中国南海硇洲岛潮间带产胞外蛋白酶的可培养海洋真菌多样性

【目的】研究中国南海硇洲岛潮间带产胞外蛋白酶的可培养海洋真菌多样性。【方法】从中国南海硇洲岛潮间带采集海水和沉积物样品,采用分离培养、蛋白酶生产菌平板检测法和基于内转录间隔区1-5.8S rRNA基因内转录间隔区2(ITS1-5.8S-ITS2)序列的系统进化分析法,研究产胞外蛋白酶真菌的多样性。【结果】采用50%海水配制的马铃薯葡萄糖琼脂(PDA)固体培养板从所采集的样品中分离、纯化并收集了198株真菌分离株,并采用ITS1-5.8S-ITS2序列PCR扩增、测序、BLAST和系统进化分析的方法成功鉴定了其中的178株。其中,有10株的ITS1-5.8S-ITS2序列与其在NCBI数据库中最匹配的ITS1-5.8S-ITS2序列的一致性<97,表明它们有可能是新的物种;其余168株的ITS1-5.8S-ITS2序列与NCBI数据库中已存在的相关ITS1-5.8S-ITS2序列的一致性均≥98%。这178株真菌归属为66个种,分布在子囊菌门和担子菌门的6纲,16个目,27个科的45个属中。其中的主要属为青霉菌属,占28.70%;其次为曲霉属,占11.24%。有83株真菌在加在脱脂奶粉的PDA固体培养板上的菌落周围有一透明圈,表明其可产生分泌胞外蛋白酶。【结论】从中国南海硇洲岛潮间带共分离、鉴定和收集了178株真菌,其中10株可能是新的物种,83株为胞外蛋白酶生产菌。     

海洋喇叭虫rRNA基因18S-ITS1序列及其系统发育分析

海洋喇叭虫Maristentor dinoferus 1996年在关岛(Guam)的珊瑚暗礁上被发现,至今尚未阐明其确切的系统发育地位.克隆到的海洋喇叭虫的18S-ITS1-5.8S rDNA序列包括222 bp的18S序列,77 bp的ITS1序列和22 bp的5.8S序列.比较分析了纤毛虫主要类群的ITS1序列后得出:短的ITS1序列可能是异毛类纤毛虫的特征.根据18S序列,利用邻接法构建,最大简约法和最大似然法构建系统发育树.其拓扑结构显示海洋喇叭虫属于异毛纲纤毛虫,但并不隶属喇叭虫科,应予以新的分类地位.     

日耳曼曲霉(Aspergillus germanicus)—曲霉属焦曲霉组一个我国新记录种

【目的】鉴定并报道我国一个曲霉新纪录种,即日耳曼曲霉。【方法】采用形态学性状及钙调蛋白和β-微管蛋白基因部分序列及核糖体DNA ITS1-5.8S-ITS2序列进行多相系统分类学分析。【结果】根据与日耳曼曲霉Aspergillus germanicus模式菌株CBS 123887的形态学和分子性状的比较分析,两株分离自中国山东泰山地区土壤的曲霉菌(AS3.15303和AS3.15304)被鉴定为日耳曼曲霉A.germanicus。【结论】参考我国迄今已报道的曲霉物种,确定日耳曼曲霉A.germanicus是曲霉属焦曲霉组的一个我国新记录种。     

冬虫夏草培养子实体ITS,5.8S的分析及系统发育研究

对野生冬虫夏草的人工培养子实体的ITS1,ITS2和5.8S间区序列进行了克隆,并结合已有的序列对ITS1,ITS2和5.8S进行了系统发育分析。结果表明,人工培养子实体与中华被毛孢(Hirsutella sinensis L95DBM-1)具有96%的同源性,在NJ邻接树上形成明显的一支,与虫草属其它类群种间具有明显差异,表明分离培养的虫草子实体为中华被毛孢。遗传距离分析结果显示,培养虫草序列与已知虫草在种内也存在一定差异,可能暗示虫草种群在不同区域具有一定的遗传分化。     

