Nanotechnological Improvement of Veterinary Anthelmintics.

Helminths infections are among the most important problems in animal health and husbandry. Moreover, zoonotic helminths endanger rural communities, particularly in developing countries. Helminthiasis are not only important in relation to the harmful effects of parasites; additional issues like anthelmintic resistance spread became more important over time. As new anthelmintic development takes many years and millions of dollars of investment, some strategies are currently focused on the modification of already available drugs, in order to improve their efficacy and overcome their limitations. In this field, nanotechnology has brought a novel approach, showing advantages like the regulation of the drug's delivery and kinetics, reaching of specific targets, and possibilities to avoid the systemic spread and side effects. Taking this into account, the present review aims to introduce some of the current knowledge in anthelmintic improvement based on nanotechnology, and how researchers could benefit from this technology in order to overcome the drugs limitations. Finally, some insights into potential field applications are discussed, based on the most important concerns of current anthelmintic therapy.

A Vaccine Based on Kunitz-Type Molecule Confers Protection Against Fasciola hepatica Challenge by Inducing IFN-γ and Antibody Immune Responses Through IL-17A Production.

Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.

First report of pre-Hispanic Fasciola hepatica from South America revealed by ancient DNA.

It is generally assumed that the digenean human liver fluke, Fasciola hepatica, gained entry to South America during the 15th century upon arrival of Europeans and their livestock. Nonetheless in Patagonia, Argentina, digenean eggs similar to F. hepatica have been observed in deer coprolites dating back to 2300 years B.P. The main objective of our present study was to identify and characterize these eggs using an ancient DNA (aDNA) study. Eggs were isolated and used for aDNA extraction, amplification and sequencing of partial regions from the cytochrome c oxidase subunit 1 and the nicotinamide adenine dinucleotide dehydrogenase subunit 1 mitochondrial genes. Also, phylogenetic trees were constructed using Bayesian and maximum likelihood. Our results confirm the presence of F. hepatica in South America from at least 2300 years B.P. This is the first report and the first aDNA study of this trematode in South America prior to the arrival of the European cattle in the 15th century. The present work contributes to the study of phylogenetic and palaeobiogeographical aspects of F. hepatica and its settlement across America.

The egg hatch test: A useful tool for albendazole resistance diagnosis in Fasciola hepatica.

In the current study, the egg hatch test (EHT) has been evaluated as an in vitro technique to detect albendazole (ABZ) resistance in Fasciola hepatica. The intra- and inter-assay variations of the EHT were measured by means of the coefficient of variation in different fluke isolates and over time; then, the results of the EHT were compared with the "gold standard" controlled efficacy test, which assesses the in vivo anthelmintic efficacy. The EHT was used later to evaluate the intra-herd variability regarding the level of ABZ resistance in calves infected by the same fluke isolate. Finally, several factors of the initial protocol were modified to improve the simplicity of the assay, including the incubation time of eggs with the drug and the use of eggs collected from faeces. The greatest uniformity between results within the assay and over time until 8 weeks after gallbladder collection (the deadline proposed for egg analysis) was obtained with an ABZ concentration of 0.5 µM. The length of exposure to ABZ was shown to be critical, as prolonged incubation (15 days) led to a change of ovicidal activity. The ABZ concentration of 0.5 µM is suggested as a possible discriminating dose to predict ABZ resistance, due to the close agreement between the results of the EHT at an ABZ concentration of 0.5 µM and those of the in vivo assays.

Pharmacokinetic profile and anthelmintic efficacy of moxidectin administered by different doses and routes to feedlot calves.

