Risk of pseudoaneurysm and bleeding complications after partial nephrectomy: comparison of tumor enucleation to standard margin technique.

Partial nephrectomy (PN) is the gold standard for the resection of amenable small renal masses. Some surgeons have adopted tumor enucleation (TE) over the standard margin PN (SPN) technique based on preservation of healthy renal parenchyma by following the tumor pseudocapsule. However, TE may also confer additional advantages due to avoidance of sharp incision including reduction in perioperative and bleeding complications. Therefore, we evaluated the rate of pseudoaneurysms and other complications following TE vs. SPN. A retrospective cohort study of patients undergoing PN (TE and SPN) between 2008 and 2020 was conducted. Baseline characteristics were compared between the TE and SPN cohorts with univariable and multivariable logistic regression models. A total of 534 patients were included, 195 (36.5%) receiving TE and 339 (63.5%) SPN. There were no differences in baseline patient demographics. There was no difference in RENAL nephrometry scores between the two groups (p = 0.47). TE had lower rates of postoperative complications (11.3 vs. 21.5%, p = 0.002). TE had less bleeding complications (2.1 vs. 8.0%, p = 0.002) with no pseudoaneurysm events following TE compared to 12 following SPN (0.0 vs. 3.5%, p = 0.008). Need for interventional radiology largely reflected pseudoaneurysm differences (0 (0.0%) TE vs. 13 (3.8%) SPN, p = 0.006. Readmission occurred less often after TE vs. SPN (4.1 vs. 8.3%, p = 0.07). Patients receiving TE experienced no clinically significant pseudoaneurysm formation and were less likely to have any bleeding complication or major complication postoperatively. TE may be preferred when minimizing morbidity aligns with patient selection and preferences.

The Current Status of Palliative Care, Hospice, and End-of-Life Health Care Utilization in Patients With Malignant Ureteral Obstruction.

INTRODUCTION: Malignant ureteral obstruction is associated with a poor prognosis, with a median survival of 3 to 7 months. These patients are ideal candidates for concurrent palliative care services, consistent with American Society of Clinical Oncology guidelines. We aimed to characterize palliative care, hospice, and end-of-life health care utilization in patients with malignant ureteral obstruction. METHODS: Patients ≥ 18 years old at our institution and diagnosed with malignant ureteral obstruction between May 2014 and August 2020 were retrospectively identified and pertinent data extracted. Palliative care, hospice, and end-of-life health care utilization was described, and factors associated with each were assessed with logistic regression models. Overall survival was assessed with Cox proportional hazard regression models. RESULTS: One hundred fifteen patients qualified for analysis; 39.1% (45/115) utilized palliative care and spent a median of 12.5 days (IQR 3-52 days) on nonhospice palliative care. On adjusted analysis Black ethnicity (aOR 3.44, 95% CI: 1.08-10.94) was associated with palliative care utilization. Of the patients, 53.9% (62/115) utilized hospice. The median time from hospice initiation to death was 12 days (IQR 5-23 days). On adjusted analysis, prior extirpative surgery (aOR 3.63, 95% CI 1.01-13.05) and palliative care utilization (aOR 4.38, 95% CI 1.70-11.31) were associated with hospice utilization. Median survival following diagnosis was 141 days (IQR 37.5-442.5). Of the patients, 43.0% (37/86) had high end-of-life health care utilization. On multivariable analysis, only hospice (aOR 0.03, 95% CI 0.01-0.14) was associated with less end-of-life health care utilization. CONCLUSIONS: Palliative care is underutilized in malignant ureteral obstruction. Hospice, but not palliative care utilization, was associated with decreased end-of-life health care utilization.

Transition from standard robotic prostatectomy to Retzius-sparing prostatectomy: feasibility and early outcomes.

BACKGROUND AND OBJECTIVE: Retzius-sparing robotic-assisted radical prostatectomy (rsRARP) has gained popularity due to superior early continence outcomes compared to standard robotic prostatectomy (sRARP). We evaluate the results of a single surgeon who transitioned from sRARP to rsRARP and compare oncologic and functional outcomes. METHODS: We retrospectively reviewed all prostatectomies performed by a single surgeon between June 2018 and October 2020. Perioperative, oncologic, and functional data were collected and analyzed. Patients who underwent sRARP were compared with those who underwent rsRARP. RESULTS: Both groups contained 37 consecutive patients each. Preoperative patient characteristics and biopsy results were similar between the two groups. Perioperative outcomes were significant for longer operative room time and higher proportion of T3 tumors in the rsRARP group. Thirty-day complication and readmission rates were similar between groups. There was no difference in early oncologic outcomes, including positive surgical margin rate, biochemical recurrence, and need for adjuvant or salvage treatments. The time to urinary continence and immediate continence rate was superior in the rsRARP group. CONCLUSIONS: The Retzius-sparing approach can be safely adopted by surgeons experienced in sRARP without compromising early oncologic outcomes and with the benefit of improved early continence recovery.

