Prevalence and subtypes of Influenza A Viruses in Wild Waterfowl in Norway 2006-2007

The prevalence of influenza A virus infection, and the distribution of different subtypes of the virus, were studied in 1529 ducks and 1213 gulls shot during ordinary hunting from August to December in two consecutive years, 2006 and 2007, in Norway. The study was based on molecular screening of cloacal and tracheal swabs, using a pan-influenza A RT-PCR. Samples found to be positive for influenza A virus were screened for the H5 subtype, using a H5 specific RT-PCR, and, if negative, further subtyped by a RT-PCR for the 3'-part of the hemagglutinin (HA) gene, encompassing almost the entire HA2, and the full-length of the neuraminidase (NA) gene, followed by sequencing and characterization. The highest prevalence (12.8%) of infection was found in dabbling ducks (Eurasian Wigeon, Common Teal and Mallard). Diving ducks (Common Goldeneye, Common Merganser, Red-breasted Merganser, Common Scoter, Common Eider and Tufted Duck) showed a lower prevalence (4.1%). In gulls (Common Gull, Herring Gull, Black-headed Gull, Lesser Black-headed Gull, Great Black-backed Gull and Kittiwake) the prevalence of influenza A virus was 6.1%. The infection prevalence peaked during October for ducks, and October/November for gulls. From the 16 hemagglutinin subtypes known to infect wild birds, 13 were detected in this study. Low pathogenic H5 was found in 17 dabbling ducks and one gull

Long-Term Safety of Intraperitoneal Radio Transmitter Implants in Brown Bears (Ursus arctos)

Intraperitoneal radio transmitters have been widely used in free-ranging wild mammals, but there are no long-term studies on their biocompatibility or technical stability within the abdominal cavity of animals. Possible negative health effects may bias results from ecological studies on instrumented animals and raise concerns over animal welfare issues. The aim of this study was to evaluate the long-term technical stability and pathological effects of Telonics intraperitoneal very high frequency (VHF) radio transmitters in brown bears (Ursus arctos). We instrumented 305 individual bears with intraperitoneal VHF radio transmitters during a 19-year period. We surgically removed devices that had been in bears for 1–9 years and collected transmitters from animals that died 1–13 years after implantation. We took biopsies for histopathology from tissue encapsulating implants in live bears. Retrieved transmitters underwent a technical inspection. Of the 125 transmitters removed from live bears, 66 were free-floating in the peritoneal cavity [a mean (SD) of 3.8 (1.5) years after implantation], whereas 59 were encapsulated in the greater omentum [4.0 (1.8) years after implantation]. Histopathology of biopsies of the 1–15 mm thick capsules in 33 individuals showed that it consisted of organized layers of connective tissue. In one third of the bears, the inner part of the capsule was characterized by a foreign body reaction. We inspected 68 implants that had been in bears for 3.9 (2.4) years. The batteries had short-circuited four (5.9%) of these devices. This resulted in the death of two animals 10 and 13 years after implantation. In two other bears that underwent surgery, we found the short-circuited devices to be fully encapsulated within the peritoneal cavity 5 and 6 years after implantation. A significant proportion of the other 64 inspected implants showed serious technical problems, such as corrosion of metal parts or the batteries (50%), detachment of the end cap (11.8%), and erosion (7.4%) or melting (5.9%) of the wax coating. We conclude that the wax coating of the transmitters was not biocompatible, that the technical quality of the devices was poor, and that these implants should not be used in brown bears

In silico exploration of Red Sea Bacillus genomes for natural product biosynthetic gene clusters

