Molecular Characterization of “Candidatus Parilichlamydia carangidicola,” a Novel Chlamydia-Like Epitheliocystis Agent in Yellowtail Kingfish,Seriola lalandi (Valenciennes), and the Proposal of a New Family, “Candidatus Parilichlamydiaceae” fam. nov. (Order Chlamydiales)

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported “Candidatus Piscichlamydia salmonis” ({"type":"entrez-nucleotide","attrs":{"text":"AY462244","term_id":"42525281","term_text":"AY462244"}}AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as “Candidatus Parilichlamydia carangidicola” and that the new family be known as “Candidatus Parilichlamydiaceae.”     

Independent emergence of Yersinia ruckeri biotype 2 in the United States and Europe

Biotype 2 (BT2) variants of the bacterium Yersinia ruckeri are an increasing disease problem in U.S. and European aquaculture and have been characterized as serovar 1 isolates that lack both peritrichous flagella and secreted phospholipase activity. The emergence of this biotype has been associated with an increased frequency of enteric redmouth disease (ERM) outbreaks in previously vaccinated salmonid fish. In this study, four independent specific natural mutations that cause the loss of both motility and secreted lipase activity were identified in BT2 strains from the United States, United Kingdom, and mainland Europe. Each of these was a unique mutation in either fliR, flhA, or flhB, all of which are genes predicted to encode essential components of the flagellar secretion apparatus. Our results demonstrate the existence of independent mutations leading to the BT2 phenotype; thus, this phenotype has emerged separately at least four times. In addition, BT2 strains from the United Kingdom were shown to have the same mutant allele found in U.S. BT2 strains, suggesting a common origin of this BT2 lineage. This differentiation of distinct BT2 lineages is of critical importance for the development and validation of alternative vaccines or other treatment strategies intended for the control of BT2 strains.     

Homology modeling, simulation and molecular docking studies of catechol-2, 3-Dioxygenase from Burkholderia cepacia: Involved in degradation of Petroleum hydrocarbons

Catechol 2, 3-dioxygenase is present in several types of bacteria and undergoes degradation of environmental pollutants through an important key biochemical pathways. Specifically, this enzyme cleaves aromatic rings of several environmental pollutants such as toluene, xylene, naphthalene and biphenyl derivatives. Hence, the importance of Catechol 2, 3-dioxygenase and its role in the degradation of environmental pollutants made us to predict the three-dimensional structure of Catechol 2, 3-dioxygenase from Burkholderia cepacia. The 10ns molecular dynamics simulation was carried out to check the stability of the modeled Catechol 2, 3- dioxygenase. The results show that the model was energetically stable, and it attains their equilibrium within 2000 ps of production MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3-dioxygenase reveals that the benzene, O-xylene, Toluene, Fluorene, Naphthalene, Carbazol, Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl makes strong hydrogen bond and Van der waals interaction with the active site residues of H150, L152, W198, H206, H220, H252, I254, T255, Y261, E271, L276 and F309. Free energy of binding and estimated inhibition constant of these compounds demonstrates that they are energetically stable in their binding cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except Pyrene all the substrates made close contact with Fe atom by the distance ranges from 1.67 to 2.43 Å. In addition to that, the above mentioned substrate except pyrene all other made π-π stacking interaction with H252 by the distance ranges from 3.40 to 3.90 Å. All these docking results reveal that, except Chrysene all other substrate has good free energy of binding to hold enough in the active site and makes strong VdW interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of catalyzing the above-mentioned substrate.     

藏药七十味珍珠丸改善APP/PS1小鼠学习记忆能力

藏药七十味珍珠丸（ratanasampil,RNSP）可改善大脑氧化应激水平,改善大脑功能,有安神和促进学习记忆的功效,然而RNSP是否可改善阿尔茨海默症（AD）小鼠的学习记忆功能,尚缺乏系统研究。本研究采用APP/PS 1转基因小鼠为研究对象,并随机将其分为实验组和对照组。对实验组进行为期12周的RNSP灌胃给药,对照组进行12周的蒸馏水灌胃,采用Morris水迷宫与开场实验评价小鼠学习记忆能力,比较小鼠体重与相关器官质量,并比较器官质量指数,通过分子生物学检测指标评价小鼠脑内老年斑数量,Aβ生成量及BACE1表达水平。本研究证实,与对照组相比,给药组小鼠定位航行潜伏期明显缩短（22.60±13.26 vs. 46.44±8.41, P<0.01, day 5）,穿越平台次数明显增加（1.29±0.37 vs. 0.54±0.29, P<0.01）,探洞次数明显增加（32.11±9.85 vs. 20.89±8.78, P<0.05）,表明RNSP提高了APP/PS 1小鼠的学习记忆能力和空间探索能力。与对照组相比,给药组小鼠大脑重量及脑质量指数均增高（0.4135±0.0102 vs. 0.3833±0.0254, P<0.05;2.04±0.08 vs. 1.84±0.15, P<0.05）,脑内老年斑数量减少（18.70±7.88 vs. 38.83±6.15, P<0.05）,Aβ1- 42水平及BACE1表达均显著降低（0.19±0.08 vs. 0.41±0.12, P<0.05; 0.136±0.04 vs. 0.206±0.02, P<0.05）,表明RNSP延缓了APP/PS 1小鼠的脑萎缩进程,降低脑内老年斑的形成,下调脑内Aβ1-42水平和BACE1裂解酶的蛋白质表达量。本研究提示,RNSP可改善APP/PS 1小鼠的学习记忆能力,其机制可能和RNSP抑制脑萎缩,降低BACE1蛋白表达以及减少脑内Aβ沉积有关。     

DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout

DNA vaccines based on the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV have been demonstrated to be very efficient in inducing a protective immune response against the respective diseases in rainbow trout. Nanogram doses of plasmid DNA delivered by intramuscular injection are sufficient to induce high levels of immunity in fingerling-size fish, whereas larger fish require more vaccine for protection. The protection is long lasting and, more surprisingly, is partly established already 4 days post vaccination. The early protection involves cross-protective anti-viral defence mechanisms, while the long duration immunity is highly specific. The nature of these immune response mechanisms is discussed and it is suggested that the efficacy of the vaccines is related to their ability to activate the innate immune system as it is activated by live virus.     

Fish DNA vaccine against infectious hematopoietic necrosis virus: efficacy of various routes of immunisation

The DNA vaccine, pIHNVw-G, contains the gene for the glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV), a major pathogen of salmon and trout. The relative efficacy of various routes of immunisation with pIHNVw-G was evaluated using 1.8 g rainbow trout fry vaccinated via intramuscular injection, scarification of the skin, intraperitoneal injection, intrabuccal administration, cutaneous particle bombardment using a gene gun, or immersion in water containing DNA vaccine-coated beads. Twenty-seven days after vaccination neutralising antibody titres were determined, and 2 days later groups of vaccinated and control unvaccinated fish were subjected to an IHNV immersion challenge. Results of the virus challenge showed that the intramuscular injection and the gene gun immunisation induced protective immunity in fry, while intraperitoneal injection provided partial protection. Neutralising antibodies were not detected in sera of vaccinated fish regardless of the route of immunisation used, suggesting that cell mediated immunity may be at least partially responsible for the observed protection.     

Polymorphisms in 16S rRNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease, which causes significant problems to aquaculture worldwide. A recent study (Soule M, Cain K, LaFrentz S, Call DR [2005] Infect Immun 73:3799-3802) identified two 16S rRNA gene sequence variants (6 base differences) within the variable stem-loop region 3 for F. psychrophilum strains ATCC 49418 and CSF 259-93. That study also hypothesized that F. psychrophilum is composed of at least 2 distinct genetic lineages (I and II) described by a microarray-based comparative genomics study. In the present study, we augmented an existing 16S rDNA microarray to detect both 16S rRNA sequence variants from F. psychrophilum. Subsequent microarray experiments showed that CSF 259-93 hybridized as expected, but ATCC 49418 was positive for both sequence variants. We then developed a PCR-restriction fragment length polymorphism (RFLP) assay (MnlI and MaeIII) to distinguish between the 2 sequences. Gel isolation of PCR-RFLP products, cloning, and sequencing confirmed that ATCC 49418 harbors both 16S rRNA sequences. Microarray experiments showed that 11 of 14 strains from genetic Lineage I harbor both the CSF 259-93 and ATCC 49418 16S rRNA sequence variants, whereas all 15 Lineage II strains were only positive for the CSF 259-93 sequence (p < 0.0001). Elastin hydrolysis and tetracycline resistance were most closely associated with the latter strains (p < 0.0001 and p = 0.024, respectively). These data support the hypothesis that F. psychrophilum is composed of at least 2 distinct genetic lineages that are closely associated with host origin.     

甘肃盐池湾黑颈鹤亚成体夏季生境选择

开展对亚成体的研究,可以更加全面了解一个物种,进而更有效地开展保护工作。甘肃盐池湾国家级自然保护区是黑颈鹤（Grus nigricollis）成体的重要繁殖地和亚成体的重要栖息地之一。为研究甘肃盐池湾黑颈鹤亚成体生境选择,于2020年7月初至8月中旬在盐池湾党河湿地展开调查,并依据Johnson对生境选择空间尺度的划分,对亚成体活动区内各类型生境和觅食微生境的生境选择进行了研究。通过遥感影像解译和卫星跟踪分别获得各栖息地类型面积以及黑颈鹤的活动位点,利用核密度分析法估计活动区面积并利用Manly研究中的设计Ⅲ来研究活动区内各类型生境选择;通过选取利用样方和对照样方,使用χ2检验、独立样本t检验和Mann-Whitney U检验,对比检验样方数据,进行微生境选择的研究。结果表明,活动区内各类型生境中亚成体选择河流,拒绝戈壁和沼泽化草甸,对沼泽既不选择也不拒绝,而成体选择湖泊,没有利用河流,同时拒绝戈壁、山脉、沼泽化草甸和盐化草甸,对沼泽既不选择也不拒绝;觅食微生境选择中,亚成体选择平均植被盖度为57.07% ± 4.53%,基质类型为泥炭,基质硬度为中,主要植被黑褐苔草（Carex atrofusca）的微生境栖息,相比成体,亚成体选择的生境基质更硬,距道路距离更近,距房屋、河流、山脉和湖泊距离更远。亚成体的栖息地选择主要受到生境质量、生境资源有限性以及成体选择等因素的影响。在这些因素的影响下,亚成体与成体产生了生态位分离,并在栖息地选择上出现了分化。这种分化对亚成体的生存和成体的繁殖都有益,可以避免种内无效的冲突和竞争,有利于亚成体和成体的适合度增加。保护黑颈鹤的栖息环境需同时考虑到亚成体的选择和生存。