斯氏并殖吸虫染色体核型分析

最早研究并殖吸虫染色体的是陈品芝(Chea,1937)系采用石蜡切片法,观察到克氏并殖吸虫(P.kellicotti)染色体2n=16,n=8,但未提到其染色体核型。以后近四十年中,未见有关并殖吸虫染色体的报道。而这些年来,国内外并殖吸虫的分类学研究却有了蓬勃的开展,并殖吸虫新种以及对人体致病的虫种不断发现。分类学研究主要以     

The localization of allergens of Paragonimus westermani by pleural exudates from patients.

The allergens of the lung fluke Paragonimus westermani were localized by indirect immunostaining in adult fluke sections using pleural exudates from 3 patients with P. westermani. Immunostaining performed by using pleural exudate with the highest level of specific IgE revealed that the P. westermani major allergen (or allergens) was located in the gut epithelium and luminal contents and that minor allergens were in the tegument and parenchyma. The antigens recognized by specific IgG were located at various sites including those recognized by specific IgE. Paragonimus westermani-specific IgE cross-reacted with only the gut of 2 other Paragonimus species, Paragonimus miyazakii and Paragonimus ohirai. The major allergen in the gut also was recognized by the other 2 pleural exudates. These results indicate that the substance present in and secreted from the gut is not only a major allergen but is also a common allergen among Paragonimus species.     

Intra- and interindividual variation in ITS1 of paragonimus westermani (Trematoda: digenea) and related species: implications for phylogenetic studies

We investigated the utility of the ribosomal first internal transcribed spacer (ITS1) for phylogenetic studies on trematodes of the genus Paragonimus. Numerous clones containing ITS1 PCR products were sequenced for P. miyazakii, P. macrorchis, and members of the P. ohirai and P. westermani species complexes. Some additional data were obtained by direct sequencing of PCR products. The ITS1 is composed of three distinct regions: the short 5' end, followed by a tract of approximately 120 nucleotides which occurs a variable number of times in tandem, and the 3' region, which lacks repeats and is referred to as the "post-repeat" fragment. Sequences from all three regions can be aligned among the species studied. Our initial hypothesis, that the post-repeat region would be valuable for phylogenetic studies within the P. westermani complex, was proved wrong. Intraindividual sequence variation in P. westermani was sometimes greater than between individuals of the species complex. In the P. ohirai species complex, however, sequence variation within individuals was minimal. Possible reasons for these observations are discussed. We also wished to determine whether the length variants sequenced were the dominant variants present in Paragonimus species. This was done by probing Southern blots of genomic digests with an ITS1 fragment which lacks repeat sequences. There is generally greater abundance of large variants, with much lower abundance of small variants, such as those sequenced. Differences in ITS1 lengths are attributed largely to differing numbers of repeats, though some exceptions (which are discussed) were found.     

Molecular identification of Paragonimus ohirai and P. westermani from Anhui Province, China

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) region were determined from seven adults of species Paragonimus collected from Jinde and Xiuning Counties, Anhui Province, China. Among these, the nucleotide sequence obtained from one Paragonimus adult (Jinde County) was identical to the ITS2 sequence of P. ohirai previously reported. In order to confirm the result, partial regions of mitochondrial cytochrome C oxidase I (COI) and NADH dehydrogenase 1 (ND1) from the putative P. ohirai sample were further sequenced. They showed a high level of similarity with those of P. ohirai, COI (99.7%) and ND1 (99.5%), supporting the result obtained from the ITS2. In addition to this, we designed P. ohirai- and P. westermani-specific primers (BDW and BD2OH) from ITS2 to identify P. westermani and P. ohirai easily and rapidly. After testing utility of the primers, they were applied to identify seven unidentified Paragonimus samples collected from Jinde and Xiuning Counties, China. All the examined samples showed P. westermani band pattern, and it was reconfirmed by sequencing their ITS2 regions that they are P. westermani. This result indicates that the two newly designed specific primers could be quite helpful for easily identifying P. westermani and P. ohirai, that most of Paragonimus in Jinde and Xiuning Counties consist of P. westermani, and that P. ohirai exists in Jinde County with minority.     

