鰤诺卡氏菌对大口黑鲈头肾巨噬细胞的侵染过程

【背景】鱼类诺卡氏菌病潜伏期和病程较长,感染率和死亡率较高,给水产养殖业带来较大的经济损失,其病原鰤诺卡氏菌(Nocardia seriolae)是胞内寄生菌,侵入细胞后引起慢性感染是主要的致病机制。【目的】构建鰤诺卡氏菌侵染大口黑鲈(Micropterus salmoides)头肾巨噬细胞体外模型,观察鰤诺卡氏菌侵染巨噬细胞的过程并探究鰤诺卡氏菌对巨噬细胞的凋亡作用。【方法】采用密度梯度离心法分离巨噬细胞,通过特异性染色和PCR扩增巨噬细胞表达基因mpeg1对细胞进行鉴定,并通过CCK-8法和氧呼吸暴发活性测定检测巨噬细胞的活性;通过倒置荧光显微镜和流式细胞术观察侵染过程中细菌与细胞的形态与数量变化;通过双荧光流式细胞术检测、乳酸脱氢酶(lactate dehydrogenase, LDH)释放试验及线粒体膜电位检测,探究鰤诺卡氏菌对巨噬细胞的凋亡作用。【结果】从大口黑鲈头肾分离获得纯度高的巨噬细胞,经染色和PCR法鉴定为巨噬细胞;筛选出最优的体外培养条件为1640培养基+1%青霉素链霉素+1%胎牛血清。在脂多糖刺激后,巨噬细胞的氧呼吸暴发能力显著提高(P<0.05)。GFP-鰤诺卡氏菌侵染细胞2 h后细菌被细胞吞噬,4 h细胞变圆且贴壁率降低,6 h细菌大量繁殖并包围细胞,8 h后细胞大量死亡。凋亡相关实验结果表明,侵染初期巨噬细胞凋亡率增加,LDH释放增加,线粒体膜电位下降;随着侵染时间延长,细胞凋亡率下降、LDH释放量及线粒体膜电位下降减少,说明鰤诺卡氏菌对巨噬细胞起先促进后抑制凋亡的作用。【结论】通过密度梯度离心法成功分离大口黑鲈头肾巨噬细胞,并通过鰤诺卡氏菌侵染细胞后初步摸清鰤诺卡细菌在细胞水平的致病机理,建立了鰤诺卡氏菌侵染大口黑鲈头肾巨噬细胞的体外模型;证实了鰤诺卡氏菌可侵染巨噬细胞并抑制细胞凋亡,从而达到在巨噬细胞内存活,为进一步开展鰤诺卡氏菌与巨噬细胞相互作用并阐明鰤诺卡氏菌的致病机制奠定了研究基础。     

9株鱼源鰤诺卡氏菌生物学特征和致病性比较

【背景】鰤诺卡氏菌(Nocardia seriolae)是一种革兰氏阳性好氧菌,易引起鲈形目鱼类结节病发生,近几年对大口黑鲈、杂交鳢等名优鱼类的危害越来越大。【目的】比较分析9株不同年份、不同地区患结节病的不同淡水鱼品种分离的鰤诺卡氏菌的种间同源关系、生长特性、致病性以及药物敏感性差异,为进一步研究制定相应的防控与治疗措施提供基础数据和科学依据。【方法】在9株试验菌生理生化实验鉴定结果基础上,采用16SrRNA基因序列比对、限制性片段长度多样性比较菌株间的同源性;扩增看家基因secA1基因序列进行不同来源菌株种内相似度比对;通过体外培养比较不同菌株的生长情况;人工注射感染分析9株试验菌对大口黑鲈的致病性差异;采用微量肉汤二倍稀释法测试9株试验菌对13种抗菌药物的最小抑菌浓度;运用PCR技术检测菌株携带毒力基因和耐药基因的情况。【结果】生理生化结果显示9株试验菌与鰤诺卡氏菌的基本理化特征相符;9株试验菌聚类分析聚为一类,种内相似度高,限制性片段长度多样性酶切图谱相同,生长特性无明显差异;9株试验菌对大口黑鲈致病性无显著差异,但毒力基因携带情况略有不同,其均携带mce1A、mig和pup基因,而dop和whiB3只在部分菌株检出;9株试验菌均对氟甲喹和氨苄西林耐药,除NS1外,都对磺胺间甲氧嘧啶耐药,对大部分抗菌药物敏感,主要携带blaTEM和sul1耐药基因。【结论】不同时间、不同地区及不同宿主分离的9株鰤诺卡氏菌在生物学特性、致病性和药物敏感性等方面无明显差异;同源性相近,提示华南地区感染淡水鱼的鰤诺卡氏菌可能为同一流行株型。研究结果为鰤诺卡氏菌的发病机制及防控技术的进一步研究奠定了基础。     

