Browsing by Author "Hanna, Robert E.B."
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ItemBio-Fabrication of Human Amniotic Membrane Zinc Oxide Nanoparticles and the Wet/Dry HAM Dressing Membrane for Wound Healing(Frontiers Media, 2021-07-28) Ramasamy, Palaniappan; Krishnakumar, Ramachandran; Rekha, Ravichandran; Vaseeharan, Baskaralingam; Saraswathi, K.; Raj, Mohan; Hanna, Robert E.B.; Brennan, Gerard P.; Dayanithi, Govindan; Vijayakumar, SekarThe preparation of unique wet and dry wound dressing products derived from unprocessed human amniotic membrane (UP-HAM) is described. The UP-HAM was decellularized, and the constituent proteins were cross-linked and stabilized before being trimmed and packed in sterile Nucril-coated laminated aluminium foil pouches with isopropyl alcohol to manufacture processed wet human amniotic membrane (PWHAM). The dry type of PD-HAM was prepared by decellularizing the membrane, UV irradiating it, lyophilizing/freeze-drying it, sterilizing it, and storing it at room temperature. The UP-HAM consists of a translucent yellowish mass of flexible membranes with an average thickness of 42 µm. PW-HAM wound dressings that had been processed, decellularized, and dehydrated had a thinner average thickness of 30 µm and lacked nuclear-cellular structures. Following successful decellularization, discrete bundle of fibrous components in the stromal spongy layers, microvilli and reticular ridges were still evident on the surface of the processed HAM, possibly representing the location of the cells that had been removed by the decellularization process. Both wet and dry HAM wound dressings are durable, portable, have a shelf life of 3–5 years, and are available all year. A slice of HAM dressing costs 1.0 US$/cm2 . Automation and large-scale HAM membrane preparation, as well as storage and transportation of the dressings, can all help to establish advanced technologies, improve the efficiency of membrane production, and reduce costs. Successful treatment of wounds to the cornea of the eye was achieved with the application of the HAM wound dressings. The HAM protein analysis revealed 360 µg proteins per gram of tissue, divided into three main fractions with MWs of 100 kDa, 70 kDa, and 14 kDa, as well as seven minor proteins, with the 14 kDa protein displaying antibacterial properties against human pathogenic bacteria. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 July 2021 | Volume 9 | Article 695710 fbioe-09-695710 July 22, 2021 Time: 16:39 # 2 Ramasamy et al. HAMP-ZnO Nanoparticles HAM Wound Dressing Wet and dry wound dressings were produced. HAM proteins were purified and analysed. The zinc oxide nanoparticles (HAMP-ZnO NP) made from HAM proteins were characterised and tested for their antibacterial activity. Wounds to the cornea of the eye healed easily when treated with HAM wound dressings. Fresh human Amniotic membrane, Serological screening, selection of disease-free HAM, reome stromal layer, preparation of HAM. UNPROCESSED HAM Cuboidal epithelial cells, basement membrane, compact layer, stromal and spongy layers containing scatted fibroblast cells are visible in hsitological analysis. The flow chart depicts the methods for processing, and preparation of wet (PWHAM) and dry (PD-HAM) wound healing dressings. HAM proteins, Nanoparticle synthesis (HAMP-ZnO NP) and analysis. Antibacterial analysis show Inhibition of growth and biofilm formation of pathogenic bacteria . Processed HAM lacked a nuclear-cellular epithelium, but it did have a distinct fibrous elements in basement membrane, stromal and spongy layers. Processed PW-HAM (Light &SEM) showed smooth epithelial surface topography with microvilli,. HAM dressing, wet/dry, packed, labelled, sterilised and processed. They are durable, portable, have long shelf life . A slice of HAM dressing costs US$ 1.0 / cm² . The wound dressings are ready to be applied. The dermal wounds and conjunctival surface can be successfully repaired using processed HAM wound dressings GRAPHICAL ABSTRACT | Flow chart depicting the methods, preparing, and characterizing, by histological, and scanning electron microscopy, of wet (PW-HAM) and dry (PD-HAM)of wound healing dressing, and preparation of nanoparticles (HAMP ZnO NP); and application of HAM wound dressing. A wide range of antibacterial activity was observed after treatment with 75 µg/ml zinc oxide nanoparticles derived from human amniotic membrane proteins (HAMP-ZnO NP), including dose-dependent biofilm inhibition and inhibition of Gram-positive (S. aureus, S. mutans, E. faecalis, and L. fusiformis) and Gram-negative bacteria (S. sonnei, P. aeruginosa, P. vulgaris, and