Applying genetic technologies to combat infectious diseases in aquaculture.

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies-sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture.

Oxidized Dietary Oil, High in Omega-3 and Omega-6 Polyunsaturated Fatty Acids, Induces Antioxidant Responses in a Human Intestinal HT29 Cell Line.

When oxidized, dietary oils generate products which have the potential to cause adverse effects on human health. The objective of the study was to investigate whether lipid oxidation products in an oxidized dietary oil can be taken up in intestinal cells, induce antioxidant stress responses and potentially be harmful. The in vitro cell model HT29 was exposed to camelina oil with different extents of oxidation, or only 4-hydroxy-2-hexenal (HHE) or 4-hydroxy-2-nonenal (HNE). The cellular content of HHE increased with an increasing extent of oxidation of the camelina oil added to the cell's growth media, whereas HNE did not show a similar trend. Deuterated HHE was taken up by the HT29 cells, with 140 µM HHE metabolized within 0.5-1 h. The low oxidation degree of the camelina oil increased the gene expression of antioxidant markers (GPX, ATF6, XBP1). The increase in the gene expression of SOD at medium oxidation levels of the oil might indicate different regulation mechanisms. Highly oxidized camelina oil and a low concentration of HHE, over time, induced SOD and catalase enzyme activity in HT29 cells. Oxidized camelina oil contains multiple oxidation products which can be responsible for the intracellular responses observed in HT29 cells, while HHE and HNE in combination with other oxidation products induce antioxidant defence responses.

Controlled Release of Volatile Antimicrobial Compounds from Mesoporous Silica Nanocarriers for Active Food Packaging Applications.

Essential oils and their active components have been extensively reported in the literature for their efficient antimicrobial, antioxidant and antifungal properties. However, the sensitivity of these volatile compounds towards heat, oxygen and light limits their usage in real food packaging applications. The encapsulation of these compounds into inorganic nanocarriers, such as nanoclays, has been shown to prolong the release and protect the compounds from harsh processing conditions. Nevertheless, these systems have limited shelf stability, and the release is of limited control. Thus, this study presents a mesoporous silica nanocarrier with a high surface area and well-ordered protective pore structure for loading large amounts of natural active compounds (up to 500 mg/g). The presented loaded nanocarriers are shelf-stable with a very slow initial release which levels out at 50% retention of the encapsulated compounds after 2 months. By the addition of simulated drip-loss from chicken, the release of the compounds is activated and gives an antimicrobial effect, which is demonstrated on the foodborne spoilage bacteria Brochothrixthermosphacta and the potentially pathogenic bacteria Escherichia coli. When the release of the active compounds is activated, a ≥4-log reduction in the growth of B. thermosphacta and a 2-log reduction of E. coli is obtained, after only one hour of incubation. During the same one-hour incubation period the dry nanocarriers gave a negligible inhibitory effect. By using the proposed nanocarrier system, which is activated by the food product itself, increased availability of the natural antimicrobial compounds is expected, with a subsequent controlled antimicrobial effect.

Biodegradable Active Packaging as an Alternative to Conventional Packaging: A Case Study with Chicken Fillets.

Innovative active packaging has the potential to maintain the food quality and preserve the food safety for extended period. The aim of this study was to discover the effect of active films based on commercially available polylactic acid blend (PLAb) and natural active components on the shelf life and organoleptic properties of chicken fillets and to find out; to what extent they can be used as replacement to the traditional packaging materials. In this study, commercially available PLAb was compounded with citral and cinnamon oil. Active films with 300 µm thickness were then produced on a blown film extruder. The PLAb-based films were thermoformed into trays. Fresh chicken breast fillets were packed under two different gas compositions, modified atmosphere packaging of 60% CO2/40% N2, and 75% O2/25% CO2 and stored at 4 °C. The effect of active packaging materials and gas compositions on the drip loss, dry matter content, organoleptic properties, and microbial quality of the chicken fillets were studied over a storage time of 24 days. The presence of active components in the compounded films was confirmed with FTIR, in addition the release of active components in the headspace of the packaging was established with GC/MS. Additionally, gas barrier properties of the packages were studied. No negative impact on the drip loss and dry matter content was observed. The results show that PLAb-based active packaging can maintain the quality of the chicken fillets and have the potential to replace the traditional packaging materials, such as APET/PE trays.

