Larval Feeding Inhibition Assay Research Articles

Review on anthelmintic resistances and its detection method on gastrointestinal nematodes of small ruminants

Antihelmintic resistance is a genetic change in the ability of an individual parasite to survive the recommended therapeutic dose which is caused by factors like under-dosing, frequent drug exposures at short intervals, continuous use of drugs with a similar mode of action and treatment when parasites have small refugee. The objective of this review article is to fill the gap of knowledge on methods of detection against antihelmintic resistance of gastrointestinal nematodes of small ruminants by using different approaches like: In vivo (fecal egg count reduction test, controlled efficacy test), in vitro (egg hatch assay, larval paralysis, larval motility assays, larval development assay, adult development assay, larval feeding inhibition assay, biochemical assays) and molecular assays (PCR assays). Their main comparison between in vivo and in vitro tests are, in vivo tests do involve slaughtering the animals for the test, sensibility, specify, reputability and easiness to develop. The molecular and genetic probes capable of determining individual susceptibility and complimentary use of some of them along with their use on a suspect would allow for an increase of sensitivity were finally recommended.

The response of soil nematode Caenorhabditis elegans on the sewage sludge-derived micropollutants

Sewage sludge application to soil is of great interest, due to required organic matter and the wide spectra of nutrients it provides. However, the presence of unpredictable content of emerging contaminants may turn this valuable raw material into a hazardous substance. In this study, three selected sewage sludges derived micropollutants from different origins; that is, one each under persistent organic pollutants (POPs), pharmaceuticals and personal care products (PPCPs) were considered. The effect of each micropollutant on the feeding activity of free-living soil nematode Caenorhabditis elegans was analysed. The analysis was performed in model soil solution using a larval feeding inhibition assay. The results showed no significant effects from selected POP—2,2′,4,4′,5-pentabromodiphenyl either and pharmaceutical—chlortetracycline on the feeding activity of tested nematodes. On the contrary, feeding activity was inhibited by PPCP—galaxolide (HHCB) with an effective concentration of 12.2 ± 2.2 mg.l-1. The calculated risk quotient for galaxolide (RQ = 0.14) demonstrated a medium ecological risk to the nematodes. Based on our findings, concentrations of micropollutants in sewage sludge treated soil pose negligible risk to feeding activity of soil nematode. However, the potential impact of musk compounds on free-living soil biota requires detailed evaluation in further research.

Impact of chemical structure of flavanol monomers and condensed tannins on in vitro anthelmintic activity against bovine nematodes.

Plants containing condensed tannins (CT) may have potential to control gastrointestinal nematodes (GIN) of cattle. The aim was to investigate the anthelmintic activities of four flavan-3-ols, two galloyl derivatives and 14 purified CT fractions, and to define which structural features of CT determine the anti-parasitic effects against the main cattle nematodes. We used in vitro tests targeting L1 larvae (feeding inhibition assay) and adults (motility assay) of Ostertagia ostertagi and Cooperia oncophora. In the larval feeding inhibition assay, O. ostertagi L1 were significantly more susceptible to all CT fractions than C. oncophora L1. The mean degree of polymerization of CT (i.e. average size) was the most important structural parameter: large CT reduced larval feeding more than small CT. The flavan-3-ols of prodelphinidin (PD)-type tannins had a stronger negative influence on parasite activity than the stereochemistry, i.e. cis- vs trans-configurations, or the presence of a gallate group. In contrast, for C. oncophora high reductions in the motility of larvae and adult worms were strongly related with a higher percentage of PDs within the CT fractions while there was no effect of size. Overall, the size and the percentage of PDs within CT seemed to be the most important parameters that influence anti-parasitic activity.

