菊花绿变病植原体在中国的发生及分子鉴定

对内蒙古农业大学校园内表现花器绿变症状的菊花样品进行采集和DNA提取,应用植原体16S rRNA基因和rp基因的引物进行巢式PCR扩增,从感病样品中分别扩增得到了长度均约为1.2 kb的片段。序列一致性分析表明,菊花绿变植原体16S rRNA基因与翠菊黄化植原体匈牙利风信子株系(GenBank登录号MN080271)、印度玉米株系(KY565571)、印度繁缕株系(KC623537)和印度马铃薯株系(KC312703)的核酸一致性最高,为99.9%,rp基因序列与翠菊黄化植原体立陶宛洋葱株系(GU228514)的核酸一致性最高,为99.8%。基于16S rRNA基因和rp基因构建系统进化树时发现,菊花绿变植原体均与16SrI-B亚组成员聚为一起。16S rRNA基因相似性系数分析表明,菊花绿变植原体与洋葱黄化植原体(AP006628)的相似性系数最高为1.00,洋葱黄化植原体(AP006628)在分类上属于16SrI-B亚组。因此,我们可以确定该菊花绿变植原体属于16SrI-B亚组。这是我国首次报道菊花绿变病的发生。     

四种短体线虫的形态和分子生物学鉴定

 采用形态学和分子生物学方法对来自荷兰的藏橐吾和铃兰、泰国的高山榕、缅甸的香蕉等种苗(球)分别鉴定出玻利维亚短体线虫Pratylenchus bolivianus、铃兰短体线虫P. convallariae、咖啡短体线虫P. coffeae和斯佩奇短体线虫 P. speijeri。对这4种线虫的形态特征进行较为详细的描述后,认为头环、口针、侧区、生殖系统和尾形等形态特征是种类鉴定的重要依据;进一步利用引物28S-D2A/28S-D3Br扩增测序得到上述4种线虫的28S rRNA基因D2/D3区序列,序列分析发现玻利维亚短体线虫从欧洲到美洲的不同种群之间的遗传距离变异较小,仅为0~0.007;铃兰短体线虫同一种群不同个体之间的遗传变异较大,为0.006~0.029;咖啡短体线虫不同种群之间的平均遗传距离为0.013;斯佩奇短体线虫不同种群之间的平均遗传距离为0.010;咖啡短体线虫P. coffeae和斯佩奇短体线虫P. speijeri亲缘关系很近,二者种间平均遗传距离仅为0.026。本研究再次证明线虫28S rRNA基因D2/D3区基因序列可作为短体线虫种间鉴定的依据。     

Using genetic methods for analysis of fish meals and feeds employed in Greek mariculture

Large quantities of high protein fish meals are needed to sustain cultured species and thus the impact to marine ecosystem has been highly discussed. The aim of this study was to apply a PCR‐cloning methodology for a robust insight into the composition of commercial fish meals and feeds for farmed species of the Greek mariculture, assessing the risk posed by aquaculture to marine ecosystems but also the risk posed by commercial fish feeds to the increase in trophic level of species farmed in Greece. 89% of the sequences were identified to species level and only 11% to genus/family level. Overall, a total of 49 taxa were identified (44 fish species/taxon, five non‐fish species/taxon). Even though small pelagic fish like Engraulis sp. were the main portion, a wide range of species constituted the fish meals and feeds. Plant and animal species were also detected as an alternative protein source. Feed products employed in Greek mariculture still contain large portions of fish meals which increase the mean trophic level of farmed species causing a farming up trend. The results emphasize that such molecular methodologies are needed to certify aquafeeds allowing fish feed producers to demonstrate their commitment to sustainable aquaculture.     

