New benzimidazole derivative compounds with in vitro fasciolicidal properties.

BACKGROUND: Control of the zoonotic food-borne parasite Fasciola hepatica remains a major challenge in humans and livestock. It is estimated that annual economic losses due to fasciolosis can reach US$3.2 billion in agriculture and livestock. Moreover, the wide distribution of drug-resistant parasite populations and the absence of a vaccine threaten sustainable control, reinforcing the need for novel flukicides. METHODS: The present work analyses the flukicidal activity of a total of 70 benzimidazole derivatives on different stages of F. hepatica. With the aim to select the most potent ones, and screenings were first performed on eggs at decreasing concentrations ranging from 50 to 5 µM and then on adult worms at 10 µM. Only the most effective compounds were also evaluated using a resistant isolate of the parasite. RESULTS: After the first screenings at 50 and 10 µM, four hit compounds (BZD31, BZD46, BZD56, and BZD59) were selected and progressed to the next assays. At 5 µM, all hit compounds showed ovicidal activities higher than 71% on the susceptible isolate, but only BZD31 remained considerably active (53%) when they were tested on an albendazol-resistant isolate, even with values superior to the reference drug, albendazole sulfoxide. On the other hand, BZD59 displayed a high motility inhibition when tested on adult worms from an albendazole-resistant isolate after 72 h of incubation. CONCLUSIONS: BZD31 and BZD59 compounds could be promising candidates for the development of fasciolicidal compounds or as starting point for the new synthesis of structure-related compounds.

In vitro assessment of the anthelmintic activity of copper oxide and zinc oxide nanoparticles on egg and adult stages of Fasciola hepatica: evidence on oxidative stress biomarkers, and DNA damage.

OBJECTIVES: Fasciolosis is of significant economic and public health importance worldwide. The lack of a successful vaccine and emerging resistance in flukes to the drug of choice, triclabendazole, has initiated the search for alternative approaches. In recent years, metallic nanoparticles have been extensively investigated for their anthelmintic effects. This study investigates the in vitro anthelmintic activity of copper oxide and zinc oxide nanoparticles against Fasciola hepatica. METHODS: The in vitro study was based on egg hatchability test (EHA), adult motility inhibition tests, DNA damage, ROS levels, as well as several biomarkers of oxidative stress, including glutathione peroxidase (GSH) and glutathione S-transferase (GST), superoxide dismutase (SOD) and malondialdehyde (MDA). For this purpose, different concentrations of copper oxide nanoparticles (CuO-NPs) and Zinc oxide nanoparticles (ZnO-NPs) (1, 4, 8, 12, and 16 ppm) were used to evaluate the anthelmintic effect on different life stages, including egg and adults of Fasciola hepatica, over 24 h. RESULTS: In vitro treatment of F. hepatica worms with both CuO-NPs and ZnO-NPs could significantly increase ROS production and oxidative stress induction (decreased SOD, GST and GSH and increased MDA) compared to control group. CONCLUSIONS: Based on the results, it seems that CuO-NPs and ZnO-NPs may be effective in the control and treatment of F. hepatica infection. Further research is needed to investigate their potential for in vivo use in the treatment of parasitic infections.

Evaluation of Th1/Th2, regulatory cytokines and transcriptional factor FoxP3 in sheep immunized with a partially protective and non-protective vaccine and challenged with Fasciola hepatica.

Gene expression for Th1/Th2 cytokines (IL-4 and IFN-É£), regulatory cytokines (TGF-ß and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-É£ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-É£ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-ß in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

Blood parameters and parasite burden in cattle with chronic fascioliasis.

