Liver Fluke Research Articles

The combination between praziquantel treatment and fatty liver disease in relation to diabetes mellitus in Northeast of Thailand

Diabetes mellitus (DM) is a growing global public health concern, including Thailand. Prior research has established an association between fatty liver disease (FLD) and DM. Moreover, the distribution of DM was also found in liver fluke endemic areas where praziquantel (PZQ) was used prevalently. However, limited information exists regarding factors associated with DM, specifically in FLD patients undergoing PZQ treatment. Our study aimed to evaluate the association between the combination of PZQ treatment and FLD diagnosis, and DM in Northeast Thailand. We conducted the study among participants in northeastern Thailand with self-reported DM diagnosis and PZQ treatment history using health questionnaires. Additionally, FLD diagnosis was confirmed through ultrasonography from the Cholangiocarcinoma Screening Program data collected from 2013-2022. We employed multilevel mixed-effect logistic regression to assess the association between the combination of PZQ treatments and FLD diagnosis, and DM, represented as adjusted odds ratios (AOR) and their 95% confidence intervals (CI). Of 756,431 participants, the overall DM prevalence was 7%. Notably, 10.45% of individuals with FLD+ and 8.23% with PZQ+ were found to have DM. Specifically, the prevalence of DM in individuals with PZQ+ and FLD-, PZQ- and FLD+, and PZQ+ and FLD+ combinations were 7.14%, 10%, and 11.81%, respectively. In comparison to PZQ- and FLD-, participants with PZQ+ and FLD-, PZQ- and FLD+, and PZQ+ and FLD+ were associated with DM (AOR = 1.27; 95% CI: 1.24-1.30, AOR = 1.8; 95% CI: 1.76-1.85, and AOR = 2.19; 95% CI: 2.12-2.27, respectively). In conclusion, the combination of PZQ and FLD plays a crucial role in the association with DM, particularly in individuals with PZQ+ and FLD+. Our findings emphasize the importance of DM screening, with special attention to individuals with a combination of PZQ treatment and FLD diagnosis, in order to promote early detection and management of DM in Northeast Thailand.

Fumarate respiration of Fasciola flukes as a potential drug target.

Fascioliasis is a neglected tropical zoonotic disease caused by liver flukes belonging to the genus Fasciola. The emergence of resistance to triclabendazole, the only World Health Organization-recommended drug for this disease, highlights the need for the development of new drugs. Helminths possess an anaerobic mitochondrial respiratory chain (fumarate respiration) which is considered a potential drug target. This study aimed to evaluate the occurrence of fumarate respiration in Fasciola flukes. We analyzed the properties of the respiratory chain of Fasciola flukes in both adults and newly excysted juveniles (NEJs). Fasciola flukes travel and mature through the stomach, bowel, and abdominal cavity to the liver, where oxygen levels gradually decline. High fumarate reductase activity was observed in the mitochondrial fraction of adult Fasciola flukes. Furthermore, rhodoquinone-10 (RQ10 Em'= -63 mV), a low-potential electron mediator used in fumarate respiration was found to be predominant in adults. In contrast, the activity of oxygen respiration was low in adults. Rotenone, atpenin A5, and ascochlorin, typical inhibitors of mitochondrial enzymes in complexes I, II, and III, respectively, inhibit the activity of each enzyme in the adult mitochondrial fraction. These inhibitors were then used for in vitro viability tests of NEJs. Under aerobic conditions, NEJs were killed by rotenone or ascochlorin, which inhibit aerobic respiration (complex I-III), whereas atpenin A5, which inhibits complex II involved in fumarate respiration, did not affect NEJs. Moreover, ubiquinone-10 (UQ10 Em'= +110 mV), which is used in oxidative respiration, was detected in NEJs, in addition to RQ10. In contrast, under anaerobic conditions, rotenone and atpenin A5, which inhibit fumarate respiration (complex I-II), were crucial for NEJs. These findings demonstrate that NEJs have active hybrid respiration, in which they can properly use both oxygen and fumarate respiration, depending on oxygen availability. Thus, fumarate respiration is a promising drug target for Fasciola flukes, because it plays an essential role in both adults and NEJs.