Background: The increasing spectrum of multidrug-resistant bacteria is a major global public health concern, necessitating discovery of novel antimicrobial agents. Here, members of the genus Bacillus are investigated as a potentially attractive source of novel antibiotics due to their broad spectrum of antimicrobial activities. We specifically focus on a computational analysis of the distinctive biosynthetic potential of Bacillus paralicheniformis strains isolated from the Red Sea, an ecosystem exposed to adverse, highly saline and hot conditions. Results: We report the complete circular and annotated genomes of two Red Sea strains, B. paralicheniformis Bac48 isolated from mangrove mud and B. paralicheniformis Bac84 isolated from microbial mat collected from Rabigh Harbor Lagoon in Saudi Arabia. Comparing the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 with nine publicly available complete genomes of B. licheniformis and three genomes of B. paralicheniformis, revealed that all of the B. paralicheniformis strains in this study are more enriched in nonribosomal peptides (NRPs). We further report the first computationally identified trans-acyltransferase (trans-AT) nonribosomal peptide synthetase/polyketide synthase (PKS/ NRPS) cluster in strains of this species. Conclusions:B. paralicheniformis species have more genes associated with biosynthesis of antimicrobial bioactive compounds than other previously characterized species of B. licheniformis, which suggests that these species are better potential sources for novel antibiotics. Moreover, the genome of the Red Sea strain B. paralicheniformis Bac48 is more enriched in modular PKS genes compared to B. licheniformis strains and other B. paralicheniformis strains. This may be linked to adaptations that strains surviving in the Red Sea underwent to survive in the relatively hot and saline ecosystems

Genotyping of <it>B. licheniformis</it> based on a novel multi-locus sequence typing (MLST) scheme

<p>Abstract</p> <p>Background</p> <p><it>Bacillus licheniformis</it> has for many years been used in the industrial production of enzymes, antibiotics and detergents. However, as a producer of dormant heat-resistant endospores <it>B. licheniformis</it> might contaminate semi-preserved foods. The aim of this study was to establish a robust and novel genotyping scheme for <it>B. licheniformis</it> in order to reveal the evolutionary history of 53 strains of this species. Furthermore, the genotyping scheme was also investigated for its use to detect food-contaminating strains.</p> <p>Results</p> <p>A multi-locus sequence typing (MLST) scheme, based on the sequence of six house-keeping genes (<it>adk, ccpA, recF, rpoB, spo0A</it> and <it>sucC</it>) of 53 <it>B. licheniformis</it> strains from different sources was established. The result of the MLST analysis supported previous findings of two different subgroups (lineages) within this species, named “A” and “B” Statistical analysis of the MLST data indicated a higher rate of recombination within group “A”. Food isolates were widely dispersed in the MLST tree and could not be distinguished from the other strains. However, the food contaminating strain <it>B. licheniformis</it> NVH1032, represented by a unique sequence type (ST8), was distantly related to all other strains.</p> <p>Conclusions</p> <p>In this study, a novel and robust genotyping scheme for <it>B. licheniformis</it> was established, separating the species into two subgroups. This scheme could be used for further studies of evolution and population genetics in <it>B. licheniformis.</it></p

Cloning and analysis of Polyhydroxyallkanoate synthase gene (phaC) from the extreme haloarchaea-Haloarcula sp. strain HLR2

