=11) were shown to belong within ST-24, with five partial profiles also likely to belong to the same ST. To date, three serovars are within ST-24 (Jalna, Bratislava and Muenchen), that belong to the Australis serogroup. This was the first study to utilise DNA extracted directly from kidney tissue to perform Leptospira MLST analysis. MLST data further emphasises a single infecting species and presents evidence for a single infecting serogroup. Further work involved full genome sequencing of ten strains not previously investigated, covering pathogenic, intermediate and saprophytic species. Sequence data for each strain was obtained using the MiSeq platform. The ‘core’ genome was identified across 17 strains (n=1,095; 28.76%), with pathogenic strains being more conserved with a greater shared core genome (n=2,859; 69.30%). Single nucleotide polymorphism (SNP) data was generated for strains (n=6), with an average of 35,346 SNPs per strain (range=686 to 55,303). L. interrogans serovar Icterohaemorrhagiae had the lowest SNP count (686) and was the only strain with a greater number of non-synonymous SNPs compared to synonymous (1.8:1); indicating close relatedness between serovars Icterohaemorrhagiae and Copenhageni. Coding sequences were identified within genome regions that may relate to antigenic differences (high SNP variation) or relate to key cellular processes (low SNP variation). Due to poor gene characterisation, hypothetical proteins make up a high proportion of coding sequences within such regions. Further work to characterise identified coding sequences may identify future therapeutic or diagnostic targets. This project aimed to investigate the current situation concerning canine and rodent Leptospira research within the UK. Results presented within this thesis demonstrate several wild rodent species within England are capable of maintaining and potentially shedding pathogenic strains known to infect both humans and dogs. Serogroup Australis (found infecting rodents) is now protected within a tetravalent canine vaccine; however annual booster vaccinations are required for optimal immunity. Extended urban sampling would be of great benefit considering the absence of positive urban samples. Suspected and confirmed canine cases are still witnessed within UK practices despite the reported majority having current vaccinations. Continued monitoring of serogroups would benefit vaccination strategies, and an emphasis on early detection within infected dogs would allow for the greatest chance of survival.

Bluetongue (BT) is a seasonal vector-borne, viral, disease that causes significant economic and welfare problems in ruminants. It is transmitted by species of Culicoides midges (Diptera: Ceratopogonidae), and as such, the distribution of the disease is restricted to regions where the vectors are present. Once restricted to tropical and subtropical regions of the world, serotypes of BT have been causing outbreaks in southern Europe, following its introduction in 1998, and in 2006, BT serotype-8 emerged in northern Europe, causing devastating economic, welfare and production consequences. The northwards expansion of BT has been attributed to a shift in the geographic limit of the Culicoides imicola Meigen vector, and the involvement of the newly implicated Palaearctic vectors, the Obsoletus and Pulicaris Groups. Little is known about the ecological characteristics of the newly implicated vectors, or indeed those believed to be non-vectors, including their distribution and abundance, making disease risk assessment and management difficult. Within this thesis, a series of field experiments were initiated on a group of farms to gain insight into the distribution and abundance of Culicoides species. The results highlighted that a very high level of variation is seen when trapping Culicoides at the local-scale, yet it is possible to build a strong model explaining this variation using a mixture of host and environmental variables, with satellite-derived ecological correlates. This high level of variation in midge catches present between farms undermines attempts to record their nationwide distribution in larger scale models. The results uniquely model Obsoletus Group abundance, and highlight a difference in host involvement between vector and non-vector models. Further field studies which showed a lack of significant variation both between years and at the within-farm level highlight the robustness of this model in predicting the distribution of the BT vectors species, such that it could prove useful for exploring targeted surveillance and control methods. Culicoides distributions do not remain static, therefore an understanding of their flight behaviour is critical to determining the distance over which an insect may transmit a disease agent and the size of the area over which control should be applied. Laboratory studies were undertaken to validate the use of commercial fluorescent dusts as a quick and effective method of marking Culicoides for both field and laboratory studies, and a ‘self-marking’ technique was conceived. Dispersal studies, using the dusts, determined the distances that Obsoletus Group females and males, as well as C. pulicaris females, are able to disperse over a set period of time. This knowledge of flight speed and distance is of utmost value as a critical component in the modelling of BT disease and other Culicoides-borne diseases. The Obsoletus Group contains four members (C. obsoletus, C. scoticus, C. chiopterus and C. dewulfi¬) which are difficult to differentiate down a microscope. Using morphometric analyses, female C. obsoletus and C. scoticus individuals could be separated under a stereomicroscope based on abdominal measurements. Studies such as those contained in this thesis, therefore, are of utmost value in providing information on critical components in the modelling of BT disease and other Culicoides-borne diseases.

Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of economically important arboviruses of livestock. Two such arboviruses, bluetongue virus (BTV) and Schmallenberg virus (SBV) have recently emerged in northern Europe inflicting unprecedented outbreaks of disease in this region. The aim of the current investigation was to explore both host seeking behaviour and surveillance methods for livestock-associated Culicoides species in the UK. To achieve this aim, a series of field-based, manipulative experiments were conducted using three farm sites in southern England. These studies demonstrated that host preference had a significant impact upon several parameters important in determining arbovirus transmission. Culicoides were found to be differentially attracted to different breeds of sheep (p<0.05) and blood feeding efficiency was shown to be determined in part by whether the sheep had been sheared (p<0.05). In addition the presence of an alternative host (a cow and its calf) was demonstrated to lead to an increased Culicoides biting rate on sheep held in close proximity (p<0.05), increasing the risk of arbovirus transmission. Preliminary studies of volatile chemicals produced by hosts illustrated that while these attracted livestock-associated Culicoides at rates higher than those recorded in un-baited traps (p<0.05), collections only represented a small proportion of those collected on hosts themselves. These studies, however, provided a platform for future investigations of this area. Finally, the use of light-emitting diode (LED) baited suction traps was trialled as a means of improving detection sensitivity in surveillance of Culicoides populations. This study found that certain Culicoides species demonstrated increased sensitivity to specific wavelengths (p<0.05) and integration of these commercially available traps could improve our understanding of the abundance, geographic distribution and behaviour of these species.

This thesis presents work to examine the genetic basis of CCL rupture in dogs. It describes research to identify causative mutations that will help to develop a genetic screening test to identify dogs that have a high risk of developing CCL rupture. Cranial Cruciate Ligament (CCL) rupture is the most common cause of hind limb lameness in dogs and is especially common in large and giant breeds such as Newfoundlands, Rottweilers and Staffordshire Bull Terriers. CCL rupture cases and from two continents (Europe n = 48 and North America n = 48) were examined using genome wide association studies (GWAS). A candidate gene study was performed using Sequenom iPlex genotyping, on cases and controls from Newfoundlands (99 cases, 172 controls) and three other susceptible breeds: Labradors (124 cases, 165 controls), Rottweilers (57 cases, 81 controls) and Staffordshire Bull Terriers (13 cases, 38 controls). One hundred and eighty-six SNPs across 26 candidate genes were investigated for association with CCL rupture. To investigate downstream events, gene expression was compared between healthy and CCL rupture tissue. An in-vitro laboratory model of CCL rupture was investigated by examining ligamentocytes induced with and without TNFα. To investigate whether there was an auto-immune component to CCL rupture, the two main loci of the dog leucocyte antigen (DLA) system were assessed for disease association. Principle component analysis of the GWAS data revealed population stratification within the Newfoundland breed, indicative of the continent of origin (Europe or North America). GWAS identified three main regions associated with CCL rupture (on chromosomes 1, 3 and 33). Significantly associated genes SORCS2 and SEMA5B function in neurological pathways; this may indicate that mechanotransduction, neurological and neuromuscular pathways play an important role in the pathogenesis and susceptibility to CCL rupture. Candidate gene analysis identified associations with two collagen genes (collagen type-V and collagen type-I) and three extracellular matrix proteins; Aggrecan (ACAN), Opticin (OPTC) and Latent transforming growth factor beta 2 (LTBP2). Gene expression analysis revealed significant differential expressions in COL1A1 and COL1A2. These results indicate that the strength and stability of the ligament is probably important in susceptibility to CCL rupture. Gene expression results also revealed that genes involved in degradation (TRAP and DIRC2) are upregulated, indicating that the cells are trying to repair themselves whilst a simultaneous degradative process is still on going. The TNFα model may be used as an in-vitro model to study CCL rupture, but may be more useful as a model for examining changes that occur after CCL rupture rather than the early stages of the disease. We showed no association with the DLA region and CCL rupture. The identified associated regions should be further investigated and refined using next generation, targeted re-sequencing and transcriptomic approaches. This could identify the specific causative mutations involved in CCL rupture susceptibility. This study confirms that there are complex multigenetic and environmental factors involved in CCL rupture susceptibility. The work has contributed to the understanding of causative factors involved in CCL rupture susceptibility and may be an important step in the development of a screening test(s) to reduce the incidence of CCL rupture in dogs and thus improve their health and welfare.