Differentiating Oncocytic Renal Tumors from Chromophobe Renal Cell Carcinoma: Comparison of Peak Early-phase Enhancement Ratio to Clinical Risk Factors and Rater Predictions.

Background: Most surgically resected benign renal tumors are found to be oncocytomas or indolent hybrid oncocytic tumors, which are difficult to differentiate from chromophobe renal cell carcinoma (chRCC) on renal mass biopsy. Both often exhibit CD117+ staining. Objective: To evaluate the ability of the peak early-phase enhancement ratio (PEER) to distinguish oncocytomas from chRCC and compare its discrimination to traditional clinical risk factors and blinded clinical raters. Design setting and participants: This was a diagnostic case-control study of patients (2006-2020) with oncocytoma or chRCC according to surgical pathology. Intervention: Partial or radical nephrectomy. Outcome measurements and statistical analysis: Three clinical raters blinded to histology measured the PEER and the presence of stellate scar and predicted the final histology for each tumor. Averaged and individual PEER values were compared to surgical pathology and assessed for interobserver variability. Subanalyses were conducted for patients with confirmed CD117+ status. Results and limitations: For the 76 patients identified, PEER was higher among the 32 (42.1%) oncocytomas than among the 44 (57.9%) chRCCs (median 0.81 vs 0.43; p < 0.001), with high correlation across raters (correlation coefficients ≥0.85). A PEER cutoff of <0.60 was strongly associated with identification of chRCC (OR 95.7 (95% CI 19.9-460.8), p < 0.001). In the overall and CD117+ cohorts, sensitivity was 93.2% and 97.0%, the negative predictive value was 90.3% and 95.5%, and the area under the receiver operating characteristic curve (AUC) on multivariable modeling was 95.0% and 98.1%, respectively. PEER outperformed models with clinical risk factors alone (AUC 70.4%) and histology predictions by three raters (AUC 51.6%, 62.5%, and 63.1%). Limitations include reliance on surgical pathology and inclusion of a mix of early contrast-enhanced phases. Conclusions: PEER reliably differentiated benign renal oncocytomas and indolent hybrid tumors from malignant chRCC with excellent diagnostic performance. A diagnostic pathway with biopsy, CD117 staining, and PEER deserves further study to potentially avoid unnecessary surgery for oncocytic renal tumors. Patient summary: We assessed a measurement called PEER on computed tomography (CT) scans and found higher values for benign and lower values for malignant kidney masses, so we were able to tell these apart. PEER was reliable for identifying tumors with positive staining for the CD117 protein biomarker as well as in the overall patient group. Our results show that PEER could be considered for use with biopsy and CD117 staining to potentially avoid unnecessary surgery for benign kidney masses.

An automated multiplexed turbidometric and data collection system for measuring growth kinetics of anaerobes dependent on gaseous substrates.

Standard methods of monitoring the growth kinetics of anaerobic microorganisms are generally impractical when there is a protracted or indeterminate period of active growth, and when high numbers of samples or replications are required. As part of our studies of the adaptive evolution of a simple anaerobic syntrophic mutualism, requiring the characterization of many isolates and alternative syntrophic pairings, we developed a multiplexed growth monitoring system using a combination of commercially available electronics and custom designed circuitry and materials. This system automatically monitors up to 64 sealed, and as needed pressurized, culture tubes and reports the growth data in real-time through integration with a customized relational database. The utility of this system was demonstrated by resolving minor differences in growth kinetics associated with the adaptive evolution of a simple microbial community comprised of a sulfate reducing bacterium, Desulfovibrio vulgaris, grown in syntrophic association with Methanococcus maripaludis, a hydrogenotrophic methanogen.

Hypoxia during incubation does not affect aerobic performance or haematology of Atlantic salmon (Salmo salar) when re-exposed in later life.

