Search

Fasciola spp. is a major livestock parasite, especially in cattle, water buffalo, sheep, and goats. Infection reduces animal productivity, e.g., meat, dairy, wool and might cause death. In Thailand, reports of Fasciola spp. infection in livestock, especially dairy and beef cattle, are sparse. Pakchong district in Nakhon Ratchasima province is one of Thailand's largest farming areas for dairy and beef cattle, but the prevalence of Fasciola spp. infection has never been reported in this district. The landscape of this area is mainly a hilltop plateau with many water sources suitable for the development of lymnaeid snails, the intermediate host of Fasciola spp., which are essential for the parasite life cycle. This study surveyed the copro- and seroprevalence of Fasciola spp. infection in dairy and beef cattle farmed in Pakchong district by microscope-based examination, PCR, and indirect ELISA. Associated risk factors and geographic information data were collected and analyzed. Paired stool and serum samples were collected from 102 dairy cattle and 99 beef cattle from April to November 2021. Sample analyses demonstrated a high prevalence of Fasciola spp. infection, especially in beef cattle. The overall copro-prevalence was 5.97%, with 0.99% in dairy cattle and 11.11% in beef cattle. The overall seroprevalence was 23.88%, with 2.94% in dairy cattle and 45.45% in beef cattle. Moreover, the data indicated that infection status was not correlated with animal sex and age whereas consumption of natural grasses, water resources, housing floor, and farming system were significant risk factors. Data analysis by a geographic information system (GIS) demonstrated that an associated risk could be farmed in lowering areas, especially in Chan Thuck, Nong Sa Rai, and Khlong Muang subdistricts. In conclusion, this study reports the prevalence of Fasciola spp. infection in cattle in a major farming area of Thailand which could be beneficial for designing parasite control policies in this region as well as adapting this knowledge to other Fasciola spp. endemic areas. [Display omitted] • Beef cattle in the vast farming area of Thailand have a high prevalence of Fasciola spp. [ABSTRACT FROM AUTHOR]

Fasciola gigantica is considered the major pathogen causing fasciolosis in Africa; however, molecular characterization of this fluke has not been adequately elucidated. It is important to scientifically elucidate the dispersal history of F. gigantica by analyzing its genetic diversity. Fasciola flukes from Nigeria were analyzed using nuclear and mitochondrial DNA markers. A total of 172 Fasciola flukes collected from cattle were identified as F. gigantica because they displayed the F. gigantica fragment pattern in multiplex PCR for the nuclear marker, phosphoenolpyruvate carboxykinase ( pepck ). In total, 70 haplotypes were detected from Nigerian F. gigantica on the basis of the concatenated sequence of mitochondrial NADH dehydrogenase subunit 1 ( nad1 ) and cytochrome c oxidase 1 ( cox1 ). The index of neutrality (Fu's Fs ) suggests rapid expansion of the Nigerian F. gigantica population. Although four haplogroups, Nigeria 1A, 1B, 2A, and 2B, were detected from Nigerian F. gigantica , a climate-specific genetic structure was not observed among F. gigantica populations from three agro-climatic regions (Sahel, Savannah, and Forest). This is probably because of the frequent transportation of livestock from one part of the country to the other. Nigeria 1A and 1B had close relationships with the Egyptian population of F. gigantica , whereas Nigeria 2A and 2B were comparatively related to the Zambian population. No haplotype was shared among the three countries, and it therefore is difficult to estimate the dispersal route of F. gigantica within the African continent. [ABSTRACT FROM AUTHOR]

Fasciola gigantica and aspermic (hybrid) Fasciola flukes are thought to be distributed in Southeast Asian countries. The objectives of this study were to investigate the distribution of these flukes from unidentified ruminants in western Java, Indonesia, and to determine their distribution history into the area. Sixty Fasciola flukes from western Java were identified as F. gigantica based on the nucleotide sequences of the nuclear phosphoenolpyruvate carboxykinase ( pepck ) and DNA polymerase delta ( pold ) genes. The flukes were then analyzed phylogenetically based on the nucleotide sequence of the mitochondrial NADH dehydrogenase subunit 1 ( nad1 ) gene, together with Fasciola flukes from other Asian countries. All but one F. gigantica fluke were classified in F. gigantica haplogroup C, which mainly contains nad1 haplotypes detected in flukes from Thailand, Vietnam, and China. A population genetic analysis suggested that haplogroup C spread from Thailand to the neighboring countries including Indonesia together with domestic ruminants, such as the swamp buffalo, Bubalus bubalis . The swamp buffalo is one of the important definitive hosts of Fasciola flukes in Indonesia, and is considered to have been domesticated in the north of Thailand. The remaining one fluke displayed a novel nad1 haplotype that has never been detected in the reference countries. Therefore, the origin of the fluke could not be established. No hybrid Fasciola flukes were detected in this study, in contrast to neighboring Asian countries. [ABSTRACT FROM AUTHOR]

