Investigation of co-infections with pathogens associated with gill disease in Atlantic salmon during an amoebic gill disease outbreak.

Gill diseases are a complex and multifactorial challenge for marine farmed Atlantic salmon. Co-infections with putative pathogens are common on farms; however, there is a lack of knowledge in relation to the potential effect co-infections may have on pathology. The objective of this study was to determine the prevalence and potential effects of Neoparamoeba perurans, Desmozoon lepeophtherii, Candidatus Branchiomonas cysticola, Tenacibaculum maritimum and salmon gill poxvirus (SGPV) during a longitudinal study on a marine Atlantic salmon farm. Real-time PCR was used to determine the presence and sequential infection patterns of these pathogens on gill samples collected from stocking until harvest. A number of multilevel models were used to determine the effect of these putative pathogens on gill health (measured as gill histopathology score), while adjusting for the effect of water temperature and time since the last freshwater treatment. Results indicate that between 12 and 16 weeks post-seawater transfer (wpst), colonization of the gills by all pathogens had commenced and by week 16 of marine production each of the pathogens had been detected. D. lepeophtherii and Candidatus B. cysticola were by far the most prevalent of the potential pathogens detected during this study. Detections of T. maritimum were found to be significantly correlated with temperature showing distinct seasonality. Salmon gill poxvirus was found to be highly sporadic and detected in the first sampling point, suggesting a carryover from the freshwater stage of production. Finally, the model results indicated no clear effect between any of the pathogens. Additionally, the models showed that the only variable which had a consistent effect on the histology score was N. perurans.

Characterization of the live salmonid movement network in Ireland: Implications for disease prevention and control.

Live fish movement is considered as having an important role in the transmission of infectious diseases. For that reason, interventions for cost-effective disease prevention and control rely on a sound understanding of the patterns of live fish movements in a region or country. Here, we characterize the network of live fish movements in the Irish salmonid farming industry during 2013, using social network analysis and spatial epidemiology methods, and identify interventions to limit the risk of disease introduction and spread. In the network there were 62 sites sending and/or receiving fish, with a total of 130 shipments (84 arcs) comprising approx. 17.2 million fish during the year. Atlantic salmon shipments covered longer distances than trout shipments, with some traversing the entire country. The average shipment of Atlantic salmon was 146,186 (SD 194,344) fish, compared to 77,928 (127,009) for trout, however, variability was high. There were 3 periods where shipments peaked (February-April, June-September, and November), which were related to specific stages of fish. The network was disconnected and had two major weak components, the first one with 39 nodes (mostly Atlantic salmon sites), and the second one with 10 nodes (exclusively trout sites). Correlation between in and out-degree at each site and assortativity coefficient were slightly low and non-significant: -0.08 (95% CI: -0.22, 0.06) and -0.13 (95% CI: -0.36, 0.08), respectively, indicating random mixing with regard to node degree. Although competing models also produced a good fit to degree distribution, it is likely that the network possesses both small-world and scale-free topology. This would facilitate the spread and persistence of infection in the salmon production system, but would also facilitate the design of risk-based surveillance strategies by targeting hubs, bridges or cut-points. Using Infomap community detection algorithms, 2 major communities were identified within the giant weak component, which were linked by only 4 nodes. Communities found had no correspondence with geographical zones within the country, which could potentially hinder the implementation of zoning strategies for disease control and eradication. Three significant spatial clusters of node centrality measures were detected, two in county Donegal (betweenness and outcloseness) and one in county Galway (incloseness), highlighting the importance of these locations as hot spots of highly central sites with a higher potential for both introduction and spread of infection. These results will assist in the design and implementation of measures to reduce the sanitary risks emerging from live fish trade within Ireland.

Risk factor analysis for sea lice, Caligus rogercresseyi, levels in farmed salmonids in southern Chile.

Sea lice, Caligus rogercresseyi, are ectoparasitic copepods, which severely affect the salmon farming industry in southern Chile, reducing the health status of fish and producing both direct and indirect economic losses. Local farmers have reported increasing infestation levels since 2004, reaching a peak in 2007. In response to this situation, the Chilean Fisheries Service (Sernapesca) developed a surveillance programme; the first step of which consisted of a general survey of salmon farms. This survey included documenting counts of parasite burdens on fish and measurements of several husbandry and environmental factors providing an evaluation of risk factors for the observed infestation levels. The information collected was analysed using a linear mixed model technique, which takes into account the clustered structure of data, decomposing the unexplained variation and assigning it to different aggregation levels of the productive system. Geographical zones, fish species, treatment against sea lice performed 1 month before sampling, stocking density, fish weight and water salinity were the variables significantly associated with sea lice burdens. In contrast, treatments performed 2-3 months before sampling, use of photoperiod in sea cages and water temperature, were not significant. There was significant unexplained variation at all aggregation levels, i.e. sub-zone, fish farm and cage level, with the fish farm level showing the greatest variation.