Fascioliasis is a trematodiasis that affects domestic and wild animals as well as humans worldwide. It is a well-recognized disease in livestock, were it produces serious economic losses. Yet in cattle, there is limited information about the burden of liver flukes and its relation to the eggs per gram shed to the environment. There is also lack of knowledge on the effect of parasite load in blood parameters of infected animals, which is important to evaluate the severity and progression of the disease. The objective of this work was to gain insight in these aspects. Cattle from Mendoza province, Argentina, were inspected at a farm and at the abattoir determining the presence or absence of Fasciola hepatica. Each animal was sampled for blood and feces and in the slaughterhouse the livers were inspected. Hematology and blood chemistry parameters were determined, feces were examined for F. hepatica eggs by a quantitative sedimentation technique and livers were thoroughly inspected to determine the number of flukes. Infected cattle presented a mild burden of liver flukes per animal, strongly correlated (r = 0.72) to the number of eggs per gram of feces. The total number of eggs (X̄=35,100) shed per animal to the environment and the type of livestock management techniques in the region exacerbate the role of cattle as efficient reservoirs of this disease. Statistically significant lower red blood cell, lymphocyte and neutrophil counts were observed in infected compared to uninfected animals. All hepatic parameters tested showed highly statistically significant differences (p < 0.001) as well as proteins by cause of rise of globulins in infected cattle. The correlation between the amount of flukes in the liver and the number of eggs per gram of faces indicates coprology as a reliable and cost-effective method to infer parasite burden. The impact of fascioliasis on blood parameters can be of aid for the veterinary practitioner on the assessment of this disease on cattle.

Eosinophilic Liver Abscess: A Case of Locally Acquired Fasciola hepatica in Alberta, Canada.

Fasciola hepatica is a trematode causing acute and chronic infection. A 33-year-old Canadian woman with eosinophilic liver abscesses and no relevant travel was diagnosed with F hepatica infection. F hepatica is reported in livestock in Alberta. This is the first case of locally acquired fascioliasis in Canada in >100 years.

Triclabendazole efficacy, prevalence, and re-infection of Fasciola hepatica in bovine and ovine naturally infected in the Andes of Ecuador.

Fasciola spp., infections are distributed worldwide including the Andes region of Ecuador, affecting cattle, sheep, porcine, humans, and other herbivores. Triclabendazole (TCBZ) is commonly used to treat animal infections. However, prospective studies on TCBZ efficacy and fascioliosis prevalence have not been studied in the highlands of Ecuador. This study was performed in a rural community at central of the Ecuadorian Andes in freely roaming bovine and ovine aimed to 1) evaluate the efficacy of TCBZ by administering a single oral dose of 12 mg/kg body weight, 2) assess the prevalence of F. hepatica infection and 3) to monitor re-infections for a follow-up period of five months. In total, 122, 86, 111, 110, 89, and 90 and 49, 34, 47, 28, 27, and 31 stool samples were collected each month from bovines and ovine, respectively. Besides, 32 stool samples from porcine were also collected at the beginning of the study. Stools were microscopically analyzed by formalin-ether concentration method to detect F. hepatica ova. The prevalence of F. hepatica infections before treatment was 55,7% and 63,3% for bovine and ovine, respectively. The infection prevalence was of 22% in porcine. The efficacity of triclabendazole was 83% and 97% in bovines and ovine, respectively, at 30 days post-treatment. The re-infection reaches to 54,4% in bovines and 61,3% in ovine after five months. TCBZ had a high efficacy and could be used for bovines and ovine Fasciola infections in the study region; however, re-infections reach the initial prevalence after five months. Therefore, we recommend integrated control strategies, including chemotherapy with a single oral dose of TCBZ, vector control, and future drug resistance studies.

The effects of Fasciola hepatica recombinant proteins (peroxiredoxin and cathepsin L1) on Crohn's disease experimental model.

The immunomodulatory potential of the excretory-secretory (E/S) proteins of the helminths has been shown in previous investigations. This study evaluated the effects of the recombinants and excretory-secretory proteins of the Fasciola hepatica on induced colitis in Balb/c mice. The F. hepatica Recombinant proteins, Cathepsin L1 and Peroxiredoxin, and E/S proteins were intraperitoneally injected into the three mice groups as the case groups, while the control groups received PBS. Colitis was induced in mice by intraluminal administration of the 2, 4, 6-Trinitrobenzenesulfonic acid solution (TNBS). After 8 h, the case groups received the second dosage of the treatments, and it was repeated 24 h later. The immunological, pathological, and macroscopic changes were evaluated 3 days after colitis induction. The macroscopic evaluation revealed significantly lower inflammatory scores in the mice treated with recombinant Peroxiredoxin (rPRX) and recombinant Cathepsin L1 (rCL1). Despite the macroscopic observation, the pathological finding was insignificant between the groups. IFN-γ secretion was significantly lower in splenocytes of the groups that received rPRX, rCL1, and E/S than the controls. IL-10 showed significantly higher levels in groups treated with rPRX and rCL1 than controls, whereas the level of IL-4 was not statistically significant. Excretory-secretory proteins of the F. hepatica showed immunomodulatory potency and the main effects observed in this study were through the reduction of inflammatory cytokine and inflammation manifestation as well as induction of anti-inflammatory cytokines.

