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。     

坛紫菜叶状体的抗生素联合除菌效果

为研究坛紫菜及其外生菌群对氯霉素、竹桃霉素和头孢噻肟3种抗生素的敏感性,通过将其与氨苄青霉素、卡那霉素和庆大霉素的组合,优化坛紫菜叶状体的除菌方法,并利用涂布平板、qRT-PCR及16S rRNA测序分析其除菌效果。结果显示,当坛紫菜先以氨苄青霉素(300 mg/L)、卡那霉素(100 mg/L)和庆大霉素(100 mg/L)混合处理18 h,再以氯霉素(50 mg/L)、头孢噻肟(200 mg/L)和竹桃霉素(50 mg/L)混合处理4 d,叶状体的健康率保持在96.3%以上,而对可培养细菌的抑制率达到99.9%。根据qRT-PCR和16S rRNA的结果可知,该种组合方法较前3种抗生素的组合,其菌总数下降41.5%,微生物的丰度和多样性指数明显下降。其中,假单胞菌、交替赤杆菌、交替单胞菌以及海杆菌等得到针对性的抑制。研究表明,多种抗生素的优化组合能够对坛紫菜叶片的菌群结构产生显著影响,使其受到抑制,但仍无法达到绝对除菌的效果。     

规模化畜禽养殖场粪便中多重耐药菌分离鉴定及其耐药特征

为了解禽畜粪便中多重耐药菌的污染特征,本研究对鸡粪、牛粪、猪粪和有机肥这4种不同样品中多重耐药菌进行了计数分析,并对不同来源的多重耐药菌株进行了分离和纯化,进一步开展了基于16S rDNA序列比对的分子生物学鉴定以确定其种属地位,以及基于药敏试验的耐药性特征分析。结果表明:不同养殖动物粪便中四环素、恩诺沙星、磺胺甲恶唑和泰乐菌素4种抗生素多重耐药菌的绝对数量和相对数量排序均为鸡粪>猪粪>牛粪,粪源有机肥中可培养的多重耐药菌的绝对数量和相对数量在堆肥后均有所下降。通过对耐药菌株鉴定和分类学分析,发现禽畜粪便中多耐药菌的菌门集中分布在Proteobacteria、Firmicutes和Actinobacteria,多重耐药菌的优势菌属为Escherichia、Corynebacterium、Kurthia;而有机肥样品中多重耐药菌的优势菌属为Staphylococcus、Glutamicibacter。菌株的药敏试验表明,3类动物粪污中的多重耐药菌都对红霉素、四环素有着较高的耐药率,均高于80%,而对阿米卡星这种抗生素表现出较好的敏感性,其耐药率低于30%。通过对Ⅰ、Ⅱ类整合子基因盒的扩增,发现PCR产物的大小从0.8 kb到1.8 kb不等,基因盒ad A2、dfr A17、dfr A1及sat2在养殖场粪污中检出率均较高。     

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.     

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.     

甘露寡糖添加方式对哺乳期犊牛瘤胃细菌菌群结构的影响

为利用16S rDNA技术研究甘露寡糖不同添加方式对哺乳期犊牛瘤胃细菌菌群结构影响,本研究选用出生日龄一致、体重接近、健康状况良好的荷斯坦公犊牛20头,随机分为4组。CR组为对照组,饮用乳及开食料中均不添加甘露寡糖;ORa组在饮用乳中添加5 g甘露寡糖,开食料中不添加甘露寡糖;ORb组在开食料中添加5 g甘露寡糖,饮用乳中不添加甘露寡糖;ORc组在饮用乳及开食料中各添加2.5 g甘露寡糖(混合添加)。应用16S rDNA测序技术,研究了甘露寡糖添加方式对犊牛瘤胃细菌构建的影响。结果表明:甘露寡糖添加方式会影响哺乳期犊牛瘤胃细菌总数,但对哺乳期犊牛瘤胃细菌菌群多样性未产生显著影响(P>0.05)。在门水平上,与对照组相比,ORb组颗粒料中添加甘露寡糖显著降低了厚壁菌门与放线菌门的丰度(P<0.05),极显著提高了变形菌门的丰度(P<0.01),优势菌门增加为3种,分别为拟杆菌门、厚壁菌门与变形菌门。在属水平上,甘露寡糖不同添加方式导致哺乳期犊牛瘤胃细菌菌群结发生了改变,其中甘露寡糖不同添加方式对小杆菌属和脱硫弧菌属在瘤胃内丰度均产生了影响,但对ORa组与ORc组犊牛瘤胃细菌影响不及ORb组;且ORb组犊牛瘤胃细菌功能性菌属普雷沃氏-7属、氨基酸球菌属、优杆菌属和琥珀酸弧菌等均发生显著变化。基于以上结果,得出如下结论:本研究条件下,甘露寡糖添加方式对哺乳期犊牛瘤胃细菌菌群多样性未产生显著影响(P>0.05),但开食料中寡糖添加方式对菌群结构产生部分影响,其中发挥瘤胃蛋白降解作用和淀粉降解作用的琥珀酸弧菌科-UCG-001属、利用乳酸的新月形单胞菌属丰度极显著提高(P<0.01),而利用乳糖的优杆菌属丰度显著降低,降解半纤维素的小杆菌属丰度极显著降低(P<0.01)。     

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。     

广州桑树植原体分子检测及多样性初探

采用植原体16S-23S rDNA区段的通用引物对P1/P7和巢式引物对Rm16F2/Rm16R1,建立了快速准确的桑树植原体巢式PCR检测技术。对广州的两个桑树品种资源圃中的部分桑树品种进行了植原体分子检测,结果在两个资源圃中均发现有植原体存在。对巢式PCR的扩增产物(16S rDNA片段)进行了限制性片断长度多态性(RFLP)分析,显示出3种RFLP带型,暗示桑树植原体存在多样性。对所得植原体16S rDNA片段进行序列测定,并与其它植物植原体作亲缘关系分析,结果表明该植原体的16S rDNA序列与其它植物病原植原体之间的同源性为83.3%~99.9%,并初步判断所检测到的桑树植原体属于16S rI组。