Hypoxia in aquatic ecosystems is becoming increasingly prevalent, potentially reducing fish performance and survival by limiting the oxygen available for aerobic activities. Hypoxia is a challenge for conserving and managing fish populations and demands a better understanding of the short- and long-term impacts of hypoxic environments on fish performance. Fish acclimate to hypoxia via a variety of short- and long-term physiological modifications in an attempt to maintain aerobic performance. In particular, hypoxia exposure during early development may result in enduring cardio-respiratory modifications that affect future hypoxia acclimation capacity, yet this possibility remains poorly investigated. We incubated Atlantic salmon (Salmo salar) in normoxia (~100% dissolved oxygen [DO, as percent air saturation]), moderate hypoxia (~63% DO) or cyclical hypoxia (100-25% DO daily) from fertilization until 113 days post-fertilization prior to rearing all groups in normoxia for a further 8 months. At ~11 months of age, subsets of each group were acclimated to hypoxia (50% DO) for up to 44 days prior to haematology, aerobic metabolic rate and hypoxia tolerance measurements. Hypoxia exposure during incubation (fertilization to 113 days post-fertilization) did not affect the haematology, aerobic performance or hypoxia tolerance of juvenile salmon in later life. Juveniles acclimated to hypoxia increased maximum aerobic metabolic rate and aerobic scope by ~23 and ~52%, respectively, when measured at 50% DO but not at 100% DO. Hypoxia-incubated juveniles also increased haematocrit and haemoglobin concentration but did not affect acute hypoxia tolerance (critical oxygen level and DO at LOE). Thus, while Atlantic salmon possess a considerable capacity to physiologically acclimate to hypoxia by improving aerobic performance in low oxygen conditions, we found no evidence that this capacity is influenced by early-life hypoxia exposure.

Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning.

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian 'greenlip' abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone 'omics website, http://abalonedb.org.

Heterozygous Mutations in SMARCA2 Reprogram the Enhancer Landscape by Global Retargeting of SMARCA4.

Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.

Physiological effects of dissolved oxygen are stage-specific in incubating Atlantic salmon (Salmo salar).

Oxygen availability is highly variable during salmonid incubation in natural redds and also in aquaculture incubation systems. Hypoxia generally decreases growth and aerobic metabolism prior to hatching, in parallel with eliciting physiological modifications that enhance oxygen delivery. However, it is less-well known whether developmental hyperoxia can drive the opposite effect. Moreover, there is insufficient understanding of stage-specific developmental windows during which ambient oxygen availability may be of greater or lesser impact to incubating embryos. Here, we tested the effects of hypoxia (50% dissolved oxygen: DO, % air saturation) and hyperoxia (150% DO) on the growth, routine aerobic metabolism ([Formula: see text]) and hypoxia tolerance (O2crit) of Atlantic salmon (Salmo salar) during seven developmental windows throughout incubation. Embryos exposed to hyperoxia (150% DO) did not differ from the normoxic group in growth, [Formula: see text] or O2crit at any developmental window. In contrast, embryos exposed to hypoxia grew slower and had a lower [Formula: see text], but had higher hypoxia tolerance (lower O2crit) than normoxic and hyperoxic counterparts. Interestingly, these differences were only apparent when the embryos were measured prior to hatching. Larvae (alevins) incubated in hypoxia following hatching grew similarly to normoxia-incubated alevins. Our results provide evidence that Atlantic salmon embryos are most sensitive to hypoxia prior to hatching, probably due to increasing (absolute) oxygen requirements concurrent with restricted oxygen diffusion through the egg. Moreover, the similarities between normoxia- and hyperoxia-incubated salmon demonstrate that embryos are not oxygen-limited under normoxic conditions.

Sex Differences in Mechanisms of Recovery after Isometric and Dynamic Fatiguing Tasks.

PURPOSE: The purpose of this study was to determine whether supraspinal mechanisms contribute to the sex difference in fatigability during and recovery from a dynamic and isometric fatiguing task with the knee extensors. METHODS: Transcranial magnetic stimulation and electrical stimulation were used to determine voluntary activation and contractile properties of the knee extensors in 14 men and 17 women (20.8 ± 1.9 yr) after a 1) 60-s sustained, maximal voluntary isometric contraction (MVIC), and 2) dynamic fatiguing task involving 120 maximal voluntary concentric contractions with a 20% MVIC load. RESULTS: There were no differences between men and women in the reduction of maximal torque during the sustained MVIC (54.4% ± 18.9% vs 55.9% ± 11.2%, P = 0.49) or in the decrease in power during the dynamic fatiguing task (14.7% ± 20.1% vs 14.2% ± 18.5%, P = 0.92). However, MVIC torque recovered more quickly for women than men after the sustained MVIC and the dynamic task (P < 0.05). The transcranial magnetic stimulation-elicited superimposed twitch was larger for men than for women during the sustained MVIC and in recovery (immediately post, R0.1: 4.7% ± 3.3% vs 2.4% ± 1.9% MVIC; P = 0.02), with no sex difference after the dynamic task (P = 0.35). The reduction in resting twitch amplitude was larger for men than for women immediately after the dynamic task (37% ± 22% vs 23% ± 18%; P = 0.016) with no sex difference after the sustained MVIC (64% ± 16% vs 67% ± 11%; P = 0.46). CONCLUSIONS: Supraspinal fatigue contributed to fatigability of the knee extensors more for men than for women after a maximal isometric task, whereas contractile mechanisms explained the sex difference in torque recovery after the fast-velocity dynamic task. The mechanisms for the sex difference in fatigability are task dependent.

