安荔赤眼蜂在中国野外首次发现及其体内Wolbachia的检测

【目的】为丰富赤眼蜂Trichogramma的种类资源,明确野外新采集的一种赤眼蜂的种类,探明该赤眼蜂所感染Wolbachia的类型。【方法】采用挂米蛾Corcyra cephalonica卵卡法在华南农业大学树木园诱集到两批赤眼蜂,通过形态鉴定和PCR扩增ITS2序列并测序分析的分子鉴定手段对野外采集的赤眼蜂材料进行种类鉴定;通过PCR扩增Wolbachia的外膜蛋白基因(wsp)序列,检测赤眼蜂体内Wolbachia的感染情况;通过PCR扩增wsp序列和多位点序列分型(multilocus sequence typing, MLST)对检测到的赤眼蜂体内的Wolbachia进行同源性分析。【结果】所诱集到的两批赤眼蜂均被鉴定为安荔赤眼蜂Trichogramma oleae Voegelé & Pointel,体内Wolbachia的感染率达100%。该Wolbachia株系与安荔赤眼蜂(前南斯拉夫品系)、短管赤眼蜂T. pretiosum(乌拉圭品系)以及T. deion(荷兰品系)体内Wolbachia亲缘关系较近,属于B超组Sib亚组,对应MLST序列型为ST486。【结论】安荔赤眼蜂T. oleae为中国野外首次发现,是完全感染Wolbachia的产雌孤雌生殖品系。本研究为害虫生物防治提供了一种新的天敌种类资源,并为进一步探明Wolbachia与赤眼蜂的互作提供了研究材料。     

Genome assisted molecular typing and pathotyping of rice blast pathogen,Magnaporthe oryzae,reveals a genetically homogenous population with high virulence diversity

Genome sequence-driven molecular typing tools have the potential to uncover the population biology and genetic diversity of rapidly evolving plant pathogens like Magnaporthe oryzae. Here, we report a new molecular typing technique -a digitally portable tool for population genetic analysis of M. oryzae to decipher the genetic diversity. Our genotyping tool exploiting allelic variations in housekeeping and virulence genes coupled with pathotyping revealed a prevalence of genetically homogenous populations within a single-field and plant niches such as leaf and panicle. The M. oryzae inciting leaf-blast and panicle-blast were confirmed to be genetically identical with no or minor nucleotide polymorphism in 17 genomic loci analyzed. Genetic loci such as Mlc1, Mpg1, Mps1, Slp1, Cal, Ef-Tu, Pfk, and Pgk were highly polymorphic as indicated by the haplotype-diversity, the number of polymorphic sites, and the number of mutations. The genetically homogenous single field population showed high virulence variability or diversity on monogenic rice differentials. The study indicated that the genetic similarity displayed by the isolates collected from a particular geographical location had no consequence on their virulence pattern on rice differentials carrying single/multiple resistance genes. The data on virulence diversity showed by the identical Sequence Types (STs) is indicative of no congruence between polymorphic virulence genes-based pathotyping and conserved housekeeping genes-based genotyping.     

Wolbachia prevalence patterns: horizontal transmission,recombination, and multiple infections in chestnut gall wasp-parasitoid communities

The Oriental chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), is a global invasive pest that causes serious damage to almost all chestnut species belonging to the Castanea genus (Fagaceae). Dryocosmus zhuili Liu et Zhu is a recently described sibling species of D. kuriphilus, which induces galls on Castanea henryi (Skan) Rehd. et Wils. There are many indigenous parasitoid species in China which play an important role in the natural regulation of their population dynamics. Wolbachia is a maternally inherited α-proteobacterium widely found in arthropods. This study screened for the presence of Wolbachia in the two chestnut gall wasps and in six parasitoid species from 12 populations, to investigate the prevalence patterns of Wolbachia in the chestnut gall wasp-parasitoid communities. We found that D. zhuili and four parasitoid species were infected with Wolbachia; among them, all individuals of the two populations of Megastigmus sp. had multiple Wolbachia infections. By using multilocus sequence types to characterize bacterial strains, three new sequence types were identified. The Wolbachia strains infecting D. zhuili (ST-507), Torymus sinensis Kamijo (ST-508), and Sycophila variegata (Curtis) (ST-508) belonged to supergroup A, whereas the Wolbachia strain infecting Megastigmus nipponicus Kamijo (ST-503) belonged to supergroup B. Our results also suggested that horizontal transmission of Wolbachia occurs between chestnut gall wasps and their parasitoids. Moreover, multiple Wolbachia infections of Megastigmus sp. may be due to gene recombination and horizontal transmission.     

基于基因组序列的脑膜炎奈瑟菌分型方法

【目的】建立并评估1种适宜的脑膜炎奈瑟菌(Neisseriameningitidis,Nm)基因组分子分型方法。【方法】本研究以125株代表性Nm菌株的基因组序列为对象,建立了基于核心基因SNP的基因组分型方法,并与pubMLST网站公布的MLST和cgMLST分型方法进行比较。【结果】基于核心基因SNP的基因组分型方法和cgMLST方法对125株Nm菌株的分型结果一致性较高,两种方法均明显优于MLST分型方法。基于SNP的基因组分型方法在认识Nm菌的种群结构、界定克隆群方面具有优势;cgMLST分型方法能够对任一菌株进行分型,但不能进行克隆群的界定和归类。【结论】基于核心基因SNP的基因组分型方法和cgMLST均明显优于MLST分型方法,未来仍有待进一步整合和提高。     

Current methods for molecular typing of Campylobacter species

Campylobacter remains one of the most common bacterial causes of gastroenteritis worldwide. Tracking sources of this organism is challenging due to the large numbers of human cases, and the prevalence of this organism throughout the environment due to growth in a wide range of animal species. Many molecular subtyping methods have been developed to characterize Campylobacter species, but only a few are commonly used in molecular epidemiology studies. This review examines the applicability of these methods, as well as the role that emerging whole genome sequencing technologies will play in tracking sources of Campylobacter spp. infection.     