African horse sickness (AHS) is a devastating disease of many equids and in the absence of effective treatment, vaccination plays an important role. Concerns of the commercially available live attenuated vaccine have stimulated the development of novel recombinant vaccines. The viral protein VP7 of AHSV induces high levels of antibody in infected horses. Both viral replication, and expressed VP7 in insect cells, results in the generation of hexagonal crystalline structures composed entirely of VP7 (~5pm in diameter). The aim of this thesis was to explore the immunogenicity of VP7 crystals and their potential as a novel vaccine delivery platform. Firstly, successful expression of AHSV-4 VP7 was characterised in vitro using recombinant baculovirus and live vaccinia vectored systems. These provided the necessary tools to explore VP7 crystal immunogenicity. Pilot VP7 vaccination studies were conducted in the equine and mouse animal model. Secondly, insertions of AHSV-4 VP2a peptide into VP7 at amino acid position 200 did not compromise the ability of the modified VP7 constructs to form crystals and allowed investigations of the immunogenicity of VP7/VP2a200 in the mouse animal model. This project demonstrated that VP7 crystals were poor immunogens in the absence of adjuvant and multiple vaccinations. However, immunogenicity was Improved when crystals were partially solubilised with urea, indicating that soluble VP7 was more immunogenic than VP7 crystals. Hence, crystal generation may provide a mechanism for immune evasion in AHSV infection. Nevertheless, both VP7/VP2a200 untreated crystals and urea treated VP7/VP2a200 crystals induced insert-specific antibodies. Importantly, VP2a peptide antibodies raised against urea treated crystals induced low levels of AHSV-4 neutralisation in vitro. Future work should include exploring the immunological and protective potential of soluble VP7 and further Investigating the use of VP7 as a vaccine platform.

This study was conducted to evaluate the effect of season on reproductive parameters in cattle in the temperate climate of the UK. In the first study, reproductive fertility data were collected from a local dairy herd. The results revealed that cows born in autumn were inseminated at an earlier age (P < 0.05) and calved earlier (P < 0.001) than spring and summer born animals. In addition, the conception rate within 90 days after calving was higher (P < 0.05) in autumn calving animals. Conception rate was higher (P < 0.05) when insemination was performed at a temperature range from 7 to 15°C compared with < 7 and >15°C and a temperature humidity index (THI) range from 40 to 59 compared with >60 units (10 days before and 21 days after insemination). In the second study, ovarian tissues were collected from a local abattoir to investigate the effect of season on follicular populations, corpus luteum (CL) development and incidence of multiple ovulations. There were no effects of season on antral follicle count. However, individual and total CL weight was heavier in the autumn. Additionally, season influenced multiple ovulations with a higher incidence (P < 0.05) in summer and autumn compared to winter and spring. The number of follicles >7mm in cows with multiple CLs was higher (P < 0.05) than cows with a single CL. The individual CL weight was heavier in single ovulation cows. However, the total luteal tissue weight and total progesterone (P4) content of luteal tissue was higher in cows with multiple ovulations. The third study investigated the effects of culturing granulosa cells (GCs) under low (5%; physiological oxygen (O2)) conditions rather than traditional culture (20%; atmospheric O2) systems. Granulosa cells from antral follicles were cultured in fibronectin coated plates in M199 for up to 144 hour (h) under physiological (5%) and atmospheric (20%) O2 tension. Melatonin was added at one of four concentrations (0, 20, 200, 2000 pg/ml). The number of viable GCs was greater (P < 0.05) under 5% O2 than 20% O2. Reactive oxygen species (ROS) generation was similar under both physiological and atmospheric O2, but was reduced (P < 0.05) by treatment with melatonin. Oestrogen concentration (P < 0.001) and aromatase activity (P < 0.014) were also influenced by O2 tension in a time dependent manner. Both oestradiol (E2) production and aromatase enzyme activity were maintained for up to 144 h of culture under 5% O2 conditions. Progesterone production was increased under 20% O2 compared with 5% O2 (P < 0.05). Additionally, the expression of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1) mRNA increased (P < 0.05) with time under 20% O2, but remained unchanged at 5% O2. Haemoglobin subunit alpha 1 (HBA) transcript was increased (P < 0.05) under 5% O2 levels. The final study quantified the effect of temperature and melatonin on GC function. Cells were cultured for up to 144 h under 5% O2 tension. Treatments commenced after 48 h of culture and consisted of two incubation temperatures (37.5 vs 40.0°C) and four melatonin treatments. Melatonin increased cell number at high temperature (40.0°C). However, BCL2-associated X protein (BAX) mRNA expression was greater (P < 0.05) in GCs cultured at 40.0°C than at 37.5°C by 144 h. Culture temperature did not affect ROS, but melatonin reduced (P < 0.001) generation of ROS. Oestradiol production increased with time (P < 0.001) and was not affected by temperature. In contrast, high temperature reduced P4 production (P < 0.001) at 144 h of culture. Similarly, the effect of melatonin treatment depended on temperature; melatonin increased P4 production at 37.5C, while reducing P4 at 40.0C. Temperature increased acetylserotonin O-methyltransferase (ASMT) mRNA expression (P < 0.05) though there was no significant effect of temperature and melatonin on tumour protein p53 (P53), HSD3B1, superoxide dismutase (SOD1 and SOD2), HBA and heat shock protein family A (Hsp70) member 1A (HSPA1A) gene expression. The results of this thesis contribute to our understanding of the effects of season on ovarian function and seasonal variation in cattle fertility particular in temperate climate regions where season influenced puberty, conception rate, incidence of multiple ovulations and CL development. In in vitro studies, low O2 (5%) enhance cell proliferation, reduced luteinisation and altered steroidogenesis as well as increasing the expression of HBA mRNA. Culture at higher temperature reduced P4 production and increased apoptotic mRNA while addition of melatonin reduced ROS generation and influenced P4 production. This new approach to culture could offer a valuable system for future investigation of the physiological function of cells in vitro.

