宽体金线蛭嗜水气单胞菌感染的病原检验*

对河北某宽体金线蛭(Whitmania pigra Whitm an)养殖场所养殖的宽体金线蛭发生的病害,进行了发病情况、临床表现、病理变化等方面的检验。同时,择代表菌株进行了16S rRNA基因的分子鉴定,测定了16S rRNA基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树。结果表明所检病例为由嗜水气单胞菌(Aerom onas hydrophila)所引起的感染。分离后做纯培养的10株嗜水气单胞菌均为同种血清型菌株;代表菌株对健康宽体金线蛭的人工感染试验表明了相     

6种水蛭的COⅠ、12S rRNA和16S rRNA基因及分子进化分析

水蛭是一种常见的传统中药,为了解常见蛭类细胞色素氧化酶亚基Ⅰ（COⅠ）、12S rRNA和16S rRNA基因特征和水蛭分子系统进化关系。对常用的入药品种日本医蛭（Hirudo nipponia）、宽体金线蛭（Whitmania pigra）、尖细金线蛭（Whitmania acranulate）和相近物种菲牛蛭（Poecilobdella manillensis）、光润金线蛭（Whitmania laevis）及八目石蛭（Erpobdella octoculata）的COⅠ、12S rRNA和16S rRNA基因进行扩增、测序,利用Mega 5.0分析基因特征、颠换率、分化年代,利用PAUP＊4.10b和MrBayes 3.1.2构建分子系统树。结果表明6种水蛭的COⅠ、12S rRNA和16S rRNA基因全长分别为1534~1536 bp、709~744 bp、1129~1173 bp,GC含量分别为32.35%~34.79%、24.42%~28.49%、24.82%~27.02%,总体颠换率为0.002%~0.760%,分化年代为3.55×106a~9.85×106a;每种水蛭为单系群的支持值均≥82。说明COⅠ、12S rRNA和16S rRNA基因具有种间特异性,可用于6种水蛭的分类鉴别。     

患病大鲵中嗜水气单孢菌的分离鉴定及其防治

2009年5月,贵州省贵定县人工饲养的大鲵(娃娃鱼)发生了以四肢和腹侧的皮肤溃烂、口腔粘膜弥漫性出血以及肝脏肿大为临床病理特征的传染病。本研究对该传染病进行了病原分离、动物回归、菌株16S rRNA基因测序检测、药物敏感性、药物治疗及灭活乳化疫苗免疫保护方面的实验研究。试验结果表明,动物回归分离菌株与病原分离菌株其形态特征及理化特性一致,分离菌基因16S rRNA测序检测与嗜水气单胞菌的同源性达到99.57%以上,因此确诊该病为鱼类嗜水气单孢菌感染。致病株具有较强的耐药性,敏感药物治疗能抗菌,但皮肤溃烂组织、深部肌肉组织抗炎效果较差。经致病菌株灭活乳化免疫和免疫保护攻毒试验表明,灭活乳化疫苗免疫平均保护率达83.33%。     

抑制嗜水气单胞菌乳酸菌的筛选、鉴定及抑菌作用

从健康成年尼罗罗非鱼（Oreochromis niloticus）肠道中分离纯化得到乳酸菌,采用牛津杯双层平板法筛选具有抑制嗜水气单胞菌(Aeromonas hydrophila)活性的乳酸菌菌株,借助16S rRNA分子鉴定乳酸菌种类。通过排除有机酸和过氧化氢干扰及蛋白酶敏感性等试验分析抑菌活性最佳乳酸菌的抑菌活性物质成分,并进行热、酸碱稳定性和扫描电镜观察等确定抑菌活性物质对嗜水气单胞菌的抑菌作用。结果显示,尼罗罗非鱼肠道中有3株乳酸菌对嗜水气单胞菌表现出良好的抑菌效果,经鉴定后分别为植物乳酸菌（Lactobacillus plantarum）和唾液乳杆菌（Lactobacillus salivarius）。其中,菌株LX3的抑菌活性最佳,抑菌圈直径可达到（25.25±0.23） mm且对多种蛋白酶敏感,经不同温度和酸碱性处理后抑菌活性物质仍保持较高的抑菌活性,特别是在pH 12和100 ℃处理后,抑菌圈直径仍达到（13.87±0.12）和（16.65±0.26） mm。此外,扫描电镜观察发现菌株LX3抑菌活性物质主要破坏了嗜水气单胞菌的细胞结构,使细胞内容物流出,致其死亡。上述结果表明,尼罗罗非鱼肠道中拥有较为丰富的抑制嗜水气单胞菌乳酸菌资源,尤其是菌株LX3显示出良好的抑菌活性与抑菌作用,这对于替代抗生素防治嗜水气单胞菌引发的相关鱼类疾病具有重要价值。     

