Streptococcus uberis is an important bovine mastitis pathogen, which places a significant financial burden upon the dairy industry. Determining the genetic diversity of a collection of field isolates and the mechanisms by which S. uberis colonises the host were the general aims of this project, in particular the determination of the basis for bacterial persistence despite antibacterial therapy. Multi-locus sequence typing identified high levels of recombination within the population, but also a single dominant clonal complex which comprised nearly all sequence types which were isolated from more than one animal. The dominant clonal complex also comprised isolates, derived, however, from both persistent and non-persistent infections, but RAPD typing demonstrated that these isolates can differ in genetic composition elsewhere in the genome. Whole genome sequencing of additional S. uberis isolates confirmed that despite significant homology between much of these genomes, novel genetic material was commonly obtained by phage insertion and horizontal gene transfer. Isolates with identical housekeeping sequences are thus highly likely to differ in their virulence gene repertoires. In this study, the potential for differentiating S. uberis isolates based instead upon protein profiles derived from mass spectrometry of disrupted whole cells was therefore also explored. Differentiation between small numbers of isolates was achieved after optimisation of this protocol, however, discriminatory ability and reproducibility were somewhat compromised when the technique was scaled up to analyse 50 Italian isolates. During the period of study, profile differences between persistent and non-persistent isolates could not be explored. Basic methods were thus also utilised in an attempt to identify factors which promoted bacterial survival in vitro; and a defined medium, representative of the udder environment, was optimised for this purpose. The use of this medium permitted the demonstration that S. uberis was reliant upon magnesium and manganese for proliferation and that, interestingly, the absence of iron did not inhibit bacterial growth. It was also shown that S. uberis had the ability to directly utilise casein, identifying a potential alternative pathway for the acquisition of essential nutrients from nutritionally-limited environments. It was also observed that to a limited extent S. uberis seemed to produce a siderophore. Although this remains to be confirmed, it may correlate with the observation that iron, although not essential for proliferation, improved the growth rate of the bacterium. It was also notable that most novel genes, identified from S. uberis genome sequences, exhibited functions for nutrient metabolism, demonstrating that flexibility in nutrient acquisition is central to the ability of the bacteria to adapt, permitting survival in vastly different environments. The use of the defined medium also demonstrated that S. uberis was able to form biofilms; this ability being variable depending on the growth conditions used and the isolate studied. Most significantly, under conditions representative of the mammary gland, there was an apparent trend for high levels of biofilm formation to correlate with isolates from persistent infections. Biofilm formation by Staphylococcus aureus is considered to be pivotal to the development of chronic mastitis, thus, biofilms may similarly play a role in S. uberis persistence. In an attempt to identify the molecular basis for S. uberis biofilm formation, genes with homology to those of the intercellular adhesion (ica) operon, well described for their involvement in Staphylococcus epidermidis and S. aureus biofilm formation, were identified in the genome sequence of S. uberis 0140J. A targeted mutagenesis protocol was optimised to ‘knock out’ these genes and observe the subsequent effects of these mutations on biofilm formation. During the course of this study, two of these potential biofilm genes (hasA and SUB 0809) were deleted from the S. uberis 0140J chromosome. Surprisingly, deletion of these genes did not retard subsequent biofilm formation, but instead biofilm formation was dramatically improved in the mutant strains. Characterisation of mastitis-causing S. uberis strains and a detailed understanding of the pathogenicity of the organism are required to further the development of a successful vaccine. The research presented in this thesis has increased the knowledge of these important research objectives and optimised techniques which will allow further advancement of knowledge in this field.