中国猪种布鲁氏菌分子流行病学特征分析

【目的】调查猪种布鲁氏菌的基因多态性和分子流行病学特征。【方法】用经典分型方法对菌株的生物型进行鉴定,分析菌株的地理分布特点;用MLVA-16分型方法对60株猪种布鲁氏菌进行基因分型,采用在线软件评估分型方法的分辨率和位点的多态性,用BioNumerics 5.0软件进行聚类分析。【结果】我国流行的猪种布鲁氏菌主要是猪种生物1型(33株)、2型(3株)和3型菌(24株);分布范围较广,包括广东、广西、内蒙古、北京、吉林、宁夏和西藏等地。MLVA-16分型方法对猪种布鲁氏菌具有极高的分辨力,多态性指数为0.992;Panel1、MLVA-11和Panel 2B均具有较高的分辨率,多态性指数分别为0.884、0.916和0.979。60株猪种布鲁氏菌聚为6大类52个基因型,5个共享基因型(GT24,GT25,GT26,GT28,GT29)包括13株布鲁氏菌,各基因型菌株间有潜在的流行病学关联,可能是分别来自相同传染源的暴发流行;另47株布鲁氏菌呈现独特的基因型,表明菌株来自无流行病学关联的零星散发病例。猪种布鲁氏菌的最小生成树表明我国菌株分别与美国、法国和波兰的菌株有完全相同的MLVA-15基因型。【结论】中国猪种布鲁氏菌有较高的遗传多态性,并与美国、法国和阿根廷的菌株有较高的遗传相似性。我国猪种布病以零星散发为主。     

安徽省部分地区2016–2018年猪圆环病毒2型遗传进化分析

【背景】猪圆环病毒(Porcinecircovirus,PCV)可以引起断奶仔猪多系统衰竭综合症(Postweaningmultisystemicwastingsyndrome,PMWS)。【目的】了解安徽省部分地区猪圆环病毒2型(PCV2)的遗传变异情况。【方法】采用PCR技术,对2016-2018年间安徽省部分地区猪场疑似PMWS感染的组织样品共计31份进行PCV2检测和全基因扩增,并通过DNAStar等生物信息学软件对所得到的PCV2毒株基因序列进行遗传进化分析。【结果】所得的8株PCV2安徽株与GenBank上已发表的国内外参考毒株相比较,核苷酸相似性为92.8%-99.0%,ORF2及其推导的氨基酸序列相似性分别为85.8%-99.6%和82.5%-100%。遗传进化树分析结果显示8株安徽株中有1株PCV2a,2株PCV2b,5株PCV2d,未发现PCV2c、PCV2e和PCV2f基因型。此外,通过对ORF2基因编码的氨基酸序列分析,发现各基因型Cap蛋白氨基酸序列上的位点具有其独特性。【结论】近年来PCV2在安徽地区的猪群中感染较为普遍,其中PCV2d基因型的感染病例增多,逐渐成为安徽地区的优势流行株。本研究为安徽地区的PCV2防控提供了一定的参考依据。     

芍药内生真菌的鉴定及产生活性次生代谢产物的评估

【目的】研究药用植物芍药(Paeonia lactiflora Pall.)内生真菌的种群多样性,同时对其可能存在的聚酮合酶(Polyketide synthase,PKS)和非核糖体多肽合成酶(Non-ribosomal peptide synthetase,NRPS)基因多样性进行评估,预测芍药内生真菌产生活性次生代谢产物的潜力。【方法】采用组织分离法获得芍药根部内生真菌菌株,结合形态学特征和ITS序列分析,进行鉴定;利用兼并性引物对内生真菌中存在的聚酮合酶(PKS)基因和非核糖体多肽合成酶(NRPS)基因进行PCR扩增及序列测定分析,构建系统发育树,明确芍药内真菌PKS基因序列和NRPS基因序列的系统进化地位。【结果】从芍药组织块中共分离得到105株内生分离物,去重复后获得52株内生真菌,菌株ITS基因序列信息显示,52株芍药内生真菌隶属于7目、13科、15属,其中小球腔菌属(Leptosphaeria)、土赤壳属(Ilyonectria)和镰孢属(Fusarium)为优势种群;从52株内生真菌中筛选获得13株含PKS基因片段的菌株,8株含NRPS基因片段的菌株,部分菌株功能基因的氨基酸序列与Gen Bank中已知化合物的合成序列具有一定的同源性,预示芍药根部内生真菌具有合成丰富多样的次生代谢产物的潜力。【结论】药用植物芍药根部具有丰富的内生真菌资源,且具有产生活性次生代谢产物的潜力,值得进一步开发研究和应用。     