斑马鱼嗜水气单胞菌的鉴定和人工感染组织病理学研究

从患病斑马鱼体内分离致病菌株并进行鉴定,观察人工感染分离菌株后组织病理学变化。通过对分离菌株ZF4形态特征、生理生化特性、保守基因和系统发育树建立等综合分析进行鉴定。结果显示,ZF4为β溶血的革兰氏阴性短杆菌,吲哚试验为阳性,发酵葡萄糖、蔗糖等糖类,AHA_0438、ASP、gyr B和16S r RNA等基因均为阳性,基于gyr B、16S r RNA基因系统发育分析结果显示ZF4为嗜水气单胞菌。人工感染后斑马鱼临床症状明显,发生肝脏组织变性、肠道内膜脱落、脾脏充血、胰腺间质纤维组织增生等组织病理学变化。斑马鱼体内分离的ZF4鉴定为致病性嗜水气单胞菌,且斑马鱼对该菌敏感,因此斑马鱼可作为研究嗜水气单胞菌的动物模型。     

鲟源病原性嗜水气单胞菌拮抗芽孢杆菌的鉴定及其生物学特性

从养殖池污泥中分离筛选了1株优良的鲟源嗜水气单胞菌拮抗芽孢杆菌G1,其对鲟源嗜水气单胞菌S1产生的抑菌圈直径为18.50 mm。通过API50CH细菌鉴定系统以及16S rRNA序列分析法,菌株G1被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),GenBank登录号HM245965.1,其16S rRNA序列与基因库中芽孢杆菌属菌株的16S rRNA序列有99%100%的同源性,而且与解淀粉芽孢杆菌Ba-74501(GenBank登录号:DQ422953.1)的亲缘关系最近。菌株G1的最适生长pH值为7,最适生长温度为30°C,其在30°C、200 r/min条件下的生长曲线为:0 6 h为生长延迟期,6 54 h为对数生长期,54 90 h为稳定期,90 h以后为衰亡期。此外,菌株G1对其他实验选用的病原性嗜水气单胞菌也表现出良好的拮抗活性。本实验结果有利于填补嗜水气单胞菌拮抗菌在分类地位、生物学特性等方面的不足,为鲟鱼嗜水气单胞菌病的生物防控提供科学资料。     

三种金线蛭颚的形态比较研究

对金线蛭属Whitmania Blanchard,1888中光润金线蛭Whitmania laevis(Baird,1869)、宽体金线蛭Whitma-nia pigra(Whitman,1884)和尖细金线蛭Whitmania acranulata(Whitman,1886)的颚运用解剖学方法以及环境扫描电子显微镜进行研究。结果表明宽体金线蛭的颚发达,表面有两列钝的齿板,排列很规则;光润金线蛭的颚不发达,表面没有齿板;尖细金线蛭的颚不发达,表面有两列钝的齿板,排列不规则。颚的形态结构很好地区分了物种,为水蛭的分类研究提供了新的形态特征。     

锦鲤中吉氏库特菌的分离与鉴定

对从患病锦鲤(Cyprinus carpio L.)肝组织中分离的1株纯培养菌(HC050630C-1)进行了形态特征、主要理化特性、对健康鲤鱼的致病作用、药物敏感性等方面的检验;同时测定了该株菌的16S rRNA基因序列,构建了系统发育树。结果表明,被检菌为吉氏库特菌(Kurthia gibsonll),对健康鲤鱼未显示明显的致病作用;所测菌株的16S rRNA基因序列长度1459bp,在GenBank中登录号为EF611423;该菌株16SrRNA基因序列与GenBank数据库中吉氏库特菌的16S rRNA基因序列同源性在99%;药敏试验结果显示,对供试的氨苄青霉素等27种药物呈现高度敏感或敏感,对头孢吡肟等6种呈低度敏感,对苯唑青霉素等4种耐药。     