Herpesviruses are ubiquitous and are found worldwide, most animal species can be infected with multiple herpesviruses. Some cause clinical disease and others remain symptomatic throughout life. Herpesviruses are found in both captive and wild animals including Asian elephants (Elephas maximus). Elephant Endothelioltropic Herpesvirus (EEHV) has been reported in both captive and wild Asian elephants, with a number of cases being reported in North America, Europe and Asia. It has been suggested that EEHV is associated with haemorrhagic disease, which has been attributed to a number of Asian elephant deaths, affecting mostly juveniles and calves. Clinical signs can vary from weight loss, lethargy, depression, cyanosis of the tongue and sudden death. Molecular testing using qPCR has enabled the detection of individual variants of EEHV, this thesis investigates the EEHV1 variant. EEHV1 has been highlighted as the variant that is more frequently associated with deaths. This thesis includes five studies investigating different aspects of EEHV. Including, the relationship between pregnancy and EEHV viral shedding, the use of an amended human protocol for culturing endothelial cells, EEHV tissue tropism, a potential genetic or familial link between EEHV associated deaths and the detection of potential co-pathogens. The main findings from this thesis include: 1) the use of a longitudinal study investigating a potential link between the physiological stress of pregnancy and EEHV viral shedding. This study suggested there was no link between pregnancy and EEHV viral shedding however other stressors may be involved. 2) Using an amended human umbilical vein endothelial cell protocol, the culture of Asian elephant endothelial cells was successful. The cells from this study may be used in subsequent drug testing and vaccine development. 3) Quantitative PCR was used to determine EEHV1 tropism in tissues from two deaths associated with the virus. Tropism appeared to be for the heart and liver. 4) This thesis provides results from a preliminary study into a potential link between EEHV associated deaths. The data from an Asian elephant genogram shows there is the possibility of a genetic or familial link, which requires further investigation. 5) A number of tissues from deaths associated with EEHV and or death from other causes were investigated for the presence of potential co-pathogens, including the presence of encephalomyocarditis virus (EMCV), using microarray technology. The results indicated there were no co-pathogens present in the tissues. This thesis adds to the current published data, and includes the first known preliminary study investigating a potential genetic link between elephant deaths due to EEHV.