Risk factors and predictors for surgical site infection after hepatic resection.

Strict control of blood glucose levels with insulin in a surgical intensive care unit reduces postoperative morbidity and mortality. The aim of this study was to identify risk factors and the predictors for the prevention of surgical site infection (SSI) in a consecutive series of hepatectomised cases in a single institution. The association between SSI and various clinical parameters was investigated in 152 patients who underwent hepatic resection at Kochi Medical School from January 2000 through March 2007. The incidence of SSI in these patients was 14.5%. Multivariate analysis identified four independent parameters correlating with the occurrence of SSI: (i) body mass index >23.6 kg/m(2); (ii) estimated blood volume loss >810 mL; (iii) presence of postoperative bile leakage of organ/space SSI; and (iv) use of the sliding scale method for postoperative glucose control. There was no observed SSI after liver resection in the group whose postoperative blood glucose levels were controlled by an artificial pancreas. This study reveals that lack of postoperative glycaemic control is associated with a significantly higher incidence of postoperative infectious complications and longer hospitalisation. Obesity and the level of intraoperative estimated blood loss and bile leakage after hepatic resection are also risk factors with predictive value for SSI. Artificial pancreas is a safe and beneficial device to perform postoperative strict glycaemic control without hypoglycaemia for patients who undergo hepatic resection for liver diseases.

Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes.

BACKGROUND: Intra-articular injection of hyaluronan (HA) has been suggested to have a disease-modifying effect in osteoarthritis, but little is known about the possible mechanisms. OBJECTIVE: To investigate the effects of HA species of different molecular mass, including 800 kDa (HA800) and 2700 kDa (HA2700), on the expression of aggrecanases (ie, ADAMTS species), which play a key role in aggrecan degradation. METHODS: The effects of HA species on the expression of ADAMTS1, 4, 5, 8, 9 and 15 in interleukin 1alpha (IL1alpha)-stimulated osteoarthritic chondrocytes were studied by reverse transcription PCR and real-time PCR. Expression of ADAMTS4 protein and aggrecanase activity and signal transduction pathways of IL1, CD44 and intracellular adhesion molecule 1 (ICAM1) were examined by immunoblotting. RESULTS: IL1alpha treatment of chondrocytes induced ADAMTS4, and HA800 and HA2700 significantly decreased IL1alpha-induced expression of ADAMTS4 mRNA and protein. IL1alpha-stimulated aggrecanase activity in osteoarthritic chondrocytes was reduced by treatment with HA2700 or transfection of small interfering RNA for ADAMTS4. A similar result was obtained when HA2700 was added to explant cultures of osteoarthritic cartilage. HA2700 neither directly inhibited nor bound to ADAMTS4. Downregulation of ADAMTS4 expression by HA2700 was attenuated by treatment of IL1alpha-treated chondrocytes with antibodies to CD44 and/or ICAM1. The increased phosphorylation of IL1 receptor-associated kinase-1 and extracellular signal-regulated protein kinase1/2 induced by the IL1alpha treatment was downregulated by enhanced IRAK-M expression after HA2700 treatment. CONCLUSION: These data suggest that HA2700 suppresses aggrecan degradation by downregulating IL1alpha-induced ADAMTS4 expression through the CD44 and ICAM1 signalling pathways in osteoarthritic chondrocytes.

Accuracy and reliability of continuous blood glucose monitor in post-surgical patients.

BACKGROUND: The STG-22 is the only continuous blood glucose monitoring system currently available. The aim of this study is to determine the accuracy and reliability of the STG-22 for continuously monitoring blood glucose level in post-surgical patients. METHODS: Fifty patients scheduled for routine surgery were studied in surgical intensive care unit (ICU) of a university hospital. After admission to the ICU, the STG-22 was connected to the patients. An attending physician obtained blood samples from a radial arterial catheter. Blood glucose level was measured using the ABL800FLEX immediately after blood collection at 0, 4, 8, and 16 h post-admission to the ICU (total of 200 blood glucose values). RESULTS: The correlation coefficient (R2) was 0.96. In the Clarke error grid, 100% of the paired measurements were in the clinically acceptable zone A and B. The Bland and Altman analysis showed that bias+/-limits of agreement (percent error) were 0.04(0.7)+/-0.35(6.3) mmol (mg/dl) (7%), -0.11(-2)+/-1.22(22) (15%) and -0.33(-6)+/-1.28(23) (10%) in hypoglycemia (<70(3.89) mmol (mg/dl), normoglycemia (3.89(70)-10(180) mmol (mg/dl), and hyperglycemia (>10(180) mmol (mg/dl), respectively. CONCLUSIONS: The STG-22 can be used for measuring blood glucose level continuously and measurement results are consistent with intermittent measurement (percentage error within 15%). Therefore, the STG-22 is a useful device for monitoring in blood glucose level in the ICU for 16 h.

