养殖刺参溃疡病杀鲑气单胞菌的分离、致病性及胞外产物特性分析

从患溃疡病的养殖刺参(Apostichopus japonicus)病灶处分离出1株优势菌H1,以浸浴、创伤浸浴、体腔注射和体壁肌肉注射等方式进行感染实验,证实菌株H1为养殖刺参溃疡病病原菌,并证明该菌通过体表创伤侵入的方式感染刺参,以创伤浸浴和体壁肌肉注射感染的LD50(半数致死量)分别为2.26×107CFU/尾和1.80×107CFU/尾。经形态学观察、生理生化特性分析和mini API系统鉴定,确定菌株H1为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ma-soucida)。提取菌株H1的胞外产物(ECP)进行致病性实验,结果表明ECP可导致刺参死亡,其对刺参的LD50为5.24μg蛋白/g体质量。H1-ECP具有酪蛋白酶、明胶蛋白酶、几丁质酶和淀粉酶活性,并具有溶血素活性;对底物偶氮酪蛋白(Azocasin)作用的酶比活力可达到674.5活力单位/mg蛋白,最适作用温度为50℃;对热不稳定,70℃作用30 min时,酪蛋白酶活性降到0;100℃作用30 min,ECP对刺参的毒性消失;ECP酶活可被10 mmol/L EDTA完全抑制,可被5 mmol/L PMSF抑制98.8%,Ca2 和Mg2 可使酶活性分别提高约9%和4%。结论认为,该病原菌通过体表创伤侵入方式感染宿主刺参,菌株H1胞外产物是其对刺参致病的因子之一。     

杀鲑气单胞菌杀日本鲑亚种胞外产物毒性及免疫原性分析

用玻璃纸平板法提取了杀鲑气单胞菌杀日本鲑亚种Aeromonassal monicida masoucida的胞外产物（ECP）。毒性试验证实,ECP对剑尾鱼Xiphophorus helleri具有致死性,其半致死量（LD50）为4.72μg蛋白/g体重。SDS-PAGE表明,ECP由13条蛋白带组成。利用大鼠抗ECP血清进行的Western-blot印迹显示,组成ECP的13条蛋白带中有7条具有免疫原性,能够引发机体的免疫应答反应产生抗体,其分子量分别为88、70、42、39、36、22和15kDa。     

导电胶合板的研究

脲醛树脂中分别添加三种导电剂压制导电胶合板，可使胶层面积电阻率降至１００Ω以下。导电胶合板外加低电压时，板面温度分布的不均匀性主要来自电极处的高温区和板子边角处的低温区，小粒度的导电剂可以改善这一不均匀性，研究表明：在添加ＨＦ－２型导电粉和混加ＭＧ型稀释剂的条件下，板面温度分布均匀，中央区域的温度极差小于４℃，变异系数小于６％，并有利于改善板子的胶合强度。     

Nucleotide polymorphism in the carrot late embryogenesis abundant gene DC8/ECP63

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In several plant species, late embryogenesis abundant (LEA) genes are reported as the embryogenesis-related genes that are specifically expressed in embryonic tissues. Several LEA genes have been isolated for carrot somatic embryogenesis. The carrot LEA genes DC8 and ECP63 have very similar nucleotide sequences, and the gene pair is likely to be a nucleotide polymorphism. In several carrot cultivars, plants in which only DC8 was detected (DD genotype), only ECP63 was detected (EE genotype), and both DC8 and ECP63 were detected (DE genotype) have been observed via genomic PCR. Furthermore, distinct frequencies of the genotypes were observed in some carrot cultivars. These results suggested that detection of the nucleotide polymorphism might be useful in defining carrot cultivars. In addition, DC8 and ECP63, which include 14 amino acid insertions/deletions and 21 amino acid substitutions, may have similar effects on somatic embryogenesis, as all genotypes (DD, DE, and EE) were detected in some embryogenic cell lines. Therefore, the nucleotide polymorphism may be a useful candidate polymorphic marker that is easily detectable by PCR.     

蟹源致病性拟态弧菌的编码鉴定及其特性分析

从病蟹体内分离到 1株致病菌 (H4株菌 ) ,经细菌编码鉴定法确定为拟态弧菌 (Vibrio mimicus)。对 H4株菌及其胞外产物的生物学特性检测结果显示 :(1) H4株菌对弧菌抑制剂 O/ 12 9、多粘菌素敏感 ,能在无盐胨水中生长 ,拉丝试验阳性 ,氧化酶阳性 ,VP阳性 ,发酵葡萄糖产酸不产气。这些生物学性状符合《伯杰氏细菌鉴定手册》中对拟态弧菌的描述 ,证实了编码鉴定结果的正确性 ;(2 ) H4株菌能粘附人及多种动物红细胞 ,使红细胞发生凝集 ,且该凝集现象不能被 D-甘露糖所抑制 ,推测红细胞膜上存在的血凝素受体不含有甘露糖结构 ;(3) H4株菌的胞外产物具有致死性、溶血性和蛋白酶活性 ,表明胞外产物中至少存在溶血素和胞外蛋白酶 2种致病因子     

