Molecular characterization and phylogenetic analysis of Fasciola hepatica from Peru

The causative agent of fasciolosis in South America is thought to be Fasciola hepatica. In this study, Fasciola flukes from Peru were analyzed to investigate their genetic structure and phylogenetic relationships with those from other countries. Fasciola flukes were collected from the three definitive host species: cattle, sheep, and pigs. They were identified as F. hepatica because mature sperms were observed in their seminal vesicles, and also they displayed Fh type, which has an identical fragment pattern to F. hepatica in the nuclear internal transcribed spacer 1. Eight haplotypes were obtained from the mitochondrial NADH dehydrogenase subunit 1 (nad1) sequences of Peruvian F. hepatica; however, no special difference in genetic structure was observed between the three host species. Its extremely low genetic diversity suggests that the Peruvian population was introduced from other regions. Nad1 haplotypes identical to those of Peruvian F. hepatica were detected in China, Uruguay, Italy, Iran, and Australia. Our results indicate that F. hepatica rapidly expanded its range due to human migration. Future studies are required to elucidate dispersal route of F. hepatica from Europe, its probable origin, to other areas, including Peru.     

Molecular identification and genetic-polymorphism analysis of Fasciola flukes in Dali Prefecture,Yunnan Province,China

This study aimed to identify species of Fasciola flukes in Dali Prefecture (Yunnan Province, China) and analyze their genetic diversity. Fasciola flukes (n = 122) were collected from cattle livers in a farmers' market in Xiaguan Town, Dali Prefecture. Nucleotide sequences of ribosomal internal transcribed spacer (ITS) as well as nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were amplified, sequenced, and subjected to homology analysis. The heterozygosity ratios of different ITS alleles were determined using the peak-height ratio of heterozygous loci. Multiplex PCR analysis of the nuclear protein coding gene, phosphoenolpyruvate carboxykinase (pepck), was used to identify Fasciola species. Multiple ND1 sequence alignments enabled further genetic diversity analysis of regional Fasciola flukes. Seven ITS sequences belonged to F. hepatica and 115 belonged to Fh/Fg heterozygous flukes. Sequencing analysis of heterozygous flukes revealed 11 heterozygous loci with double peaks, with significantly variable ratios among individuals. ND1 and CO1 results indicated that one specimen was identical to F. hepatica, while 121 specimens were identical to F. gigantica or contained one variable site. Multiplex PCR results for pepck showed that double bands for F. hepatica and F. gigantica were amplified from Dali Fasciola specimens; hence, they were all heterozygous. By combining ITS, ND1, and CO1 sequences with multiplex pepck PCR results, all 122 specimens were identified as Fh/Fg heterozygous Fasciola flukes. Our experimental results preliminarily confirmed a high degree of Fh/Fg heterozygosity among Fasciola flukes in the Dali area. Selecting multiple molecular markers for concurrent analysis will provide more comprehensive and accurate genetic information.     

Molecular characterization revealed Fasciola specimens in Ecuador are all Fasciola hepatica,none at all of Fasciola gigantica or parthenogenic Fasciola species

All 225 Fasciola flukes obtained from domestic animals (73 cattle, 7 sheep and 1 pig) of 18 distinct geographic areas in Ecuador-South America, were identified as Fasciola hepatica, based on molecular analyses of nuclear pepck and pold genes, and mitochondrial nad1gene as well as the morphological observation of sperm within the seminal vesicles. Fasciola gigantica and parthenogenic Fasciola forms endemic to Asian countries were not found in this study, although zebu cattle and water buffalos have introduced into South America from Asia; this could be due to the absence of suitable intermediate host snails. The results of pepck analysis using multiplex PCR developed previously showed that 32 of the flukes could not be confirmed as F. hepatica, suggesting that the method is unreliable for the accurate discrimination of F. hepatica, and that pepck gene of the species consists of multiple loci, not a single locus. The results of genetic diversity, phylogenetic, and network analyses based on mitochondrial nad1 sequences suggest that F. hepatica populations in South America, including Ecuador, formed from the ancestral F. hepatica individuals introduced into the continent along with anthropogenic movement of livestock infected with the species.     

Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein-coding genes

Fasciolosis is an economically important disease of livestock caused by Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola flukes. The aspermic Fasciola flukes have been discriminated morphologically from the two other species by the absence of sperm in their seminal vesicles. To date, the molecular discrimination of F. hepatica and F. gigantica has relied on the nucleotide sequences of the internal transcribed spacer 1 (ITS1) region. However, ITS1 genotypes of aspermic Fasciola flukes cannot be clearly differentiated from those of F. hepatica and F. gigantica. Therefore, more precise and robust methods are required to discriminate Fasciola spp. In this study, we developed PCR restriction fragment length polymorphism and multiplex PCR methods to discriminate F. hepatica, F. gigantica, and aspermic Fasciola flukes on the basis of the nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and DNA polymerase delta, which are single locus genes in most eukaryotes. All aspermic Fasciola flukes used in this study had mixed fragment pattern of F. hepatica and F. gigantica for both of these genes, suggesting that the flukes are descended through hybridization between the two species. These molecular methods will facilitate the identification of F. hepatica, F. gigantica, and aspermic Fasciola flukes, and will also prove useful in etiological studies of fasciolosis.     

Molecular characterization and phylogenetic analysis of Fasciola hepatica from high- plateau and steppe areas in Algeria

A previous study based on mitochondrial DNA markers reported the presence of Fasciola hepatica in Algeria. However, a precise species identification is still required. In this report, a total of 68 Fasciola isolates, collected from high-plateau (Bordj-Bou-Arreridj) and steppe (Djelfa) areas of Algeria, were identified at the species level by multiplex PCR and PCR-restriction fragment length polymorphism (RFLP) for nuclear phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), respectively. The result of the multiplex PCR conflicted with that of the PCR-RFLP; however, subsequent nucleotide sequencing of pepck clearly showed that all isolates should be classified as F. hepatica. The two mitochondrial markers, NADH dehydrogenase subunit I (nad1) and cytochrome c oxidase subunit 1 (cox1), revealed a close relationship between the parasite populations from the plateau and those from the steppe. A dispersal direction from the high plateau to the steppe was indicated because the former population was more diversified than the latter. Moreover, these populations were more closely related to populations from Spain than those from Egypt or Afghanistan. Given the population characteristic of F. hepatica in Spain and the history of cattle trade, it seems likely that the parasite was introduced to Algeria from Europe through a route across the Mediterranean Sea.     

Molecular phylogenetic identification of Fasciola flukes in Nepal

Eighty-one Fasciola flukes collected from 8 districts in Nepal were analyzed for their species identification on the basis of their spermatogenic status and nuclear ribosomal internal transcribed spacer 1 (ITS1) and for their phylogenetic relation with Fasciola flukes from other Asian countries on the basis of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene. Sixty-one flukes (75.3%) were aspermic Fasciola sp., and 20 flukes (24.7%) were identified as Fasciola gigantica. All of the aspermic flukes displayed the Fh/Fg type in ITS1, which was predominant in aspermic Fasciola sp. from China, and most (60 flukes) displayed the Fsp-ND1-N1 haplotype in the nad1, which had an identical nucleotide sequence to the major haplotype (Fg-C2) of the aspermic flukes from China. These results suggest that aspermic Fasciola sp. was introduced into Nepal from China. Furthermore, the results of the diversity indices, neutrality indices, and median-joining network analysis with reference haplotypes from Asian countries suggest that aspermic Fasciola sp. rapidly expanded its distribution. In contrasts, F. gigantica displayed 10 nad1 haplotypes, which showed higher population diversity indices than the haplotypes of aspermic flukes, indicating that the F. gigantica population was clearly distributed in Nepal earlier than the aspermic Fasciola population. Although the F. gigantica haplotypes from Nepal formed a star-like phylogeny consisting of a main founder haplotype (Fg-ND1-N1), together with some F. gigantica haplotypes from Myanmar and Thailand, the Nepal population differed genetically from F. gigantica populations of neighboring countries as each country had distinct founder haplotype(s).     

