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.

A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens.

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/µL for DNA samples diluted in water, 10 fg/µL for Fasciola/snail DNA scramble, and 100 fg/µL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.

Fasciolosis in ruminants in Brazil.

This review aims to promote discussion about the situation of fasciolosis in ruminants in Brazil. The disease is still found more frequently in the South and Southeast regions, but reports outside these areas show the spread of the disease, including human cases. Many studies have been published on the diagnosis and control of fasciolosis, but development of field diagnosis methods and drugs that control all stages of the parasite is still a challenge. Studies should be carried out of new distribution areas and alternatives for control in Brazil, which depends on understanding the complex interactions between of the environment, ecosystems and hosts of this trematode.

Esta revisão tem como objetivo levantar uma discussão sobre a situação da fasciolose em ruminantes no Brasil. A enfermidade ainda se encontra mais frequentemente nas regiões Sul e Sudeste do país, entretanto relatos fora dessas regiões evidenciam a expansão da enfermidade, inclusive casos em humanos. Muitos estudos têm sido publicados sobre o diagnóstico e controle da fasciolose, entretanto, o diagnóstico a campo e fármacos que combatam todas as fases evolutivas do parasito ainda são desafios. Novos estudos devem ser realizados sobre novas áreas de distribuição e alternativas de controle em condições brasileiras, o que depende da compreensão das complexas interações entre meio ambiente, ecossistemas e hospedeiros desse trematódeo.

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.

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.

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.

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.

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).

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.

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.

Triclabendazole resistance in Fasciola hepatica: First report in sheep from the Santa Cruz province, Argentinian Patagonia.

In the fall of 2022, decreased triclabendazole (TCBZ) efficacy against F. hepatica was suspected in a sheep farm located in the Santa Cruz province, Argentinian Patagonia. Since TCBZ-resistance in F. hepatica has never been reported in this province, this study aimed to confirm potential TCBZ-resistance in F. hepatica and to evaluate the efficacy of closantel (CLO) and nitroxinil (NTX), through faecal egg count reduction test (FECRT), and the efficacy of albendazole (ABZ) through the in vitro egg hatch test (EHT) in sheep. Sixty-eight (68) animals were selected from a herd of eighty (80) female Merino naturally infected with F. hepatica based on eggs per gram of F. hepatica (EPGFh) counts and assigned into four (4) groups (n = 17 per group): Group Control, animals did not receive anthelmintic treatment; Group TCBZ, animals were orally treated with TCBZ (12 mg/kg); Group CLO, animals were orally treated with CLO (10 mg/kg); and Group NTX, animals were subcutaneously treated with NTX (10 mg/kg). The fluke egg output was monitored on days 0 and 21 post-treatment. For the EHT, liver fluke eggs were isolated from faecal samples (approx. 50 g) collected from animals of the control group. TCBZ efficacy against liver fluke was 53.4%, confirming the presence of TCBZ-resistant isolates on the farm. CLO and NTX were highly effective (100%) for the treatment of F. hepatica on this farm. The EHT was carried out in two different laboratories, in which was observed an ABZ efficacy of 95.8 (Bariloche) and 96.5% (Tandil). These results indicate the ABZ susceptibility of this F. hepatica isolate and the inter-laboratory precision of the test.

In vitro and in vivo studies on a group of chalcones find promising results as potential drugs against fascioliasis.

About a third of the world population is infected by helminth parasites implicated in foodborne trematodiasis. Fascioliasis is a worldwide disease caused by trematodes of the genus Fasciola spp. It generates huge economic losses to the agri-food industry and is currently considered an emerging zoonosis by the World Health Organization (WHO). The only available treatment relies on anthelmintic drugs, being triclabendazole (TCBZ) the drug of choice to control human infections. The emergence of TCBZ resistance in several countries and the lack of an effective vaccine to prevent infection highlights the need to develop new drugs to control this parasitosis. We have previously identified a group of benzochalcones as inhibitors of cathepsins, which have fasciolicidal activity in vitro and are potential new drugs for the control of fascioliasis. We selected the four most active compounds of this group to perform further preclinical studies. The compound's stability was determined against a liver microsomal enzyme fraction, obtaining half-lives of 34-169 min and low intrinsic clearance values (<13 µL/min/mg), as desirable for potential new drugs. None of the compounds were mutagenic or genotoxic and no in vitro cytotoxic effects were seen. Compounds C31 and C34 showed the highest selectivity index against liver fluke cathepsins when compared to human cathepsin L. They were selected for in vivo efficacy studies observing a protective effect, similar to TCBZ, in a mouse model of infection. Our findings strongly encourage us to continue the drug development pipeline for these molecules.

Prevalence of flukes (Fasciola hepatica and paramphistomids) in cattle in south-eastern Mexico.

