Prevalence of Fasciola hepatica infection in slaughtered sheep from Northwest Tunisia and its risk factors: Association with gastrointestinal helminths infection and anaemia.

We investigated herein the prevalence of Fasciola hepatica infection in sheep at Sejnane slaughterhouse, governorate of Bizerte, Northwest of Tunisia, using three different diagnostic techniques (liver dissection, bile examination, and coprology). Faeces, liver, gall bladder as well as blood samples were collected from 603 slaughtered sheep in two seasons: winter and summer. Faecal egg counts of F. hepatica were estimated using sedimentation technique. Livers were examined for the presence of flukes, and bile collected from gall bladder was examined by sedimentation technique for the presence of F. hepatica eggs. Faecal egg counts of gastrointestinal helminths were estimated using flotation followed by the McMaster technique. Blood samples were used to estimate blood cell count (RBC) (×106/mL), haemoglobin (Hb) (g/dL), and haematocrit (Ht) (%) levels. A total of 1714 F. hepatica flukes were collected from 68 infected livers, the number of flukes per sheep ranged between naught and 195. Bile examination (16.78% ± 1.83; 51/310) showed the higher infection prevalence, followed by liver dissection (11.28% ± 1.17; 68/603) and coprology (9.12% ± 1.08; 55/603) (p = 0.015). Infection prevalences were significantly higher in young sheep aged of less than 1 year (8.13% ± 1.22; 49/498), in cross-bred sheep (10.61% ± 1.39%; 64/478), and in summer (7.13% ± 1.82; 43/293) (p < 0.05). There was no significant difference in infection prevalence by gastrointestinal helminths in F. hepatica-infected and F. hepatica-non-infected animals (p > 0.05). The overall prevalence of F. hepatica-infected anaemic sheep was higher (22.73% ± 4.47; 20/88) than F. hepatica-non-infected anaemic sheep (p < 0.05). Fasciola hepatica infection is frequent in sheep from Sejnane representing hence an important constraint for the development of the sheep industry in this region. Therefore, it is necessary to establish and implement a specific control programme to reduce fasciolosis infection risks including animal owners' education.

Risk of invasion and disease transmission by the Australasian freshwater snail Orientogalba viridis (Lymnaeidae): a field and experimental study.

BACKGROUND: Biological invasions pose risks to the normal functioning of ecosystems by altering the structure and composition of several communities. Molluscs stand out as an extensively studied group given their long history of introduction by either natural or anthropogenic dispersal events. An alien population of the lymnaeid species Orientogalba viridis was first sighted in 2009 in southern Spain. In its native range (Australasian), this species is one of the main intermediate hosts of Fasciola hepatica, a major worldwide trematode parasite largely affecting humans, domestic animals and wildlife. METHODS: We collected field populations of O. viridis from its native (Malaysia) and invaded (Spain) ranges. We performed detailed morphoanatomical drawings of the species and screened for natural infection of parasites. Individuals were molecularly characterized using ITS2 for comparison with existing sequences in a fine phylogeography study. We founded experimental populations at two different conditions (tropical, 26 °C and temperate, 21 °C) to study the life-history traits of exposed and non-exposed individuals to different F. hepatica isolates. RESULTS: We found a 9% natural prevalence of trematode infection (98% similarity with a sequence of Hypoderaeum conoideum [Echinostomatidae]) in the Spanish field population. The haplotypes of O. viridis found in our study from Spain clustered with Australian haplotypes. Experimental infection with F. hepatica was successful in both experimental conditions but higher in tropical (87% prevalence) than in temperate (73%). Overall lifespan, however, was higher in temperate conditions (mean 32.5 ± 7.4 weeks versus 23.3 ± 6.5) and survivorship remained above 70% during the first 20 weeks. In parasite-exposed populations, life expectancy dropped from an overall 37.75 weeks to 11.35 weeks but still doubled the time for initial cercariae shedding. Cercariae shedding started at day 23 post-exposure and peaked between days 53 and 67 with an average of 106 metacercariae per snail. CONCLUSIONS: Whether O. viridis will succeed in Europe is unknown, but the odds are for a scenario in which a major snail host of F. hepatica occupy all available habitats of potential transmission foci, ravelling the epidemiology of fasciolosis. This research provides a comprehensive understanding of O. viridis biology, interactions with parasites and potential implications for disease transmission dynamics, offering valuable insights for further research and surveillance.

