Comparative evaluation of real-time PCR and ELISA for the detection of human fascioliasis.

Fascioliasis is a zoonotic parasitic infection caused by Fasciola species in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validating methods for the diagnosis of fascioliasis in humans. Serological techniques are convenient assays that significantly improves the diagnosis of Fasciola infection. However, a more sensitive method is required. The aim of this study was to compare the Real-Time PCR technique with the indirect-ELISA for the detection of Fasciola hepatica in human. Using a panel of sera from patients infected with Fasciola hepatica (n = 51), other parasitic infections (n = 7), and uninfected controls (n = 12), we optimized an ELISA which employs an excretory-secretory antigens from F. hepatica for the detection of human fascioliasis. After DNA extraction from the samples, molecular analysis was done using Real-Time PCR technique based on the Fasciola ribosomal ITS1 sequence. Of 70 patient serum samples, 44 (62.86%) samples were identified as positive F. hepatica infection using ELISA and Real-Time PCR assays. There was no cross-reaction with other parasitic diseases such as toxoplasmosis, leishmaniasis, taeniasis, hydatidosis, trichinosis, toxocariasis, and strongyloidiasis. The significant difference between the agreement and similarity of the results of patients with indirect ELISA and Real-Time PCR was 94.4% and 99.2%, respectively (Cohen's kappa ≥ 0.7; P = 0.02). Based on the Kappa agreement findings, the significant agreement between the results of ELISA and Real-Time PCR indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in humans.

Advances in diagnostic approaches to Fasciola infection in animals and humans: An overviews.

Fasciolosis, caused by Fasciola hepatica and F. gigantica, is an impediment to the livestock industry's expansion and has a massively negative socio-economic impact due to its widespread prevalence in livestock. It is a waterborne zoonosis affecting human populations in the countries where rural economies are associated with livestock rearing. Conventional diagnosis of Fasciola infection is done by detecting parasite eggs in the faeces of infected animals or by immunological methods. Accurate and quick immunodiagnosis of Fasciola infection in animals and humans is based on the detection of antibodies and specific antigens expressed in the prepatent stage of the parasite. Both molecular and serodiagnostic tests developed thus far have enhanced the reliability of Fasciola diagnosis in both man and animals but are not widely available in resource-poor nations. A pen-side diagnostic test based on a lateral flow assay or a DNA test like loop-mediated isothermal amplification (LAMP) would be simple, fast, and cost-effective, enabling clinicians to treat animals in a targeted manner and avoid the development of drug resistance to the limited flukicides. This review focuses on the recent advances made in the diagnosis of this parasite infection in animals and humans.

Reconstruction of the TGF-ß signaling pathway of Fasciola gigantica.

In the present study, we reconstructed the transforming growth factor beta (TGF-ß) signaling pathway for Fasciola gigantica, which is a neglected tropical pathogen. We defined the components involved in the TGF-ß signaling pathway and investigated the transcription profiles of these genes for all developmental stages of F. gigantica. In addition, the presence of these components in excretory and secretory products (FgESP) was predicted via signal peptide annotation. The core components of the TGF-ß signaling pathway have been detected in F. gigantica; classical and nonclassical single transduction pathways were constructed. Four ligands have been detected, which may mediate the TGF-ß signaling pathway and BMP signaling pathway. Two ligand-binding type II receptors were detected, and inhibitory Smad7 was not detected. TLP, BMP-3, BMP-1, and ActRIb showed higher transcription in 42-day juvenile and 70-day juvenile, while ActRIIa, Smad1, ActRIIb, Smad8, KAT2B, and PP2A showed higher transcription in egg. TLM, Ski, Smad6, BMPRI, p70S6K, Smad2, Smad3, TgfßRI, Smad4, and p300 showed higher transcription in metacercariae. Four ligands, 2 receptors and 3 Smads are predicted to be present in the FgESP, suggesting their potential extrinsic function. This study should help to understand signal transduction in the TGF-ß signaling pathway in F. gigantica. In addition, this study helps to illustrate the complex mechanisms involved in developmental processes and F. gigantica - host interaction and paves the way for further characterization of the signaling pathway in trematodes.

Fasciola Infection Unexpectedly Found During Cholecystectomy: Review on How to Avoid Increasing Surgery Interventions in Non-human Endemic Areas.

