In vitro activity of the nonsteroidal anti-inflammatory drug indomethacin on a scuticociliate parasite of farmed turbot

The scuticociliatosis produced by the endoparasite Philasterides dicentarchi is a severe parasitic infection of farmed turbot (Scophthalmus maximus) characterized by several histopathological effects including extensive inflammation. Indomethacin is a nonsteroidal anti-inflammatory drug that specifically inhibits synthesis of the proinflammatory mediator prostaglandins. The effect of indomethacin on the in vitro growth of P. dicentrarchi was investigated. In vitro growth of the scuticociliate was significantly inhibited by treatment with 100 microM indomethacin for 48 h. Higher concentrations of indomethacin (mM levels) did not affect the gelatinolytic activity of the cysteine proteinases of P. dicentrarchi. In vitro treatment with 25, 50 or 100 microM indomethacin for 3 days did not significantly affect the enzymatic activity of cysteine proteinases, as assayed with p-nitroanilide as substrate. Immunoblot analysis with anti-cysteine proteinase antibodies revealed an increase in proteinase expression (molecular weights of 80, 32 and 40-45 kDa) in parasite lysates originating from in vitro cultures incubated with 25 microM indomethacin for 72 h. Degradation of genomic DNA of the ciliates was observed in cultures incubated with 100 microM indomethacin for 1, 3 and 7 days. The results suggest that indomethacin is capable of inhibiting in vitro growth of the scuticociliate P. dicentrarchi by a mechanism related to the induction of programmed cell death, without affecting the enzymatic activation of parasite proteinases, which demonstrates the potential therapeutic use of this drug in the control of turbot scuticociliatosis.     

In vitro growth requirements for the fish pathogen Philasterides dicentrarchi (Ciliophora,Scuticociliatida)

Philasterides dicentrarchi is a scuticociliate causing fatal disease in farmed turbot and sea bass. In view of its high virulence and endoparasitic location, this parasite cannot be effectively controlled by formalin baths, and no systemic chemotherapeutic treatments have yet proved effective; immunoprophylaxis may thus be an attractive alternative approach. Since vaccine development is greatly facilitated by axenic culture of the pathogen, we have developed a simple axenic culture system based on commercially available Leibovitz L-15 medium, supplemented with fetal bovine serum, lipids (lecithin and Tween 80), nucleosides and glucose. After 1 week's culture under optimal conditions (salinity 10 per thousand, pH 7.2, temperature between 18 and 23 degrees C), yields of 1-2 x 10(5)cells/ml were obtained. Even cultures with seeding densities as low as 20 cells/ml were found to produce a good yield of ciliates (about 6 x 10(4)cells/ml) after 11 days of incubation. The ciliates thus obtained were free of contamination by other microorganisms, enabling preparation of pure P. dicentrarchi antigens for vaccine development studies.     

Effect of a mixed herb-enriched diet on the innate immune response and disease resistance of Paralichthys olivaceus against Philasterides dicentrarchi infection

We investigated the effect of a mixed herb-enriched diet obtained from pomegranate Punica granatum, Dalmatian chrysanthemum Chrysanthemum cinerariaefolium, and mastic-leaved prickly-ash Zanthoxylum schinifolium on innate immune mechanisms (e.g., phagocytosis activity, respiratory burst activity, alternative complement activity, lysozyme activity, and disease resistance) of olive flounder Paralichthys olivaceus against the scuticociliate Philasterides dicentrarchi. All experimental groups were challenged with P. dicentrarchi (1 x 10(5) ciliates/mL) through intraperitoneal administration of the pathogen (50 microL) on day 1. On day 7, the infected groups were fed 0, 5, 50, and 100 mg/kg of the enriched diets. The innate immune parameters, cumulative mortality, and relative percent survival (RPS) were assessed at weeks 1, 2, 3, and 4. Administration of 50 or 100 mg/kg of the herbal-enriched diet enhanced immunity throughout the experimental period. However, at the 5-mg/kg dosage, the enriched diet did not enhance the innate immune estimates at any time. At doses of 50 and 100 mg/kg, administration of the diet preceding the challenge with P. dicentrarchi decreased the percentage cumulative mortality in the experimental groups and thereby increased RPS values. This study reports that administration of 50 or 100 mg/kg mixed herbal-enriched diet could positively influence the innate immune response to P. dicentrarchi and enhance the health status of olive flounder with respect to this microbe.     