Molecular analyses confirm the coexistence of Fasciola gigantica and parthenogenetic Fasciola in the Philippines

Fasciola flukes collected from domestic buffalos and cattle in the Philippines were confirmed as Fasciola gigantica and parthenogenetic Fasciola based on DNA analyses of nuclear pepck and pold genes, and the mitochondrial ND1 gene. This study is the first to elucidate that F. gigantica and parthenogenetic Fasciola coexist in the Philippines with prevalences of 90.6% and 9.4%, respectively. The F. gigantica population showed a high genetic diversity with 25 ND1 haplotypes, suggesting that F. gigantica has existed in the Philippines for a long time. In contrast, parthenogenetic Fasciola flukes showed a single ND1 haplotype (Fsp-ND1-P1), which was identical to the founder haplotype, Fg-C2 of parthenogenetic Fasciola in China. These results indicate that parthenogenetic Fasciola in the Philippines is a recently introduced population from a neighboring continent.     

Molecular characterization and phylogenetic analyses of Fasciola gigantica of buffaloes and goats in Punjab,Pakistan

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in production losses of millions of dollars in the livestock industry. Understading the dispersal origin and the patterns of spread of F. gigantica is important. A total of 53 Fasciola flukes collected from buffaloes and goats in Punjab, Pakistan between 2017 and 2018 were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference between F. gigantica from buffaloes and goats was indicated by the genetic analyses of mitochondrial markers, NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes identified in Pakistan were clustered in haplogroup A. fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, probably from neighbouring Middle East countries. However, cautious interpretation is required due to the very limited samples size of this study. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica.     

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.     

Molecular characterization of Fasciola hepatica from Sardinia based on sequence analysis of genomic and mitochondrial gene markers

The aim of the present study is to investigate for the first time the genetic diversity of samples identified morphologically as Fasciola hepatica (Platyhelminthes: Trematoda: Digenea) (n = 66) from sheep and cattle from two localities of Sardinia and to compare them with available data from other localities by partial sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes, the mitochondrial cytochrome c oxidase subunit I (COI), and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) genes. Comparison of the sequences from Sardinia with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. hepatica. The nucleotide sequencing of ITS rDNA showed no nucleotide variation in the ITS-1, 5.8S and ITS-2 rDNA sequences among all Sardinian samples, comparing with two ITS-2 haplotypes in standard F. hepatica, showing a substitution C/T in 20 position 859, reported previously from Tunisia, Algeria, Australia, Uruguay and Spain. The present study shows that in Sardinian sheep and cattle there is the most frequent haplotype (FhITS-H1) of F. hepatica species from South Europe. Considering NDI sequences, the phylogenetic trees showed reliable grouping among the haplotypes of F. hepatica from Sardinia and the mitochondrial lineage I, including the main N1 haplotype, observed previously from Europe (Russia, Belarus, Ukraine and Bulgaria), Armenia, West Africa (Nigeria), America (Uruguay and USA), Asia (Turkey, Japan, and China), Georgia, Turkmenistan, Azerbaijan and Australia. Furthermore, common haplotypes FhCOI-H1 and FhCOI-H2 of F. hepatica from Sardinia also corresponded mostly to the first lineage including the main C1 haplotype reported previously from Eastern European and Western Asian populations, they belonged just to a phylogenically distinguishable clade, as F. hepatica from Australia, France, Turkey, Uruguay, Russia, Armenia, Ukraine, Belarus, Turkmenistan, USA, Tunisia and Algeria, indicating that this is the main haplotype involved in the spread of F. hepatica throughout all continents.     

Identification of new polymorphic positions in rDNA sequences of the “intermediate” Fasciola forms

Fascioliasis is a foodborne zoonotic disease generally caused by the parasitic flukes Fasciola gigantica and Fasciola hepatica in class Trematoda. An “intermediate” Fasciola forms between F. gigantica and F. hepatica has been shown to exist. However, the relationships among F. gigantica, F. hepatica, and “intermediate” Fasciola forms remain unclear. In this study, we found five new polymorphic positions in 18S and 28S rDNAs sequences of “intermediate” Fasciola forms. According to the high-throughput sequencing results, all known 16 polymorphic positions of “intermediate” Fasciola forms show a clear and consistent tendency for F. gigantica or F. hepatica, and the percentages of the most frequently occurring bases were different in specimens. In the three ITS sequence fragments, hybrid-type base combinations of the polymorphic positions were detected, and the percentages of the most frequent base combinations were different in specimens too. In addition, interestingly, the newly detected ITS-802 position was not a traditional polymorphic position in “intermediate” Fasciola forms, and the bases in ITS-802 position are not same as the allele bases of F. gigantica or F. hepatica. Our results will be helpful to investigations into the molecular taxonomy, population genetics, and ecology of F. gigantica, F. hepatica, and “intermediate” Fasciola forms.     

