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.

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.

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.

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.

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.

Validation of the natural sedimentation technique in the diagnosis of chronic fasciolosis.

There are various diagnostic techniques available for chronic fasciolosis in ruminants. However, many of them exhibit low specificity and sensitivity, making them impractical for field use and in low-resource laboratories. The present study evaluates the usefulness of the Natural Sedimentation technique in diagnosing chronic fasciolosis in three domestic species conducted at the Laboratorio de Parasitología y Enfermedades Parasitarias, Facultad de Ciencias Veterinas, Universidad Nacional de Cajamarca. Fecal samples were collected from n = 323 cattle, n = 362 sheep, and n = 231 swine for Fasciola hepatica fecal egg counts. The visualization of adult parasites in animal livers post-mortem was considered the gold standard. Additionally, the sensitivity of the technique was evaluated using five different amounts of feces. In cattle, a sensitivity of 0.93 ± 0.03, specificity of 0.91 ± 0.06, positive predictive value of 0.96 ± 0.03, and negative predictive value of 0.86 ± 0.07 were obtained. In sheep, a sensitivity of 0.79 ± 0.05, specificity of 0.83 ± 0.07, positive predictive value of 0.90 ± 0.04, and negative predictive value of 0.66 ± 0.08 were observed. In swine, a sensitivity of 0.92 ± 0.06, specificity of 1.00 ± 0.00, positive predictive value of 1.00 ± 0.00, and negative predictive value of 0.96 ± 0.03 were found. There was no statistical difference in egg counts when using 1, 2, 3, 4, and 5 g of feces (p = 0.907). Furthermore, 1 to 688 fecal eggs of F. hepatica were counted in 1 g of feces. The Natural Sedimentation technique has both qualitative and quantitative applications with satisfactory results when using 1 g of feces in the diagnosis of chronic fasciolosis in domestic animals. Due to its simplicity, it can be implemented in field conditions and low-resource laboratories.

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.

In-herd prevalence of Fasciola hepatica and Calicophoron / Paramphistomum spp. infections in German dairy cows with comparison of two coproscopical methods and establishment of real-time pyrosequencing for rumen fluke species differentiation.

Infections with liver and rumen flukes are among the most frequent parasitic diseases in cattle worldwide. In Europe, the predominant liver fluke species is Fasciola hepatica, and the recently rapidly spreading rumen flukes are mostly Calicophoron daubneyi and occasionally Paramphistomum leydeni. In this study, 1638 faecal samples from individual dairy cows from 24 northern and 18 southern German farms as well as one central German farm, all preselected for potential F. hepatica infection, were examined to determine in-herd prevalences of liver and rumen fluke infections. Furthermore, individual faecal egg counts (FECs) were determined in the northern and central German cows. On farms with patent F. hepatica infections, the mean in-herd prevalence was 15.8% in northern Germany, 41.6% in southern Germany and 14.0% in the central German farm. Rumen fluke infections resulted in high in-herd prevalences in all regions with a mean prevalence of 46.0% in northern, 48.4% in southern and 40.0% in central Germany. Individual FECs varied between 0.1 and 4.1 (mean 0.4) eggs per gram faeces (EPG) for F. hepatica and between 0.1 and 292.4 (mean 16.9) EPG for rumen flukes. Mean in-herd prevalence and mean FECs did not differ significantly between mono- and coinfected farms for either fluke species. Comparison of the classical sedimentation technique and the Flukefinder® method on a subset of 500 faecal samples revealed a similar number of positive samples, however, Flukefinder® mean FECs were three to four times higher for liver and rumen fluke eggs, respectively, with an increasing gap between EPG levels with rising egg counts. Fluke egg size measurement confirmed P. leydeni eggs on average to be larger in length and width (161.0 µm x 87.1 µm) than those of C. daubneyi (141.8 µm x 72.9 µm). However, due to overlap of measurements, morphological species identification based on egg size proved unreliable. For accurate identification, a real-time pyrosequencing approach was established, offering the advantage over classical Sanger sequencing of unambiguously identifying rumen fluke mixed species infections. Real-time pyrosequencing confirmed C. daubneyi (78.1% [50/64]) as the predominant rumen fluke species in Germany, while P. leydeni was detected in 12.5% (8/64) of sampled cows. A total of 9.4% (6/64) cows were infected with both C. daubneyi and P. leydeni, representing the first finding of a mixed infection in domestic ruminants in Europe to date.

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.

Idiopyrgus souleyetianus (Gastropoda: Tomichiidae) as an intermediate host of the liver fluke Amphimerus sp. (Trematoda: Opisthorchiidae) in Brazil.

Despite the importance of fish-borne trematodes of the family Opisthorchiidae as causative agents of human liver fluke disease, studies on these parasites outside Asia are relativally scarce. In South America, human focus of amphimerosis is known in Ecuador since the mid-20th century, and Amphimerus spp. have also been reported in wild and domestic mammals. Nevertheless, the knowledge on the snails that act as the first intermediate host of these potentially zoonotic parasites are scarce. Herein, a new cercaria of the pleurolophocercous morphotype found in the freshwater snail Idiopyrgus souleyetianus from Brazil was subjected to morphological and molecular studies. Multigene phylogenetic analyses based on 28S, 5.8S-ITS-2 and Cox-1 sequences enabled the identification of Amphimerus sp., a species distinct from that reported in humans from Ecuador. This cercariae was morphologically compared with other opisthorchiid cercariae known. The possible occurrence of human amphimerosis in Brazil is discussed.

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

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

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.

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

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

Fasciola hepatica infection modifies IgG1 specific immune response to foot-and-mouth disease virus induced by vaccination.

Fasciola hepatica, a worldwide distributed helminth, has a robust immunoregulatory effect in the host, increasing the susceptibility to secondary infections. Foot and mouth disease (FMD) is a highly contagious acute vesicular viral disease effectively controlled by vaccination in endemic regions. Despite the evidence of immunoregulatory effects, the impact of fasciolosis on the immune response induced by FMD vaccination in cattle has never been assessed. Our objective was to evaluate whether the infection by F. hepatica in cattle influences the long-term immunity elicited by the currently used commercial FMD-inactivated vaccines. Aberdeen Angus steers negative for F. hepatica were vaccinated twice against FMD virus (FMDV) during the first 6 months of age using a commercial oil vaccine formulated with A24/Cruzeiro and O1/Campos strains. When maternal antibodies against F. hepatica were weaned (18--20 months of age) animals were divided into groups of 12 and infected or mock-infected with 500 metacercariae/animal. Individual serum samples were collected at 0-, 28-, 59-, 87- and 157-days post-infection (dpi). Indirect ELISAs were used to detect A24/Cruzeiro specific bovine IgG and IgG subtypes. The total IgG antibody levels and avidity against FMDV did not show significant differences between all the groups. The commercial vaccine induced higher IgG2 than IgG1 titers in vaccinated animals. Anti-FMDV IgG1 levels significantly decreased in the infected group at 28 dpi. In addition, the avidity of IgG1 FMDV-specific antibodies at day 28 in the infected group was reduced compared to the control. These results show that F. hepatica infection modified anamnestic responses against FMDV, reducing serum IgG1 titers and avidity. To our knowledge, this is the first report of immune-regulation of F. hepatica altering the immune response of FMD vaccines, one of the most globally used animal vaccines.

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.