Yazmin Alcala-Canto

In Silico Fasciolicide Activity of Three Experimental Compounds in Sheep

The aim of the present study was to evaluate the fasciolicide activity of three experimental drugs, selected by an in silico system called TOMOCOMD–CARDD, in sheep. Drugs were identified by the computer system, and, after statistical selection, 24 Pelibuey sheep were infected on days 0 and 30, each with 200 metacercariae of Fasciola hepatica. When the infection reached 8 and 4 weeks of age, respectively, four groups of six animals each were formed. Group 1 received thiacetazone 150 mg/animal/p.o. Group 2 was treated with 3,5,5, trimethyloxazolidine 2,4‐dione at 450 mg/animal/p.o. G3 received guanabenz acetate at a dose of 1.5 mg/animal/p.o. G4 served as an untreated control. Monitoring of the animals was followed by individual coprological examinations and slaughter of the animals 15 days after treatment to collect and count flukes from the liver. Efficacy was measured as the reduction in the percentage of flukes of treated animals relative to untreated controls. Results indicated an efficacy of 80.0, 43.8, and 100% for 8‐week‐old flukes and 62.1, 57.9, and 100% for 4‐week‐old flukes in the three experimental groups, respectively. Even though guanabenz acetate showed a high efficacy, it was highly toxic since two animals died approximately 24 h after being treated. We conclude that further investigations should be conducted to perform computer‐aided prediction of drugs aimed to detect fasciolicide activity.

Downregulation of the goat β-defensin-2 gene by IL-4 in caprine intestinal epithelial cells infected with Eimeria spp.

Defensins are antimicrobial peptides that are produced by leukocytes and epithelial cells. Recent advances indicate that these peptides play an important role in innate immune responses. Nonetheless, the role of defensins in caprine eimeriosis remains unknown. Therefore, this study investigated the expression of a goat β-defensin, named GBD-2 in caprine intestinal epithelial cells (CIEC) stimulated with recombinant bovine interferon-gamma (IFN-γ) in the presence or absence of recombinant bovine interleukin-4 (IL-4) by a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. GBD-2 mRNA was clearly expressed in IFN-γ-stimulated CIEC. On the other hand, the direct addition of IL-4 showed no significant effect on GBD-2 expression in CIEC. However, when supernatants from peripheral blood mononuclear cells (PBMC) cultured with IL-4 were added to CIEC, the expression of GBD-2 decreased. To elucidate if IFN-γ functions as a signaling molecule that facilitates the generation of GBD-2 against Eimeria spp. in goats, anti- IL-4 was added to PBMC from Eimeria-infected goats and levels of IFN-γ in culture supernatants were determined by an enzyme-linked immunosorbent assay test. Results showed that IFN-γ secretion increased when anti-IL-4 was added to PBMC. It then appears safe to suggest that IL-4 may be a further factor in the pathogenesis of goat coccidiosis and its induction may be part of an evasion strategy of the parasite to avoid pro-inflammatory responses.

Anticoccidial efficacy of naringenin and a grapefruit peel extract in growing lambs naturally-infected with Eimeria spp

The current study aimed to determine the anti-Eimeria efficacy of an extract of grapefruit peels (GF) and commercial naringenin (NAR) in naturally-infected lambs, as well as the influence of these flavonoids on the oxidative status during ovine coccidiosis. Pharmacokinetic profiles were also determined. Extracts were administered per os to Eimeria naturally infected growing lambs during 90 consecutive days. The commercial anticoccidial drug toltrazuril (TTZ) was included in this trial as a standard. Twenty-four lambs were divided into four groups: NAR, lambs given a daily dose of 5mg of a commercial naringenin extract of 98% higher purity per kg body weight; GF, lambs that recived a daily dose of 5mg of ethanolic extract of grapefruit peels per kg body weight; TTZ, lambs treated with 20mg of toltrazuril/kg body weight on days 0 and 15 of the experiment; and CTRL, untreated lambs that received daily dose of 30ml of water. Daily doses of GF and NAR were dissolved in 30ml of water and orally given to animals; whereas toltrazuril was administered as a single dose of an undiluted suspension to lambs of the TTZ group. The CTRL group received 30ml of water; as well as the TTZ group for the period after the single dose administration. Fecal and serum samples were collected from all lambs. Anticoccidial efficacy was estimated by coprological techniques. Generation of nitric oxide levels and the antioxidant capacity of the experimental compounds were determined by the Griess and ABTS assays, respectively. The pharmacokinetic parameters of NAR and the GF extract were obtained. On day 30 post-ingestion, anticoccidial efficacy was 91.76% (NAR) and 89.65% (GF); whereas 99.63% of efficacy was achieved with TTZ 15days after treatment. NAR, GF and TTZ significantly reduced oxidative stress in infected animals. The mean daily weight gain for each group was 122g (NAR), 122g (GF), 143g (TTZ) and 98g (CTRL). Following the oral administration of NAR and GF, values in plasma approached maximum concentrations within 2.1 to 2.5h. In conclusion, the administration of NAR and the GF extract reduced Eimeria oocyst output, oxidative stress and promoted higher mean daily weight gains in infected lambs.