The objective of this study was to determine the risk factors and prevalence of trematodes in south-eastern Mexico. The prevalence of trematodes was determined in 1010 bovines. The study was carried out from October 2018 (n=291) to December 2019 (n=719). Only in 2019 rumen and liver fluke eggs were differentiated. Faecal samples (n=311) were obtained from farms in southeast Mexico located in Tabasco, Chiapas and Campeche. In addition, the presence of flukes in liver and rumen from slaughtered cattle in abattoirs was recorded with a total of 408 samples. A logistic procedure was used to obtain the prevalence and the effect of main risk factors such as land physiography (flooded areas and hills), year, sex, animals' age and type of sample obtained (eggs in faeces and flukes). The general prevalence of flukes in cattle was 32.3 % in 2018 and 41.7 % in 2019. Prevalence of F. hepatica (liver fluke) was 18.6 % (134/719) and that of paramphistomids (rumen fluke) was 33.4 % (240/719). The infected cattle from the slaughterhouse indicated a lower prevalence of F. hepatica (1 %) and rumen fluke (26.7 %) than in farms detected by egg in faeces (41.8 % and 42.1 %, respectively). The physiographic zone was decisive in the presence of F. hepatica and rumen fluke, while sex did not represent a risk factor (P > 0.05). The environmental conditions of the Mexican southeast favour the presence of both liver and rumen fluke.

Serological diagnosis of fasciolosis (Fasciola hepatica) in humans, cattle, and sheep: a meta-analysis.

Fasciola hepatica can cause problems in both animals and humans. Fasciolosis can be diagnosed through the indirect ELISA immunodiagnostic test. Serological diagnosis of Fasciola is based on recombinant antigens secreted by this worm. We used PubMed and Google Scholar databases to review the published literature on 'antigens with immunogenic potential' used in serological tests to identify antibodies against F. hepatica in humans, cattle, and sheep. Studies that investigated diagnostic tests with common reference standards were included in the sensitivity and/or specificity bivariate meta-analysis. In the quality and susceptibility to bias analysis of the 33 included studies, 26 fulfilled at least six (75%) of the eight QUADAS criteria and were considered good-quality papers. We found that most of the studies used native excretory-secretory antigens and recombinant cathepsin in ELISA tests for serological diagnosis of fascioliasis in humans, cattle, and sheep. The meta-analysis revealed that all antigens demonstrated good accuracy. The best results in terms of sensitivity [0.931-2.5% confidence interval (CI) and 0.985-97.5% CI] and specificity (0.959-2.5% CI and 0.997-97.5% CI) were found in human FhES. FhrCL-1, FhES, and FhrSAP-2 antigens gave the best results for the serum diagnosis of human and animal fasciolosis.

Incidence of endemic fascioliasis in animals and estimation of the financial loss due to seizure of livers in Andahuaylas, Peru / Incidencia de fascioliasis endémica en animales y estimación de la pérdida económica por comiso de hígados en Andahuaylas, Perú

ABSTRACT This study aimed to determine the frequency of fascioliasis in animals slaughtered in slaughterhouses in the province of Andahuaylas and the financial loss due to the seizure of livers. The research was quantitative at a basic level, analytical design, observational type, and cross-sectional. It was carried out between October and December 2016 in the slaughterhouses of the districts of Andahuaylas, San Jerónimo, and Talavera. At the same time, a retrospective study corresponding to the years 2011-2016 was conducted. Fascioliasis was diagnosed through post-mortem sanitary inspection of the viscera. The statistical examination was carried out with the software Epidat 4.2, using Pearson's Chi-square test to verify differences between proportions; and the t-test to estimate equality of means, assuming a confidence level of 95% and confidence intervals of < = 0,05. Most of the infected animals came from Pampachiri (p < 0,01); the most affected species was bovine (51,9%; 95% CI = 49,9 - 53,9). In the 2011-2016 period, the frequency of fascioliasis was 46% (95% CI = 45,8 - 46,2) with a total financial loss for the seizure of livers of USD 238.551.1. The spring season registered the lowest frequency of fascioliasis and the lowest financial loss (p < 0,01). The most significant losses occurred in 2014, in March and August, in the Talavera slaughterhouse (p < 0,01). The study concludes that Andahuaylas is mesoendemic for fascioliasis but hyperendemic in cattle.

RESUMEN El objetivo del estudio fue determinar la frecuencia de fascioliasis en animales faenados en mataderos de la provincia de Andahuaylas y la pérdida económica por decomiso de hígados. La investigación fue cuantitativa de nivel básico, diseño analítico, tipo observacional y corte transversal. Se realizó entre octubre y diciembre de 2016 en los mataderos de los distritos de Andahuaylas, San Jerónimo y Talavera. Al mismo tiempo, se realizó un estudio retrospectivo correspondiente a los años 2011-2016. La fascioliasis fue determinada a través de la inspección sanitaria post mórtem de las vísceras. El examen estadístico se realizó con el programa Epidat 4.2, utilizando la prueba de Chi-cuadrado de Pearson para comprobar diferencias entre proporciones y la prueba t para estimar igualdad de medias, con un nivel de confianza de 95% e intervalos de confianza de < = 0,05. La mayoría de los animales infectados procedieron de Pampachiri (p < 0,01); la especie más afectada fue la bovina (51,9%; IC 95% = 49,9-53,9). En el periodo 20112016, la frecuencia de fascioliasis fue de 46% (IC 95% = 45,8-46,2) con una pérdida económica total por decomiso de hígados de USD 238.551.1. La menor frecuencia de fascioliasis y la menor pérdida económica (p < 0,01) se registró en primavera. Las mayores pérdidas se presentaron en 2014, en marzo y agosto, en el matadero de Talavera (p < 0,01). El estudio concluyó que Andahuaylas es mesoendémico para fascioliasis, pero hiperendémico en bovinos.