Molecular characterization of Fasciola flukes using mitochondrial 28S rRNA gene in Naimi Saudi sheep

Fasciolosis is a parasitic disease of medical and economic importance. This retrospective study was conducted on 110 Fasciola flukes collected from livers of 14 infected Naimi sheep slaughtered at Riyadh abattoir in Saudi Arabia during winter season of 2016. Collected specimens were analyzed for their species identification on the basis of partial sequences of mitochondrial 28S rRNA gene. Results have shown the presence of both Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica) species. Where Fasciola hepatica was predominate (80%). Both intra-species and interspecies genetic distance was studied and results showed that the intraspecific variability among individuals of both species i.e., F. hepatica and F. gigantica, ranging between 0 and 1% while the interspecific diversity between F. hepatica and F. gigantica was only 1%. In conclusion, mitochondrial 28S rRNA gene is a proved as a good marker in identifying Fasciola of different species. Where, the F. hepatica and F. gigantica are present in sheep breed in Riyadh region, Saudi Arabia.     

Monitoring of Fasciola Species Contamination in Water Dropwort by cox1 Mitochondrial and ITS-2 rDNA Sequencing Analysis

Fascioliasis, a food-borne trematode zoonosis, is a disease primarily in cattle and sheep and occasionally in humans. Water dropwort (Oenanthe javanica), an aquatic perennial herb, is a common second intermediate host of Fasciola, and the fresh stems and leaves are widely used as a seasoning in the Korean diet. However, no information regarding Fasciola species contamination in water dropwort is available. Here, we collected 500 samples of water dropwort in 3 areas in Korea during February and March 2015, and the water dropwort contamination of Fasciola species was monitored by DNA sequencing analysis of the Fasciola hepatica and Fasciola gigantica specific mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 500 samples assessed, the presence of F. hepatica cox1 and 1TS-2 markers were detected in 2 samples, and F. hepatica contamination was confirmed by sequencing analysis. The nucleotide sequences of cox1 PCR products from the 2 F. hepatica-contaminated samples were 96.5% identical to the F. hepatica cox1 sequences in GenBank, whereas F. gigantica cox1 sequences were 46.8% similar with the sequence detected from the cox1 positive samples. However, F. gigantica cox1 and ITS-2 markers were not detected by PCR in the 500 samples of water dropwort. Collectively, in this survey of the water dropwort contamination with Fasciola species, very low prevalence of F. hepatica contamination was detected in the samples.     

Genotypic characterization and species identification of Fasciola spp. with implications regarding the isolates infecting goats in Vietnam

Ribosomal RNA sequences (361 or 362 bp) of the second internal transcribed spacer 2 (ITS-2) and a portion of mitochondrial cox1 (423 bp) for Fasciola spp. obtained from specimens collected in indigenous and hybrid goats and sheep in Vietnam were characterized for genotypic status and hybridization/introgression. Alignment of 48 ITS-2 sequences (also those from goats and sheep in this study) indicates that F. gigantica and F. hepatica differ typically from each other at seven sites whereas one of these is a distinguishing deletion (T) at the 327th position in F. gigantica relative to F. hepatica. The isolates from the mountainous goats in the North of Vietnam (Yen Bai province) showed the ITS-2 composition relatively identical to that of F. hepatica. The ITS-2 sequences from populations of Fasciola isolates in goats had probably experienced introgression/hybridization as reported previously in other ruminants and humans. All Vietnamese goat-of-origin specimens had high pairwise percentage of mitochondrial cox1 sequences to F. gigantica (97-100%), and very low identity to F. hepatica (91-93%), suggesting their maternal linkage to be traced to F. gigantica. The presence of hybrid and/or introgressed populations of liver flukes bearing genetic material from both F. hepatica and F. gigantica in the goats/sheep in Vietnam, regardless of indigenous or imported hosts, appears to be the first demonstration from a tropical country.     