Bacterial communities associated with natural and commercially grown rooibos (Aspalathus linearis)

Aspalathus linearis is a commercially important plant species endemic to the Cape Floristic Region of South Africa and is used to produce a herbal tea known as rooibos tea. Symbiotic interactions between A. linearis and soil bacteria play an important role in the survival of Aspalathus plants in the highly nutrient-poor, acidic fynbos soil. The aim of this study was to characterize and compare rhizosphere and bulk soil bacterial communities associated with natural and commercially grown A. linearis, as well as the effect of seasonal changes on these communities. Bacterial communities were characterized using high throughput amplicon sequencing, and their correlations with soil chemical properties were investigated. The N-fixing bacterial community was characterized using terminal restriction fragment length polymorphism and real time quantitative polymerase chain reaction. Actinobacteria, Proteobacteria, and Acidobacteria were the most dominant bacterial phyla detected in this study. Highly similar bacterial communities were associated with natural and commercially grown plants. Significant differences in the bacterial community were observed between rhizosphere and bulk soils collected in the dry season, while no significant differences were detected in the wet season. This study provides insights into bacterial community structure and potential factors shaping bacterial community structure with commercially important A. linearis.     

Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm

Ortholinea labracis n. sp. is described and its life cycle is inferred from a Southern Portuguese fish farm, with basis on microscopic and molecular procedures. This myxosporean parasite infects the urinary bladder of the European seabass Dicentrarchus labrax and the intestinal epithelium of a marine oligochaete of the genus Tectidrilus. Myxospores subspherical in valvular view and ellipsoidal in sutural view measuring 7.6 ± 0.3 (6.8–8.7) μm in length, 7.2 ± 0.2 (6.7–7.7) μm in width and 6.5 ± 0.4 (5.8–7.7) μm in thickness. Two polar capsules, 3.0 ± 0.2 (2.6–3.4) μm long and 2.4 ± 0.1 (2.0–2.9) μm wide, located at the same level, but with divergent orientation and opening to opposite sides of the suture line. Sequencing of the SSU rRNA gene revealed a similarity of 100% between the analysed myxospores and triactinomyxon actinospores. The phylogenetic setting of O. labracis n. sp. shows subgrouping in correlation with tissue tropism, but identifies this parasite as another exception to the main division of Myxosporea into the main freshwater and marine lineages.     

Changes in the gut microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White spot syndrome virus (WSSV) infection

Intestinal microorganisms play important roles in maintaining host health, but their functions in aquatic animal hosts have yet to be fully elucidated. The Chinese mitten crab, Eriocheir sinensis, is one such example. We attempted to identify the shift of gut microbiota that occurred in response to infection of white spot syndrome virus (WSSV), an emerging viral pathogen in the crab aquaculture industry. The microbiota may exert some control over aspects of the viral pathogenesis. We investigated the changes in composition and structure of the crab gut microbiome during various WSSV infection stages of 6 h post‐infection (hpi) and 48 hpi, using a 16S rRNA approach on the MiSeq Illumina sequencing platform. Four phyla (Firmicutes, Proteobacteria, Tenericutes and Bacteroidetes) were most dominant in the gut of E. sinensis regardless of the WSSV infection stages. However, further analysis revealed that over 12 bacterial phyla, 44 orders and 68 families were significantly different in abundance at various states of WSSV infection. Several intriguing aspects of E. sinensis gut bacteria that had not been previously reported were also uncovered, such as class Mollicutes was dominant here, but absent in crabs from Yangtze River estuary and Chongming Islands. Overall, this study provided the first evidence that changes in gut microbiome were closely associated with the severity of WSSV infection and that indicator taxa could be used to evaluate the crab health status.     

微生物菌群培养组学在动物医学中的应用

实验室条件下可培养的微生物约占自然界中微生物总数的1%，这限制了人们对99%未知微生物的认识和利用，而研究表明，那些“不可培养的微生物”是可以被开发和利用的，未能被纯培养的微生物才是未知微生物的主体。微生物培养组学探索利用多种培养条件和长时间的培养，结合基质辅助激光解吸电离飞行时间质谱法（MALDI-TOF-MS）和16S核糖体RNA（rRNA）测序可以大规模鉴定各种微生物，同时利用全基因组测序和宏基因组测序手段对未知微生物进行深入分析。本文综述了国内外近年来微生物菌群培养组学在反刍动物胃肠道、禽类盲肠及家畜鼻腔微生物菌群研究中的最新进展，探讨将动物体内菌群培养组学方法应用于动物疾病防治领域的可行性。作为一个新兴的研究方法，尽管该培养组学还存在一些不够成熟的方面，但它的发展前景十分广阔，微生物菌群培养组学方法和其他研究方法的互补已经逐渐成为发展兽医微生物学新的突破口。     