Assessing the microbiota of the snail intermediate host of trematodes, Galba truncatula

BackgroundThe microbiome is known to play key roles in health and disease, including host susceptibility to parasite infections. The freshwater snail Galba truncatula is the intermediate host for many trematode species, including the liver and rumen flukes Fasciola hepatica and Calicophoron daubneyi, respectively. The snail-parasite system has previously been investigated. However, the specific interaction between the snail-associated microbiota and intra-snail developmental stages of trematodes has yet to be explored.MethodsGalba truncatula snails were collected from farms in Northern Ireland and trematode infection was diagnosed using PCR. High-throughput sequencing analysis of the bacterial 16S ribosomal DNA V3-V4 hypervariable regions was subsequently applied to characterise the microbiota of both uninfected and infected snails.Results We first showed that the snail harboured microbiota that was distinct for its environment. The microbiota of infected snails was found to differ significantly from that of uninfected snails. In particular, the bacterial genera Mycoplasma and Methylotenera were significantly more abundant in infected snails, while genera Sphingomonas and Nocardioides were predominantly associated with uninfected snails. ConclusionThese findings pave the way to future studies on the functional roles of bacteria in host-parasite relationships.Graphical

Development of a qPCR Duplex Assay for simultaneous detection of Fascioloides magna and Galba truncatula in eDNA samples: Monitoring beyond boundaries

Parasites constitute a significant economic burden and highly impact environmental, public, and animal health. The emergence of many parasitic diseases is environmentally mediated and they share the same biogeography with humans and both domestic and wild animals. American liver fluke, Fascioloides magna – a trematode parasite of domestic and wild ungulates – is an example of the anthropogenic introduction of an “invasive alien species” in Italy and Europe. Multiple introductions to Europe have led to the biogeographical expansion of the parasite across the Danube region mainly provided by the presence of suitable habitats for all hosts involved in the parasite's life cycle, human-assisted transport, and drastic environmental events such as flooding. In Italy, it was introduced and established in La Mandria Regional Park (LMRP) near Turin in 1865 along with imported wapitis (Cervus elaphus canadensis) from North America (Bassi, 1875), but with no reported expansion to the surrounding areas. LMRP isolated F. magna focus, poses an important threat of possible expansion since the enclosed area is vulnerable to occasional bidirectional passage of roe deer. Additionally, tributary rivers to the Po river system, traversing the enclosed area, could further bolster the possibility of such spread. In this study, we developed a duplex qPCR assay for F. magna and its principal intermediate host Galba truncatula optimized for testing eDNA samples to meet the needs for surveillance of the parasite. Moreover, we validated the developed assay in natura by testing samples derived from filtered water and sediments collected inside and outside LMRP's fenced-off area. Our findings for the first time demonstrate the presence of F. magna's eDNA outside the park's internal fenced-off area.

Evaluation of LAMP for Fasciola hepatica detection from faecal samples of experimentally and naturally infected cattle

Fasciola hepatica causes liver fluke disease in production animals and humans worldwide. Faecal egg counts (FEC) are the most common diagnostic tool for the diagnosis of liver fluke disease. However, FEC has low sensitivity and is often unreliable for the detection of patent infection. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of Fasciola hepatica infection, with the aim of increased sensitivity and making it suitable for on-farm application. LAMP was initially conducted under laboratory conditions, optimised to enable visual detection using calcein dye. DNA extraction based on bead-beating was developed to enable on-farm application. LAMP results were compared to FEC and polymerase chain reaction (PCR). Under laboratory conditions, LAMP was conducted using two incubation methods: a conventional PCR thermocycler and a field-deployable LAMP instrument. When compared to a ‘rigorous’ FEC protocol consisting of multiple counts using a comparatively large volume of faeces and with infection confirmed post-mortem, LAMP was highly sensitive and specific (using silica membrane DNA extraction sensitivity 88 %, specificity 100 %; using sieving and beat-beating DNA extraction sensitivity 98.9 %, specificity 100 %). When applied on-farm, LAMP was compared to conventional FEC, which suggested high sensitivity but low specificity (sensitivity 97 %, specificity 37.5 %). However, further analysis, comparing field LAMP results to laboratory PCR, suggested that the low specificity was likely the outcome of the inability of conventional FEC to detect all true F. hepatica positive samples. Based on the high sensitivity and specificity of LAMP compared to a ‘rigorous’ FEC protocol and its ability to be used in field settings, the study demonstrates the potential of LAMP for diagnosing F. hepatica infection in agriculture.

Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini-infected hamsters.Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov-M60-like-2 possess a mucinase activity, together with Ov-M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct.

Clonorchiasis and opisthorchiasis: epidemiology, transmission, clinical features, morbidity, diagnosis, treatment, and control.

Clonorchis sinensis, Opisthorchis viverrini, and Opisthorchis felineus are important liver flukes that cause a considerable public health burden in eastern Asia, southeastern Asia, and eastern Europe, respectively. The life cycles are complex, involving humans, animal reservoirs, and two kinds of intermediate hosts. An interplay of biological, cultural, ecological, economic, and social factors drives transmission. Chronic infections are associated with liver and biliary complications, most importantly cholangiocarcinoma. With regard to diagnosis, stool microscopy is widely used in epidemiologic surveys and for individual diagnosis. Immunologic techniques are employed for screening purposes, and molecular techniques facilitate species differentiation in reference laboratories. The mainstay of control is preventive chemotherapy with praziquantel, usually combined with behavioral change through information, education and communication, and environmental control. Tribendimidine, a drug registered in the People's Republic of China for soil-transmitted helminth infections, shows potential against both C. sinensis and O. viverrini and, hence, warrants further clinical development. Novel control approaches include fish vaccine and biological control. Considerable advances have been made using multi-omics which may trigger the development of new interventions. Pressing research needs include mapping the current distribution, disentangling the transmission, accurately estimating the disease burden, and developing new diagnostic and treatment tools, which would aid to optimize control and elimination measures.

Liver Flukes: Clonorchis and Opisthorchis.

Clonorchis sinensis, Opisthorchis viverrini and O. felineus are liver flukes of human and animal pathogens occurring across much of Europe and Asia. Nevertheless, they are often underestimated compared to other, better known neglected diseases in spite of the fact that many millions of people are infected and hundreds of millions are at risk. This is possibly because of the chronic nature of the infection and disease and that it takes several decades prior to a life-threatening pathology to develop. Several studies in the past decade have provided more information on the molecular biology of the liver flukes which clearly lead to better understanding of parasite biology, systematics and population genetics. Clonorchiasis and opisthorchiasis are characterized by a chronic infection that induces hepatobiliary inflammation, especially periductal fibrosis, which can be detected by ultrasonography. These chronic inflammations eventually lead to cholangiocarcinoma (CCA), a usually fatal bile duct cancer that develops in some infected individuals. In Thailand alone, opisthorchiasis-associated CCA kills up to 20,000 people every year and is therefore of substantial public health importance. Its socioeconomic impacts on impoverished families and communities are considerable. To reduce hepatobiliary morbidity and CCA, the primary intervention measures focus on control and elimination of the liver fluke. Accurate diagnosis of liver fluke infections in both human and other mammalian, snail and fish intermediate hosts is important for achieving these goals. While the short-term goal of liver fluke control can be achieved by praziquantel chemotherapy, a comprehensive health education package targeting school children is believed to be more beneficial for a long-term goal/solution. It is recommended that transdisciplinary research or multisectoral control approach including one health and/or eco health intervention strategy should be applied to combat the liver flukes and hence contribute to reduction of CCA in endemic areas.

Dicrocoeliidae Family: Major Species Causing Veterinary Diseases.