Human African trypanosomiasis (HAT) is a parasitic disease caused by the protozoan parasites T. b. rhodesiense and T. b. gambiense. The disease is currently endemic in 36 sub-Saharan countries with an estimated 60 million people at risk from the infection. The disease progresses through two stages; an early or haemolymphatic stage where the parasites are confined to the peripheral compartment and a late or encephalitic stage where the parasites penetrate the blood-brain barrier (BBB) and invade the CNS. Without treatment the disease is invariably fatal but at present chemotherapy is reliant on a small handful of drugs. Pentamidine and suramin are available for the treatment of the early stage of the disease while the CNS stage of the disease is treated with a combination of nifurtimox and eflornithine known as NECT therapy or melarsoprol. NECT therapy is only effective in the treatment of T. b. gambiense infections meaning treatment of T. b. rhodesiense infections is completely dependant on the trivalent arsenical melarsoprol. Melarsoprol is an extremely toxic compound, the administration of which is very painful and associated with numerous adverse reactions. The most series of which is a post treatment reactive encephalopy (PTRE). The PTRE occurs in up to 10% of all patients given melarsoprol of which 50% die as a result of the complication. This gives melarsoprol an overall fatality rate of 5% which is unacceptably high. There is therefore an urgent need for new trypanocides, which are safe and easily administrable. To improve the physiochemical and pharmacokinetic properties of melarsoprol the drug was complexed with two cyclodextrin molecules, hydroxypropyl-cyclodextrin (HPCD) and randomly methylated-cyclodextrin (RAMCD) to produce; mel/HPCD and mel/RAMCD. Cyclodextrins are cyclic oligosaccharides, widely used within the pharmaceutical industry to improve the solubility and oral bioavailability of poorly soluble lipophilic drugs. In this study, the trypanocidal activity of the melarsoprol cyclodextrin complexes was investigated in-vitro and in an in-vivo CNS stage model of T. b. brucei infection. The trypanocidal activity of melarsoprol is retained following its complexation with HPCD and RAMCD. The in-vitro trypanocidal activity of the melarsoprol cyclodextrin complexes against bloodstream T. b. brucei trypanosomes was comparable to that of contemporary melarsoprol. Furthermore, in an in-vivo murine model of CNS stage T. b. brucei the melarsoprol cyclodextrin complexes, mel/HPCD and mel/RAMCD produced 100% cure rates when administered orally at a dose of 0.05mmol/kg, daily, for seven consecutive days. Contemporary melarsoprol when administered by the same route and schedule only cured 33.3% of the animals. The cyclodextrins HPCD and RAMCD thus increase the oral bioavailability of melarsoprol whilst retaining the compounds trypanocidal activity. An oral administrable, water soluble formulation of melarsoprol instantly eliminates the problems associated with the intravenous administration of conventional melarsoprol. Furthermore, an orally available formulation would be of great benefit in the resource poor, isolated settings in which HAT occurs, as patients would not require hospitalisation during treatment thus alleviating the pressure on local hospitals. In the current investigation quantitative taqman PCR was utilised to investigate the rate of parasite clearance from the CNS during complexed melarsoprol treatment. Both mel/HPCD and mel/RAMCD were rapidly trypanocidal. Twenty-four hours after administration of one dose the number of trypanosomes within the brain was reduced by greater than 80% and all trypanosomes were eliminated from the brain by twenty-four hours after administration of four doses of mel/HPCD and five doses of mel/RAMCD. The elimination of all trypanosomes from the CNS following four doses of mel/HPCD and five doses of mel/RAMCD, indicates that it may be possible to reduce the dosage schedule from seven daily doses to four daily doses of mel/HPCD and five doses of mel/RAMCD. A short, simple, easily administrable treatment protocol is an essential requirement of any new trypanocide as if the treatment schedule is prolonged and complicated patients are unlikely to comply. CNS stage trypanosome infection is associated with a breakdown of the blood-brain barrier (BBB). Ideally following successful chemotherapy BBB function should be restored. In this investigation the effect of curative mel/HPCD treatment on the BBB was investigated in a murine model of CNS T. b. brucei infection using small bore MRI analysis. Mel/HPCD treatment results in a rapid restoration of BBB function as by twenty-four hours after the completion of mel/HPCD therapy the integrity of the BBB was fully restored. However, a very mild neuroinflammatory reaction persisted in the brain for up to fifteen days after completion of chemotherapy. This suggests that the BBB damage observed in trypanosome infection may be due to either the parasites directly or their secretory products and not as a result of the ongoing neuroinflammatory reaction. Despite melarsoprol being in use for over 60 years its pharmacokinetics are poorly understood and a sensitive assay by which to quantify the concentration of arsenic reaching tissues following administration of the compound is not available. In this study a gas chromatography mass spectrometry (GC-MS) technique was developed to quantify the concentration of arsenic reaching the plasma and brain following oral and intravenous administration of the melarsoprol cyclodextrin complexes, mel/HPCD and mel/RAMCD. The GC-MS assay had a limit of detection of 5ng/ml and a precision (expressed as