Norwegian Food Safety Authority

Oslo, Norway

Norwegian Food Safety Authority

Oslo, Norway
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News Article | May 8, 2017

In chronic-wasting disease (CWD), a cousin of mad-cow disease and already present in North America, deer brains turn spongy, causing the animal to lose weight and die. It is contagious among deer and reindeer but not known to pass from animals to humans. The disease was detected for the first time in Europe last year in Norway, with three known cases of reindeer infected in a single herd and two other cases among moose—though the latter cases were considered to be of less concern since moose do not live in herds. To prevent the spread of the disease, the Norwegian Food Safety Authority—which oversees animal health issues—called for the slaughter of the affected herd, which has between 2,000 and 2,200 wild reindeer living in the southwestern mountainous region of Nordfjella. In a letter sent Monday to the authority, the agriculture ministry gave the green light "based on the knowledge we have today and the unanimous recommendations of experts." The herd, which represents about six percent of Norway's total reindeer population, is to be eradicated by May 1, 2018, the ministry said. The Food Safety Authority has until June 15 to present an action plan. Possibilities being considered include rounding up the animals for slaughter, or extending hunting in the region. Explore further: Lightning strike kills more than 300 reindeer in Norway

News Article | May 18, 2017

The Norwegian Seafood Council has downplayed the risk of contracting anisakiasis from raw fish used in sushi, after reports hit news headlines around the world. Norwegian Seafood Council's (NSC) UK representative Jack-Robert Moller told Undercurrent News that although wild fish may carry anisakid nematodes -- the worms which cause anisakiasis -- when raw fish for sushi is prepared properly it carries no risk to consumers. "Fish in the wild contract the parasite through what they eat -- it's in the food chain," he said. He added that some fish are more prone to contracting the parasite than others. But, paraphrasing the Norwegian Food Safety Authority, he said: "It is a general requirement that fishery products intended to be consumed raw must have been frozen before consumption, if the presence of parasites in the product presents a health hazard. The objective of this treatment is to kill viable parasites." Moller noted the parasite has never been detected in farmed salmon from Norway, the world's biggest salmon producer. "The reason is that feed itself is controlled in farming operations, so you know you are feeding the fish with controlled feed. You know fish is safe to eat raw," Moller said. The US Food and Drug Administration's guidelines (available here and here) also advise the freezing method, but notes its effectiveness "depends on several factors". It notes: "The effectiveness of freezing to kill parasites depends on several factors, including the temperature of the freezing process, the length of time needed to freeze the fish tissue, the length of time the fish is held frozen, the species and source of the fish, and the type of parasite present." "The temperature of the freezing process, the length of time the fish is held frozen, and the type of parasite appear to be the most important factors. For example, tapeworms are more susceptible to freezing than are roundworms. Flukes appear to be more resistant to freezing than roundworms." FDA guidelines also support NSC's comments about farmed fish being safer: "Species that normally have a parasite hazard as a result of consuming infected prey apparently do not have the same parasite hazard when raised only on pelleted feed in an aquaculture operation. You need not consider such aquacultured fish as having a parasite hazard." However, the FDA cautions that farmed fish fed on processing waste, fresh fish, or plankton may have a parasite hazard, even when wild caught fish of that species do not normally have a parasite hazard. "Pellet fed fish that sometimes depend on wild-caught prey to supplement their diet may have a parasite hazard. In addition, fish raised in freshwater may have a parasite hazard from trematodes because these parasites enter the fish through the skin rather than in the food." It concludes: "You should verify the culture methods used by your aquaculture producers before eliminating parasites as a significant hazard." Molluscs, large non-farmed tuna (as these don't have parasites), or fish eggs also pose no threat, it states. US FDA agrees that some fish are more susceptible to acquiring parasites than others, meaning fish processors should be wary of product substitution. For instance, Spanish mackerel can be inappropriately labelled as kingfish. But while Spanish mackerel carries the threat of parasites, histamine and ciguatera fish poisoning, kingfish is associated with none of these hazards (see full table of substitution hazards below).

Guzman-Herrador B.,Norwegian Institute of Public Health | Vold L.,Norwegian Institute of Public Health | Berg T.,Norwegian Institute of Public Health | Berglund T.M.,Norwegian Food Safety Authority | And 4 more authors.
Epidemiology and Infection | Year: 2016

In 2005, the Norwegian Institute of Public Health established a web-based outbreak rapid alert system called Vesuv. The system is used for mandatory outbreak alerts from municipal medical officers, healthcare institutions, and food safety authorities. As of 2013, 1426 outbreaks have been reported, involving 32913 cases. More than half of the outbreaks occurred in healthcare institutions (759 outbreaks, 53 2%). A total of 474 (33 2%) outbreaks were associated with food or drinking water. The web-based rapid alert system has proved to be a helpful tool by enhancing reporting and enabling rapid and efficient information sharing between different authorities at both the local and national levels. It is also an important tool for event-based reporting, as required by the International Health Regulations (IHR) 2005. Collecting information from all the outbreak alerts and reports in a national database is also useful for analysing trends, such as occurrence of certain microorganisms, places or sources of infection, or route of transmission. This can facilitate the identification of specific areas where more general preventive measures are needed. © Cambridge University Press 2015.

Westrell T.,Centers for Disease Control and Prevention | Dusch V.,Ministry of Agriculture | Ethelberg S.,Statens Serum Institute | Harris J.,Public Health England | And 6 more authors.
Eurosurveillance | Year: 2010

This paper reports on several simultaneous outbreaks of norovirus infection linked to the consumption of raw oysters. Since January 2010, 334 cases in 65 clusters were reported from five European countries: the United Kingdom, Norway, France, Sweden and Denmark. The article describes the available epidemiological and microbiological evidence of these outbreaks.

