镰形扇头蜱唾液腺、中肠和卵的菌群结构

采用PCR-DGGE技术分析了镰形扇头蜱雌成蜱中肠内容物、唾液腺、虫卵的菌群结构。无菌条件下采集镰形扇头蜱雌成蜱中肠内容物、唾液腺、虫卵,提取细菌总DNA;以通用引物扩增细菌16SrDNA V3区;DGGE电泳,回收、克隆、测定DGGE优势条带。结果表明,饱血和半饱血雌蜱中肠菌群结构相同,中肠内容物、唾液腺和虫卵部分细菌相同;9条明显条带的序列分别与柯克斯体属(Coxiella sp.)、假单胞菌属(Pseudomonas sp.)芽孢杆菌属(Bacillus sp.)、葡萄球菌属(Staphylococcus sp.)、涅斯捷连科菌属(Nesterenkonia)、共生菌(Symbiotic bacteria)和未培养土壤细菌的16SrDNA V3区序列高度相似。分离到2株细菌,为模仿葡萄球菌和蜡样芽孢杆菌。     

牵牛花花蜜细菌多样性研究

对牵牛花花蜜中细菌进行分离、纯化和培养,共获得29株细菌菌株,以传统方法通过形态学、培养性状以及生理生化特征对其进行鉴定,结果表明:29株细菌分别属于芽孢杆菌属(Bacillus sp.)、泛生菌属(Pantoea sp.)、柠檬酸细菌属(Citrobacter sp.)、微杆菌属(Microbacterium sp.)、假单胞菌属(Pseudomonas sp.)、土壤杆菌属(Agrobacterium sp.)、鞘氨醇单胞菌属(Sphingomonas sp.)。芽孢杆菌分离获得的几率最高。对牵牛花花蜜中细菌多样性进行研究,可为花蜜中细菌多样性研究提供基础资料,为进一步研究蜜源植物花蜜中细菌对蜜蜂体内细菌结构的影响提供一定的参考数据。     

黑广肩步甲(Calosoma maximociczi)成虫肠道细菌的分离及产脂肪酶菌株的筛选与鉴定

以柞蚕害虫黑广肩步甲(Calosoma maximociczi Morawitz)的成虫为材料,分离其肠道细菌并从中筛选产脂肪酶的菌株,了解害虫肠道细菌菌群结构,寻找具有应用价值的高产脂肪酶的微生物资源。从黑广肩步甲成虫的肠道中共分离出21个好氧细菌分离株,进一步利用选择性培养基筛选产脂肪酶的菌株并检测酶活力,获得4株产脂肪酶的优势菌株。经过生理生化特性测试和16S r DNA序列分析,确定4株产脂肪酶的菌株分别属于变形杆菌属(Proteus sp.)、沙雷氏菌属(Serratia sp.)和芽孢杆菌属(Bacillus sp.)。研究结果表明,黑广肩步甲成虫肠道内的细菌种类丰富,分离的4株产脂肪酶菌株的产酶活力较高,可进一步研究评价其利用价值。     

桑树根际固氮细菌的分离鉴定及固氮酶活力测定

利用固氮细菌可降低桑园化肥使用量和提高桑叶产量与品质。采用选择性培养基,从桑树根际分离获得24个具有固氮能力的细菌分离株,以rep-PCR基因指纹分析聚类为18个聚类群。经固氮酶活性测定,PA19、PA2和PK1菌株具有较强的固氮酶活性。利用菌落形态特征观察及16S rDNA碱基序列测定和同源性分析,对3株细菌进行鉴定的结果是:PA19菌株为中慢生根瘤菌属(Mesorhizobium sp.),PA2菌株为假单胞菌属(Pseudomonas sp.),PK1菌株为土壤杆菌属(Agrobacterium sp.)。     

猫爪草内生真菌的分离、鉴定及抑菌活性的初步研究

植物内生真菌是一类具有开发潜力的新型微生物资源。为了从药用植物猫爪草中分离有生物活性的内生真菌,试验采用常规组织块分离法分离内生真菌,形态学及分子鉴定结合进行菌株分类,圆纸片法和对峙培养法检测抑菌活性。结果表明:从栽培猫爪草的根、茎、叶中分离纯化获得23株内生真菌,形态学初步鉴定为7个属;检测到有8株内生真菌对病原细菌金黄色葡萄球菌和病原真菌核盘菌、灰葡萄孢菌都有抑制作用。同属镰刀菌(Fusarium sp.)的菌株R1、L6、L11,刺盘孢菌(Colletotrichum sp.)P2及链格孢菌(Alternaria sp.)L2菌株具有开发生物防治药物的潜力,值得进一步深入研究。     

