灭钉螺微生物的选育及抑螺效果观察

【目的】筛选出一种高效、持久、安全及价廉的灭螺微生物,对其进行鉴定并观察其抑螺功效。【方法】从钉螺孳生的土壤中筛选出4株灭螺活性较强的菌株(B8、B27、B36、B59),显微镜观察菌株形态和革兰氏染色均为G+杆菌。不同分离胶浓度的SDS-PAGE分析其灭螺活性成分;优势菌株经16s rRNA扩增后,PCR产物测序,序列比对,构建系统发育树鉴定其种属。【结果】灭螺结果表明,各试验组中发酵上清液各菌株间灭螺效果差异有统计学意义(χ2=21.286,P=0.002);细菌发酵液各菌株间差异也有统计学意义(χ2=17.298,P=0.008);菌体悬液各菌株间差异无统计学意义(χ2=7.579,P=0.271);此外,B59菌株的灭螺效果优于其它菌株,尤其是其发酵上清液浸泡48 h和72 h钉螺死亡率高达73.3%和96.7%。SDS-PAGE发现在B59细菌上清液中无蛋白带出现,推测其灭螺活性物质可能是其他成分;分子系统发育分析结果显示B59菌株位于Bacillus cereus (CP001746)分支上,一致性达100%。【结论】B59菌株的发酵上清液灭螺效果最好,其灭螺活性物质可能不是蛋白质,B59菌株被鉴定为Bacillus cereus。     

具杀线虫活性植物内生细菌的筛选和活性产物

【目的】植物寄生线虫是危害植物的重要病原物,为了筛选到能在植物体内稳定定殖并且对植物寄生线虫具有较高杀线虫活性的植物内生细菌生防菌。【方法】以松材线虫为靶标,用直接触杀法进行筛选。对高活性菌株采用正交实验优化发酵条件,测定发酵液杀线虫活性的稳定性,并对菌株进行鉴定。【结果】从6种植物中分离筛选出13株对松材线虫具有较高杀线虫活性的植物内生细菌菌株,这些菌株的发酵上清液对松材线虫处理24h杀线虫率均达到了100%;其中BCM2、SZ5、CCM7和DP1这4个菌株的杀线虫活性较高,发酵上清液稀释3倍处理24h杀线虫率均达到95%以上,DP1和SZ5菌株达到了100%;并发现部分菌株发酵液能使线虫虫体发生渗漏或消解。发酵条件优化后能使发酵液杀线虫效果提高4倍。4株高活性菌株产生的杀线虫物质均对蛋白酶稳定、耐热不耐酸碱且长时间保藏活性不下降。经过鉴定DP1和CCM7是枯草芽孢杆菌(Bacillus subtilis),BCM2和SZ5是蜡样芽孢杆菌(Bacillus cereus)。【结论】经济作物体内存在一定数量的能产生杀线虫活性物质的内生细菌,其中一些细菌产生的杀线虫物质具有较强的稳定性。认为杀线虫活性的植物内生细菌具有很大的生防潜力。     

植物内生真菌杀螺活性菌株筛选

分别从醉鱼草和水菖蒲中分离出99、103株内生真菌,按照WHO杀螺剂浸泡试验法观察不同内生真菌以及不同浓度发酵液杀螺效果.用河虾急性毒性试验评价其对非靶水生生物毒性.结果表明LL3026、S38、S21菌株发酵液有较高的杀螺活性,发酵液浓度为10%时,钉螺死亡率为100.0%;对河虾急性毒性较小.     

抗多杀性巴氏杆菌植物乳杆菌细菌素的生物学特性研究

目的 针对多杀性巴氏杆菌耐药性不断增强的情况,寻找替抗产品。方法 使用MRS培养基分离酸菜中的乳酸菌菌株,采用16S rRNA基因测序鉴定分离菌株。对分离菌株进行发酵培养,研究其无菌发酵上清液对酶的敏感性、热稳定性、酸碱稳定性和其抑菌谱。结果 分离得到了1株优势乳酸菌YWH-4,经过16S rRNA基因测序鉴定该菌株为植物乳杆菌。植物乳杆菌YWH-4发酵上清液对胃蛋白酶具有高敏感性,推测其发酵上清液中具有抗菌活性物质细菌素。该细菌素具有良好的热稳定性,经100℃处理2 h后仍有较强抑菌活性;具有酸碱稳定性,在pH值3.0～5.0之间保持良好抑菌活性。结论 植物乳杆菌YWH-4所产细菌素对多杀性巴氏杆菌具有良好的抗菌活性。     