Sesquiterpene lactone containing extracts from two cultivars of forage chicory (Cichorium intybus) show distinctive chemical profiles and in vitro activity against Ostertagia ostertagi

The study investigated direct anthelmintic effects of sesquiterpene lactones (SL)-containing extracts from forage chicory against free-living and parasitic stages of Ostertagia ostertagi. Freeze-dried leaves from chicory cultivars ‘Spadona’ and ‘Puna II’ were extracted using methanol/water. Total SL were further fractionated by solid-phase extraction and resulting extracts were characterised by high-performance liquid chromatography (HPLC). O. ostertagi eggs from faeces of mono-infected calves were hatched and L1 were used in a larval feeding inhibition assay (LFIA), while cultured L3 were used in a larval exsheathment inhibition assay (LEIA). Adult worms were immediately recovered after slaughter and used for motility inhibition assays (AMIA). Electron microscopy (EM) was performed on adult O. ostertagi exposed to 1000 μg extract mL−1 of both chicory cultivars. In all assays, decreasing concentrations of SL-containing extracts in PBS (1% DMSO) were tested in replicates with 1% DMSO in PBS as negative controls. HPLC demonstrated similar concentrations of most SL in both extracts. However, Spadona-extract contained significantly higher concentrations of 11, 13-dihydro-8-deoxylactucin (P = 0.01), while Puna II-extract had increased levels of 11, 13-dihydrolactucin (P < 0.0001). In the LFIA, both extracts reduced larval feeding at increasing concentrations, but Spadona-extract showed higher potency confirmed by significantly lower EC50 (P < 0.0001). In the LEIA, neither of the two extracts interfered with the exsheathment of L3 (P > 0.05). In the AMIA, both SL-containing extracts induced a dose-dependent effect but Spadona-extract showed greater activity and exerted faster worm paralysis than Puna II-extract with significantly lower EC50 (P < 0.0001). No cuticular damage was observed by EM in worms exposed to any of the extracts. We have demonstrated that SL-containing extracts from forage chicory can inhibit feeding of free-living larvae and exert direct effects against parasitic stages of O. ostertagi. Our results may contribute to the identification of natural anti-parasitic compounds and to interpret the in vivo anthelmintic effects of forage chicory.

In vitro effects of extracts and purified tannins of sainfoin (Onobrychis viciifolia) against two cattle nematodes

Sainfoin (Onobrychis viciifolia) is a condensed tannin (CT)-containing legume and has anthelmintic potential against gastrointestinal nematodes of ruminants. This study investigated in vitro effects of acetone/water extracts and derived CT fractions from different types of sainfoin (i.e. accessions) against larvae of Cooperia oncophora and Ostertagia ostertagi by applying the larval feeding inhibition assay (LFIA). Seven sainfoin accessions were extracted and tested with L1 larvae at 10 and 40μg extract/ml. In addition, CT in extracts from 4 accessions were fractionated according to polymer size and tested by LFIA at two concentrations (2 and 10μg CT fraction/ml). All sainfoin extracts caused significant inhibition of L1-feeding of both C. oncophora and O. ostertagi with varying intensity compared to the control (phosphate buffered saline). For both nematode species the in vitro effect was positively correlated with CT content in the extracts, but not with any of the structural CT parameters. In contrast, the 16 CT fractions revealed significant correlations between in vitro effect and CT content, polymer size (mean degree of polymerisation, mDP) and monomeric composition (prodelphinidin percentage, % PD). These differences between crude extracts and purified fractions may stem from the fact that extracts contain complex CT mixtures, which may mask and thus suppress CT structural effects. This study provides the first indication that, apart from CT and % PD content, polymer size also contributes to anthelmintic activity of CTs. The results, therefore, suggest that the inter-accession variability in CT content and composition needs to be taken into account in future plant breeding programmes which seek to enhance the anthelmintic properties of sainfoin.

The present status of anthelmintic resistance in gastrointestinal nematode infections of sheep in the northwest of Spain by in vivo and in vitro techniques

The aim of this study was to update the anthelmintic resistance (AR) status in sheep flocks infected by gastrointestinal nematodes (GIN) by means of in vivo and in vitro methods in the northwest of Spain. With this objective, we studied the efficacy of benzimidazoles (BZs), imidazothiazoles (IMs) and macrocyclic lactones (MLs), between 2006 and 2011. The sampling area was the Autonomous Community of Castilla y León but the majority of the flocks were located in the province of León. When the mean of GIN eggs per gram (epg) in faeces in a flock was higher than 150, the in vivo Faecal Egg Count Reduction Test (FECRT) was carried out. According to this test, AR was present in 63.6% of flocks, independently of the anthelmintic used. Flocks were mainly resistant to levamisole (LEV) (59.0%), followed by ivermectin (IVM) (27.3%) and albendazole (13.6%). Multidrug-resistance was also observed in 27.2% of the flocks, one of them being resistant to all anthelmintic families, including long-acting moxidectin. Comparing the evolution of AR in the last decade, between 1999 and 2011, the level of resistance to BZs and MLs was fairly constant throughout the time by means of the FECRT. However, the resistance to LEV increased significantly in only one decade since during the period 1999–2003 the percentage was 38.5%. The AR status was also measured by in vitro techniques in those flocks with an egg output lower than 150 epg. The prevalence of AR to BZs reached the 35.3% by Egg Hatch Assay. However, the level of resistance reported for LEV and IVM was 61.5% and 23.5%, respectively, by using the Larval Feeding Inhibition Assay, percentages very similar to those reported with the FECRT.

