In the present study, the second nuclear internal transcribed spacer (ITS-2) rDNA of Schistosoma japonicum isolates in mainland China was amplified, sequenced, and assessed for inferring the intra- and inter-species phylogenetic relationships of trematodes in the order Strigeata. The fragment containing ITS-2 rDNA was obtained from 24 S. japonicum isolates from eight epidemic provinces in mainland China. The length polymorphisms were observed among these ITS-2 rDNA sequences, ranging from 343 to 346 bp, and the intra- and inter-population variations in ITS-2 sequence were 0.0-2.1% among S. japonicum isolates in China. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods revealed that the ITS-2 rDNA sequence is not a suitable marker for studying inter- and intra-population variation in S. japonicum. However, phylogenetic analysis of trematodes in the order Strigeata indicated that the ITS-2 rDNA sequence provides an effective molecular marker for studying inter-species phylogenetic relationships among trematodes in this order.

In the present study, sequence-related amplification polymorphism (SRAP) was utilized to study the genetic variability among Schistosoma japonicum isolates from different provinces in China, using Schistosoma mansoni from Puerto Rico for comparison. Five out of ten tested SRAP primer combinations displayed significant polymorphisms among S. japonicum isolates from China, namely ME2/EM1, ME4/EM1, ME4/EM6, ME5/EM4 and ME5/EM5. Analysis of the 61 S. japonicum samples from China with five SRAP primer combinations identified a total of 83 reproducible polymorphic fragments. The number of fragments using each primer combination ranged from 14 to 19, with an average of 16 polymorphic bands per primer pair, and the size of fragment ranged approximately from 100 to 1000 bp. Representative-specific SRAP fragments were excised from the gels, and confirmed by PCR amplification of genomic DNA using primers designed and based on the sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair-group method with arithmetic averages (UPGMA) dendrogram was constructed. UPGMA clustering algorithm categorized S. japonicum isolates from China into nine clades and two lineages (representing the mountainous and lake/marshland regions). These results indicate the usefulness of the SRAP technique for revealing genetic variability among S. japonicum isolates from China, and the SRAP technique should be applicable to other living organisms.

The intergenic spacer (IGS) between the 28S and 18S ribosomal RNA genes was PCR-amplified, sequenced and characterized for Schistosoma japonicum from mainland China, and compared with those of other Schistosoma species. Excluding flanking portions of the 28S and 18S genes, the IGS in the longest sequenced amplicon from S. japonicum IGS was 1457bp in length. However, intra-specific and intra-individual variation was noted. The IGS region of S. japonicum is strikingly different in structure from those of African Schistosoma species for which data are available. S. japonicum has a shorter IGS and largely lacks a long region of complex repeats seen in the African species. However, careful comparisons with African species highlighted the presence of a few shared repeat motifs that were not apparent from study of African species only. Such motifs presumably have functional significance. Discovery of such motifs may in general be aided by comparisons of relatively distant taxa rather than of sibling taxa.

In the present study, restriction site-amplified polymorphism (RSAP) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different endemic provinces in mainland China. Of the 45 pairs of primers screened, 10 RSAP markers showed a clear banding pattern with good resolution; however, only six exhibited a polymorphism among different isolates. Among six RSAP markers, one pair of primers (R8 + R10) was able to differentiate male and female parasites, and amplified one constant specific band for female S. japonicum isolates. The specific band was recovered, re-amplified and sequenced, and a sequence of 162 bp was obtained. Based oil this sequence, a pair of specific primers was designed and used to develop sequence characterized amplified region (SCAR)-PCR assay for identification and differentiation of female S. japonicum isolates. The SCAR-PCR assay allowed the specific identification of female S. japonicum, with no amplicons being amplified from male S. japonicum, Fasciola hepatica, Clonorchis sinensis, S. mansoni (male and female parasite). DNA sequencing confirmed the identity of the amplified products. The minimum amount of DNA detectable using SCAR-PCR assay was 0.3 ng for female S. japonicum. The SCAR-PCR was able to differentiate effectively the male and female S. japonicum worms collected from 12 geographical origins in eight endemic provinces, the gender of which was known based on the morphological and biological features. These results showed that SCAR-PCR provides an effective tool for the sex differentiation studies of S. japonicum, identification of female S. japonicum, diagnosis and epidemiological survey of S. japonicum infections in animals and human.