This chapter analyses the taxonomic position of the Dicrocoeliidae family and several of its genera and species. The chapter reviews the biology of major veterinary disease-causing species, including Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium chinensis, Eurytrema pancreaticum and Platynosomum fastosum. All these species have indirect life cycles with two intermediate hosts: molluscs as the first host and ants, grasshoppers and lizards as the second host. Dicrocoelium dendriticum is a widespread liver fluke found in ruminants across Europe, Asia, North Africa and North America. It can also infect humans. Dicrocoelium hospes is widely distributed in the savanna areas of Africa south of the Sahara, while D. chinensis is mainly found in ruminants in East Asia and some European countries (probably imported from Asia). Eurytrema pancreaticum is a common parasite that lives in the bile ducts, gall bladder, pancreatic ducts and intestines of ruminants. It is found in Europe, Madagascar, Asia and South America. Adults of P. fastosum live in the liver, gall bladder and pancreas of birds and mammals in Europe, Africa, Asia, North, Central and South America. Information on the epidemiology, pathology, clinical aspects, diagnosis, treatment, control, prevention and economic impact of Dicrocoeliosis caused by D. dendriticum, Eurytrematodosis and Platynosomiosis have been included.

Transcriptome changes of liver fluke Opisthorchis viverrini in diabetic hamsters.

A recent study in hamsters showed that infection with the liver fluke Opisthorchis viverrini in diabetic hosts worsens the severity of hepatobiliary disease. However, the effects of diabetes on the worm's phenotype and gene expression pattern remain unknown. This study investigated the impact of diabetes on the global gene expression and development of O. viverrini in diabetic hamsters. Parasitological parameters were assessed, and mRNA sequencing with bioinformatic analysis was performed. The study revealed that worm establishment rates in diabetic hamsters were directly correlated with fasting plasma glucose levels. Interestingly, worms collected from diabetic hosts exhibited stunted growth and reduced egg production. Transcriptomic analysis revealed significant alterations in gene expression, with 4314 and 567 differentially expressed genes at 21- and 35-days post-infection, respectively. Gene ontology enrichment analysis highlighted changes in biological processes related to stress response, metabolism, and cellular organization. Notably, genes associated with parasite virulence, including granulin, tetraspanins, and thioredoxins, showed significant upregulation in diabetic hosts. These findings demonstrate the profound impact of host diabetic status on O. viverrini development and gene expression, providing insights into the complex interplay between host metabolism and parasite biology, including molecular adaptations of O. viverrini in hosts. This study contributes to our understanding of opisthorchiasis in the context of metabolic disorders and may inform future strategies for disease management in diabetic human populations.

Identificação molecular de espécies de Fasciola com base emsequências de DNA ribossômico e sequências de DNA ribossômico e mitocondrialemRiad, ArábiaSaudita

ABSTRACT Fasciola species are the causative agents of liver fluke disease (fasciolosis) which is primarily found in domestic and wild ruminants but also are causal agents of fascioliasis in humans. Few reports for the genotyping of Fasciola species in Saudi Arabia were available. This study aimed to identify the Fasciola species infecting sheep using morphology and molecular tools. For that, livers of 500 imported sheep were slaughtered at abattoirs located in Riyadh (Saudi Arabia), and parasite species were isolated from 28 liver samples and then preserved for subsequent stages in this study. These parasite species were identified as Fasciola hepatica and Fasciolagigantica via morphology and confirmed via the 28S rRNA, ITS-1, COI, and NDI sequences with a close identity for other Fasciola species within the family Fasciolidae. Phylogenetic analysis of the obtained sequences showed that Fasciola isolates from the current study were clustered in one subclade closely related to isolates from Iran, Vietnam, Australia, South Africa, Eastern Europe, India, Egypt, Sudan, Japan, Poland, Spain, Armenia, and Turkey. A combination of data from morphology and molecular analysis could be considered a useful tool for identifying Fasciola species infecting sheep in Saudi Arabia.

Machine-Learning-Based Forest Classification and Regression (FCR) for Spatial Prediction of Liver Fluke Opisthorchis viverrini (OV) Infection in Small Sub-Watersheds