自然界斜纹夜蛾核型多角体病毒的基因型多态性

【目的】从基因组序列角度进一步揭示自然界斜纹夜蛾核型多角体病毒（Spodoptera litura nucleopolyhedrovirus, SpltNPV）的基因型多态性。【方法】病毒克隆A5, F1, X3 和 X15分别以活体克隆法分离自SpltNPV埃及株、 日本福冈株和日本小笠原株。根据SpltNPV基因组全序列（GenBank登录号: AF325155）和海灰翅夜蛾核型多角体病毒（S. littoralis NPV, SpliNPV）部分基因序列（GenBank登录号: X99377, X99376 和X98924）设计引物, PCR扩增获得A5, F1, X3 和 X15的多角体蛋白（polyhedrin, polh）基因和ORF18~ORF23序列。【结果】根据多角体蛋白基因序列, X3和X15属于SpltNPV型, 而A5和F1属于SpliNPV型。将A5, F1, X3 和 X15的ORF18~ORF23与SpltNPV和SpliNPV相应的基因序列进行同源性比较。结果发现, F1与SpliNPV以及X3与SpltNPV的核苷酸序列相似性高, 但X3的ORF20在172~558 nt处缺失387 bp。尽管依据多角体蛋白基因序列X15属于SpltNPV型, 但对于ORF18~ORF23序列, X15与SpliNPV的相似性高于与SpltNPV的相似性。同样, A5属于SpliNPV型, ORF18~ORF20与SpliNPV相应的核苷酸序列相似性高, 但ORF21与SpltNPV相应的核苷酸序列一致性为100%, 特别是ORF22, SpltNPV的特有序列出现在A5的基因组中, 而且与SpltNPV的ORF22一致性为100%; 反过来, ORF23又与SpliNPV相应的核苷酸序列相似性高。【结论】所有这些都表明, SpltNPV在自然界不仅存在基因型多态性, 而且即使属于同一基因型, 它们的基因组序列也有显著差异。可利用SpltNPV在自然界的这种异质性筛选适宜防治斜纹夜蛾幼虫的株系。     

新疆羊粪便戊型肝炎病毒RNA的检测与序列分析

【目的】为了了解新疆地区羊群中是否存在戊型肝炎的感染,我们从戊型肝炎病毒(Hepatitis E virus,HEV)IgG检测阳性的新疆某羊场采集54份1-3岁的羊粪便,【方法】利用逆转录套式聚合酶链方法(RT-nPCR),检测HEVRNA,其中6份为阳性,阳性率11.11%。【结果】将PCR扩增产物克隆,测序并进行序列分析,结果表明,6株羊源HEV检测株在HEV ORF2 189bp 99.38%-100%,为同一基因型;与HEVⅠ、Ⅱ、Ⅲ和Ⅳ型的同源性分别为78.67%-85.33%、81.33%-82.67%、78.67%-84.00%和84.67%-95.36%,与Ⅳ型最高同源性达94.04%-95.36%。以该189bp片段绘制进化树,发现与新疆牛源分离株、中国大陆猪源分离株(FJ610232)和中国大陆人源分离株(AJ272108)在同一分枝上,同属基因Ⅳ型;【结论】提示新疆羊群中可能存在HEV感染,并且羊可能是人类HEV传染源中除猪之外的新宿主。     