Fasciola flukes from eastern India were characterized on the basis of spermatogenesis status and nuclear ITS1. Both Fasciola gigantica and aspermic Fasciola flukes were detected in Imphal, Kohima, and Gantoku districts. The sequences of mitochondrial nad1 were analyzed to infer their phylogenetical relationship with neighboring countries. The haplotypes of aspermic Fasciola flukes were identical or showed a single nucleotide substitution compared to those from populations in the neighboring countries, corroborating the previous reports that categorized them in the same lineage. However, the prevalence of aspermic Fasciola flukes in eastern India was lower than those in the neighboring countries, suggesting that they have not dispersed throughout eastern India. In contrast, F. gigantica was predominant and well diversified, and the species was thought to be distributed in the area for a longer time than the aspermic Fasciola flukes. Fasciola gigantica populations from eastern India were categorized into two distinct haplogroups A and B. The level of their genetic diversity suggests that populations belonging to haplogroup A have dispersed from the west side of the Indian subcontinent to eastern India with the artificial movement of domestic cattle, Bos indicus , whereas populations belonging to haplogroup B might have spread from Myanmar to eastern India with domestic buffaloes, Bubalus bubalis . [ABSTRACT FROM AUTHOR]

The number of wild sika deer ( Cervus nippon yesoensis ) continues to increase in Hokkaido Prefecture, Japan. The major concern for the livestock industry is the transmission of pathogens between sika deer and cattle. Fasciolosis is an important disease that can occur in both animals. The aim of this study was to examine the possible mutual transmission of this disease in Hokkaido Prefecture. A total of 105 Fasciola flukes were obtained from sika deer and 96 from domestic cattle. The Fasciola flukes in Japan are reported to possess no mature sperm. However, in this study, 14 flukes from sika deer and eight flukes from cattle contained mature sperm in their seminal vesicles. All the Fasciola flukes from the two host animals had Fh/Fg type in nuclear phosphoenolpyruvate carboxykinase ( pepck ) gene, with a mixed fragment pattern derived from F. hepatica and F. gigantica , which are considered to be hybrid Fasciola flukes. However, almost all the flukes had Fsp1 haplotype in NADH dehydrogenase subunit 1 ( nad1 ) gene, indicating that their maternal lineage was F. hepatica . A new haplotype, Fsp3, was detected in one fluke obtained from cattle and differed in one nucleotide from Fsp1. Therefore, the Fasciola flukes detected in both host species had almost identical molecular characteristics. These findings suggest the mutual transmission of Fasciola flukes between sika deer and domestic cattle in Hokkaido. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Fasciola spp. in Myanmar were characterized on the basis of spermatogenesis status and DNA markers of nuclear internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1). We collected 88 adult flukes from Yangon, Lashio, and Myitkyina. Spermatogenesis status was analyzed by the presence of sperm in the seminal vesicles, and 8 aspermic and 80 spermic flukes were detected. The flukes were identified on the basis of spermatogenesis status and ITS1 types which were analyzed by a PCR-RFLP method, and 80 spermic flukes were identified as F. gigantica. A very low detection rate of aspermic Fasciola sp. indicated that they are not established in Myanmar. In phylogenetic analyses, the 7 aspermic Fasciola sp. from Myitkyina displayed a haplotype in nad1 sequence, which was identical to that of aspermic Fasciola sp. from other Asian countries including China. Therefore, they were probably introduced from China through an infected domestic ruminant. On the other hand, 17 nad1 haplotypes detected in F. gigantica belonged to 2 clades unique to Myanmar, each with a distinct founder haplotype in a network analysis. This indicated a unique history of F. gigantica introduction into Myanmar involving ancient artificial movements of domestic ruminants. [Copyright &y& Elsevier]