Infection of liver flukes (Opisthorchis viverrini) is partly due to their suitability for habitats in sub-basin areas, which causes the intermediate host to remain in the watershed system in all seasons. The spatial monitoring of fluke at the small basin scale is important because this can enable analysis at the level of the factors involved that influence infections. A spatial mathematical model was weighted by the nine spatial factors X1 (index of land-use types), X2 (index of soil drainage properties), X3 (distance index from the road network, X4 (distance index from surface water resources), X5 (distance index from the flow accumulation lines), X6 (index of average surface temperature), X7 (average surface moisture index), X8 (average normalized difference vegetation index), and X9 (average soil-adjusted vegetation index) by dividing the analysis into two steps: (1) the sub-basin boundary level was analyzed with an ordinary least square (OLS) model used to select the spatial criteria of liver flukes aimed at analyzing the factors related to human liver fluke infection according to sub-watersheds, and (2) we used the infection risk positional analysis level through machine-learning-based forest classification and regression (FCR) to display the predictive results of infection risk locations along stream lines. The analysis results show four prototype models that import different independent variable factors. The results show that Model 1 and Model 2 gave the most AUC (0.964), and the variables that influenced infection risk the most were the distance to stream lines and the distance to water bodies; the NDMI and NDVI factors rarely affected the accuracy. This FCR machine-learning application approach can be applied to the analysis of infection risk areas at the sub-basin level, but independent variables must be screened with a preliminary mathematical model weighted to the spatial units in order to obtain the most accurate predictions.

Parasite eggs in 16th-18th century cesspits from Granada (Spain)

The study of parasites from archaeological materials can yield information on socioeconomic conditions, as well as hygiene and waste management. The investigation of contemporaneous texts offers a complementary approach to understanding health in the past. Finding ancient parasites has proved important for analyzing structures related to waste management, such as latrines, cesspits and sewer drains. The aim of this study was to analyze the sediment in four cesspits from the early modern period (16th-18th century CE) in the city of Granada, Spain. After rehydration, homogenization, and micro-sieving (RHM) with subsequent visualization under optical microscopy, roundworm (Ascaris sp.) and whipworm (Trichuris sp.) eggs were detected in all four cesspits, with liver fluke (Fasciola sp.) eggs also being found in one cesspit. These findings are consistent with written sources from this period, which describe waste management challenges as a cause of water contamination and reveal the possible utilization of human fecal material as fertilizer. The spread of parasites would have been favored by overcrowding in the city. This study offers the first analysis of cesspits from the early modern period in the Iberian Peninsula, and demonstrates that ineffective sanitation led to widespread infection of the population by intestinal worms.

Hepatic vascular changes associated with Opisthorchis felineus infection in Syrian hamsters and humans

The liver fluke Opisthorchis felineus is a foodborne zoonotic pathogen endemic to Russia, Kazakhstan, and several European countries. The adult flukes affect the hepatobiliary system of piscivorous mammals and humans, thereby causing numerous complications, including liver fibrosis. Detailing the mechanisms of progression of the fibrotic complications is a hot topic in the field of research on opisthorchiasis pathogenesis. Pathologic angiogenesis appears to be associated with the fibrogenic progression due to active participation in the recruitment of inflammatory cells and many factors involved in the modulation of the extracellular matrix. The aim of the study was to evaluate neoangiogenesis and amyloid deposits in liver tissues of model animals and patients with confirmed chronic opisthorchiasis. In addition, we assessed a possible correlation of neoangiogenesis with liver fibrosis. We found a significant increase in the number of newly formed vessels and amyloid deposits in the liver of people with chronic opisthorchiasis compared to that of uninfected ones. Thus, for the first time we have demonstrated neoangiogenesis and amyloid deposits during O. felineus infection in a Mesocricetus auratus model. Regression analysis showed that CD34+ newly formed vessels correlate with fibrosis severity in the course of the infection. Our results indicate the potential contribution of angiogenesis to the progression of liver fibrosis, associated with O. felineus infection.

Prevalence and associated risk factors assessment of bovine fasciolosis in the Imbo Region, Burundi

Fasciolosis is a zoonosis that limits the productivity of ruminants worldwide, but there is a lack of information on its occurrence in Burundi. Therefore, this study aimed to fill the information gap by determining the prevalence and risk factors associated with bovine fasciolosis in the Imbo Region of Burundi. Two prevalence studies were conducted in parallel in the five communes of the five provinces in the Imbo region. In the first study, a total of 426 fecal samples were collected from randomly selected cattle farms and microscopically examined to determine Fasciola egg burden. Survey data on cattle husbandry were collected from owners of these cattle and analyzed to determine the risk factors for bovine fasciolosis. In the second study, 467 cattle were randomly selected in abattoirs and their livers were examined postmortem to determine liver fluke burdens. Data were entered separately into Microsoft Excel and analyzed using R software. The overall prevalence of bovine fasciolosis was 47.7% (42.9–52.4, 95% CI) for microscopic examination and 33.2% (28.9–37.5, 95% CI) for postmortem examinations. The majority of positive cattle (60.6%) had light intensity infections as determined by eggs per gram of feces (epg). Postmortem examinations corroborated these results and indicated that 80% of cattle had light intensity infections. Chi-square analysis showed a statistical association with the presence of bovine fasciolosis and the age, sex, and origin of cattle and the practices of cattle owners (P < 0.05).