Severe scuticociliate (Philasterides dicentrarchi) infection in a population of sea dragons (Phycodurus eques and Phyllopteryx taeniolatus)

Scuticociliatosis is a disease of fish induced by ciliated parasites of the genus Scuticociliatida. It has been described in sea horses (Hippocampus sp.), flounders (Paralichthys olivaceus), and turbots (Scophthalmus maximus). Here we present a case study of a population of sea dragons chronically infected with scuticociliates identified as Philasterides dicentrarchi by histopathology and PCR. Beginning in 2004, over a period of 19 months, 10 sea dragons (Phycodurus eques and Phyllopteryx taeniolatus) were found dead in an aquarium of the Zoological Garden Basle, Switzerland. Clinically, the animals showed only faint symptoms of disease over a short period of time. At necropsy, macroscopic lesions were confined to the skin with multiple, often hemorrhagic, ulcerations. Histologically, epidermal ulcers were associated with necrosis and inflammation of the underlying dermis and musculature. Numerous ciliates, with a morphology consistent with scuticociliates, were present in these lesions. In several animals these ciliates had invaded blood vessels and were detected in gills and internal organs including kidney, thyroid gland, and central nervous system (CNS). In these organs, mild degenerative lesions and inflammatory reactions were evident. The ciliates were identified as Philasterides dicentrarchi based on small-subunit ribosomal RNA (SSUrRNA) gene sequences obtained by polymerase chain reaction (PCR) on DNA extracted from paraffin-embedded tissue sections. Our report shows that scuticociliate infections of sea dragons can develop into a systemic infection and that both species of sea dragons can be affected.     

Korean mistletoe enriched diet enhances innate immune response in kelp grouper, Epinephelus bruneus against Philasterides dicentrarchi

The present study investigated the immunostimulatory effect of Korean mistletoe Viscum album extract (KM-E) on innate immune response in kelp grouper Epinephelus bruneus against Philasterides dicentrarchi. Kelp grouper were divided into four groups of 25 each and fed with 0 (control), 0.5, 1.0, and 2.0% enriched diets with Korean mistletoe extract (KM-E). After feeding for 30 days, the fish were injected intraperitoneally (i.p.) with 100 μl of P. dicentrarchi (4.2 × 10(7)ciliates/ml) to study the immune responses at weeks 1, 2, and 4. The respiratory burst activity did not significantly enhance when fed with 0.5% and 1.0% supplementation diets on week 1 when compared to control diet. On weeks 2 and 4, the respiratory burst activity significantly increased with 1.0% and 2.0% diets. The phagocytic activity significantly enhanced with 1.0% and 2.0% diets, but not with 0.5% diet at any time. When fed with 1.0% KM-E-diet the lysozyme activity did not significantly vary at any week whereas with 1.0% and 2.0% diets it was significantly enhanced. The total protein level significantly increased with 1.0% and 2.0% KM-E-diets from weeks 1 to 4 as compared to control. The present study suggests that 1.0% or 2.0% KM-E-supplementation diet positively enhances the innate immune response in E. bruneus against P. dicentrarchi infection.     

Molecular characterization of Malassezia nana isolates from cats

Malassezia nana (M. nana) is a lipid-dependent yeast that has been isolated from cats and cows. Some sequence variability has been observed in the large subunit (LSU) and internal transcribed spacer (ITS) regions between strains isolated from cats and cows though these regions in M. nana isolates from cats alone have proven to be relatively conserved. In the present study, microsatellite PCR fingerprinting and β-tubulin gene sequence analysis were carried out on M. nana isolates from cats to investigate the genetic diversity of this species. Although a relatively small number of isolates were available, the similarity in the sequences of the β-tubulin and the microsatellite profiles indicate that a particular M. nana genotype colonizes cats. Moreover, all isolates obtained from animals with otitis externa had the same microsatellite fingerprinting pattern. Further studies of a wider population of M. nana isolates from other hosts and status disease are needed to establish that M. nana is a genetically homogeneous species. This is the first report of the characterization of the β-tubulin gene in Malassezia spp.     

