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.

Benzimidazole-resistant gastrointestinal nematodes in indigenous Chiapas and Pelibuey sheep breeds from Chiapas, Mexico.

Because of the natural adaptation of Mexican sheep, the aim of the present study was to identify the presence or absence of gastrointestinal parasitic nematodes (GIN) resistant to benzimidazole (BZ) in both Chiapas and Pelibuey sheep breeds on local farms. Both male and female GIN-infected grazing sheep of the two breeds were selected. Sheep faecal samples were collected to obtain infective larvae (L3). This evolving stage of the parasite was used for taxonomic identification of the genus, based on its morphological characteristics. BZ anthelmintic resistance was evaluated using a nematode-compound in vitro interaction bioassay and the allele-specific polymerase chain reaction technique to detect mutations of residues 198 and 200 on isotype 1 of the ß-tubulin gene. Three BZ-based compounds (febendazole (FBZ), tiabendazole (TBZ) and albendazole (ABZ)) at concentrations of 1, 0.5, 0.25, 0.125, 0.062 and 0.03 mg/ml were used to estimate the anthelmintic efficacy and lethal dose (LD50, LD90 and LD99) of the drugs. Two parasitic nematodes, Haemonchus and Teladorsagia, were identified in both isolates. Also, the proportions of anthelmintic resistance identified in GIN of the two sheep breeds were 68% in isolates from the Chiapas breed and 71.8% in the Pelibuey breed. The specific lethal activity obtained with FBZ was higher than 90%. However, TBZ and ABZ showed a lethal activity lower than 50%. High variability in the discriminating dose values was found among the BZ drugs. For example, FBZ LD ranged from 0.01 to 1.20 mg/ml; on the other hand, TBZ and ABZ required a dose ranging from 0.178 to 759 mg/ml. In addition, amino acid changes of Phe (TTC) to Tyr (TAC) at codon 200 of the ß-tubulin gene, showing resistance to BZ, and no changes at codon 198 Glu (GAA) to Ala (GCA) were observed for both isolates. These results confirmed the presence of a genetic mutation associated with BZ in both Chiapas and Pelibuey nematode isolates.

Evaluation of predation of the mite Lasioseius penicilliger (Aracnida: Mesostigmata) on Haemonchus contortus and bacteria-feeding nematodes.

Predation by the mite Lasioseius penicilliger was studied on three nematode species, i.e. infective larval stages (L3) of Haemonchus contortus and adults of Panagrellus redivivus and Rhabditis sp. Experiments were carried out in 5.5-cm diameter Petri dishes containing 2% water-agar over a period of 5 days. Batches of up to 1500 third-stage larvae (L3) of H. contortus and 1000 adult nematodes of P. redivivus and Rhabditis sp. were exposed to five mites in separate Petri dishes. Upon contact, each mite used its pedipalp and legs to identify and hold its prey and then used its chelicerae to feed upon the prey. Predation by L. penicilliger was chance dependent but mites became aggregated around any injured/damaged prey, thereby suggesting some form of chemoperception. The rate of predation on the three species of nematodes was high but L3 of H. contortus and adult Rhabditis sp. were preferred.

Biochemical characterization of two purified proteins of the IB-16 Bacillus thuringiensis strains and their toxicity against the sheep nematode Haemonchus contortus in vitro.

The aim of this study was to characterize the biochemical composition of two main IB-16 Bacillus thuringiensis proteins and to determine their toxicity on the blood-feeder nematode, Haemonchus contortus. The soluble toxin of IB-16 strain of B. thuringiensis produces five main bands of proteins, the chemical composition of which might play an important role on the lethal activity. Two bands of proteins around 25 and 70 kDa were chosen and purified by HPLC using a hydrogel column and sephadex-beads G-50. Biochemical analysis was carried out to determine enzyme and carbohydrate moieties on purified fractions of the 25 and 70 kDa proteins. In addition, in vitro assays were carried out using H. contortus histiotropic larvae (L(4)) and the purified fractions. Biochemical results showed only enzyme activity in the purified fraction of the 25 kDa protein using gelatine as the substrate. In contrast, carbohydrate moieties were only observed in the purified fraction of the 70 kDa protein. Moreover, IB-16 B. thuringiensis purified fractions of 70 and 25 kDa showed lethal activity of 67.1% and 17.3% of toxicity on H. contortus L(4), respectively.

Anthelmintic effects of Prosopis laevigata n-hexanic extract against Haemonchus contortus in artificially infected gerbils ( Meriones unguiculatus).