Hypoxic acclimation leads to metabolic compensation after reoxygenation in Atlantic salmon yolk-sac alevins.

Hypoxia is common in aquatic environments and has substantial effects on development, metabolism and survival of aquatic organisms. To understand the physiological effects of hypoxia and its dependence on temperature, metabolic rate ( [Formula: see text] ) and cardiorespiratory function were studied in response to acute hypoxia (21→5kPa) at different measurement temperatures (Ta; 4, 8 and 12°C) in Salmo salar alevins that were incubated under normoxic conditions (PO2=21kPa) or following hypoxic acclimation (PO2=10kPa) as well as two different temperatures (4°C or 8°C). Hypoxic acclimation lead to a developmental delay manifested through slower yolk absorption. The general response to acute hypoxia was metabolic depression (~60%). Hypoxia acclimated alevins had higher [Formula: see text] s when measured in normoxia than alevins acclimated to normoxia. [Formula: see text] s were elevated to the same degree (~30% per 4°C change) irrespective of Ta. Under severe, acute hypoxia (~5kPa) and irrespective of Ta or acclimation, [Formula: see text] s were similar between most groups. This suggests that despite different acclimation regimes, O2 transport was limited to the same degree. While cardiorespiratory function (heart-, ventilation rate) was unchanged in response to acute hypoxia after normoxic acclimation, hypoxic acclimation led to cardiorespiratory changes predominantly in severe hypoxia, indicating earlier onset and plasticity of cardiorespiratory control mechanisms. Although [Formula: see text] in normoxia was higher after hypoxic acclimation, at the respective acclimation PO2, [Formula: see text] was similar in normoxia and hypoxia acclimated alevins. This is indicative of metabolic compensation to an intrinsic [Formula: see text] at the acclimation condition in hypoxia-acclimated alevins after re-exposure to normoxia.

Developmental Hypoxia Has Negligible Effects on Long-Term Hypoxia Tolerance and Aerobic Metabolism of Atlantic Salmon (Salmo salar).

Exposure to developmental hypoxia can have long-term impacts on the physiological performance of fish because of irreversible plasticity. Wild and captive-reared Atlantic salmon (Salmo salar) can be exposed to hypoxic conditions during development and continue to experience fluctuating oxygen levels as juveniles and adults. Here, we examine whether developmental hypoxia impacts subsequent hypoxia tolerance and aerobic performance of Atlantic salmon. Individuals at 8°C were exposed to 50% (hypoxia) or 100% (normoxia) dissolved oxygen (DO) saturation (as percent of air saturation) from fertilization for ∼100 d (800 degree days) and then raised in normoxic conditions for a further 15 mo. At 18 mo after fertilization, aerobic scope was calculated in normoxia (100% DO) and acute (18 h) hypoxia (50% DO) from the difference between the minimum and maximum oxygen consumption rates ([Formula: see text] and [Formula: see text], respectively) at 10°C. Hypoxia tolerance was determined as the DO at which loss of equilibrium (LOE) occurred in a constantly decreasing DO environment. There was no difference in [Formula: see text], [Formula: see text], or aerobic scope between fish raised in hypoxia or normoxia. There was some evidence that hypoxia tolerance was lower (higher DO at LOE) in hypoxia-raised fish compared with those raised in normoxia, but the magnitude of the effect was small (12.52% DO vs. 11.73% DO at LOE). Acute hypoxia significantly reduced aerobic scope by reducing [Formula: see text], while [Formula: see text] remained unchanged. Interestingly, acute hypoxia uncovered individual-level relationships between DO at LOE and [Formula: see text], [Formula: see text], and aerobic scope. We discuss our findings in the context of developmental trajectories and the role of aerobic performance in hypoxia tolerance.