PURPOSE: Fascioliasis is caused by Fasciola hepatica of almost worldwide distribution and F. gigantica in wide regions of Asia and Africa. Their adult stage develops in the biliary canals and gallbladder. Infection follows an initial, 3-4 month long invasive, migratory or acute phase, and a several year-long biliary, chronic or obstructive phase. METHODS: The unexpected finding of a fasciolid inside the gallbladder during a cholecystectomy for obstructive lithiasis suspicion in a patient is reported from an area of Iran where human infection had been never reported before and studies on fascioliasis in livestock are absent. RESULTS: The fluke obtained was phenotypically classified as F. hepatica by morphometry and genotypically as F. gigantica by mtDNA cox1 fragment sequencing, although with F. hepatica scattered mutations in species-differing nucleotide positions. The clinical, radiological, and biological signs observed at the acute and chronic phases often lead to some misdiagnosis. Serological methods may be useful in cases of negative coprology. Diagnostic techniques with insufficient resolution leading to unnecessary invasive interventions are analyzed. The way to avoid unnecessary surgery is described, including analyses to be made, diagnostic tools to be used, and aspects to be considered. CONCLUSION: Reaching a correct diagnosis in the confusing presentations avoids procedure delays and unnecessary surgery. A correct drug treatment may be sufficient. Except in extreme pathological presentations, lesions decrease in number and size and finally disappear or calcify after a successful treatment. Finally, the need to increase awareness of physicians about fascioliasis is highlighted, mainly in non-human endemic areas.

First morphometric and molecular characterization of Fasciola spp. in Northwest Tunisia.

The aim of this study was to characterize the Tunisian Fasciola spp. flukes by morphometric and molecular analyses. Flukes were collected from livers of sheep slaughtered in Sejnane slaughterhouses (Bizerte gouvernorate, Northwest Tunisia) between January and March 2021.Five morphometric parameters were determined for all the liver flukes, as follows: (i) total body length (BL), (ii) distance between ventral sucker and the tail (VS-T), (iii) distance between oral sucker and ventral sucker (OS-VS), (iv) abdomen diameter (AD), (v) tail diameter (TD) and the body length to width ratio (BL/BW). Molecular identification of the fluke specimens was carried out by polymerase chain reaction, restriction fragment polymorphism (PCR-RFLP) of a 680 bp sequence of the internal transcribes spacer 1 (ITS1) gene and by amplification, sequencing, and phylogenetic analysis of a 500 bp sequence of the ITS2 gene. Morphometric measurements showed that the mean of the total body length of the adult flukes was 21.1 ± 2.7 mm with minimum and maximum lengths of 13 and 31 mm, respectively. The PCR-RFLP analysis revealed a single profile consisting of three bands of approximately 370, 100, and 60 bp. Fasciola sequences described in the present study (GenBank numbers: OQ457027 and OQ457028) showed 99.58-100% identity to Fasciola hepatica. In conclusion, the results of this study show that molecular and phylogenetic analyses confirm the presence of a single species of F. hepatica in the Sejnane region Northwest of Tunisia. However, further studies are needed to identify the occurrence of Fasciola species in other Tunisian regions.

Morphological and molecular characterization of Fasciola isolates from livestock in Golestan province, northern Iran.

BACKGROUND: Fascioliasis, caused by the liver flukes Fasciola hepatica and Fasciola gigantica, is a global zoonotic helminthic disease. The livestock and human are the final hosts of the parasites. Northern Iran is an important endemic region for fascioliasis. Few studies have been conducted on the characterization of Fasciola isolates from eastern regions of the Caspian littoral of the country. OBJECTIVE: The aim of the present study was to identify F. hepatica, F. gigantica and intermediate/hybrid forms of Fasciola isolates from livestock in Golestan province, northern Iran, using morphometric and molecular tools. METHODS: Livestock livers naturally infected with Fasciola spp. were collected from Golestan slaughterhouse during 2019-2020. The worms were morphometrically studied using a calibrated stereomicroscope. Genomic DNA was extracted from all samples, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on internal transcribed spacer (ITS1) region using Rsa1 restriction enzyme. All the isolates were then analysed by multiplex PCR on Pepck region. RESULTS: A total of 110 Fasciola isolates were collected from the infected livers, including 94 sheep, 12 cattle and 4 goats. Morphometric analysis of 61 adult Fasciola isolates indicated that, 44 and 17 isolates belonged to F. hepatica and F. gigantica, respectively. Eighty-one and 29 isolates belonged to F. hepatica and F. gigantica using ITS1-RFLP, respectively. However, Pepck Multiplex PCR indicated 72 F. hepatica, 26 F. gigantica and 12 intermediate/hybrid forms. All 12 hybrid isolates were found in sheep host. Two isolates were identified as F. gigantica using morphometry and F. hepatica using both molecular methods. CONCLUSION: The present study confirmed the existence of both F. hepatica and F. gigantica species and reported the first molecular evidence of hybrid Fasciola isolates in ruminants of Golestan province.

