浙江省某猪场暴发II型猪链球菌感染及病原诊断和药敏试验

报告 2 0 0 3年秋季浙江省某猪场II型猪链球菌感染猪群的微生物学检查和药物敏感试验结果 ,以及采取的控制疫情措施及其效果。从 112头病、死猪中分离出 71株猪链球菌疑似菌株 ,经革兰染色镜检、生化反应和特异性诊断血清的玻片凝集 ,均鉴定为II型猪链球菌。上述II型猪链球菌分离株对阿莫西林等 5种抗生素敏感 ,但对庆大霉素等 7种抗生素耐药。采取饲养环境和饲料消毒、发病猪隔离、阿莫西林和环丙沙星治疗、未发病猪疫苗接种等措施 ,有效地控制了疫情 ,未发生人的感染。

Diversity of filamentous fungi in organic layers of two forests in Zijin Mountain

A study was conducted to evaluate the cultivable filamentous fungal diversity in organic layers (L, F, and H layers) and A1 layer of two main forest types, Pinus massoniana and Liguidambar formasana mixed forest and Quercus variabilis forest, in Zijin Mountain(325?N, 11848?E), Nanjing, China. A total of 67 taxa comprising 56 Deuteromycetes, 3 Zygomycetes, 5 Asco-mycetes and 3 unidentified fungi were recognized from samples from the forest floor of the two forest types. The most abundant group was Deuteromycetes. The dominant genera in both forests were Alternaria sp., Aspergillus sp., Cladosporium sp., Mucor sp., Penicillium sp., Rhizopus sp., Gliocladium sp. and Trichoderma spp. The fungal diversity was higher in the mixed forest than that in Q. variabilis forest. For both forest types, the maximum fungal diversity was found in layer F and there existed significantly different in fungal diversity between layer F and layer L. In the mixed forest, richness of fungi isolated from needle litter (P. massoniana) was lower than that from leaf litter (L. formasana). The richness of fungi from needle litter increased with the in-crease of forest floor depth, but for leaf litter, the fungal diversity decreased with the depth of forest floor. The co-species of fungi from the two forest types, as well as from two kinds of litters in mixed forest, increased with the depth of the forest floor. The succession of fungi along with the process of decomposition was discussed here. The results also showed that litter quality was a critical factor affecting fungal diversity.

A Preliminary Study on Genetic Variation of g E Gene of an Epidemic Pseudorabies Virus Strain and Its Pathogenicity to Piglets

[Objective] This study aimed to investigate the genetic variation of g E gene of an epidemic pseudorabies virus（PRV） strain and its pathogenicity to piglets. [Method] By serial passage in Vero cells, a PRV strain was isolated from the brain tissues of stillborn fetuses delivered by sows with suspected PRV infection and preliminarily identified by PCR. g E gene of the isolated PRV strain was amplified and sequenced for phylogenetic analysis. In addition, the pathogenicity of the isolated PRV strain to 6-week-old piglets was evaluated. [Result] A PRV strain was successfully isolated and named PRV N5 B strain, which could proliferate in Vero cells and TCID50 of the 15 thgeneration virus liquid reached 10^7.125/0.1 ml. Specific bands could be amplified by PCR. g E gene in the isolated PRV strain was 1 740 bp in length. A phylogenetic tree was constructed based on full-length g E sequences, which showed that PRV N5 B strain and PRV strains isolated since 2012 were clustered into the same independent category and shared 99.7%-100% homology of nucleotide sequences. Compared with related sequences published previously, there were insertions of three consecutive bases at two loci. Animal experiments showed that intranasal inoculation of 6-week-old piglets with 2 ml of PRV N5 B strain（10^6/0.1 ml） led to a mortality rate of 100%. [Conclusion] In this study,genetic variability of g E gene in PRV N5 B isolate and its pathogenicity to piglets were analyzed, which provided a theoretical basis for the development of new vaccines to prevent and control porcine pseudorabies.

Isolation and Identification of Black Spot Pathogen in Psidium guajava

[Objective]The aim was to isolate and identify the pathogen causing black spot disease in guava（Psidium guajava）,so as to determine its taxonomic status.[Method]The fungus was identified by determining its pathogenicity,observing its morphology characteristics and analyzing its ITS sequence.[Result]The pathogen causing black spot disease in guava was a strain of Guignardia mangiferae.[Conclusion]This study will provide theoretical basis for curing black spot disease of guava.