News Article | September 13, 2016

The study analysed epidemiological data collected while handling LA-MRSA in Norwegian herds of pigs, from the first discovery in 2013 until 2015. In addition, the researchers performed genetic testing of bacterial isolates from all individuals identified with LA-MRSA since 2008, and they collected samples from all animals, people and pig farm environments that were affected by outbreaks in 2013 and 2014. These findings show that pig farm workers are the principal source for the introduction of LA-MRSA in Norwegian herds of swine. This transmission route was previously unknown. "This is an important discovery and herds must be monitored if they are to remain free of MRSA, particularly in countries where there is little or no import of live pigs," says Petter Elstrøm, researcher at the Norwegian Institute of Public Health. The strategy's goal has been to prevent LA-MRSA from being introduced and spread among Norwegian herds, thereby preventing pig herds from becoming a major source of MRSA dissemination to the general population. The strategy has been effective and any further transmission from animals or humans in the affected farms to the general population has not been detected. Recommendations about who should be tested for LA-MRSA before contact with livestock have been issued to prevent transmission from farm workers to pigs. "MRSA rarely causes severe infections among otherwise healthy people but a rising incidence of MRSA in the population will contribute to an increased infection burden for vulnerable patients in the health services," says Elstrøm. Since 2014, the Norwegian Food Safety Authority and the Norwegian Veterinary Institute have continuously monitored all pig herds in Norway, in close collaboration with the swine industry, and this surveillance will continue. "Our strategy of slaughtering pig herds where LA-MRSA bacteria are detected plus farm disinfection was developed in a close collaboration between the authorities and swine industry. It is an excellent example of the "One Health" approach," says Elstrøm. An ongoing study is analysing data on the effect of each control measure within the strategy, but the present article shows that the Norwegian LA-MRSA strategy has so far been a success. Norway is currently the only country that has managed to stop these bacteria from establishing among pig herds, thereby preventing further dissemination to the general population and the health service. In other countries with a low MRSA prevalence, such as Denmark and the Netherlands, the spread of LA-MRSA among pig herds contributed to a significant increase in MRSA prevalence in the population. Explore further: The pig of the future might be free of diseases that can infect people More information: Carl Andreas Grøntvedt et al, MRSA CC398 in humans and pigs in Norway: A "One Health" perspective on introduction and transmission, Clinical Infectious Diseases (2016). DOI: 10.1093/cid/ciw552