仲彬草属物种细胞质基因组PCR-RFLP分析

应用PCR-RFLP技术对32份仲彬草属和2份外类群共34份材料细胞质基因组DNA进行遗传变异分析。选用的16个引物中4个叶绿体引物和5个线粒体引物可扩增出稳定而明显的PCR产物,其扩增产物用15种限制性内切酶进行酶切。结果发现,所有的135种引物/酶组合中,得到清晰稳定的83种引物/酶组合,其中在32份仲彬草属材料中有40种引物/酶组合(占48.2%)能检测到多态性,但多态性水平较低。32份仲彬草属物种间的遗传相似系数在0.833以上,平均值为0.903。聚类分析表明,细胞质基因组PCR-RFLP标记能将全部供试材料区分开,32份仲彬草属材料聚为4类。同种不同居群细胞质间的遗传变异很小,分别聚在一起;种间的细胞质遗传差异大于种内不同居群间的遗传差异;而形态相似、地理分布一致的物种有聚类在一起的倾向。因此,利用细胞质基因组PCR-RFLP标记不仅可检测到仲彬草属种间多态性,也可检测到种内多态性。细胞质基因组PCR-RFLP标记能为仲彬草属物种系统学研究提供有价值的资料。     

基于线粒体细胞色素b基因的细颈囊尾蚴种群遗传多样性研究

为探讨我国细颈囊尾蚴种群之间的遗传相似性及与其他带科带属绦虫的亲缘关系,用线粒体细胞色素b(Cyt b)基因全序列研究了分离自我国四川和云南2省7地区猪、山羊和绵羊共33个细颈囊尾蚴的遗传变异,并用MEGA 4.0程序NJ法和PAUP 4.0程序MP法绘制种系发育树.本研究结果显示:细颈囊尾蚴分离株的Cyt b基因全长为1 068 bp,共检测到22个单倍型,单倍型间核苷酸相似性较高(97.3％～99.9％),其与带科带属其他5种绦虫的相似性均在82％以下.系统发育树显示,7个不同地域的细颈囊尾蚴聚为一支,分为3个亚群,且与地理区域、宿主没有直接的相关性,呈现混杂的分布格局,未形成明显的地理分支或宿主分支.研究结果表明:细颈囊尾蚴Cytb基因存在一定的遗传差异,但种内相对保守,与其他带科绦虫种间差异较大,故可作为种间遗传变异研究的标记,并为细颈囊尾蚴的种群遗传学研究及其与其他带科绦虫病的鉴别诊断的建立奠定基础.     

Ribotype diversity of porcine Pasteurella multocida from Australia

OBJECTIVE: To use the technique of ribotyping to investigate the genetic diversity of Australian isolates of Pasteurella multocida associated with outbreaks of clinical disease in Australian pigs. DESIGN: One hundred and seven porcine P multocida isolates were analysed by ribotyping using the restriction enzymes HpaII and HindIII. The genetic population structure of the Australian porcine P multocida isolates was determined through statistical analysis of the joint ribotype patterns, and this was then compared with biochemical and epidemiological data available for the population. RESULTS: A total of 25 combined ribotypes were recognised, which were grouped into five ribotype clusters. Despite the deliberate selection of diverse isolates, the study revealed only a limited degree of genetic diversity. Fourteen of the ribotypes contained multiple isolates, and 12 of these ribotypes were present on more than one farm. Three of the seven biovars analysed in the study showed very limited diversity. All fifteen biovar 2 isolates (subsp multocida) were found in a single cluster (III), while all four biovar 8 isolates, which correspond to P multocida subsp gallicida, were allocated by themselves to a single cluster (IV). All nine of the biovar 12 isolates (lactose-positive subsp multocida) were assigned to a single cluster (I), together with the single biovar 14 isolate, which was the only other lactose-positive isolate in the population (ODC-negative). CONCLUSION: A limited number of ribotypes of P multocida are associated with Australian pigs. The majority of these ribotypes are widely distributed across multiple farms, and across multiple states. Individual farms can possess multiple ribotypes of P multocida. Some of the unusual biochemical variants of P multocida present in Australian pigs have a very limited genetic diversity. The nature of pig production in Australia, primarily involving continuous flow systems with few closed herds, has possibly contributed to the widespread distribution of a limited number ribotypes.     

