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.

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.

Pharmacokinetic behavior in sheep and cattle of 5-chloro-2-(methylthio)-6-(1-naphthyloxy)-1H-benzimidazole, a new fasciolicide agent.

The physicochemical properties, pK(a), Log P and solubility of compound alpha, (5-chloro-2-(methylthio)-6-(1-naphthyloxy)-1H-benzimidazole), a new fasciolicide agent, were characterized using conventional methods. Also, its pharmacokinetics was evaluated in sheep and cattle. In both species an oral dose of 12 mg/kg was administered. Blood samples were collected during 144 h and analyzed by using an HPLC assay. Results showed that compound alpha is a weak base with a pK(a) value of 2.87 and log P of 1.44. The solubility was very low in aqueous solvents. Pharmacokinetic studies showed that in both species compound alpha could not be detected at any sampling time. The mean half-life (t(1/2)) values of alpha sulphoxide in sheep and cattle were 19.86 and 29.87 h, while the half-life values of alpha sulphone were 19.43 and 46.32 h respectively. C(max) values of alpha sulphoxide did not differ between species while alpha sulphone values were higher in cattle. Plasma protein binding of alpha sulphoxide was between 82% and 86%. These results, combined with the previous efficacy studies, suggest that compound alpha could be a promising fasciolicide agent.

Cytokine gene expression and NF-kappaB activation following infection of intestinal epithelial cells with Eimeria bovis or Eimeria alabamensis in vitro.

In cattle, Eimeria produces clinical disease with different degrees of severity, depending on the dominant species. Eimeria bovis triggers severe intestinal damage, while E. alabamensis causes minimal damage. Cytokines and other factors derived from epithelial cells play important roles in inflammatory and immune responses in intestinal tissue. This study aimed to obtain a detailed view of IFN-gamma and IL-4 mRNA expression as well as of activation of NF-kappaB p50 and p65 subunits induced by E. bovis or E. alabamensis in intestinal epithelial cells by means of a RT-PCR assay and a NF-kappaB p50/p65 ELISA-based kit, respectively. Our results demonstrate that infection by both Eimeria species enhances IL-4 mRNA expression in intestinal cells. However, IL-4 was expressed more intensely in cells incubated with E. bovis whereas IFN-gamma levels were higher and detected at an earlier time in cells infected with E. alabamensis. NF-kappaB was activated in infected cells irrespective of species, yet the activity of the p50 subunit was significantly higher in cells incubated with E. bovis. Our results suggest that the intensity of host-cell responses triggered by these two Eimeria species could be considered as potential determinants of pathogenicity.

Downregulation of the goat beta-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 beta-defensin, named GBD-2 in caprine intestinal epithelial cells (CIEC) stimulated with recombinant bovine interferon-gamma (IFN-gamma) 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-gamma-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-gamma 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-gamma in culture supernatants were determined by an enzyme-linked immunosorbent assay test. Results showed that IFN-gamma 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.

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).

Serine protease activity demonstrated in the larval stage of the pentastomid Linguatula serrata.

Linguatula serrata is a cosmopolitan parasite whose intermediate hosts are cattle, goats, sheep, and other ruminants. The adult form is found in the nasal airways, frontal sinuses, and tympanic cavity of canines and felines, and it produces hemorrhages and breathing difficulties. To elucidate if L. serrata produces enzymes that are capable of degrading tissues from the intermediate host, proteolytic activities in larval products were studied. Using the zymography technique, one major protease was detected in parasite in vitro-released products with an approximate molecular weight of 75 kDa. This enzyme was inhibited with phenylmethylsulfonyl fluoride, suggesting that it is a serine protease, which was also shown to degrade type I collagen. The serine protease exhibited maximal activity at alkaline pH and temperatures varying from 37 to 45 degrees C. To gather evidence about the physiological roles of the enzyme, further biochemical and functional studies are suggested.

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.

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.

Efficacy of combined chemotherapy against gastrointestinal nematodes and Fasciola hepatica in cattle.