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. All mouse experiments were approved by the Animal Research Committee, the Norwegian Food Safety Authority (NFDA), and conducted in accordance with the rules and regulations of the Federation of European Laboratory Animal Science Associations (FELASA). C57BL6/CBA mice were housed in IVC SealSafe Plus Greenline cages in an Aero IVC Greenline system at SPF status. They were maintained on a 12 h light, 12 h dark cycle with ad libitum access to food and water. Four- to eight-week-old donors were injected with 5 units of pregnant mare serum gonadotropin (for oocytes and 2-cells at 14:00; for 8-cells at 15:00, 100 μl of 50 I.U. (international units) ml−1 solution) followed by 5 units of human chorionic gonadotropin (hCG) (for oocytes and 2-cells at 11:00; for 8-cells, 15:00, 100 μl of 50 I.U. ml−1 solution) 45 h (for oocytes and 2-cells) or 48 h (for 8-cells) after injection of pregnant mare serum gonadotropin. For 2-cells and 8-cells collection, females were transferred to cages with males for breeding immediately after hCG injections. Donor mice were killed by cervical dislocation 18 h after hCG injection (no mating). Oviducts were transferred to a clean dish with M2 (Sigma) medium. The ampulla was identified under a stereomicroscope, and the oocytes released followed by removal of cumulus mass by room temperature incubation in M2 containing 0.3 mg ml−1 hyaluronidase. The oocytes were further washed in M2. Donor mice were killed by cervical dislocation 45 h after hCG injection. Oviducts were transferred to a clean dish with M2 medium. Infundibulum was identified and the 2-cells were released by placing a syringe containing M2 inside the infundibulum opening, followed by flushing the M2 through the whole oviduct. The 2-cells were further washed in M2 medium. Donor mice were killed by cervical dislocation 68 h after hCG injection/mating. Oviducts were transferred to a clean dish with M2 medium. Infundibulum was identified and the 8-cells were released by placing a syringe containing M2 inside the infundibulum opening, followed flushing the M2 through the whole oviduct. The 8-cells were further washed in M2 medium. The oocytes, 2-cells and 8-cells were transferred to a 150 μl drop of Acidic Tyrode’s solution (Sigma), and further transferred to a drop of M2 immediately after the zona had been removed. 5 steps of washing in M2 were carried out, and the oocytes, 2-cells and 8-cells were ready for fixation. Immature oocytes were isolated from 12-day-old and 15-day-old prepubertal CD-1 mice (RjOrl:SWISS) as follows. Ovaries were removed with fine scissors and carefully freed from surrounding tissues with a 25G needle. Batches of five ovaries were placed in 800 μl DPBS in a 60 mm culture dish, 400 μl of Trypsin-EDTA (0.05%) (Gibco) was added immediately before fine mincing of the ovaries with a scalpel. After mincing, 5 μl of DNase I (10 U μl−1) (Sigma, 04716728001) was added and the minced ovaries were incubated at 37 °C for 20 min. Next, 20 μl of Collagenase Type II (100 mg ml−1) (Sigma, C9407), 800 μl of DBPS and 400 μl of Trypsin-EDTA (0.05%) was added and the dish was incubated for 10 min at 37 °C. Mechanical dissociation with a pipette then resulted in denuded oocytes. To remove any possible traces of somatic contaminants, and to remove the zona, oocytes were washed four times in M2 medium, incubated in two consecutive drops of M2 containing 0.3 mg ml−1 hyaluronidase, washed two times in M2 medium, then in two drops of Acidic Thyrode’s solution (Sigma) and again washed four times in M2 medium. Batches of oocytes to be analysed for DNA methylation were washed once in WGBS lysis solution (20 mM Tris-HCl, 20 mM KCl, 2 mM EDTA), transferred in a volume of maximum 5 μl to a 1.5 ml tube, snap-frozen in liquid nitrogen and stored at −80 °C before further processing. Batches of oocytes for ChIP−seq were treated as described below. Mouse embryos were immunostained using an adapted protocol from ref. 31 in 96-well plates. Briefly, embryos were subjected to thinning of the zona pellucida using acidic DPBS (pH 2.5), and fixation in 2% paraformaldehyde for 30 min. Embryos were permeabilized in 0.3% BSA, 0.1% Triton X-100, 0.02% NaN PBS solution. Blocking was carried out in 0.3% BSA, 0.01% Tween-20, and 0.02% NaN in PBS. Embryos were incubated in blocking solution with 1:200 H3K4me3 antibody (Merck Millipore, 04-745) for 60 min at room temperature. After further blocking, embryos were finally incubated with goat anti-rabbit Alex Fluor 488 (Invitrogen, A-11008) or Alexa Fluor 568 for morpholino injected embryos (Invitrogen, A-21069) at 1:200 dilution and placed on a slide in SlowFade Gold with DAPI (Invitrogen). Quantitative measurements of H3K4me3 were obtained using a Zeiss Axio Observer epi-fluorescence microscope with a Coolsnap HQ2 camera. Confocal images were obtained with a Zeiss Axio Observer LSM 710 confocal microscope. Images were processed and quantified in Axiovision and ImageJ software. Isolated zygotes were injected at 0.5 dpc (days post coitum) with either fluorescein-tagged morpholino oligonucleotides (Gene-tools) targeted at Kdm5a (5'-TGACGGCCACCAAAGCCCTCTCA-3') and Kdm5b (5'-AGCACAGGGCAGGCTCCGCAACC-3') or five base mismatch control morpholinos for Kdm5a (5'-TGAaGGaCACaAAAcCCCTaTCA-3') and Kdm5b (5'-AcCAaAGGGaAGGaTCCGaAACC-3'). Embryos were cultured in G1 plus media (Vitrolife) until late 2-cell (35 h after hCG) and fixed in 2% PFA. Embryos were further treated as described above. Lysates from 132 two-cell embryos were prepared adding lysis buffer (20 mM Tris-HCl, pH 7.4, 20% glycerol, 0.5% NP40, 1 mM MgCl , 0.150 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF, 1× PIC, 1% SDS) to a final volume of 7 μl. Embryos were lysed on ice for 30 min with occasional vortexing and spinning. Embryos were vortexed and spun down at the end and were frozen on dry ice. Samples were subsequently thawed, sonicated on ultrasound bath for 1 min and centrifuged at 16,000g for 10 min followed by transfer of 5 μl supernatant to a new tube. The simple western immunoblots were performed on a PeggySue (ProteinSimple) using the Size Separation Master Kit with Split Buffer (12–230 kDa) according to the manufacturer’s standard instruction, using the following abtibodies: anti-Kdm5A (CellSignalling, 3876), anti-Kdm5b (Abcam, 181089) and anti-β-actin (Abcam, ab8227). The Compass software (ProteinSimple, version 2.7.1) was used to program the PeggySue-robot and for presentation (and quantification) of the western Immunoblots. Output data was displayed from the software-calculated average of seven exposures (5–480 s). Human NCCIT pluripotent embryonal carcinoma cell line was obtained from ATCC (CRL-2073), and cultured according to ATCC specifications. Mouse E14 ES cells were obtained from a stock at passage P2 equal to what was used for the mouse ENCODE project, and cultured according to that specified by the mouse ENCODE project ( Cell lines were validated by ChIP–seq confirming species and a highly conserved profile. Cell lines were never passaged passed passage 15 for the work described here. Mycoplasma testing was carried out on a regular basis and both of the cell lines were free for Mycoplasma. Cross-linking of oocytes, 2-cell or 8-cell embryos. We added 50 μl M2 medium to a 0.6-ml tube. Embryos were then added and let settle to the bottom. Volume was controlled by eye by comparing to another 0.6-ml tube with 50 μl M2 medium and adjusted with mouth pipette to 50 μl. 50 μl of PBS with 2% formaldehyde was added to get a 1% final concentration and vortexed carefully, incubated at room temperature for 8 min, and vortexed once more. 12 μl of 1.25 M glycine stock (final concentration 125 mM) was added, mixed by gentle vortexing, incubated for 5 min at room temperature, and vortexed once during the incubation step. This was centrifuged at 700g for 10 min at 4 °C in a swinging-bucket rotor with soft deceleration settings and washed twice with 400 μl ice-cold PBS. A volume of 10 μl was left after the last wash, snap-frozen in liquid nitrogen and stored at −80 °C. Binding of antibodies to paramagnetic beads. The stock of paramagnetic Dynabeads Protein A was vortexed thoroughly to ensure the suspension was homogenous before pipetting. 