Efficacy of a single-dose albendazole against lancet liver fluke Dicrocoelium dendriticum and liver enzymes activity in naturally infected sheep

Infections with D. dendriticum are distributed worldwide and mostly associated with ruminant livestock. Depending on the length and strength of the infection it can be manifested with losses in milk production, reductions in milk and wool quality, decreased weight gains, reproductive performance and poor carcass quality. The objective of this study was to determine the efficacy of albendazole (ABZ) against the lancet liver fluke Dicrocoelium dendriticum in naturally infected sheep using parasitological methods. Twenty-four sheep were divided into four groups: two untreated control groups (C1, C2) and two treated groups (T1, T2), with six animals in each group. The sheep in the treated groups were administered a single oral dose (15 mg/kg bwt) of ABZ suspension. After ABZ treatment the animals were slaughtered on Day 14 (groups C1, T1) and Day 30 (groups C2, T2) and were necropsied. Coprological therapeutic ABZ efficacy reached 92.4% on Day 14 (P < 0.001) and 88.5% on Day 30 (P < 0.001). On Day 30, the serum activities of hepatic and cholestatic enzymes including serological analysis of total protein concentration (TP) and protein fractions were evaluated. Significant decrease of aspartate aminotransferase (AST) (P < 0.01) and gamma-glutamyltransferase (GGT) (P < 0.05) activity by 36.9% and 34.6%, respectively, were detected for sheep in T2 group. These enzymes showed a strong positive correlation to fluke burden: AST (r = 0.654) and GGT (r = 0.768), respectively (P < 0.05). Additionally, the electrophoretic analysis of serum total protein and protein fraction concentrations revealed minimal hypoproteinemia and hyperalbuminemia after ABZ treatment. The decrease of liver enzyme activities and their correlation with fluke burden may indicate recovery of hepatocellular and biliary damage following the reduction of fluke burdens after ABZ therapy. A decline in AST and GGT activity could serve as a valuable adjunct bioindicator of liver damage and fluke reduction after treatment of dicrocoeliosis in naturally infected sheep.

Effects of temperature on the life history traits of intermediate host snails of fascioliasis: A systematic review.

The impact of climate change has led to variations in various biological processes, leading to altered transmission dynamics of infectious diseases, including snail-borne diseases (SBDs). Fascioliasis is one of the neglected zoonotic tropical snail-borne diseases caused by the trematode of the genus Fasciola. This review focused on laboratory experimental and model studies that evaluate the potential effect of temperature change on the ecology and biology of the intermediate host snails (IHS) of Fasciola. A literature search was conducted on Google Scholar, EBSCOhost, and PubMed databases using predefined medical subject heading terms, Boolean operators, and truncation symbols in combination with direct keywords: Fasciolosis AND Temperature, Lymnaea OR Austropeplea OR Radix OR Galba OR Fossaria OR Pseudosuccinea AND growth, fecundity, AND survival at the global scale. Other search terms used were (Fascioliasis AND Temperature), (Lymnaea AND Temperature), (Austropeplea AND Temperature), (Fossaria AND Temperature), (Galba AND Temperature), (Pseudosuccinea AND Temperature), and (Radix AND Temperature). The final synthesis included thirty-five published articles. The studies reviewed indicated that temperature rise may alter the distribution, and optimal conditions for breeding, growth, and survival of IHS, ultimately resulting in changing the transmission dynamics of fascioliasis. The literature also confirmed that the life history traits of IHS and their interaction with the liver fluke parasites are driven by temperature, and hence climate change may have profound outcomes on the population size of snails, parasite density, and disease epidemiology. We concluded that understanding the impact of temperature on the growth, fecundity, and survival of IHS may broaden our knowledge of the possible effects of climate change and hence inform fascioliasis control programs.