A controlled trial of the efficacy of several anthelmintic compounds as a combined therapy in the treatment of gastrointestinal nematodes (GIN) and Fasciola hepatica (F. hepatica) in naturally infected cattle was carried out. Twenty crossbred calves, 8-18 months old, were selected for inclusion in the trial based on finding eggs of F. hepatica and GIN in the faeces. They were blocked in four groups of five animals each according to GIN fecal egg counts on day 0. Treatments were sequentially allocated to animals in each block as follows: Group 1 served as non-treated control; Group 2 was treated with netobimin orally at 20 mg/kg; Group 3 received triclabendazole orally at 12 mg/kg and levamisole was applied intramuscularly at 5.5 mg/kg; Group 4 received clorsulon administered subcutaneously (s.c.) at 2 mg/kg and ivermectin s.c. at 200 microg/kg. Six to eight days after treatment the animals were euthanatized in order to collect and identify the parasites. Results showed a reduction of GIN by 87.3, 95.8 and 99.5% in Groups 2, 3 and 4, respectively. The percentage reduction of immature flukes was 0.0, 72.5, and 67.5% and for adult flukes 91.0, 97.5 and 100% for Groups 2, 3 and 4, respectively. Compounds indicated against nematodes showed high efficacy and products directed against F. hepatica acceptably removed adult flukes. However, efficacy against immature stages was generally not satisfactory.

[Fasciolinip-1: experimental fasciolicidal activity in sheep]. / Fasciolinip-1: eficacia fasciolicida experimental en ovinos.

The aim of the present study was to evaluate the flukicide efficacy of the 2-amino-5(6)-chloro-1-methoxycarbonylbenzimidazole (II), named Fasciolinip-1, against eight and twelve-week old flukes in sheep. In a first experiment 35 young sheep were infected each with 300 metacercariae of Fasciola hepatica. Eight weeks postinfection five groups of seven animals each were treated as follows: Groups 1, 2 and 3 were given 2.5, 5 and 10 mg/kg of Fasciolinip-1. Groups 4 and 5 remained as non-treated and vehicle controls, respectively. In a second study, 32 sheep were infected each with 150 metacercariae. Twelve weeks postinfection, the animals were divided into 4 groups of 8 sheep each. Groups 1, 2 and 3 were treated orally with 5, 10 and 15 mg/kg of Fasciolinip-1, respectively. Group 4 remained as non-treated control. Fifteen days after treatment, all sheep from both experiments were killed to collect the flukes present in liver. Results indicated efficacies of 19.1%, 49.6% and 65.2% for groups 1, 2 and 3 of the first study and 48.3%, 64.8 and 90.6% for sheep of the second experiment, respectively. It was concluded that Fasciolinip-1 removed 8-week old flukes in limited numbers and 12-week old flukes in 90.6% when used at 15 mg/kg body weight.

[Evaluation of chemotherapeutic agents against cichlidogyriasis in tilapia (Oreochromis hornorum) in Mexico]. / Evaluación de quimioterapéuticos contra la ciclidogiriasis en tilapia (Oreochromis hornorum) en México.

To evaluate the efficacy of seven compounds against the cichlidogyriasis of tilapia fish (Oreochromis hornorum), two experiments were carried out. In one, 160 naturally infected fish with a mean burden of 33.4 parasites/animal were used. In another, 1600 fish with a burden of 49.9 parasites/fish were used. Fish were randomly divided into eight equal groups and received three treatments as submersion baths: methylene blue, malachite green, potassium permanganate, sodium chloride, formaldehyde, copper sulfate and triclorfon, and a non-treated control. Five days after the last treatment, all fish were killed and dissected to quantify the remaining parasites. All data were submitted to an ANOVA analysis. All groups compared to the control showed statistical difference (P < 0.01), with better efficacy at higher doses. Sodium chloride, potassium permanganate and triclorfon are highly efficient in the control of cichlidogyriasis of tilapia fish, but care should be taken with the toxicity of the two first compounds.

[Evaluation of 4 vermifuges against Bothriocephalus acheilognathi in carp]. / Evaluación de cuatro vermicidas contra Bothriocephalus acheilognathi en carpas.

With the aim to evaluate the efficacy of four vermicides, praziquantel, mebendazole, nitroscanate and triclorfon in the control of Bothriocephalus acheilognathi in carps, one hundred and sixty, two year-old carps (Ctenopharyngodon idellus) with an average weight of 120 g and a mean parasitic burden of 115.5 parasites/fish were used. Five groups of 30 fish each were randomly formed. Each group was then subdivided in three groups, each of 10 individuals/pond. The three subgroups of group A received 30, 50 and 70 mg/Kg of praziquantel, respectively. Groups B and C received 2.2, 4.4, and 6.8 mg/Kg of mebendazole suspension and mebendazole solution respectively; groups D and E received 10, 30, 50 mg/Kg; and 2.0, 4.0, 6.0 mg/Kg of nitroscanate and trichlorfon respectively. Group F of 10 fish remained as non-treated control. Praziquantel showed 100% of efficacy for all doses, therefore it was excluded from the statistical analysis. The subgroups B, C, D and E compared with the non-treated control were statistically significant (P < 0.01); showing in all treatments an additive effect, the higher the dose the better efficacy, except trichlorfon which showed a different tendency, having no explanation on that matter.