100 μl of Dynabeads stock solution was transferred into a 1.5-ml tube, which was placed in a magnetic rack and the beads captured on the tube wall. The buffer was discarded, and the beads washed twice in 500 μl of RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate) and resuspended in RIPA buffer to a final volume of 100 μl. 96 μl of RIPA buffer was aliquoted into 200-μl PCR tubes on ice, the washed beads were vortexed thoroughly, and 2 μl of bead suspension and 2 μl of either antibody against H3K4me3 (Merck Millipore, 04-745) or to H3K27ac (Active Motif, catalogue number AM39133) was added to each of the 200 μl PCR tubes. This was then incubated at 40 r.p.m. on a ‘head-over-tail’ tube rotator for at least 4 h at 4 °C. Chromatin preparation. The desired number of cross-linked and frozen pools of embryos was removed from −80 °C storage and placed on dry ice in an insulated box (for example, four tubes with a total number of 1,000 2-cell embryos). 10 minutes of cross-linking was carried out during thawing as follows: one tube was moved at the time from dry-ice to ice for 5 s, and any frozen droplets quick pelleted by a brief spin in a mini-centrifuge. 100 μl of 1.1% formaldehyde solution was added (PBS with 1 mM EDTA, 1.1% formaldehyde, 20 mM sodium butyrate, 1 mM PMSF and protease inhibitor cocktail). The tubes were incubated for 10 min at room temperature and vortexed gently twice. 7 μl was added of 2.5 M glycine, vortexed gently and incubated for 5 min before the tube was moved to ice. Tubes were centrifuged at 750g for 10 min at 4 °C in a swinging-bucket rotor with soft deceleration settings, then washed twice with 400 μl PBS with 1 mM EDTA, 20 mM sodium butyrate, 1 mM PMSF and protease inhibitor cocktail. A volume of 10 μl was kept after the last wash. For four tubes, a total of 120 μl of 0.8% SDS lysis buffer with 20 mM sodium butyrate, 1 mM PMSF and protease inhibitor cocktail was used. First 60 μl, then 2 × 30 μl was used for two consecutive rounds of washing through the four tubes by pipetting. The same tip was used and the entire volume (160 μl) left in the last of the four tubes. The sample was sonicated for 5 × 30 s using a UP100H Ultrasonic Processor (Hielscher) fitted with a 2-mm probe. We allowed 30 s pauses on ice between each 30 s session, using pulse settings with 0.5 s cycles and 27% power. 170 μl RIPA Dilution buffer (10 mM Tris-HCl pH 8.0, 175 mM NaCl, 1 mM EDTA, 0.625 mM EGTA, 1.25% Triton X-100, 0.125% Na-deoxycholate, 20 mM sodium butyrate, 1 mM PMSF and protease inhibitor cocktail) was added. The sample was centrifuged at 12,000g in a swinging-bucket rotor for 10 min at 4 °C and the supernatant transferred to a 1.5-ml tube. 200 μl of RIPA Dilution buffer was added to the pellet and sonicated 3 × 30 s. The sample was centrifuged at 12,000g in a swinging-bucket rotor for 8 min, then the supernatant was removed and mixed well with the first supernatant, resulting in a total volume of about 530 μl of ChIP-ready chromatin. Immunoprecipitation and washes. Pre-incubated antibody–bead complexes were washed twice in 130 μl RIPA buffer by vortexing roughly. The tubes were centrifuged in a mini-centrifuge to bring down any solution trapped in the lid and antibody–bead complexes were captured in a magnetic rack cooled on ice. 250 μl of chromatin was added to each of anti H3K4me3 or H3K27ac reactions, and 25 μl kept for input control. 2 μl of cross-linked recombinant histone octameres and 1.25 μg of non-immunized rabbit IgG was immediately added to ChIP reactions, then incubated at 4 °C, 40 r.p.m. on a ‘head-over-tail’ rotator for 30 h. The chromatin–antibody–bead complexes were washed four times in 100 μl ice-cold RIPA buffer. The concentration of SDS and NaCl was titrated for each antibody to find optimal conditions for maximized signal-to-noise ratio. For H3K4me3, we washed 1× RIPA buffer with 0.2% SDS and 300 mM NaCl, 1× RIPA buffer with 0.23% SDS and 300 mM NaCl followed by 2× RIPA buffer with 0.2% SDS and 300 mM NaCl. For H3K27ac, we washed 4× RIPA buffer with 0.1% SDS and 140 mM NaCl. Each wash involved rough vortexing on full speed, repeated twice with pauses on ice in between. Next, a wash in 1 × 100 μl TE and tube shift was carried out as previously described32, 33. DNA isolation and purification. We removed TE and added 150 μl ChIP elution buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM EDTA. 1% SDS, 30 μg RNase A) and incubated at 37 °C, 1 h at 1,200 r.p.m. on a Thermomixer. 1 μl of Proteinase K (20 mg ml−1 stock) was added to each tube and incubated at 68 °C, 4 h at 1,250 r.p.m. Eluate was transferred to a 1.5-ml tube. A second elution with 150 μl was performed for 5 min and pooled with the first supernatant. ChIP DNA was purified by phenol-chloroform isoamylalcohol extraction, ethanol-precipitated with 10 μl acrylamide carrier as described previously32, 33 and dissolved in 10 μl EB (10 mM Tris-HCl). Library preparation and sequencing. ChIP and input library preparations were carried out according to the ThruPLEX (Rubicon Genomics) procedure with some modifications, including increased incubation times for the library purification and size selection. 12 ChIP libraries were pooled before AMPure XP purification and allowed to bind for 10 min after extensive mixing. Increased elution time, thorough mixing and the use of a strong neodymium bar magnet allowed for high recovery in elution volumes of 25 μl buffer EB. Sequencing procedures were carried out as described previously according to Illumina protocols with minor modifications (Illumina,). We sequenced all P12, P15, oocyte, 2-cell, and 8-cell ChIP–seq libraries as paired-end and all NCCIT ChIP–seq libraries as single-end. Mouse ES cell ChIP–seq libraries were sequenced as paired-end and single-end and the results were combined after mapping for analysis. Single-end and paired-end library information for all samples have been deposited in the GEO database (GSE72784). Sequence read alignment. We aligned single- and paired-end μChIP–seq reads from H3K27ac and H3K4me3 experiments to the mm10 reference genome by using BWA-mem34. For human ChIP–seq samples performed with human NCCIT cells, we aligned reads to the hg19 reference genome using BWA-mem. Unmapped and non-uniquely mapped reads were removed. We also removed PCR duplicate reads with Picard. H3K4me3 ChIP–seq data for heart, liver, and cerebellum were downloaded from the mouse ENCODE project26. H3K4me3 ChIP–seq data for sperm was downloaded from GEO database under accession number GSE42629 (ref. 23). Culture and collection of embryos. 2-cell stage embryos (2 × 25 embryos) were transferred to 350 μl of Buffer RLT (QIAgen RNaseay) (including β-Me according to manufacturer’s description) in a 1.5 ml low-binding tube and snap-frozen in liquid nitrogen and stored at −80 °C. Zygotes were cultured in M16 medium. For α-amanitin treatment the medium contained 10 μg ml−1 α-amanitin (Sigma, A2263). RNA extraction with QIAGEN RNeasy Micro Kit. RNAs were extracted by following QIAGEN RNeasy Micro handbook. Briefly, 25 embryos were disrupted by addition of buffer RLT followed by homogenization of the lysate. One volume of 70% EtOH was added to the lysate, transferred to an RNeasy MinElute spin column, and centrifuged for 15 s. Next, 350 μl of buffer RW1 from QIAGEN RNeasy Micro Kit was added to wash the RNeasy MinElute spin column by 15 s centrifugation. 