Automated Non-invasive Video-Microscopy of Oyster Spat Heart Rate during Acute Temperature Change: Impact of Acclimation Temperature.

We developed an automated, non-invasive method to detect real-time cardiac contraction in post-larval (1.1-1.7 mm length), juvenile oysters (i.e., oyster spat) via a fiber-optic trans-illumination system. The system is housed within a temperature-controlled chamber and video microscopy imaging of the heart was coupled with video edge-detection to measure cardiac contraction, inter-beat interval, and heart rate (HR). We used the method to address the hypothesis that cool acclimation (10°C vs. 22°C-Ta10 or Ta22, respectively; each n = 8) would preserve cardiac phenotype (assessed via HR variability, HRV analysis and maintained cardiac activity) during acute temperature changes. The temperature ramp (TR) protocol comprised 2°C steps (10 min/experimental temperature, Texp) from 22°C to 10°C to 22°C. HR was related to Texp in both acclimation groups. Spat became asystolic at low temperatures, particularly Ta22 spat (Ta22: 8/8 vs. Ta10: 3/8 asystolic at Texp = 10°C). The rate of HR decrease during cooling was less in Ta10 vs. Ta22 spat when asystole was included in analysis (P = 0.026). Time-domain HRV was inversely related to temperature and elevated in Ta10 vs. Ta22 spat (P < 0.001), whereas a lack of defined peaks in spectral density precluded frequency-domain analysis. Application of the method during an acute cooling challenge revealed that cool temperature acclimation preserved active cardiac contraction in oyster spat and increased time-domain HRV responses, whereas warm acclimation enhanced asystole. These physiologic changes highlight the need for studies of mechanisms, and have translational potential for oyster aquaculture practices.

A method to analyze, sort, and retain viability of obligate anaerobic microorganisms from complex microbial communities.

A high speed flow cytometric cell sorter was modified to maintain a controlled anaerobic environment. This technology enabled coupling of the precise high-throughput analytical and cell separation capabilities of flow cytometry to the assessment of cell viability of evolved lineages of obligate anaerobic organisms from cocultures.

Erosion of functional independence early in the evolution of a microbial mutualism.

Many species have evolved to function as specialized mutualists, often to the detriment of their ability to survive independently. However, there are few, if any, well-controlled observations of the evolutionary processes underlying the genesis of new mutualisms. Here, we show that within the first 1,000 generations of initiating independent syntrophic interactions between a sulfate reducer (Desulfovibrio vulgaris) and a hydrogenotrophic methanogen (Methanococcus maripaludis), D. vulgaris frequently lost the capacity to grow by sulfate respiration, thus losing the primary physiological attribute of the genus. The loss of sulfate respiration was a consequence of mutations in one or more of three key genes in the pathway for sulfate respiration, required for sulfate activation (sat) and sulfate reduction to sulfite (apsA or apsB). Because loss-of-function mutations arose rapidly and independently in replicated experiments, and because these mutations were correlated with enhanced growth rate and productivity, gene loss could be attributed to natural selection, even though these mutations should significantly restrict the independence of the evolved D. vulgaris. Together, these data present an empirical demonstration that specialization for a mutualistic interaction can evolve by natural selection shortly after its origin. They also demonstrate that a sulfate-reducing bacterium can readily evolve to become a specialized syntroph, a situation that may have often occurred in nature.

Respirometry: correcting for diffusion and validating the use of plastic multiwell plates with integrated optodes.

Polystyrene multiwell plates with integrated optodes act as multiple closed-system respirometers that enable the simultaneous measurement of oxygen consumption in small animals. However, the diffusion of oxygen through polystyrene needs to be taken into consideration. Here we provide an equation that accounts for the empirically determined rate of oxygen through a polystyrene well when calculating the instantaneous rate of oxygen consumption. Furthermore, we describe a novel method of calibrating a small respirometer for accuracy using micro-osmotic pumps containing an oxygen scavenger, which is delivered at a constant rate and therefore yields a constant rate of oxygen consumption in an airtight system.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways.

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways.

Transcriptome analyses of amoebic gill disease-affected Atlantic salmon (Salmo salar) tissues reveal localized host gene suppression.

