In-vivo analysis of Protec™ and ß-glucan supplementation on innate immune performance and intestinal health of rainbow trout.

Functional diets are often given to fish during key stages to improve health through the interaction of the feed components with the host intestine. The additional factors added in these diets are known to modulate the immune response and as such may also offer protection against pathogenic challenges. The present study was undertaken to evaluate whether ß-glucan supplementation for 6 weeks can alter the magnitude of immune response to immunological challenges and subsequently offer an improved innate immune response to bacterial challenge in rainbow trout. Two experimental diets were used to study these effects: a basic commercial diet supplemented with ß-glucan and a commercially available functional diet (Protec™) that has ß-glucan as a functional component in addition to other components were compared to a basic commercial control diet. No significant differences were observed in biometric data. Histological analysis revealed a significantly greater number of goblet cells in the fish fed Protec™ and ß-glucan diets compared to those fed a control diet. Cell marker gene expression of distal intestine leucocytes indicated higher expression of T- and B-cells marker genes to both the ß-glucan containing diets in comparison to control. The Protec™ diet demonstrated modulation of innate immune markers after 6 weeks of feeding with key antimicrobial genes (SAA, HAMP, IL-1ß and TNFα) showing significant increases compared to the other diets. After stimulation with both PAMPs and an immune challenge with A. salmonicida fish fed the ß-glucan diet and the Protec™ exhibited modulation of the innate immune response. An immune challenge with A. salmonicida was carried out to identify if dietary composition led to differences in the innate immune response of rainbow trout. Modulation of the magnitude of response in some immune genes (SAA, IL-1ß and HAMP) was observed in both the distal intestine and head kidney in the Protec™ and ß-glucan fed fish compared to those fed the control diet.

Gut Associated Lymphoid Tissue (GALT) primary cells and stable cell lines as predictive models for intestinal health in rainbow trout (Oncorhynchus mykiss).

The use of functional feeds for farmed fish is now regarded as a key factor in improving fish health and performance against infectious disease. However, the mechanisms by which these nutritional components modulate the immune response are not fully understood. The present study was undertaken to identify the suitability of both primary gut-associated lymphoid tissue (GALT) leucocyte cells and established rainbow trout cell lines as potential alternative methods to test functional feed ingredients prior to full fish feeding trials that can take months to complete. In addition to the primary GALT culture cells, the two rainbow cell lines RTS11 and RTgutGC which are from macrophage and gut epithelial cells, respectively. The cells were stimulated with a variety of pathogen associated molecular patterns (PAMPs) (PHA and Poly I:C) and recombinant rainbow trout IL-1ß (rIL-1ß), a proinflammatory cytokine, additionally two forms of ß-glucan, a prebiotic commonly used aquafeeds were used as stimulants. From this, the suitability of cell models as a health screen for functional feeds was assessed. GALT leucocytes were deemed most effective to act as a health screen over the 4hr time point demonstrating responses to Poly I:C, PHA, and rIL-1ß. RTS11 and RTgutGC also responded to the stimulants but did not give a strong T-cell response, most likely reflecting the nature of the cell type as opposed to the mixed cell populations from the primary GALT cell cultures. When stimulated with both forms of ß-glucan, GALT leucocytes demonstrated a strong proinflammatory and T-cell response.

Immune responses to prebiotics in farmed salmonid fish: How transcriptomic approaches help interpret responses.

Within aquaculture, prebiotics are composed of complex carbohydrate molecules that cannot be digested by the fish directly but are metabolised by the microbial communities within the host gut, with the desire that "healthy" bacterial species are promoted with subsequently improved performance of the fish, there are likely some direct responses of intestinal cells to these dietary components. The sources and processing of prebiotics, which fall under the overarching theme of "functional feeds" are highly varied between species and types of prebiotics administered. How these feeds exert their effect, and the host responses are hard to determine, but new technologies and the development of high-throughput technologies (omics) are enabling the mechanisms and methods of action to be further understood. The recent advances in the availability of 'omics' technologies with the transition from single gene assays to microarray and RNA-seq in fish health have enabled novel functional ingredients to be analysed. This review will focus on recent studies on targeted gene expression and 'omics' technologies to characterize immune responses. Comparisons between the immunomodulatory effect of different prebiotics have been made and specific examples of how transcriptomics techniques have been used to identify immune responses to prebiotics are given.

Diabetes care and pregnancy outcomes for women with pregestational diabetes in Ireland.