Early stage Fasciola hepatica infection in growing cattle: impact on weight gain and liver weight determined in flukicide efficacy studies.

Although the negative impact of liver fluke (Fasciola hepatica) infection on production and health in cattle is generally accepted, results of individual research have been variable, ranging from important negative impacts on the animal to minimal or no impact. To add information on the impact of F. hepatica infection in growing cattle, weight gain and liver weight of young experimentally infected animals from seven controlled efficacy studies were analyzed. In each study, fluke naïve animals were inoculated with approximately 450 to 500 F. hepatica encysted metacercariae, blocked on body weight and randomly assigned into one untreated group (controls) and groups which were administered an experimental flukicide when the flukes were 4 weeks old (migrating) and sacrificed 8 weeks thereafter (12 weeks after inoculation). Data of groups which demonstrated >90% reduction of fluke counts following treatment and groups left untreated (total 103 and 47 animals, respectively) were compared. There was a significant (p < 0.0001) negative association between fluke count and weight gain while fluke count and liver weight and fluke count and relative liver weight were positively associated (p < 0.0001). Over the 8-week post-treatment period, flukicide-treated cattle had almost 15% more weight gain than the controls (50.9 kg vs. 44.4 kg; p = 0.0003). Absolute and relative liver weight was significantly (p < 0.0001) lower in flukicide-treated compared to untreated cattle. Overall, this analysis provided evidence of a substantial negative effect of early (migrating) liver fluke infection on the growth of young cattle, likely due to pathology of the liver and associated reduction in its function as the central organ for bioenergy and protein metabolism.

Mitochondrial genome of the fluke pond snail, Austropeplea cf. brazieri (Gastropoda: Lymnaeidae).

BACKGROUND: Lymnaeid snails of the genus Austropeplea are an important vector of the liver fluke (Fasciola hepatica), contributing to livestock production losses in Australia and New Zealand. However, the species status within Austropeplea is ambiguous due to heavy reliance on morphological analysis and a relative lack of genetic data. This study aimed to characterise the mitochondrial genome of A. cf. brazieri, an intermediate host of liver fluke in eastern Victoria. METHODS: The mitochondrial genome was assembled and annotated from a combination of second- and third-generation sequencing data. For comparative purposes, we performed phylogenetic analyses of the concatenated nucleotide sequences of the mitochondrial protein-coding genes, cytochrome c oxidase subunit 1 and 16S genes. RESULTS: The assembled mt genome was 13,757 base pairs and comprised 37 genes, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The mt genome length, gene order and nucleotide compositions were similar to related species of lymnaeids. Phylogenetic analyses of the mt nucleotide sequences placed A. cf. brazieri within the same clade as Orientogalba ollula with strong statistical supports. Phylogenies of the cox1 and 16S mt sequences were constructed due to the wide availability of these sequences representing the lymnaeid taxa. As expected in both these phylogenies, A. cf. brazieri clustered with other Austropeplea sequences, but the nodal supports were low. CONCLUSIONS: The representative mt genome of A. cf. brazieri should provide a useful resource for future molecular, epidemiology and parasitological studies of this socio-economically important lymnaeid species.

Evaluation of Reproductive Histology Response of Adult Fasciola hepatica in Goats Vaccinated with Cathepsin L Phage-Exposed Mimotopes.