Intraspecific variation in the 16S rRNA gene sequences of Mycoplasma agalactiae and Mycoplasma bovis strains

Intraspecific variation in the 16S rRNA genes of 17 Mycoplasma agalactiae and eight Mycoplasma bovis isolates was investigated to determine the degree of sequence variation in these two species and to determine whether the polymorphisms in the 16S rRNA genes could be used for the construction of an evolutionary tree and as epidemiological markers. A high degree of variation was found within isolates (between operons) and between isolates of both species. In contrast to M. capripneumoniae no distinct evolutionary pattern could be seen, probably because there are functional systems for gene conversion in M. agalactiae and M. bovis. However, the non-European isolates of M. agalactiae shared three characteristic nucleotides and European isolates from the same or neighbouring countries were very similar. Differences within isolates included both polymorphic positions and sequence length differences between operons. The amount of variation within isolates of the respective species ranged from zero to seven polymorphisms for M. agalactiae and from zero to four polymorphisms for M. bovis. The high degree of variation suggests the potential for misdiagnosis of species in diagnostic PCR assays based on the 16S rRNA gene sequences. All isolates of both species had a thymidine in position 912 (E. coli numbering) that causes streptomycin resistance in several bacterial species and which is characteristic for the members of the hominis group. As expected, when five M. agalactiae and three M. bovis isolates were tested for streptomycin susceptibility, they all demonstrated streptomycin resistance. M. agalactiae and M. bovis were found to have high intraspecific variation in their 16S rRNA gene and the polymorphisms patterns indicate that gene conversion takes place.     

河南省狗牙根种质资源表型遗传多样性研究

基于7个表型性状对河南省自然分布的15个狗牙根居群的表型遗传变异及其与环境因子的关系进行研究,结果表明狗牙根供试性状总的变异系数从大到小依次为草层高度(40.18％)>匍匐茎叶长(36.46％)>节间长(33.20％)>直立茎叶长(32.77％)>匍匐茎叶宽(31.81％)>直立茎叶宽(28.57％)>节间直径(27....     

Morphological and molecular characterization of Pseudocohnilembus longisetus Thompson, 1965 from farmed black rockfish Sebastes schlegelii in Korea

The morphology, infraciliature, silverline system, and the small subunit ribosomal RNA (SSU rRNA) of the little-known marine scuticociliate Pseudocohnilembus longisetusThompson, 1965 from the diseased black rockfish Sebastes schlegelii in Korea were studied. This scuticociliate possessed the typical characteristics of the genus Pseudocohnilembus, but could be discriminated from Pseudocohnilembus hargisi, and Pseudocohnilembus persalinus in terms of the body size, shape, the number of somatic kineties and kinetids in somatic kinety 1, and the number/position of contractile vacuole pores. The SSU rRNA gene of P. longisetus was sequenced in order to gain a better understanding of appropriate phylogenetic classification. The SSU rRNA was 1754 bp and the sequence was deposited in GenBank under accession number FJ899594. The SSU rRNA gene sequences of P. longisetus had an identity of 98.1%, 96.8% and 95.3% with P. hargisi, P. persalinus, and Pseudocohnilembus marinus SSU rRNA sequences, respectively. Our population of P. longisetus belonged to the genus Pseudocohnilembus and was in an isolated position based on the SSU rRNA gene tree, which was consistent with the conclusions based on the morphological studies. However, further investigation is required to determine the pathogenicity of this species.     