the inter-day coefficient of variation) of 13.2%. The concentration of arsenic within the brain following the oral and intravenous administration of mel/HPCD was below the limit of quantification of the assay. The pharmacokinetics of mel/HPCD and mel/RAMCD could therefore not be determined in the present study. This study demonstrates that the melarsoprol cyclodextrin complexes mel/HPCD and mel/RAMCD are highly trypanocidal with no overt signs of toxicity and more importantly orally available. Following the oral administration of mel/HPCD or mel/RAMCD the melarsoprol is slowly released over a prolonged period of time from the cyclodextrin cavity. Patients are therefore not exposed to a ‘bolus’ of the drug as is the case in the intravenous administration of contemporary melarsoprol. The slow and sustained release of melarsoprol from the cyclodextrins should result in less adverse reactions and a decreased incidence of the PTRE. Furthermore, the complexed melarsoprol treatment protocol is shorter than the currently used 10 day concise melarsoprol treatment schedule therefore the total amount of melarsoprol administered to patients will be reduced. Patients should therefore experience fewer adverse reactions. In conclusion the results from this study demonstrate that the melarsoprol cyclodextrin complexes mel/HPCD and mel/RAMCD are promising oral candidates for the treatment of HAT.

As veterinary graduates will take up an ethically challenging role, initiatives fostering reflective thinking and moral development are being increasingly promoted in the veterinary curriculum. The aim of this study was to develop and validate a structured, reflective learning tool to promote ethical awareness in pre-clinical veterinary students. The Animal Welfare Associated Reflective Exercise (AWARE) focused on the ethical content of animal welfare related issues witnessed by pre-clinical students during extra mural study (EMS) placements. The AWARE had five sections: demographic information, animal welfare related event, personal reflection, ethical reflection and round up. Students were invited to identify, and give details of, a relevant incident that had an animal welfare impact. The AWARE guided students to reflect on their emotional reaction to the event, and its ethical basis, with reference to three well established ethical frameworks. A computer based teaching package was created to accompany the AWARE. The AWARE was piloted with 25 first year veterinary undergraduate students. Most students reflected on an experience on a lambing placement and feedback from the pilot study was positive with the majority of students self-reporting that their awareness of animal welfare and ethical issues had improved. Validation of the AWARE was then completed with a full cohort of first year veterinary students using a mixed-methods approach. Qualitative analysis revealed that students exhibited higher levels of reflection in the AWAREs than they did in the unstructured reflections previously completed by students following EMS placements. Ethically relevant text was also significantly increased in the AWAREs than in the unstructured reflections. However, completion of the AWARE did not improve scores on standardised measures of ethical sensitivity or moral reasoning, two components of moral development. Following validation, the AWARE was adapted for use in clinical EMS contexts. Fourth year veterinary students completed either the AWARE using a clinical situation which impacted animal welfare or a modified version of the AWARE, the Reflection on Professional Ethics (ROPE) which focused on a professional ethical dilemma. Three different frameworks were utilised in the ROPE – RCVS’s ten guiding principles, the bioethical principles and virtue ethics. Engagement with the AWARE was similar in clinical and pre-clinical students but fewer clinical students left responses blank and more considered their future actions. Findings from analyses of the ROPEs indicated that veterinary surgeons struggled to meet all of their ethical obligations in difficult situations, that respect for client autonomy was met in the majority of cases, and that virtue ethics was poorly understood by students completing the exercise. Investigations into moral reasoning abilities of vet students at various points in the curriculum were also carried out, using a well-established measure, the Defining Issues Test (DIT). First year students were found to have a wide range of moral reasoning abilities but their mean scores were similar to that expected for students of their age and stage. The moral reasoning scores of clinical stage veterinary students were no higher than those of first year veterinary students. Application of the DIT to qualified veterinary surgeons also revealed a wide range of moral reasoning ability, with practising veterinarians scoring no higher than members of the public and over a quarter relying primarily on a basic form of moral reasoning, normally reserved for pre-adolescent children. These findings raise important questions regarding the impact of veterinary education on moral reasoning and concern for animal welfare and veterinary well-being. Ethical development is an area where both undergraduates and qualified veterinarians could benefit from improved training of ethical skills. Collectively, the findings show that the AWARE reliably elicits ethically relevant content, is viewed positively by students and has several learning benefits including improved ability to recognise and reflect on animal welfare and ethical issues. The AWARE now forms part of the veterinary curriculum at the University of Glasgow and is available to other UK vet schools.