Abstract: Thioredoxin-glutathione reductase (TGR) was purified from the adult stage of the liver fluke Fasciola hepatica. At 38°C and pH 7.8, specific activity values were 10.2U mg−1 and 64.5U mg−1, with DTNB or GSSG as substrates, respectively. Under the same conditions, apparent Km values were 46±8μM (DTNB) and 30±5μM (GSSG). The enzyme was also able to catalyze thiol/disulfide exchange reactions. A subunit Mr of 61,000 was obtained. Like the homologous enzyme from the tapeworms, a lag time was observed in the enzyme assays at moderate or high concentrations of the substrate GSSG. The hysteretic behavior was reverted in the presence of GSH and was notably dependent on pH, such that the magnitude of the lag time increased with the acidity of the medium. These results strongly suggest that a hysteretic kinetic is a common feature of TGR from any parasitic flatworm. A sequence comparison revealed the structural cysteine residues proposed to be in the origin of the peculiar kinetic behavior of TGR are absent from the F. hepatica enzyme. Based on these observations, the model proposed recently to explain the GSSG-dependent hysteretic kinetic of TGR, which assumes the covalent modification of specific cysteine residues through glutathionylation [Bonilla M. et al. (2008) J Biol Chem 283: 17898] needs to be reevaluated. [Copyright &y& Elsevier]

Abstract: Fascioliasis has a negative impact on the farming industry in both developed and developing countries, rather than a public health challenge. This study was performed to identify Fasciola sp. from different definitive hosts (buffalo, cattle, and sheep) based on the molecular parameters and spermatogenesis. Ninety-one adult flukes were collected from livers of slaughtered animals at abattoirs in different prefectures in Egypt. Microscopic examination of the analyzed flukes showed many normal spermatozoa in the seminal vesicles (spermic), suggesting that they have the ability of spermatogenesis. This study showed that no parthenogenic Fasciola species occurred in Egypt. Molecular analysis was performed utilizing genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers. Whereas 16 animals proved to have infection with a single Fasciola species, 2 were infected with both F. hepatica and F. gigantica. The results indicated that sheep were prone to F. hepatica (8 out of 10 animals) more than F. gigantica infection. Sequences of ITS1 and ITS2 ribosomal region indicated that the flukes were categorized into 3 groups F. hepatica-type (47), F. gigantica-type (42) and 2 flukes possessed sequences of both types indicating an existence of different alleles at the same loci. Unique overlapping of T/C bases were detected in both ITS1 (Position 96) and ITS2 (Position 416). Based on results of mitochondrial gene markers (NDI and COI), flukes were classified into F. hepatica-type and F. gigantica-type. Extensive intra-sequence polymorphism was detected at both markers. NDI and COI sequences of Egyptian strain of F. gigantica showed pronounced diversity compared with relevant sequences at database. [ABSTRACT FROM AUTHOR]

Abstract: We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%–99.9%), 100% (95% CI: 98.6%–100%) and 0.997 (95% CI: 0.987–1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R 2 =0.84; p <0.001). The agreement between results obtained with pairs of serum and milk samples was very high: there was matching classification in 96% (910/947) of paired samples (kappa=0.92; p <0.001). Individual milk samples may therefore be used, instead of serum samples, in the MM3-SERO ELISA, for reliable detection of seropositive cows. Testing bulk tank milk samples enabled detection of infected herds, even when the within-herd prevalence of infection was as low as 12%. We conclude that the MM3-SERO ELISA is a sensitive and highly specific test for serodiagnosis of bovine fasciolosis, and can be used with individual samples of either serum or milk. Use of the assay with bulk milk samples enables estimation of the within-herd prevalence of infection. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Fascioliasis is a zoonotic disease caused by parasites of the genus Fasciola spp. which cause an important loss to the livestock industry. The objectives of this study were: to estimate the prevalence of fascioliasis in three provinces of Amazonas, to evaluate possible risk factors of infection in cattle and to genetically characterize the Fasciola haplotypes circulating in this area. According to the results the prevalence of fascioliasis in cattle was 90.13% (712/790). Odds ratio results showed a significant association between fascioliasis and the Brown Swiss breed (OR = 2.62; 95% CI: 1.57–4.35; p < 0.001), and with female cattle older than 30 months (OR = 1.71; 95% CI: 1.05–2.79; p < 0.031). According to the molecular genetic studies using the gene marker NAD1 , six haplotypes of Fasciola hepatica were found in the 35 infected livers collected. The results obtained in this study are concerning due to the high prevalence presented and it reveals the necessity of a continuing monitoring because of the high risk of transmission to humans. • Molecular characterization of fascioliasis in bovines from Amazon region, Peru. • The Fasciola hepatica haplotypes HAP4, HAP5 and HAP6 were found in the study area. • Very high prevalence of fascioliasis (90,13%) in cattle from Amazonas, Peru. • Fascioliasis should be monitored in bovines and humans continuously. [ABSTRACT FROM AUTHOR]