In vitro ovicidal and larvicidal activity of methanolic leaf extract of Manihot esculenta (cassava) on susceptible and resistant strains of Trichostrongylus colubriformis

This study aimed to represent the first report of the ovicidal and larvicidal activity of the methanolic leaf extract of Manihot esculenta (cassava) against eggs and larvae of susceptible and resistant strains of Trichostrongylus colubriformis. As well as, to determine the total tannin compounds, antioxidant activity and toxicity of the extract. The egg hatch test was used to evaluate ovicidal activity against unembryonated eggs, whereas larval feeding inhibition assay and MTT-formazan assay were used to evaluate larvicidal activity against first (L1) and infective (L3) larvae, respectively. The results showed no significant differences were detected between the sensitivities of susceptible and resistant strains of T. colubriformis to the extract. Eggs, L1 and L3 were significantly affected (P<0.001) compared with negative control, and L1 were more sensitive than the eggs and L3. The total tannin compounds were investigated using tannin quantification assay and determined by 254.44TAE/mg. The antioxidant activity was evaluated using the DPPH radical scavenging assay and the median inhibition concentration (IC50) was determined by 2.638mg/ml. Acute oral toxicity at dose of 5000mg/kg, and sub-chronic oral toxicity at 500 and 1000mg/kg of the extract were observed in male and female Sprague-Dawley (SD) rats. The acute oral toxicity revealed that the median lethal dose (LD50) of methanolic extract of cassava leaves on SD rats was greater than 5000mg/kg, whereas the sub-chronic oral toxicity did not show observed adverse effects at 500 and 1000mg/kg per day for 28days. In conclusion, the methanolic extract of cassava leaves has direct ovicidal and larvicidal activity against T. colubriformis strains with a safety margin for animals, and it may be potentially utilized as a source of natural antioxidants.

Characterization of a multidrug resistant Teladorsagia circumcincta isolate from Spain

The aim of this work was to know the anthelmintic resistance (AR) status of a Spanish sheep flock infected by gastrointestinal nematodes (GIN) and the possible cross resistance among anthelmintics of the macrocyclic lactones (ML) family. The Faecal Egg Count Reduction Test (FECRT) was carried out to check the efficacy of albendazole (Zodalben®), levamisole (LEV) (Endex®) and an oral formulation of ivermectin (IVM) (Oramec®), at the recommended dose rates. Then, the study was extended to check the cross resistance between drugs of the ML family: injectable IVM (Ivomec®), oral moxidectin (Cydectin®), injectable moxidectin (Biodectin®) and doramectin (Dectomax®), at the recommended dose rates. The GIN species were identified after faecal cultures in all groups. The FECRT showed the resistance of a Teladorsagia circumcincta isolate against LEV (39-58%), IVM (88-92%) and doramectin (85%). This study is the first report to confirm the side resistance between these MLs, which belong to the avermectin chemical group, in a Spanish sheep flock. The in vitro efficacy of LEV and IVM was measured by the Larval Feeding Inhibition Assay (LFIA) using the IC(50) measurement (concentration needed to inhibit the ingestion of 50% L1). The values of the multidrug resistant isolate were 0.25 μg/ml for LEV and 3 ng/ml for IVM. Both results were higher than the values obtained with a susceptible isolate, which could be indicative of AR. However, further research examining the response of a greater range of susceptible and resistant nematodes isolates should be carried out to establish a discrimination threshold.