Molecular characterization of Fasciola hepatica in endemic regions of Colombia.

Fasciola hepatica is a zoonotic trematode that affects a wide range of hosts, including cattle, sheep, and goats. The economic impact of the parasite on the cattle industry is significant, with high losses reported worldwide. While its impact on human health was previously underestimated, recent years have seen a rise in fascioliasis cases, leading to increased interest among researchers globally. To characterize the genetic diversity and intraspecific variation of this parasite in South America, specifically in Colombia, we collected 105 adult parasites from cattle bile ducts in seven Colombian departments (Antioquia, Boyacá, Santander, Cauca, Cundinamarca, Nariño, Norte de Santander, and Santander) to assess the parasite's phenotypic analyses, genetic diversity, and population structure. A computer image analysis system (CIAS) was applied based on standardized morphological measurements. Liver-fluke size was studied by principal component analysis (PCA). DNA sequences were obtained for nuclear markers such as the 28S, ß-tubulin 3, ITS1, ITS2, and the mitochondrial marker Cytochrome Oxidase I (COI). Multiple statistical tests were performed, and the parasite's population structure was analyzed. Maximum Likelihood (ML) phylogenetic reconstructions were carried out using the sequences obtained herein and sequences available in GenBank. Morphological results revealed that all the obtained individuals matched F. hepatica's morphology. There was no evidence of high genetic diversity, and the absence of genetic structure at the country-level was notable, possibly caused by a demographic expansion of this trematode in Colombia or the low resolution of the molecular markers employed. Future studies are still needed to unveil the genetic population structure of F. hepatica across the country.

A promising new target to control fasciolosis: Fasciola hepatica leucine aminopeptidase 2.

Parasite M17 leucine aminopeptidases (LAPs) have been associated with critical roles in different key functions such as the nutrition, migration, and invasion of the natural host. Native or recombinant LAP used as a vaccine antigen has proved effective to elicit protection against Fasciola hepatica infection in sheep, pointing to potential vaccine candidates against fascioliasis in ruminant species. Previously, the FhLAP1, abundantly secreted in vitro by the mature adult parasite was used as a vaccine antigen obtaining promising protection results against F. hepatica challenge in small ruminants. Here, we report the biochemical characterization of a second recombinant LAP (FhLAP2) associated with the juvenile stage of F. hepatica. FhLAP2 showed aminopeptidase activity using different synthetic substrates, including leucine, arginine, and methionine and was increased in the presence of Mn+ 2 and Mg+ 2. The activity was inhibited by bestatin, 1,10-phenanthroline, and EDTA, specific inhibitors of aminopeptidase and/or metalloproteases. Finally, the recombinant FhLAP2 functional form was tested in combination with Freund's incomplete adjuvant in an immunization trial in mice followed by an experimental challenge with F. hepatica metacercariae. The immunization with FhLAP2/FIA resulted in a significant reduction of parasite recovery compared to control groups. The immunized group elicited total specific IgG and subclasses IgG1 and IgG2 antibody responses. This study highlights the potential of a new candidate vaccine formulation with potential applications in natural ruminant hosts, especially those targeting the juvenile stage.

Protein Modelling and Molecular Docking Analysis of Fasciola hepatica ß-Tubulin's Interaction Sites, with Triclabendazole, Triclabendazole Sulphoxide and Triclabendazole Sulphone.

PURPOSE: Fasciola hepatica is a globally distributed trematode that causes significant economic losses. Triclabendazole is the primary pharmacological treatment for this parasite. However, the increasing resistance to triclabendazole limits its efficacy. Previous pharmacodynamics studies suggested that triclabendazole acts by interacting mainly with the ß monomer of tubulin. METHODS: We used a high-quality method to model the six isotypes of F. hepatica ß-tubulin in the absence of three-dimensional structures. Molecular dockings were conducted to evaluate the destabilization regions in the molecule against the ligands triclabendazole, triclabendazole sulphoxide and triclabendazole sulphone. RESULTS: The nucleotide binding site demonstrates higher affinity than the binding sites of colchicine, albendazole, the T7 loop and pßVII (p < 0.05). We suggest that the binding of the ligands to the polymerization site of ß-tubulin can lead a microtubule disruption. Furthermore, we found that triclabendazole sulphone exhibited significantly higher binding affinity than other ligands (p < 0.05) across all isotypes of ß-tubulin. CONCLUSIONS: Our investigation has yielded new insight on the mechanism of action of triclabendazole and its sulphometabolites on F. hepatica ß-tubulin through computational tools. These findings have significant implications for ongoing scientific research ongoing towards the discovery of novel therapeutics to treat F. hepatica infections.