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.

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep.

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 µg, 200 µg and 400 µg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 µg, 200 µg and 400 µg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.

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.

Duddingtonia flagrans chlamydospores in nutritional pellets: effect of storage time and conditions on the trapping ability against Haemonchus contortus larvae.

The study evaluated the effect of storage time and conditions of nutritional pellets (NP) containing Duddingtonia flagrans chlamydospores on its in vitro trapping ability against Haemonchus contortus L3 larvae. The treated batch (200 NP) contained 4 × 106 chlamydospores of the FTH0-8 strain, whereas the control batch (200 NP) was produced without spores. Both NP batches were exposed to four experimental storage conditions: (T1) shelves (indoors); (T2) refrigeration (4°C); (T3) outdoors under a roof; and (T4) 100% outdoors. Each group comprised 48 NP with spores and 48 NP without spores (control). The ability of D. flagrans spores to trap H. contortus L3 larvae was evaluated for 8 weeks for each storage condition. For that purpose, six randomly selected NP with spores were compared to their respective control NP. Each NP was individually crushed. The crushed material (1 g) was placed on the surface of a 2% water agar plate with 200 H. contortus L3 larvae. Plates were sealed and were incubated at room temperature for 8 days. The whole content of every plate was transferred to a Baermann apparatus to recover the remaining larvae. There was a clear larval reduction in the NP with spores, compared to the respective control NP in the four storage conditions (P< 0.05). The mean reductions ( ± SEM) of the storage conditions were 67 ± 4.9 (T2), 77 ± 6.1 (T1), 81.5 ± 3.8 (T4) and 82.1 ± 2.5 (T3). Larval reductions were similar at all times and were not affected by storage conditions or storage time (R 20.05). The long-term shelf-life of the chlamydospores in the NP suggests that this spore dosage technology is a viable option.

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.

Proteolytic activity in Fasciola hepatica is reduced by the administration of cathepsin L mimotopes.

The objective of this study was to assess the proteolytic activity of Fasciola hepatica cathepsins in liver sections from mice vaccinated with phage clones of cathepsin L mimotopes, using the film in situ zymography technique. Female BALB/c mice were immunized three times with 2.5 x 10¹¹ phage particles without adjuvant. Animals vaccinated with phage clones produced high titres of anti-mimotope antibodies and a significant reduction in fluke burden was observed following challenge with metacercariae of F. hepatica. The proteolytic activity in hepatic tissue was reduced after the immunization with phage clones.

Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library.

An M13 phage random 12-mers peptide library was used to screen cathepsin L mimotopes of Fasciola hepatica and to evaluate their immunogenicity in sheep. Seven clones showed positive reactivity to a rabbit anti-cathepsin L1/L2 antiserum in ELISA, and their amino acid sequences deduced by DNA sequencing were tentatively mapped on the protein. Twenty sheep were randomly allocated into 4 groups of 5 animals each, for immunization with 1x10(14) phage particles of clones 1, 20, a mixture of 7 clones and PBS, without adjuvant at the beginning, and 4 weeks later. All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. The mean worm burdens after challenge were reduced by 47.61% and 33.91% in sheep vaccinated with clones 1 and 20, respectively; no effect was observed in animals inoculated with the clone mixture. Also, a significant reduction in worm size and burden was observed for those sheep immunized with clone 1. Animals receiving clone 20, showed a significant reduction in egg output. Immunization induced a reduction of egg viability ranging from 58.92 to 82.11%. Furthermore, vaccinated animals produced clone-specific antibodies which were boosted after challenge with metacercariae of F. hepatica.

Frequency of Toxoplasma gondii infection in sheep from a tropical zone of Mexico and temporal analysis of the humoral response changes.

An indirect ELISA and an immunoblot were standardized to detect anti-Toxoplasma gondii antibodies in sheep, and were compared with a commercial ELISA as reference. Sensitivity was 92.0 and 96.0%, and specificity 88.0 and 75.0%, respectively. Then the serum samples of 103 sheep on a ranch located in the Eastern region of Mexico were analysed. A frequency ranging from 77 to 84% was observed, with a heterogeneous pattern among the animals by immunoblotting. Ten months later 56 sheep were sampled and tested again. Six animals became negative while 1 case was negative the first time and strongly positive 10 months later. Considering this to be a new case we calculated an incidence rate of 2.1% (CI 95% 0.6-4.8%). IgG avidity ELISA was performed on 36 positive samples, 33 being of high-avidity at both times; slight increases in 2 samples and conservation of low-avidity in 1 sheep were also observed. Higher prevalence rates of toxoplasmosis in a moist warm compared to a cold atmosphere was attributed to the long viability of T. gondii oocysts. This may explain the high frequency of T. gondii in this region, which apparently has favourable climatic conditions for the transmission of this protozoan, besides the presence of both domestic and wild cats.

Prevalence of Fasciola hepatica (ELISA and fecal analysis) in ruminants from a semi-desert area in the northwest of Mexico.

