Diagnosis of fasciolosis antibodies in Brazilian cattle through ELISA employing both native and recombinant antigens.

Bovine fasciolosis is a parasitic disease with a global reach. Coprological based on egg detection in fecal samples and liver inspection to evaluate the presence of the parasite is currently the gold standard for diagnosing chronic fasciolosis in cattle. However, these techniques are labor-intensive and ineffective during the acute phase of the disease. Serodiagnosis using native and recombinant antigens has become an interesting alternative in efforts to identify cattle fasciolosis. We evaluated cattle from abattoir (n = 139) and farms (n = 500) through liver inspection and coprological examination, respectively. Our laboratory team optimized and validated enzyme-linked immunosorbent assay tests based on somatic antigen, excretory/secretory proteins, and the recombinant antigen cathepsin L-1 to detect serum antibodies against fasciolosis in cattle. For animals from abattoir, 10 were positive for fasciolosis according to liver inspection. Both FhES and FhrCL-1 presented an area under the receiver operating characteristic (AUROC) curve of 0.80, with a sensitivity of 0.80 (95% CI: 0.46-0.95) and 0.70 (95% CI: 0.38-0.90) and specificity of 0.81 (95% CI: 0.73-0.87) and 0.87 (95% CI: 0.80-0.92), respectively. For those cattle from farms, 28 were positive only for fasciolosis according to coprological examination. In this scenario, FhES gave the best performance, with an AUROC of 0.84, sensitivity of 0.79 (95% CI: 0.60-0.90), and specificity of 0.86 (95% CI: 0.82-0.89). In conclusion, our study highlights the potential of serodiagnosis for accurately screening cattle fasciolosis. The promising sensitivity and specificity values of FhES when compared to liver inspection or coprological examination enhance its importance for cattle fasciolosis diagnosis. IMPORTANCE: The aim of this article was to identify antibodies against fasciolosis in cattle in Brazil. The methodology was reproduced in our laboratory and applied for the first time to the Brazilian cattle herd. The antigens tested can be used as a screening test and thus speed up the diagnosis of bovine fascioliasis.

Exploring the utility of circulating miRNAs as diagnostic biomarkers of fasciolosis.

Effective management and control of parasitic infections on farms depends on their early detection. Traditional serological diagnostic methods for Fasciola hepatica infection in livestock are specific and sensitive, but currently the earliest detection of the parasite only occurs at approximately three weeks post-infection. At this timepoint, parasites have already entered the liver and caused the tissue damage and immunopathology that results in reduced body weight and loss in productivity. Here, we investigated whether the differential abundance of micro(mi)miRNAs in sera of F. hepatica-infected sheep has potential as a tool for the early diagnosis of infection. Using miRNA sequencing analysis, we discovered specific profiles of sheep miRNAs at both the pre-hepatic and hepatic infection phases in comparison to non-infected sheep. In addition, six F. hepatica-derived miRNAs were specifically identified in sera from infected sheep. Thus, a panel of differentially expressed miRNAs comprising four sheep (miR-3231-3p; miR133-5p; 3957-5p; 1197-3p) and two parasite miRNAs (miR-124-3p; miR-Novel-11-5p) were selected as potential biomarkers. The expression of these candidates in sera samples from longitudinal sheep infection studies collected between 7 days and 23 weeks was quantified using RT-qPCR and compared to samples from age-matched non-infected sheep. We identified oar-miR-133-5p and oar-miR-3957-5p as promising biomarkers of fasciolosis, detecting infection as early as 7 days. The differential expression of the other selected miRNAs was not sufficient to diagnose infection; however, our analysis found that the most abundant forms of fhe-miR-124-3p in sera were sequence variants (IsomiRs) of the canonical miRNA, highlighting the critical importance of primer design for accurate diagnostic RT-qPCR. Accordingly, this investigative study suggests that certain miRNAs are biomarkers of F. hepatica infection and validates miRNA-based diagnostics for the detection of fasciolosis in sheep.

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.

Increased specificity of Fasciola hepatica excretory-secretory antigens combining negative selection on hydroxyapatite and salt precipitation.

A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.

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

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

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.

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

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

A delayed diagnosis of fascioliasis: The importance of appropriate fecal diagnostic method.

Fascioliasis, a zoonotic helminthiasis, occurs sporadically in Japan. In this report, we describe a case of fascioliasis that was initially difficult to diagnose because the fecal examination method was negative for the Fasciola sp. eggs. A 64-year-old man living in Shimonoseki City, Japan, presented with fatigue and anorexia. Laboratory tests showed hepatic dysfunction and eosinophilia. Abdominal dynamic contrast-enhanced computed tomography and magnetic resonance cholangiopancreatography suggested intrahepatic biliary cysts. Thereafter, fever and night sweats persisted, and positron emission tomography and biopsy of the porta hepatis lymph node were performed on suspicion of malignancy. However, histopathological diagnosis found non-specific inflammation. As fascioliasis was suspected due to eosinophilia and the multiple hepatic masses, fecal egg examination was performed by an external private laboratory, which adopted the flotation method and reported the absence of parasite eggs. However, fecal examination was retried in our laboratory using the formalin-ether concentration method, and we detected Fasciola sp. eggs. This case suggests that misdiagnosis may occur depending on the fecal examination method; thus, it is necessary to choose a suitable method for certain parasite species.