微生物生長於營養不平衡的狀態下會將體內多餘的碳源以聚羥基烷酯(Polyhydroxyalkanoate, PHA)的方式於體內累積。所累積的PHA會在生物渡過逆境後，再降解為乙醯輔酶A (acetyl-CoA)，並經由代謝途徑提供生物生長所需的碳源及能量來源。利用全基因定序已完成的極端高鹽太古生物Haloarcula marismortui ATCC 43049被命名為PHA合成酶的基因 (poly-β-hydroxyalkanoate synthase, phaC)的ORF序列設計引子，以苗栗縣通霄鎮曬鹽場的鹽山所純化出的Haloarcula sp. strain HLR2的染色體DNA進行聚合酶連鎖反應 ﹙PCR﹚得到其phaCHLR2。phaCHLR2基因全長為1434 bp，G＋C %為61.7 %，預估等電點為3.7，可轉譯出477個胺基酸，預估分子量為53.3 kDa。phaCHLR2與Haloarcula marismortui的PHA synthase胺基酸序列有97.1 %的相似度，且其序列上具有與PHA聚合反應相關的(Cys-162)-(Asp-317)-(His-346)所組成之catalytic triad residues，因此應與真細菌型PHA synthases同歸屬於α/β-hydroxylase superfamily，證實此phaCHLR2確為PHA synthase基因。另與已知的真細菌型PHA合成酶進行胺基酸序列比對，亦發現PhaCHLR2胺基酸序列的Glu-110, Thr-166, Try-263, Val-276, Tyr-283 and Glu-284等6個可能與受質鍵結的位置。此PhaCHLR2是首次被選殖且分析的極端高鹽太古生物的PHA合成酶基因。經由系統演化分析所有已知的PHA合成酶胺基酸序列，發現所有已知太古生物的PHA合成酶獨立成一群，且和真細菌第三型PHA合成酶位於同一群集 (cluster)但不具第三型PHA合成酶特有的Cyanobacterial box。高鹽太古生物的極端酵素具耐鹽、耐高溶劑濃度、抗氧化且高溫穩定的特性，因此，適合工業與胞外 (試管)生產應用，太古生物的PHA合成酶基因的開發研究，不僅可提高phaC基因庫以供構築更多樣化的PhaC及更多新型的PHA，並可開發利用高鹽太古生物的極端酵素。Many microorganisms have been demonstrated to transform and accumulate the excess carbon source into the polyhydroxyalkanoate (PHA) granules as energy storage materials while encountering the nutrient-limiting stress. When the supply of the limiting nutrient is restored, the PHA can be depolymerized and subsequently metabolized as carbon and energy source with acetyl-CoA as intermediate. Extreme halophilic archaeon- Haloarcula strain HLR2 was isolated from the salt crystals of the salt mountain at Miao-Li, Taiwan. The unique triangle shape of strain HLR2 grew at 45 ℃ in NHB medium contained 25 % NaCl. Analysis of 16S rDNA sequence revealed that strain HLR2 was closely related to Haloarcula marismortui ATCC 43049. Based on the annoated polyhydroxyalkanoate synthase gene (phaC) of Haloarcula marismortui genome, the specific primers were designed for PCR amplification of phaCHLR2. The phaCHLR2 contains 1434 bp with G+C at 61.7 %. The total 477 amino acids could be translated with predicted molecular weight of 53,300 and the isoelectric point is 3.7. The amino acid similarity of PhaCHLR2 was 97.1 % identity with PhaC of Haloarcula marismortui. The amino acids of (Cys-162)-(Asp-317)-(His-346) was detected at PhaCHLR2 which is correspondent to the (Cys-149)-(Asp-302)-(His-331) occurred at α/β hydrolase superfamily among bacteria that was proposed as the catalytic nucleophile for PHA polymerization. Additionally, six amino acids as possible substrate binding site in bacterial PHA stynthases was also detected at PhaCHLR2 as Glu-110, Thr-166, Try-263, Val-276, Tyr-283 and Glu-284. These results confirmed that phaCHLR2 is a gene for PHA stynthase and this is the first haloarchaeal phaC gene has been cloned, sequenced and expressed in Escherichia coli. Phylogenetic analysis of all known PHA synthases indicated that all archaeal PhaC formed independent group and was clustered with eubacterial type III PHA synthase. The enzymes of extreme halophilic archaea