猪生物发酵床保育期垫料中效应细菌的筛选及鉴定

本试验对猪生物发酵床保育期垫料样品中效应细菌的组成和作用进行了研究,共获得了6株效应细菌;经纯培养后观察其个体与群体形态,并进行生化特征鉴定以及16S rRNA基因分析.结果表明,所得的6株效应细菌中,芽孢杆菌属(Bacillus sp.)细菌有4株,分别为枯草芽孢杆菌(Bacillus subtilis)、嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)、蜡样芽孢杆菌(Bacillus cereus)、地衣芽孢杆菌(Bacillus licheniformis);沙雷氏菌属(Serratia sp.)有1株,为粘质沙雷氏菌(Serratia marcescens);假单胞菌属(Pseudomonas sp.)有1株,为假单胞杆菌(Pseudomonas).     

桑树内生细菌的分离及生防益菌的筛选

对不同品种健康桑树植株组织中的内生细菌进行分离,共得到229个菌株。探讨了较合理的内生细菌分离纯化方法,对内生细菌数量进行了测定。结果表明,桑树根、茎、叶柄、叶片、花蕾等组织内均存在大量的内生细菌,且不同品种及组织中内生细菌的数量均存在差异。离体抑菌作用测定表明,229株桑树内生细菌中,有42株(18.3%)菌株对桑树炭疽病菌有拮抗作用,有25株(10.9%)菌株对桑粘格孢菌有拮抗作用;以上67株菌种又有8株菌株对多种病原真菌都有抑菌作用,表现出较强抑菌活性,具有作为生防菌的应用潜能。对8株内生拮抗菌株进行了细菌学鉴定,结果表明,菌株G21、G49、Y12和J26归属于芽孢杆菌属(Bacillussp.),G82和J50归属于假单孢菌属(Pseudom onas sp.),Y33归属于欧文氏菌属(Erwinia sp.),B19归属于短小杆菌属(Curtobacterium sp.)。     

瑞香狼毒内生真菌的分离与鉴定

为探明瑞香狼毒(Stellera chamaejasme)内生真菌的种类及其种属分布特点,进一步揭示瑞香狼毒内生真菌与毒性物质合成、化感作用以及抗逆性形成之间的相互关系,作者对采自4省5个地区瑞香狼毒的茎、叶、花部位进行内生真菌的分离和纯化,通过形态学和分子生物学方法鉴定种属。结果表明,瑞香狼毒中共分离到21株内生真菌,19株菌分属7纲8目9科8属,2株未鉴定种属,所有从瑞香狼毒分离到的内生真菌中链格孢属(Alternariasp.)8株,占总分离菌株的42%,为瑞香狼毒的优势菌株,除1株粪壳菌纲(Sordariomycetes sp.)和8株链格孢属(Alternariasp.)外,其余菌属均首次从瑞香狼毒中分离得到。     

不同饱血状态微小牛蜱中肠和唾液菌群结构的分析

旨在探明微小牛蜱随吸血时间的延长,中肠和唾液微生物菌群结构的差异,以及优势菌属在中肠和唾液中增殖和迁移的特点。在无菌条件下采集半饱血、饱血微小牛蜱唾液和中肠内容物;提取细菌总DNA,PCR扩增其16SrDNA V3-V4区,Illumina MiSeq双端测序;对序列进行拼接和过滤、OTUs聚类、物种注释及多样性分析。结果显示,共得到134 317条序列,聚类后获得1 304个OTUs,半饱血雌成蜱唾液(SP)、饱血雌成蜱唾液(SF)、半饱血雌成蜱中肠内容物(MP)、饱血雌成蜱中肠内容物(MF)分别获得336、332、269、289个OTUs,其中152个OTUs为4个样本共有;4个样品在门水平上,以变形菌门和厚壁菌门为优势菌门,变形菌门在中肠中的含量大于唾液,而厚壁菌门反之;在属水平上,4个样本以柯克斯体属、不动杆菌属、甲基杆菌属、短波单胞菌属、立克次体属、埃希菌属为优势菌属,其中柯克斯体属、不动杆菌属在4个样本中含量均较大,柯克斯体属在半饱血状态下,含量高于饱血状态;不动杆菌属在唾液中的含量高于中肠。结果表明,微小牛蜱中肠和唾液中有复杂的微生物菌群结构;细菌在不同组织中的分布不同,其含量随吸血时间的延长而发生变化。     