Cellular senescence is an end point of a signal transduction programme leading to irreversible cell cycle arresst accompanied by characteristic alterations to cell morphology, biochemical properties and gene expression profile. This phenotype can be triggered by a variety of stimuli including telomere shortening, DNA damage or activated oncogenes. Senescence is now recognised as a tumour suppressor mechanism mediated by p53 and pRB pathways which act to prevent the proliferatio of cells that are at risk of tumourigenic transformation. RUNX1 is a transcription factor essential for definitive hematopoiesis and is frequently targeted in human leukaemias by chromosomal rearrangements. RUNX1 has been also demonstrated to act as a dominant oncogene in mice and the ectopic expression of RUNX1 in murine embryonic fibroblasts has been shown to cause senescence. The central aim of this study was to investigate the mechanism of senescence induction by RUNX1 and its fusion derived leukaemogenic oncoproteins in primary fibroblasts. My work showed that RUNX1 induces a strong senescence-like response in murine and human primary fibroblasts that requires intact DNA binding, CBFB interaction and C-terminal transcriptional activation/repression domains. However, surprising differences were found between the major RUNX1 fusion oncoprotein derivatives. The N-terminal fusion protein TEL-RUNX1 fails to induce senescence despite retention of a virtually full-lenght RUNX1 moiety, while the senescence-inducing potential is exaggerated in the truncated C-terminal fusion protein RUNX1-ETO (AML1-ETO). The potential to drive senescence is retained by the deletion mutant RUNX1-ETO[]469 which lacks critical corepressor binding sites suggesting that the repression of target genes may be a primary mechanism implicated in RUNX1-ETO induced senescence. Interestingly, CBFB-MYH11 fusion oncoprotein that affects RUNX1 indirectly by targeting CBFB cn also induce senescence when ectopically expressed in human primary cells. The RUNX1 and RUNX1-ETO induced senescent phenotypes differ from archetypal H-Ras [superscript v12] as arrest occurs without a preliminary phase of proliferation and the arrested cells lack prominent foci of DNA strand breaks and chromatin condensation. Notably however, RUNX1 and RUNX1-ETO display differences in their potency and the extent of engagement of p53 and Rb effector pathways. RUNX1-ETO is highly dependent on p53 function and unlike RUNX1 drives senescence in cells lacking intact p16Ink4a. RUNX1-ETO appears to exert its unique effects through potent induction of reactive oxygen species and p38MAPK phosphorylation. These findings illustrate the heterogeneous manifestations of senescence-like growth arrest and elucidate the distinctive biology and oncogenic properies of RUNX1 and its fusion derivatives.

Infection of bovine leukocytes by the intracellular protozoan parasites Theileria annulata and Theileria parva represents a unique model of reversible host cell transformation; characterised by uncontrolled proliferation and protection against apoptosis. Host cell transformation is known to involve parasite dependent manipulation of a number of signalling pathways that result in constitutive activation of pro-inflammatory transcription factors. Activation of these factors has been shown to confer protection against apoptosis or influence the metastatic potential of the infected cell, respectively. However, transcription factors have the potential to be detrimental as well as beneficial to the infected cell and the infected animal. This study investigated the potential of the parasite to manipulate the outcome of cellular activation by comparing the outcome of stimulating Theileria-infected and uninfected bovine cells with LPS. The results clearly indicate that prolonged stimulation of uninfected cells generated a phenotype associated with loss of proliferation, cell death and activation of NF-B. In contrast, infected TBL20 cells were refractory to stimulation, despite displaying high levels of constitutively activated NF-B.

