Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1.

Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis.

Motility of Fasciola hepatica miracidia assessed with a computer-assisted sperm analyser.

The motility parameters of Fasciola hepatica miracidia were assessed at different temperatures and times post-hatching using computer-assisted sperm analysis. Eggs were incubated at 22 °C or 25 °C for 14 days. Five motion parameters were evaluated at different incubation temperatures up to 10 h post-hatching. No differences were observed in the percentage that hatched after incubation at the two different temperatures. However, the straight-line velocity of miracidia following incubation at 22 °C was significantly different from that observed at 25 °C (P< 0.01). All miracidium motion parameters at different post-hatching temperatures showed an overall decrease at the end of the experiment. Those miracidia hatching from eggs incubated at 25 °C had a higher velocity of 1673.3 µm/s compared with 1553.3 µm/s at 22 °C. Velocity parameters increased as the post-hatching temperature increased from 22 °C to 37 °C.

Gastrointestinal nematodes in rotationally grazing ewes in the mountainous region of central Mexico.

The aims of this study were to determine the frequency of egg shedding (percentage of egg-positive faecal samples) and faecal egg counts (FEC) over 13 months in two different breeds of ewes, both pregnant and non-pregnant, in a mountainous region of central Mexico. Additionally, the effect of ivermectin and albendazole treatments on FEC reduction was recorded. The study also aimed to relate temperature and rainfall to FEC. The gastrointestinal nematode (GIN) third-stage larvae genera recovered from both faeces and grassland pastures in a temperate region were also assessed. Faecal samples were collected from ewes at monthly intervals for 13 months to investigate the FEC population of GIN larvae, their concentration and genera in grass samples collected from grazed and rested pastures. Egg-shedding frequency ranged from 0 to 92% and FEC from 0 to 12,000 eggs per g faeces (epg), with counts in Suffolk higher than in Dorset ewes. The identified genera were Haemonchus, Trichostrongylus, Teladorsagia, Cooperia, Oesophagostomum, Bunostomum, Nematodirus and Strongyloides. Haemonchus and Trichostrongylus were the most common genera. The number of L3 was higher in grazing lands than in those at rest. The highest FEC were recorded in the dry season due to peripartum, but the highest L3 counts were recorded in the rainy season. The coexistence of species of different geographical distributions at this site may be because there is a confluence of Nearctic and Neotropical geographic regions; thus, despite the temperate climate, tropical species can be found. Additionally, this study suggests that increasing temperatures could favour the presence of different tropical GIN species together with typical temperate-zone GIN species.

Comprehensive diagnosis of parasites in sheep kept under different zootechnical management in a region temperate in Mexico.

Parasites cause losses in animal production. Parasite infection in ruminants has been estimated to be a major problem causing more than 3 billion USD per year, from which 60% corresponds to the sheep industry. Treatment is based on the use of synthetic anthelmintics; however, repeated application or under dosage have resulted in the selection of nematodes resistant to anthelmintics. The objective of the present work was to perform a diagnosis of gastrointestinal parasites in sheep kept under different zootechnical management. Ninety female sheep were used, most of them pregnant. Sampling was performed monthly from December 2015 to June 2016 (flock 5 until April). Fecal samples were collected from the rectum; the McMaster technique was performed, morphological characteristics were observed, oocysts and eggs were counted per gram of feces (opg and epg), frequency and intensity were obtained. Faecal culture was performed for feces that had a positive result, infective larvae were obtained and taxonomically identified. At the end of the study, a dewormer (fenbendazole) was administered and its effect was measured. The frequency of gastrointestinal parasites was 100%. The highest opg was 3,600 (flock 3, March, 2016), the epg for cestodes was 2800 (flock 1, January, 2016) and for gastrointestinal nematodes (GIN) was 25,000 (flock 1, May, 2016); the intensity was variable and it was increased by peripartum. Protists (Eimeria spp), cestodes (Moniezia) and nematodes (Haemonchus, Trichostrongylus, Cooperia, Chabertia ovina. Teladorsagia, Oesophagostomum, Nematodirus and Trichuris ovis) were identified. No previous diagnosis is performed in flocks, and sometimes dewormers are administered, even though resistance to ivermectin and benzimidazole is suspected. Flock management, its feeding system and its conditions were determinant for the observed results; therefore, it is necessary to count with a diagnosis that provides information about the parasitic population and its dynamic, in order to carry out a selective and comprehensive control that has an impact on the animal, human and environmental health.

Exposure to Fasciola hepatica miracidia increases the sensitivity of Lymnaea (Fossaria) humilis to high and low pH.

Humidity and temperature have been considered important factors affecting the infectivity of Fasciola hepatica to its molluscan host. One hundred and thirty laboratory-reared Lymnaea humilis were exposed for 4 hr to the miracidia of F. hepatica over a pH range from 4.0 to 10.0, and their rates of survival were compared with 130 similarly treated but unexposed control snails. All control snails died within 24 hr at pH 4.0, but they showed better survival at pH 5.0-10.0. Their sensitivity to solutions with high and low pH, however, was increased if kept in the presence of F. hepatica miracidia. Snails exposed at pH 5.0 died within 24 hr, whereas most other pHs also affected survival such that by day 18 only those snails exposed at pH 7.2 remained alive. The increased sensitivity of the snails to pH could be explained by a damage-mediated release of parasite enzymes, because infectivity was highest at pHs associated with the lowest host mortality.

Seasonal transmission of Fasciola hepatica in cattle and Lymnaea (Fossaria) humilis snails in central Mexico.

A 19-month study on the prevalence of fasciolosis in 30 naturally infected cows, the presence of infected and non-infected Lymnaea (Fossaria) humilis snails, and variation in soil temperature and humidity is reported. The prevalence of fasciolosis in cattle declined from around 50% in March to 30% in July, then, it increased from August, reaching a plateau of 100% in November-January, before gradually declining thereafter. A rise in soil humidity and temperature in June and July, respectively, which peaked between August and November was observed. In July, L. (F.) humilis snails appeared, but the infection could only be found in these in August and November. The number of infected snails did not reflect the infestation rate in cows, even though the infestation kinetics in both hosts behaved as predicted from the life cycle of the parasite.