Fish-isolated Naegleria strains and their phylogeny inferred from ITS and SSU rDNA sequences

Effort was made to identify Naegleria strains isolated from organs of fish, using phylogenetic analyses of SSU rDNA and ITS sequences. Eighteen fish-isolated strains studied enlarged substantially the so far available set of Naegleria strains characterized by both molecular markers. The phylogenetic analyses of separate and concatenated SSU rDNA and ITS sequences revealed phylogenetic relationships of strains under study; however, they failed to solve classification of fish-isolated strains into species. The sequence similarity of strain-representatives of Naegleria species as well as data obtained on intragenomic variation of ITS sequences discouraged the authors from the definition of new species. The results of the present study provide evidence of a need to re-evaluate the current practice of setting boundaries between species of the genus Naegleria. Sequences obtained in this study have been deposited in GenBank with accession numbers DQ768714-DQ768743.     

Viability of pathogenic Naegleria and Acanthamoeba isolates during 10 years of cryopreservation

This is a follow-up report on the viability of pathogenic Naegleria fowleri, Naegleria australiensis and Acanthamoeba castellanii isolates during 5 to 10 years of cryopreservation at -70 degrees C. The greatest decrease in viability occurred with N. fowleri and the least occurred with N. australiensis. At 10 years of cryostorage, viability was 21% for N. fowleri, 32% for A. castellanii and 51% for N. australiensis.     

New data on aetiology of nodular gill disease in rainbow trout, Oncorhynchus mykiss

We studied amoebae associated with nodular gill disease (NGD) outbreaks in rainbow trout Oncorhynchus mykiss (Walbaum) in fish farms in South-Western Germany. Gills of 12 diseased rainbow trout were examined in fresh, by isolation attempts, histologically and using in situ hybridisation (ISH). A total of nine amoeba strains of the genera Acanthamoeba (1), Hartmannella (2), Naegleria (1), Protacanthamoeba (1) and Vannella (4) were isolated and determined using light microscopical, ultrastructural and molecular methods. Specific molecular probes designed from the SSU rDNA sequences of individual amoeba strains were used for non-radioactive ISH in histological sections. Association of Naegleria sp. with NGD and a direct ISH proof of Naegleria trophozoites attached to hyperplastic gill epithelium are novel findings, expanding the number of possible agents of NGD and supporting the hypothesis on multicausal aetiology of this disease.     

Pathogenesis of pathogenic Naegleria amoeba.

In brain sections of the Naegleria-caused cases of primary amoebic meningoencephalitis, extensive demyelinization was found in the white matter, besides the severe histopathological changes and large clusters of trophozoites in the grey matter. The myelinoclasis appeared to be a result of a specific phospholipolytic effect, unlike that in post-viral encephalomyelitis, which has been attributed to vascular blockade or hemorrhages. In monkey kidney cell cultures a very early cytopathic effect was observed and traced to the cytolytic property of the seeding culture fluid. Rat brain slices inoculated with Naegleria culture exhibited amoebic growth and demyelinization in 28-52 hours incubation at 35 degrees C. In a chemically defined medium containing sphingomyelin, casein and glucose, the Naegleria produced a limited growth parallelling the clearance of the lipid turbidity during a 72 hour incubation at 35 degrees C. Chromatographic analysis of the turbidity-cleared cultures revealed decomposition of sphingomyeline with liberation of choline, sphingosine and fatty acids. It is, hence, concluded that the pathogenicity of cytopathic effect of pathogenic Naegleria can be attributed to the latter's capacity to liberate a phospholipolytic enzyme or factor during active growth, which "makes holes" in the lipid-rich cytoplasmic membrane of cells as well as demyelinizes nerve tissue.     

The effects of some factors on the growth and morphology of Naegleria sp. and three strains of the genus Acanthamoeba.