The in vitro diagnosis of anthelmintic resistance in cyathostomins

Cyathostomins are the primary parasitic pathogens of equids. For over 40 years, these nematodes have been controlled using broad spectrum anthelmintics. Three classes of anthelmintic are currently available for this use but, unfortunately, resistance to each of these has now been recorded in cyathostomin populations. As part of an optimal strategy to control cyathostomin infections in the field, it will be important to identify drug-resistant worms at as early a stage as possible. This objective needs to be supported by methodologies that will allow the accurate comparison of anthelmintic resistance in different nematode populations. At present, the faecal egg count reduction test is considered the most suitable method for initial screening for anthelmintic resistance in equine nematode populations. However, in its current state, this test lacks sensitivity. It is also costly and time-consuming to perform. Laboratory-based techniques, such as the egg hatch assay, larval development assay, larval migration inhibition assay and the larval feeding inhibition assay offer alternative options for assessing anthelmintic resistance in nematode populations. All of these tests have been investigated for their utility in measuring drug resistance in sheep nematode populations and some have proven useful. The egg hatch assay, larval development assay and larval migration inhibition assay have been investigated for use in measuring levels of drug resistance in equine nematode populations. However, at best, the results obtained thus far indicate that these tests require further refinement.

Anthelmintic activity of Cymbopogon martinii, Cymbopogon schoenanthus and Mentha piperita essential oils evaluated in four different in vitro tests

Anthelmintic resistance is a worldwide concern in small ruminant industry and new plant-derived compounds are being studied for their potential use against gastrointestinal nematodes. Mentha piperita, Cymbopogon martinii and Cymbopogon schoenanthus essential oils were evaluated against developmental stages of trichostrongylids from sheep naturally infected (95% Haemonchus contortus and 5% Trichostrogylus spp.) through the egg hatch assay (EHA), larval development assay (LDA), larval feeding inhibition assay (LFIA), and the larval exsheathment assay (LEA). The major constituent of the essential oils, quantified by gas chromatography for M. piperita oil was menthol (42.5%), while for C. martinii and C. schoenanthus the main component was geraniol (81.4% and 62.5%, respectively). In all in vitro tests C. schoenanthus essential oil had the best activity against ovine trichostrongylids followed by C. martini, while M. piperita presented the least activity. Cymbopogon schoenanthus essential oil had LC50 value of 0.045mg/ml in EHA, 0.063mg/ml in LDA, 0.009mg/ml in LFIA, and 24.66mg/ml in LEA. The anthelmintic activity of essential oils followed the same pattern in all in vitro tests, suggesting C. schoenanthus essential oil could be an interesting candidate for nematode control, although in vivo studies are necessary to validate the anthelmintic properties of this oil.

Condensed tannins act against cattle nematodes

The use of natural plant anthelmintics was suggested as a possible alternative control of gastrointestinal nematodes (GIN) in ruminants. Direct anthelmintic effects of tannin-containing plants have already been shown in sheep and goat GIN. These anthelmintic properties are mainly associated with condensed tannins. In the present study, we evaluated possible in vitro effects of three tannin-containing plants against bovine GIN. Effects of Onobrychis viciifolia, Lotus pedunculatus and Lotus corniculatus condensed tannin (CT) extracts on Cooperia oncophora and Ostertagia ostertagi were determined by a larval feeding inhibition assay (LFIA) and a larval exsheathment assay (LEA). In the LFIA, all three plant extracts significantly inhibited larval feeding behaviour of both C. oncophora and O. ostertagi first stage larvae in a dose-dependent manner. The L. pedunculatus extract, based on EC50 (effective concentration for 50% inhibition), was the most effective against both nematodes, followed by O. viciifolia and L. corniculatus. The effect of CT extracts upon larval feeding behaviour correlates with CT content and procyanidin/prodelphidin ratio. Larval exsheathment of C. oncophora and O. ostertagi L3 larvae (third stage larvae) was also affected by CT extracts from all three plants. In both in vitro assays, extracts with added polyvinylpolypyrrolidone, an inhibitor of tannins, generated almost the same values as the negative control; this confirms the role of CT in the anthelmintic effect of these plant extracts. Our results, therefore, indicated that tannin-containing plants could act against cattle nematodes.