Molecular detection of Fasciola, Schistosoma and Paramphistomum species from freshwater snails occurring in Gauteng and Free State provinces, South Africa.

Trematodiases are diseases caused by snail-borne trematode parasites that infect both animals and humans. Fascioliasis, schistosomiasis and paramphistomosis are some of these diseases and they affect millions of livestock, leading to significant economic losses. The aim of the study was to document freshwater snails occurring in selected study sites in the Free State and Gauteng provinces as well as identify and detect larval trematodes that they harbour. Samples were collected from a total of five study sites within two provinces of South Africa. Morphological features were used to identify snail species and were further confirmed genetically by polymerase chain reaction (PCR), sequencing and phylogenetic analysis. The larval trematodes were also detected by PCR, PCR-Restriction Length Fragment Polymorphism (PCR-RLFP), sequencing and phylogenetic analysis. A total of 887 freshwater snails were collected from Free State (n = 343) and Gauteng (n = 544). Five different genera of snails as well as species in the Succineidae family were documented. The snails in descending order of abundance were identified as: Physa (P.) spp. (51%), Succineidae spp. (20%), Galba (G.) truncatula (12%), Pseudosuccinea (Ps.) columella (10%), Planorbella (Pl.) duryi (6%) and Bulinus (B.) truncatus (1%). Approximately 272 DNA pools were created for genetic identification of snails and detection of trematode parasites. Schistosoma species were not detected from any of the snail species. A total prevalence of 46% was obtained for Fasciola hepatica in the identified snail species across all study sites. Overall, the highest prevalence of F. hepatica was obtained in Physa species (24%), whilst the lowest was observed in B. truncatus snails (1%). Forty three percent (43%) of the snail samples were PCR positive for Paramphistomum DNA. This is the first report of P. mexicana in South Africa. Fasciola hepatica was confirmed from all obtained snail species per study site. This is the first reported detection of F. hepatica in Pl. duryi and P. mexicana snails as well as the first confirmation of natural infection from P. acuta in South Africa.

Differentiating paramphistome species in cattle using DNA barcoding coupled with high-resolution melting analysis (Bar-HRM).

Paramphistomosis is caused by paramphistome or amphistome parasites, including Fischoederius elongatus, Gastrothylax crumenifer, Orthocoelium parvipapillatum, and Paramphistomum epiclitum. The control and prevention of these parasite outbreaks are difficult because of the wide occurrence of these species. Besides, the clinical manifestations and their egg characteristics are similar to those of other intestinal flukes in the paramphistome group, leading to misdiagnosis. Here, we employed DNA barcoding using NADH dehydrogenase (ubiquinone, alpha 1) (ND1) and cytochrome c oxidase subunit I (COI), coupled with high-resolution melting analysis (Bar-HRM), for species differentiation. As a result, ParND1_3 and ParCOI4 resulted in positive amplification in the paramphistomes and Fasciola gigantica, with significantly different melting curves for each species. The melting temperatures of each species obtained clearly differed. Regarding sensitivity, the limit of detection (LoD) for all species of paramphistomes was 1 pg/µl. Our findings suggest that Bar-HRM using ParND1_3 is highly suitable for the differentiation of paramphistome species. This approach can be used in parasite detection and epidemiological studies in cattle.

Assessment of genetic markers for multilocus sequence typing (MLST) of Fasciola isolates from Iran.