The effects of various biophysical and chemical factors on the cytology of vegetative stages of Naegleria sp., Vitek strain, Acanthamoeba culbertsoni, Acanthamoeba castellanii, Neff strain and Acanthamoeba polyphaga, No. 1289, were studied. The amoebae were cultured in a liquid medium under axenic conditions. The optimum temperature was 37 degrees C for pathogenic strains of Naegleria sp. and Acanthamoeba culbertsoni and 20 degrees C for A. castellanii. No changes were observed in the growth of A. polyphaga at the temperatures 20 degrees and 37 degrees C. The strains investigated grew at pH values of 5.6 to 7.7 using Soerensen's buffer. At the limit values the growth was inhibited and the morphology of cells was markedly changed. All of the four strains grew still at pH 8.4 kept by NaHCO3. A. polyphaga grew at partial anaerobiosis. The three tested strains of the genus Acanthamoeba grew in liquid axenic medium with 0.89% NaCl. The growth of Naegleria sp., Vitek was inhibited already at 0.2% concentration of this salt. The addition of 3 X 10(-2) m KCl to the culture medium had a harmful effect on the growth and morphology of three tested strains, except A. polyphaga. In the culture medium containing 2 X 10(-3) m CaCl2 the encystment of both pathogenic strains was stimulated. The cytological changes under experimental conditions were manifested by atypical movement of trophozoits and their intracellular structure.     

Flagella number among Naegleria flagellates.

Scanning electron microscopy was used to determine the number of flagella on the flagellates of Naegleria australiensis, N. fowleri, N. gruberi, and N. jadini. Although the majority of flagellates had 2 flagella, there was considerable variation among individual cells. The number of flagella per flagellate varied from 1-8, with 2.4 being the average number per cell. For the different species, the average number of flagella per cell ranged from 2.0 in N. jadini to 3.1 for N. australiensis. The greatest amount of variation occurred in N. australiensis, with only 43% of the cells having 2 flagella. By contrast, 92% of N. fowleri cells had 2 flagella. Naegleria jadini and N. gruberi were intermediate with 80% and 74% biflagellates, respectively.     

Some further characteristics of the growth of Naegleria fowleri and N. gruberi in axenic culture.

The effects of pH, various viscosity of the medium, changed ratio between the concentrations of dissolved and corpuscular components in the medium, and dissolved inorganic salts on the growth of axenic cultures of Naegleria fowleri and N. gruberi have been studied. The cultures were grown in liquid CALYG and BCS media. The pH optimum was 6.5 for N. fowleri and 6.0--6.5 for N. gruberi. No negative influence on the growth of N. fowleri was observed even at 0.5% concentration of highly viscous methylcellulose, whereas the growth of N. gruberi was distinctly inhibited by more than 0.2% of methycellulose. N. fowleri preferred the osmotorphic and N. gruberi phagotrophic nutrition in the given system of cultivation. The growth of both Naegleria species was inhibited by 0.1 N concentration of sodium chloride and potassium chloride without any significant difference in the tolerance. The inhibitory effect of these salts correlated primarily with the concentration of chloride anion. The ability to grow in a medium with increased viscosity and the preference for osmotrophic nutrtion are, besides the higher temperature optimum determined earlier, further characteristics of the pathogenic species N. fowleri.     

Mammalian cell cultures affected by Naegleria gruberi

Amebae of 8 strains of Naegleria gruberi were able to destroy 10 established mammalian cell lines including lung, kidney, ovary, connective tissue, neuroblastoma, and laryngeal and cervical carcinoma cells. The strains of N. gruberi varied in their ability to produce a destructive effect (DE) in African green monkey kidney (Vero) cell cultures. However, cell line susceptibility was found to be equivalent when tested with the considerably destructive 1518/l strain of N. gruberi. The Vero cell line proved to be a useful indicator culture for assessing the destructive potential of N. gruberi strains. Other factors affecting the extent of DE produced were ameba to mammalian cell ratio and the length of time that amebae were maintained in cell culture.     