The present study studied the genetic variation among Schistosoma japonicum isolates from different endemic regions in mainland China and examined the phylogenetic relationships of zoonotic trematodes using the combined mitochondrial 16S and 12S ribosomal DNA sequences. The fragments of 16S and 12S rDNA were amplified from 22 S. japonicum isolates, and sequenced, and the relevant sequences of other nine trematode species belonging to six genera in four families were downloaded from GenBank, and their phylogenetic relationships were re-constructed by unweighted pair-group method with arithmetic averages analyses using the combined 16S and 12S rDNA sequences, with Trichinella spiralis as outgroup. The results showed that the partial sequences of mitochondrial 16S and 12S rDNA of S. japonicum were 757 and 797 bp, respectively, and they were quite conserved among the S. japonicum isolates. Phylogenetic analysis revealed that the combined 16S and 12S rDNA sequences were not able to distinguish S. japonicum isolates in mountainous areas from those in lake/marshland areas in mainland China. However, the combined sequences could distinguish different species of zoonotic trematodes. Therefore, the combined mitochondrial 16S and 12S rDNA sequences provide an effective molecular marker for the inter-species phylogenetic analysis and differential identification of zoonotic trematodes.

In order to study the genetic polymorphism and reconstruct phylogenetic relationship among Schistosoma japonicum(S.japonicum) isolated in mainland China based on mitochondrial cytochrome b(cytb) gene,cytb gene was amplified from S.japonicum isolated from 11 epidemic origins,and sequenced.Nucleotide sequences of partial cytb gene were aligned using the ClustalⅩ 1.81,MP and NJ trees,constructed using the software Paup version 4.0,and ML tree was also reconstructed using PUZZLE version 4.1.Sequence homology analysis was performed and the nucleotide composition,transition and transversion were accounted using the Megalign program in the software DNAStar version 5.0.The results indicated that cytb gene of S.japonicum was 419 bp,and 7 mutation sites were detected(1.67%).Phylogenetic analysis revealed that cytb gene was helpful to distinguish validly between S.japonicum isolated from mountainous areas and lake areas in mainland China.The cytb gene could also distinguish S.japonicum from other trematodes belonging to the family Schistosomatidae.Therefore,the cytb gene might provide a sui-table marker for phylogenetic analysis and differential identification of S.japonicum from other Schistosomes.

The present study examined sequence variation in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 (cox3), NADH dehydrogenase subunits 4 and 5 (nad4 and nad5), among Schistosoma japonicum isolates from different endemic regions in China, and their phylogenetic relationships were re-constructed. A portion of the cox3 gene (pcox3), a portion of the nad4 and nad5 genes (pnad4 and pnad5) were amplified separately from individual trematodes by polymerase chain reaction (PCR) and the amplicons were subjected to direct sequencing. In the mountainous areas, sequence variations between parasites from Yunnan and those from Sichuan were 0.3% for pcox3, 0.0-0.1% for pnad4, and 0.0-0.2% for pnad5. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0-0.3% for pcox3, 0.0-0.7% for pnad4, and 0.0-1.6% for pnad5. Sequence variations between S. japonicum from mountainous areas and those from lake/marshland areas were 0.0-0.5% for pcox3, 0.0-0.7% for pnad4, and 0.0-1.6% for pnad5. Phylogenetic analyses based on the combined sequences of pcox3, pnad4 and pnad5 revealed that S. japonicum isolates from mountainous areas (Yunnan and Sichuan provinces) clustered together. For isolates from the lake/marshland areas, isolates from Anhui and Jiangsu provinces clustered together and was sister to samples from Jiangxi province, while isolates from Hubei and Zhejiang province clustered together. However, isolates from different geographical locations in Hunan province were in different clades. These findings demonstrated the usefulness and attributes of the three mtDNA sequences for population genetic studies of S. japonicum, and have implications for studying population biology, molecular epidemiology, and genetic structure of S. japonicum, as well as for the effective control of schistosomiasis.