10 μl DNase I mix was added to the RNeasy MinElute spin column membrane (10 μl DNase + 70 μl buffer RDD) and incubated at room temperature for 15 min. The spin column membrane was washed twice with 500 μl buffer RPE from the QIAGEN RNeasy Micro Kit followed by 500 μl of 80% EtOH. 14 μl RNase-free water was added directly to the centre of the spin column membrane and centrifuged to elute RNA. RNA amplification with NuGEN Ovation RNA-seq system V2. Extracted RNA was amplified by following the NuGEN ovation RNA-seq handbook. Briefly, step 1 was first-strand cDNA synthesis. 2 μl of First Strand Primer Mix from NuGEN Ovation RNA-seq system V2 was added to a PCR tube followed by addition of 5 μl of total RNA sample, and the thermal cycler for primer annealing was run (65 °C for 2 min, held at 4 °C). 3 μl of the First Strand Master Mix from NuGEN Ovation RNA-seq system V2 was added to each tube and thermal cycler for first strand synthesis was run (4 °C for 1 min; 25 °C for 10 min; 42 °C for 10 min, 70 °C for 15 min, held at 4 °C). Step 2 was second-strand cDNA synthesis. 10 μl of the second-strand mix from NuGEN Ovation RNA-seq system V2 was added to each first-strand reaction tube and the thermal cycler was run (4 °C for 1 min; 25 °C for 10 min; 50 °C for 30 min; 80 °C for 20 min; hold at 4 °C). Step 3 was purification of cDNA with Agencourt RNAClean XP beads. Step 4 was SPIA amplification. 40 μl of the SPIA master Mix from NuGEN Ovation RNA-seq system V2 was added to each tube containing the double-stranded cDNA bound to the Agencourt RNAClean XP beads, and the thermal cycler was run to amplify double-stranded cDNA (4 °C for 1 min; 47 °C for 60 min; 80 °C for 20 min, held at 4 °C). The tubes were transferred to the magnet and 40 μl of the supernatant containing the SPIA cDNA was transferred to a new tube, followed by SPIA cDNA purification with QIAGEN MinElute reaction cleanup kit. Library preparation and sequencing. The volume and concentration of purified SPIA cDNA to 500 ng in 100 μl and sonicated SPIA cDNA with Covaris M220 ultrasonicator was adjusted to 400 bp DNA fragment size. Library preparation was carried out according to TruSeq library preparation. Sequencing procedures were carried out as described previously according to Illumina HiSeq2500 protocols with minor modifications (Illumina). DNA methylation libraries of growing oocytes obtained from day 12 (P12) and day 15 (P15) mice were constructed with a modified library protocol from ref. 2. Briefly, 100-500 embryos were lysed in 5 μl lysis buffer (20 mM Tris, 2 mM EDTA, 20 mM KCl, 1 mg ml−1 proteinase K (QIAGEN)) for 1.5 h at 56 °C. followed by heat-inactivation for 30 min at 75 °C. 45 μl nuclease-free water and 0.5% Lamda DNA (Promega) spike-in was added into the lysate. DNA was fragmented with Covaris M220 ultrasonicator and incubated at 37 °C to reduce volume to 30 μl. The fragmented DNA was end-repaired by incubating with 5 μl end-repair enzyme mixture (3.5μl T4 DNA ligase buffer (NEB), 0.35 μl 10 mM dNTP, 1.15 μl NEBNext End Repair Enzyme Mix (NEB)) for 30 min at 20 °C, followed by heat-inactivation for 30 min at 75 °C. Then, 5 μl of dA-tailing mixture (0.5 μl T4 DNA ligase buffer, 1 μl Klenow exo- (NEB), 0.5 μl 100 mM dATP and 3 μl nuclease free water) was added and incubated for 30 min at 37 °C, followed by heat-inactivation for 30 min at 75 °C. Finally, 10 μl ligation mixture (1 μl T4 DNA ligase buffer, 0.5 μl 100 mM ATP, 1.5 μl 50 mM cytosine methylated Illumina adaptor, 2 μl T4 DNA ligase (NEB) and 5 μl nuclease-free water) was added and incubated at 16 °C overnight. 100 ng Carrier RNA (Ambion) was added into the tube. Bisulfite conversion reaction was performed with the EZ DNA methylation-Gold Kit (Zymo Research) according to the manufacturer’s instructions. The purified DNA was then amplified with 6 cycles PCR by using KAPA HiFi HotStart Uracil+ DNA polymerase (KAPA). Amplified DNA was purified with Ampure XP beads (Beckman) to discard the short fragments and adaptor-self ligations. Then, another round of 6–8 cycles of PCR was performed to obtain sufficient molecules for sequencing. Sequencing procedures were carried out as described previously according to Illumina HiSeq2500 protocols with minor modifications (Illumina). Reads were trimmed by Trimmomatic35 with default parameters to remove the reads containing adapters and showing low quality. Trimmed reads were aligned by using Bismark (V12.5)36 Bisulfite Mapper against the mouse reference genome mm10 with parameters: -N 1 –score_min L,0,-0.6. Duplicate reads were removed with Picard after splitting aligned reads into Watson and Crick strands. CpG methylation level was extracted with Samtools mpileup. Strands were merged to calculate the CpG methylation level per dinucleotide CpG site. Methylation level was calculated for each site spanned by at least 4 reads. During RNA-seq data analysis we used GENCODE gene annotation v3. We considered all level 1 and 2 genes and included level 3 protein-coding genes. To define gene expression levels, mouse oocytes, 2-cell, and 8-cell stage embryos RNA-seq data sets were downloaded from the GEO database with accession number GSE44183 (ref. 8). Mouse ES cell RNA-seq data were downloaded from GEO database with accession number GSE39619. RNA-seq reads were aligned to the mm10 reference genome using BWA-mem. Unmapped and non-uniquely mapped reads were removed. Gene expression values were obtained based on GENCODE annotation v3 and normalized to fragments per kilobase of transcript per million mapped (FPKM) values using Cufflinks37. Whole-genome bisulfite sequencing (WGBS) data from sperm was obtained from GEO database with accession number GSE56697 (ref. 2). Oocyte DNA methylation data was obtained from GEO database under accession number GSE56879 (ref. 19). We combined all data from 12 individual MII oocytes and the bulk oocyte sample. Deeply sequenced results were used for MII oocyte with number 2 and 5. WGBS data for GVO and NGO stage oocytes were obtained through personal communication with the authors22. We performed broad peak calling for H3Kme3 in oocytes based on MACS2 broad peak calling algorithm with default parameters (–format = BAM -g mm -m 5 50 -p 1e-5 –broad) followed by combining adjacent peaks within 5 kb. We determined the optimal distance to combine adjacent peaks on the basis of the number of broad H3K4me3 domains at varying distance thresholds. At 5 kb distance threshold, the number of broad domains became stable as shown in Extended Data Fig. 5b. On the basis of the location of transcription start sites (GENCODE v3), we classified broad H3K4me3 domains into two groups as TSS-containing and non-TSS-containing domains. The basic idea of RPKM values is to calculate relative ChIP signal enrichment for a given genomic region compared to the entire genome to normalize different sequencing depth between samples. This approach is reasonable when the total amount of ChIP DNA is similar between samples, and in general the fraction of genomic regions covered by each histone modification mark is similar between samples such as that H3K4me3 marks around 1–3% of the human genome in cells/tissues assessed to date. Therefore by using RPKM values, one can simply avoid a potential bias caused by different sequencing depth. However, if a sample shows an extraordinary ChIP signal distribution, the sample with much larger genomic regions covered with, for example, H3K4me3 tends to show relatively lower RPKM values owing to the large amount of total ChIP signal. In oocytes, we observed such notably broadly distributed H3K4me3 signals, resulting in lower RPKM values than other samples when we consider the top-ranked promoter regions in terms of H3K4me3 signal (Extended Data Fig. 5d). In this regard, to compare H3K4me3 signals fairly between samples, we need to adjust H3K4me3 RPKM values in each cell type. In order to adjust H3K4me3 RPKM values between samples, we used the top-5,000 ranked promoters in terms of H3K4me3 level as internal control regions during H3K4me3 normalization. We calculated H3K4me3-adjustment scaling factors on the basis of the H3K4me3 ChIP signals at the top 5,000 ranked promoters, with