桑树根际硅酸盐细菌的分离鉴定及解钾能力测定

利用选择性培养基,从桑树根际土样中分离获得29个具有解钾能力的细菌分离株,经rep-PCR DNA指纹分析得到24株硅酸盐细菌。通过解钾能力测定,筛选出FK2、FK3、FK8、FK11、FK4′、FK23和PK187个具有较强解钾能力的菌株,其中FK2菌株的解钾能力最强,有效态钾增长41.79%。对这7株具有较强解钾能力的细菌进行菌落形态特征观察及16S rDNA序列测定和同源性分析:FK2菌株为假单胞菌属(Pseudomonas sp.),FK3和FK23菌株为中华根瘤菌属(Sinorhizobium sp.),FK11和FK8菌株为根瘤菌属(Rhizobiumsp.),FK4′菌株为中慢生根瘤菌属(Mesorhizobium sp.),PK18菌株为屈挠杆菌属(Flexibacter sp.)。     

Theileria orientalis, a blood parasite of cattle. First report in New Zealand

A Theileria sp. piroplasm has been found in cattle from 10 Northland herds. Transmission studies, involving two splenectomized calves, led to its identification as T. orientalis, which has not been previously found in New Zealand. This piroplasm is relatively benign hut can cause severe anaemia in heavily parasitized animals. The cattle tick Haemaphysalis longicornis is considered to be the likely vector.     

Molecular cloning of two caspase-like genes from the hard tick Haemaphysalis longicornis

We identified two caspase-like genes from the midgut cDNA library of the hard tick, Haemaphysalis longicornis. Sequence analysis showed that these cDNAs encoded homologues of caspase-2 and caspase-8 that were categorized as apoptosis initiators. The H. longicornis caspase-2 (Hlcaspase-2) cDNA encodes 340 amino acid residues with a predicted molecular weight (Mw) of 38.5 kDa. Another cDNA identified as the H. longicornis caspase-8 (Hlcaspase-8) encodes 306 amino acid residues with an estimated Mw of 35.3 kDa. A catalytic active site was highly conserved in Hlcaspase-8 but not in Hlcaspase-2. RT-PCR analysis showed that both Hlcaspase-2 and Hlcaspase-8 were expressed in tick midgut and salivary glands. This is the first report of the molecular cloning of apoptosis-related genes in the tick.     

Gastritis and intestinal metaplasia in Syrian hamsters infected with Helicobacter aurati and two other microaerobes

Chronic gastritis and intestinal metaplasia associated with naturally occurring colonization by Helicobacter aurati and two other microaerobic species were observed in Syrian hamsters. Thirty-five hamsters, between 7 and 12 months of age, were evaluated from two research and three commercial facilities. Microaerobic bacteria were cultured from the hamster stomachs. These bacteria included H. aurati, a fusiform, urease-positive species; a second novel helical, urease-negative Helicobacter sp.; as well as a smaller, urease-negative Campylobacter sp. Southern blot analysis detected Helicobacter spp. DNA in the gastric tissues of all 35 hamsters; 15 hamsters also had Campylobacter sp. DNA in their gastric tissues. When examined by light microscopy, argyrophilic bacteria consistent with H. aurati or the second Helicobacter sp. were present in antral sections of 12 out of the 15 hamsters where bacteria were seen, while 9 out of the 15 hamsters had bacteria resembling the Campylobacter sp. The presence of Helicobacter spp. but not the presence of Campylobacter sp. was significantly correlated to gastritis severity (P < 0.0001 for Helicobacter spp., P = 0.6025 for Campylobacter sp.) and intestinal metaplasia, as measured by numbers of goblet cells (P = 0.0239 for Helicobacter spp., P = 0.5525 for Campylobacter sp.). Severely affected hamsters also had Giardia sp. within their metaplastic gastric pits. Hamsters with naturally occurring helicobacter-associated gastritis provide a model for studying the development of intestinal metaplasia and gastric giardiasis in H. pylori-infected humans.     