Isolation,Identification and in vitro Antimicrobial Susceptibility of Pathogenic Aeromonas veronii from Soft-shelled Turtles

In order to effectively control diseases caused by Aeromonas veronii,pathogenic bacteria were isolated and incubated from infected soft-shelled turtles with traditional bacterial isolation method. Four strains of pathogenic bacteria were isolated and identified with traditional biochemical identification method and modern molecular biological identification techniques. According to the results, four strains of pathogenic bacteria were identified as A. veronii biovar sobria. Drug sensitivity test and in vitro antimicrobial test against Bacillus amyloliquefaciens were performed with agar diffusion method. The results showed that B. amyloliquefaciens and several antibiotics such as ofloxacin and gentamicin exerted strong antimicrobial effects on four isolates. B. amyloliquefaciens could be used for the prevention and control of diseases caused by A. veronii in aquaculture.

Isolation and Identification of Anthracnose Pathogen from Fruit of Red Fuji Apple

The fruits of Red Fuji apple with anthracnose symptoms were collected and submitted to tissue isolation and culture. One strain of anthracnose pathogen （numbered as Acgl） was obtained, and it was identified by both morphological and molecular biological methods. According to the morphological characteristics of the colony and conidia and the results of rDNA-ITS sequence analysis, the Acgl strain was identified as Colletotrichum gloeosporioides.

Screening and Isolation of Highly Efficient Cellulose-degrading Microorganisms and Construction of Complex Microbial System

This study almed to screen a highIy efficient ceI uIose-degrading compIex microbial system from soiI and rotten straw and then study its appIication to natural ceI uIose. The isoIated stralns were preIiminariIy screened with Congo red stalning and the ceI uIases activities were determined with DNS method. The non-antagonis-tic highIy efficient ceI uIos-degrading stralns were seIected and cuItured combinedIy for deveIoping a ceI uIose-degrading compIex microbial system. The resuIts showed that the CMC enzyme activity of the mixed cuIture of the three fungi stralns was higher than those of the cuItures of the three singIe stralns. The morphoIogical and moIecuIar bioIogical identification indicated that F1 was Botryosphaeria, F2 was Rhi-zopus oryzae, and F5 was Fusarium oxysporum. When straw was used as carbon source, the CMC enzyme activities of F1, F2 and F5 were 39.2, 31.4 and 40.6 IU/mI, respectiveIy; whiIe the CMC enzyme activity of the mixed cuIture of F1＋F2＋F5 was 50.12 IU/mI, which was increased by 23% compared to that of the singIe straln of F5. The experimental resuIts indicated that the ceI uIose-degrading effect of the compIex microbial system was better than those of the singIe fungi stralns, and the singIe stralns of F1, F2 and F5 had certaln deveIopment potentials.

cDNA Cloning, Expression and Characterization of Taxadiene Synthase, a Diterpene Cyclase from Taxus chinensis

Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.

Isolation and Identification of Ecliptasaponin A andEcliptasaponin B from Eclipta alba(L.)Hassk

Two new triterpenoid glucosides ecliptasaponin A(3)and ecliptasaponin B(4)were isolated together with echinocystic acid(1)and oleanolic acid(2)from Eclipta alba(L.) Hassk.Their structures were deduced as 3β,16a-dihydroxy olean-12-ene-28-oic acid-3β-O-β-D-glu-copyranoside(3)and 3β-O-[β-D-glucopyranosyl(1-4)]-β-D-glucopyranosyl-16a-hydroxy olean-12-ene-28-oic acid 28-O-β-D-glucopyranoside(4),based on spectral analysis and chemical evidences as well as results ofhydrolysis.

Isolation and Characterization of a Mitochondrial atp6 Gene from Soybean (Glycine max)

Mitochondrial ATPase (mtATPase) complex plays vital roles in higher plants. It consists of a few subunits. In the present study, a new copy of the mtATPase subunit 6 (EC 3.6.1.34) gene (atp6) was cloned and characterized from Glycine max (L.) Merr., which has the shortest opening reading frame of 223 amino acids in all organisms examined and designated as the atp6 copy3 (atp6_3). PCR amplifications of the atp6_3 from 9 soybean cultivars combined with sequencing analysis suggested its wide occurrence in G. max . RFLP analysis of a RILs population implied that paternal inheritance of the atp6_3 might occur in G. max at undetermined frequency. Under salicylic acid (SA)_treated condition, the expression of the atp6 gene was significantly inhibited. The possible role of this inhibition was discussed.