the assumption that the promoters with the highest H3K4me3 levels in each cell type represent fully H3K4me3-modified promoters and have similar H3K4me3 signal levels. In support of this assumption, all oocyte and embryo samples represent highly homogenous cell populations, thus it is plausible that most or all cells carry the H3K4me3 mark at the cell-type-specific top-ranked promoters. Furthermore, ChIP conditions were kept the same for all samples. As expected, we observed very similar H3K4me3 signals between samples exhibiting only canonical H3K4me3 patterns when we consider the same number of top-ranked promoters (Extended Data Fig. 5e). On the basis of this observation, we calculated H3K4me3 RPKM adjustment scaling factors for different numbers of the top-ranked promoters (Extended Data Fig. 5f). The scaling factors were calculated by dividing median H3K4me3 RPKM values at the top-ranked promoters in each sample by median H3K4me3 RPKM value at the top-ranked promoters in mES cells. Importantly, the scaling factors are very robust regardless of the number of promoters analysed, indicating that there is a systematic bias caused by different genomic coverage of H3K4me3. Indeed, the adjustment scaling factors are supported by the qPCR-quantified amount of ChIP DNA that is precipitated in each experiment (Supplementary Table 2). Therefore, in this study, we defined the scaling factors based on the top 5,000 most highly ranked promoters. We downloaded a list of maternally expressed genes from GEO database under accession number GSE45719 (ref. 7). We excluded all genes expressed in oocytes to allow us to distinguish maternally expressed genes in the early embryo. We considered genes with less than 0.3 FPKM values as not expressed. On the basis of the extracted genes, we tested whether maternally expressed genes are enriched within broad H3K4me3 domains. The number of genes expected by chance was calculated on the basis of the fraction of all genes located within broad H3K4me3 domains. The significance of enrichment of maternally expressed genes was calculated by Fisher-exact tests. P values were 1.9−8, 2.9−9, 1.6−4, 8.2−6, 2.8−4, 8.0−4 for zygote, early 2-cell-, mid 2-cell-, late 2-cell-, 4-cell-, and 8-cell-stage embryos, respectively. We used a predefined ZGA gene list obtained from a previous study5. The list of oocyte-specific genes (denoted as maternal RNA) was also obtained from the same study after excluding any genes showing less than 0.3 FPKM values in oocytes. Visualization and preceding analysis was done using EaSeq and its integrated tools38. Heat maps were generated using the ‘HeatMap’ tool, and superimposed tracks were generated using the ‘FillTrack’ tool. Data were imported using default settings and all values were normalized to FPKM and scaled as described above (see ‘An adjustment of H3K4me3 RPKM values’). Distances from and orientation of each TSS to the nearest domain centre were calculated using the ‘Colocalize’ tool, and the ‘Sort’ tool was used to order the TSS in the heat maps according to these distances or for ordering heat maps according to domain size. We only considered broad H3K4me3 domains that span more than 5 kbp DNA to avoid any overlapping information at domain boundaries between 5′ and 3′ends of domains. Clustering of domain boundaries was carried out by: (1) quantifying normalized and scaled H3K4me3 RPKM values for P12, P15, and oocyte samples at a set of regions corresponding to the most proximal 2 kbp within the boundary and average DNA methylation frequency for NGO, P12, P15, GVO and oocyte samples at a set of regions corresponding to the most proximal 2 kbp outside of the domain boundaries using the EaSeq (ref. 38) ‘Quantify’-tool (settings: ‘Start = Center, offset = -1000, Fixed width’, ‘End = Center, offset = 1000, Fixed width’, ‘Normalize to reads pr. million, checked’, ‘Normalize to signal size of, unchecked’, ‘Normalized counts to fragments, checked’, ‘Present values as Z-scores, unchecked’); then (2) clustering the boundaries based on this quantified signal using EaSeq’s ‘ClusterP’-tool (settings: ‘Log-Transform, unchecked’, ‘Normalize parameters to average signal’, ‘k-means clustering, checked’, ‘k = 10’, ‘g = 0’). The order of the clusters was changed manually. Distances from each boundary to nearest CGI were calculated using a set of CGIs downloaded from the UCSC table browser and ‘Colocalize’-tool. We predicted distal cis-regulatory elements on the basis of H3K27ac μChIP–seq results. We combined two biological replicates for each cell type and called H3K27ac peaks using MACS2 with the following parameters (–format = BAM -g mm -m 5 50 -p 1e-5). To directly compare the activity of distal cis-regulatory elements between cell types, we defined putative distal cis-regulatory elements by combining all H3K27ac peaks from oocytes, 2-cell and 8-cell embryos and ES cells after excluding chrY, chrM, and any peaks within 2.5 kb from known transcription start sites (GENCODE v3). The activity for each distal cis-regulatory element in each cell type was defined by taking the log ratio between H3K27ac ChIP–seq and input RPKM values. On the basis of the activity of distal cis-regulatory elements, we performed k-means clustering. 20 clusters were defined with Euclidian distance metric followed by reordering clusters manually. On the basis of the clustered patterns, we identified stage-restricted distal cis-regulatory elements. We defined nearby genes of each cRE when the distance between gene TSS and each distal cRE is less than 15 kb. Similarly, in order to define nearby distal cREs for ZGA genes, we combined all distal cREs within 15 kb from each ZGA gene TSS. We used HOMER to find enriched transcription factor motif sequences in distal cREs for each developmental stage. We also performed GREAT39 analysis for each class of stage-restricted distal cREs using the settings ‘single nearest gene’, ‘within 300 kb’ of the enriched H3K27ac region, and no curated regions. In order to identify genes with a certain transcription factors in nearby cREs, we carried out STORM40 motif search with –f –t 0.9 parameters for nearby cREs within 15 kb from each TSS. Each transcription factor motif position weight matrix was obtained from HOMER motif search41 results. The genes with a certain transcription factor in nearby cREs were called if any cREs within 15 kb from the TSS matched with the corresponding transcription factor motif sequence. We called downregulated genes between Kdm5a and Kdm5b MO injected and control MO injected 2-cell embryos when gene FPKM values were 1.5-fold or more reduced in both of the two biological replicates. Additionally, we called experimental stage specific ZGA genes when gene FPKM values were twofold or more reduced in α-amanitin treated embryos as compared to control MO injected embryos. The rational for identifying the experimental stage-specific ZGA genes comes from the observation that the composition of the transcriptome changes dramatically and rapidly during the 2-cell stage7. Although α-amanitin-treated embryos blocked polymerase II transcription from the early 1-cell stage onwards, de novo transcription-independent degradation of maternal RNA may still occur. Therefore, they provide a well-suited control for defining the experimental stage-specific ZGA genes when compared to the control morpholino-injected embryos. As a result, we identified 7,132 putative experimental stage-specific ZGA genes and these genes are significantly overlapped with the ZGA gene list obtained from a previous study5 (hypergeometric P value is 0). KDM5A- and KDM5B-depleted embryos showed that 1,303 ZGA genes are downregulated among 7,132 experimental stage-specific ZGA genes, whereas 980 non-ZGA genes are downregulated among 25,155 genes. We visualized ChIP–seq and RNA-seq data on the basis of raw read depth after converting aligned bam files to wig files using genomeCoverageBed and wigTobigWig utilities.