Ceratomyxa seriolae n. sp. and C. buri n. sp. (Myxozoa: Myxosporea) from the gall-bladder of cultured yellowtail Seriola quinqueradiata

Ceratomyxa seriolae n. sp. and C. buri n. sp. (Myxozoa: Myxosporea) were found in the gall-bladder of cultured yellowtail Seriola quinqueradiata Temminck & Schlegel (Carangidae) in Japan. Mature spores of C. seriolae n. sp. were elongate and 6.5 (6.0-7.5) m long and 33.7 (28.0-41.5) m thick. Disporous plasmodia of C. seriolae n. sp., 40-100 m in size, were amoeboid to spherical. C. buri n. sp. were elliptical with a flattened posterior end, 6.5 (5.5-7.5) m long and 14.3 (11.0-16.5) m thick. Spherical plasmodia of C. buri n. sp., 15-20 m in diameter, were disporous. In periodical sampling of yellowtail bile from August, 1999 to February, 2000, the two new species of Ceratomyxa, as well as Myxobolus spirosulcatus Maeno, Sorimachi, Ogawa & Kearn 1995, first appeared in October, and the prevalences were very variable (20-100%) during the study period.     

加州鲈源鰤鱼诺卡氏菌的分离鉴定及致病性

[背景] 鰤鱼诺卡氏菌（Nocardia seriolae）是一种严重危害水产养殖业的病原菌,可引起以体表溃疡、出血及组织器官形成结节为特征的鱼类慢性肉芽肿疾病,目前尚无有效的防治方法。[目的] 明确引起安徽省临泉县某养殖场加州鲈（Micropterus salmonoides）结节病的病原菌,探讨其致病性,为该病的有效防治提供科学依据。[方法] 取肝脏结节病灶接种于TSB培养基分离优势细菌,利用表型检查结合分子生物学方法鉴定分离菌株。进一步通过检测分离菌株的毒力基因、测定其对加州鲈的半数致死量（LD₅₀）以及所感染加州鲈的组织病理学变化与组织载菌量,分析其致病性。[结果] 从病鱼体内分离到一株优势菌株NI,综合NI分离株的表型特性、16S rRNA基因序列与鰤鱼诺卡氏菌参考株相应序列的一致性以及特异性PCR扩增结果,确定其为鰤鱼诺卡氏菌。鰤鱼诺卡氏菌NI分离株携带毒力基因gapA、ibeA和mip,人工回归感染后加州鲈出现与自然病例相似的症状,其对加州鲈的LD₅₀为2.58×10⁶ CFU/尾。组织病理学观察到头肾、心脏、肝脏、胃和脾脏均出现慢性肉芽肿病变,肠管肌层疏松、肠绒毛脱落,肌肉组织中肌纤维疏松、间隙增宽。qPCR检测结果显示,组织中鰤鱼诺卡氏菌载量由高到低依次为头肾、心、肝、胃、脾、肠和肌肉。[结论] 鰤鱼诺卡氏菌是引起此次加州鲈结节病的病原菌,对该菌致病性的研究为加州鲈诺卡氏菌病的防控提供了理论依据。     