AIMS: Pre-gestational diabetes mellitus (PGDM) is associated with adverse outcomes. We aimed to examine pregnancies affected by PGDM; report on these pregnancy outcomes and compare outcomes for patients with type 1 versus type 2 diabetes mellitus; compare our findings to published Irish and United Kingdom (UK) data and identify potential areas for improvement. METHODS: Between 2016 and 2018 information on 679 pregnancies from 415 women with type 1 Diabetes Mellitus and 244 women with type 2 diabetes was analysed. Data was collected on maternal characteristics; pregnancy preparation; glycaemic control; pregnancy related complications; foetal and maternal outcomes; unscheduled hospitalisations; congenital anomalies and perinatal deaths. RESULTS: Only 15.9% of women were adequately prepared for pregnancy. Significant deficits were identified in availability and attendance at pre-pregnancy clinic, use of folic acid, attaining appropriate glycaemic targets and appropriate retinal screening. The majority of pregnancies (n = 567, 83.5%) resulted in a live birth but the large number of infants born large for gestational age (LGA) (n = 280, 49.4%), born prematurely <37 weeks and requiring neonatal intensive care unit (NICU) admission continue to be significant issues. CONCLUSIONS: This retrospective cohort study identifies multiple targets for improvements in the provision of care to women with pre-gestational DM which are likely to translate into better pregnancy outcomes.

An Irish National Diabetes in Pregnancy Audit: aiming for best outcomes for women with diabetes.

AIMS: The purpose of this study was to identify the number of pregnancies affected by pre-gestational diabetes in the Republic of Ireland; to report on pregnancy outcomes and to identify areas for improvement in care delivery and clinical outcomes. METHODS: Healthcare professionals caring for women with pre-gestational diabetes during pregnancy were invited to participate in this retrospective study. Data pertaining to 185 pregnancies in women attending 15 antenatal centres nationally were collected and analysed. Included pregnancies had an estimated date of delivery between 1 January and 31 December 2015. RESULTS: The cohort consisted of 122 (65.9%) women with Type 1 diabetes and 56 (30.3%) women with Type 2 diabetes. The remaining 7 (3.8%) pregnancies were to women with maturity-onset diabetes of the young (MODY) (n = 6) and post-transplant diabetes (n = 1). Overall women were poorly prepared for pregnancy and lapses in specific areas of service delivery including pre-pregnancy care and retinal screening were identified. The majority of pregnancies 156 (84.3%) resulted in a live birth. A total of 103 (65.5%) women had a caesarean delivery and 58 (36.9%) infants were large for gestational age. CONCLUSIONS: This audit identifies clear areas for improvement in delivery of care for women with diabetes in the Republic of Ireland before and during pregnancy.

Effect of starvation and refeeding on the hepatopancreas of whiteleg shrimp Penaeus vannamei (Boone) using computer-assisted image analysis.

Under normal farming conditions, shrimp can experience starvation periods attributable to disease outbreaks or adverse environmental conditions. Starvation leads to significant morphological changes in the hepatopancreas (HP), being the main organ for absorption and storage of nutrients. In the literature, limited research has described the effect on the HP of periods of starvation followed by refeeding and none in whiteleg shrimp (Penaeus vannamei) using computer-assisted image analysis (CAIA). This study describes the effect of starvation and starvation followed by refeeding on the HP of whiteleg shrimp using CAIA. Visiopharm® software was used to quantify the following morphological parameters, measured as ratio to the total tissue area (TLA): total lumen area (TLA:TTA), haemocytic infiltration area in the intertubular spaces (HIA:TTA), B-cell vacuole area (VBA:TTA), lipid droplet area within R cells (LDA:TTA) and F-cell area (FCA:TTA). Significant changes were measured for HIA:TTA and LDA:TTA during starvation (increase in HIA:TTA associated with decrease in LDA:TTA) and starvation followed by refeeding (decrease in HIA:TTA associated with increase in LDA:TTA). In the future, HIA:TTA and LDA:TTA have the potential to be used in a pre-emptive manner to monitor the health of the HP, facilitate early diagnosis of diseases and study the pathophysiology of the organ.

Use of computer-assisted image analysis for semi-quantitative histology of the hepatopancreas in whiteleg shrimp Penaeus vannamei (Boone).