Fasciolosis, a globally re-emerging zoonotic disease, is mostly caused by the parasitic infection with Fasciola hepatica, often known as the liver fluke. This disease has a considerable impact on livestock productivity. This study aimed to evaluate the fluke burdens and faecal egg counts in goats that were administered phage clones of cathepsin L mimotopes and then infected with F. hepatica metacercariae. Additionally, the impact of vaccination on the histology of the reproductive system, specifically related to egg generation in adult parasites, was examined. A total of twenty-four goats, which were raised in sheds, were divided into four groups consisting of six animals each. These groups were randomly assigned. The goats were then subjected to two rounds of vaccination. Each vaccination involved the administration of 1 × 1013 phage particles containing specific mimotopes for cathepsin L2 (group 1: PPIRNGK), cathepsin L1 (group 2: DPWWLKQ), and cathepsin L1 (group 3: SGTFLFS). The immunisations were carried out on weeks 0 and 4, and the Quil A adjuvant was used in combination with the mimotopes. The control group was administered phosphate-buffered saline (PBS) (group 4). At week 6, all groups were orally infected with 200 metacercariae of F. hepatica. At week 22 following the initial immunisation, the subjects were euthanised, and adult F. hepatica specimens were retrieved from the bile ducts and liver tissue, and subsequently quantified. The specimens underwent whole-mount histology for the examination of the reproductive system, including the testis, ovary, vitellaria, Mehlis' gland, and uterus. The mean fluke burdens following the challenge were seen to decrease by 50.4%, 62.2%, and 75.3% (p < 0.05) in goats that received vaccinations containing cathepsin L2 PPIRNGK, cathepsin L1 DPWWLKQ, and cathepsin L1 SGTFLFS, respectively. Animals that received vaccination exhibited a significant reduction in the production of parasite eggs. The levels of IgG1 and IgG2 isotypes in vaccinated goats were significantly higher than in the control group, indicating that protection is associated with the induction of a mixed Th1/Th2 immune response. The administration of cathepsin L to goats exhibits a modest level of efficacy in inducing histological impairment in the reproductive organs of liver flukes, resulting in a reduction in egg output.

Water temperature and microenvironmental factors predict the presence and detection of the snail host of Fasciola hepatica in Andean Patagonia.

The transmission of Fasciola hepatica occurs only where there are -or recently were- aquatic or amphibious snails of the Lymnaeidae family, the intermediate host of this parasite. Direct detection of these snails is time-consuming and imprecise, hindering accurate and detailed mapping of transmission risk. To identify which microenvironmental factors could be used as proxies for the occurrence of the lymnaeid snail Galba viator, a major intermediate host in South America, a total of 183 1-m2 quadrants across diverse water bodies in an endemic area in Andean Patagonia were manually timed-searched for snails and microenvironmental variables were registered. Data was analyzed using a Bayesian hierarchical occupancy model that assessed the effects of the microenvironmental variables on the presence of snails while considering imperfect snail detection. The model estimated that G. viator predominantly inhabits shallow aquatic environments, in the presence of grasses, where snails of the genus Biomphalaria are also detected, and with scarce tree canopy cover. Physical factors affecting occupancy presumably act as proxies for the average water temperature, while the temperature at the time of sampling was found to affect snail detectability. The identified variables are easy, fast, and inexpensive to measure, and can complement management decisions and risk maps based on coarser remote-sensing data, particularly relevant in a context of growing resistance to anthelminthic drugs.

Bioclimatic analysis and spatial distribution of fascioliasis causative agents by assessment of Lymnaeidae snails in northwestern provinces of Iran.