BACKGROUND: Several markers have been described to characterise the population structure and genetic diversity of Fasciola species (Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica). However, sequence analysis of a single genomic locus cannot provide sufficient resolution for the genetic diversity of the Fasciola parasite whose genomes are ∼1.3 GB in size. OBJECTIVES: To gain a better understanding of the gene diversity of Fasciola isolates from western Iran and to identify the most informative markers as candidates for epidemiological studies, five housekeeping genes were evaluated using a multilocus sequence typing (MLST) approach. METHODS: MLST analysis was developed based on five genes (ND1, Pepck, Pold, Cyt b and HSP70) after genomic DNA extraction, amplification and sequencing. Nucleotide diversity and phylogeny analysis were conducted on both concatenated MLST loci and each individual locus. A median joining haplotype network was created to examine the haplotypes relationship among Fasciola isolates. RESULTS: Thirty-three Fasciola isolates (19 F. hepatica and 14 F. gigantica) were included in the study. A total of 2971 bp was analysed for each isolate and 31 sequence types (STs) were identified among the 33 isolates (19 for F. hepatica and 14 for F. gigantica isolates). The STs produced 44 and 42 polymorphic sites and 17 and 14 haplotypes for F. hepatica and F. gigantica, respectively. Haplotype diversity was 0.982 ± 0.026 and 1.000 ± 0.027 and nucleotide diversity was 0.00200 and 0.00353 ± 0.00088 for F. hepatica and F. gigantica, respectively. There was a high degree of genetic diversity with a Simpson's index of diversity of 0.98 and 1 for F. hepatica and F. gigantica, respectively. While HSP70 and Pold haplotypes from Fasciola species were separated by one to three mutational steps, the haplotype networks of ND1 and Cyt b were more complex and numerous mutational steps were found, likely due to recombination. CONCLUSIONS: Although HSP70 and Pold genes from F. gigantica were invariant over the entire region of sequence coverage, MLST was useful for investigating the phylogenetic relationship of Fasciola species. The present study also provided insight into markers more suitable for phylogenetic studies and the genetic structure of Fasciola parasites.

Identification and differentiation of Fasciola hepatica and F. gigantica using multiplex PCR technique.

We aimed to present an alternate method instead of PCR-RFLP and also develop an optimized method for rapid, time-saving and affordable molecular-based approach to discriminate species of liver fluke, Fasciola hepatica and F. gigantica. Seventy-six samples of F. hepatica and 28 F. gigantica were collected from the slaughterhouses of endemic regions in Iran. Following a comprehensive analysis of the mitochondrial complete sequences of both F. hepatica and F. gigantica, the extracted DNAs from all samples were used as templates in multiplex PCR reactions containing two sets of primers specific for cytochrome c oxidase I (cox I) gene of both species. In a parallel experiment, PCR-RFLP was performed for each sample using internal transcribed spacer (ITS1) sequence. Furthermore, following a PCR amplification for cox I gene, the amplicons were purified for sequencing. To assess the validity of the multiplex PCR approach, the obtained data from the multiplex PCR and PCR-RFLP experiments were compared with each other. By sequence analysis of 104 samples, 76 and 28 samples were identified as F. hepatica and F. gigantica, respectively. Results revealed 100% and 92% of accuracy as for multiplex PCR and PCR-RFLP. The designed multiplex PCR strategy offers a valid alternative approach to the conventional methods with distinctive features including convenience, cost-effectiveness, time-saving (3 hours from sampling to obtain final results) and high efficacy.

New perspectives for fascioliasis in Upper Egypt's new endemic region: Sociodemographic characteristics and phylogenetic analysis of Fasciola in humans, animals, and lymnaeid vectors.

BACKGROUND: Fascioliasis is a significant vector-borne disease that has emerged in numerous tropical and subtropical countries causing severe health problems. Egypt is one of the fascioliasis endemic regions; however, the current situation in Upper Egypt is understudied, with only sporadic human cases or outbreaks. This study aims to highlight the sociodemographic characteristics of human fascioliasis in a newly emerged endemic area in Upper Egypt, along with risk factors analysis and the molecular characteristics of the fasciolid population in humans, animals, and lymnaeid snails. METHODOLOGY/PRINCIPAL FINDINGS: The study reported Fasciola infection in patients and their close relatives by analyzing the risk of human infection. Morphological and molecular characterization was performed on lymnaeid snails. Multigene sequencing was also used to characterize fasciolids from human cases, cattle, and pooled snail samples. The study identified asymptomatic Fasciola infection among family members and identified the presence of peridomestic animals as a significant risk factor for infection. This is the first genetic evidence that Radix auricularia exists as the snail intermediate host in Egypt. CONCLUSIONS/SIGNIFICANCE: This study revealed that Assiut Governorate in Upper Egypt is a high-risk area for human fascioliasis that requires additional control measures. Fasciola hepatica was the main causative agent infecting humans and snail vectors in this newly emerged endemic area. In addition, this is the first report of R. auricularia as the snail intermediate host transmitting fascioliasis in Upper Egypt. Further research is required to clarify the widespread distribution of Fasciola in Egypt's various animal hosts. This provides insight into the mode of transmission, epidemiological criteria, and genetic diversity of fasciolid populations in Upper Egypt.\.