Fosfatriclaben, a prodrug of triclabendazole: Preparation, stability, and fasciolicidal activity of three new intramuscular formulations.

In this study, we present the preparation, stability, and in vivo fasciolicidal activity of three new intramuscular formulations in sheep of a prodrug based on triclabendazole, named fosfatriclaben. The new formulations were ready-to-use aqueous solutions with volumes recommended for intramuscular administration in sheep. The use of poloxamers (P-407 and P-188) and polysorbates (PS-20 and PS-80) in the new formulations improved the aqueous solubility of fosfatriclaben by 8-fold at pH 7.4. High-performance liquid chromatography with UV detection was used to evaluate the stability of fosfatriclaben in the three formulations. High recovery (> 90%) of fosfatriclaben was found for all formulations after exposure at 57 ± 2 °C for 50 h. The three intramuscular formulations showed high fasciolicidal activity at a dose of 6 mg/kg, which was equivalent to the triclabendazole content. The fasciolicidal activity of fosfatriclaben was similar to commercial oral (Fasimec®) and intramuscular (Endovet®) triclabendazole formulations at a dose of 12 mg/kg. In the in vivo experiments, all formulations administered intramuscularly reduced egg excretion by 100%, and formulations F1, F2, and F3 presented fasciolicidal activities of 100%, 100%, and 99.6%, respectively.

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.

Bayesian hierarchical modelling of the geospatial distribution of fasciolosis in dairy cattle and the impact on production: Application to the main milk-producing region (Galicia) in Spain.

Fasciolosis caused by Fasciola hepatica is a common parasitic infection among cattle in many countries. Although infected adult cows rarely show overt clinical signs, milk production may be impaired. Thus, significant production losses may occur in dairy herds with a high prevalence of fasciolosis. In this study, Bayesian hierarchical modelling was used to estimate the geospatial distribution of dairy cattle fasciolosis and its impact on milk production. The study was conducted in Galicia, the main milk producing region in Spain and a geographically heterogeneous area. The aims were: 1) to model the geospatial distribution of fasciolosis in dairy herds in the study area, 2) to identify clusters of herds with a high prevalence of fasciolosis, and 3) to assess the effect of fasciolosis on milk yield and quality. A large number of dairy cattle farms (n = 4907), of which 1660 provided production records, were surveyed. Fasciola infection status was determined by applying the MM3-SERO ELISA test to bulk tank milk samples. A high probability of infection was predicted in several zones, particularly in the centre, northeast and southeast of Galicia. Conversely, the predicted probability was very low in some parts of the northwest of the region. Infections with high within-herd prevalence (> 25% lactating cows infected) predominated. High within-herd prevalence was associated with loss of milk production (-1.387 kg/cow/ day, on average). No association between Fasciola infection and either milk fat or protein content was observed. This study has generated the first maps of the spatial distribution of the probability of Fasciola infection in dairy cattle herds in Galicia. The maps presented here can be used for reference purposes, enabling the design of better targeted fasciolosis control programmes in the region. Use of Bayesian hierarchical statistical analysis enabled us to ascertain the uncertainty of the predictions and to account for the spatial autocorrelation in the data. It also enabled us to generate maps showing the residual spatial variation in milk production, a topic that may deserve more detailed study.

Fasciola hepatica GST mu-class suppresses the cytokine storm induced by E. coli-lipopolysaccharide, whereas it modulates the dynamic of peritoneal macrophages in a mouse model and suppresses the classical activation of macrophages.