红河橙叶表型遗传多样性研究

红河橙（Citrus hongheensis Ye et al.）是云南特有的珍稀柑橘种质资源,仅在元江、红河南岸有零星分布,其单身复叶具有极为发达的翼叶。本研究对红河橙在绿春县、红河县和元江县的11个居群进行调查和取样,采用巢式方差分析、多重比较、主成分分析等方法对12个叶表型的变异和遗传多样性进行分析。结果表明：红河橙12个叶表型在居群间和居群内均表现为极显著差异（P<0.01）,其叶表型遗传变异水平较高;同一叶表型性状在不同居群间的变异程度不同,其中翼叶宽/本叶宽这个叶表型的变异程度最大（91.36 %）,而叶总长的变异程度最小（8.24 %）,其表现相对最为稳定;叶片的绝对大小的变异程度（如叶总长、翼叶长度、翼叶宽度、本叶长度、本叶宽度、叶柄长等）普遍低于叶片形状（如翼叶长/宽、本叶长/宽）和叶片相对大小（如翼叶长/叶总长、翼叶长/本叶长、翼叶宽/本叶宽、本叶长/叶总长、本叶长/翼叶长、本叶宽/翼叶宽等）的变异程度,且表现出叶片形状变异>叶片相对大小变异>叶片绝对大小变异的变异规律;红河橙12个叶表型性状在居群间和居群内的方差分量百分比分别为12.762%和34.718%,平均表型分化系数为21.581%,居群内变异是红河橙叶表型变异的主要来源;主成分分析表明前3个主成分（叶片绝对大小因子、叶片相对大小因子、叶形指数因子）的累计贡献率达85.258%。研究结果可为红河橙的群体遗传进化及种质资源保护和利用等提供形态学方面的参考。     

中国野生老芒麦形态多样性研究与种质利用潜力分析

为揭示野生老芒麦形态变异特征和遗传背景，对我国野生老芒麦主要分布区域不同生态类型的104个居群520份野生老芒麦种质进行了23个形态指标测定和遗传多样性分析。结果表明：不同生境老芒麦材料的形态特征存在显著差异，数量性状变异系数为9.49%～49.56%；聚类分析将104个居群分为具有各自明显特征和开发利用潜力的4类；形态特征与地理环境因子的相关性分析发现，野生老芒麦抽穗早晚及茎秆叶鞘基部小刺等特殊性状与其海拔呈显著相关；主成分分析发现，株高、抽穗、茎节数、单株干鲜重、旗叶长宽等指标代表了老芒麦67.79%形态多样性，是造成老芒麦形态特征变异的主要因素，可作为老芒麦形态分化的重要参考指标。     

Defining and interpreting intraspecific molecular variation.

Defining the extent and character of intraspecific genetic variation provides important information about gene function and organismal history. Powerful tests may be applied to sequenced alleles in order to critically examine whether natural selection is responsible for limiting or elevating intraspecific polymorphism in particular genes. Unconventional patterns of sequence variation and unusual allelic frequency distributions can be used to test whether genes encoding parasite antigens are being diversified by immune selection. The strikingly limited genetic variation in the falciparum malaria genome, and in human chromosomes encoding resistance to severe malaria, date the emergence of this disease to within the last few thousand years, illustrating the power of population genetic analysis to elucidate the history of host-parasite interactions. Coupling phylogenetic and geographic information and analyzing the rate of diversification in intraspecific gene trees provides new and rich sources of information on microbial evolution and epidemiology.     

尼日利亚非洲蜂的形态特征研究

测定本地意蜂（安徽肥东）、西非尼日利亚非洲蜂、本地意蜂与非洲蜂杂交第一代的蜂群工蜂吻长、右前翅长、右前翅宽、肘脉指数、第三背板宽度和第四背板宽度,并对测定的性状特征数据进行数学分析,结果表明,本地意蜂（安徽肥东）（A）、西非尼日利亚非洲蜂（B）、本地意蜂与非洲蜂杂交第一代的蜂群（C）的吻长、右前翅长、前翅面积、前翅肘脉a、第三节背板宽、第四节背板宽都存在极显著的差异,右前翅宽存在显著差异,而三者的前翅肘脉b以及翅脉指数差异不显著。这三种蜜蜂种群内遗传变异丰富,种群形态性状的多态性、多型性及其生态地理变异式样,具有生态适应意义。     