Phylogenetic Characteristics of Fasciola hepatica Isolated from a Korean Patient.

Fascioliasis is a parasitic infection caused by liver flukes. Although several cases have been reported in Korea, phylogenetic analysis of isolates is lacking. In this study, a 66-year-old woman with right upper quadrant (RUQ) abdominal pain was diagnosed as fascioliasis involving abdominal muscle by imaging study. She received praziquantel treatment, but symptoms were not improved. Lateral movement of the abscess lesion was followed. Trematode parasite was surgically removed from the patient's rectus abdominis muscle. The fluke was identified as Fasciola hepatica based on sequence analysis of 18S rDNA. To determine the phylogenetic position of this Fasciola strain (named Korean Fasciola 1; KF1), the cox1 gene (273 bp) was analyzed and compared with the genes of 17 F. hepatica strains isolated from cows, sheep, goats, and humans from various countries. Phylogenetic analysis showed that KF1 was closely related with the isolates from China goat.

Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene.

BACKGROUND: Multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) for nuclear phosphoenolpyruvate carboxykinase (pepck) and polymerase delta (pold), respectively, have been used to differentiate Fasciola hepatica, F. gigantica, and hybrid Fasciola flukes. However, discrimination errors have been reported in both methods. This study aimed to develop a multiplex PCR based on a novel nuclear marker, the fatty acid binding protein type I (FABP) type I gene. METHODS: Nucleotide sequence variations of FABP type I were analyzed using DNA samples of F. hepatica, F. gigantica, and hybrid Fasciola flukes obtained from 11 countries in Europe, Latin America, Africa, and Asia. A common forward primer for F. hepatica and F. gigantica and two specific reverse primers for F. hepatica and F. gigantica were designed for multiplex PCR. RESULTS: Specific fragments of F. hepatica (290 bp) and F. gigantica (190 bp) were successfully amplified using multiplex PCR. However, the hybrid flukes contained fragments of both species. The multiplex PCR for FABP type I could precisely discriminate the 1312 Fasciola samples used in this study. Notably, no discrimination errors were observed with this novel method. CONCLUSIONS: Multiplex PCR for FABP type I can be used as a species discrimination marker in place of pepck and pold. The robustness of the species-specific primer should be continuously examined using a larger number of Fasciola flukes worldwide in the future since nucleotide substitutions in the primer regions may cause amplification errors.

Dispersal direction of Malaysian Fasciola gigantica from neighboring southeast Asian countries inferred using mitochondrial DNA analysis.

Fasciola gigantica and hybrid Fasciola flukes, responsible for the disease fasciolosis, are found in Southeast Asian countries. In the present study, we performed molecular species identification of Fasciola flukes distributed in Terengganu, Malaysia using multiplex PCR for phosphoenolpyruvate carboxykinase (pepck) and PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). Simultaneously, phylogenetic analysis based on mitochondrial NADH dehydrogenase subunit 1 (nad1) was performed for the first time on Malaysian Fasciola flukes to infer the dispersal direction among neighboring countries. A total of 40 flukes used in this study were identified as F. gigantica. Eight nad1 haplotypes were identified in the F. gigantica population of Terengganu. Median-joining network analysis revealed that the Malaysian population was related to those obtained from bordering countries such as Thailand and Indonesia. However, genetic differentiation was detected using population genetics analyses. Nevertheless, the nucleotide diversity (π) value suggested that F. gigantica with the predominant haplotypes was introduced into Malaysia from Thailand and Indonesia. The dispersal direction suggested by population genetics in the present study may not be fully reliable since Fasciola flukes were collected from a single location in one state of Malaysia. Further studies analyzing more samples from many locations are required to validate the dispersal direction proposed herein.