are generally temperature, salt and solvent stable and could be a good resource of industrial enzymes. The investigation of PHA synthase gene (phaC) and protein from the extreme halophilic archaea should increase the PhaC gene library to construct more diverse PHA synthetases and produce more novel PHAs as we desired. Results of these studies will not only increase the diversity of PHAs and its application potentials but also explore the utilization of extremozyme of halophilic archaea.中文摘要 i 英文摘要 ii 目次 iii 表與圖目次 vi 壹、前言 1 貳、前人研究 4 一、 極端高鹽太古生物 6 二、 聚羥基烷酯(Polyhydroxyalkanoate, PHA) 6 (一)真細菌型PHA 6 (二)極端高鹽太古生物型PHA 8 三、 PHA生合成途徑 9 (一)真細菌型PHA生合成途徑簡介 9 (二)真細菌型PHA形成所需具備的基因 10 (三)真細菌型PHA帶有三鍵結構的合成途徑 11 四、 PHA合成酶 11 (一)α/β hydrolase superfamily 11 (二)真細菌型PHA合酶之催化機制 12 (三)真細菌型PHA合成酶受質專一性的辨識與PHA的碳鏈延長作用 13 (四)真細菌型PHA合成酶的PHA顆粒形成機制 14 (五)極端高鹽太古生物型PHA合成酶 14 五、 真細菌型PHA合成的代謝調控機制 15 (一)酵素層次的調控機制 15 (二)轉譯層次的調控機制 16 (三)PHA降解作用的調控機制 16 六、 醱酵槽生產PHA 16 (一)以醱酵槽系統生產真細菌型PHA 16 (二)以醱酵槽系統生產極端高鹽太古生物型PHA 17 七、 PHA的生物降解性 18 八、 PHA應用 19 九、 商業生產PHA 20 十、 極端高鹽太古生物Haloterrigenia thermotolerans strain H13的 PHA (hPHA)具有的優勢 21 參、材料與方法 23 一、 菌種及質體 23 二、 極端高鹽太古生物培養基組成及製備 23 三、 極端高鹽太古生物之接種、培養及保存 23 四、 大腸桿菌培養基組成及製備 25 五、 大腸桿菌之培養與保存 25 六、 PHA的生成與定量 26 七、 核酸膠體電泳分析與紀錄 27 八、 極端高鹽太古生物染色體DNA萃取 27 九、 篩選極端高鹽太古生物Haloterrigenia thermotolerans strain H13 及Haloarcula sp. strain HLR2的PHA合成酵素基因phaC 29 十、 PCR產物回收與純化 31 十一、DNA黏合反應 31 十二、勝任細胞的製備及質體的轉形作用 31 十三、質體快速篩選法 32 十四、質體的抽取與純化 32 十五、核酸序列定序及分析 33 十六、南方墨漬分析法 34 十七、核酸序列分析 37 十八、表現質體pET-21b-phaCHLR2的構築 37 十九、全細胞蛋白質之製備 38 二十、蛋白質電泳(SDS-PAGE) 38 二十一、PhaC蛋白質的大量表現與製備細胞萃取液 39 二十二、利用Ni SepharoseTM 6 Fast Flow resin回收純化PhaC蛋白質 40 二十三、西方墨漬法 (Western blotting) 41 肆、結果與討論 44 一、 生產極端高鹽太古生物Haloterrigenia thermotolerans strain H13的生物聚酯hPHA 44 二、 以聚合酶鏈鎖反應(PCR)及隱蔽型聚合酶鏈鎖反應(nested-PCR) 偵測Haloterrigenia thermotolerans strain H13的PHA synthase 合成基因phaCH13 45 三、 Haloarcula sp. strain HLR2的PHA synthase基因的選殖與序列分析 46 四、 以phaCHLR2探針以南方墨漬分析法偵測36株極端高鹽太古生物的 PHA合成酶基因 47 五、 PhaCHLR2蛋白質 48 六、 PhaC演化樹圖 49 七、 質體pET-21b-phaCHLR2選殖及誘導表現 50 伍、結論與展望 52 陸、圖與表 53 柒、參考文獻 6

Body mass, age (years) and drug doses (mg) used for anesthesia of brown bears during winter and summer.

*<p>Denotes the bears that had the best quality of anesthesia. For bears requiring several darts to be anesthetized in summer^#, the dose presented is the total dose and the induction time is not included in the mean.</p>§<p>Escaped from rock dens, darted while running.</p>•<p>Induction not observed (ran 200 meters), not included in the mean.</p>1<p>Captured in 2010.</p>2<p>Captured in 2011.</p

Physiological variables and blood gas results from seven brown bears anesthetized during winter and summer 2010 and 2011.

<p>Variables corrected to rectal temperature are marked with an *. Statistically signficant differences using a paired two-tailed t-test are denoted by;</p>a<p>Between winter and summer sample 1,</p>b<p>winter and summer sample 2.</p>c<p>winter sample 1 and 2 and d. summer sample 1 and 2.</p