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).

Evaluation of the seroprevalence and the demographic and clinical findings of fascioliasis patients in the Dicle River Basin in Turkey: a nine-year experience at a university hospital.

OBJECTIVE: Fasciola hepatica and Fasciola gigantica are liver trematodes that cause fascioliasis in humans and animals. In Turkey, the medical importance of fascioliasis has been increasing in humans, and it continues to cause great economic loss in the field of animal husbandry. Therefore, it is important to diagnose fascioliasis quickly and reliably. The aim of this study is to show that the ELISA test is a reliable and specific method for diagnosing fascioliasis both in the early stage and in the acute stage. PATIENTS AND METHODS: In this study, 640 individuals aged 7-75 years who showed one or more symptoms of fascioliasis, such as abdominal pain, fever, weight loss, weakness, fatigue, headache, sweating, nausea, vomiting, allergic urticaria, liver mass, hypereosinophilia, or liver enzyme elevation, were recruited from the Dicle University Research and Application Hospital in southeastern Turkey. Serum and fecal samples were taken from them to investigate if the Fasciola hepatica IgG antibody was present in the serum and if eggs were present in the feces. To detect the IgG antibodies, an enzyme-linked immunosorbent assay (ELISA) kit was used. The stool samples were analyzed for three consecutive days in mini Parasep fecal parasite concentrator tubes using the native-lugol and sedimentation methods. Abdominal ultrasonography and computed tomography were performed in all the patients. RESULTS: Among the subjects of this study, 90 (14%) were positive for fascioliasis, of whom 85 (94.4%) were adults and 5 (5.5%), children; 73 (81.1%) were women and 17 (18.8%), men; 57 (63.3%) lived in the rural areas and 33 (36.6%), in the city center; 90 (14%) were positive for Fasciola hepatica IgG antibodies; (20%) had helminth eggs in their stools; and 85 (94.4%) had a history of eating watercress. CONCLUSIONS: According to the epidemiological classification for fascioliasis by Mas-Coma, the Dicle Basin, which is the setting of this study, is indeed a hyperendemic region. Thus, ELISA is a reliable and specific method of diagnosing fascioliasis, both in the early phase and in the acute phase, when the eggs are no longer seen in the stool.

Seroprevalence and coprological prevalence of liver fluke Fasciola hepatica in cattle and sheep from Santander department, Colombia.

Fasciola hepatica is a parasite with a worldwide distribution that affects several mammals, including humans, and is considered a public health problem. Therefore, the aim of this study was to determine the prevalence of Fasciola hepatica in humans, cattle and sheep, as well as to evaluate factors associated with the prevalence. A total of 185 serum samples from sheep, 290 from cattle, and 114 from humans were collected and processed using an in-house developed ELISA to detect IgG antibodies against F. hepatica. Additionally, 185 stool samples from sheep and 290 from cattle were examined using a Dennis sedimentation technique. Risk factors were analyzed using epidemiological surveys. The overall seroprevalence was 46.5% (86/185) in sheep, 32.5% (94/289) in cattle, and no humans tested positive for the infection. The coprological prevalence was 47.7% (86/180) in sheep and 33.7% (98/290) in cattle. Female gender and cattle living with alternate grazing management showed 2.5 and 6.5 times higher probability of infection, respectively. Bovines coexisting with sheep exhibited a higher risk of infection (odds ratio [OR]=4.3) compared to those without sheep. We concluded that F. hepatica in cattle and sheep has an endemic behavior, and therefore represents a problem of public health for rural communities.

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

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

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.

Emerging Human Fascioliasis: A Retrospective Study of Epidemiological Findings in Dali, Yunnan Province, China (2012-2021).

BACKGROUND Human fascioliasis is an emerging zoonotic disease caused by the trematodes, or flatworms, Fasciola hepatica and Fasciola gigantica, also known as liver flukes. This retrospective study aimed to report the epidemiological findings in 95 cases of human fascioliasis in Dali, Yunnan Province, southwestern China, diagnosed between 2012 and 2021. MATERIAL AND METHODS The epidemiologic and clinical data of 95 patients diagnosed with human fascioliasis in Dali area from January 2012 to December 2021 were collected and retrospectively analyzed. The diagnosis of fascioliasis was based on the Chinese National Standard of Diagnosis of Fascioliasis (WS/T566-2017). RESULTS The mean age of patients was 38.54±15.68 years, and there were more female patients than male (61.05% vs 38.95%). The high-incidence seasons were identified as summer and autumn. The patients with human fascioliasis lived in pastoral areas or were infected F. gigantica by consuming contaminated vegetables or water containing metacercaria. Meanwhile, human fascioliasis was diagnosed by positive serologic tests (1: 640), and Fasciola eggs (144-180×73-96 µm) were detected in stool samples of 6 patients. The most common clinical features were abdominal pain (70.53%), accompanied by elevated eosinophils in 89.5% of these patients. Antiparasitic treatment with triclabendazole at 10 mg/kg/day for 2 days led to symptom relief in all patients. CONCLUSIONS The findings from this observational epidemiological study have highlighted the importance of recognizing, diagnosing, and managing fascioliasis, which is an emerging zoonosis associated with increased human proximity to plant-eating domestic and farmed animals.