Fasciola hepatica is responsible for substantial economic losses and animal welfare issues within the agricultural sector worldwide. The increasing incidence of fasciolosis, coupled with the emergence of flukicide resistance, makes vaccination an attractive alternative control strategy. Hidden antigens extracted from the gut of blood feeding parasites have proven to be excellent vaccine candidates against haematophagous parasites, most notably Haemonchus contortus and Rhipicephalus (Boophilus) microplus. Here, as a first step towards a prototype liverfluke vaccine an attempt to identify hidden gut antigens in F. hepatica was made. Proteomic analysis on extracts of adult F. hepatica was used to identify molecules exclusively found within the membrane-bound fraction including four proteases; cathepsin B2, legumain-2, a putative lysosomal pro-x-carboxypeptidase precursor and a saposin-like protein. Histological sections of adult F. hepatica were screened with a panel of 21 lectins to identify those with an affinity for glycoproteins on the parasite’s gut and to inform subsequent lectin affinity chromatography. Seven lectins showed affinity for the gut region, with peanut (PNA) and jacalin (JAC) lectins binding to glycoproteins on either the gastrodermal cells or gut lamellae, respectively. PNA and JAC were then used to purify glycoproteins from the crude S3 extract by affinity chromatography. The resultant fractions were separated by SDS-PAGE and the protein profiles analysed by mass spectrometry. The enriched lectin-binding fractions shared a number of proteins but one of note that was exclusively identified in the PNA-binding fraction was a cathepsin D-like aspartyl protease, which had not previously been studied in F. hepatica. The proteolytic activities of three somatic extracts of adult F. hepatica were therefore investigated. The ability of the respective fractions to digest haemoglobin, a potential food source, was measured in the presence/absence of class-specific enzyme inhibitors. These analyses confirmed the presence of cathepsin D-like aspartyl protease activity capable of digesting haemoglobin optimally at pH 2 - 2.5. Further characterisation of the cathepsin D-like aspartyl (FhCatD) protease revealed it to be highly conserved within trematodes, to be localized to the gastrodermis of immature (10 day) and adult fluke, and to be more highly expressed, at the RNA level, in the Newly Excysted Juveniles (NEJ) than adult stages. Western blot analysis of native somatic extracts, enriched lectin-binding fractions and recombinant FhCatD using antisera from naturally infected sheep, showed some recognition of the recombinant FhCatD but did not provide clear evidence that the cathepsin D is strongly antigenic during natural infection.

Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. While aquatic birds and ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, anatomical distribution of influenza virus receptors (sialic acid SAα2,3-Gal and SA α2,6-Gal) in key organs of both species was studied using lectin histochemistry with linkage specific lectins, and receptor binding assays with swine H1N1 (classical A/sw/Iowa/15/30) and avian H2N3 (A/mallard duck/England/7277/06) influenza viruses. Widespread presence of both SAα 2,6-Gal and SAα2,3-Gal receptors were found in all major organs examined in both chickens and ducks. Interestingly, the predominant receptor type in chicken tracheal epithelium (TE) was SAα2,6-Gal whereas SAα2,3-Gal receptors were most abundant in duck TE. Paradoxically, infection of primary cell cultures (duck and chicken lung cells and embryo fibroblasts) with the swine H1N1, the low pathogenicity avian H2N3, and a highly pathogenic H5N1 (A/turkey/England/50-92/91) virus resulted in more extensive and rapid cell death in duck cells than in chicken cells. Infected duck cells displayed morphological features of apoptosis, increased DNA fragmentation and activation of caspase-3/7. Infected duck cells produced comparable levels of viral RNA but less infectious virus than infected chicken cells. Notably, such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 virus (A/turkey/Turkey/1/05) which has been shown to be fatal to ducks. Gene expression profiling of infected chicken and duck cells, 24hrs post-infection, with a chicken Affymetrix microarray platform revealed differential transcription of many genes between the two avian species. In particular, the array results suggested a possible role of BCoR, HSPA-9, STAT-3, AVEN, BCLAF1, IL-18, IFN-α, and TNF-α genes in mediating the contrasting species phenotypic response to influenza infections. In summary, rapid cell death in duck cells, mediated at least in part by apoptosis, results in reduced infective virus production and may well be an important protective host response of resistant ducks. By contrast, longer surviving infected chicken cells produce much higher infective virus load along with high levels of pro-inflammatory cytokines which could account for the susceptibility of chickens to influenza infections.