Fasciola hepatica proteolytic activity in liver revealed by in situ zymography

Fasciola hepatica secretes cysteine proteases that play a role in facilitating parasite migration. The aim of this study was to detect the inhibition of the proteolytic activity of F. hepatica cysteine proteases in the liver of C57BL/6 cathepsin B knockout mice (cat B−/−) and wild-type controls (cat B+/+) by intraperitoneal administration of N-[ N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine, (E-64) using the film in situ zymography (FIZ) technique and image analysis. The FIZ technique revealed that intraperitoneal administration of E-64 dramatically reduced (85%) F. hepatica proteolytic activity in the liver of experimentally infected mice with no discernable side effects. These results suggest the usefulness of the FIZ for determining in vivo activity of F. hepatica proteases, as well as their inhibition by intraperitoneal administration of E-64 in hepatic tissue of infected mice.

Pharmacodynamic evaluation of a reference and a generic toltrazuril preparation in broilers experimentally infected with Eimeria tenella or E. acervulina

Abstract 1. The aim of this study was to investigate the effects on pigmentation, faecal oocyst output, immune responsiveness and reactive oxygen species (ROS) generation following treatment with either the reference toltrazuril (Baycox) or a generic preparation (gen-TTZ), during an experimental Eimeria tenella (Et) or E. acervulina (Ea) infection of 210 Ross broiler chickens. 2. Results showed a significant difference on the anticoccidial efficacy 6 d after treating infected animals with Baycox (Et: 99.69% and Ea: 99.52%) or gen-TTZ (Et:85.71% and Ea 81.81%). 3. Gen-TTZ-treated animals were less strongly carotenoid-pigmented than Baycox-treated broilers. Mean plasma carotenoid concentrations were significantly higher in groups treated with Baycox than in broilers given gen-TTZ. 4. Treatment of animals with Baycox led to a significant decrease in ability of the peripheral blood mononuclear cells to produce ROS in contrast to gen-TTZ-treated groups. Baycox, but not generic toltrazuril, increased IL-10 and decreased tumour necrosis factor alpha (TNF-α) concentrations in chickens infected with E. tenella and E. acervulina. 5. It is suggested that differences in anticoccidial efficacy may be observed when using a generic toltrazuril product. Hence, in addition to plasma profiles of drugs, standardised clinical control tests may be necessary for generic formulations, particularly if other parameters are important to achieve a better control of coccidiosis.

Influence of Curcumin (Curcuma Longa) as a Natural Anticoccidial Alternative in Adult Rabbits: First Results

Coccidiosis is one of the most common ailments in rabbits farming and is usually treated with drugs that can produce resistance; therefore, a natural alternative was sought. The objective of the current study was to evaluate the efficacy of the aqueous extract of curcumin (Curcuma longa) on the excretion of oocysts of Eimeria spp. in New Zealand white rabbits. Twenty-four eight-month-old rabbits were divided into four groups of six animals to be C. longa extract administered at 0 (Control), 10, 25 or 40 mg/kg body weight (BW). Rabbit weights were recorded and faeces samples were collected on d 0, 7, 14, 21, 28, 35 and 42. The McMaster technique was used for quantifying Eimeria spp. oocysts. Results were analysed using multivariate analysis of variance for repeated observations. Statistically significant differences (P<0.05) from d 28 were observed among the Control, the group of 25 mg/kg BW and that of 40 mg/kg BW. At d 42, statistically difference (P<0.05) among the Control group and the other three groups was observed. It could be concluded that C. longa decreased Eimeria spp. oocysts excretion efficiently at a dose of 40 mg/kg BW with 80.1, 63.7 and 64.9% for d 28, 35 and 42, respectively, with reducing concentration of eggs per gram of faeces with about 20.1, 15.6 and 17.8 for d 14, 21 and 35, respectively. However, further studies are needed to assess and confirm the antiparasitic activity of C. longa.