Identification and expression of a transforming growth factor beta (TGF-ß) homologue in the tropical liver fluke Fasciola gigantica.

Liver flukes, Fasciola spp., are veterinary and medically important parasites infecting numerous species of economically important animals in addition to humans on a global scale. The components of transforming growth factor beta (TGF-ß) signalling are widely distributed throughout the animal kingdom and are considerably conserved. Through shared common signal transduction mechanisms, crosstalk of TGF-ß signalling between a host and the parasite during infection is possible. Herein, we have identified and undertaken the molecular characterisation of a putative TGF-ß homologue from the tropical liver fluke F. gigantica (FgTLM). A FgTLM cDNA was 3557 bp in length, it encoded for 620 amino acid polypeptide which consisted of 494 amino acids of prodomain and 126 amino acids comprising the mature protein. FgTLM displayed characteristic structures of mammalian TGF-ß ligands that were unique to the inhibin-ß chain, monomer of activin. A phylogenetic analysis revealed the high degree of conservation with TGF-ß molecules from trematode species. Interestingly, the sequence of amino acid in the active domain of FgTLM was completely identical to FhTLM from F. hepatica. FgTLM expressed throughout the lifecycle of F. gigantica but was highly expressed in developmental active stages. The dynamics of expression of FgTLM during the developmental stages of F. gigantica was comparable to the pattern of TGF-ß expression in F. hepatica. Our findings demonstrated that FgTLM exhibits a high level of similarity to FhTLM in the context of both amino acid sequence and the life stage expression patterns. These similarities underline the possibility that the FgTLM molecule might have the same properties and functions as FhTLM in biological processes of the immature parasites and host immune evasion. Consequently, the specific biological functions of FgTLM on either parasite or relevant hosts need to be defined experimentally.

Omics tools enabling vaccine discovery against fasciolosis.

In the past decade significant advances in our understanding of liver fluke biology have been made through in-depth interrogation and analysis of evolving Fasciola hepatica and Fasciola gigantica omics datasets. This information is crucial for developing novel control strategies, particularly vaccines necessitated by the global spread of anthelmintic resistance. Distilling them down to a manageable number of testable vaccines requires combined rational, empirical, and collaborative approaches. Despite a lack of clear outstanding vaccine candidate(s), we must continue to identify salient parasite-host interacting molecules, likely in the secretory products, tegument, or extracellular vesicles, and perform robust trials especially in livestock, using present and emerging vaccinology technologies to discover that elusive liver fluke vaccine. Omics tools are bringing this prospect ever closer.

Exploiting Comparative Omics to Understand the Pathogenic and Virulence-Associated Protease: Anti-Protease Relationships in the Zoonotic Parasites Fasciola hepatica and Fasciola gigantica.

The helminth parasites, Fasciola hepatica and Fasciola gigantica, are the causative agents of fasciolosis, a global and economically important disease of people and their livestock. Proteases are pivotal to an array of biological processes related to parasitism (development, feeding, immune evasion, virulence) and therefore their action requires strict regulation by parasite anti-proteases (protease inhibitors). By interrogating the current publicly available Fasciola spp. large sequencing datasets, including several genome assemblies and life cycle stage-specific transcriptome and proteome datasets, we reveal the complex profile and structure of proteases and anti-proteases families operating at various stages of the parasite's life cycle. Moreover, we have discovered distinct profiles of peptidases and their cognate inhibitors expressed by the parasite stages in the intermediate snail host, reflecting the different environmental niches in which they move, develop and extract nutrients. Comparative genomics revealed a similar cohort of peptidase inhibitors in F. hepatica and F. gigantica but a surprisingly reduced number of cathepsin peptidases genes in the F. gigantica genome assemblies. Chromosomal location of the F. gigantica genes provides new insights into the evolution of these gene families, and critical data for the future analysis and interrogation of Fasciola spp. hybrids spreading throughout the Asian and African continents.

Population structure, molecular characterization, and phylogenetic analysis of Fasciola gigantica from two locations in Uganda.