C. freundii). ItemCase Report of Puffinosis in a Manx Shearwater (Puffinus puffinus) Suggesting Environmental Aetiology(MDPI, 2022-12-07) Esmonde, Niamh P.G.; Hanna, Robert E.B.; Patel, Jignasha G.; Smyth, Victoria; Caplat, Paul; Smyth, Wesley; Jaggers, Paris; Padget, Oliver; Guilford, Tim; Perrins, Chris; Reid, NeilPuffinosis is a disease of a range of seabirds characterised by dorsal and ventral blistering of their webbed feet, conjunctivitis, dry necrosis, leg spasticity, head shaking, loss of balance, tremors, and death. It is associated with Manx shearwaters (Puffinus puffinus), frequently affecting chicks within their underground nesting burrows. The aetiology of the disease is unclear but has been attributed to a type-2 coronavirus associated with Neotombicula mites as a potential vector. However, there is some uncertainty given potential laboratory contamination with mouse hepatitis virus and failure to fulfil Koch’s postulates, with birds injected with isolates remaining healthy. We describe a detailed case report of puffinosis in a Manx Shearwater covering necropsy, histology, bacteriology, and metagenomics including viral sequencing. We found no evidence of viral infection or parasites. Our results are consistent with an entirely environmental aetiology, with caustic faecal ammonia in damp nesting burrows causing conjunctivitis and foot dermatitis breaking the skin, allowing common soil bacteria (i.e., Flavobacterium, Staphylococcus and Serratia spp., Clostridia perfringens and Enterococcus faecalis) to cause opportunistic infection, debilitating the bird and leading to death. A similar condition (foot pad dermatitis or FPD) has been reported in broiler chickens, attributed to caustic faeces, high humidity, and poor environmental conditions during indoor rearing, preventable by adequate ventilation and husbandry. This is consistent with puffinosis being observed in Shearwater nesting burrows situated in tall, dense, vegetation (e.g., bracken Pteridium aquilinum) but rarely reported in burrows situated in well-ventilated, short coastal grasslands. This proposed environmental aetiology accounts for the disease’s non-epizootic prevalence, spatial variation within colonies, and higher frequency in chicks that are restricted to nesting burrows. ItemControl of Nematodirus spp. infection by sheep flock owners in Northern Ireland(BMC, 2017-10-19) McMahon, Connor; Edgar, Hillary W.J.; Barley, Jason; Hanna, Robert E.B.; Brennan, Gerard P.; Fairweather, IanBackground To address a lack of information on the control of ovine helminth parasites in Northern Ireland (NI), a number of research projects have been undertaken, dealing with gastrointestinal nematodes, tapeworms and liver fluke. This investigation concerns Nematodirus and concentrates on three aspects of disease: farm management strategies for its control, derived from the results of a Questionnaire; the efficacy of treatment used by farmers, as determined by a coprological survey; and the hatching requirements of Nematodirus eggs, that is, whether prolonged chilling is a pre-requisite for hatching. Results A Questionnaire was sent to 252 sheep farmers in NI in March 2012 (covering the years 2009–2012) and replies were received from 228 farmers. Under-dosing, inaccurate calibration of equipment and inappropriate product choice were poor practices identified. Following this survey, the efficacy of treatment of Nematodirus spp. in sheep flocks was evaluated in April and May 2012. Sampling kits were sent to 51 flock owners, all of whom returned pre- and post-anthelmintic dosing faecal samples to the laboratory for analysis. At the time of treatment, 41 flocks were positive for Nematodirus (as diagnosed by the presence of eggs). Reduced benzimidazole efficacy was detected in 35.7% of flocks tested (n = 28). Although only involving a small number of flocks, reduced efficacy of levamisole treatment was detected in 50%, of avermectins in 33% and of moxidectin in 75% of flocks tested (n = 2, 6 and 4, respectively). In the egg hatch experiment, carried out under “chilled” and “non-chilled” conditions, 43% of the eggs in the “non-chilled” group were able to hatch, compared to 100% in the “chilled” group. Conclusions The identification of inefficient control strategies argues for continued education of stockholders, in order to improve their management programmes. This is particularly important where the practices might impact on the development of anthelmintic resistance, which has been shown to exist on NI farms. The appropriate choice of anthelmintic is a vital part of this plan. The ability of eggs to hatch under