Feasibility of on/at Line Methods to Determine Boar Taint and Boar Taint Compounds: An Overview.

Classification of carcasses at the slaughter line allows an optimisation of its processing and differentiated payment to producers. Boar taint is a quality characteristic that is evaluated in some slaughter plants. This odour and flavour is mostly present in entire males and perceived generally by sensitive consumers as unpleasant. In the present work, the methodologies currently used in slaughter plants for boar taint classification (colorimetric method and sensory quality control-human nose) and the methodologies that have the potential to be implemented on/at the slaughter line (mass spectrometry, Raman and biosensors) have been summarized. Their main characteristics are presented and an analysis of strengths, weaknesses, opportunities and threats (SWOT) has been carried out. From this, we can conclude that, apart from human nose, the technology that arises as very promising and available on the market, and that will probably become a substitute for the colorimetric method, is the tandem between the laser diode thermal desorption ion source and the mass spectrometry (LDTD-MS/MS) with automation of the sampling and sample pre-treatment, because it is able to work at the slaughter line, is fast and robust, and measures both androstenone and skatole.

Chicken fillets subjected to UV-C and pulsed UV light: Reduction of pathogenic and spoilage bacteria, and changes in sensory quality.

We have compared the efficacy of continuous ultraviolet (UV-C) (254 nm) and pulsed UV light in reducing the viability of Salmonella Enteritidis, Listeria monocytogenes, Staphylococcus aureus, enterohemorrhagic Escherichia coli, Pseudomonas spp., Brochothrix thermospacta, Carnobacterium divergens, and extended-spectrum ß-lactamase producing E. coli inoculated on chicken fillet surface. Fluences from 0.05 to 3.0 J/cm2 (10 mW/cm2, from 5 to 300 s) used for UV-C light resulted in average reductions from 1.1 to 2.8 log cfu/cm2. For pulsed UV light, fluences from 1.25 to 18.0 J/cm2 gave average reductions from 0.9 to 3.0 log cfu/cm2. A small change in the odor characterized as sunburnt and increased concentration of volatile compounds associated with burnt odor posed restrictions on the upper limit of UV treatment, however no sensory changes were observed after cooking the meat. Treatments under modified atmosphere conditions using a UV permeable top film gave similar or slightly lower bacterial reductions. PRACTICAL APPLICATIONS: Ultraviolet (UV) light may be used for decontaminating the surface of food products and reduce viability of pathogenic and spoilage bacteria. Exposure of raw chicken fillet surface to various doses of continuous UV-C or pulsed UV light proposed in the present work represent alternatives for microbiological improvement of this product. Chicken fillets can be treated in intact packages covered with UV permeable top film, thus avoiding recontamination of the meat. UV-C light treatment is a low cost strategy with low maintenance, whereas pulsed UV light involves more elaborate equipment, but treatment times are short and less space is required. Both methods can be helpful for producers to manage the safety and quality of chicken fillets.

Formation of Malondialdehyde, 4-Hydroxynonenal, and 4-Hydroxyhexenal during in Vitro Digestion of Cooked Beef, Pork, Chicken, and Salmon.

Red meat high in heme iron may promote the formation of potentially genotoxic aldehydes during lipid peroxidation in the gastrointestinal tract. In this study, the formation of malondialdehyde (MDA) equivalents measured by the thiobarbituric acid reactive substances (TBARS) method was determined during in vitro digestion of cooked red meat (beef and pork), as well as white meat (chicken) and fish (salmon), whereas analysis of 4-hydroxyhexenal (HHE) and 4-hydroxynonenal (HNE) was performed during in vitro digestion of cooked beef and salmon. Comparing products with similar fat contents indicated that the amount of unsaturated fat and not total iron content was the dominating factor influencing the formation of aldehydes. It was also shown that increasing fat content in beef products caused increasing concentrations of MDA equivalents. The highest levels, however, were found in minced beef with added fish oil high in unsaturated fat. This study indicates that when ingested alone, red meat products low in unsaturated fat and low in total fat content contribute to relatively low levels of potentially genotoxic aldehydes in the gastrointestinal tract.

Effects of environmental factors on edible oil quality of organically grown Camelina sativa.