仔猪雷极氏普罗菲登斯菌的分离鉴定及系统进化分析

为了解猪雷极氏普罗菲登斯菌(P.rettgeri)的生物学特性、致病性及16SrRNA基因系统进化关系,从发生严重腹泻、血便的哺乳仔猪群的内脏器官中分离到1株病原菌,根据形态特征、培养特性、生化特性、16SrRNA基因序列分析鉴定为P.rettgeri。小鼠攻毒试验证实,该分离菌株对试验小鼠有较强致病力,哺乳仔猪回归试验可复制出与临床上相似的典型症状,并从发病死亡小鼠及哺乳仔猪中分离到攻毒菌株。系统进化分析表明,分离菌株与雷极氏普罗菲登斯菌系统进化关系最为密切,其16SrRNA基因序列与雷极氏普罗菲登斯菌代表菌株的同源性在97.3%~99.6%之间。药敏试验结果表明,分离菌株对头孢哌酮钠、头孢噻肟钠、头孢曲松钠、头孢唑啉、头孢呋肟、头孢吡肟、阿莫西林、链霉素、氨曲南、诺氟沙星、左氟沙星、恩诺沙星、卡那霉素、氨苄青霉素、米诺环素、链霉素、阿米卡星等多数药物敏感。首次报道了雷极氏普罗菲登斯菌可以引起哺乳仔猪严重腹泻,提示在仔猪腹泻中应注意该菌感染的诊断、监测和防控。     

肺炎克雷伯氏菌强毒株的分离鉴定及16-23SrRNAITS序列分析

为确诊疑似仔猪肺炎克雷伯氏菌(K.pneumonia)感染,并研究其病原的致病性、耐药性、16-23SrRNA ITS系统进化特征,本研究从云南因肺炎、腹泻而大量死亡的仔猪中分离到1株革兰氏阴性短粗杆菌,命名为KP14013,对其进行生化鉴定、16SrRNA鉴定,研究其对小白鼠和仔猪的致病性,并对其16-23SrRNA ITS基因进行测序和遗传进化分析。结果显示,KP14013分离株生化特征与肺炎克雷伯氏菌相符,其16SrRNA与GenBank中23株肺炎克雷伯氏菌代表株之间的同源性均为99%,将KP14013鉴定为肺炎克雷伯氏菌。KP14013对小白鼠半数致死量(LD50)为3×101.8 CFU,腹腔注射3×108 CFU可使仔猪100%致死。16-23SrRNA ITS系统进化关系结果表明,KP14013与GenBank中收录的15株肺炎克雷伯氏菌形成进化树的一个分支,属于同一个亚群,它们之间的核苷酸同源性为98.4%~99.2%。本研究证实了肺炎克雷伯氏菌是该起仔猪腹泻大量死亡的病原;KP14013分离株为毒力极强菌株,具有多重耐药性,其16-23SrRNA ITS与GenBank中收录的肺炎克雷伯氏菌代表株之间核苷酸存在差异,可用于肺炎克雷伯氏菌菌株间的鉴别。     

Study on Biological Characteristics and Drug Sensitivity of Haemophilus parasuis Isolated in Sichuan Province

The study was aimed to investigate the biological characsteristics and drug sensitive tests of Haemophilus parasuis isolated in Sichuan province.This paper reported the biological characteristics,16S rRNA gene PCR and drug sensitive test of the isolates by technique of microbiological and molecular biology.The results showed that the isolates had micro anaerobic and could produce satellite phenomenon,the isolates were just same in biochemical characteristics with field isolates in other area.All the isolates amplified 821 bp 16S rRNA gene.And drug sensitive tests showed that the isolates were highly sensitive to cefradine,ampicillin,cefradine,amoxicillin,cefaclor,ofloxacin,cefotaxime and furadantin,the sensitive rate was 75.61% to 87.80%,but the isolates were high resistance to lincomycin,carbenicillin,enrofloxacin,amikacin and sulfamethoxazole,and multiple-drug resistance was found among the isolates,the number of bacterial strains resistant to antibiotics were more between five and eight,the resisitance strains were 21.95% to 30.49%,7 isolates were even resistant to 13 antibiotics.The results showed the phenomenon of drug resistance of the isolates was very serious.