IMPORTANCE: Sepsis is the consequence of a systemic bacterial infection that exacerbates the immune cell's activation via bacterial products, resulting in the augmented release of inflammatory mediators. A critical factor in the pathogenesis of sepsis is the primary component of the outer membrane of Gram-negative bacteria known as lipopolysaccharide (LPS), which is sensed by TLR4. For this reason, scientists have aimed to develop antagonists able to block TLR4 and, thereby the cytokine storm. We report here that a mixture of mu-class isoforms from the F. hepatica GST protein family administered intraperitoneally 1 h prior to a lethal LPS injection can modulate the dynamics and abundance of large peritoneal macrophages in the peritoneal cavity of septic mice while significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock. These results suggest that native F. hepatica glutathione S-transferase is a promising candidate for drug development against endotoxemia and other inflammatory diseases.

Spatial analysis and risk mapping of Fasciola hepatica infection in dairy cattle at the Peruvian central highlands.

This study aimed to develop maps for Fasciola hepatica infection occurrence in dairy cattle in the districts of Matahuasi and Baños in the Peruvian central highlands. For this, a model based on the correlation between environmental variables and the prevalence of infection was constructed. Flukefinder® coprological test were performed in samples from dairy cattle from 8 herds, during both the rainy and wet season. Grazing plots were geo-referenced to obtain information on environmental variables. Monthly temperature, monthly rainfall, elevation, slope, normalized difference vegetation index (NDVI), enhanced vegetation index (EVI), normalized difference water index (NDWI), distance to rivers, urban areas and roads were obtained by using remote sensor images and ArcGIS®. Multilayer perceptron Artificial Neural Networks modeling were applied to construct a predictive model for the occurrence of fasciolosis, based on the relationship between environmental variables and level of infection. Kappa coefficient (k > 0.6) was used to evaluate concordance between observed and forecasted risk by the model. Coprological results demonstrated an average prevalence from 20% to 100%, in Matahuasi, and between 0 and 87.5%, in Baños. A model with a high level of concordance between predicted and observed infection risk (k = 0.77) was obtained, having as major predicting variables: slope, NDWI, NDVI and EVI. Fasciolosis risk was categorized as low (p < 20%), medium (20% < p < 50%) and high (p ≥ 50%) level. Using ArcGIS 10.4.1, risk maps were developed for each risk level of fasciolosis. Maps of fasciolosis occurrence showed that 87.2% of Matahuasi area presented a high risk for bovine fasciolosis during the dry season, and 76.6% in the wet season. In contrast, 21.9% of Baños area had a high risk of infection during the dry season and 12.1% during the wet season. In conclusion, our model showed areas with high risk for fasciolosis occurrence in both districts during both dry and rainy periods. Slope, NDWI, NDVI and EVI were the major predictors for fasciolosis occurrence.

Fasciolosis: pathogenesis, host-parasite interactions, and implication in vaccine development.

Fasciola hepatica is distributed worldwide, causing substantial economic losses in the animal husbandry industry. Human fasciolosis is an emerging zoonosis in Andean America, Asia, and Africa. The control of the disease, both in humans and animals, is based on using anthelmintic drugs, which has resulted in increased resistance to the most effective anthelmintics, such as triclabendazole, in many countries. This, together with the concerns about drug residues in food and the environment, has increased the interest in preventive measures such as a vaccine to help control the disease in endemic areas. Despite important efforts over the past two decades and the work carried out with numerous vaccine candidates, none of them has demonstrated consistent and reproducible protection in target species. This is at least in part due to the high immunomodulation capacity of the parasite, making ineffective the host response in susceptible species such as ruminants. It is widely accepted that a deeper knowledge of the host-parasite interactions is needed for a more rational design of vaccine candidates. In recent years, the use of emerging technologies has notably increased the amount of data about these interactions. In the present study, current knowledge of host-parasite interactions and their implication in Fasciola hepatica vaccine development is reviewed.

Evaluation of the ovicidal activity and fasciolicidal activity of the extract of ethyl acetate from Artemisia ludoviciana Nutt. spp. mexicana and of artemisinin against adult parasites of Fasciola hepatica.