The anthelmintic effect of Prosopis laevigata (mezquite) n-hexanic extract was evaluated against Haemonchus contortus endoparasitic stages in artificially infected gerbils (Meriones unguiculatus). Prosopis laevigata leaves were collected from the Sierra de Huautla, Ecological Reserve of the Biosphere, in Morelos State, Mexico; dehydrated under shade and macerated with n-hexane for 3 days, followed by distillation for 8 h. This procedure was repeated three times and the final extract was kept at 4 degrees C. The in vivo effect of the plant extract was evaluated in gerbils artificially infected with H. contortus. Plant extract concentration was 40 mg/ml. Three groups of gerbils were as follows: group 1 (n = 7), P. laevigata extract at 100 microl intraperitoneally (IP); group 2 (n = 6), control--Tween 20 in water at a single dose of 100 microl IP; group 3 (n = 8) also served as a control, receiving water only, to determine the mortality due to causes other than the plant extract. An additional group of seven gerbils (group 4) was administered fenbendazole, as a positive control. Five days later the animals were euthanized and stomach and mucosa removed to quantify the nematodes. Data were analysed using the Student's t-test to compare the mean of nematodes obtained in groups 1, 2 and 3. The parasite population in the plant extract treated group 1 was reduced by 42.5% (P < 0.05) with respect to the control group 2; and when control group 3 was used for comparison the parasitic reduction was estimated as 53.11%. This study shows the in vivo anthelmintic effect of P. laevigata n-hexane extract for the first time, using gerbils as an in vivo model, with potential use in sheep.

In vitro nematicidal effects of medicinal plants from the Sierra de Huautla, Biosphere Reserve, Morelos, Mexico against Haemonchus contortus infective larvae.

Twenty extracts from plants from Sierra de Huautla Biosphere Reserve, Morelos, Mexico were evaluated against Haemonchus contortus infective larvae in an in vitro assay. The plant species evaluated were Bursera copallifera, B. grandifolia, Lippia graveolens, Passiflora mexicana, Prosopis laevigata, Randia echinocarpa and Urtica dioica. The plants were separated into their parts and macerated with different solvents (n-hexane, acetone, ethanol and methanol). An in vitro assay was used to evaluate the anthelmintic activity against unsheathed third stage H. contortus infective larvae. The experiment was carried out in 24-well cell culture plates at room temperature with three replicates per treatment and using a concentration of 20 mg ml- 1. Ten 5 microl aliquots were taken from the corresponding wells and deposited on a slide for microscopical observation at 24, 48, 72 and 96 h post-exposure. The evaluation criteria were based on the average numbers of live and/or dead larvae in the different treatments. Alive and dead larval numbers were statistically analysed through the ANOVA test (P>0.01). The Tukey test was used as a complementary tool to determine which treatment was different from the other treatments (P>0.05). The highest mortality was observed with P. laevigata hexanic extract from stem and leaves combined, which produced 51%, 81% and 86% larval mortality at 24, 48 and 72 h post-exposure, respectively. On the other hand, B. copallifera stem acetonic extract exhibited 18%, 59% and 66% nematicidal activity after 24, 48 and 72 h of exposure, respectively.

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%.

The predatory capability of three nematophagous fungi in the control of Haemonchus contortus infective larvae in ovine faeces.

The effect of oral administration of three different nematode-trapping fungi, in aqueous suspension containing either Dactylaria sp. or Arthrobotrys oligospora conidia or Duddingtonia flagrans chlamydospores, on the number of Haemonchus contortus infective larvae in sheep faeces, was evaluated. The three selected species of fungi produce three-dimensional adhesive nets in the presence of nematodes. Sixteen Creole sheep were divided into four groups of four animals each. Groups 1 and 2 were orally drenched with a suspension containing 2x10(7) conidia of either A. oligospora or Dactylaria sp. Group 3, received a similar treatment, with D. flagrans chlamydospores, instead of conidia, being administered, at the same dose. Group 4 acted as control, without any fungi. Faecal samples were collected directly from the rectum of each sheep and faecal cultures were prepared and incubated at 15 and 21 days. Larvae were recovered from faecal cultures and counted. The highest reduction of the nematode population occurred in the D. flagrans group, reaching reductions of 96.3% and 91.4% in individual samplings in plates incubated for 15 and 21 days, respectively. Arthrobotrys oligospora showed moderate reductions in the faecal larval population, ranging between 25-64% at 15 days incubation. In general, Dactylaria sp., was less efficient in its trapping ability. Despite the inconsistent results with Dactylaria sp., reduction percentages of 73.4% and 80.7% were recorded in individual samplings during the first and second days, in plates incubated for 15 days. Duddingtonia flagrans, was shown to be a potential biological control agent of H. contortus infective larvae.