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. • We identified and characterized species of Fasciola flukes in the Dali Prefecture. • ITS, ND1, and CO1 DNA sequences were sequenced and analyzed for homology. • The analysis of pepck revealed that all the specimens were heterozygous Fasciola flukes. • The predominant ND1 haplotype was N-Fga type. • N-Fga type was identical to that of "aspermic" flukes previously reported. [ABSTRACT FROM AUTHOR]

Experimental infection with Fasciola hepatica and parthenogenetic Fasciola sp. in laboratory animals have been conducted in rats and rabbits. Inoculation of less than 5 metacercariae into rat-like hamsters, Tscherskia triton, is sufficient to establish Fasciola infections. The prepatent period of F. hepatica and the parthenogenetic Fasciola sp. in T. triton was shorter than that in rats and rabbits, suggesting that T. triton is a suitable experimental model for these flukes. In contrast, F. gigantica infection in T. triton did not yield adult flukes; T. triton, is therefore, considered to be an unsuitable host for F. gigantica. The cotton rat, Sigmodon hispidus, was an unsuitable host for the parthenogenetic Fasciola sp. [Copyright &y& Elsevier]

Protections against Fasciola gigantica infection in mice immunized with the individual and combined cathepsin L1H and cathepsin B3 vaccines were assessed. The vaccines comprised recombinant (r) pro-proteins of cathepsin L1H and B3 (rproFgCatL1H and rproFgCatB3) and combined proteins which were expressed in Pichia pastoris. The experimental trials were performed in ICR mice (n = 10 per group) by subcutaneous injection with 50 μg of the recombinant proteins combined with Alum or Freund's adjuvants. At two weeks after the third immunization, mice were infected with 15 F. gigantica metacercariae per mouse by oral route. The percents of protection of rproFgCatL1H, rproFgCatB3 and combined vaccines against F. gigantica were approximately 58.8 to 75.0% when compared with adjuvant-infected control. These protective effects were similar among groups receiving vaccines with Alum or Freund's adjuvants. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th1 and Th2 immune responses, it was found that both Th1 and Th2 humoral immune responses were significantly increased in vaccinated groups compared with the control groups, with higher levels of IgG1 (Th2) than IgG2a (Th1). Mice in vaccinated groups showed reduction in liver pathological lesions when compared with control groups. This study indicates that the combined rproFgCatB3 and rproFgCatL1H vaccine had a high protective potential than a single a vaccine, with Alum and Freund's adjuvants showing similar level of protection. These results can serve as guidelines for the testing of this F. gigantica vaccine in larger economic animals. [ABSTRACT FROM AUTHOR]

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola , in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Pakistani Fasciola flukes were identified as F. gigantica by nuclear pepck and pold. • Mitochondrial nad1 and cox1 were used for phylogenetic characterizations. • Genetic difference between F. gigantica from buffaloes and goats was suggested. • Two origins of F. gigantica in Pakistan were indicated by the network analysis. [ABSTRACT FROM AUTHOR]

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 nad1 gene 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. Unlabelled Image • All 225 Fasciola flukes obtained from Ecuador were identified as Fasciola hepatica. • Fasciola gigantica and parthenogenic Fasciola flukes were not found in Ecuador. • Multiplex PCR method targeting pepck gene is unsuitable for detecting F. hepatica. • Fasciola hepatica populations in South America, including Ecuador, formed from the ancestral F. hepatica individuals introduced into the continent. [ABSTRACT FROM AUTHOR]

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola , has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis. Unlabelled Image • Seroprevalence of anti- Fasciola antibody was investigated in Hokkaido sika deer. • The recombinant cathepsin L1 was used as an antigen. • The average seroprevalence of 1109 deer was 43.9% among the 20 locations. • Mature deer possessed higher seroprevalence than younger individuals. • Monitoring the risk of the animal as a reservoir of Fasciola flukes is important. [ABSTRACT FROM AUTHOR]