Virtual Microscopy for Studying Veterinary Parasitology

Abstract The Parasitology Department of the Faculty of Veterinary Medicine, Universidad Nacional Autónoma de Mexico, digitized its glass slide collection to create a virtual microscope to help students learn key elements of parasite structures at times when the regular light microscopes are not available. The immediate aims of this project were to scan the glass slides of protozoans, helminths, and arthropods and to carry out a survey among students to evaluate the virtual slides in a side-by-side comparison with the regular microscope laboratory. Results indicated that the virtual microscope was enthusiastically received, and survey questions revealed improved time and increased student collaboration.

Ixodicide activity of Eysenhardtia polystachya against Rhipicephalus (Boophilus) microplus

The objective of this study was to evaluate the acaricidal effect of 2 compounds extracted from Eysenhardtia polystachya in vitro, namely coatlines and matlalines. Each extract was obtained by aqueous extraction in a nitrogen atmosphere. Engorged adult females of an amitraz-resistant strain of Rhipicephalus (Boophilus) microplus were used to produce larvae and to carry out the adult immersion test (AIT), while larvae were assayed using the larval packet test (LPT). After exposure to coatlines or matlalines, the mortality of larvae and engorged females, as well as the reproductive parameters of adult ticks were assessed. Coatlines were not lethal against larvae and did not decrease reproductive parameters. Conversely, matlalines showed a 90 to 100% efficacy against adults and larvae at all concentrations tested and a reduction of 76.41 to 80.64% oviposition and a 48.02 to 54.86% reduction in egg hatchability. Therefore, the acaricidal activity of matlalines was more efficient. Further studies are required to elucidate both the mode of action of matlalines as well the structure-activity relationships responsible for the observed differential efficacy between these 2 related isoflavans against the cattle tick.

Assessment of the effective dose of an experimental intramuscular formulation against immature and adult Fasciola hepatica in sheep

The effective dose of an injectable prodrug, named compound alpha prodrug, against immature and adult Fasciola hepatica in experimentally infected sheep was determined. In a first experiment, 30 sheep were infected with Fasciola hepatica on day 0 and 50. After microscopic detection of faecal eggs on day 80, groups (n = 6) 1 to 3 were treated with 6, 8 and 10 mg/kg of the experimental water-soluble prodrug compound alpha intramuscularly, respectively. Group 4 was treated with closantel and group 5 remained untreated. Copromicroscopical examinations were made on day 0, 80 and 108. On day 110, trematodes were collected from the bile ducts. Fasciolicide efficacy was assessed as a percentage of fluke-egg and adult-fluke reduction. Fluke length was also recorded. In a second experiment aimed to assess the fasciolicide activity of compound alpha prodrug against four-week-old flukes, 12 sheep were infected on day 0 and allocated into two groups (n = 6). On day 50 post infection, group A was treated with the experimental water-soluble prodrug compound alpha at 6 mg/kg/IM and B remained untreated. Fasciolicide activity was assessed on day 80 after collection, microscopic observation and measurement of flukes present in the parenchyma for immature stages and on day 108 for adults. Egg output decreased 91.2, 96.0, 98.8 and 94.9% for groups 1, 2, 3 and 4, respectively. Compound alpha prodrug cleared 97.6%, 98.51% and 100% of adult stages in a dose-dependent manner. Closantel killed 81.95% flukes. Regarding the second experiment, 81.2% efficacy was achieved. Immature flukes were significantly smaller in the treated group. It is concluded that the intramuscular application of compound alpha prodrug exerted fasciolicide efficacy against adults of Fasciola hepatica.

Design, synthesis, in vitro evaluation and preliminary SAR studies of N-(2-(heteroaryloxy)propyl)phenothiazines against Rhipicephalus microplus cattle tick.

A family of 15 N-substituted phenothiazines was designed, synthesized and their acaricidal activity against Rhipicephalus microplus was determined in vitro. The synthetic methodology is simple and can be employed in multigram scale. The rationale for the structure-based design of these compounds is the potential for azines and phenothiazine to engage in π-π interactions; these fragments, joined together by a short, flexible alkoxide linker, structurally resemble phenothiazine-based cholinesterase inhibitors, while their weak basicity implies a neutral active form, rather than a cationic one, thus facilitating penetration of the cuticle of ticks. One compound displayed excellent acaricidal activity (LD50=0.58 μg/mL). Preliminary SAR analysis suggests that the activity is influenced by the presence of a weakly basic nitrogen atom, as well as the substitution pattern within the heterocycles.