一株高毒力致病杆菌CB6的鉴定

从北京郊区果园采集的小卷蛾斯氏线虫（Steinernema carpocapsae）肠道内分离到一株具有较强杀虫和抑菌活性的致病杆菌菌株CB6。形态特征及生理生化特征测定结果表明,CB6菌株与致病杆菌属（Xenorhabdus）中的嗜线虫致病杆菌（X. nematophila）种的特征基本一致。测定了该菌株的16S rRNA序列并根据16S rRNA序列构建了系统发育树;在系统发育树中,CB6菌株与嗜线虫致病杆菌其他4个菌株形成一个类群,序列同源性大于99%。但CB6菌株的酪氨酸酶、脂酶（蛋黄）的产生、核糖产酸等生化特征与嗜线虫致病杆菌种内的其他菌株存在一定的差异,且具有更强的杀虫和抑菌活性。因此认为CB6菌株是嗜线虫致病杆菌的一个变种,命名为嗜线虫致病杆菌北京变种（X. nematophila var. pekingensis）。     

草鱼出血病混合感染的嗜水气单胞菌的分离、鉴定与理化特性

从患出血病草鱼的肝脏病灶中分离筛选出2株致病菌。取病鱼样品组织过滤液接种CIK细胞、培养, 电镜下观察到细胞质中含有草鱼呼肠孤病毒样颗粒和包涵体, 病毒颗粒大小65 nm~ 70 nm, 包涵体0.46 μm~1.81 μm。人工回归感染实验显示分离的菌株及细胞毒悬液均能使草鱼致病死亡。对分离菌株进行细胞形态学、理化特性分析及药敏试验, 初步判定所分离的2株菌均为嗜水气单胞菌。进一步对菌株进行DNA分子鉴定, 结果显示2株菌的16S rRNA基因、促旋酶亚单位蛋白(gryB)基因均与GenBank上的嗜水     

牙鲆迟钝爱德华氏菌感染症及其病原的研究

对 7起牙鲆迟钝爱德华氏菌感染病例进行了发病情况、临床特征、病理变化等方面的检验 ,经对细菌的分离与鉴定表明所检病例均为迟钝爱德华氏菌的单独感染 ,系统归纳了该感染症的主要特点。同时 ,对所分离后做纯培养的 130株迟钝爱德华氏菌进行了主要生物学性状、血清型的测定 ,表明除在生化试验的吲哚项目中表明有株间差异 (阴性的 2 0株、阳性的 110株 )外 ,130株对其他所测内容的结果一致 ,130株均为同种血清型。从每起病例分离并鉴定的各 1个代表菌株做对健康牙鲆的人工感染试验 ,表明了相应的原发病原学意义及较强的致病作用。药敏试验结果表明 ,对供试 37种抗菌药物中的头孢唑啉等 19种药物敏感、对青霉素G等 5种药物耐药、对氨苄青霉素等 13种药物表现了株间差异。经以荧光抗体技术对纯培养物、人工感染病死鱼肝脏中细菌的检验 ,初步表明了荧光抗体技术在对迟钝爱德华氏菌检验中作为辅助检验手段的可行性。     

牙鲆病原秦皇岛弧菌主要生物学性状研究

从秦皇岛某海水养殖场牙鲆(ParalichthysolivaceusL.)细菌性败血感染症的病例中,分离到了相应的病原细菌。经对12株纯培养菌进行形态特征、培养特性、理化特性等方面较系统的表观分类学指征及代表菌株DNA中G+Cmol%的测定,表明为弧菌属(VibrioPacini1854)细菌的一个新种,并定名为秦皇岛弧菌(Vibrioqinhuangdaorasp.nov.)。同时对该菌的血清型进行了检定,表明12株菌具有同种的K抗原和同种的O抗原(血清同源);另外,经以37种抗菌类药物做敏感性测定,结果显示对供试的头孢唑啉等32种药物敏感、对青霉素G等5种耐药,在不同菌株间无明显的敏感与耐药差异。       

Mutagenicity of 2-amino-N6-hydroxyadenine at the tk locus in L5178Y strains differing in repair capabilities and karyotype