枫杨(Pterocarya stenoptera)水浸液灭螺实验研究

采用枫杨的新鲜根皮、茎皮和叶的水浸液处理钉螺,并用五氯酚钠溶液处理和清水饲养钉螺实验作对照．结果表明,５％以上的枫杨各部水浸液对钉螺具有２０％～１００％的毒杀致死作用．处理时间在２４０ｈ左右时,其死螺率可达９３．５％～１００％,含２．５％的新鲜枫杨叶和２．５％的新鲜乌桕叶、池杉叶、樟叶、羊蹄全株、青蒿全株等的混浸液,处理钉螺２４０ｈ亦分别具有９６．７％、９３．３％、８０％、１００％、８６．７％的灭螺效果     

枫杨水浸液灭螺实验研究

采用枫杨的新鲜根皮、茎皮和叶的水浸液处理螺,并用五氯酚钠溶液处理和清水饲养钉螺实验作对照,结果表明,５％以上的枫杨各部水浸液对钉螺个有２０－１００％的毒杀致死作用,处理时间在２４０ｈ左右时,其经可达９３．５－１００％,含２．５％的新鲜枫杨叶和２．５％的新鲜乌桕叶、池杉叶、樟叶、羊蹄全株、青蒿全株等的混浸液,处理钉螺２４０ｈ亦分别具有９６．７％、９３．３％、８０％、１００％、８６．７％的灭螺效果。     

樟树水浸液的灭螺效果

研究了新鲜樟树的茎皮、根皮和叶的水浸液对钉螺的杀灭效果。结果表明：通过一定时间的处理,樟树水浸液对钉螺有明显的毒杀作用。其中,0．5％以上的茎皮和根皮的水浸液对钉螺具有35％-100％的毒杀作用;处理时间在72h时,死螺率可达80％-100％。0．5％的樟树根皮水浸液浸泡72h时,死螺率为95％;1％的樟树根皮水浸液浸泡72h时,死螺率为100％。当用同种水浸液浸泡时,钉螺死亡率随水浸液浓度的增加而升高;随着浸泡时间的延长,钉螺的死亡率也升高。     

动物乳杆菌的分离鉴定及其抑菌蛋白的特性分析

通过滤纸片法从健康肉猪猪大肠、小肠中分离得到212株抗生物质产生菌, 以杯碟法复筛, 得到1株对溶壁微球菌(Micrococcus lysodeikticus)、金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、蜡状芽孢杆菌(Bacillus cereus)等革兰氏阳性菌和大肠杆菌(Escherichia coli)、鼠伤寒沙门氏菌(Salmonella typhimurium)等革兰氏阴性菌以及部分真菌如禾谷镰刀霉(Fusarium graminearum)均有强烈抑制作用的乳酸菌。经形态学、生理生化特征及16S rDNA序列同源性分析等手段鉴定该菌株为动物乳杆菌(Lactobacillus animalis)。排除酸和过氧化氢的干扰后, 该菌株的发酵上清液对指示菌仍有明显抑菌活性; 用蛋白酶处理该菌株的发酵上清液后, 抑菌活性丧失; 发酵液粗提物具有较好的热稳定性(经121°C处理20 min仍有较强抑菌活性)以及较宽的抑菌活性pH值范围(3.5~5.5), 因此初步认为该菌株产生一类具有广谱抑菌活性的类细菌素物质。     

筛选产类细菌素乳酸菌及类细菌素特性研究

从健康鸡肠道内容物及新鲜粪便中分离到21株乳酸菌,通过单层琼脂平板扩散实验,筛选出1株对藤黄微球菌(Micrococcus luteus)、金黄色葡萄球菌(Staphylococcus aureus)和枯草杆菌(Bacillus subtilis)等革兰氏阳性菌和大肠杆菌(Escherichia coli)等革兰氏阴性菌有明显抑菌活性代谢产物的乳酸菌,经细菌鉴定为乳杆菌属。排除酸和过氧化氢的干扰后,发酵液上清对指示菌仍有抑菌活性;用胰蛋白酶和蛋白酶K处理后抑菌活性明显降低,而胃蛋白酶对其活性无影响;用过氧化氢酶作用上清液,抑菌效果不变,说明过氧化氢未起作用;pH在2.0~7.0时,发酵上清液有抑菌活性;培养液粗提物经120℃处理20 min仍有部分活性,表明培养上清中有蛋白质类细菌素。     