The objective of this work was to evaluate the effect of the ethyl acetate extract from A. ludoviciana (EALM) and artemisinin against adult parasites and eggs of F. hepatica. For the ovicidal assay, cell culture plates with 24 wells were used, and 90 to 110 F. hepatica eggs were placed in each well. The eggs were exposed to concentrations of 100, 200, 300, 400, and 500 mg/L EALM and incubated for 16 days. Additionally, triclabendazole (TCBZ) was used as a reference drug at concentrations of 10 and 50 mg, and the response of artemisinin at concentrations of 10 and 20 mg was simultaneously assessed. Adult flukes were exposed to concentrations of 125, 250, 375, and 500 mg/L EALM. The results of the ovicidal action of EALM on the eggs showed that concentrations greater than 300 mg/L were significant, with ovicidal percentages greater than 60% observed on day 16 of incubation (p < 0.05). The maximum efficiency of EALM on adult flukes was reached 72 h post-exposure at a concentration of 125 mg/L (p < 0.05).

Fasciola hepatica soluble antigens (FhAg) induce ovine PMN innate immune reactions and NET formation in vitro and in vivo.

Fasciola hepatica causes liver fluke disease, a worldwide neglected and re-emerging zoonotic disease, leading to hepatitis in humans and livestock. In the pathogenesis, flukes actively migrate through liver parenchyma provoking tissue damage. Here, parasites must confront leukocytes of the innate immune system in vivo. Polymorphonuclear neutrophils (PMN) are the most abundant granulocytes and first ones arriving at infection sites. PMN may display neutrophil extracellular traps (NETs), consisting of nuclear DNA, decorated with histones, enzymes, and antimicrobial peptides. We investigated for the first time whether F. hepatica soluble antigens (FhAg) can also trigger NETosis and innate immune reactions in exposed ovine PMN. Thus, isolated PMN were co-cultured with FhAg and NET formation was visualized by immunofluorescence and scanning electron microscopy analyses resulting in various phenotypes with spread NETs being the most detected in vitro. In line, NETs quantification via Picogreen®-fluorometric measurements revealed induction of anchored- and cell free NETs phenotypes. Live cell 3D-holotomographic microscopy revealed degranulation of stimulated PMN at 30 min exposure to FhAg. Functional PMN chemotaxis assays showed a significant increase of PMN migration (p = 0.010) and intracellular ROS production significantly increased throughout time (p = 0.028). Contrary, metabolic activities profiles of FhAg-exposed PMN did not significantly increase. Finally, in vivo histopathological analysis on F. hepatica-parasitized liver tissue sections of sheep showed multifocal infiltration of inflammatory cells within liver parenchyma, and further fluorescence microscopy analyses confirmed NETs formation in vivo. Overall, we hypothesized that NET-formation is a relevant host defence mechanism that might have a role in the pathogenesis of fasciolosis in vivo.

Efficacy of flukicides against Fasciola hepatica and first report of triclabendazole resistance on German sheep farms.

Fasciola hepatica infections lead to severe health problems and production losses in sheep farming, if not treated effectively. Triclabendazole has been used extensively over decades due to its unique efficacy range against all definitive hostfluke stages but published data about the susceptibility of F. hepatica to anthelmintics in Germany are lacking. This study aimed to identify current F. hepatica infections in German sheep flocks by coproscopic examinations and to evaluate the efficacy of anthelmintics with a focus on triclabendazole in a field study conducted from 2020 to 2022. Initial screening included 71 sheep farms, many of them with known history of fasciolosis. In this highly biased sample set, the frequency of F. hepatica infection at individual sheep and farm level were 12.8% and 35.2%, respectively. Additionally, eggs of Paramphistominae were found at frequencies of 4.8% and 15.5% at individual sheep and farm level, respectively. Due to low egg shedding intensity, faecal egg count reduction (FECR) tests could only be conducted on a few farms. The efficacy of triclabendazole was tested on 11 farms and albendazole on one farm, including 3-53 sheep/farm. Individual faecal samples were collected before and two weeks after treatment to evaluate the FECR using the sedimentation or FLUKEFINDER® or a modified FLUKEFINDER® method. On all farms a coproantigen reduction test was conducted in parallel. Lacking efficacy of triclabendazole even at double dosage was shown on one farm associated with a high number of animal losses due to acute fasciolosis. On this farm, the Fasciola miracidium development test was additionally performed, revealing a high in vitro ovicidal activity of albendazole while closantel was effective in vivo. On all other farms, sufficient efficacy of triclabendazole was observed. In conclusion, triclabendazole resistance appears not to be widespread on German sheep farms but, when present, can have serious effects on animal health.

