Characterization of the Toxoplasma gondii hsp60 gene sequences from different hosts and geographical locations.

The intracellular protozoan Toxoplasma gondii is one of the most successful parasites, with the ability to invade all warm-blooded animals, including humans. T. gondii heat shock protein 60 (TgHSP60) plays an important role in intracellular survival and in the differentiation of the parasite, and is also recognized as being associated with its virulence. In the present study, we examined sequence variation in the hsp60 coding region among five T. gondii isolates from different hosts and geographical regions, which were compared with the corresponding sequences of strains ME49, 76K, and GT1 available in the ToxoDB databases. The length of the T. gondii hsp60 sequence was 1728 bp for all strains, and the A+T content ranged from 41.96 to 42.13%. The sequence alignment of the 8 T. gondii strains identified 20 variable positions (0-1.44%) and showed 1.16% overall sequence variation, suggesting a relatively considerable sequence diversity. Phylogenetic analysis of hsp60 sequences using Bayesian inference and maximum parsimony differentiated the two major clonal lineage types into their respective clusters, and thus separated atypical strains from classical genotypes. The results of the present study suggested that the coding region of the hsp60 gene may represent a novel genetic marker for intraspecies phylogenetic analyses of T. gondii.

The second transcribed spacer rDNA sequence: an effective genetic marker for inter-species phylogenetic analysis of trematodes in the order Strigeata.

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.

Genetic diversity and relatedness of Fasciola spp. isolates from different hosts and geographic regions revealed by analysis of mitochondrial DNA sequences.

The present study examined sequence variability in a portion of the mitochondrial cytochrome c oxidase subunit 1 (pcox1) and NADH dehydrogenase subunits 4 and 5 (pnad4 and pnad5) among 39 isolates of Fasciola spp., from different hosts from China, Niger, France, the United States of America, and Spain; and their phylogenetic relationships were re-constructed. Intra-species sequence variations were 0.0-1.1% for pcox1, 0.0-2.7% for pnad4, and 0.0-3.3% for pnad5 for Fasciola hepatica; 0.0-1.8% for pcox1, 0.0-2.5% for pnad4, and 0.0-4.2% for pnad5 for Fasciola gigantica, and 0.0-0.9% for pcox1, 0.0-0.2% for pnad4, and 0.0-1.1% for pnad5 for the intermediate Fasciola form. Whereas, nucleotide differences were 2.1-2.7% for pcox1, 3.1-3.3% for pnad4, and 4.2-4.8% for pnad5 between F. hepatica and F. gigantica; were 1.3-1.5% for pcox1, 2.1-2.9% for pnad4, 3.1-3.4% for pnad5 between F. hepatica and the intermediate form; and were 0.9-1.1% for pcox1, 1.4-1.8% for pnad4, 2.2-2.4% for pnad5 between F. gigantica and the intermediate form. Phylogenetic analysis based on the combined sequences of pcox1, pnad4 and pnad5 revealed distinct groupings of isolates of F. hepatica, F. gigantica, or the intermediate Fasciola form irrespective of their origin, demonstrating the usefulness of the mtDNA sequences for the delineation of Fasciola species, and reinforcing the genetic evidence for the existence of the intermediate Fasciola form.

Combined mitochondrial 16S and 12S rDNA sequences: an effective genetic marker for inter-species phylogenetic analysis of zoonotic trematodes.

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.

Heterogeneity of class I and class II MHC sequences in Schistosoma japonicum from different endemic regions in mainland China.

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.

Genetic variability among Schistosoma japonicum isolates from different endemic regions in China revealed by sequences of three mitochondrial DNA genes.

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.

Survey of intestinal parasites in pigs from intensive farms in Guangdong Province, People's Republic of China.

The prevalence of intestinal parasites was investigated in intensive pig farms in Guangdong Province, China between July 2000 and July 2002. Faecal samples from 3636 pigs (both sexes and five age groups) from 38 representative intensive pig farms employing different parasite control strategies were examined for the presence of helminth ova and protozoan oocysts, cysts and/or trophozoites using standard techniques. Of the 3636 pigs sampled, 209 (5.7%) were infected with Trichuris suis, 189 (5.2%) with Ascaris, 91 (2.5%) with Oesophagostomum spp., 905 (24.9%) with coccidia (Eimeria spp. and/or Isospora suis) and 1716 (47.2%) with Balantidium coli. These infected pigs were mainly from farms without a strategic anti-parasite treatment regime. Concurrent infection of multiple parasites was common, and T. suis was the most common nematode infecting breeding, young and mature pigs. The results of the present investigation provide relevant 'base-line' data for assessing the effectiveness of control strategies against intestinal parasitism in intensively raised pigs in Guangdong Province, China.