海洋链霉菌GB-2发酵产物的抗细菌活性及性质研究

从连云港海域潮间带采集的样品中筛选得到一株产高活性抗细菌物质的链霉菌GB-2。该菌的发酵产物对蜡样芽孢杆菌AS1.1846、金黄色葡萄球菌ATCC25923及6株耐药性金黄色葡萄球菌等11株革兰氏阳性菌,大肠杆菌AS1.487、荧光假单胞菌AS1.1802等4株革兰氏阴性菌有显著拮抗作用。纸层析对抗细菌物质分析结果表明,菌株GB-2所产抗细菌物质是中性的水溶性物质,其产生与海水的存在有显著相关性。发酵液稳定性研究表明,该物质在121℃,pH1和pH12条件下抑菌活性均不变;紫外线照射也不影响其抑细菌活性。显示菌株GB-2产物在生防、食品及医药方面潜在的应用价值。     

中国灭螺技术的研究进展

本文就我国在化学药物灭螺、环境改造灭螺、利用食螺动物和靶螺竞争生物灭螺及植物灭螺的研究进展进行了综述,以期为灭螺技术的开发和应用提供依据.     

湘江长沙段洲滩钉螺防治后的种群动态

采用以系统抽样结合环境抽查的查螺方法,对湘江长沙段洲滩进行连续6年的螺情调查.2003-2008年跟踪调查了湘江长沙段24个洲滩,其中20个洲滩有钉螺分布,占洲滩总数的83.3%,4个洲滩从未查见钉螺.活螺洲滩数分别占当年调查洲滩的62.50%、66.67%、66.67%、70.83%、33.33%、37.50%;2008年与2003年相比,活螺洲滩数下降了40.00%,有螺框出现率、钉螺平均密度、活螺率分别下降了99.10%、99.74%、51.09%.经统计学处理,各洲滩有螺框出现率(Z=91.56,P<0.01)、活螺率(Z=65.13,P<0.01)不相同,但年度下降趋势明显.在20个有螺洲滩中,有感染性钉螺分布的洲滩5个,占有螺洲滩总数的25.00%;有感染性钉螺分布的洲滩逐年减少,平均密度和感染率逐年降低,2007-2008年所有洲滩均无感染性钉螺分布.湘江长沙段有螺洲滩采取的各种防治措施中,药物与环改灭螺相结合的效果远好于单纯的药物灭螺.各种环改灭螺措施中,又以抬洲降滩、翻耕垦种的灭螺效果更持久,更彻底.2007、2008年有螺洲滩数、钉螺总数急剧减少,有螺框出现率、活螺率大幅度下降还町能与水位和气候有关.湘江长沙段洲滩的螺情得到了较好的控制.     

Transplantation of Schistosoma japonicum daughter sporocysts in Oncomelania hupensis

Schistosoma japonicum daughter sporocysts obtained from infected Oncomelania hupensis hupensis were successfully transplanted to parasite-free O. hupensis hupensis. Survival and infection rates of recipient snails were 80% and 75% respectively. Intramolluscan development of transplanted daughter sporocysts in recipient snails appears to proceed in a similar manner as those reported for transplanted S. mansoni and S. haematobium in their respective snail intermediate hosts. Complete colonization of the digestive gland of recipient snails by sporocysts was observed 80 days after transplantation. Cercarial production during a 10-day observation from recipient snails was characterized by periods of high and low and irregular daily emissions. The average daily cercarial production was 150 per snail. Cercariae produced by recipient snails were infective to mice. Of those cercariae exposed to mice, approximately 30% developed to adult schistosomes. These results have definitive utility in the maintenance of S. japonicum in the laboratory.     

Malic dehydrogenase patterns in different strains of Schistosoma japonicum and subspecies of Oncomelania hupensis.

Malic dehydrogenase (MDH) of the Formosan Philippine and Indonesian Strains of Schistosoma japonicum and 3 subspecies of snails, Oncomelania hupensis formosana, O. h. quadrasi, and O. h. lindoensis were identified by disc electrophoresis with 10.5% acrylamide gel columns using tris-malic buffer. Male worm extracts of S. japonicum demonstrated 3, 2, and 2 clear MDH patterns and female worm extracts showed patterns of 2, 1 and 1 for the Formosan, Philippine and Indonesian strains of the parasite, respectively. MDH patterns were the same for all 3 subspecies of snails; one major and one minor band being found in all.     