[In vitro predatory activity of 8 fungal isolates against the nematode Panagrellus redivivus]. / Acción depredadora in vitro de ocho aislados de hongos contra el nematodo Panagrellus redivivus.

The aim of this study was to evaluate the predacious capacity in vitro of eight isolates of nematophagous fungi: four of Arthrobotrys sp., one of Arthrobotrys oligospora, one of Duddingtonia flagrans, one of Dactylaria sp. and one Monacrosporium eudermatum. Nine groups of Petri dishes with 13 repetitions each were set up. The fungi were seeded in fluor-corn-agar media, following this each Petri dish was added with 150 larvae of the free living nematode Panagrellus redivivus. Five days after larval addition these were collected by Baermannization and were quantified. A significant difference (p < 0.05) between all treated group was observed respect with the control. Isolates FTHO-8 D. flagrans, R6 M. eudermatum, DAC Dactylaria sp. as well as FTHO-4 and FTHO-6 Arthrobotrys sp., showed an excellent predatory activity (> 90%) and they could be considered as potential bio-control agents in future field trials.

[In vitro nematophagous capacity of Duddingtonia flagrans maintained under 2 conditions of preservation]. / Capacidad nematófaga in vitro de Duddingtonia flagrans Mantenido en dos Condiciones de Preservación.

One trial was carried out to evaluate the nematophagous capacity of two Duddingtonia flagrans cultures, one maintained during one year at laboratory temperature and the other one was a recent culture, twelve Petri dishes with flour-corn-agar media were seeded with the 1YC another 12 Petri dishes were inoculated with the RC. Both were added with 150 larvae/dish of the free living nematode Panagrellus redivivus and 12 fluor-corn-agar dishes only with the free living nematode were used as a control. The results showed that the nematophagous capacity of both cultures were similar but it was statistically different (p < 0.05) with respect to the control group. It was concluded that the nematophagous capacity of D. flagrans was not affected in spite of being kept one year at laboratory temperature.

Biological control of Haemonchus contortus infective larvae in ovine faeces by administering an oral suspension of Duddingtonia flagrans chlamydospores to sheep.

A single oral dose of an aqueous suspension containing 11,350,000 chlamydospores of a Mexican isolate of Duddingtonia flagrans (FTHO-8) given to sheep, resulted in a maximum reduction of 88% (range 86.7-90.4%) of the population of Haemonchus contortus infective larvae in the faeces. The effect of this treatment continued for 4-5 days after administration of the suspension. The possible use of this treatment as a method of control of ovine haemonchosis is discussed.

Cryopreservation of infective larvae of Haemonchus contortus.

Three freeze protectants were evaluated to preserve H. contortus infective larvae. Freezing solutions used: A) saline solution phosphate buffer pH 7.2 (PBS); B) 10% DMSO (dimethyl sulphoxide); C) 10% glycerol. Fifty thousand infective larvae were put into each of 10 vials per freeze protectant and then stored into liquid nitrogen. Results were based on the motility of the larvae under a light microscope at 30, 90, 180, and 360 days of freezing. Ten vials of each freeze protectant were removed from the liquid nitrogen at these times and immediately were put on water at 37 C during a minute. Motility percentages obtained were as follows: PBS: 36%, 20%, 7% and 39%; DMSO,: 87%, 69%, 46% and 85%; glycerol: 67%, 62%, 29% and 55%; at 30, 90, 180 and 360 days respectively. Inoculation of infectiva larvae from DMSO and glycerol to calves was successful after 28 days. DMSO was a better freeze preserver for H. contortus.

Development and recovery of Haemonchus contortus first larval stages on experimental plots in Mexico.

The objective of this study was to determine the influence of relative humidity, environmental temperature and precipitation on the development of Haemonchus contortus from egg to infective larvae on experimental plots in a subtropical subhumid climate of Mexico. The study was of 1 year duration, with the highest rates of recovery of first-stage (L1) and second-stage larvae (L2) obtained at the end of the rainy season, and of third-stage larvae (L3) in the post-rainy season during the autumn. The lowest rate of recovery of L1 was in winter, whereas that of L2 and L3 mainly occurred in the dry season. The shortest development period for L1 was 12 h, for L2 18 h, and for L3 36 h. The longest development period for L1 was 66 h, for L2 156 h and L3 216 h. Statistical analysis of the data obtained showed significant differences in the time of development from eggs to infective larvae with respect to the season of the year.