Relationship between the resistance of cattle to Haemaphysalis longicornis and to Boophilus microplus

Cattle that had been exposed to Haemaphysalis longicornis were as susceptible to Boophilus microplus as cattle that had never been exposed to either species of tick. Cattle with acquired resistance to both species ranked consistently for levels of resistance to each when infested separately. Concurrent infestation with H. longicornis had no effect on ranking for resistance to B. microplus. The coefficient of concordance between the rankings of individuals on their levels of resistance to both species of tick was positive, but was not statistically significant. We conclude that the tick antigens that stimulate host resistance are species-specific and do not cross protect. The apparent correlation in rankings for resistance to the 2 species may be a consequence of either an individual's immunological responsiveness to tick antigens or to non-specific host factors which determine levels of resistance. The apparent correlation suggests that co-selection for resistance to different tick species is practicable.     

Survey of ixodid tick species on domestic cats in Japan

Various species of ixodid ticks, attached to domestic cats in Japan, were identified in spring (April to June) and autumn (September to November). In the spring, a total of 282 ticks, including 61 larvae, 70 nymphs, 127 females and 24 males were collected from 126 cats. Of these, 264 were identified up to the species level. In the spring, Haemaphysalis longicornis was the most frequently (39.7%, 50/126) found tick species on feline hosts, followed by Ixodes ovatus (35.0%, 44/126), Ixodes nipponensis (15.9%, 20/126) and Haemaphysalis flava (9.5%, 12/126). Small numbers of Haemaphysalis megaspinosa, Haemaphysalis japonica, Ixodes persulcatus, Ixodes granulatus and Amblyomma testudinarium were also recovered. H. longicornis was the most frequently found tick species on cats around riversides or river basins, while I. ovatus and I. nipponensis were more frequently found on cats kept near woodland or related areas. I. nipponensis was more frequently found on castrated males. No major statistical differences in the frequency of tick attachment among sex, age or hair length for the three major tick species were found. Of 205 ticks including 173 (84.4%) larvae, 27 (13.2%) nymphs, 4 (2.0%) females and 1 (0.5%) male recovered from 62 cats in autumn, only 32 (15.6%) were identified. Most of the larvae were fully- or partly-engorged Haemaphysalis spp., and it was difficult to identify them further by morphological characterization.     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

Maturation of Theileria sergenti in salivary glands of Haemaphysalis longicornis induced by temperature stimulation

The parthenogenetic Haemaphysalis longicornis larvae engorged on cattle naturally infected with Theileria sergenti were reared at 24 degrees C. The resultant nymphal ticks were incubated at 37 degrees C to clear the effect of incubation on the development and maturation of sporozoites. The sporozoites in the salivary glands of the nymphal ticks exposed to 37 degrees C for 16 days were observed by the methyl green pyronin staining method. The ticks exposed to 37 degrees C were ground up in a mortar and the supernatant of the tick suspension in PBS was inoculated into cattle. The cattle showed parasitemia and specific antibody response 18 days after inoculation. Consequently, the parasites in the tick salivary glands became infective to cattle by incubating infected. H. longicornis nymphs at 37 degrees C.     

Identification of genes encoding cement-like antigens expressed in the salivary glands of Haemaphysalis longicornis

A cDNA expression library from the salivary glands of hard tick, Haemaphysalis longicornis, was constructed. Immunoscreening was performed using sera of the rabbit repeatedly infested with ticks and seventeen positive clones were obtained. A BLASTP search suggested that 8 sequences matched with that of hypothetical H. longicornis sequence and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no match to any sequence reported in the database. The proteins expected from these novel sequences possess common characteristics with cement proteins which assist ticks in their attachment to the host during blood feeding. The expression of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences showed to be upregulated upon blood feeding. These immunodominant antigens are of particular interest as candidates for future cement protein based-tick vaccine.     

Production and characterization of monoclonal antibodies against midgut of ixodid tick,Haemaphysalis longicornis

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.     

The numbers of bush ticks, Haemaphysalis longicornis, parasitic on grazing cattle before and after the acquisition of host resistance

The numbers of bush ticks, Haemaphysalis longicornis, on Bos taurus steers in south east Queensland was recorded regularly from July 1971 until January 1974. The steers had no prior exposure to the tick. The parasitic tick population was large during the first year, but declined to low levels in the second and third years, apparently as a result of the steers acquiring resistance to tick feeding.