The aim of the present study was to evaluate the potential for the production of edible oil from organically grown camelina ( Camelina sativa L. Crantz), focusing on the influence of environmental factors on nutritional quality parameters. Field experiments with precrop barley were conducted in Norway in the growing seasons 2007, 2008, and 2009. Trials were fully randomized with two levels of nitrogen (N) fertilization, 0 and 120 kg total N ha(-1), and two levels of sulfur (S) fertilization, 0 and 20 kg total S ha(-1). Weather conditions, that is, temperature and precipitation, were recorded. Additional experiments were performed in the years 2008 and 2009 to evaluate the effects of replacing precrop barley with precrop pea. Seed oil content was measured by near-infrared transmittance, and crude oil compositions of fatty acids, phytosterols, tocopherols, and phospholipids were analyzed by chromatography and mass spectrometry. Results showed significant seasonal variations in seed oil content and oil composition of fatty acids, tocopherols, phytosterols, and phospholipids that to a great extent could be explained by the variations in weather conditions. Furthermore, significant effects of N fertilization were observed. Seed oil content decreased at the highest level of N fertilization, whereas the oil concentrations of α-linolenic acid (18:3n-3), erucic acid (22:1n-9), tocopherols, and campesterol increased. Pea compared to barley as precrop also increased the 18:3n-3 content of oil. S fertilization had little impact on oil composition, but an increase in tocopherols and a decrease in brassicasterol were observed. In conclusion, organically grown camelina seems to be well suited for the production of edible oil. Variations in nutritional quality parameters were generally small, but significantly influenced by season and fertilization.

Threshold detection of boar taint chemicals using parasitic wasps.

UNLABELLED: Surgical castration has been long used to prevent consumers from experiencing taint in meat from male pigs, which is a large problem in the pig husbandry industry. Due to obvious animal welfare issues, the EU now wants an alternative for castration, suggesting an urgent need for novel methods of boar taint detection. As boar taint is only a problem when taint chemicals exceed a well-defined threshold, detection methods should be concentration-specific. The wasp, Microplitis croceipes' ability to learn and respond to particular concentrations of the boar taint compounds, skatole, androstenone, and indole was tested. Also tested was the wasps' ability to discriminate between known concentrations of indole, skatole, and androstenone in real boar fat samples at room temperature. Wasps were trained using associative learning by providing food-deprived wasps with sucrose-water in the presence of specific odor concentrations. Trained wasps' responses were tested to a range of concentrations of 3 compounds. Wasps showed unidirectional generalization of learned concentration responses, whereby the direction of concentration generalization was shown to be chemical-dependent. Through both positive (sucrose) and negative feeding experiences (water only) with varying compound concentrations, the wasps can also be conditioned to respond to concentrations exceeding a defined threshold, and they were successful in reporting low, medium, and high concentrations of indole, skatole, and androstenone in boar fat at room temperature. The need for threshold detection rather than simple detection of absence/presence applies to many food quality issues, including the detection of spoilage or pest damage in crops or stored foods. PRACTICAL APPLICATION: An inexpensive and reliable means of detecting boar tainted pork at slaughter to avoid tainted meat on the market and dissatisfied consumers.

Oxidised fish oil does not influence established markers of oxidative stress in healthy human subjects: a randomised controlled trial.

Intake of fish oil reduces the risk of CHD and CHD deaths. Marine n-3 fatty acids (FA) are susceptible to oxidation, but to our knowledge, the health effects of intake of oxidised fish oil have not previously been investigated in human subjects. The aim of the present study was to investigate markers of oxidative stress, lipid peroxidation and inflammation, and the level of plasma n-3 FA after intake of oxidised fish oil. In a double-blinded randomised controlled study, healthy subjects (aged 18-50 years, n 54) were assigned into one of three groups receiving capsules containing either 8 g/d of fish oil (1.6 g/d EPA+DHA; n 17), 8 g/d of oxidised fish oil (1.6 g/d EPA+DHA; n 18) or 8 g/d of high-oleic sunflower oil (n 19). Fasting blood and morning spot urine samples were collected at weeks 0, 3 and 7. No significant changes between the different groups were observed with regard to urinary 8-iso-PGF2α; plasma levels of 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and α-tocopherol; serum high sensitive C-reactive protein; or activity of antioxidant enzymes in erythrocytes. A significant increase in plasma level of EPA+DHA was observed in both fish oil groups, but no significant difference was observed between the fish oil groups. No changes in a variety of in vivo markers of oxidative stress, lipid peroxidation or inflammation were observed after daily intake of oxidised fish oil for 3 or 7 weeks, indicating that intake of oxidised fish oil may not have unfavourable short-term effects in healthy human subjects.