Field Evaluation for Anthelmintic-Resistant Ovine Gastrointestinal Nematodes by In Vitro and In Vivo Assays

A coprological survey to analyze the presence of flock resistance to benzimidazoles (BZ) and macrocyclic lactones (ML) was performed in sheep under field conditions. Fecal samples were collected from 2,625 sheep in 72 commercial farms from Galicia (NW Spain). The in vitro (FECRT, fecal egg count reduction test) and in vivo (EHA, egg hatch assay, and LFIA, larval feeding inhibition assay) tests were used to assess the efficacy of these anthelmintics. Coprocultures were also developed to obtain knowledge on the main genera of trichostrongylid nematoda prior to, and after, the administration of the anthelmintics. By using the FECRT, BZ resistance was observed in 13 (18%) flocks, whereas ML resistance was only detected in 2 (3%) farms. The number of resistant flocks to BZ was 21 (29%) by using the EHA and 7 (10%) by means of the LFIA. None of the flocks used in this study showed simultaneous resistance to both employed anthelmintics. The results from the in vitro and in vivo tests revealed that 92% of the flocks FECRT resistant to BZ were also resistant with the EHA. The LFIA confirmed all the farms resistant to ML by using the in vivo test. After the administration of BZ, nematode larvae belonging to Teladorsagia circumcincta (32.2%), Trichostrongylus spp. (29%), Nematodirus spp. (6.5%), and Chabertia ovis (3.2%) were identified. In the flocks receiving ML, only T. circumcincta was identified (57%). We recommend the use of in vitro tests because they are more efficient. As the use of macrocyclic lactones is increasing in this region, further investigation is needed for detecting resistance to the anthelmintic family compounds by the LFIA.

Application of in vitro anthelmintic sensitivity assays to canine parasitology: Detecting resistance to pyrantel in Ancylostoma caninum

Resistance of the canine hookworm Ancylostoma caninum to anthelmintic therapy with pyrantel is an emerging problem in canine veterinary practice. Detecting anthelmintic resistance in parasites of pets is problematic because traditional resistance-monitoring techniques used with livestock parasites, such as the faecal egg count reduction test, are often impractical for use in small animals. We used two field-collected isolates of A. caninum in an abbreviated critical trial to test their pyrantel resistance status. The strains showed high-level and low-level resistance, with in vivo pyrantel efficacies of 28% and 71%, respectively. We noted a distinct worm density dependence effect on faecal egg count during the critical trial; egg counts in the dogs containing the low-level resistant isolate were 41% higher 6 days after drug treatment, despite the removal of 71% of the adult worms by the drug treatment. We then assessed four candidate in vitro assays for their ability to detect pyrantel resistance in A. caninum larvae, using these two isolates. The assays included a new format termed the larval arrested morphology assay (LAMA), based on observation of the effects of pyrantel on the body shape adopted by infective stage A. caninum larvae in vitro. Our data suggests that three of these assays, the LAMA, the larval motility assay (LMA), and larval feeding inhibition assay (LFIA) show promise with regards to detection of pyrantel resistance in A. caninum, but the complexity of the LFIA would likely limit its suitability for field studies. In vivo pyrantel efficacies of 28% and 71% in the two A. caninum isolates were associated with a 17-fold shift in the in vitro IC 50 values measured using the LAMA. Further testing with isolates of varying degrees of resistance is required to determine which of these assays is suitable as a rapid in vitro laboratory test for pyrantel resistance in A. caninum. The present study also indicates that potential exists for the novel LAMA or the LMA to be of use in detecting pyrantel resistance in the human hookworms, Necator americanus and Ancylostoma duodenale.

The larval feeding inhibition assay for the diagnosis of nematode anthelmintic resistance

A larval feeding assay for detection of nematode anthelmintic resistance to macrocyclic lactones and imidazothiazoles is described. The estimated concentration of anthelmintic required to inhibit larval feeding in 50% of L1’s (IC 50) that were resistant to either macrocyclic lactones or imidazothiazoles were significantly higher ( P ⩽ 0.05) than those of susceptible isolates. Some variations in IC 50 values were observed during the patent period of infection in all strains, although the pattern of the IC 50 followed the same course. IC 50 values varied in larvae developing from eggs shed throughout the patent period, with low values in the earliest larvae followed by higher values as the infection progressed, before decreasing at 70–90 days post-infection, although the low values of the first part of the patent period were not reached. However, the IC 50 differences between all resistant and susceptible strains were significant throughout the whole patent period for ivermectin and levamisole. These results suggest that this technique may provide an alternative in vitro to detect anthelmintic resistance.