Advances in the knowledge of amphizoic amoebae infecting fish

Free-living amoebae infecting freshwater and marine fish include those described thus far as agents of fish diseases, associated with other disease conditions and isolated from organs of asymptomatic fish. This survey is based on information from the literature as well as on our own data on strains isolated from freshwater and marine fish. Evidence is provided for diverse fish-infecting amphizoic amoebae. Recent progress in the understanding of the biology of Neoparamoeba spp., agents responsible for significant direct losses in Atlantic salmon and turbot industry, is presented. Specific requirements of diagnostic procedures detecting amoebic infections in fish and taxonomic criteria available for generic and species determination of amphizoic amoebae are analysed. The limits of morphological and non-morphological approaches in species determination are exemplified by Neoparamoeba, Vannella and Platyamoeba spp., which are the most common amoebae isolated from fish gills, Acanthamoeba and Naegleria spp. isolated from various organs of freshwater fish, and by other unique fish isolates of the genera Nuclearia, Thecamoeba and Filamoeba. Advances in molecular characterisation of SSU rRNA genes and phylogenetic analyses based on their sequences are summarised. Attention is particularly given to specific diagnostic tools for fish-infecting amphizoic amoebae and ways for their further development.     

The role of blood vessels and lungs in the dissemination of Naegleria fowleri following intranasal inoculation in mice

Primary amoebic meningoencephalitis (PAM) was induced in mice by intranasal inoculation of Naegleria fowleri (Singh et Das, 1970) to study the role of the blood vessels and lungs in the early and later stages in this disease. Upon culturing blood and lung tissue obtained at 24-, 36-, 48-, 72-, 96-, and 120-hour time periods, it was found that amoebae grew only from blood and lung tissue obtained at the 96 and 120 hour time periods. Paraffin sections of the head revealed small foci of acute inflammation and amoebae within the olfactory bulb of the central nervous system (CNS) at 24 hours. Amoebae were not observed within blood vessels of the CNS until 96 and 120 hours. Also, amoebae were observed within the connective tissue surrounding blood vessels and sutures of the skull, bone marrow, and venous sinusoids between the skull bone tables at 96 and 120 hours. No amoebae or acute inflammatory reactions were observed in the lung sections from any time period and indirect immunofluorescence microscopy was negative for N. fowleri. This study provides evidence that neither blood vessels nor lungs provide routes for N. fowleri to the CNS during the early stages of PAM and that amoebae enter veins of the CNS and bone marrow during later stages of the disease.     

Detection of PSTVd and TCDVd in seeds of tomato using real‐time RT‐PCR

Worldwide outbreaks of pospiviroids in potato and tomato have increased the need for a reliable test for the detection of pospiviroids in seeds. This study describes the development and validation of a sensitive and fast test for the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in tomato seeds. The test is based on RNA isolation using a commercial kit and is suitable for routine application. The test is able to detect one PSTVd or TCDVd contaminated seed in sub samples of 1000 seeds and results were both repeatable and reproducible.     

收获期豌豆籽粒离散元仿真参数标定

针对收获期豌豆籽粒在脱粒与清选仿真模拟中缺乏准确离散元仿真参数的难题,采用物理试验和仿真试验相结合的方法对豌豆籽粒仿真参数进行标定。以籽粒堆积角为评价指标,通过Plackett-Burman试验筛选显著性参数,借助最陡爬坡试验和Box-Behnken试验得到显著性参数最优组合,通过仿真试验标定非显著性参数。结果表明：籽粒间碰撞恢复系数、静摩擦系数和滚动摩擦系数分别为0.364、0.519和0.444;豌豆籽粒-钢碰撞恢复系数、静摩擦系数和滚动摩擦系数分别为0.505、0.462和0.090;物理和仿真试验堆积角分别为19.841°和19.714°,其相对误差为0.64%。研究可为豌豆机械化收获过程离散元仿真分析提供参考。     