Boar taint detection using parasitoid biosensors.

The off-flavor boar taint associated with the substances skatole, androstenone, and possibly indole represents a significant problem in the pig husbandry industry. Boar taint may occur in meat from uncastrated sexually mature male pigs; consumers commonly show a strong aversion to tainted meat. Consequently, there is a need for rapid methods to sort out and remove tainted carcasses at the slaughterline. We tested the ability of wasps, Microplitis croceipes to perceive and learn the 3 boar taint compounds both individually and in combination using classical conditioning paradigms. We also established the effectiveness and reliability of boar taint odor detection when wasps were used as biosensors in a contained system called the "wasp hound" using a cohort of trained wasps. We found that the wasps are able to successfully learn indole, skatole and to also detect them when presented a 1:1:1 mixture of all 3 compounds. This was shown for both a single hand-manipulated wasp bioassay and when using the "wasp hound" detector device. In contrast, the wasps showed a weak conditioned response to androstenone at the concentration tested. The estimated gas phase concentrations that the wasps perceived during training were in the range of 10 ± 0.4 pg/s for skatole and indole, and 2 ± 0.5 pg/s for androstenone. We conclude that use of these wasps as biosensors presents a promising method for boar taint detection and discuss future training paradigms that may improve their responses to compounds such as androstenone. Practical Application: The development of a perceptive, inexpensive, and reliable means of detecting boar taint before the product is presented to sensitive consumers.

Monitoring of warmed-over flavour in pork using the electronic nose - correlation to sensory attributes and secondary lipid oxidation products.

Sensory analysis of meatballs was carried out to monitor the warmed-over flavour (WOF) development in cooked, cold-stored (at 4°C for 0, 2 and 4days) and reheated meatballs derived from M. longissimus dorsi (LD) and M. semimembranosus (SM) of pigs fed a standard diet supplemented with either 3% of rapeseed oil or 3% of palm oil. This was performed in combination with measurement of volatile compounds using a solid-state-based gas sensor array system (electronic nose) and gas chromatography/mass spectrometry together with measurement of thiobarbitoric acid reactive substances (TBARS). Subsequently, to elucidate the relations and predictability between the obtained data, the gas sensor responses were correlated with chemical (volatile and non-volatile secondary lipid oxidation products) and sensory data (flavour and odour attributes), using partial least squares regression modelling (PLSR). The TBARS, hexanal, pentanal, pentanol and nonanal all correlated to the sensory attributes associated to WOF formation. Moreover, the responses from eight of the MOS (metal oxide semiconductor) sensors within the electronic nose proved to be significantly related to WOF characteristics detected by both sensory and chemical analysis, while six of the MOSFET (metal oxide semiconductor field effect transistor) sensors were related to freshly cooked meat attributes determined by sensory analysis. The obtained results show the potential of the present gas sensor technology to monitor WOF formation in pork.

Volatile compounds suitable for rapid detection as quality indicators of cold smoked salmon (Salmo salar).

Volatile compounds in cold smoked salmon products were identified by gas chromatography to study their suitability for rapid detection as indicators to predict sensory quality evaluated by quantitative descriptive analysis. Smoked salmon odour contributed by guaiacol, boiled potato- and mushroom-like odours characteristic for fish lipid degradation and sweet odours associated with the microbial metabolites 3-methyl-butanal and 3-hydroxybutanone were the most intense odours. Other key volatiles were present in high levels but contributed less to the odours. These included furan-like compounds originating from the smoking, spoilage compounds like ethanol, 3-methyl-1-butanol, 2-butanone, and acetic acid along with oxidatively derived compounds like 1-penten-3-ol, hexanal, nonanal and decanal. Partial least square regression models based on data from storage studies of cold smoked salmon from Iceland and Norway verified that selected key volatile compounds performed better as predictors to explain variation in sensory attributes (smoked, sweet/sour rancid and off odour and flavour) than traditional chemical and microbial variables.

Association of hereditary prostate cancer gene polymorphic variants with sporadic aggressive prostate carcinoma.