Despite the increasing use of novel molecular techniques in pathology, histology remains the standard method for monitoring tissue alterations and for assessing pathology. Histopathological evaluation is generally laborious and subjective with risk of discrepancies in semi-quantitative scoring between pathologists. In contrast, computer-assisted image analysis (CAIA) is potentially faster, more objective and thus suitable for routine screening. Limited research has been carried out on CAIA in crustacean histopathology, and the methods described were not fully automated. Therefore, the aim of this study was to develop CAIA in whiteleg shrimp (Penaeus vannamei) for the study of the hepatopancreas. Paraffin sections were immunohistochemically stained with monoclonal antibodies WSH8 against haemocytes and counterstained with Mayer's haematoxylin for detection of haemocytes and B-cell vacuoles, and modified toluidine blue protocol was used for detection of F-cells; frozen sections were stained with Oil Red O for detection of lipid droplets within R-cells. Visiopharm® software was used to develop and validated protocols for the quantification of morphological parameters (areas of haemocyte infiltration, F-cells, B-cell vacuoles, lipid droplets and their ratios to total tissue area and total lumen area). These protocols enable the future use of CAIA for determination of the nutritional and pathological condition of this organ.

Optimization of fixation methods for image analysis of the hepatopancreas in whiteleg shrimp, Penaeus vannamei (Boone).

Pathology in penaeid shrimps relies on histology, which is subjective, time-consuming and difficult to grade in a reproducible manner. Automated image analysis is faster, objective and suitable for routine screening; however, it requires standardized protocols. The first critical step is proper fixation of the target tissue. Bell & Lightner's (A Handbook of Normal Penaeid Shrimp Histology, 1988, The World Aquaculture Society, Baton Rouge) fixation protocol, widely used for routine histology of paraffin sections, is not optimized for image analysis, and no protocol for frozen sections is described in the available literature. Therefore, the aim of this study was to optimize fixation of the hepatopancreas (HP) from whiteleg shrimp (Penaeus vannamei) for both paraffin and frozen sections using a semiquantitative scoring system. For paraffin sections, four injection volumes and three injection methods were compared, for frozen sections, four freezing methods and four fixation methods. For paraffin sections, optimal fixation was achieved by increasing threefold the fixative volume recommended by Bell and Lightner, from 10% to 30% of the shrimp body weight, combined with single injection into the HP. Optimal fixation for frozen sections was achieved by freezing the cephalothorax with liquid nitrogen, followed by fixation of the section with 60% isopropanol. These optimized methods enable the future use of image analysis and improve classical histology.

A primary phosphorus-deficient skeletal phenotype in juvenile Atlantic salmon Salmo salar: the uncoupling of bone formation and mineralization.

To understand the effect of low dietary phosphorus (P) intake on the vertebral column of Atlantic salmon Salmo salar, a primary P deficiency was induced in post-smolts. The dietary P provision was reduced by 50% for a period of 10 weeks under controlled conditions. The animal's skeleton was subsequently analysed by radiology, histological examination, histochemical detection of minerals in bones and scales and chemical mineral analysis. This is the first account of how a primary P deficiency affects the skeleton in S. salar at the cellular and at the micro-anatomical level. Animals that received the P-deficient diet displayed known signs of P deficiency including reduced growth and soft, pliable opercula. Bone and scale mineral content decreased by c. 50%. On radiographs, vertebral bodies appear small, undersized and with enlarged intervertebral spaces. Contrary to the X-ray-based diagnosis, the histological examination revealed that vertebral bodies had a regular size and regular internal bone structures; intervertebral spaces were not enlarged. Bone matrix formation was continuous and uninterrupted, albeit without traces of mineralization. Likewise, scale growth continues with regular annuli formation, but new scale matrix remains without minerals. The 10 week long experiment generated a homogeneous osteomalacia of vertebral bodies without apparent induction of skeletal malformations. The experiment shows that bone formation and bone mineralization are, to a large degree, independent processes in the fish examined. Therefore, a deficit in mineralization must not be the only cause of the alterations of the vertebral bone structure observed in farmed S. salar. It is discussed how the observed uncoupling of bone formation and mineralization helps to better diagnose, understand and prevent P deficiency-related malformations in farmed S. salar.

Investigating the underlying mechanisms of temperature-related skin diseases in Atlantic salmon, Salmo salar L., as measured by quantitative histology, skin transcriptomics and composition.