In the present study, molecular characterization of Fasciola flukes from Spain was performed to reveal the relation with the previously reported Peruvian F. hepatica population. The nuclear DNA markers, phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), were used for species identification of Fasciola flukes. A total of 196 Fasciola flukes were identified as F. hepatica by pepck and pold , and 26 haplotypes were detected in mitochondrial NADH dehydrogenase subunit 1 (nad1). Only one of them was previously found in Spanish samples; which indicates the existence of high genetic diversity and population structure in F. hepatica from Spain. Three haplotypes were identical to those from Peruvian F. hepatica. The pairwise fixation index value confirmed a relatively close relationship between the Spanish and Peruvian F. hepatica samples. The Spanish samples showed clearly higher genetic variability than the Peruvian population. These results are discussed in relation with the hypothesis of the introduction of the parasite in America from Europe and recent evidence of pre-Hispanic F. hepatica from Argentina revealed by ancient DNA. Unlabelled Image • Spanish Fasciola flukes were identified as F. hepatica by nuclear pepck and pold. • Phylogenetic analyses were performed using mitochondrial nad1. • High level of genetic diversity was observed in Spanish F. hepatica. • Some Spanish F. hepatica showed a close relation with that in Peru. [ABSTRACT FROM AUTHOR]

Fasciola spp. are responsible for over 3 billion US dollars of production loss annually in livestock and cause widespread zoonotic disease. Nevertheless, understating of the emergence and spread of the trematode species is poor. The multiplicity of F. gigantica infection and its spread is potentially influenced by multiple factors, including the abundance of suitable intermediate hosts, climatic conditions favouring the completion of the parasite's lifecycle, and translocation of infected animals, or free-living parasite stages between regions. Here we describe the development of a 'tremabiome' metabarcoding sequencing method to explore the numbers of F. gigantica genotypes per infection and patterns of parasite spread, based on genetic characteristics of the mitochondrial NADH dehydrogenase 1 (mt-ND-1) locus. We collected F. gigantica from three abattoirs in the Punjab and Balochistan provinces of Pakistan, and our results show a high level of genetic diversity in 20 F. gigantica populations derived from small and large ruminants consigned to slaughter in both provinces. This implies that F. gigantica can reproduce in its definitive hosts through meiosis involving cross- and self-breeding, as described in the closely related species, Fasciola hepatica. The genetic diversity between the 20 populations derived from different locations also illustrates the impact of animal movements on gene flow. Our results demonstrate the predominance of single haplotypes, consistent with a single introduction of F. gigantica infection in 85% of the hosts from which the parasite populations were derived. This is consistent with clonal reproduction in the intermediate snail hosts. Unlabelled Image • To confirm the species identity of recovered Fasciola spp. • To identify the presence of single or multiple genotypes per infection (multiplicity of infection) • Demonstrate the spread of F. gigantica mt-ND-1 haplotypes [ABSTRACT FROM AUTHOR]

Recently, phosphoenol pyruvate carboxykinase (pepck) and DNA polymerase delta (pold) were established as reliable nuclear markers for species identification of Fasciola spp. in multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism-based assays, respectively. Currently, little is known about Fasciola species distribution in Central Asia. Therefore, the objective of the present study was to perform precise molecular species identification of liver flukes from Afghanistan and to reveal their dispersal route(s) via phylogenetic analysis based on mitochondrial nad1 haplotypes. Ninety-two Fasciola flukes collected from sheep in Kabul, Afghanistan, were identified as F. hepatica based on pepck and pold screening. Although the pepck fragment pattern obtained via multiplex PCR analysis could not distinguish the species of the seven Fasciola flukes, the pepck nucleotide sequence data confirmed that they were F. hepatica.The 20 nad1 haplotypes detected among the Afghani liver flukes were closely related to those from China and Egypt, with the F ST value (−0.003, P =.41) between the F. hepatica populations from Afghanistan and China confirming a very close relationship. Nucleotide diversity was greater in the population from Afghanistan compared with that from China, indicating that the Afghani population was older, and that the dispersal direction of F. hepatica was from Afghanistan to China. The results of the present study contribute to our understanding of the dispersal of F. hepatica from its predicted origin, the Fertile Crescent. Unlabelled Image [ABSTRACT FROM AUTHOR]