Lund V.,Norwegian Institute of Public Health | Anderson-Glenna M.,Norwegian Institute of Public Health | Skjevrak I.,Norwegian Food Safety Authority | Skjevrak I.,Statoil | Steffensen I.-L.,Norwegian Institute of Public Health
Journal of Water and Health | Year: 2011

The objectives of this study were to investigate migration of volatile organic compounds (VOCs) from cross-linked polyethylene (PEX) pipes used for drinking water produced by different production methods, and to evaluate their potential risk for human health and/or influence on aesthetic drinking water quality. The migration tests were carried out in accordance with EN-1420-1, and VOCs were analysed by gas chromatography-mass spectrometry. The levels of VOC migrating from new PEX pipes were generally low, and decreasing with time of pipe use. No association was found between production method of PEX pipes and concentration of migration products. 2,4-di-tert-butyl phenol and methyl tert-butyl ether (MTBE) were two of the major individual components detected. In three new PEX pipes, MTBE was detected in concentrations above the recommended US EPA taste and odour value for drinking water, but decreased below this value after 5 months in service. However, the threshold odour number (TON) values for two pipes were similar to new pipes even after 1 year in use. For seven chemicals for which conclusions on potential health risk could be drawn, this was considered of no or very low concern. However, odour from some of these pipes could negatively affect drinking water for up to 1 year. © IWA Publishing 2011.