BACKGROUND: Snails of the Lymnaeidae family are the intermediate hosts of Fasciola species, the causative agents of fascioliasis. The purpose of this study was to determine the prevalence of Fasciola species in lymnaeid snails and to investigate the association of geoclimatic factors and Fasciola species distribution in northwestern provinces of Iran using geographical information system (GIS) data. METHODS: A total of 2000 lymnaeid snails were collected from 33 permanent and seasonal habitats in northwestern Iran during the period from June to November 2021. After identification by standard morphological keys, they were subjected to shedding and crushing methods. Different stages of Fasciola obtained from these snails were subjected to the ITS1 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for species identification. The associations of weather temperature, rainfall, humidity, evaporation, air pressure, wind speed, elevation, and land cover with the distribution of Fasciola species were investigated. Geographical and statistical analysis was performed using ArcMap and SPSS software, respectively, to determine factors related to Fasciola species distribution. RESULTS: Of the 2000 snails collected, 19 were infected with Fasciola hepatica (0.09%), six with F. gigantica (0.03%), and 13 with other trematodes. Among geoclimatic and environmental factors, mean humidity, maximum humidity, and wind speed were significantly higher in areas where F. hepatica was more common than F. gigantica. The altitude of F. hepatica-prevalent areas was generally lower than F. gigantica areas. No significant relationship was observed between other investigated geoclimatic factors and the distribution of infected snails. CONCLUSIONS: The present study showed the relationship of humidity and wind speed with the distribution of snails infected with F. hepatica or F. gigantica in the northwestern regions of Iran. In contrast to F. gigantica, F. hepatica was more prevalent in low-altitude areas. Further research is recommended to elucidate the relationship between geoclimatic factors and the presence of intermediate hosts of the two Fasciola species.

Anthelmintic effects of some medicinal plants on different life stages of Fasciola hepatica: Evidence on oxidative stress biomarkers, and DNA damage.

Fasciolosis caused by Fasciola hepatica is a major public health and economic problem worldwide. Due to the lack of a successful vaccine and emerging resistance to the drug triclabendazole, alternative phytotherapeutic approaches are being investigated. This study investigated the in vitro anthelmintic activity of Lavender (Lavandula angustifolia) and carob (Ceratonia siliqua L.) essential oils (EOs) against F. hepatica. The in vitro study was based on an egg hatch assay (EHA), adult motility inhibition assays, DNA damage, reactive oxygen species (ROS) level along with several oxidative stress biomarkers including glutathione peroxidase (GSH), and glutathione-S-transferase (GST), superoxide dismutase (SOD) and malondialdehyde (MDA). To this end, different concentrations of L. angustifolia and C. siliqua EOs (1, 5, 10, 25 and 50 mg/mL) were used to assess anthelmintic effects on different life stages including egg, and adults of F. hepatica for 24 hrs. The results indicated that these EOs play a significant role as anthelminthics, and the effect was dependent on time and concentration. The in vitro treatment of F. hepatica worms with both L. angustifolia and C. siliqua EOs increased DNA damage, ROS production and induction of oxidative stress (decreased SOD, GST and GSH, and increased MDA), significantly compared to control. Therefore, it can be concluded that L. angustifolia and C. siliqua EOs have the potential to be used as novel agents for the control and treatment of F. hepatica infections. Further studies are required to investigate their pharmacological potential and effectiveness in vivo for the treatment of parasitic infections.

Misclassification of Opisthorchis viverrini and Minute Intestinal Fluke Eggs by Routine Laboratory Staff Using Images from the Kato-Katz Method.

