大片吸虫成虫cDNA表达文库的构建及其表达序列标签初步分析

为发现潜在的抗大片吸虫病的候选疫苗分子,控制大片形吸虫病,本研究以大片吸虫成虫为材料,应用SMARTTcDNA文库构建技术,构建了以表达载体λTriplEx2为基础的大片吸虫成虫cDNA表达文库。经测定,库容量为1·08×106PFU/ml ,重组率为96·6 %,扩增后的文库滴度为2·41×109PFU/ml ,插入片段平均大小约为1 000 bp;经大肠杆菌BM25·8质粒化后,从文库中随机挑选40个重组克隆测序,获得32条有效ESTs ;经BLASTX和BLASTn程序检索和分析,发现有9条ESTs代表已知基因, 16条ESTs相似性较低或无匹配,列为新基因。9条已知基因代表了半胱氨酸蛋白酶、卵壳蛋白、钙连接蛋白等三类功能蛋白,其余新基因也暗示与信号传导、蛋白合成、免疫刺激等基因相关,具有潜在的研究价值[动物学报51 (5) : 879 -883 , 2005]。     

Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam

The two species common of liver fluke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis. Hybrids between these species, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia. Here, we report the presence of hybrid and/or introgressed liver flukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoonotic disease. Specimens examined came from domestic stock (cattle and buffalo) at slaughter and also from human patients. DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial protein-coding genes. Mitochondrial sequences in every case were similar to those of Fasciola gigantica. Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm. This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia.     

Immunoexpression of intermediate filaments and morphological changes in the liver and bile duct of rats infected with Fasciola hepatica

We investigated the immunoexpression of the intermediate filament proteins, cytokeratin and desmin, and the morphological changes in the liver of rats during experimental fasciolosis at 4, 7 and 10 weeks post-infection. Rats were infected with 30 Fasciola hepatica metacercariae. Paraffin sections of the liver were stained using H & E, PAS and azan stains. Immunohistochemical reactions were performed using antibodies against cytokeratin and desmin. The experimental F. hepatica infection led to fibrosis and cirrhosis of the liver, and to inflammation of the common bile ducts. The expression of cytokeratin was increased in the epithelial cells of both the liver bile ductules at 4, 7 and 10 weeks post-infection and in the common bile ducts at 7 and 10 weeks post-infection compared to uninfected rats; expression in the common bile ducts was more intense. The myofibroblasts of the liver and smooth myocytes of the interlobular bile ducts and common bile ducts, showed a slight increase in desmin expression compared to the uninfected rats. The increased expression of cytokeratins in the hyperplastic rat common bile duct epithelium during the biliary phase of fasciolosis at 7 and 10 weeks post-infection may be explained by mechanical irritation by the parasite and an inflammatory reaction in the bile duct epithelium and in periductal fibrous tissue.     

Molecular characterization of Fasciola spp. from the endemic area of northern Iran based on nuclear ribosomal DNA sequences

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered as the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the endemic regions of the North of Iran, Fasciola hepatica and Fasciola gigantica have been previously characterized on the basis of morphometric differences, but the use of molecular markers is necessary to distinguish exactly between species and intermediate forms. Samples from buffaloes and goats from different localities of northern Iran were identified morphologically and then genetically characterized by sequences of the first (ITS-1) and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the ITS of the northern Iranian samples with sequences of Fasciola spp. from GenBank showed that the examined specimens had sequences identical to those of the most frequent haplotypes of F. hepatica (n = 25, 48.1%) and F. gigantica (n = 20, 38.45%), which differed from each other in different variable nucleotide positions of ITS region sequences, and their intermediate forms (n = 7, 13.45%), which had nucleotides overlapped between the two Fasciola species in all the positions. The ITS sequences from populations of Fasciola isolates in buffaloes and goats had experienced introgression/hybridization as previously reported in isolates from other ruminants and humans. Based on ITS-1 and ITS-2 sequences, flukes are scattered in pure F. hepatica, F. gigantica and intermediate Fasciola clades, revealing that multiple genotypes of Fasciola are able to infect goats and buffaloes in North of Iran. Furthermore, the phylogenetic trees based upon the ITS-1 and ITS-2 sequences showed a close relationship of the Iranian samples with isolates of F. hepatica and F. gigantica from different localities of Africa and Asia. In the present study, the intergenic transcribed spacers ITS-1 and ITS-2 showed to be reliable approaches for the genetic differentiation of Fasciola spp., providing bases for further studies on F. hepatica, F. gigantica and their intermediate forms in the endemic areas in Asia.     