Skin integrity is recognized as of vital consideration for both animal welfare and final product quality of farmed fish. This study examines the effects of three different rearing temperatures (4, 10 and 16 °C) on the skin of healthy Atlantic salmon post-smolts. Changes in skin condition were assessed by the means of skin composition analyses, quantitative histology assessments and transcriptome analysis. Level of protein, vitamin C and vitamin E was significantly higher at 16 °C compared with 4 °C. Quantitative histology measurements showed that the epidermal thickness decreased from low to high temperature, whereas the epidermal area comprising mucous cells increased. The difference was only significant between 4 and 16 °C. Both high and low temperature exhibited significant changes in the skin transcriptome. A number of immune-related transcripts responded at both temperatures. Contrary to well-described immunosuppressive effects of low water temperature on systemic immunity, a subtle increase in skin-mediated immunity was observed, suggesting a pre-activation of the mucosal system at 4 °C. Upregulation of a number of heat-shock proteins correlating with a decrease in epidermal thickness suggested a stress response in the skin at high temperature. The results demonstrate distinctive temperature-related effects on the skin of Atlantic salmon.

Intensive care in a general hospital: demographics, utilization and outcomes.

INTRODUCTION: Intensive care units (ICU) in Irish academic centres are known to fare as well as their international counterparts. Our aim in this study was to characterise the role and outcomes of an ICU in a smaller Irish hospital and to compare these to international best practice. METHODS: We reviewed admissions of patients to the ICU of St. Luke's Hospital, Kilkenny. Patient demographics, indications for admission, and outcomes were all recorded and analysed. Sequential organ failure assessment (SOFA) scores were calculated. RESULTS: Forty-three patients were included in our study, 33 (76.7 %) of which were emergency admissions. Median length of stay was 2 days. The observed mortality rate in our cohort was 20.9 %. The median SOFA score in patients admitted was 7. Higher median SOFA scores on admission were predictive of mortality. The ICU occupancy rate during the duration of our study was 98 %, with only 15 (35.7 %) of admissions to ICU occurring within core working hours. CONCLUSION: Critical care can be provided safely and in line with current best practice in smaller Irish hospitals. There is a cohort of patients for whom care may be best provided in a tertiary centre, how best to provide for these patients will likely be achieved by early identification (e.g. with SOFA score). Bed capacity issues remain problematic.

Reducing unnecessary outpatient attendances.

PURPOSE: The purpose of this paper is to explore unnecessary outpatient department attendances and to improve efficiency by actively pre-assessing patient charts and pre-specifying management plans before scheduled outpatient visits. DESIGN/METHODOLOGY/APPROACH: All charts were reviewed by a senior clinician two weeks before the clinic and a brief, written management plan made. FINDINGS: A significant reduction in clinic sizes by about 40 per cent can be expected. There was a non-significant increase in the ratio of new to review patients and no significant reduction in waiting lists. RESEARCH LIMITATIONS/IMPLICATIONS: Pre-assessing patient charts leads to cancelling unnecessary outpatient reviews, which can increase outpatient department effectiveness. ORIGINALITY/VALUE: Clinical efficiency is becoming increasingly important. This work describes a simple strategy to improve outpatient department productivity.

Bradykinin infusion in chronic cardiac failure and the effects of captopril.

BACKGROUND: Patients with chronic cardiac failure (CCF) have abnormal vascular responses. Bradykinin (BK) is thought to contribute to the vasodilator effects of ACE inhibitors, but the effect of BK itself in patients with CCF has not been examined. METHODS: We studied the responses to infused BK at 10, 30 and 100 pmol min(-1) in patients with CCF (n=10) and controls (n=10). The slope of the dose-response curve was used for comparisons between the groups. Forearm blood flow (FBF) was measured by venous occlusion plethysmography. RESULTS: Following BK, vasodilatation was observed in both groups as the slopes were positive in all, but the difference between the groups was not significant (P=0.77). The study was repeated with the co-administration of 4 micromol min(-1) of N(G)-monomethyl L-arginine (L-NMMA). The vasodilator response to BK was reduced in both groups, and the effect was somewhat greater in the patient group (P=0.23). The vasodilator response to the endothelium-independent vasodilator sodium nitroprusside was slightly less in the patient group (P=0.08). The patients only then underwent repeat infusion of BK before and after a single oral dose of captopril 12.5 mg or placebo. Following captopril, the vasodilator response to BK was unchanged when compared to placebo (difference between slopes, P=0.53). CONCLUSIONS: BK produces dose-dependent vasodilatation in both patients with CCF and controls; there was no difference in the responses, which were antagonised by L-NMMA and therefore in part NO (endothelium)-dependent. The responses were also unchanged after administration of an ACE inhibitor (captopril).