Substantial genetic divergence between morphologically indistinguishable populations of Fasciola suggests the possibility of cryptic speciation

The liver flukes, Fasciola hepatica and Fasciola gigantica, are considered to be sister species and between them present a major threat worldwide to livestock production. In this study sequence data have been employed from informative regions of the nuclear and mitochondrial genomes of over 200 morphologically F. hepatica-like or F. gigantica-like flukes from Europe, sub-Saharan Africa and South Asia to assess genetic diversity. Evidence is presented for the existence of four well-separated clades: African gigantica-like flukes, Indian gigantica-like flukes, European hepatica-like flukes and African high-altitude hepatica-like flukes. Application of the Biological Species Concept to trematodes is problematic; however, the degree of separation between these groups was sufficient for them to be considered as distinct species using the four times rule for speciation.     

Molecular characterization of Fasciola gigantica from Mauritania based on mitochondrial and nuclear ribosomal DNA sequences

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n = 60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene.Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries.     

The stimulation of resistance in sheep to Fasciola hepatica by infection with Cysticercus tenuicollis

Infection of sheep with Cysticerus tenuicollis for 12 weeks generated a high level of protection (> 95%) against intra-ruminal challenge with metacercariae of Fasciola hepatica as measured by recovery of flukes from liver and bile ducts and counts of fluke eggs in faeces. The animals were resistant to Fasciola whether challenge was superimposed upon the cestode infection or after removal of the cestode with mebendazole.Previous infection with C. tenuicollis also protected against the pathogenic effects of challenge infection with F. hepatica. Liver fibrosis was much less extensive in resistant sheep than controls and PCV's were not affected although these were reduced during fluke infection in the control animals.     

Phylogenetic relationships between Lymnaeidae in relation to infection with Fasciola sp. in Hokkaido,Japan

ABSTRACT

In this paper, lymnaeid snails collected from 107 localities in Hokkaido, Japan were examined for infection by Fasciola larvae. DNA sequences from the snails were analysed phylogenetically based on their mitochondrial cytochrome c oxidase (COI) gene. Based on shell morphology, the snails were identified as Galba truncatula, Radix auricularia, and Austropeplea ollula and were divided into distinct clades in the COI tree. No Fasciola larvae were detected in any of the collected snails. The 58 haplotypes of Galba truncatula which were found in Hokkaido included five detected previously in snails infected with Fasciola larvae, and constituted a closely related haplogroup (Gt-c1), suggesting that Gt-c1 snails would be susceptible to Fasciola sp. The Gt14 haplotype snails were considered to be Lymnaea schirazensis, an intermediate host of F. hepatica. The COI haplotypes of A. ollula belonged to a single clade, and were considered conspecific, whereas those of R. auricularia constituted three genetically distinct haplogroups, each of which was possibly a distinct species. This study suggests that morphologically-identified lymnaeid snails possibly include additional species. Further study of these snails is required to clarify their susceptibility to Fasciola sp.     

Occurrence of spermic diploid and aspermic triploid forms of Fasciola in Vietnam and their molecular characterization based on nuclear and mitochondrial DNA

Fasciola spp. found in Asian countries are diversified in nature, and they should therefore be characterized by spermatogenesis, ploidy and genetic differentiation as well as morphology. The present study showed that spermic diploid and aspermic triploid forms of Fasciola occurred in Vietnam. The spermic diploid specimens were accurately identified as F. gigantica, while the aspermic triploids could not be identified on the basis of their morphology by the ratio of body length and width and DNA sequences of nuclear ribosomal ITS1 and mitochondrial NDI and COI genes. The molecular data also indicated that Vietnamese aspermic triploids might be hybrids and/or their offspring between Fasciola hepatica and F. gigantica, because they showed the ITS1-Fh/Fg haplotype, which had chimeric sequences of the two species. Furthermore, the aspermic triploids seem to have originated in countries other than Vietnam and to have rapidly spread to that country with infected animals.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke,Fasciola hepatica

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica.2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP.3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries.4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.