Evidence for metacercarial polymorphism in lung flukes, Paragonimus ohirai and Paragonimus iloktsuenensis

Cross breeding experiments between 2 species of lung fluke, Paragonimus ohirai and Paragonimus iloktsuenensis, were carried out using metacercarial characteristics as distinguishing markers. All metacercariae of F1 obtained were identical to those of P. ohirai. In the F2 and BF1 of F1 X P. iloktsuenensis, both the P. ohirai and P. iloktsuenensis types of metacercariae appeared. In the BF1 of F1 X P. ohirai, however, only the P. ohirai type metacercariae were produced. No intermediate type between the 2 species appeared. The results obtained demonstrate that the differences in the metacercariae, which were previously regarded as the most important characteristic for specific discrimination between these 2 flukes, are only a hereditary phenomenon within a single species. The metacercarial form, number of cyst layers, and body size seem to be controlled by a couple of alleles or very closely linked genes following simple Mendelian inheritance. Furthermore, we confirmed that reproduction in the lung flukes depends on cross-fertilization.     

Phylogenetic relationship of ribosomal ITS2 and mitochondrial COI among diploid and triploid Paragonimus westermani isolates

We compared patterns of intraspecific polymorphism of two markers with contrasting modes of evolution, nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA), in the lung fluke, diploid and triploid Paragonimus westermani from three geographical regions of Korea. The genetic distances between three populations of Korean diploid and triploid P. westermani showed no significant difference in the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (mtCOI) and ribosomaal second internal transcribed spacer (ITS2) genes. A highly resolved strict-consensus tree was obtained that illustrated phylogenetically useful information of the ITS2 and mtCOI sequences from diploid and triploid P. westermani.     

我国松阳和宽甸地区卫氏并殖吸虫的同工酶分析

作者采用Disc-PAGE电泳对我国浙江松阳和辽宁宽甸夹皮沟二倍体型和三倍体型卫氏并殖吸虫的乳酸脱氨酶(LDH)、苹果酸脱氢酶(MDH)、酯酶(EST)同工酶进行了比较。结果显示两型卫氏并殖吸虫的LDH、MDH和EST同工酶酶带的数目、Rf值、优势同工酶带的位置存在明显的差异。这些差异是否属种的特征尚待进一步研究。     

Comparison of gene representation between diploid and triploid Paragonimus westermani by expressed sequence tag analyses

Expressed sequence tag (EST) analysis of the diploid and triploid Paragonimus westermani genes was done to have a rapid and informative outlook of the gene-expression profiles of the parasites. Totals of 506 and 505 ESTs were generated from the diploid and triploid P. westermani cDNA libraries. Based on the BLASTx search results of the diploid P. westermani ESTs, 308 (60.9%) matched significantly with formerly identified genes and 198 (39.1%) showed no significant homology in the GenBank database. A similar homology pattern was shown from the triploid EST BLASTx search results with 346 (68.5%) sharing homology with previously identified genes and 159 (31.5%) showing no significant homology. The EST data from both libraries were analyzed and grouped into 9 categories. Comparison of the 2 EST pools revealed high similarities among the categories of the significantly matched genes. Single genes matched repeatedly were also observed in the 2 EST data. Some genes were found that are not yet characterized in P. westermani; these genes were matched by both the diploid and triploid ESTs. Further study of these genes may provide us with more understanding on the parasite's biology and their specific functions in the 2 strains.     

Triploidy in Paragonimus westermani

The finding of triploid and diploid forms of Paragonimus westermani in Japan has caused tremendous controversy over the identity and speciation of this parasite. Should these two genetically different forms be given different specific names? Hirohiso Hirai and Takeshi Agatsuma have studied the lung flukes by using cytogenetic techniques and zymodeme analysis. In this article, they briefly compare triploid P. westermani with the diploid form and develop a hypothesis for the mechanism and origin of triploidy.     