The aim of the present study was to estimate the prevalence of Fasciola hepatica in cattle, goats and sheep from the municipalities of Guaymas and Cajeme, Sonora State, Mexico using the indirect ELISA and Benedeck's sedimentation tests. A total of 2,936 serum and fecal samples from 1,346 bovines, 1,199 goats and 381 sheep were analyzed. Other parameters such as breed and age were also taken into consideration. The results indicated a moderate percentage of prevalence for fasciolosis in these municipalities. In bovines, a prevalence of 11.4 +/- 0.9 was found using the sedimentation test and 24.4 +/- 1.2 for the indirect ELISA. In goats, a prevalence of 24.5 +/- 1.2 was found for the fecal analysis and 43 +/- 1.5 for the indirect ELISA. In sheep, the prevalence for both tests was 19.4 +/- 2.0 and 30.6 +/- 2.7, respectively. Results showed a greater proportion of positive animals when the ELISA test was used than when the Benedeck's sedimentation test was applied. Moderate agreement was observed between both laboratory tests for the three species of ruminants included in the study (Cohen's kappa, K; cattle K=0.527, goats K=0.541 and sheep K=0.539).

Efficacy of an experimental fasciolicide against immature and mature Fasciola hepatica in artificially infected calves.

The efficacy of 5-chloro-2-methylthio-6-(1-naphthyloxy)- 1H-benzimidazole, called "alpha", was tested against Fasciola hepatica. Fluke-free calves ( n=32) were divided into 8 groups and infected with 150 metacercariae per animal. All animals subsequently received a second infection with another 150 metacercariae, given at different time intervals aimed to produce flukes of differing ages within the experimental animals. When the flukes reached the required age in the animals, four groups were treated with a single oral dose of 12 mg/kg of compound alpha and the remaining ones served as non-treated controls. Two weeks after treatment the animals of all groups were sacrificed and the livers were removed to determine the numbers of parasites present in the treated and untreated controls. In the treated groups the fluke reduction for the 3 day/2 week group was 100%, for the 3 week/4 week group it was 96.4%, for the 6 week/8 week group it was 99.2% and for the 10 week/12 week group it was 100%.

Field trial on the efficacy of an experimental fasciolicide compared with some commercial compounds in naturally infected cattle.

The efficacy of 5-chloro-2-methylthio-6-(1-napthyloxy)-1H-benzimidazole was evaluated with three commercial fasciolicides in terms of the percentage of egg reduction in cattle. Fifty Swiss cows were selected for inclusion in the trial based on finding eggs of Fasciola hepatica in their feces. On day 0, they were blocked in five groups (G) of ten animals each according to fecal egg counts. G1 received compound alpha at 12 mg/kg p.o.; G2 triclabendazole at 12 mg/kg p.o.; G3.closantel at 3.5 mg/kg s.c.; G4 clorsulon at 2.0 mg/kg s.c. G5 animals served as untreated controls. Fecal analysis was performed on days 0, 7, 14, 21, 28, 60 and 90. Efficacy was measured on days 14 and 21. In addition, the extension and intensity effects were determined on day 60. The percentage efficacy for groups 1-4 was 98.1, 98.7, 98.2 and 97.9 on day 14 and 98.5, 97.9, 97.7 and 97.9 on day 21, respectively. No statistical differences were observed between treated groups.

Comparison of the trapping ability of Arthrobotrys robusta and Monacrosporium gephyropagum on infective larvae of Strongyloides papillosus.

In an in vitro trial, the trapping ability of Arthrobotrys robusta and Monacrosporium gephyropagum against Strongyloides papillosus infective larvae on corn meal agar plates, was evaluated after seven days of interaction at 25 degrees C. Monacrosporium gephyropagum trapped 93.1% of the larvae whereas A. robusta trapped only 32.3%. Variability in the capture of S. papillosus infective larvae by both trapping fungi is discussed.

Interaction between the nematode-destroying fungus Arthrobotrys robusta (Hyphomycetales) and Haemonchus contortus infective larvae in vitro.

In an in vitro trial, the effect of the nematode-destroying fungus Arthrobotrys robusta on Haemonchus contortus infective larvae was evaluated in petri dishes containing corn meal agar. After seven days incubation at 25 degrees C, 92.33% (+/- 4.1) predation was recorded.

Non-specific resistance of sheep against Haemonchus contortus with Freund's complete adjuvant.

Six groups of four ovines each were injected intraperitoneally with Freund's complete adjuvant (FCA), at various times before, during, or after infection with 10 000 larvae (L3) of Haemonchus contortus per sheep. Animals in groups A and B received FCA on days 14 and 7, respectively, before infection. Ovines in group C were treated with FCA on the same day of infection; while sheep in groups D, E, and F were injected with the adjuvant on days 7, 14, and 21, respectively, after infection. Significant reductions in adult worm numbers compared with non-treated controls, as determined 42 days after infection, were of 30%, 34%, 45%, 52%, 56%, and 55% in groups A, B, C, D, E, and F, respectively. A significant correlation, between the time of FCA administration (referred to the day of infection) and the number of worms recovered at necropsy, was also observed.