Studies on 5-lipoxygenase inhibitors. II. Discovery, optical resolution and enantioselective synthesis of FR110302, a highly potent non-redox type 5-lipoxygenase inhibitor.

A novel series of 2,2-dialkyl-5-(2-quinolylmethoxy)-1,2,3, 4-tetrahydro-1-naphthols was synthesized and evaluated as 5-lipoxygenase (5-LO) inhibitors. Systematic optimization led to identification of several highly potent non-redox type 5-LO inhibitors with nanomolar IC50s as racemic mixtures. Optical resolution of racemate 50 indicated that its 5-LO inhibitory activity was enantiospecific and due to the (+)-enantiomer. An efficient synthetic route to the (+)-enantiomers via asymmetric reduction of tetralone intermediates was established. The best compound, (+)-2,2-dibutyl-5-(2-quinolylmethoxy)-1,2,3,4-tetrahydro-1-naphtho l (FR110302, (+)-50), showed potent inhibitory activity against leukotriene (LT) biosynthesis by intact neutrophiles in rats (IC50 4.9 nM) and in humans (IC50 40 nM). Furthermore oral administration of FR110302 significantly inhibited neutrophil migration in the rat air pouch model at 1 mg/kg.

In vivo biodistribution of a radiotracer for imaging serotonin-1A receptor sites with PET:[11C]LY274601.

LY274601 [R-(+)8-thiomethyl-2-(di-n-propyl-amino)tetralin], a full agonist of the 5-HT1A receptor with high affinity and selectivity, was labeled with 11C and 3H, and its in vivo behavior was studied to evaluate [11C]LY274601 as a PET radiotracer for imaging 5-HT1A receptor sites in living brain. Following intravenous tail injection into mice, [11C]LY274601 showed high blood-brain barrier permeability and accumulated in regions known to have high densities of 5-HT1A receptor sites such as the brain stem including the raphe nuclei. The binding of the radiotracer in target tissues is blocked by pre-injection of the 5-HT1A receptor selective ligand 8-OH-DPAT (1 mg/kg, s.c.), suggesting that the binding is specific to 5-HT1A receptor sites. Using ex vivo autoradiography, the target tissues such as hippocampus CA1-4 fields, piriform cortex, dorsal raphe nucleus and lateral septum were visualized as hot spots. These tissues were observed to have binding 2-2.7 times greater than the cerebellum. The distribution of the radiotracer agrees well with the distribution of 5-HT1A receptors revealed by in vitro autoradiography with [3H]8-OH-DPAT. However, the radiotracer was metabolized quickly and cleared from target tissues with a half life of approximately 15 min. [11C]LY274601 showed high non-specific binding in regions with low number of 5-HT1A receptor sites such as cerebellum.

Studies on 5-lipoxygenase inhibitors. I. Synthesis and 5-lipoxygenase-inhibitory activity of novel hydroxamic acid derivatives.

A series of novel hydroxamates has been prepared and tested for inhibitory activity towards rat polymorphonuclear leukocyte (PMN) 5-lipoxygenase (5-LO) in vitro and towards neutrophil migration in the rat air pouch model of inflammation in vivo. Many 3,4-dihydronaphthyl compounds were potent inhibitors of 5-LO, and several compounds were potent inhibitors of neutrophil migration. The most potent 3,4-dihydronaphthyl compound, N-[[(3,4-dihydro-5-phenoxy)-2-naphthyl]methyl]-N-hydroxy-N'-ethylurea (FR122788, 18) had an IC50 of 25 nM in the 5-LO assay, and strongly reduced neutrophil migration in the rat air pouch model at 1 mg/kg (p.o.). FR122788 also had an ameliorating effect in a rat hepatitis model induced by D-galactosamine, with an ED50 values of 14.6 mg/kg (p.o.) for glutamate oxaloacetate transaminase (GOT) and 16.8 mg/kg (p.o.) for glutamate pyruvate transaminase (GPT).

A 5-lipoxygenase inhibitor, FR110302, inhibits ozone-induced airway hyperresponsiveness in guinea pigs and dogs.