基于ITS条形码序列的刺桐姬小蜂分子鉴定

【目的】刺桐姬小蜂Quadrastichus erythrinae Kim体型小,传统的形态学鉴定方法难以快速准确识别。【方法】本研究测定了刺桐姬小蜂的rDNA ITS1和ITS2序列,根据18S rDNA部分序列,利用MEGA的最大相似法(Maximum Likehood)构建系统发育树。根据刺桐姬小蜂ITS1和ITS2序列设计了特异引物,应用特异引物对单只刺桐姬小蜂进行PCR扩增,可稳定地扩增出明显的目的DNA条带。【结果】研究表明,基于ITS基因的DNA条形码技术可以用于刺桐姬小蜂的快速准确鉴定。【结论】因此,采用ITS1和ITS2区的特异性引物可对刺桐姬小蜂进行快速分子鉴定。     

Genetic heterogeneity in internal transcribed spacer genes of Balantidium coli (Litostomatea, Ciliophora)

The species Balantidium coli is the only ciliate that parasitizes humans. It has been described in other primates, and it has been proposed that the species B. suis from pigs and B. struthionis from ostriches are synonyms of B. coli. Previous genetic analysis of pig and ostrich Balantidium isolates found a genetic polymorphism in the ITS region but its taxonomic relevance was not established. We have extended the genetic analysis to Balantidium isolates of pig, gorilla, human and ostrich origin. We have PCR-amplified and sequenced the ITS region of individual Balantidium cells. The predicted ITS secondary structures of the sequences obtained were transferred by homology modelling to the sequences of other Trichostomatia ciliates (Isotricha, Troglodytella, Lacrymaria and Spathidium) and compared to determine the importance of the differences in the primary sequences. The results show that the ITS2 secondary structure of the species considered follows the general pattern of other ciliates, although with some deviations. There are at least two main types of ITS sequence variants in B. coli which could be present in the same cell and they are common to the mammal and avian hosts studied. These data do not support B. suis and B. struthionis as distinct species.     

Phenotypic and genotypic characterization of Salmonella spp. Isolated from pigs and their farm environment in Korea

This study's objective was to determine the prevalence of Salmonella spp. in pigs and their farm environments in Korea, and to investigate the relationship between the strains based on their phenotypic and genotypic characteristics. A total of 36 Salmonella spp. were isolated in this study: 18 isolates from 492 pigs (3.7%) and 18 isolates from 418 (4.3%) farmhouse environmental samples from 16 different pig farms. Of the Salmonella strains isolated from the numerous environmental samples, the highest prevalence was observed in slurry or manure, followed by partitions, farmer's hands, floors, water/ nipples, ventilation sources, and feed, respectively. All the Salmonella isolates originating from different farms were genetically distinct. In three farms, however, identical phage types and pulse-field gel electrophoresis patterns were observed among Salmonella isolates from pig feces and environmental samples. This study suggests that environments contaminated with Salmonella could pose an infection risk to pigs on pig farms.     

致病性大肠杆菌和蜡样芽胞杆菌的分离鉴定

【背景】猪只消化道疾病是养猪业上一大重要疾病,给养猪业带来一定的经济损失。大肠杆菌是引起猪腹泻的一种常见病原菌,可以引起不同日龄的猪腹泻,但主要以幼龄猪为主。【目的】旨在分离鉴定引起四川省眉山市一规模化养猪场病猪大规模腹泻的病原菌。【方法】采用常规细菌分离方法结合16S rRNA基因序列的分析方法从发病猪肝脏、胃以及污染的饲料分离鉴定细菌,并对分离株进行小鼠致病性试验、16S rRNA基因遗传进化树分析、毒力基因的检测、药物敏感试验。【结果】从腹泻猪肝脏中分离到一株致病性大肠杆菌,胃中分离到一株蜡样芽胞杆菌,并且追溯到传染源是该猪场饲料。通过检测这两株菌相应的毒力基因发现大肠杆菌不属于肠外致病性型,蜡样芽胞杆菌检测到了nheA、nheB、nheC、bceT、entFM 5种毒力基因;药敏试验表明常规的氨基糖苷类和头孢类抗生素对大肠杆菌抑菌效果较好,红霉素、氟苯尼考、头孢氨苄、头孢哌酮对蜡样芽胞杆菌抑菌效果较好,而蜡样芽胞杆菌对青霉素、阿莫西林等常规药物不敏感。【结论】饲料存在大肠杆菌和蜡样芽胞杆菌的混合污染。     