The present study examined sequence variation in class I and class II major histocompatibility complex (MHC) genes among Schistosoma japonicum isolates from different endemic regions in mainland China and assessed the level of horizontal gene transfer and sequence similarity between parasites and their hosts. S. japonicum cercariae were used to infect male adult rabbits to obtain adult S. japonicum samples. A portion of the class I MHC gene (pMHC I) and class II MHC genes (pMHC II) were amplified separately from individual adult trematodes by polymerase chain reaction and sequenced. Among all the examined isolates of S. japonicum, sequence differences between male and female parasites were 0.0–26.6% for pMHC I and 0.0–7.0% for pMHC II. Sequence variations between male and female parasites among different geographical locations from the mountainous areas were 1.1–26.6% for pMHC I and 1.5–3.0% for pMHC II. Sequence variations between samples from Yunnan and those from Sichuan were 2.7–23.5% for pMHC I and 1.1–3.7% for pMHC II. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0–25.0% for pMHC I and 0.0–7.0% for pMHC II. Sequence variations between S. japonicum isolates from mountainous areas, and those from lake/marshland areas were 0.0–26.1% for pMHC I and 0.4–6.1% for pMHC II. BLASTN analysis indicated that all the pMHC II sequences showed high homology to a portion of exon 3 in rabbit MHC class II DP beta gene with more than 89% similarity, and all the pMHC I sequences except isolates in Yunnan (Eryuan) revealed high homology to the portion of exon 2 in rabbit MHC I gene with more than 81% similarity. Phylogenetic analysis showed no specific clustering comprising parasites from single geographical or endemic regions, and the paired parasites were even found in different clusters. These results demonstrated that pMHC I and II of S. japonicum isolates in mainland China existed heterogeneity, but the pMHC I, II, or combined sequences were not suitable markers for examining genetic relationship among different isolates from endemic regions in mainland China.

In the present study, inter-simple sequence repeats (ISSRs) markers were used to examine the genetic variability of Schistosoma japonicum isolates from different provinces in mainland China, using S. japonicum from Japan and S. mansoni from Puerto Rico for comparison. Of the 30 primers screened, 4 produced highly reproducible ISSR fragments. Using these primers, 107 discernible DNA fragments were generated with 105 (98.13%) being polymorphic, indicating considerable genetic variation among the examined S. japonicum isolates. The percentage of polymorphic bands among S. japonicum isolates from mainland China and Japan was 82.24%, 43.93% among mountainous type isolates and 64.49% among lake/marshland type isolates from mainland China. UPGMA analysis revealed that all of the S. japonicum samples were grouped into two clades, the first contained isolates from mainland China, and the other one contained samples from Japan. Within the cluster of S. japonicum isolates from mainland China, isolates from mountainous Sichuan and Yunnan provinces grouped together, whereas isolates from lake/marshland regions (Anhui, Jiangsu and Hubei provinces) clustered together. The results of present study demonstrated that the ISSR markers are useful for studying genetic diversity and population structure of S. japonicum isolates from mainland China.

A series of 4-(2-methoxyphenyl)-2-oxo-butyl-quinazolinones were designed and synthesized based on the structure of febrifugine. The structures of the new compounds were confirmed by H NMR, C NMR, IR spectra and HRMS. The biological activity test results indicated that they exhibited anticoccidial activities against Eimeria tenella in the chicken diet with a dose of 9 mg/kg. Compared with halofuginone, these compounds have the advantages of shorter synthetic routes and lower cost.

日本血吸虫是一种重要的人兽共患寄生虫,能够寄生于人和40多种哺乳动物门静脉系统中,具有重要的公共卫生和社会经济意义[1-2].根据日本血吸虫的生物学、形态学、免疫学和地理分布等特征,将其分为中国大陆、中国台湾、菲律宾、日本和印度尼西亚5个地域品系(geographic strains);但对于分布在中国大陆长江流域以南12个省、市、自治区的日本血吸虫是否为同一品系-中国大陆品系的问题,国内外学者曾进行了大量的研究.本文综述国内外关于中国大陆日本血吸虫种群遗传结构的研究进展,以供借鉴.

2004年5月,广东省博罗县某肉鸡养殖户饲养的3000只21日龄肉仔鸡开始发病.病鸡拉白色或绿色粪便,并出现死亡.用泰乐菌素、长乐霉素和庆大霉素治疗均无效,7d后死亡300多只.经现场调查及临床症状、病理剖检和实验室检查等,确诊为肉仔鸡大肠杆菌病.