We evaluated the comparative plasma disposition kinetics and efficacy of moxidectin (MXD), administered by the intraruminal (IR) or subcutaneous (SC) route at two different dosage levels (0.2 and 1 mg/kg) in feedlot calves. Additionally, the efficacy was compared to an ivermectin (IVM, SC administration) treated group. This study was divided into two separate studies, the "Pharmacokinetic (PK) study" and the "Efficacy study". The "PK study" involved 24 calves free of gastrointestinal nematodes (GIN), which were allocated into 4 groups (n = 6) and treated with MXD by either the SC or the IR route at the therapeutic (MXDSC0.2, MXDIR0.2, respectively) or at fivefold the therapeutic dose (MXDSC1.0, MXDIR1.0, respectively). Blood samples were collected from 3 h up to 14 days post-treatment. MXD concentrations in plasma samples were analyzed by HPLC. The "Efficacy study" included 125 calves naturally infected with GIN, which were allocated into five experimental groups (n = 25 each); the same four MXD-treated groups described for the "PK study", and an additional group treated by the SC route with IVM (IVMSC0.2). The efficacy of IVM given at its therapeutic dose and the different MXD groups at the therapeutic and fivefold the therapeutic dose was calculated by analysis of the individual efficacy using the package eggCounts-2.1-1' on the R software environment, version 3.5.0 (R Core Team, 2018). Daily weight gain (DWG) was also measured over the first 47 days of the fattening cycle. Independently of the administration route, MXD peak plasma concentration (Cmax) and area under the concentration-time curve (AUC) were higher in groups treated with the higher dose (1.0 mg/kg), whereas a longer time to reach Cmax (Tmax) was observed after the IR treatments. The observed MXD efficacies were 85% (MXDSC0.2), 94% (MXDSC1.0), 84% (MXDIR0.2) and 99% (MXDIR1.0), at day +27. At day +27, all MXD-treated groups showed higher efficacies than the group having received IVM (45%). The post-treatment Cooperia spp. L3 counts were particularly low in the groups MXDSC1.0 and MXDIR1.0. All of the groups treated with MXD showed better DWG than the IVMSC0.2 group (P = 0.01). Dose and administration route modifications effectively improved the anthelmintic and productive performance of MXD. A high dose of MXD improved the control of IVM-resistant GIN in feedlot calves. However, this practice must be taken with caution, since MXD resistance could rapidly emerge, especially in grazing cattle.

Efficacy of oxyclozanide against adult Paramphistomum leydeni in naturally infected sheep.

The aim of the current study was to assess oxyclozanide (OCZ) efficacy against Paramphistomum leydeni in naturally infected adult sheep. OCZ concentrations in blood stream and gastrointestinal fluids collected from treated animals were also measured. Fifteen P. leydeni naturally infected sheep were randomly divided into two groups: untreated control (n=5) and treated (n=10). The treated group was orally drenched with OCZ (20mg/kg, day 0). A second dose was administered 72 h later. Faecal samples were taken at days 0, +3 and +5. Five sheep from both groups were slaughtered at day +5. At necropsies, rumen, abomasum and small intestine were examined for adult and immature flukes. All recovered flukes were counted and the treatment efficacy was estimated. Additionally, serum and gastrointestinal fluid content (ruminal, abomasal and small intestine) samples, obtained from five treated animals at day +5, were analyzed by HPLC to measure OCZ concentrations. OCZ showed high efficacy (99%) against mature P. leydeni. The post-treatment egg reduction was also high after the first dose with values ranging from 98.4% (day +3) to 99.5% (day +5). The highest OCZ concentrations were measured in serum (20.7 ± 11.5 µg/mL) followed by the small intestinal fluid (6.00 ± 4.50 µg/mL). Very low OCZ concentrations (ranging between 0.05 and 0.02 µg/mL) were measured in ruminal and abomasal fluids. OCZ administered to sheep twice (20mg/kg) by the oral route was highly efficacious against mature stages of P. leydeni in naturally infected sheep. Despite a high drug concentration at the intestinal fluid, OCZ efficacy against immature stages could not be assessed. OCZ efficacy and assessment of its concentration profiles in different tissues are considered a contribution to the scarce information available on this ruminant fluke.

Fasciola hepatica: variations in redial development and cercarial production in relation to the geographic origin of the parasite.

Two hundred Galba truncatula, originating from a French population, were subjected to single-miracidium infections with an allopatric isolate (origin, Argentina) of Fasciola hepatica. The control group was constituted by 100 snails coming from the same population and exposed to sympatric miracidia of F. hepatica according to the same protocol. Snail samples were collected bimonthly from both groups between day 14 and day 112 p.e. (at 20 °C) and snail dissections were performed to count free rediae, intraredial morulae and free cercariae. Third and fourth generation rediae were significantly more numerous in the allopatric group, while the number of first generation rediae was significantly lower. In the sympatric group, the decrease in the number of intraredial morulae in the first, second and third redial generations was significantly faster. Free cercariae within the snail body were significantly more numerous in the sympatric than in the allopatric groups, whatever the date of snail dissection. The changes in redial development and cercarial production noted in the Argentinean group might be due to the evolution of South American flukes in a divergent way after the introduction of foreign infected ruminants in this continent from the 15th century.

Metaphylactic effect of Diclazuril 0.25% in suckling beef calves, during a coccidiosis outbreak in extensive farming.