The transcriptome response of Atlantic salmon (Salmo salar) displaying advanced stages of amoebic gill disease (AGD) was investigated. Naïve smolt were challenged with AGD for 19 days, at which time all fish were euthanized and their severity of infection quantified through histopathological scoring. Gene expression profiles were compared between heavily infected and naïve individuals using a 17 K Atlantic salmon cDNA microarray with real-time quantitative RT-PCR (qPCR) verification. Expression profiles were examined in the gill, anterior kidney, and liver. Twenty-seven transcripts were significantly differentially expressed within the gill; 20 of these transcripts were down-regulated in the AGD-affected individuals compared with naïve individuals. In contrast, only nine transcripts were significantly differentially expressed within the anterior kidney and five within the liver. Again the majority of these transcripts were down-regulated within the diseased individuals. A down-regulation of transcripts involved in apoptosis (procathepsin L, cathepsin H precursor, and cystatin B) was observed in AGD-affected Atlantic salmon. Four transcripts encoding genes with antioxidant properties also were down-regulated in AGD-affected gill tissue according to qPCR analysis. The most up-regulated transcript within the gill was an unknown expressed sequence tag (EST) whose expression was 218-fold (+/- SE 66) higher within the AGD affected gill tissue. Our results suggest that Atlantic salmon experiencing advanced stages of AGD demonstrate general down-regulation of gene expression, which is most pronounced within the gill. We propose that this general gene suppression is parasite-mediated, thus allowing the parasite to withstand or ameliorate the host response.

Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.).

The association between major histocompatibility (MH) polymorphism and the severity of infection by amoebic gill disease (AGD) was investigated across 30 full sibling families of Atlantic salmon. Individuals were challenged with AGD for 19days and then their severity of infection scored by histopathological examination of the gills. Fish were then genotyped for the MH class I (Sasa-UBA) and MH class II alpha (Sasa-DAA) genes using polymorphic repeats embedded within the 3' untranslated regions of the Sasa-UBA and Sasa-DAA genes. High variation in the severity of infection was observed across the sample material, ranging from 0% to 85% gill filaments infected. In total, seven Sasa-DAA-3UTR and ten Sasa-UBA-3UTR marker alleles were identified across the 30 families. A significant association between the marker allele Sasa-DAA-3UTR 239 and a reduction in AGD severity was detected. There was also a significant association found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes. While the associations between MH allele/genotypes and AGD severity reported herein may be statistically significant, the small sample sizes observed for some alleles and genotypes means these associations should be considered as suggestive and future research is required to verify their biological significance.

18S ribosomal DNA-based PCR identification of Neoparamoeba pemaquidensis, the agent of amoebic gill disease in sea-farmed salmonids.

Neoparamoeba pemaquidensis is a parasomal amoeboid protozoan identified as the agent of amoebic gill disease (AGD) in Atlantic salmon Salmo salar reared in sea-pens in Tasmania, Australia, and coho salmon Oncorhynchus kisutch farmed on the west coast of the USA. Outbreaks of AGD caused by immunologically cross-reactive paramoebae have also been reported in sea-farmed salmonids in several other countries. Complete 18S rDNA sequences were determined for respective paramoebae isolated from infected gills of salmon from Tasmania and Ireland, and N. pemaquidensis isolates from the USA and UK, including representative free-living isolates. Alignments over 2110 bp revealed 98.1 to 99.0% sequence similarities among isolates, confirming that paramoebae implicated in AGD in geographically distant countries were homologous and belonged to the same species, N. pemaquidensis. The results supported previous findings that N. pemaquidensis exists as a widely distributed, amphizoic marine protozoan. Partial 18S rDNA sequences were obtained for the ultrastructurally similar species, N. aestuarina, and for the morphologically similar but non-parasomal amoeba Pseudoparamoeba pagei. N. aestuarina had 95.3 to 95.7% sequence similarities with N. pemaquidensis strains, which distinguished 2 closely related but separate species. Neoparamoeba spp. were not analogous to P. pagei or to other marine Gymnamoebia. We designed 4 oligonucleotide primers based on elucidated 18S rDNA sequences and applied them to single-step and nested 2-step PCR protocols developed to identify N. pemaquidensis to the exclusion of apparently closely related and non-related protistan taxa. Nested PCR was able to detect the AGD parasite from non-purified, culture-enriched net microfouling samples from Atlantic salmon sea-pens in Tasmania, and confirmed that N. pemaquidensis was also responsible for AGD in chinook salmon O. tshawytscha in New Zealand. Our sequence and PCR analyses have now shown that AGD affecting 3 different salmonid species farmed in 4 countries are associated with N. pemaquidensis. A species-specific diagnostic PCR provides for the first time, a highly specific detection and identification assay for N. pemaquidensis that will facilitate future ecological and epidemiological studies of AGD.