假俭草杂交后代部分外部性状的遗传分析

选用假俭草2份外部性状存在差异的种源材料E102和E092(1)进行杂交,获得F₁分离群体,应用植物数量性状主基因+多基因混合遗传模型分析方法对F₁群体的叶片长度与宽度、节间长度与直径及草层高度等外部性状进行遗传分析,以初步明确假俭草上述性状的遗传特性。结果表明,1)在调查的杂交后代5个外部性状中,变异系数由大到小依次为叶片长度(23.39%)＞节间长度(21.89%)＞草层高度(15.32%)＞节间直径(7.41%)＞叶片宽度(6.77%),变异系数最大的为叶片长度,最小的为叶片宽度。2)叶片长度的最佳遗传模型为A-1模型,即1对主基因模型,该主基因表现为加性和部分显性或超显性, 主基因遗传率为68.98%;节间长度的最佳遗传模型为A-4模型,1对主基因模型,该主基因表现为负向完全显性,主基因遗传率为47.37%;草层高度的最佳遗传模型为B-6模型,即2对主基因模型,主基因表现为等显性模型,主基因遗传率为47.20%;叶片宽度和节间直径的最佳遗传模型均为A-0模型,即无主基因模型。     

内蒙古野生披碱草属牧草居群穗部形态多样性研究

采用14项穗部形态学性状对内蒙古地区披碱草属牧草进行形态多样性研究,结果表明,14项穗部形态性状均存在较大的变异,平均变异系数为27．32％,变异系数最大为旗叶与穗基部长度（64．23％）,最小为第一内稃长（12．25％）。14项穗部形态性状中小穗数的居群总多样性指数最高为2．051,最低的为每穗轴小花数（1．294）。披碱草属牧草种质资源遗传多样性丰富,居群内多样性指数最高为1．652（AE050）,最低为1．019（AE027）;居群总平均多样性指数为1．903,居群内平均多样性指数为1．407,表型分化系数为22．77％。14项穗部性状中穗长、穗宽、穗长、小穗数、第一颖长、每穗轴小花数和第一外稃长是引起表型变异的主要性状,这些性状与披碱草属牧草分类及种子产量密切有关。聚类分析表明,地理来源不同的同种材料能集中地聚在一起,说明相同物种即使地理位置相距较远,其亲缘关系仍然是最为接近,肥披碱草存在变异现象。此外,还提出了我国披碱草属牧草保护的策略。     

Supplemental diagnosis of a myxozoan parasite from common carp Cyprinus carpio: synonymy of Thelohanellus xinyangensis with Thelohanellus kitauei

Thelohanellus kitauei Egusa et Nakajima, 1981, was described from common carp Cyprinus carpio L. in Japan. In China, a myxosporean infecting the intestinal tissue of the same host species was described as Thelohanellus xinyangensis Xie, Gong, Xiao, Guo, Li et Guo, 2000, despite many similarities to T. kitauei. To examine the potential conspecificity of these species, a morphological and molecular investigation of T. xinyangensis was conducted. Comparing myxospore morphology, the mean spore length and width of each species is not identical between species, but ranges of dimensions overlap. These data are more suggestive of intraspecific variation than distinct species. Comparison of relative ratios of spore length to polar capsule length and spore width to polar capsule width of T. xinyangensis and T. kitauei reveal no differences and scanning electron microscopy reveals a smooth spore surface of T. xinyangensis, which is consistent with that of T. kitauei. Most convincingly, DNA sequences of the small subunit ribosomal RNA (ssrRNA) gene of the two species were identical. From the morphological and molecular biological data, we propose T. xinyangensis from the intestine of common carp is not a distinct species and is synonymous with T. kitauei.     

披碱草属植物形态多样性及其主成分分析

以我国9种披碱草的40个居群为材料,对28个形态特征进行聚类和主成分分析。结果表明:以遗传距离系数0.78为尺度把40个披碱草居群分为两大类,第1类主要包括垂穗组披碱草种类,第2类大多数包括穗直立的披碱草种类。披碱草属每个种的各居群基本能够聚在一起,并和地理条件有一定的关系;披碱草(Elymus dahuricus)、圆柱披碱草(E.cylindricus)和紫芒披碱草(E.purpuraristatus)变异性很大,而垂穗披碱草(E.nutans)、老芒麦(E.sibiricus)归类比较清晰明显;在形态分化的指标中,与生产应用及分类功能联系密切的17个特征向量可反映总变异85%以上的信息,根据贡献的大小分为10个不同的层次;单株重、单穗重、分蘖数、生殖枝长、旗叶与穗基部长度、穗色、小穗长、外稃、芒长、小穗宽、穗宽、生长速度(出苗-幼苗)、颖宽、植株绒毛分级、生长速度(幼苗-分蘖)、叶片颜色和株高是披碱草形态分化的主要指标。