产志贺样毒素和肠毒素大肠杆菌分子流行病学

[目的]人和动物腹泻的主要病原菌为大肠杆菌,本文主要研究贵州省致腹泻大肠杆菌毒力因子的分布类型.[方法]采用PCR技术对各毒力因子的基因分布进行研究.[结果]共分离到333株大肠杆菌,其中产肠毒素大肠杆菌(ETEC)在腹泻的人、猪、牛群中占优势,分别为:人群73(n=112),猪群82(n=106),牛群18(n=115).在ETEC菌株中检测到热敏肠毒素(lt)和不耐热肠毒素(st)基因,还存在lt/st并存现象.从人、猪、牛群中还检测到产志贺样毒素大肠杆菌(STEC),其中源自猪的STEC的检出率最高.大部分STEC同时携带lt、st或lt和st同时并存.编码F18菌毛的主亚基由fedA基因编码.对所分离大肠杆菌F18菌毛进行的研究结果表明,fedA基因主要与肠毒素基因共存,与stx基因并存的类型较少,25份猪源STEC菌株中仅有4份检测到fedA基因.[结论]贵州省人群、猪群和牛群致腹泻病原菌中以带F18菌毛的ETEC为主,STEC主要分布在腹泻的猪群中.     

Changes in antimicrobial resistance in fecal bacteria associated with pig transit and holding times at slaughter plants

Fecal coliforms, fecal streptococci, and antimicrobial resistance (AMR) associated with various pig transit and holding times were investigated at slaughter plants. Changes in the relative abundance of two biotypes of Streptococcus faecium were associated with transit and holding of pigs, although approximately 20% of the isolates were unidentified. The greatest variety of coliforms was isolated from porcine feces after short transit (2 h) or holding (3 h) times and was qualitatively similar to those from pigs on farms. Isolates from pigs with longer average transit or holding times were almost all Escherichia coli (four biotypes). Streptococcal resistance to most antimicrobial agents was significantly greater (P less than 0.05) in isolates from live pigs at slaughter plants than in those from pigs at farms and was apparent after a short transit time (2 h). Streptococci from pigs held an average of 15 h were less resistant to most antimicrobial agents than those from pigs held 3 or 43 h. When compared with short transit times, moderate transit times (6 h) were associated with significantly decreased (P less than 0.05) coliform resistance and decreased resistance transfer but a greater diversity of AMR patterns. Holding pigs overnight (14 h) was associated with lowered coliform resistance to several antimicrobial agents, compared with the resistance of isolates from pigs held 3 or 39 h. A substantial increase (18 to 48%) in the ability to transfer streptomycin resistance was demonstrated in coliforms from pigs held 39 h, when compared with those from pigs held 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in antimicrobial resistance in fecal bacteria associated with pig transit and holding times at slaughter plants.

Fecal coliforms, fecal streptococci, and antimicrobial resistance (AMR) associated with various pig transit and holding times were investigated at slaughter plants. Changes in the relative abundance of two biotypes of Streptococcus faecium were associated with transit and holding of pigs, although approximately 20% of the isolates were unidentified. The greatest variety of coliforms was isolated from porcine feces after short transit (2 h) or holding (3 h) times and was qualitatively similar to those from pigs on farms. Isolates from pigs with longer average transit or holding times were almost all Escherichia coli (four biotypes). Streptococcal resistance to most antimicrobial agents was significantly greater (P less than 0.05) in isolates from live pigs at slaughter plants than in those from pigs at farms and was apparent after a short transit time (2 h). Streptococci from pigs held an average of 15 h were less resistant to most antimicrobial agents than those from pigs held 3 or 43 h. When compared with short transit times, moderate transit times (6 h) were associated with significantly decreased (P less than 0.05) coliform resistance and decreased resistance transfer but a greater diversity of AMR patterns. Holding pigs overnight (14 h) was associated with lowered coliform resistance to several antimicrobial agents, compared with the resistance of isolates from pigs held 3 or 39 h. A substantial increase (18 to 48%) in the ability to transfer streptomycin resistance was demonstrated in coliforms from pigs held 39 h, when compared with those from pigs held 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genotyping of Escherichia coli from environmental and animal samples