non-chilled conditions demonstrates a flexibility in hatching behaviour. This may represent an adaptation to climate change and account for the recent emergence of a second, autumnal peak of infection. ItemFasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism(MDPI, 2022-05-15) Bennett, Adam P. S.; de la Torre-Escudero, Eduardo; Dermott, Susan S. E.; Threadgold, Lawrence T.; Hanna, Robert E.B.; Robinson, Mark W.The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host’s blood. Despite their importance in the host–parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze– thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite. ItemHighly pathogenic avian influenza A(H5N1) virus infection in foxes with PB2-M535I identified as a novel mammalian adaptation, Northern Ireland, July 2023(European Centre for Disease Prevention and Control, 2023-10-19) Lagan, Paula; McKenna, Robyn; Baleed, Salam; Hanna, Robert E.B.; Barley, Jason; McConnell, Shirley; Georgaki, Anastasia; Sironen, Tarja; Kauppinen, Ari; Gadd, Tuija; Lindh, Erika; Ikonen, Niina; McMenamy, Michael J.; Lemon, Ken; Animal Health and WelfareWe report cases of mammalian infection with highly pathogenic avian influenza (HPAI) virus A(H5N1) clade 220.127.116.11b in Northern Ireland. Two common gulls (Larus canus) and two red fox kits (Vulpes vulpes), were found dead in close vicinity. Comparison of viral whole genome sequences obtained from the animals identified a novel mammalian adaptation, PB2-M535I. Analysis of genetic sequences from other recent mammalian infections shows that this mutation has arisen on at least five occasions in three European countries since April 2023. ItemA Novel Prototype Biosensor Array Electrode System for Detecting the Bacterial Pathogen Salmonella typhimurium.(MDPI, 2022-06-04) Ramasamy, Palaniappan; Dakshinamoorthy, Gajalakshmi; Jayashree, Shanmugam; Prabhu, Dhamodharan; Rajamanikandan, Sundararaj; Velusamy, Palaniyandi; Dayanithi, Govindan; Hanna, Robert E.B.Salmonellosis caused by Salmonella sp. has long been reported all over the world. Despite the availability of various diagnostic methods, easy and effective detection systems are still required. This report describes a dialysis membrane electrode interface disc with immobilized specific antibodies to capture antigenic Salmonella cells. The interaction of a specific Salmonella antigen with a mouse anti-Salmonella monoclonal antibody complexed to rabbit anti-mouse secondary antibody conjugated with HRP and the substrate o-aminophenol resulted in a response signal output current measured using two electrode systems (cadmium reference electrode and glassy carbon working electrode) and an agilent HP34401A 6.5 digital multimeter without a potentiostat or applied potential input. A maximum response signal output current was recorded for various concentrations of Salmonella viz., 3, 30, 300, 3000, 30,000 and 300,000 cells. The biosensor has a detection limit of three cells, which is very sensitive when compared with other detection sensors. Little non-specific response was observed using Streptococcus, Vibrio, and Pseudomonas sp. The maximum response signal output current for a dialysis membrane electrode interface disc was greater than that for gelatin, collagen, and agarose. The device and technique have a range of biological applications. This novel detection system has great potential for future development and application in surveillance for microbial pathogens. ItemRabbit Haemorrhagic Disease Virus 2 (RHDV2; GI.2) in Ireland Focusing on Wild Irish Hares (Lepus timidus hibernicus): An Overview of the First Outbreaks and Contextual Review.(MDPI, 2022-02-22) Byrne, Andrew W.; Marnell, Ferdia; Barrett, Damien; Reid, Neil; Hanna, Robert E.B.; McElroy, Maire C.; Casey, MichaelRabbit haemorrhagic disease virus 2 (RHDV2; GI.2) is a pathogenic lagovirus that emerged in 2010, and which now has a global distribution. Outbreaks have been associated with local population declines in several lagomorph species, due to rabbit haemorrhagic disease (RHD)-associated mortality raising concerns for its potential negative impact on threatened or vulnerable wild populations. The Irish hare (Lepus timidus hibernicus) is endemic to Ireland, and is of conservation interest. The first cases of RHDV2 in Ireland were reported in domestic rabbits (Oryctolagus cuniculus) in 2016, soon followed by the first known case in a wild rabbit also in 2016, from a population reported to be experiencing high fatalities. During summer 2019, outbreaks in wild rabbits were confirmed in several locations throughout Ireland. Six cases of RHDV2 in wild hares were confirmed