Genetic diversity of Bartonella quintana in macaques suggests zoonotic origin of trench fever

Bartonella quintana is a bacterium that causes a broad spectrum of diseases in humans including trench fever. Humans were previously considered to be the primary, if not the only, reservoir hosts for B. quintana. To identify the animal reservoir and extend our understanding of the ecological and evolutionary history of B. quintana, we examined blood samples from macaques and performed multilocus sequence typing (MLST) analysis. We demonstrated the prevalence of B. quintana infection was common in macaques from main primate centres in mainland China. Overall, 18.0% (59/328) of rhesus macaques and 12.7% (39/308) of cynomolgus macaques were found to be infected with B. quintana by blood culture and/or polymerase chain reaction. The infection was more frequently identified in juvenile and young monkeys compared with adult animals. In contrast with the relatively low level of sequence divergence of B. quintana reported in humans, our investigation revealed much higher genetic diversity in nonhuman primates. We identified 44 new nucleotide variable sites and 14 novel sequence types (STs) among the B. quintana isolates by MLST analysis. Some STs were found only in cynomolgus macaques, while some others were detected only in rhesus macaques, suggesting evidence of host‐cospeciation, which were further confirmed by phylogenetic analysis and Splits decomposition analysis. Our findings suggest that trench fever may primarily be a zoonotic disease with macaques as the natural hosts.     

沙门氏菌分型研究进展

沙门氏菌是重要的致病菌之一,该菌属型别繁多。沙门氏菌的分型方法可分为以表型特征为依据的表型分型方法和以基因特征为依据的分子分型方法。沙门氏菌的表型分型主要有血清分型和噬菌体分型。分子分型主要有分子血清分型、脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)、多位点序列分析(Multi-locus sequence typing,MLST)、多位点可变重复序列分析(Multi-locus variable numbers tandem repeat analysis,MLVA)以及成簇的规律间隔的短回文重复序列分析(Clustered regularly interspaced short palindromic repeats,CRISPR)等。与表型分型相比,分子分型技术具有快速、准确、重复性好等特点,现已得到广泛应用。其中,分子血清分型是鉴定沙门氏菌血清型的新方法,PFGE被认为是病原微生物分子分型的"金标准",MLST和MLVA以其分辨率高、可重复性好和可比性强等优势满足了全球流行病学发展的要求,能实现序列数据交换和共享,成为新一代的分子分型新工具,而近年发现的CRISPR分型对同源性较高的同种血清型具有较高的分型能力。但每种分型方法也有各自的优缺点和使用条件及适用范围,因此可以根据菌株特性、分型目的和实验室拥有的条件选择最合适的分型方法。     

Real‐time PCR detection of Enterococcus faecalis associated with amyloid arthropathy

Aims: To develop a RT‐PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Methods and Results: Bacteria were selected from lesions including AA in layers. The primers were designed based on the phosphate ATP binding cassette transporter (pstS) and xanthine phosphoribosyltransferase (xpt) genes and first tested against three isolates with known base pairs at the specific sites. Subsequently, 12 isolates were selected from our collection by one researcher, and RT‐PCR was performed blinded. The sequence type (ST) was then confirmed by multilocus sequence analysis. Two single‐nucleotide polymorphisms in the pstS and xpt genes allowed an unambiguous identification of ST82. As an alternative to DNA extraction, a boiling method for release of DNA from cells was used. Conclusions: The real‐time PCR targeting ST82 enables rapid screening of Ent. faecalis cultured from suspect cases with results available after a few hours, much faster than multilocus sequence typing and pulse field gel electrophoresis. Significance and Impact of the Study: The new method allows a rapid screening of isolates with results available after only few hours. This RT‐PCR method could be a useful tool for molecular epidemiological studies on the spread of arthropathic and amyloidogenic Ent. faecalis within and between birds more efficiently.     

Development of a new MLST scheme for differentiation of Fusarium solani Species Complex (FSSC) isolates

Fungi belonging to the Fusarium solani Species Complex (FSSC) are well known plant pathogens. In addition to being the causative agent of some superficial infections, FSSC has recently emerged as a group of common opportunistic moulds, mainly in patients with haematological malignancies. Molecular typing methods are essential in order to better understand the epidemiology of such opportunistic agents with the final goal of preventing contamination. A three-locus typing scheme has thus been developed for FSSC; based on polymorphisms in the domains of the ITS, EF-1 alpha, and RPB2 genes. This method is now considered to be a useful reference for phylogenetic and taxonomic studies. In other significant clinical fungi (e.g., Candida sp., Cryptococcus neoformans, and Aspergillus fumigatus), genes coding for metabolic enzymes have been widely used and proven to be very informative for diagnosis and epidemiology. The contribution of these genes has never been evaluated for Fusarium sp. and more specifically for F. solani Species Complex.Here, we have evaluated the contribution of 25 genes for diagnosis and epidemiological purposes. We then report a new five-locus MLST scheme useful for diagnosis and typing of clinical FSSC isolates. The method has been validated on 51 epidemiologically unrelated strains of FSSC and presents a high power of discrimination calculated at 0.991.