Proteomic analysis of glutathione transferases from the liver fluke parasite, Fasciola hepatica

The parasite Fasciola hepatica causes major global disease of livestock, with increasing reports of human infection. Vaccine candidates with varying protection rates have been identified by pre-genomic approaches. As many candidates are part of protein superfamilies, sub-proteomics offers new possibilities to systematically reveal the relative importance of individual family proteins to vaccine formulations within populations. The superfamily glutathione transferase (GST) from liver fluke has phase II detoxification and housekeeping roles, and has been shown to contain protective vaccine candidates. GST were purified from cytosolic fractions of adult flukes using glutathione- and S-hexylglutathione-agarose, separated by 2-DE, and identified by MS/MS, with the support of a liver fluke EST database. All previously described F. hepatica GST isoforms were identified in 2-DE. Amongst the isoforms mapped by 2-DE, a new GST, closely related to the Sigma class enzymes is described for the first time in the liver fluke. We also describe cDNA encoding putative Omega class GST in F. hepatica.     

In vivo studies of the early, peritoneal, cellular and free radical response in rats infected with Fasciola hepatica by flow cytometric analysis

Early recruitment of the peritoneal cell population was observed during migration of newly excysted juvenile flukes. The peritoneal lavages were examined for T cells, cytotoxic NK cells (CNK) and free radicals production of rats at an early stage of infection by Fasciola hepatica. Male Sprague–Dawley rats were infected with 50 metacercariae of F. hepatica and non-infected controls were euthanized 2, 4 and 7 days post infection (d.p.i.), respectively. The peritoneal fluid of experimental animals was analyzed by flow cytometry to estimate cell phenotypes. The peritoneal areas were infiltrated by inflammatory cells, particularly from numerous neutrophils, eosinophils and CD4+ lymphocytes, which were significantly higher for infected rats than non-infected. CNK cells dominated in the peritoneal fluid of infected rats as early as 2 d.p.i. However, after 4 d.p.i. there was a decreased level of CNK cells which may indicate a change from a cytotoxic natural killer (NK) to a regulatory NK response. The challenged group generated very high in vivo levels of inducible nitric oxide (NO) from eosinophils. Superoxide expression was very high in macrophages and neutrophils compared to the uninfected control. In conclusion, our studies suggest that early F. hepatica infection could directly affect lymphoid cells and generate a high in vivo NO production by eosinophils in the peritoneal cavity. Moreover juvenile flukes could stimulate the macrophages and neutrophils to generate H₂O₂ radicals. The host parasite interactions resulting from immune response regulation by effector cells and immune evasion are discussed.     

The late Pleistocene climates of the Lake Zeribar region (Kurdistan,western Iran) deduced from the ecology and pollen production of nonarboreal vegetation

The concept of an arid pleniglacial in the Middle East depends primarily on the interpretation of pollen diagrams including those of Lake Zeribar in the Zagros Mountains of western Iran. It has been assumed that Lake Zeribar was surrounded by a Chenopodiaceae-Artemisia steppe and that the climate was therefore dry. Both assumptions are questioned. The environment of Pleistocene Lake Zeribar may have been similar to the tragacanthic or alpine zone of the modern Zagros Mountains. The dominance by pollen of Chenopodiaceae and Artemisia is explained by low pollen production of high-altitude vegetation, preferential incorporation of pollen of late-blooming plants into the sediments, and high production and long-distance transport of lowland pollen. In any case, high percentages of Chenopodiaceae and Artemisia pollen do not necessarily indicate low annual precipitation but a highly seasonal climate with cold winters and hot, dry summers. Such a climatic regime was in effect continuous except for a period beginning about 10600 B. P. during which summer rainfall or reduced summer drought occurred. This change in seasonality resulted in the dominance of Poaceae pollen and the initial increase in arboreal pollen. A moisture curve based on the ratio between Chenopodiaceae and Artemisia pollen indicates a pleniglacial climate with wet winters and a late-glacial and early-Holocene climate with periods of intense aridity. The climatic history presented here is compatible with non-palynological evidence of regional late Pleistocene climates and with seasonality changes suggested by climatic modelling based on orbital parameters.Abbreviations C/A Chenopodiaceae-Artemisia ratio     