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens

B. FONNESBECH, H. FRØKIAER, L. GRAM AND C. MOSBY JESPERSEN. 1993. Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria ( Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum ). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.     

Miracidia of Schistosoma japonicum: approach and attachment to the snail host

Schistosoma japonicum miracidia swim directed along a chemical gradient toward the snails Oncomelania hupensis and Biomphalaria glabrata, and they turn back when the concentration of attractive chemicals decreases. The host signal for this chemotactic response has a molecular weight of more than 30,000. When swimming miracidia encounter the surface of O. hupensis or agar containing O. hupensis snail-conditioned water (SCW) they perform the host-specific responses "contact with return," "repeated investigation," and "attachment," but they do not exhibit such behavior when encountering B. glabrata surface or agar containing B. glabrata SCW. Thus S. japonicum miracidia respond to different host signals when they approach snails than when they attach to snails.     

Isolation and Characterization of Bacteria from the Baltic Sea

A bacteriological examination was done on samples of water and sediment from three localities in the Baltic. The highest numbers of bacteria were recovered from areas subjected to pollution. The isolates included members of the family Enterobacteria-ceae, the genus Pseudomonas and strains of Aeromonas hydrophila, Alteromonas putrefaciens and some Gram positive bacteria. It is suggested tentatively that H2S production in the black sediments was caused by Alt. putrefaciens. None of the isolates had an absolute requirement for NaCl, although all of them were salt-tolerant to varying degrees, and most were able to grow aerobically at salinities comparable with those found in seawater. Isolates belonging to the family Enterobacteriaceae were, however, unable to grow anaerobically under comparable conditions. Freshwater strains of several genera of the family Enterobacteriaceae and of Aeromonas hydrophila and Aer. sobria displayed salt tolerance identical with that of the Baltic isolates. One strain each of Escherichia coli, Klebsiella pneumoniae and Yersinia enterocolitica survived well during three weeks at 17°C in artificial seawater lacking both carbon and nitrogen sources. These results suggest the need for a re-evaluation of the persistence of potentially pathogenic bacteria in the sea.     

Susceptibility of Oncomelania hupensis formosana recombinants and hybrids with Oncomelania hupensis nosophora to infection with Schistosoma japonicum

Chiu J.-K., Ong S.-J., Yu J.-C., Kao C.-Y. and Iuima T. 1981. Susceptibility of Oncomelania hupensis formosana recombinants and hybrids with Oncomelania hupensis nosophora to infection with Schistosoma japonicum. International Journal for Parasitology11: 391–397. Three generations of Oncomelania hupensis formosana recombinants were produced by mating the Kaohsiung race with the Ilan and Changhua races of the snails. F₂ and F₃ recombinants were produced by back-crossing F₁ and F₂ with Kaohsiung O. h. formosana. Subsequently, susceptibility of recombinants to infection with the original strain of Schistosoma japonicum, Ilan or Changhua strain, was investigated. Results indicated that susceptibility of recombinants declined steadily generation by generation. Marked decline of infectivity was observed for Kaohsiung-Ilan recombinants as compared with Kaohsiung-Changhua recombinants. For example, the overall infection rate of Kaohsiung-Ilan F₁ recombinants was 7.3 % with a 51.4 % of control snails. The same figures for F₂ were 4.2 and 52.6%, and 1 and 40.3 % for F₃. On the other hand, the overall infection rate of Kaohsiung-Changhua F₁ recombinants was 21.9% with a 46.9% of control snails; and 11.9 and 50.3 % for F₂; and 7.6 and 33.2% for F₃. The F₃ hybrids of Oncomelania hupensis nosophora from Japan and O. h. formosana from Kaohsiung and Ilan were also produced, and susceptibility with the Japanese strain of S. japonicum was studied. A highly significant decline of susceptibility was observed among hybrids (4.4%) in contrast with control snails (85.6 %).Feasibility of applying O. h. formosana in biological control of S. japonicum was discussed. One must determine in the laboratory, prior to application, which race of O. h. formosana should be used depending on the Oncomelania snails of an endemic area. For S. japonicum prevalent in Yamanashi, Japan, the Ilan race of O. h. formosana was found to be better choice than the Kaohsiung race of the snails.