BACKGROUND: The Kato-Katz method is a commonly used diagnostic tool for helminth infections, particularly in field studies. This method can yield inaccurate results when samples contain eggs that are similar in appearance, such as Minute Intestinal Fluke (MIF) and Opisthorchis viverrini (OV) eggs. The close resemblance of eggs can be problematic and raises the possibility of false diagnoses. The objectives were to compare the diagnostic performance of the Kato-Katz method for accurately identifying MIF and OV and to provide evidence of possible misclassification.  Methods: Based on questionnaire responses from 15 (young parasitologists and public health staff), the test comprised 50 MIF egg images and 50 OV egg images, for a total of 100 Google Form questionnaires. RESULTS: The morphology of MIF and OV eggs found size and shape similarity and found that the shoulder rims were small, while the OV egg found the knobs had disappeared. The opercular conjunction was apparent, the shoulder rims and miricidium were prominent. The average percentage of correctly classified infections was 61.6 ± 12.1%. The accuracy percentages for both public health staff and young parasitologists in identifying were found to be 59.0 ± 14.8 and 66.8 ± 2.8, respectively. There was no significant difference observed in both groups. CONCLUSION: These findings highlight the need for improving the accuracy of parasite identification. Preserving stool samples before the Kato-Katz method can help mitigate the potential degradation or distortion of parasite eggs. The incorrect classification of both eggs had an impact on treatment plans and the policy of parasite control programs.

Neoblast-like stem cells of Fasciola hepatica.

The common liver fluke (Fasciola hepatica) causes the disease fasciolosis, which results in considerable losses within the global agri-food industry. There is a shortfall in the drugs that are effective against both the adult and juvenile life stages within the mammalian host, such that new drug targets are needed. Over the last decade the stem cells of parasitic flatworms have emerged as reservoirs of putative novel targets due to their role in development and homeostasis, including at host-parasite interfaces. Here, we investigate and characterise the proliferating cells that underpin development in F. hepatica. We provide evidence that these cells are capable of self-renewal, differentiation, and are sensitive to ionising radiation- all attributes of neoblasts in other flatworms. Changes in cell proliferation were also noted during the early stages of in vitro juvenile growth/development (around four to seven days post excystment), which coincided with a marked reduction in the nuclear area of proliferating cells. Furthermore, we generated transcriptomes from worms following irradiation-based ablation of neoblasts, identifying 124 significantly downregulated transcripts, including known stem cell markers such as fgfrA and plk1. Sixty-eight of these had homologues associated with neoblast-like cells in Schistosoma mansoni. Finally, RNA interference mediated knockdown of histone h2b (a marker of proliferating cells), ablated neoblast-like cells and impaired worm development in vitro. In summary, this work demonstrates that the proliferating cells of F. hepatica are equivalent to neoblasts of other flatworm species and demonstrate that they may serve as attractive targets for novel anthelmintics.

Estimation of diagnostic sensitivity and specificity of abattoir registrations and bulk tank milk ELISA as herd-level tests for Fasciola hepatica using Bayesian latent class modelling.

The common liver fluke, Fasciola hepatica, is a trematode parasite found worldwide, typically with a focal distribution due to its requirement for suitable climatic and environmental conditions to complete its lifecycle. Bovine fasciolosis causes suboptimal production and economic losses, including liver condemnation at slaughter. The lack of reliable diagnostic methods is a disadvantage to the increasing demand for surveillance and control. The aim of this study was to evaluate the diagnostic accuracy of bulk tank milk (BTM) antibody testing and aggregated abattoir registrations (AAR) of liver fluke as herd-level tests for F. hepatica infection using Bayesian latent class models. Data from the abattoirs in 2019-2021 and BTM, sampled in the winter of 2020/2021, were collected from 437 herds on the southwest coast of Norway. The BTM samples were analysed with the SVANOVIR® F. hepatica-Ab ELISA test, with results given as an optical density ratio (ODR), and later dichotomized using the recommended cut-off value from the test manufacturer (ODR ≥0.3). Based on the BTM ELISA test, 47.8% of the herds tested positive. The AAR test was defined as the herd-level proportion of female slaughtered animals registered with liver fluke infection during the study period. For this test, three cut-offs were used (a proportion of 0.05, 0.1 and 0.2). The herds were split into two subpopulations ("Coastal" and "Inland"), which were expected to differ in true prevalence of F. hepatica infection based on climate-related and geographical factors. The diagnostic accuracies of both tests were estimated using Bayesian latent class models with minimally informative priors. Post-hoc analysis revealed that the maximum sum of sensitivity (Se) and specificity (Sp) of the tests was achieved with a herd-level proportion of ≥0.1 registered with liver fluke as the AAR test. Using this cut-off, the median estimate for the diagnostic accuracy of the BTM ELISA was 90.4% (84.0-96.2 95% Posterior Credible Interval (PCI)) for Se and 95.3% (90.6-100% PCI) for Sp, while the median estimate of Se for AAR was 87.5% (81.4-93.1% PCI) and the median estimate of Sp for AAR was 91.0% (85.2-96.5% PCI). The cut-off evaluation of the SVANOVIR® F. hepatica-Ab ELISA test for BTM confirmed the manufacturer's recommended cut-off of ODR ≥0.3 to denote positive and negative herds. This study suggests that AAR and BTM ELISA test can be used as herd-level tools to monitor liver fluke infection, so that appropriate interventions against infection can be implemented as necessary.