Airway hyperresponsiveness is a key feature of asthma, and attenuating airway hyperresponsiveness is an important part of asthma therapy. In the present study we examined the inhibitory effect of a potent 5-lipoxygenase inhibitor, FR110302, on airway hyperresponsiveness induced by ozone exposure in guinea pigs and dogs. Respiratory resistance (Rrs) was measured by a forced oscillation method. Airway responsiveness was determined from the dose-response curve of Rrs to acetylcholine. Guinea pigs were exposed to 2.5 ppm ozone for 1 h. In a control group of guinea pigs, delta log PC100 (the index of the ozone-induced airway hyperresponsiveness) was 0.58 +/- 0.04 (log mg/ml). Treatment with FR110302 (10 or 100 mg/kg p.o.) significantly diminished delta log PC100 (10 mg/kg: 0.22 +/- 0.10; 100 mg/kg; 0.11 +/- 0.06). Dogs were exposed to 3 ppm ozone for 2 h. In a control group of dogs, delta log Dmin (another index of the ozone-induced airway hyperresponsiveness) was 1.24 +/- 0.15 (log unit). Treatment with FR110302 (1 or 3.2 mg/kg p.o.) significantly diminished delta log Dmin (1 mg/kg: 0.60 +/- 0.18; 3.2 mg/kg: 0.27 +/- 0.12). These results suggest that FR110302 may be a useful drug for attenuating airway hyperresponsiveness in asthmatic patients.

A circular dichroism study on thermal denaturation of a dimeric globular protein, Streptomyces subtilisin inhibitor.

Thermal denaturation of Streptomyces subtilisin inhibitor was studied by means of circular dichroism (CD) measurements in the far-UV and near-UV regions. The denaturation was found to be largely reversible; the partial irreversibility was associated with a slight loss of the inhibitory activity. Difference CD spectra in the far-UV region clarified the existence of two distinct steps in the thermal transition of the secondary structure. The first step below 80 degrees C is attributable to a partial conformational change in the alpha-helix portion, whereas the second step between 80 degrees C and 94 degrees C is attributable to a major conformational change involving the beta-sheet portion. On the assumption that the major denaturation involves dissociation of the SSI into its subunits, the enthalpy and entropy changes were determined to be 216 kcal X mol-1 and to be 603 cal X deg-1 X mol-1, respectively.

[Quality control of radioimmunoassay by bivariate analysis].

Automatic calculation of control charts for precision and accuracy of radioimmunoassay was reported by Faure, et al. Duplicate control samples independently measured was assumed to have a bivariate normal distribution. In this case they assumed that the correlation coefficient between each value of the pairs of control samples is zero. Our experience using this method revealed that a considerable number of assayed samples distributed outside the calculated control limits in case of "accuracy control". It was considered that this happened because in radioimmunoassay the between-assays precision is usually larger than the within-an-assay precision and there is a significant correlation between values of duplicates. We also found equal probability density did not make a true circle but a long circle. Therefore in the present paper we proposed for control charts of radioimmunoassay an equal probability long circle calculated by bivariate analysis of Mahalanobis' generalized distance. It was found that a Mahalanobis' long circle could explain the density distribution of radioimmunoassay with a reasonable percent of samples outside the calculated control limits. What happened here can be interpreted by a large between-assays variability shown by some commercial kits. This automatic calculation method could be applied not only for quality control but also for evaluation and comparison of radioimmunoassay system or commercial kits. Control survey could also be analyzed by such a method.

[Analysis of RIA standard curve by log-logistic and cubic log-logit models (author's transl)].

In order to improve goodness-of-fit in RIA standard analysis, programs for computing log-logistic and cubic log-logit were written in BASIC using personal computer P-6060 (Olivetti). Iterative least square method of Taylor series was applied for non-linear estimation of logistic and log-logistic. Here "log-logistic" represents Y = (a--d)/(1+log(X)/c)b)+d As weights either 1, 1/var(Y) or 1/sigma 2 were used in logistic or log-logistic and either Y2(1--Y)2, Y2(1-Y)2/var(Y), or Y2(1--Y)2/sigma 2 were used in logistic or log-logistic and either Y2(1--Y)2, Y2(1--Y)2/var(Y), or Y2(1--Y)2/sigma 2 were used in quadratic or cubic log-logit. The term var(Y) represents squares of pure error and sigma 2 represents estimated variance calculated using a following equation log(sigma 2 + 1) = log(A)+J log(y). As indicators for goodness-of-fit, MSL/Se2, CMD% and WRV (see text) were used. Better regression was obtained in case of alpha-fetoprotein by log-logistic than by logistic. Cortisol standard curve was much better fitted with cubic log-logit than quadratic log-logit. Predicted precision of AFP standard curve was below 5% in log-logistic instead of 8% in logistic analysis. Predicted precision obtained using cubic log-logit was about five times lower than that with quadratic log-logit. Importance of selecting good models in RIA data processing was stressed in conjunction with intrinsic precision of radioimmunoassay system indicated by predicted precision.