Fasciola gigantica is a major pathogen that causes fasciolosis in Africa. A recent study in Uganda demonstrated that Fasciola flukes were present in 65.7% of slaughtered cattle. However, molecular identification of Fasciola species has not yet been performed in the country. In the present study, 292 Fasciola flukes were collected from Kampala and Gulu, Uganda. The samples were identified as F. gigantica using a multiplex polymerase chain reaction (PCR) assay for phosphoenolpyruvate carboxykinase (pepck) and a PCR-restriction fragment length polymorphism (RFLP) assay for DNA polymerase delta (pold). A significant genetic difference between F. gigantica obtained from cattle slaughtered at Kampala and Gulu was observed by analyzing the mitochondrial markers NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Fasciola collected from Gulu had a more diversified population than that collected from Kampala, probably because of differences in livestock management systems. One of the possible reasons for this observation is that cattle slaughtered in Gulu were reared under an extensive communal grazing system, which is suitable for maintaining parasite diversity, whereas cattle slaughtered in Kampala mainly originated from fenced/closed farms, which limits parasite diversity. However, the cause of the difference between these two locations was not clearly defined by the results of this study. The F. gigantica population from Uganda was related to that obtained from Zambia. A star-like phylogeny was detected in a median-joining network analysis, which indicated rapid population expansion and suggested that the F. gigantica populations from both countries are maintained by domestic ruminants in eastern Africa. Interestingly, the F. gigantica population from Uganda was not related to those from Egypt and Nigeria. The results of the present study suggest that F. gigantica populations in African countries are indigenous to each country or region.

Body condition scores, fluke intensity, liver pathology, and carcass quality of different dairy cattle genotypes infected with Fasciola species at high throughput abattoirs in South Africa.

Milk is an essential commodity whose demand far exceeds supply. However, dairy animal productivity is constantly hampered by parasitic diseases such as fasciolosis, affecting milk production. Despite the negative impact of liver fluke on milk production, there is little information on liver fluke infection and associated abattoir losses (body weight, condition score, liver pathology, and carcass quality) in culled dairy cattle. This study aimed to determine body condition scores, fluke intensity, liver pathology, and carcass quality of different cattle genotypes infected with Fasciola species at three commercial abattoirs. A longitudinal study was conducted from September 2019 to October 2020 to determine body condition score, liver fluke intensity, liver pathology in 3065 dairy cattle slaughtered in CA1, CA2, and CA3, of the Eastern Cape Province South Africa. Liver fluke intensity significantly increased with cattle age (P < 0.0001). Cattle ≥ 7 years old (59.93 ± 6.42) and those 4 to 6 years old (49.78 ± 9.98) had higher infection than those 2 to 3 years old (27.55 ± 13.68). The liver fluke infection was significantly (P < 0.001) the highest when sampling was conducted in summer, followed by autumn and winter, and least for spring. The differences in carcass weights or body condition scores decreased by 0.99 units (P < 0.0001) or 0.97 units (P < 0.0001) respectively. Therefore, this study suggests that fluke infection could be responsible for considerable economic and production losses mainly due to condemnation and weight loss in dairy cattle. This study recommended a combination of holistic and grazing management to control infection rates in dairy herds.

Molecular identification of cercaria Fasciola gigantica in lymnaeid snails in Kulon Progo, Yogyakarta.

Lymnaeid snails play an essential role in transmitting fasciolosis as intermediate hosts. Therefore, this study aims to use the molecular method to identify liver fluke in lymnaeid snails. A total of 320 lymnaeid snails were collected from a rice field. The samples were dissected to collect cercaria and identified using polymerase chain reaction. Furthermore, the internal transcribed spacer 2 (ITS2) was used as the target gene to identify the species of cercaria. The result showed that 3.75% (12/320) of the snails were infected by Fasciola gigantica, while the phylogenetic tree based on ITS2 showed that the cercaria in this study was monophyletic and similar to species from several countries in Southeast Asia, including China. Furthermore, the haplotype network showed that all four cercaria samples were similar with sequences from several countries. This study suggests that the F. gigantica cercaria isolated from lymnaeid snails in Kulon Progo, Yogyakarta, Indonesia, has a sequence similar to that of other species in Southeast Asian countries, although no hybrid type was detected in these sequences. This is the first report on the molecular identification of cercaria F. gigantica isolated from lymnaeid snails in Yogyakarta, Indonesia.