Fcγ receptor-mediated phagocytosis pathway was involved in phagocytosis of mIgM+ B lymphocytes from largemouth bass (Micropterus salmoides)

The potential for phagocytosis has been proven in teleost B cells, but the research on the regulatory mechanism of phagocytosis remains lacking. In this study, three largemouth bass (Micropterus salmoides) (15 ± 5 g) were injected intraperitoneally with Nocardia seriolae (10⁵ CFU/100 μl/fish) in vivo, and their spleen was collected at 72 h post-infection for mRNA-seq. After the de novo assembly of the paired-end reads, 73,622 unigenes were obtained. Gene expression profiling revealed that 2043 unigenes were differentially expressed after N. seriolae infection, comprising 1285 upregulated and 758 downregulated unigenes (q-value <0.05, log2FC > |2|) of which 181 genes were involved in phagocytosis. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis demonstrated that 12 differentially expressed genes (DEG) associated with phagocytosis were enriched in the Fcγ receptor-mediated phagocytosis signalling pathway. In vitro, the phagocytic ability of mIgM⁺ B lymphocytes was validated using indirect immunofluorescence assay (IIFA) and fluorescence activating cell sorter (FACS), and the phagocytosis rates of the mIgM⁺ B lymphocytes incubated with a Lyn inhibitor had decreased from 18.533 ± 6.00% to 11.610 ± 4.236% compared with the unblocked group. These results suggested that the Fcγ receptor-mediated phagocytosis signalling pathway had participated in the phagocytosis of B cells and provide further insight into the role of B cells in innate immunology.     

African Hepatics: VI. Euosmolejeunea

Abstract

(1) The ratio of ¹⁴CO₂ uptake of fully expanded capsules to that of corresponding gametophytes in the species studied was about 1 : 15 in Mnium hornum, 1 : 35 in Pleurldium acuminatum and around 1 : 1 in Funaria hygrometriea.

(2) Translocation of photosynthate from gametophyte to sporophyte took place within 1 to 4 days of exposure to ¹⁴CO₂; in expanded capsules the final activity was at least equal to that remaining in the gametophyte.

(3) In M hornum there was no evidence of reverse translocation from sporophyte to gametophyte.

(4) Translocation from gametophyte continued at a high level throughout the expansion and differentiation of the capsule in M. hornum. The activity reaching the sporophyte was approximately proportional to its fresh weight.

(5) Both in situ photosynthesis and translocation to the capsule of F. hygrometrica rose to a maximum during the latter part of capsule expansion and ensuing differentiation, subsequently declining as the green tissues of the capsule senesced.

(6) Fully expanded (but premeiotic) capsules of M. hornum contributed approximately 20% of the assiinilate necessary for their growth. The corresponding figure for P. acuminatum was about 10%, and for F. hygrometrica about 50%.     

Morphological and Molecular Characterization of Philometroides seriolae from Japanese Amberjack Seriola quinqueradiata caught in East Sea,Republic of Korea

The Japanese amberjack Seriolae quinqueradiata is one of the most consumed fish species among the Koreans. However, information regarding parasitic infection in Japanese amberjack is scarce. This study described the morphological and molecular characteristics of a species of philometrid nematode, Philometroides seriolae, which was recovered from Japanese amberjack. This fish was caught in the sea of Goseong-gun, Gangwon-do, Republic of Korea (Korea). Six P. seriolae (Nematoda: Philometridae) were recovered from 2 Japanese amberjacks. These parasites were subgravid female which were 325–420 mm long and 2.95–3.27 mm wide. Furthermore, they had typical papillae distributed on their body surface with 14 papillae at the apical view. Sequence analysis of the small subunits of ribosomal RNA (SSU rRNA) showed high sequence identity (99.8%, 1,607/1,611-bp) with that of P. seriolae (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ155811","term_id":"205318602"}}FJ155811). This nematode species has been newly added to the Korean nematode fauna.     