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.

A first report of Pseudosuccinea columella (Say, 1817), an alien intermediate host for liver fluke, in Malawi.

Starting in October 2021, quarterly malacological surveys have been undertaken in Malawi, with the sampling of 12 specified freshwater habitats throughout a calendar year. Each survey monitors the presence of aquatic intermediate snail hosts of medical and veterinary importance. In March 2023, the alien lymnaeid species Pseudosuccinea columella was encountered for the first time in the surveys, in Nsanje District. This species identity was later confirmed upon DNA analysis of mitochondrial ribosomal 16S sequences. In July 2023, P. columella was also noted at single sites within Mangochi and Chikwawa Districts, and again in Nsanje District, with an additional location observed. Of particular importance, our sampled location in Mangochi District was directly connected to Lake Malawi, which expands the species list of invasive molluscs in this lake. While P. columella is a well-known intermediate snail host for human and animal fascioliasis, screening collected snails for trematode cercariae, alongside molecular xenomonitoring, did not yield equivocal evidence of active fluke infection. However, the newly recognized presence of this alien intermediate snail host within Lake Malawi, and along the Shire River Valley, flags a new concern in altered local transmission potential for human and animal fascioliasis.

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.

Designing and Developing Serological Test for the Diagnosis of Human Fascioliasis Using a New Recombinant Multi-epitope.

PURPOSE: Fascioliasis is a common parasitic disease in humans and herbivores which is caused by Fasciola hepatica and Fasciola gigantica and has a worldwide distribution. Serological tests such as the enzyme-linked immunosorbent assay (ELISA) technique play a prominent role in the fast diagnosis of the disease. However, there are diagnostic limitations, including cross-reactivity with other worms, which decline the specificity of the results. This study aimed to evaluate the structure of a recombinant multi-epitope antigen produced from linear and conformational B-cell epitopes of three parasitic proteins with sera of individuals with fasciolosis, healthy controls, and those with other diseases to gain accurate sensitivity and specificity. METHODS: After designing the multi-epitope structure of cathepsin L1, FhTP16.5, and SAP-2 antigens and then synthesizing, cloning, and expressing, the extracted purified protein was evaluated by indirect ELISA to detect IgG antibodies against Fasciola hepatica parasite among the sera of 39 serum samples of Fasciola hepatica, 35 healthy individual samples, and 20 samples of other types of parasitic diseases. The synthesized multi-epitope produced from cathepsin L1, FhTP16.5, and SAP-2 antigens was evaluated using the indirect ELISA. RESULTS: The analysis of the samples mentioned for IgG antibody diagnosis against Fasciola hepatica showed 97.43% (95% confidence interval, 94.23-100%) sensitivity and 100% (95% confidence interval, 97-100%) specificity. CONCLUSION: The recombinant B-cell multi-epitope with high antigenic potency may increase the specificity of epitopic peptides and ultimately help improve and develop indirect ELISA commercial kits for the diagnosis of fascioliasis in humans.