Veterinary communication is a core clinical skill and is believed to have a positive impact on client satisfaction, trust and adherence to patient management recommendations. Veterinary communication skills training has therefore been incorporated into veterinary undergraduate and postgraduate education. This thesis focuses on the topic of veterinary communication and comprises two studies. The aim of the first study was to gain a current understanding of the state, adequacy, and relevance of veterinary communication skills and training in the United Kingdom (UK) and United States of America (USA). This was done by conducting a survey of a sample of veterinary surgeons in each country about communication skills and training in the context of a veterinary consultation. A quantitative and qualitative analysis of the data from the survey was undertaken. Key findings were that 98 percent of respondents (1,708/1,748) believed communication skills to be equal in importance to, or more important than, clinical knowledge, whereas only 40 percent (705/1,759) were interested in further communication skills training. Barriers to participation in communication CPD appear to include lack of time and/or employer support, and a belief among some practitioners that communication training could no longer benefit them or was inadequately matched to real-world communication challenges. The aim of the second study was to assess several factors that may impact on communication dynamics during a consultation. Fifty-five video-recorded veterinary consultations in the UK and USA were analysed as follows: 1. The complexity of the consultations was assessed using a tool previously validated for recording information via direct observation of consultations. Elements recorded included details on the patient(s) and reasons for the visit, problems investigated, body systems involved, tests performed, diagnoses, and outcomes. Categorical data statistics were recorded as whole numbers and percentages and Chi-Square calculations were done to measure differences between UK and USA data. Continuous data statistics were recorded as median, range, and interquartile ratio (IQR) and Mann-Whitney U tests were performed to measure UK versus USA differences. (Continuous data for the remaining elements in the study were analysed in the same manner.) Key findings were that consultations were complex, involving multiple problems, body systems, tests, diagnoses, and outcomes. 2. Consultations were analysed for alignment with two consultation models, the Calgary-Cambridge Model for Veterinary Consultations (GCCVM) and the Patient-centred Clinical Method, by coding elements of each consultation model in the consultation transcripts. The frequency and proportion of model elements demonstrated in each consultation were assessed, as was the alignment of the consultations to each model, defined by the percent of possible model elements demonstrated in each consultation. There was 86.67% alignment with the GCCVM and 62.50% alignment with the Patient-centred Clinical Method. Veterinary surgeons in the study spent more time gathering information and explaining than empathising or soliciting client input. 3. Consultations were also analysed for dominance of medical versus lifeworld dialogue using the Mishler Discourse Analysis, and medical dialogue dominated over lifeworld dialogue (65.62% to 34.48%). 4. Client/relationship centredness was evaluated using a novel application of a tool in veterinary communication research, the Verona Patient-centred Communication Evaluation Scale (VR-COPE). Results suggested a relatively high degree of client/relationship centredness (a median score of 76/100), though with somewhat lower scores for elements related to client emotions and the veterinary surgeon responding to them. 5. Client satisfaction was evaluated using the previously validated Client Satisfaction Quotient (CSQ). There was a high degree of satisfaction expressed by clients (median score of 94/114), though average scores were slightly lower for topics related to cost and expression of interest in the client’s opinion. Limitations of the research included the low response rate of US veterinary surgeons to the survey, the small, convenience-based sample used in the consultation study, the reliance on the researcher for maintaining quality and validity, and the scoring of client/relationship-centredness with a tool that heretofore had not been used in veterinary medicine and was not subjected to extensive inter-rater variability testing. The findings in this thesis support the contention that communication skills are important for veterinary practitioners. The work also highlights the need for making communication training a priority in undergraduate veterinary education and an accessible and relevant component of postgraduate CPD. The findings also suggest a need to equip veterinary students and practitioners for communication during consultations that are relatively complex with highly iterative flow between topics, as well as for addressing emotions and inviting input of clients. Elements of the GCCVM and other models may help provide a framework for training in these competencies.

In carnivorous fish farming industry, there are progressive increase demands for the finite resource of fish meal. A potential alternative to fish meal is to use legume meals which are free of enzyme inhibitors. In selected underutilised legumes the most effective processing method for eliminating trypsin (TIA), chymotrypsin (CIA) and alpha-amylase (AIA) inhibitors, without affecting the crude protein content, was investigated. These methods included soaking (S), wet heating (W), autoclaving (A) and dry freezing (D). No single method was effective at removing all the inhibitors. In all legumes tested, the combined processing methods which involved A were most effective in reducing CIA and AIA (p < 0.05), but not TIA. However, in adzuki bean both TIA and CIA were reduced by the D+A combined method (p < 0.05), whereas AIA of soybean and adzuki bean was decreased by combined methods of S+A (84.7 % and 99.3 % reduction respectively, p < 0.05) or A+D (99.1 % and 72.6 % reduction respectively, p < 0.05). All the processing methods retained 86.5 – 90.5 % of crude protein. Replacement of 10 % (w/w) of fish meal with D+A treated legume meal (either bambara groundnut or adzuki bean) for 28 days showed no significant difference in growth performance or inflammatory effects in Danio rerio or Lates calcarifer. Compared to Lates calcarifer given feed containing unprocessed adzuki bean meal, those on feed containing processed adzuki bean meal had increased hepatic gene expression of alanine aminotransferase (p < 0.01), indicating an enhanced ability to utilise amino acids. The project identified specific food processing methods which are effective at removing enzyme inhibitors in legumes, thereby facilitating the application of legumes as aquafeed ingredients. Future studies are required to examine what inclusion level of treated legume meal can promote growth performance in specific commercial fish species.