Two Bisxanthone C-glycosides from Swertia calycina Franch

首次从抱萼獐牙菜全草中分得两个双酮碳式化合物。经理化常数和光谱分析鉴定为紫药双酮甙；3－O－去甲禁药双酮甙。

Isolation,Screening and Primary Identification of a Keratin-degrading Fungus

[Objective] The paper was to provide new germplasm sources for efficient and economical degradation and utilization of animal keratin.[Method] The keratin-degrading fungus was isolated,screened and primarily identified by using the combination method of traditional isolation and screening,solid culture-medium degradation and animal test.[Result] A strain of non-pathogenic filamentous fungi with high degradation efficiency was obtained,which was preliminarily identified to be a species in Mucoraceae.[Conclusion] The discovery of the strain enriched the family members of keratin-degrading fungus,and provided new germplasm resources for degradation and utilization of animal keratin.

Isolation and Identification of Soil Actinobacteria from Mangrove forest in Beihai,Guangxi Province

[Objective] The aim was to identify the species diversity of Actinomycetes from Mangrove forest in Beihai,Guangxi Province. [Method] 10 strains of typical Actinomycetes were isolated from Mangrove forest soil,and the Actinomycetes genomic DNA was successful extracted. 16S rDNA was amplified by PCR and sequenced by Sanger dideoxy sequencing method. [Result] All the sequences were blasted in genbank,eight strains belonged to the genus of Streptomyces (80%),and two strains belonged to the genus of Nocardiopsis (20%). [Conclusion] There are many different Actinomycetes species in Mangrove forest soil samples in Beihai,Guangxi Province.

Isolation and Identification of a Taxol-producing Endophytic Fungus Identified from Taxus media

[Objective] The aim was to isolate and identify a taxol-producing endophytic fungus from Taxus media. [Method] 32 strains of endophytic fungi were identified form the inner bark of T. media,and their fermentation products were detected by high performance liquid chromatography (HPLC). [Result] Through the screening,a strain of taxol-producing endophytic fungi M57 was obtained,which could produce 45-50 μg/L of taxol,and M57 was defined as Rhizopus sp. through morphological observation and 18S rDNA sequence analysis. [Conclusion] The finding of Rhizopus sp. M57 provided a promising strain for producing taxol with taxol-producing fungi fermentation process.

Isolation of Carbendazim-degrading Bacterium XJ-D and Analysis on Its Characteristics

[Objective] The aim was to isolate the Carbendazim-degrading bacterium, so as to provide reference for the bioremediation of carbendazim contaminated soil. [Method] A carbendazim-degrading bacterium was isolated from a vineyard which has been applied with carbendazim for a long term; then the strain was identified using Biolog automatic analysis system and phylogenetic analysis based on 16S rDNA sequence. [Result] The strain XJ-D was identified as Rhodococcus erythropolis. It can use carbendazim as the sole carbon or nitrogen source, and degrade 99.0% of carbendazim at concentrations of 600 mg/L in mineral salt medium within 11 d. In addition, it showed a high average degradation rate of 52.87 mg/（L·d）. [Conclusion] The carbendazim-degrading bacterium XJ-D has a wide application prospect in bioremediation of pesticide-polluted soil.

Isolation and Characterization of Platelet-Activating Factor (PAF) Inhibitors from Zanthoxylum schinifolium

Two new compounds,schinifolin Ⅰ and acetoxyschinifolin Ⅱ,along with five known compounds(auraptenⅢ,dictamnine Ⅳ,scoparone Ⅴ,skimmianine Ⅵ,and β-sitosterol Ⅶ)were isolated from the roots of Zanthoxylum schinifolium Sieb.et Zucc.col- lected in Yixing County,Jiangsu Province.The structure determination was based upon spectroscopic analysis(UV,IR,MS,PMR,CMR,2D NMR).The structures of schinifolin Ⅰ and acetoxyschinifolin Ⅱ were elucidated as 7[(3′,7′-dimethyl-2′,6′-octadienyl)] oxy-8-methoxy-2H-1-benzopyran-2-one,and 7[(3′,7′-dimethyl-5′-acetoxy-2′, 6′-octadienyl)]oxy-8-methoxy-2H-1-benzopyran-2-one,respectively. In the test of platelet aggregation caused by PAF,compounds Ⅰ,Ⅱ,Ⅲ and Ⅴ showed inhibitory activity.