In vitro cultivation of Paragonimus miyazakii and P. ohirai

Excysted metacercariae of Paragonimus miyazakii and P. ohirai were cultured in various media at 37.5 C in a 5% CO2 atmosphere. Paragonimus miyazakii grew rapidly and showed a well-developed ovary, uterus, and testes at 172 days in NCTC 109 supplemented with 30% rabbit serum, 50% egg yolk-109, and rabbit red blood cells (RBC's). However, none of the worms formed yolk or eggs in these cultures. On the other hand, P. ohirai grew to the adult stage, in which vitellaria and imperfect ova were formed, in NCTC 109 supplemented with 30% dog serum, 10% yeast extract Earle's solution (YLE), and dog RBC's at 252 days. The maximum body length of these worms measured 7.0 mm (mean 5.5 mm) at 252 days. The dog RBC's were an essential ingredient of the culture medium for the development of P. ohirai. Additions of liver concentrate, chick embryo extract (CEE), and egg yolk-109 in the medium did not provide any additional benefits for the development of worms. Using this supplemented medium, adult worms of P. ohirai removed from rats were maintained in vitro to examine their ability to lay eggs. Egg laying occurred during the first 10-13 days for worms that survived more than 60 days. The number of eggs deposited in this medium was about 2 times that found when Hanks' BSS and NCTC 109 were used.     

并殖吸虫成虫形态结构特征排序

对9种并殖吸虫成虫形态特征进行主成分分析及聚类分析,结果表明:在并殖吸虫成虫的6项主要特征中,体形因素及卵巢类型是重要的分类依据。在被分析的9个种中,怡乐村并殖吸虫、斯氏狸殖吸虫、巨睾狸殖吸虫、三平正并殖吸虫、异盘并殖吸虫、陈氏并殖吸虫与卫氏并殖吸虫7个种具有种的独立地位,而泡囊狸殖吸虫和扁囊并殖吸虫未能分别从斯氏狸殖吸虫和卫氏并殖吸虫中分开。     

Biodiversity of flukes

As many others parasites, speciation of flukes depends on the genetic characteristics and on ploidia. Ploidia of flukes can be different in a same species. In Asia, diploid, triploid and hybrid (2n/3n) populations are encountered. The comparison of morphological parameters between diploid and triploid flukes showed that they were morphologically different. Nevertheless, a genetic relationship between parthenogenetic organisms would exist regardless of their ploidia. In the Fasciola genus, the main consequence of the high level of diversity is the frequent probability of development of resistance to anthelmintics and fast adaptation to climatic changes. In the Paragonimus genus, diversity can enhance different forms of pathogenicity, can also be related to the species of intermediate hosts, and to the definitive host. The strain of flukes plays a part in the visceral localization of P. westermani adults.     

二倍体型与三倍体型卫氏并殖吸虫同工酶谱的发育变异及其分类学意义

采用生趣板状聚丙烯酰胺胶是民泳法分析比较了二倍体型与三倍体型卫氏并殖吸虫童虫、成虫的醛缩酶、已糖激酶和葡萄糖－６－磷酸脱氢酶的同工酶谱,结果显示２ｎ型与３ｎ型卫氏并殖吸虫在不同发育期之间及两型吸虫相应各发育期之间ＡＬＤ、ＨＫ及Ｇ６ＰＤ同工酶谱均显著变异,变异表现为两种方式,一种是在同一个基因座位区的同工酶的酶带数目     

Occurrence of a diploid type and a new first intermediate host of a human lung fluke, Paragonimus westermani, in Korea

The biology, chromosome number, and karyotype of a lung fluke, Paragonimus westermani (Kerbert, 1878) collected in Haenam, Haenam-gun Chollanam-do, Korea were analyzed. We compared the size of metacercariae from Haenam with those taken from a crayfish collected at Youngam, Youngam-gun, Chollanam-do, Korea. The mean length of P. westermani metacercariae from Haenam was 300.3 microm and that from Youngam was 362.0 microm. Adult worms were recovered from the lungs of experimentally infected dogs. The mean egg sizes obtained from adult flukes were 72.1 x 46.8 microm from Haenam and 93.5 x 54.2 microm from Youngam. Semisulcospira tegulata collected in the Youngam area were found to be infected with cercariae of P. westermani, one of the snail-borne human lung fluke trematodes in Korea. Of 4218 snails studied, 5 (0.12%) harbored P. westrermani larvae. This is the first report of S. tegulata serving as the initial intermediate host of P. westermani. The chromosome numbers of P. westermani from Haenam and Youngam were 2n = 22 and 3n = 33. The diploid type of P. westermani has not been previously reported in Korea.     