Molecular Characterization of Liver Fluke Isolated from Sheep, Goat and Cattle in Sulaymaniyah, Iraq.

We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular technique of DNA sequencing and restriction fragment length polymorphism (RFLP). The samples were collected from November 2021 to May 2022. The flukes were collected from infected livers of livestock at the slaughterhouse of Sulaymaniyah Governorate, Iraq. A total of 205 flukes were collected from 56 hosts, cattle (n=22), sheep (n=28), and goats (n=6). The specific primers for FCOX1 and 28S rDNA gene amplification were used. The PCR products were subjected to restriction fragment polymorphism (RFLP) assay using Hpy188III and Dra II restriction enzymes, besides DNA sequencing. The results showed the genetic polymorphisms among the flukes. Three patterns of RFLP were observed Fasciola hepatica, F. gigantica, and F. intermediate, where 28 of them displayed F. hepatica (sheep, n=14, goat, n=3 and cattle, n= 11), whereas 24 samples displayed the F. gigantica (sheep, n=12, goat, n=3 and cattle, n= 9), and only four samples belonged to F. intermediate (sheep n=3 and cattle, n=1). In addition, the result of the ribosomal DNA (28S rDNA) sequencing confirmed that the isolated flukes belonged to F. hepatica, F. gigantica and F. intermediate. All three main species are present in the study area and F. hepatica predominated among the animal species in this area also, our results concluded that PCR-RFLP is a rapid and reliable method for liver fluke species identification.

Determination of Clorsulon and its related substances in commercial bulk drug substance batches by a rapid ion-pair UPLC method

Clorsulon is a potent anthelmintic agent and is widely used for the treatment and control of parasites including adult liver flukes in cattle and sheep. A rapid ion-paired reversed phase ultraperformance liquid chromatography (IP-UPLC) method has been developed and validated for determination of Clorsulon and its related substances in bulk drug substance batches of Clorsulon with a short octadecyl column. Analytes were eluted by a gradient elution on a Acquity UPLC® BEH C18 column (50 mm × 2.1 mm i.d., 1.7 µm particle size). Column temperature was maintained at 55 °C and all analytes were monitored by UV detection at 268 nm. Mobile phase-A constitutes 3 mM tetrabutylammonium hydroxide in H2O and mobile phase-B constitutes acetonitrile/methanol (50/50, v/v), respectively. All analytes of interest were adequately separated within 5 min. The analytes were quantitated against an external reference standard of Clorsulon. The chemical structures of degradation products of Clorsulon were proposed based on two-dimensional UPLC-MS data. The IP-UPLC method has been successfully validated and demonstrated to be accurate, sensitive, robust, and specific.

Isolation and molecular identification of liver fluke cercariae in freshwater snails of Chaharmahal and Bakhtiari province, Iran

ABSTRACT Liver flukes are parasitic flatworms that can infect humans and other mammals. In this study, freshwater snails from Chaharmahal and Bakhtiari province in Iran were investigated for the presence of liver fluke larvae. The snails were collected from various rivers and analysed using standard morphological and molecular techniques. Additionally, molecular identification of the collected snails was conducted through polymerase chain reaction (PCR) and sequencing of the COI gene. The freshwater snails were identified as Physella acuta (Draparnaud, 1805), Galba truncatula (O.F. Müller, 1774), Radix euphratica (Mousson, 1874), Radix auricularia (Linnaeus, 1758) and Planorbis planorbis (Linnaeus, 1758). The presence of larval stages of Fasciola spp. in individual snails was examined using stereomicroscopy and a crushing method. Morphological and molecular identification based on the ITS-1 marker for identifying Fasciola spp. revealed that 10.9% (59 out of 549) of R. auricularia and 5.8% (37 out of 630) of P. acuta were infected. A blastn analysis of the collected samples revealed that all isolates belonged to F. hepatica, indicating a potential risk of liver fluke infection in the study area. These findings emphasise the importance of regular surveillance and control measures to prevent the spread of these parasites.