Isolation of E.tenella JL Strain Single-oocyst and PCR Identification

[Objective] In order to get a purified strain to carry out the relative molecular biology research about E.tenella. [Method] The single-oocyst isolation method was improved and the isolated single-oocyst which was put into capsule was fed to chickens. At the same time, the collected oocysts were identified by PCR method. [Result] The oocysts were isolated from feces of 15 chickens among that of 20 chickens and the infection rate was 75%. The PCR results demonstrated that the single-oocyst strain was E.tenella. [Conclusion] The inoculation of single oocyst capsule was simple, besides, this method did not only save time but also declined inoculation difficulty, increased infection rate and provided good materials for biological research of coccidian.

Isolation and Identification of Saccharomycetes in Wine Grape Region of Chateau Changyu Moser XV

Objective] This study was conducted to further explore the diversity of Saccharomyces cerevisiae_from Chateau Changyu Moser XV and realize better de-velopment and utilization of Saccharomyces cerevisiae resources. [Method] ln this study, the wine grape regions of Chateau Changyu Moser XV were taken as the research object. The Saccharomycetes_in the soil was isolated, screened and ob-served in the natural fermentation process of grape berry epidermis and the fruit. The 32 Saccharomycetes strains were preliminarily classified based on WL nutrient agar, and 26S rDNA D1/D2 sequence analysis was conducted. [Result] Total y, 4 kinds of Saccharomycetes were identified in this study, including Pichia kluyveri, Hanseniaspora uvarum, Saccharomyces cerevisiae_and Cryptococcus magnus. [Con-clusion] The main species of Saccharomycetes in the wine grape region of Chateau Changyu Moser XV were preliminarily determined, which provides theoretical basis and research basis for the brewing of wine with special characteristics.

Genetic Variation of Porcine Circovirus Type 2 Isolate 201105ZJ

[Objective] This study aimed to investigate the genetic variation of porcine circovirus type 2 （PCV2） in China. [Method] The strain was isolated from infected samples by cel passage and preliminarily identified by PCR and IFA. Ful-length genome of the isolated strain was obtained by specific amplification for homology and phylogenetic analysis. [Result] A PCV2 strain was successful y isolated and named 201105ZJ, which could proliferate in PK15 cel lines. Specific fragments could be amplified by specific PCR assay. According to results of IFA assay, specif-ic immunofluorescence was observed; the TCID50 was low （102.67）; the ful-length genome sequence of the isolated strain was 1 768 bp, sharing 94.1%-96.8% ho-mology with 13 reference strains; to be specific, the isolated strain exhibited the highest homology of 96.8% with AF055392PCV2a; the isolated strain 201105ZJ and reference strain AF055392 belonged to genotype PCV2a, exhibiting a distant genetic relationship with genotype PCV2c. [Conclusion] Characteristics of genetic variation of PCV2 isolate 201105ZJ provided theoretical basis for vaccine development, investi-gation of PCV2 pathogenesis, and prevention and control of porcine circovirus-as-sociated diseases （PCVAD） in East China.

Isolation and Identification of a Flocculant Producing Strain TS-1

[Objective] The purpose was to select out and identify a flocculant producing strain which could produce high active flocculant.[Method] The strain producing high active flocculant was isolated out and purified through medium culture and the selected strain was identified through observing its culture characters and determining its physiological and biochemical property.[Result] Fourteen strains of bacteria with flocculant producing function were isolated from tested soil samples through isolation,purification and preliminary screening using dilution-spread plate method and plate streaking method.Five strains of flocculant producing bacteria showing higher flocculation activity were selected out after second screening and their flocculation rates were higher than 70%;the flocculation activity of one strain among them was still stable after multiple subculturings,its flocculation rate was always above 90% and it was marked as TS-1.TS-1 was encapsulated Gram-positive bacillus and there was no lipid in it,such as poly-β-hydroxybutyric acid.TS-1 was Bacillus amyloliquefaciens,so it was named Bacillus TS-1.[Conclusion] The strain selected out in this experiment could be used in the flocculation and biochemical treatment of wastewater from starch industry.