between July and November 2019, at four locations. Overall, 27 cases in wildlife were confirmed in 2019 on the island of Ireland, with a predominantly southern distribution. Passive surveillance suggests that the Irish hare is susceptible to lethal RHDV2 infection, and that spillover infection to hares is geographically widespread in eastern areas of Ireland at least, but there is a paucity of data on epidemiology and population impacts. A literature review on RHD impact in closely related Lepus species suggests that intraspecific transmission, spillover transmission, and variable mortality occur in hares, but there is variability in reported resistance to severe disease and mortality amongst species. Several key questions on the impact of the pathogen in Irish hares remain. Surveillance activities throughout the island of Ireland will be important in understanding the spread of infection in this novel host ItemThe effect of naturally acquired rumen fluke infection on animal health and production in dairy and beef cattle in the UK(Frontiers Media, 2022-08-18) Atcheson, Erwan; Lagan, Bernard; McCormick,Ross; Edgar, Hilary; Hanna, Robert E.B.; Rutherford, Naomi; McEvoy, Amanda; Huson, Kathryn; Gordon, Alan; Aubry, Aurelie; Vickers, Mary; Robinson, Mark W.; Barley, JasonThe incidence of paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, has greatly increased within Europe in the last 15–20 years. However, the production impacts of this disease are poorly understood. This study firstly aimed to investigate the prevalence of rumen fluke in England and Northern Ireland (NI) by conducting an abattoir survey of dairy and beef cattle which also allowed the impact of rumen fluke on carcass weight, conformation and fat classification to be assessed. Secondly, an experiment aimed to assess the impact of C. daubneyi infection on diarrhea score, production loss and welfare in dairy heifers, while also evaluating the impacts of treating infected heifers with oxyclozanide. Rumen fluke prevalence was greater in NI than in England, with 53.8% (95% CI 51.9 - 55.9%) of the NI cattle carcases sampled being infected compared to 16.3% (95% CI 15.8 - 16.8%) and 17.9% (95% CI 17.4 - 18.4%) detected at the two abattoirs in England. However, there was no significant difference (P > 0.05) in the cold carcass weight between infected and non-infected cattle. Similarly, carcass conformation and fat classification were unaffected (P > 0.05) by the presence of rumen fluke. In the second experiment, daily live weight gain (DLWG), diarrhea score and welfare score were also unaffected (P > 0.05) by rumen fluke infection and by oxyclozanide treatment against rumen fluke. The farms in this experiment were managed to a high standard and animals had no intercurrent disease. Therefore, these findings suggest that on well–managed farms, production losses (growth rates) should not be compromised as a result of sub-clinical rumen fluke infection. ItemTranscriptome and secretome analysis of intra-mammalian life-stages of the emerging helminth pathogen, Calicophoron daubneyi reveals adaptation to a unique host environment.(The American Society for Biochemistry and Molecular Biology, Inc., 2020-10-20) Huson, Kathryn M.; Atcheson, Erwan; Oliver, Nicola A.M.; Best, Philip; Barley, Jason; Hanna, Robert E.B.; McNeilly, Tom N.; Fang, Yongxiang; Haldenby, Sam; Paterson, Steve; Robinson, Mark W.Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock which has seen a rapid rise in prevalence throughout Western Europe in recent years. Following ingestion of metacercariae (parasite cysts) by the mammalian host, newly-excysted juveniles (NEJs) emerge and invade the duodenal submucosa which causes significant pathology in heavy infections. The immature larvae then migrate upwards, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients and to avoid the host immune response. Here, transcriptome analysis of four intra-mammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic disease respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that whilst a family of cathepsins B with varying S2 sub-site residues (indicating distinct substrate specificities) are differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is up-regulated in adult worms, although they are underrepresented in the secretome. The most abundant proteins in adult fluke secretions were helminth defence molecules (HDMs) that likely establish an immune environment permissive to fluke survival and/or neutralise pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognise antigens from other helminths commonly found as co-infections with rumen fluke.