The complete mitochondrial genome of Neobenedenia melleni (Platyhelminthes: Monogenea): mitochondrial gene content,arrangement and composition compared with two Benedenia species

The complete mitochondrial (mt) genome sequences of Neobenedenia melleni were determined and compared with those of Benedenia seriolae and B. hoshinai. This circular genome comprises 13,270 bp and includes all 36 typical mt genes found in flatworms. Total AT content of N. melleni is 75.9 %. ATG is the most common start codon, while nad4L is initiated by GTG. All protein-coding genes are predicted to terminate with TAG and TAA. N. melleni has the trnR with a TCG anticodon, which is the same to B. seriolae but different from B. hoshinai (ACG). The mt gene arrangement of N. melleni is similar to that of B. seriolae and B. hoshinai with the exception of three translocations (trnF, trnT and trnG). The overlapped region between nad4L and nad4 was found in the N. melleni mt genome, which was also reported for the published Gyrodactylus species, but it was not found in those of B. seriolae and B. hoshinai, which are non-coding regions instead. The present study provides useful molecular characters for species or strain identification and systematic studies of this parasite.     

First record of Unicapsula seriolae (Myxozoa: Multivalvulida) from the muscle of Malabar grouper Epinephelus malabaricus in Japan

Unicapsula seriolae (Myxozoa; Multivalvulida) was found in the trunk muscle of Malabar grouper Epinephelus malabaricus caught off Wakayama Prefecture, Japan. Numerous filamentous or sesamoid brown to black lesions were observed in the skeletal muscle. Histopathological observation indicated that the lesions were myxosporean plasmodia encapsulated by a fibrous layer, accompanied by melanin deposition. Spores having one large and two rudimentary polar capsules were subspherical in shape and 6.6 × 6.9 μm in size. Scanning electron microscopy revealed that spores were composed of three spore valves. Morphological characteristics were consistent with U. seriolae, which is reported to cause myoliquefaction in yellowtail kingfish Seriola lalandi in Australia. Molecular analysis of the SSU and LSU rDNA supported identification of the species as U. seriolae. This is the first report of Unicapsula in Japan.     

Antioxidant acitivity in vitro and in vivo of the capsule polysaccharides from Streptococcus equi subsp. zooepidemicus

Crude capsule polysaccharides (CCP) were prepared from the culture of Streptococcus equi subsp. zooepidemicus C55129 and were partially purified through an anion-exchange column chromatography to afford partially purified capsule polysaccharides (PCP). The main component of CCP and PCP was hyaluronic acid. In vitro antioxidant assay, the capsule polysaccharides showed strong inhibition of lipid peroxidation and hydroxyl radical scavenging activity and moderate 1,1-diphenyl-2-picryldydrazyl radical scavenging activity. In addition, CCP exhibited much stronger reductive power than PCP. For antioxidant testing in vivo, CCP and PCP were orally administrated over a period of 15 days in a d-galactose induced aged mice model. As results, administration of capsule polysaccharides inhibited significantly the formation of malondialdehyde in mice livers and serums and raised the activities of antioxidant enzymes and total antioxidant capacity in a dose-dependent manner. However, the antioxidant activity of CCP was lower than that of PCP. The results suggest that the capsule polysaccharides from Streptococcus equi subsp. zooepidemicus C55129 have direct and potent antioxidant activities.     

Rotala pseudojuniperina sp. nov. (Lythraceae) from India

A new species, Rotala pseudojuniperina, is described and illustrated. It grows in a seasonal pond on a high altitude lateritic plateau ecosystem of the northern Western Ghats, India. It resembles the African species R. juniperina in having trimerous flowers, but differs in longer sepal appendages, ovoid capsule and ellipsoid seeds. Amongst the Indian species, R. pseudojuniperina shows affinities towards R. densiflora but differs in trimerous flowers, ovoid capsule and longer and ellipsoid seeds.     