Correlation analysis of Normalized Different Vegetation Index (NDVI) difference series and climate variables in the Xilingole steppe, China from 1983 to 1999

There is a crucial need in the study of global change to understand how terrestrial ecosystems respond to the climate system. It has been demonstrated by many researches that Normalized Different Vegetation Index (NDVI) time series from remotely sensed data, which provide effective information of vegetation conditions on a large scale with highly temporal resolution, have a good relation with meteorological factors. However, few of these studies have taken the cumulative property of NDVI time series into account. In this study, NDVI difference series were proposed to replace the original NDVI time series with NDVI difference series to reappraise the relationship between NDVI and meteorological factors. As a proxy of the vegetation growing process, NDVI difference represents net primary productivity of vegetation at a certain time interval under an environment controlled by certain climatic conditions and other factors. This data replacement is helpful to eliminate the cumulative effect that exist in original NDVI time series, and thus is more appropriate to understand how climate system affects vegetation growth in a short time scale. By using the correlation analysis method, we studied the relationship between NOAA/AVHRR ten-day NDVI difference series and corresponding meteorological data from 1983 to 1999 from 11 meteorological stations located in the Xilingole steppe in Inner Mongolia. The results show that: (1) meteorological factors are found to be more significantly correlation with NDVI difference at the biomass-rising phase than that at the falling phase; (2) the relationship between NDVI difference and climate variables varies with vegetation types and vegetation communities. In a typical steppe dominated by Leymus chinensis, temperature has higher correlation with NDVI difference than precipitation does, and in a typical steppe dominated by Stipa krylovii, the correlation between temperature and NDVI difference is lower than that between precipitation and NDVI difference. In a typical steppe dominated by Stipa grandis, there is no significant difference between the two correlations. Precipitation is the key factor influencing vegetation growth in a desert steppe, and temperature has poor correlation with NDVI difference; (3) the response of NDVI difference to precipitation is fast and almost simultaneous both in a typical steppe and desert steppe, however, mean temperature exhibits a time-lag effect especially in the desert steppe and some typical steppe dominated by Stipa krylovii; (4) the relationship between NDVI difference and temperature is becoming stronger with global warming. __________ Translated from Acta Phytoecologica Sinica, 2005, 29(5): 753–765 [译自: 植物生态学报]     

Correlation analysis of Normalized Different Vegetation Index (NDVI)difference series and climate variables in the Xilingole steppe,China from 1983 to 1999

There is a crucial need in the study of global change to understand how terrestrial ecosystems respond to the climate system.It has been demonstrated by many researches that Normalized Different Vegetation Index (NDVI)time series from remotely sensed data,which provide effective information of vegetation conditions on a large scale with highly temporal resolution,have a good relation with meteorological factors.However,few of these studies have taken the cumulative property of NDVI time series into account.In this study,NDVI difference series were proposed to replace the original NDVI time series with NDVI difference series to reappraise the relationship between NDVI and meteorological factors.As a proxy of the vegetation growing process,NDVI difference represents net primary productivity of vegetation at a certain time interval under an environment controlled by certain climatic conditions and other factors.This data replacement is helpful to eliminate the cumulative effect that exist in original NDVI time series,and thus is more appropriate to understand how climate system affects vegetation growth in a short time scale.By using the correlation analysis method,we studied the relationship between NOAA/AVHRR ten-day NDVI difference series and corresponding meteorological data from 1983 to 1999 from 11 meteorological stations located in the Xilingole steppe in Inner Mongolia.The results show that:(1)meteorological factors are found to be more significantly correlation with NDVI difference at the biomass-rising phase than that at the falling phase;(2)the relationship between NDVI difference and climate variables varies with vegetation types and vegetation communities.In a typical steppe dominated by Leymus chinensis,temperature has higher correlation with NDVI difference than precipitation does,and in a typical steppe dominated by Stipa krylovii,the correlation between temperature and NDVI difference is lower than that between precipitation and NDVI difference.In a typical steppe dominated by Stipa grandis,there is no significant difference between the two correlations.Precipitation is the key factor influencing vegetation growth in a desert steppe,and temperature has poor correlation with NDVI difference;(3)the response of NDVI difference to precipitation is fast and almost simultaneous both in a typical steppe and desert steppe,however,mean temperature exhibits a time-lag effect especially in the desert steppe and some typical steppe dominated by Stipa krylovii;(4)the relationship between NDVI difference and temperature is becoming stronger with global warming.