ELISA of rat sera infected with Paragonimus iloktsuenensis

Enzyme-linked immunosorbent assay (ELISA) of paragonimiasis iloktsuenensis rat sera was performed using crude antigens of Paragonimus iloktsuenensis (PIA), P. westermani (PWA) and Clonorchis sinensis (CSA). Three crude antigens (PIA, PWA, CSA) were prepared to saline homogenated supernatants of whole adult worms. Infected rat sera were obtained biweekly from the albino rats fed 50-80 metacercariae of P. iloktsuenensis through gastric catheter. Experimental groups were divided into 4 groups: GI (controls), GII, GIII and GIV according to 1-7 worms as GII, 10-19 worms as GIII and 22-40 worms as GIV, respectively. In ELISA, the mean OD values of each group for the homologous antigen (PIA) were increased significantly compared to the control sera at the 4th week of infection. With the progress of duration of infection, the mean OD values of infected sera of GII & GIV continuously increased up to the 12th week (last week), but in GIII the mean OD value increased until the 10th week. No significance was noted among the infection dose groups (GII, GIII and GIV), after the 6th week of infection. Also, the OD values of all infected rats did not show any proportional relationships to the number of worms recovered. In brief, the antibody productivity of individual rats were strongly different. The rat sera infected with P. iloktsuenensis cross-reacted with those infected with P. westermani or C. sinensis, as identified by OD values.     

The origin of the triploid in Paragonimus westermani on the basis of variable regions in the mitochondrial DNA

Triploid, parthenogenetic forms of the lungfluke, Paragonimus westermani, occur in Japan, Korea and China. The origin(s) of triploidy has been debated over the years. Sequences of two regions in the mitochondrial DNA, i.e. partial lrRNA (16S), and a portion of the non-coding region, were obtained from natural populations of P. westermani. All triploid individuals (Japan, Korea, China) and a single tetraploid individual (China) had identical sequences in the 16S region studied. Some sequence variation was observed among diploids, with those from Taiwan being distinct from the remainder. Both neighbour joining and parsimony trees using the 16S region placed diploid individuals from southwestern Japan close to the triploids and the tetraploid. The fragment amplified from the mitochondrial non-coding region showed dimorphism. One form (type A) consisted of 239 bp comprising two identical tracts of 70 bp separated by a tract of 93 bp. The second form (Type B) consisted of only a single 70 bp tract. All diploid individuals from Taiwan, China and Korea possessed type A, while those from Japan were polymorphic; individuals from Oita and Hyogo had type B, those from Chiba had type A, but both types were found in Mie. On the other hand, all of the triploid individuals and two tetraploid individuals possessed type B. Both the form present in the non-coding region and the 16S sequence suggest an affinity between a south-eastern group of diploid populations in Japan and the triploid form. A possible mechanism responsible for the origin of the triploid is discussed.     

Novel C-banding patterns in Paragonimus ohirai (Trematoda: Platyhelminthes)

The C-banding pattern for the karyotype of Paragonimus ohirai representing individuals in a new population is reported. The short arm of chromosome 4 consisted of a large pericentromeric proximal C-band block and euchromatic tip. This pattern has not been observed previously and is designated as type E. Other new observations were: chromosome 5 was composed of pericentromeric heterochromatin, a lightly stained intercalary band at the middle portion of the short arm, and a lightly stained interstitial band at the terminal region of the long arm. Chromosome 7 consisted of pericentromeric heterochromatin and a lightly stained telomeric band at the short arm.     

Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64.     

Genetic diversity in parthenogenetic triploid Paragonimus westermani

Paragonimus westermani is a medically important foodborne trematode occurring throughout southeast Asia. We have used molecular techniques to test the hypothesis that the parthenogenetic triploid form of P. westermani has arisen only once. Sources of data for comparison were: (a) restriction fragment length polymorphisms (RFLPs) of ribosomal internal transcribed spacers (ITS); and (b) 'fingerprint' patterns observed when genomic digests were probed with simple sequence repeats (ATT)10 and (ATGT)7. In all cases there were distinct differences among triploid isolates from southwest Japan, northeast China and Korea. These findings are considered in the context of previous cytogenetic, allozyme, mitochondrial-RFLP and partial cytochrome c-oxidase subunit I (COI) sequence studies and indicate that triploid lineages may have arisen independently on more than one occasion. We favour this view. An alternative explanation is that the triploids did have a single origin, but that different clonal lineages have undergone subsequent mutations.