Quantitative Trait Loci (QTL) Associated with Resistance to a Monogenean Parasite (Benedenia seriolae) in Yellowtail (Seriola quinqueradiata) through Genome Wide Analysis

Benedenia infections caused by the monogenean fluke ectoparasite Benedenia seriolae seriously impact marine finfish aquaculture. Genetic variation has been inferred to play a significant role in determining the susceptibility to this parasitic disease. To evaluate the genetic basis of Benedenia disease resistance in yellowtail (Seriola quinqueradiata), a genome-wide and chromosome-wide linkage analyses were initiated using F₁ yellowtail families (n = 90 per family) based on a high-density linkage map with 860 microsatellite and 142 single nucleotide polymorphism (SNP) markers. Two major quantitative trait loci (QTL) regions on linkage groups Squ2 (BDR-1) and Squ20 (BDR-2) were identified. These QTL regions explained 32.9–35.5% of the phenotypic variance. On the other hand, we investigated the relationship between QTL for susceptibility to B. seriolae and QTL for fish body size. The QTL related to growth was found on another linkage group (Squ7). As a result, this is the first genetic evidence that contributes to detailing phenotypic resistance to Benedenia disease, and the results will help resolve the mechanism of resistance to this important parasitic infection of yellowtail.     

中国新记录——毛齿藓短蒴变种(新拟)

该研究首次报道了毛齿藓短蒴变种(新拟)在中国的分布和毛齿藓属在新疆的分布。研究表明：(1)毛齿藓短蒴变种的主要识别特征为：叶基部鞘状,突然狭窄成线状披针形,背仰,叶缘平展,中肋充满叶上部,背面粗糙,远轴面有厚壁细胞束分化,叶细胞长方形至短长方形;有假根生芽胞;孢蒴椭球形,稍弓形弯曲,蒴齿线状披针形,二裂至近基部。(2)毛齿藓属的3个分类单位在中国均有分布,毛齿藓短蒴变种以较短的孢蒴和蒴柄区别于原变种,以有根生芽胞、叶基部呈明显鞘状、叶缘平展区别于云南毛齿藓的无假根生芽胞、叶基部略呈鞘状、叶缘背卷。(3)在牛毛藓科中有许多类群与毛齿藓短蒴变种形态相似,但仅有毛齿藓属的叶在横切面上主细胞的远轴面有厚壁细胞束,而其他类群的叶在横切上主细胞的近轴面和远轴面均有厚壁细胞束。该变种的叶背仰不同于牛毛藓科其他属的叶直立;以具鞘部的叶、表面光滑的孢蒴、内弯的蒴齿区别于无鞘部的叶、表面有纵沟槽的孢蒴、直立的蒴齿的牛毛藓属。(4)该变种属北极 高山分布类型,表现出了明显的欧洲 亚洲 北美洲间断分布模式,可作为研究气候变化、海陆变迁的重要材料。     

Microencapsulation of yeast cells in the calcium alginate membrane

Summary Cells of Saccharomyces cerevisiae (ATCC 24858) were encapsulated in the calcium alginate membrane and cultured. Swelling of the capsule was prevented by adding 0.2 g CaCl₂ to 1 L growth medium. The dry cell concentration based on the inner volume of the capsule reached 309 g/L, which was much higher than could be obtained by cell entrapment. All the cells remained inside the capsule during the cultivation. The flux of CO₂ through the capsule membrane increased approximately twice by adding a nonionic surfactant to the CaCl₂ solution during the step of capsule formation.     

Role of the pyruvate metabolic network on carbohydrate metabolism and virulence in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen that must adapt to unique nutritional environments in several host niches. The pneumococcus can metabolize a range of carbohydrates that feed into glycolysis ending in pyruvate, which is catabolized by several enzymes. We investigated how the pneumococcus utilizes these enzymes to metabolize different carbohydrates and how this impacts survival in the host. Loss of ldh decreased bacterial burden in the nasopharynx and enhanced bacteremia in mice. Loss of spxB, pdhC or pfl2 decreased bacteremia and increased host survival. In glucose or galactose, loss of ldh increased capsule production, whereas loss of spxB and pdhC reduced capsule production. The pfl2 mutant exhibited reduced capsule production only in galactose. In glucose, pyruvate was metabolized primarily by LDH to generate lactate and NAD⁺ and by SpxB and PDHc to generate acetyl-CoA. In galactose, pyruvate metabolism was shunted toward acetyl-CoA production. The majority of acetyl-CoA generated by PFL was used to regenerate NAD⁺ with a subset used in capsule production, while the acetyl-CoA generated by SpxB and PDHc was utilized primarily for capsule biosynthesis. These data suggest that the pneumococcus can alter flux of pyruvate metabolism dependent on the carbohydrate present to succeed in distinct host niches.     