The weight gain performance and oocysts reduction, as response to the metaphylactic treatment with Diclazuril 0.25% at the start of a coccidiosis outbreak, was studied by a cases-controls transverse study. Fifty-eight suckling calves of approximately 90 days old were randomly selected from an infected beef herd on extensive farming. The calves were weighted and individual faecal samples were taken for oocyst per gram count (OPG). Out of those, 29 were drenched with 1 mg kg(-1) of Diclazuril in one oral dose (group T), since the other 29 remained as control group (group C). Samples for OPG and weights were measured again at days 7 and 21 after treatment, respectively. Later, the groups were divided (by the median) in higher or lower OPG counts, and were compared in the same way, in order to remove those without apparent infection (lower OPG counts). The faecal oocysts reduction reached 99.5% (p<0.0001), for the treated group. Along the three weeks of study, an increment of 2.65 kg in 21 days (125 g day(-1), p=0.036), was seen in treated group respect to controls, but this difference increased to 3.94 kg in 21 days (p<0.0001), when only the calves with higher OPG were taken into account. These results highlight the magnitude of the subclinical impact of coccidiosis, biased by the individual susceptibility to infection, which leads to get heavier infections and express higher oocysts output.

Identification of a field isolate of Fasciola hepatica resistant to albendazole and susceptible to triclabendazole.

The experiments described here were designed to characterize the status of susceptibility/resistance to albendazole (ABZ) and triclabendazole (TCBZ) of a Fasciola hepatica isolate (named CEDIVE isolate) recovered from infected sheep (Gualeguay, Argentina) and maintained under laboratory conditions. Two separate clinical efficacy experiments were performed on sheep artificially infected with the CEDIVE isolate. Experiment 1: sheep were randomly distributed either in an untreated control group or in an ABZ (7.5 mg/kg) treated group (n=4 each). Additionally, the systemic exposure of ABZ metabolites was assessed in those ABZ-treated infected animals. In Experiment 2, an untreated control group and a TCBZ (10 mg/kg) treated group was included (n=4 each). The fluckicidal efficacy of ABZ and TCBZ was assessed by the comparison of the number of flukes recovered from untreated and treated sheep at 15 days post-treatment. The efficacy against the CEDIVE isolate of F. hepatica was 29% (ABZ) and 100% (TCBZ). The plasma drug exposure (expressed as AUC and C(max)) observed in the ABZ treated animals (Experiment 1) was in agreement with data obtained in the previous studies, which indicate that the low ABZ efficacy was not related to the quality of the pharmaceutical product and/or to a low systemic availability of the active drug/metabolite. The results reported here clearly show that the CEDIVE isolate of F. hepatica behaves as resistant to ABZ and susceptible to TCBZ.

Molecular characterization of the ITS-2 fragment of Paramphistomum leydeni (Trematoda: Paramphistomidae).

Paramphistomosis has shown an increased spread over the last years in Argentina, being in some regions an emerging parasitosis, which has motivated their study and identification. Even when morphological features were reported, molecular characterization appears as a complementary and reliable tool. Samples of Paramphistomum leydeni collected since 2002 from different origin, definitive hosts, natural or experimental infections and preservation method were subjected to PCR-RFLP for ITS2+ fragment, and compared to Fasciola hepatica and Notocotilidae cercarie. Frozen and ethanol fixed samples amplified fragments of 500 bp for P. leydeni and Notocotilidae samples, while F. hepatica eggs produced a 540 bp amplicon. Restriction fragments obtained from endonucleases HinfI, HhaI, BsuRI, TaqI, and TruI were identical for all P. leydeni samples, supporting the morphological classification previously performed. Four selected amplicons were sequenced and reported at GenBank, given a consensus sequence ITS-2+ of 441 bp. This first report of molecular characterization for P. leydeni, improves the current knowledge of the genus and establishes precedents for further specimen classifications.

Quiste hidatídico cerebral / Cerebral hydatid cyst

El antecedente epidemiológico asociado a una evolución crónica de signos focales y de hipertensión endocraneana, deben hacer sospechar el diagnóstico de quiste hidatídico cerebral. A esto se le suman los análisis complementarios, que gracias al avance de la tecnología disponemos con mayor facilidad de estudios como TAC, que ayudan a realizar un diagnóstico más rápido o por lo menos a un diagnóstico presuntivo. Si bien no debemos olvidar que la confirmación diagnóstica se obtiene a través del estudio anatomopatológico. No por ello se debe restarle importancia a los análisis laboratoriales de rutina, ya que aportan información extra que completa el cuadro y sobre todo ayudan a descartar otras patologías, y así, pasar de tener diagnósticos presuntivos a tener un solo diagnóstico final.