Biological control of gastrointestinal parasitic nematodes using Duddingtonia flagrans in sheep under natural conditions in Mexico.

This investigation was aimed to evaluate the use of an oral bio-preparation containing Duddingtonia flagrans chlamydospores for the control of sheep gastrointestinal parasitic nematodes under the Mexican cold high plateau conditions. Two groups of gastrointestinal parasitic nematode naturally infected sheep, were randomly selected and located into two free-gastrointestinal nematode larvae paddocks. Group 1 received once a week a supplement containing D. flagrans chlamydospores mixed with oats and molasses. Group 2 received a similar supplement without any fungal material. After 5 months grazing animals were discarded from the experiment and two groups of free-nematode "tracer" sheep were located into the same paddocks to collect larvae from the contaminated pastures. Animals were slaughtered and necropsied and the nematodes were obtained and counted. A screening of the number of gastrointestinal nematode larvae present on the grass was performed and compared between the two grazing areas. The results showed 56% reduction in the Ostertagia (Teladorsagia) circumcincta and 94% reduction in the Nematodirus sp. population of the "tracer" sheep who grazed on the D. flagrans-treated sheep area, compared to the nematode population in animals grazed on the non-treated area. The results of the number of larvae on the grazing pastures showed a 51.1% reduction for H. contortus, and 100% for Cooperia sp. in the area with fungi. In the case of Trichostrongylus sp. no reduction was observed, when compared to the control group.

Arrested development of Ostertagia ostertagi: effect of the exposure of infective larvae to natural spring conditions of the humid pampa (Argentina).

The aim of this study was to determine the effect of environmental conditions and the time of exposure to the conditions required for Ostertagia ostertagi to become inhibited in development at the early fourth larval stage in the host. Two comparable experiments were conducted from September to January, experiment I in 1997-1998 and experiment II in 1999-2000. Twenty-thousand third-stage larvae (L3), freshly obtained from coprocultures, were spread in different parasite-free grass plots at the beginning of September, October and November in each experiment and exposed to environmental conditions throughout spring and early summer. Duplicate plots for each exposure period were grazed for 3 days by two dewormed tracer calves after 1, 2, 3, 4 weeks of exposure during the corresponding month, and the remaining plots were grazed for 3 days at monthly intervals until the end of the experimental period. For each month in both experiments, control animals were inoculated orally with 20,000 L3 newly recovered from coprocultures (week 0 animals; infection controls). The control and tracer calves were sacrificed and their parasite burdens analysed. The time required to obtain greater than 50% inhibited larvae (IeL4) in the tracer animals during September and October was 3 weeks, whereas during November around 60% of the parasites were inhibited after one week of exposure. During the period tested, greater than 50% inhibition was found in concurrence with a photoperiod ranging between 13 and 14 h. The highest proportion of IeL4 (75% average) in the animals was found concomitant with a 14 h 43 min photoperiod. A high correlation between the percentage of inhibition and day length was established (0.870 p < 0.001 and 0.815 p < 0.001 for experiment I and II, respectively). In both years, the capacity for developmental arrest was lost by the end of December, when the photoperiod begins to decrease, suggesting either a disappearance of the induction stimulus, or that an excess of the stimulus could block the mechanism of inhibition. The induction time was extended 2 weeks in all months tested when the coprocultures were maintained in the dark (experiment II), suggesting that accumulation of the light stimulus contributes to shortening of the induction time. The data presented here would suggest that photoperiod is a key environmental factor for the induction of hypobiosis.

Hypobiosis induction alters the protein profile of Ostertagia ostertagi (Nematoda: Trichostrongylidae).

The appearance of variations in the protein profile of Ostertagia ostertagi (Stiles, 1892) infective larvae (L3), which were induced by hypobiosis triggering factors, was evaluated by means of SDS-PAGE and densitometric analysis. Area integration analyses of their protein profiles was carried out between 66 and 77 kDa. Important quantitative variations were identified in the protein levels of the induced larvae, where a 5.25 fold increase compared to the control was observed. Two 75.4 and 70 kDa protein bands were found which exceeded the control profile by 4.5 and 44 fold, respectively. This fact suggests that the changes brought about in the process of hypobiosis induction are restricted. This work demonstrates changes at a molecular level corresponding with biological changes induced by conditions causing O. ostertagi hypobiosis.

Study on the inductive factors of hypobiosis of Ostertagia ostertagi in cattle.

Two experiments were carried out to determine the causes producing the Ostertagia ostertagi hypobiosis phenomenon in cattle. In the first experiment, the effect of time on third-stage larvae in the environment was studied during a 2-year period. Three experimental paddocks contaminated with O. ostertagi eggs at different times of the year were used, and the levels of hypobiosis were recorded by using 'indicator' and 'tracer' calves. The results suggest that time as such is not a hypobiosis-inductive factor. The second experiment was conducted under laboratory conditions, where the effects of temperature and light on infective larvae were studied. Infective larvae were subjected to different conditions of temperature and light during 6 weeks, and then inoculated to parasite-naive calves, which were slaughtered after 4 weeks. Percentages of hypobiotic larvae in these calves varied from 3.5 to 94.8%, depending on the different storage conditions the larvae underwent before inoculation. Results suggest that increasing temperature and increasing time of light exposure simulating spring conditions would be the factors which act upon third-stage larvae inducing them to a later hypobiotic stage in the host.

Evaluation of a larval development assay for the detection of anthelmintic resistance in Ostertagia circumcincta.

Two experiments were carried out to evaluate a larval development assay for the detection of anthelmintic resistance in O. circumcincta. In Experiment I, the dose responses to levamisole (LEV), thiabendazole (TBZ) and ivermectin (IVM) of 8 isolates of O. circumcincta were measured 34 days after infection (DAI). Four of these isolates were shown to be resistant to 1 or more anthelmintics. With 2 exceptions, all isolates considered to be resistant had higher LD50 values than the susceptible isolates for that anthelmintic. One exception was isolate RM8, which was considered to be resistant to all 3 anthelmintics based on faecal egg count reduction tests in goats, but the LD50 value for LEV did not differ from that for the susceptible isolates. The other exception was an isolate considered to be susceptible to TBZ which had a relatively high LD50 value. In an unrelated trial that was prompted by this finding, this isolate was confirmed to be benzimidazole-resistant. Isolate RM8 and an isolate susceptible to all 3 anthelmintics (SK2) were used in the second experiment, which was conducted to monitor changes in the LD50 values of LEV, TBZ and IVM over time following a single infection of 35,000 infective larvae in young sheep. Faecal samples were collected weekly from 24 to 115 DAI. With all 3 anthelmintics, the LD50 values increased with time to a peak around 50-60 DAI, and then declined to levels similar to those observed soon after patency. This trend was consistent for both isolates. The highest mean LD50 values for isolates SK2 for IVM and TBZ and RM8 for IVM and RM8, respectively, were 1.7 and 1.8 times, and 2.2 and 2.9 times higher than the initial mean LD50 values. There was a clear distinction in LD50 values between isolates at each sampling day for both IVM and TBZ. However, as a consequence of the changes in LD50 values with time, the peak LD50 values of IVM for isolate SK2 were higher than the minimum LD50 values of isolate RM8. As there was no apparent difference in LEV efficacy between these 2 isolates, the data were pooled. The highest mean LD50 value was 2.3 times higher than the initial LD50 value.