BACKGROUND: ELAC2, MSR1, and RNASEL are candidate genes for hereditary prostate carcinoma (HPC). While, studies have demonstrated that single nucleotide polymorphisms (SNPs) in these genes are associated with sporadic disease as well as HPC, these results are often not replicated in follow-up studies. Given that the majority of patients studied had localized disease and up to 50% of localized prostate cancer is clinically insignificant, the inability to replicate the initial findings may reflect that some subjects had indolent tumors. Herein, we examine patients with metastatic disease to determine if an association exists between HPC SNPs and unambiguously significant prostate cancer. METHODS: We examined polymorphisms within ELAC2 (S217L, A541T, E622V), MSR1 (P275A, R293X, aIVS5-59c), and RNASEL (E265X, R462Q, D541E) in 150 European-Americans with metastatic prostate cancer and 170 prostate cancer-free controls using pyrosequencing assays. RESULTS: Only ELAC2 217L (37% cases vs. 29% controls (P=0.034)) and RNASEL 541E (61% cases vs. 53% controls (P=0.045)) were over-represented. Analysis of genotypes revealed that presence of the leucine ELAC2 allele (OR 1.54: 95% CI=0.99-2.41, SS vs. SL, LL) and homozygosity for the glutamic acid RNASEL allele (OR 1.68: 95% CI=1.04-2.70, EE vs. DE, DD) were associated with increased risk. Patients with both genotypes were of particularly high-risk (OR 2.66: 95% CI=1.36-5.19). CONCLUSIONS: These results suggest that, in a European-American population, ELAC2 217L and RNASEL 541E are associated with metastatic sporadic disease. ELAC2 and RNASEL SNP analysis may prove useful in determining which patients are at risk for developing clinically significant prostate carcinoma.

Application of an electronic nose for measurements of boar taint in entire male pigs.

An electronic nose based on ion mobility spectrometry was used for boar taint measurements of entire male pig samples varying in androstenone and skatole levels (0.09-0.88µg/g fat and 0.01-0.26µg/g fat, respectively). Sensory perceptible boar taint (especially boar odour) was found to be more related to androstenone than to skatole, whereas a rancid note was determined more related to skatole than to androstenone. Multivariate models implementing some generally prescribed cut-off limits for androstenone (0.50µg/g) and skatole (0.21µg/g) indicated that the e-nose could be used for ordering samples with respect to low and high levels of androstenone and skatole. Studying the direct relationships between e-nose data, sensory data, androstenone and skatole showed better predictivity of the chemical compounds (androstenone: r=-0.948, RMSEP=0.309; skatole: r=-0.629, RMSEP=0.069) than for single sensory descriptors (boar odour r=0.409, RMSEP=0.789). The results thus suggest that the e-nose technology based on ion mobility spectrometry as in the MGD-1 may have a potential for future rapid sorting of boars at the slaughter line. The study provides new knowledge of the applicability of ion mobility spectrometry for measuring boar taint and also confirms the challenge of measuring boar taint using chemically determined cut-off limits for a sensory perceptible phenomenon. Thus, future development should be more devoted to developing holistic approaches rather than focusing on the influence of single variables on boar taint.

Lipid oxidation in herring fillets (Clupea harengus) during ice storage measured by a commercial hybrid gas-sensor array system.

Volatile compounds released from herring fillets (Clupea harengus) during 15 days of storage on ice have been measured with a commercial hybrid gas-sensor array system. Using partial least-squares regression modeling, the sensor responses were correlated with data from chemical analyses (lipid oxidation products and antioxidants) and sensory analyses (odor). Eight of the 16 sensors proved significant in the correlation studies: 6 metal oxide semiconductor field effect transistor (MOSFET) sensors and 2 Taguchi type sensors. Correlation coefficients for chemical and sensory data ranged from 0.9 to 0.98 and from 0.49 to 0.92, respectively, with 0.92 referring to both "sharp/acrid" and "rancid" odors. Prediction errors ranged from 8 to 14% and from 11 to 25% for the chemical and sensory measures, respectively. That the prediction errors for oxidation product formation (5-9%) were close to the analytical errors of the chemical reference methods indicated close to "optimum" performance of the gas-sensor system. The sensor system predicted the storage time of the herring with a 1-day error. Results illustrate high potential of the gas-sensor technology in rapid nondestructive quality determination of ice-stored herring.