A Histological Study of the Effect of Feeding bySmicronyxspp. (Coleoptera: Curculionidae) Larvae on Seed Production byStriga hermonthica(Scrophulariaceae)

Experiments were conducted in Burkina Faso to determine the effect of feeding bySmicronyxspp. (Coleoptera: Curculionidae) larvae on the development of fruit capsule and seed production inStriga hermonthica(Scrophulariaceae). In treated plotsSmicronyxadults were released in the cages while in untreated plots (= control) noSmicronyxwere released. TheStrigacapsules were collected, fixed, and prepared for histological examination. UninfestedStrigacapsules developed normally. In infested capsules, three types of galls were distinguished: they reflected the development of capsule and the seeds. There was a synchrony between seed development and larval development in the same capsule. In most instances, one egg or larva was found per gall.Smicronyxlarvae completely destroyedStrigaseeds in the developing capsule by two mechanisms: (1) by directly eating most of the seeds and (2) by eating the placenta that normally feed the seeds.     

Rungia sinothailandica sp. nov. (Acanthaceae) from China and Thailand

Rungia sinothailandica (Acanthaceae), a new species from Yunnan, China and Chiang Rai, Thailand is described and illustrated. A colour plate, line drawing and distribution map of R. sinothailandica are provided. Rungia sinothailandica is similar to R. pinpienensis H. S. Lo, but differs in the broadly ovate sterile bracts and obovate‐elliptic fertile bracts, the crispate and tawny bracts margin, the 2‐lobed upper lip of the corolla and puberulent capsule. It is also similar to R. burmanica (C. B. Clarke) B. Hansen, but is easily distinguished by pubescent stems and leaves, the cymbiform‐oblong bracteoles and the 2‐lobed upper lip of corolla.     

The ultrastructure of the cuticle of Nematoda. II. The cephalic cuticle of Stilbonematinae (Adenophorea,Desmodoridae)

Summary  The ultrastructure of the cephalic cuticle of 6 of 7 representative genera of Stilbonematinae (Eubostrichus, Catanema, Laxus, Robbea, Leptonemella, Stilbonema) is investigated using SEM and TEM techniques. Additionally, one species of Spirinia (Spiriniinae) and one of Desmodora (Desmodorinae) were studied for outgroup comparison. Most of the studied species show modifications of the cephalic cuticle. Furthermore, at least four different pathways have been developed to reinforce the head within Stilbonematinae. Species with a coarsely annulated somatic cuticle (Leptonemella sp., Stilbonema majum, and Desmodora ovigera) developed a rigid, non-annulated cephalic capsule by modifying the main constructing element of the median zone, the ’’ring body.’’ In faintly annulated Laxus oneistus, the annulated cephalic capsule results from a newly inserted ’’block layer’’ between the median and basal zone. The non-annulated cephalic capsule of Robbea sp. is formed by both the block layer and the ring body element. The annulated capsule of Catanema sp. stems from a doubled number of fiber layers within the basal zone. In Spirinia sp., only the amphidial region is strengthened in what could be termed an amphidial shield. All forms with cephalic capsules show mechanisms to keep the oral region pliable. Only Eubostrichus topiarius lacks a reinforcement of the cephalic cuticle. A comparison with the literature is made to elucidate corresponding structures within the cephalic capsules of representatives of Desmodorida. It is demonstrated that the presence of a cephalic cuticle is of no systematic value above the genus level. Accepted: 3 March 1996