Haemonchus contortus is a blood-feeding Strongylid parasite that is economically significant worldwide. Due to the increasing problem of anthelmintic resistance, alternative approaches are urgently required for parasitic nematode control. H. contortus cathepsin B gut cysteine proteases have received attention as potential vaccine candidates because of their proposed role in blood feeding. The increasing amount of H. contortus genome information has now enabled detailed identification and annotation of cathepsin B protease gene families. In this study H. contortus BAC 18f22 was annotated and found to encode eight tandemly arranged cysteine proteases related to the previously identified AC family, but with six novel genes identified. Annotation of supercontig and scaffold sequence identified many more members of the HmCP and GCP-7 cathepsin B families. In total this work has shown that the H. contortus genome encodes at least 41 cathepsin B protease genes, more than in other nematodes, to date. In contrast, Hc-cpr-6 is present as a single copy gene that is highly conserved in a number of species, suggesting an important conserved function. Further work examined regulation of gut gene expression in H. contortus, in particular the H. contortus ELT-2 GATA transcription factor (TF), as it has been shown to be the major TF in C. elegans controlling gut gene expression. A high throughput assay was developed and used to screen an integrated C. elegans worm strain expressing GFP in the gut and hypodermis (Ce-cpl-1::gfp) against 594 chemical compounds. Compounds were identified that specifically cause a decrease in gut GFP expression, affect larval development and show a degree of lethality. Further work on two of the compounds identified an embryonic effect, with a significant decrease in number of progeny. To conclude, this thesis identified a number of novel cathepsin B genes as well as two compounds potentially interfering with TF activity and gut gene expression which may be of use as novel anthelmintics.

Working equids, vital to many of the world’s most economically vulnerable people, face many challenges to their health, welfare and productivity. In The Gambia, West Africa, appropriate nutrition, husbandry and veterinary assistance are limited, while infectious disease is a constant threat, particularly the parasitic disease trypanosomosis. The prevalence of generalised trypanosomosis in working equids attending the Gambia Horse and Donkey Trust show in 2013 using PCR was 55.4%. Trypanosoma congolense was most prevalent (47.0%), followed by T. vivax (15.7%) and T. brucei s.l. (2.4%). Mixed infections were common (9.4%) and T. congolense/ T. vivax coinfection appeared to have the greatest clinical effect. Spread of T. brucei parasites to the central nervous system (CNS), confirmed using immunohistochemistry and PCR, causes severe CNS dysfunction. Horses showed spastic paraparesis that rapidly progressed to recumbency, while donkeys more often displayed somnolence and cranial nerve dysfunction with a slower deterioration. The disease was fatal in all cases. Histopathology revealed diffuse lymphocytic-plasmacytic meningoencephalo-myelitis with marked perivascular cuffing, particularly in the white matter. T cells were prominent in this first study of lymphocyte distribution in equine CNS trypanosomosis. Extensive reactive astrocytosis was also demonstrated. Currently, a reliable diagnosis of equine CNS requires post mortem samples. The loop-mediated isothermal amplification (LAMP) assay was assessed for the diagnosis of equine T. brucei infection for the first time in both blood and cerebrospinal fluid (CSF). An entomological survey showed that Glossina morsitans submorsitans was common in dry woodland areas while G. palpalis gambiensis was found in riverine habitats. The prevalence of T. brucei in the midguts of Glossina specimens was 1.7% and equine DNA was found in tsetse bloodmeals, providing evidence for ongoing interaction between host, parasite and vector. Atylotus agrestis, vector of T. vivax and T. congolense, was present in large numbers in village areas. Equine DNA was detected in one A. agrestis specimen, however, no evidence of T. brucei in association with these flies was found. Finally, microsatellite genotyping was used for the first time to investigate T. brucei populations in equine trypanosomosis in The Gambia. The results revealed a heterogenous population, providing further evidence for a tsetse-transmitted mode of transmission. No evidence of population clustering by disease type or host species was detected, suggesting that host factors determine pathogenesis. Initial evidence for the involvement of the tsetse vector supports evaluation of vector control methods although further analysis of T. brucei populations in insect vectors and their relationships with those infecting equids is recommended. The clinicopathological descriptions will be of use in further study of equine CNS trypanosomosis and the development of new therapeutics and LAMP has the potential to facilitate research, especially in the study of CNS infection which has, up to now, relied on post mortem confirmation.