Vatn S.,Animalia Norwegian Meat and Poultry Research Center | Hektoen L.,Animalia Norwegian Meat and Poultry Research Center | Hoyland B.,Animalia Norwegian Meat and Poultry Research Center | Reiersen A.,Norwegian Food Safety Authority | And 2 more authors.
Small Ruminant Research | Year: 2012

Norway was regarded to be free from footrot until the detection of Dichelobacter nodosus in a flock suffering from severe lameness in 2008. D. nodosus was subsequently shown to be prevalent throughout the country. However, virulent strains were only isolated from sheep in one out of 19 counties. Severe footrot has been diagnoses in a total of 97 sheep flocks. An elimination program was established, based on clinical examination, slaughter of selected animals, foot bathing with zinc sulphate, judicious use of clean pastures and ongoing clinical monitoring, with the aim of eliminating severe footrot. The elimination program has so far been carried out in 35 flocks with severe footrot and preliminary results indicate a success rate of 65-70%. The continued success of the program is important to ensure economic productivity and high standards of animal welfare. © 2012 Elsevier B.V.

Bernhoft A.,National Veterinary Institute | Clasen P.-E.,National Veterinary Institute | Kristoffersen A.B.,National Veterinary Institute | Torp M.,Norwegian Food Safety Authority
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment | Year: 2010

A total of 602 samples of cereals, consisting of organically and conventionally produced barley, oats and wheat, were collected at harvest during 2002-2004 in Norway. Organic and conventional cereals were sampled in comparable numbers regarding cereal species, localisation and harvest time, and analysed for Fusarium mould and mycotoxins. Fusarium infestation and mycotoxin content were dependent on cereal species and varied year-by-year. However, in all cereal species, Fusarium infestation and levels of important mycotoxins were significantly lower when grown organically than conventionally. Concerning the most toxic trichothecenes, HT-2 and T-2 toxin, lower concentrations were found in organic oats and barley. Wheat was not contaminated by HT-2 and T-2, but lower concentrations of deoxynivalenol (DON) and moniliformin (MON) were found when organically produced. For mycotoxins considered to constitute the main risk to humans and animals in Norwegian cereals, i.e. HT-2 in oats and DON in oats and wheat, the median figures (mean levels in brackets) were as follows: HT-2 in organic and conventional oats were <20 (80) and 62 (117) μ/kg, DON in organic and conventional oats were 24 (114) and 36 (426) μ/kg, and DON in organic and conventional wheat were 29 (86) and 51 (170) μ/kg, respectively. Concentrations of HT-2 and T-2 in the samples were strongly correlated (r=0.94). Other mycotoxins did not show a significant correlation to each other. Both HT-2 and T-2 concentrations were significantly correlated with infestation of F. langsethiae (r=0.65 and r=0.60, respectively). Concentrations of DON were significantly correlated with F. graminearum infestation (r=0.61). Furthermore, nivalenol (NIV) was significantly correlated with infestation of F. poae (r=0.55) and MON with F. avenaceum (r=0.37). As lower Fusarium infestation and mycotoxin levels were found in organic cereals, factors related to agricultural practice may reduce the risk of contamination with Fusarium mycotoxins. Studies of these issues will be presented separately. © 2010 Taylor & Francis.

MacDonald E.,Norwegian Institute of Public Health | Heier B.T.,Norwegian Institute of Public Health | Nygard K.,Norwegian Institute of Public Health | Stalheim T.,Norwegian Food Safety Authority | And 6 more authors.
Emerging Infectious Diseases | Year: 2012

In 2011, an outbreak of illness caused by Yersinia enterocolitica O:9 in Norway was linked to ready-to-eat salad mix, an unusual vehicle for this pathogen. The outbreak illustrates the need to characterize isolates of this organism, and reinforces the need for international traceback mechanisms for fresh produce.

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