一种农药药液润湿性测试卡的制备及其性能分析

本论文介绍一种农药药液润湿性测试卡的制作,即将指示剂涂布在特定图案的纸卡上,再覆盖一层石蜡.使用时,把液滴加到测试卡上,液滴铺展所到之处发生显色反应,根据显色面积读数,以此判断溶液的润湿性.选用不同浓度有机硅表面活性剂Silwet 408水溶液,对其表面张力,在测试卡上的铺展系数和接触角,在黄瓜、番茄、油菜等6种植物叶片表面的接触角分别进行了测定,初步分析了该测试卡的润湿性能.结果表明,该种测试卡具有与其他自然植物叶片相似的性质.浓度0.003％的Silwet 408水溶液铺展系数为3.3,对应表面张力26.79 mN/m,在小麦表面的接触角66.5°,可见测试卡能反映药液的铺展系数、表面张力、接触角等多种因素,为田间喷雾助剂的选用提供了快速直观的判别工具.     

Side-effects of 107 pesticides on the whitefly parasitoid Encarsia formosa, studied and evaluated according to EPPO guideline no. 142

The side-effects of plant protection products on beneficial arthropods have been studied by the Netherlands Plant Protection Service since 1974. Laboratory test methods were developed in the context of IOBC/WPRS for Encarsia formosa , a natural enemy of glasshouse whitefly, Trialeurodes vaporariorum. These methods were elaborated by EPPO into a sequential decision-making scheme and published as an official EPPO guideline in 1989. The scheme includes a residual toxicity test on adults, a direct contact test on pupae, a persistence test on adults, all in the laboratory, and a field test. Following this guideline, the Netherlands Plant Protection Service tested and evaluated 107 pesticides at 307 concentrations over 10 years. Test details and complete test results are reported, including the risk assessments according to the EPPO scheme and according to the IOBC/WPRS conventions. These results are further summarized per type of pesticide, kind of test and risk classification. The efficiency of the scheme in classifying pesticide concentrations for risk to E. formosa is analysed. The scheme was found to be reasonably practical and efficient except for pesticides in the range between safe and hazardous. Suggestions for improvement are given. Also the possible need for changing the original IOBC/WPRS-criterion for harmlessness (effect < 50%) is discussed. No reasons for lowering the criterion to 30% were found. The decision-making scheme fits into the general approach for environmental risk assessment of plant protection products, developed recently by EPPO and the Council of Europe.     

An evaluation of a virobacterial agglutination test for the detection of plant viruses

Agglutination of Staphylococcus aureus bacteria particles, conjugated with specific virus antibodies, has been used to identify a wide range of plant viruses in crude sap extracts. The test distinguishes between seven different potyviruses in homologous and heterologous reactions, but does not distinguish different strains of bean yellow mosaic virus. The sensitivity of the virobacterial agglutination (VBA) test compares favourably with virus detection in ISEM particle trapping and local lesion assay and is only slightly less sensitive than direct ELISA tests. The test is more sensitive and simpler to user than latex particle agglutination tests.     

基于EDEM的沙棘冻浆果振动清选装置参数优化与分析

针对沙棘冻浆果清选加工中存在清洁率低,筛网易堵塞的问题,采用离散元EDEM软件对振动清选装置的结构和运动参数进行优化。以速冻沙棘枝条果机械脱果后的冻浆果、枝条和果梗等颗粒为试验物料,测定物料的尺寸特征,进行振动清选装置动力学分析和筛上颗粒受力分析。分析筛网不同形状以及尺寸的筛孔组合对振动清选装置清洁率和透筛概率的影响,确定筛网设计参数为第一层筛网为圆孔、孔径为11 mm,第二层筛网为圆孔、孔径为9 mm,第三层筛网为长孔、孔径为5 mm×25 mm。单因素试验结果表明：振幅的改变未显著影响洁净率和透筛率,在保证高清洁率时选取筛体振幅为6 mm。选取振动频率,振动方向角,筛面倾角为试验因素,清洁率和透筛概率为试验指标,设计二次正交旋转组合试验,建立各因素与指标间的数学模型,优化结果最优组合为：振动频率为17.52 Hz、振动方向角为44.7°、筛面倾角为1.56°时,振动清选装置清洁率为99.62%、透筛概率为33.42%。通过台架试验和仿真试验对最优参数组合进行验证,结果表明：台架试验和仿真试验结果基本一致,清洁率的相对误差为1.13%,颗粒透筛数量的相对误差为1.68%。该研究可为沙棘冻浆果振动清选装置的优化设计提供理论依据。     