The cytotoxicity and mutagenicity of 2-amino-N6-hydroxyadenine (AHA) were measured in strains of L5178Y differing in repair capabilities and karyotype. Strain LY-R83 is monosomic for chromosome 11 and is therefore hemizygous for the tk gene, while strains LY-R16 and LY-S1 are TK+/- heterozygotes. Both strain LY-R83 and LY-R16 are sensitive to UV light and are presumed to be deficient in the excision of pyrimidine dimers as shown for the parental strain, LY-R (Hagen et al., 1988; Szumiel et al., 1988). Strain LY-S1 is sensitive to the cytotoxic effects of ionizing radiation and is presumed to be defective in the repair of radiation-induced DNA double-strand breaks, as shown for the parental strain, LY-S (Evans et al., 1987a; Wlodek and Hittelman, 1987). The sensitivities of the three strains to the cytotoxic effects of AHA were similar. After a 4-hour treatment with AHA at 37 degrees C, the D37 for all three strains was approximately 35 ng/ml. The AHA-induced mutant frequency was similar for the hemizygous TK+ strain LY-R83 and the heterozygous TK +/- strain LY-R16, but was slightly higher for strain LY-S1 than for either LY-R strain at an AHA concentration of 100 ng/ml. The proportion of AHA-induced LY-S1 TK -/- mutants forming colonies with diameters less than 0.3 mm was much lower than following treatment with X radiation (24% vs. 61% for AHA and X radiation, respectively). These results indicate that the vast majority of AHA-induced TK -/- mutants harbor single gene mutations. AHA did not result in cyanide-insensitive oxygen uptake, and treatment with this compound did not induce a significant number of DNA single-strand breaks, DNA alkali labile lesions, or DNA degradation in either strain. However, two hours after AHA removal, DNA single-strand breaks and/or alkali-labile lesions, possibly due to the occurrence of DNA repair, were apparent in the DNA of both strain LY-R16 and strain LY-S1.     

Nitrogen-Fixing Bacteria from Warty Lenticellate Bark of a Mangrove Tree, Bruguiera gymnorrhiza (L.) Lamk

Detached warty lenticellate bark of a mangrove tree species, Bruguiera gymnorrhiza (L.) Lamk. from Iriomote Island, Okinawa, a subtropical region of Japan, showed development of acetylene reduction activity when incubated in a mineral nutrient solution lacking nitrogen under an atmosphere consisting of 5% O(2), 90% N(2), and 5% C(2)H(2). The bacteria responsible for nitrogen fixation were isolated from the bark, and their capacity for acetylene reduction and the incorporation of N(2) into the bacterial cells was confirmed. Four representative strains of the isolates were subjected to taxonomic classification. Two strains were similar to Enterobacter cloacae, and another resembled Enterobacter aerogenes. The characteristics of the fourth strain were similar to those of Klebsiella planticola (Bagley et al., Curr. Microbiol. 6:105-109, 1981). The results of this investigation suggest that the acetylene reduction activity of lenticellate warts of mangrove trunk bark is due to the presence in the warts of nitrogen-fixing bacteria belonging to the family Enterobacteriaceae.     

The complete mitochondrial genome sequence of Whitmania pigra (Annelida, Hirudinea): the first representative from the class Hirudinea

The mitochondrial genome is a significant tool for investigating the evolutionary history of metazoan animals. The currently available mitochondrial genome data in GenBank is limited to understand the detail evolutionary relationship among the metazoan animals, especially in the phylum Annelida. Here we present the mitochondrial gene organization, gene order and codon usage of the leech Whitmania pigra (Annelida), which is the first representative from the class Hirudinea. It is a circular molecule of 14,426bp, and encodes 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. All 37 genes of W. pigra mitochondrial genome are transcribed from the same strand, which is identical to studied annelids, two echiurans, two sipunculans and many other lophotrochozoans. Five conserved gene clusters can be found in mitochondrial genomes of nine studied annelids, including (1) cox1-N-cox2; (2) cox3-Q-nad6-cob-W-atp6; (3) H-nad5-F-E-P-T-nad4L-nad4; (4) srRNA-V-lrRNA; and (5) nad3-S(1)-nad2. Compared with that of other studied annelids, translocations of transfer RNAs were found in the gene arrangement of W. pigra mitochondrial genome. Phylogenetic analysis strongly support that the species from Hirudinina and Oligochaeta form a monophyletic group Clitellata (BPM=100, BPP=100), which is consistent with previous research based on morphological and other molecular data. Both gene order data and amino acid sequences reveal that echiurans are derived annelids and sipunculans should be clustered with annelids and echiurans.     