The triplex PCR of Clermont et al. [Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic groups. Appl. Environ. Microbiol. 66, 4555-4558.] was used to genotype E. coli isolates from the Mid-Atlantic region of the USA, obtained from freshwater, animal internal organs, and feces. Of 445 isolates subjected to genotyping, 118 isolates (26%) were genotype A, 111 (25%) genotype D, 140 (31%) genotype B1, and 76 (17%) genotype B2. All four genotypes were present in three sets of freshwater stream samples. When isolates from chicken cecal ingesta, cecal mucosa, and tracheal mucosa were screened, there was selective distribution of genotypes in these organs. Genotype D was rarely encountered in feces, milk, and intestinal tissues of dairy cows, while all four genotypes were represented in goose feces. Isolates from the feces of zoo animals reared in the US demonstrated a predominance of genotype B1. Thirty-six of the A isolates in our overall collection were subgenotype A(0), in which none of the three amplicons are observed; confirmation that these isolates were E. coli was done using an ancillary lacZ PCR assay. We conclude that the genotyping triplex PCR assay, used in combination with traditional culture methods, can be useful in categorizing E. coli from environmental and veterinary sources in the Mid-Atlantic region of the USA.     

Age-dependent Occurrence of the Intestinal Ciliate Balantidium coli in Pigs at a Danish Research Farm

A cross sectional study of the prevalence and intensity of Balantidium coli in pigs was carried out on a Danish research farm. The prevalence of B. coli infection increased from 57% in suckling piglets to 100% in most pig groups ≥4 weeks old. The mean number of cysts per gram faeces (CPG) of pigs aged 12 weeks and younger were ≤206, whereas pigs aged 28 weeks and >52 weeks had significantly higher counts of ≥865 CPG. Although some lactating sows had very high CPG’s, no significant differences in CPG could be detected between the intensities of pregnant sows, lactating sows and empty and dry sows. No human cases of B. coli infection have been published in Denmark though it is zoonotic.     

A comparison of features and the microbial constitution of the fresh feces of pigs fed diets supplemented with or without dietary microbes

The features and the constitution of the microbial population of fresh feces were compared between pigs fed a diet supplemented with dietary microbes and pigs given nonsupplemented diets. The former were reared on farm C and the latter on farms A and B. The concentrations of ammonia-N, indole, and skatole of fresh feces were not significantly different between pigs reared on farm C and those raised on farms A and B, but the concentrations of ammonia-N and the skatole of fresh feces were significantly different between pigs reared on farms A and B. The total VFA (volatile fatty acids) concentration of fresh feces in pigs on farm C was slightly lower than in those on farms A and B. Moreover, the molar proportion of the acetic acid in feces in pigs on farm C was lower; inversely, that of propionic and butyric acids was higher in comparison with those on farms A and B. No differences were evident in the total viable counts of feces among pigs reared on the three different farms. Clostridium perfringens was abundant in feces of pigs raised on farms A and B, but it was not detected in pigs reared on farm C. Megasphaerae, bifidobacteria, and clostridia except for C. perfringens were more abundant in the feces of pigs fed a diet supplemented with dietary microbes on farm C, compared with pigs given the nonsupplemented diets on farms A and B.     

stx2vha is the dominant genotype of Shiga toxin-producing Escherichia coli O157:H7 isolated from patients and domestic animals in three regions of China

Shiga toxin-producing Escherichia coli(STEC) O157: H7 strains were isolated from domestic animals and patients from Xuzhou City, Jiangsu Province, China and the bordering Anhui and Henan Provinces and were examined for the stx genotype. Of 390 strains, 277 were identified as genotype stx2vha ; 41, stx2 ; 51, stx2-stx1 ; 1, stx2-stx2vha-stx1 ; 5, stx2-stx2vha ; and 15 were un-typeable. Of the 277 stx2vha-bearing isolates, 116 were isolated from goats; 42, cattle; 38, hens, and 35 from pigs. The study shows stx2vha is the dominant genotype and goats are an important reservoir.