Virulence properties of Moritella viscosa extracellular products

Moritella viscosa is the causative agent of winter ulcer disease of marine fish. Knowledge of its pathogenicity is limited and there are no reports comparing the virulence properties of a collection of bacterial isolates. The in vivo and in vitro virulence of the extracellular products (ECP) of 22 M. viscosa isolates was screened. Two non-virulent Canadian isolates and a Norwegian isolate with reduced virulence produced non-lethal ECP. Correlation was obtained between cytotoxin and haemolysin production of M. viscosa. Isolates from salmon produced ECP with lower cytotoxic and haemolytic activities than ECP of isolates originating from other hosts. Correlation was not found between lethality of ECPs in salmon and cytotoxic or haemolytic activities. All isolates secreted esterases and a metallopeptidase (MvP1), degraded starch and produced siderophores. Variable levels of ECP protein concentration, different enzymatic activities and siderophore production could not explain differences in virulence. The results show that virulent M. viscosa isolates secrete a lethal toxic factor of unknown nature and that cytotoxin production may reflect host adaptation. Cell-culture models may not be optimal for determining the virulence of M. viscosa, as no association between cytotoxicity and bacterial virulence was obtained. Non-virulent strains may be useful in future research on M. viscosa virulence, as construction of mutants has not been successful.     

Predicted truly absorbed protein supply to dairy cattle from hulless barley (Hordeum vulgare L.) with altered carbohydrate traits with multi-year samples

Hulless barley breeding lines varying in amylose (1–20% DM) and β-glucan content (5–10% DM) have been developed at the Crop Development Centre, Canada. The objectives of this large-scale study were to 1) determine and confirm the effect of these new hulless barley lines (zero-amylose waxy, CDC Fibar; 5%-amylose waxy, CDC Rattan; normal-amylose, CDC McGwire and high-amylose, HB08302) with altered carbohydrate traits on 1) metabolic characteristics of protein; 2) intestinal digestion of various nutrients and 3) modeling nutrient supply from these barley varieties by using NRC Dairy 2001 model and DVE/OEB system. CDC Copeland was included as a hulled barley control. Among the hulless barley lines, CDC Fibar contained the highest and CDC McGwire contained the lowest total digestible protein (TDP: 147 vs. 116 g/kg DM). HB08302 was greater (P < 0.05) in intestinal digestible protein (IDP: 40.6% RUP) but relatively lower (P < 0.05) in total digestible protein (TDP: 120 g/kg DM). Compared with hulless barley, hulled barley showed relatively lower (P < 0.05) intestinal digestible protein (38 vs. 53 g/kg DM) and total digestible protein (102 vs. 129 g/kg DM). In modeling nutrient supply from the DVE/OEB system, the results showed hulled barley was lower (P < 0.01) in true protein supplied to the small intestine (TPSI: 127 vs. 142 g/kg DM), lower in truly absorbed rumen bypassed feed protein in small intestine (ABCP^DVE: 43 vs. 58 g/kg DM), lower in truly absorbed protein in the small intestine (DVE: 95 vs. 111 g/kg DM), and lower in degraded protein balance (OEB^DVE: −39 vs. −23 g/kg DM) than the hulless barley lines but greater (P < 0.01) in undigested inorganic matter (9 g/kg DM). From NRC Dairy 2001 model, CDC Fibar was greater (P < 0.05) in degraded protein balance (OEB^NRC: −30 g/kg DM) and metabolizable protein (MP: 118 g/kg DM) than the other hulless barley lines, while hulled barley was relatively lower (P < 0.01) in total metabolizable protein (MP: 83 vs. 105 g/kg DM). Our correlation results suggested that TDP was negatively correlated to amylose (r = −0.85, P < 0.001) but positively correlated to β-glucan level (r = 0.74, P < 0.001) in hulless barley cultivars. The DVE and OEB^DVE as well as MP were negatively correlated (P < 0.05) to amylose level but positively correlated to β-glucan level (P < 0.05). In conclusion, altered carbohydrate traits in the hulless barley varieties have the potential to increase intestinal nutrient availability to ruminants and significantly improved the truly absorbed protein supply to dairy cattle compared to hulled barley. Hulless barley with lower amylose and higher β-glucan level could provide greater (P < 0.05) truly digested protein in the small intestine, better synchronized available energy and N and increase metabolizable protein supply to ruminants.