Toxocara canis and Fasciola hepatica Co-Infection Leading to Hepatic Abscess: A Case Report.

Toxocariasis is a zoonotic disease caused by ingesting eggs from soil contaminated with Toxocara canis and Toxocara cati, commonly found in feces of infected dogs and cats, leading to a range of clinical symptoms including fever, abdominal pain and gastrointestinal manifestations. Fascioliasis is also a zoonotic disease caused by liver flukes Fasciola hepatica and Fasciola gigantica, which can be contracted through consumption of contaminated water or aquatic plants, leading to various clinical features. Here, we report a case of a 39-year-old woman diagnosed with a liver abscess caused by co-infection of T. canis and F. hepatica, as confirmed by serological tests. Although the existence of a pet dog and an experience of eating raw water dropwort are potential clues for diagnosis, it cannot be determined as the source of infection because the source of infection has not been clearly identified. After administrating albendazole and triclabendazole sequentially, the patient showed improvement in blood test and imaging findings. Clinicians should be aware of parasitic co-infection and take appropriate management.

Development of a novel method for diagnosis of fasciolosis based on cathepsin L7 in ruminants.

Fasciolosis is a widely distributed zoonosis reported over 81 countries around the world. Good and early diagnostic method is critical in controlling this disease and prevention of injury to the liver and bile ducts. In this study, we identified a novel member (cathepsin L7) of cathepsin family from Fasciola spp.. Firstly, the biological character of CL7 was analyzed according to the information of cathepsin L family, and then rCL7 was expressed and purified, a new iELISA based on CL7 was developed. The results exhibited CL7 iELISA had 100% sensitivity 100% specificity in sheep (cut-off 1.329) and 100% sensitivity 93.75% specificity in cattle (cut-off 0.756). Moreover, anti-Fasciola CL7 antibodies could be detected in early Fasciola gigantica infected buffaloes, as early as 3 week-post-infection (WPI). In conclusion, it is suggested that CL7 with low cost, early detection, good specificity and sensitivity could be used as a candidate antigen for detection of ruminant fasciolosis.

Comparison of Mini-FLOTAC, Flukefinder and sedimentation techniques for detection and quantification of Fasciola hepatica and Calicophoron daubneyi eggs using spiked and naturally infected bovine faecal samples.

BACKGROUND: Fasciolosis (Fasciola hepatica) and paramphistomosis (Calicophoron daubneyi) are two important infections of livestock. Calicophoron daubneyi is the predominant Paramphistomidae species in Europe, and its prevalence has increased in the last 10-15 years. In Italy, evidence suggests that the prevalence of F. hepatica in ruminants is low in the southern part, but C. daubneyi has been recently reported at high prevalence in the same area. Given the importance of reliable tools for liver and rumen fluke diagnosis in ruminants, this study evaluated the diagnostic performance of the Mini-FLOTAC (MF), Flukefinder(R) (FF) and sedimentation (SED) techniques to detect and quantify F. hepatica and C. daubneyi eggs using spiked and naturally infected cattle faecal samples. METHODS: Briefly, negative bovine faecal samples were artificially spiked with either F. hepatica or C. daubneyi eggs to achieve different egg count levels: 10, 50 and 100 eggs per gram (EPG) of faeces. Moreover, ten naturally infected cattle farms from southern Italy with either F. hepatica and/or C. daubneyi were selected. For each farm, the samples were analysed individually only with MF technique and as pools using MF, FF and SED techniques. Bayesian latent class analysis (LCA) was used to estimate sensitivity and accuracy of the predicted intensity of infection as well as the infection rate in the naturally infected farms. RESULTS: The outcome of this study showed that the highest number of eggs (F. hepatica and C. daubneyi) recovered was obtained with MF, followed by FF and SED in spiked infected samples at 50 and 100 EPG, while at lower infection levels of 10 EPG, FF gave the best results. Moreover, the sensitivity for all the techniques included in the study was estimated at > 90% at infection levels > 20 EPG for both F. hepatica and C. daubneyi eggs. However, MF was the most accurate of the three techniques evaluated to estimate fluke infection intensity. Nevertheless, all three techniques can potentially estimate infection rate at farm level accurately. CONCLUSIONS: Optimization and standardization of techniques are needed to improve the FEC of fluke eggs.