Infection Rates of Fasciola Intermediate Host Snail Species and Their Distribution in Africa: A Systematic Review and Meta-Analysis.

This systematic review and meta-analysis aimed to collate the infection rates of Fasciola spp. in intermediate host snails and their distribution in Africa. The overall infectivity prevalences of Galba truncatula, Radix natalensis, and Pseudosuccinea columella are 52%, 8%, and 3%, respectively. The intermediate host snails native to Africa (R. natalensis and G. truncatula) have been examined more than the invasive P. columella. The studies included in the review ranged from 1999 to 2022. North Africa has the highest prevalence of G. truncatula, with an infection rate of 52%. The review reveals that naturally infected intermediate host snails (G. truncatula, R. natalensis, and P. columella) are found in various regions of Africa. G. truncatula accounts for 22% (from three countries) of the studies included in the review and it was only found in the North African region with the highest overall infection rate of 52%. More studies on infection rate and distribution are needed to effectively control and prevent future transmissions.

Molecular Characterization of Fasciola hepatica in Sheep Based on DNA Sequences of Ribosomal ITS-1.

Introduction: World Health Organization (WHO) considers Fascioliasis as a neglected tropical disease that requires global efforts for disease control. Data from the genetic characterization of Fasciola population shed light on the spread of infections among animals which could help in the development of effective parasite control. The aim of the present work was to genetically characterize Fasciola adult worms isolated from sheep in Saudi Arabia by sequence analysis of ITS-1 region. Methods: A total of 12,653 slaughtered sheep in Jeddah city, Saudi Arabia were examined for the presence of Fasciola spp. adult worms. The ITS-1 region of all parasites was amplified and sequenced. Results: Overall, 12 variants DNA sequences were obtained. The variance of isolates ranged from 0.00771 to 0.34405. BLAST search showed that all obtained sequences were Fasciola hepatica and had >99.3% similarity with F. hepatica isolates from Spain and USA (from different hosts other than sheep). Phylogenetic analysis showed that Fasciola isolates were closely related to isolates from different countries. Discussion: The current study showed that F. hepatica was the only spp. isolated from sheep in Jeddah. Further studies from different localities in Saudi Arabia are needed to help in the development of disease control.

The use of cathepsin L1 (FhCL1) serological ELISA in sentinel screening for liver fluke on sheep farms.

Fasciola hepatica is a parasitic helminth (worm) that poses a significant economic threat to the ruminant livestock industry worldwide. The disease, fasciolosis, can result in a range of clinical signs including anaemia, weight loss and death, with the most severe symptoms attributed to early acute infection when the parasite is migrating through the liver. Early diagnosis and intervention are essential for the control and management of the disease to prevent productivity losses. The traditional gold standard method of diagnosis uses faecal egg counts (FEC) that is limited to detecting patent infections from 10 to 12 weeks post infection (WPI). In contrast, serological assays can detect pre-patent infections as we have shown that enzyme-linked immunosorbent assays (ELISA) using the F. hepatica cysteine peptidase cathepsin L1 (FhCL1) can detect liver fluke infections from 3 to 4 WPI. Here, we used FEC and ELISA to monitor liver fluke infections in sentinel lambs from three commercial farms in Ireland from September 2021 to March 2022. All three farms showed a significant increase in FhCL1 antibody levels and FEC over this time, with a substantial rise in positive infection detection between late November and January. However, ELISA screening detected infection at least two months prior to FEC (September). This suggests that the regular screening of sentinel lambs for F. hepatica seroconversion in a "test and treat" approach could mitigate the negative damaging impact of early fasciolosis on flock health, welfare and productivity and inform management strategies. In addition, we show that whole blood samples taken on Whatman® protein saver cards could replace conventional serum blood tubes for blood collection. Cards can be stored at room temperature for long periods of time and samples revisited at any time for re-analysis. The adoption of these cards on farm together with the FhCL1 ELISA would provide a simpler, cost-effective, and eco-friendly method for testing sentinel lambs for liver fluke disease.