Comparison of different PCR tests to identify Monilinia fructicola

Monilinia fructicola was until very recently a regulated pest in the European Union, and EU countries were requested to monitor its presence on their territories. As accredited laboratories should use validated tests, the mycological laboratory of CRA‐PAV carried out a validation process for the multiplex based PCR test (Coté et al., 2004 ), that is one of the most widely used tests for the identification of M. fructicola, although this test is not described in the EPPO diagnostic protocol PM 7/18 (2) because the validation data were lacking. The performance characteristics of this multiplex PCR test were established according to the EPPO Standard PM 7/98 (1) and the test was compared in a collaborative study with the end point PCR test (Ioos & Frey, 2000 ), considered as the ‘standard test’. The validation data were obtained using different isolates of M. fructicola, M. laxa, M. fructigena and Monilia polystroma, as well as different fruit tissues. Four series of the DNA target at different concentration, repeated three times, were analyzed in four Italian laboratories. The results showed that the multiplex PCR detection test (Coté et al., 2004 ) was fit for diagnostic purpose, although the analytical sensitivity was significantly lower compared to the conventional PCR ‘standard test’.     

农药登记环境影响试验试材使用现状及管理建议

农药环境影响试验数据是开展农药环境风险评估的重要依据,而试验试材又是影响试验数据可靠性的关键因素。农药生态毒理学和环境归趋试验涉及的试材种类繁多,主要包括土壤试材和20多种生物试材,规范化管理难度较大。本文归纳总结了国内外农药环境影响试验准则中对试材的要求,调研了目前国内农药环境影响实验室的试材使用情况,并对试材规范化管理提出了建议。     

Assessment of partial resistance of potato to, and pathotype and virulence differences in, potato cyst nematodes

Results are reported for several international collaborative experiments which examined methods of assessing degrees of partial resistance in potato and virulence in potato cyst nematode (PCN, Globodera pallida). It was demonstrated that absolute rates of multiplication can be extremely variable on both susceptible and partially resistant clones, even when the same population and test procedures are employed. It was therefore concluded that, on clones with quantitatively inherited resistance (i.e. partially resistant), absolute rates of multiplication cannot be used to separate pathotypes. Expression of these rates as percentages of those on the susceptible controls reduced the absolute differences between tests, but the values obtained were still too variable for statutory use. However, whatever the environment or nematode population used, it was observed that the resistance of the test clones and virulence of the nematode populations were generally ranked in a similar order. The main exceptions to this were: (1) a Petri-dish test where cv. Vantage was less resistant than in canisters or pots and (2) a pot test where cv. Darwina tended to be more interactive with environmental factors than the other test clones. It was also observed that with some populations of PCN in pots the susceptible cv. Bintje was a less good host than cv. Désirée. On the basis of these results it is suggested that certain partially resistant clones should be used as internal references in statutory, recommended list and breeders' assessment tests against which the resistance of the test clones are compared. For international comparability it is necessary that the different centres conducting such tests use the same reference clones and nematode populations, and similar test methods.     

地面灌溉条件下高产棉田水肥耦合的产量效应

以中棉43号为材料,采用"3414"方案,在新疆沙雅新垦农场,通过田间小区试验,研究了地面灌溉条件下不同水肥耦合水平对棉花产量的影响,并建立了棉花产量的水肥回归数学模型.结果表明:不同水肥耦合水平对棉花产量都有显著影响;各因素对棉花产量的效应顺序为:施磷量＞灌溉量＞施氮量;各因素间耦合效应顺序为:氮、磷＞氮、水＞磷、水;水肥调控的最佳组合为:氮肥用量为545.55 kg/hm2,磷肥用量为199.8 kg/hm2,灌溉量为6 429.3 m3/hm2.