Assessment of the sensitivity of Neisseria meningitidis strains to benzylpenicillin using the disc diffusion method]

The sensitivity of 235 N. meningitidis strains to 5 antibiotics was estimated by the diameter of growth inhibition zones according to the criteria recommended by a Laboratory (Marseilles, France) collaborating with the WHO. All the strains proved to be sensitive to benzylpenicillin when disks containing 10 and 2 IU of the antibiotic were used. The strains were also shown to be sensitive to chloramphenicol and tetracycline. 95.7 and 7.7 per cent of the strains were sensitive to rifampicin and oleandomycin, respectively. When the strain sensitivity was assayed with the disks containing 10 and 2 IU of benzylpenicillin by the more severe criteria recommended by J. Saez-Nieto et al., significant changes were detected: meningococci with relative resistance to benzylpenicillin were detected in various regions of this country and the number of such strains was found to have a tendency to slightly increase.     

Evaluation of nitric oxide production by lactobacilli

Six strains of Lactobacillus fermentum and Lactobacillus plantarum were investigated for nitric oxide (NO) production. First, the potential presence of NO synthase was examined. None of the strains of L. fermentum and L. plantarum examined produced NO from L-arginine under aerobic conditions. Interestingly, all L. fermentum strains expressed strong L-arginine deiminase activity. All L. fermentum strains produced NO in MRS broth, but the NO was found to be chemically derived from nitrite, which was produced by L. fermentum from nitrate present in the medium. Indeed all L. fermentum strains express nitrate reductase under anaerobic conditions. Moreover, one strain, L. fermentum LF1, had nitrate reductase activity under aerobic conditions. It was also found that L. fermentum strains JCM1173 and LF1 possessed ammonifying nitrite reductase. The latter strain also had denitrifying nitrite reductase activity at neutral pH under both anaerobic and aerobic conditions. The LF1 strain is thus capable of biochemically converting nitrate to NO. NO and nitrite produced from nitrate by lactobacilli may constitute a potential antimicrobial mechanism. studied in a rat acute liver injury model (Adawi et al. 1997). The results indicate that Lactobacillus plantarum DSM 9842 may possess NOS (Adawi et al. 1997). However, NO production from L-arginine has not been investigated in pure cultures of L. plantarum. According to the results of a 15N enrichment experiment, traces of (NO2-+NO3-)-N (total oxidised nitrogen: TON), which seemed to be formed by the resting cells of Lactobacillus fermentum IFO3956, appeared to be derived from L-arginine (Morita et al. 1997). Therefore, it was suggested that L. fermentum may possess a NOS. However, NO produced from L-arginine was not directly measured and a NOS inhibitor test was not performed by Morita et al. (1997). It is known that L-arginine deiminase (ADI) in bacteria may convert L-arginine to NH4+ (Cunin et al. 1986), which may be further oxidised to TON via nitrification by bacteria. Therefore, 15N enrichment experiments could not definitely conclude that L. fermentum possess NOS to convert L-arginine directly to NO. In this study, six Lactobacillus strains belonging to L. plantarum and L. fermentum were measured for NO production in MRS broth. The metabolism of nitrate and L-arginine by the Lactobacillus cell suspensions was also studied. The possibility that NO and nitrite production by lactobacilli may be a potential probiotic trait is also discussed.     

Comparative Study on Coat Proteins of an Attenuated, a Temperature Sensitive and Their Parent Strains of Tobacco Mosaic Virus

An attenuated strain (L₁₁A) of tobacco mosaic virus (TMV) induces no remarkable symptoms on tomato plants (Goto and Nemoto 1971) and has been used to protect tomato against virulent strains of TMV (Oshima 1981), A temperature sensitive strain (Ls1) of TMV was isolated and found to have a malfunction of virus movement from cell to cell (NISHI-GUCHI et al. 1978, 1980). Those two strains are derived from a wild virulent strain (L). Coat proteins of them were compared with one another and with that of Dahlemense (D) strain of TMV, in order to see whether coat protein was associated with their respective characters. The coat proteins of the four strains behaved similar in both SDS-polyacrylamide gel and 8 M urea polyacrylamide gel electrophoresis, suggesting that they are similar in molecular weight and charging effect in the gels. There was no significant difference in chromatographic pattern of tryptic peptides among the four strains. Amino acid compositions of tryptic peptides revealed that three strains, L₁₁A, Ls1 and L, were identical to one another and that they differed from D slightly. These results suggest that coat protein is related neither to virus attenuation of L₁₁A nor to the malfunction of Ls1.