In vitro detection of benzimidazole resistance in Haemonchus contortus: Egg hatch test versus larval development test

AbstractThe anthelmintic effects of flubendazole (FLU), its two main metabolites reduced flubendazole (FLU-R) and hydrolyzed flubendazole (FLU-H), and thiabendazole (TBZ) were compared using an in vitro larval development test in two isolates of Haemonchus contortus, a fully susceptible isolate (HCS) and a multi-resistant isolate (HCR). Results were quantified as 50 % lethal concentration (LC50), 99 % lethal concentration (LC99), efficacy factor (EF), and resistance factor (RF). For HCS, both LC50 and LC99 of FLU were lower than those of the reference TBZ. The anthelmintic activity of FLU-R in HCS and HCR was 13 and 6 times lower than the activity of FLU, respectively. The anthelmintic activity of FLU-H was negligible (approximately 363–853 times lower) compared to that of FLU. Although a marked resistance of the HCR isolate to TBZ was confirmed, only a low tolerance to FLU-R and slightly higher tolerance to FLU were found.

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In Vitro Anthelmintic Activity of Four Plant-Derived Compounds against Sheep Gastrointestinal Nematodes

Veterinary Sciences ◽

10.3390/vetsci5030078 ◽

2018 ◽

Vol 5 (3) ◽

pp. 78 ◽

Cited By ~ 9

Author(s):

Federica Giovanelli ◽

Matteo Mattellini ◽

Gianluca Fichi ◽

Guido Flamini ◽

Stefania Perrucci

Keyword(s):

Larval Development ◽

Larval Mortality ◽

Anthelmintic Activity ◽

Gastrointestinal Nematodes ◽

Experiment Data ◽

Egg Hatch ◽

Pure Compounds ◽

Petri Dishes ◽

Larval Development Test

By using the egg hatch test (EHT), the larval development test (LDT) and the larval mortality/paralysis test (LMT), the in vitro anthelmintic activity on sheep gastrointestinal strongyles (GIS) of four plant-derived pure compounds, mangiferin (at 0.25%, 0.125% and 0.0625%), rutin (at 1%, 0.75%, 0.5%), quercetin (at 1%), and β-sitosterol (at 1%, 0.75%, 0.5%), was investigated. For comparison, untreated and treated (0.1% thiabendazole, 0.1% TBZ) controls were used. Six repetitions were made throughout the experiment. Data were statistically elaborated using the χ2 test. The concentration able to inhibit the development of the 50% of L1s to L3s and causing the mortality of the 50% of L3s (EC50) was also calculated. L3s recovered from untreated Petri dishes were identified at the genus level. In EHT, all tested compounds at all concentrations significantly (p < 0.01) inhibited the hatch of the eggs when compared to the untreated controls, but none of them was as effective as 0.1% TBZ. In LDT, rutin (at 1%, 0.75% and 0.5%), mangiferin (at 0.25% and 0.125%), β-sitosterol (at 1%) and 0.1% TBZ completely prevented the larval development from L1 to L3 in respect to the untreated controls (p < 0.01). In LMT, all tested compounds significantly (p < 0.01) increased the death of L3s compared to the untreated controls, except for β-sitosterol at 0.5%. However, only rutin at all concentrations and 0.25% and 0.125% mangiferin were as effective as 0.1% TBZ. Haemonchus, Trichostrongylus, Chabertia and Teladorsagia/Ostertagia GIS genera, were identified.

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Does the in vitro egg hatch test predict the failure of benzimidazole treatment in Haemonchus contortus?

Parasite ◽

10.1051/parasite/2021059 ◽

2021 ◽

Vol 28 ◽

pp. 62

Author(s):

Michal Babják ◽

Alžbeta Königová ◽

Michaela Urda Dolinská ◽

Tomas Kupčinskas ◽

Jaroslav Vadlejch ◽

...

Keyword(s):

Haemonchus Contortus ◽

Gastrointestinal Parasites ◽

Resistant Isolate ◽

Egg Hatch ◽

Larval Stages ◽

Tubulin Isotype ◽

Faecal Egg Count ◽

Egg Hatch Test

Considerable research has been directed towards optimising in vitro tests that can diagnose resistance in pre-parasitic stages of parasites. The objective of this study was to compare the in vivo faecal egg count reduction test (FECRT), the in vitro egg hatch test (EHT), and the molecular determination of the frequency of a codon 200 allele of β-tubulin isotype 1 associated with benzimidazole resistance in larval stages of Haemonchus contortus obtained from infected goats. Animals were infected with composite infective doses representing 10, 20, 30, 40, 60, and 80% resistant alleles. Faecal samples for the EHT were collected on 28, 33, and 35 days post-infection. The results of the in vivo FECRT indicated that albendazole treatment reduced infections consisting of composite doses of 10, 20, 30, 40, 60, and 80% larvae of the resistant isolate by 91.3, 78.0, 63.3, 48.4, 36.5, and 41.4%, respectively. The drug concentration at which 50% of the eggs were prevented from developing hatching larvae (ED50) in the in vitro EHT varied from 0.09 ± 0.01 to 15.63 ± 12.10 μg/mL thiabendazole. The results of the in vitro EHT indicated that the test could estimate in vivo resistance well. The EHT could thus accurately estimate the in vivo efficacy of the drug and percentage of the resistance allele in the population using hatching parameters in delineation doses. This finding was also supported by comparing the FECRT data to the hatching percentages in the EHT on 30 goat farms in Slovakia with natural mixed infections of gastrointestinal parasites.

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Anthelmintic resistance of Haemonchus contortus from sheep flocks in Brazil: concordance of in vivo and in vitro (RESISTA-Test©) methods

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-296120201093 ◽

2021 ◽

Vol 30 (2) ◽

Author(s):

Yousmel Alemán Gainza ◽

Isabella Barbosa dos Santos ◽

Amanda Figueiredo ◽

Leonardo Aparecido Lima dos Santos ◽

Sérgio Novita Esteves ◽

...

Keyword(s):

Larval Development ◽

Haemonchus Contortus ◽

Anthelmintic Resistance ◽

Perfect Agreement ◽

Fecal Egg Count ◽

And Control ◽

Total Agreement ◽

Larval Development Test

Abstract This study evaluated the resistance status of Haemonchus contortus from sheep flocks in the state of São Paulo, Brazil, through comparison between the fecal egg count reduction test (FECRT) and the larval development test (LDT). For the FECRT, 35 sheep were selected in each of five flocks and divided into groups treated with: benzimidazole, levamisole, ivermectin, monepantel and control. Feces were collected for EPG and fecal cultures. The LDT was performed using thiabendazole (TBZ), levamisole (LEV), ivermectin aglycone (IVM-A) and Zolvix (ZLV). Resistance to all drugs was detected using FECRT in 100% of the flocks, except in relation to ZLV (40% resistant and 20% suspected of resistance). LDT indicated resistance to TBZ and IVM-A in all flocks, to LEV in 80% of flocks and to ZLV in 10%. Total agreement was obtained between the two tests for TBZ and IVM (k = 1.0), while for LEV (k = 0.8) and ZLV (k = 0.9), substantial and almost perfect agreement were obtained, respectively. The concordance between the tests was significant, thus showing that it is possible to use the outcome of the LDT to predict the FECRT, and hence validating the former as a fast diagnostic test for use by sheep farmers in Brazil.

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Detection of benzimidazole resistance in gastrointestinal nematode parasites of sheep in the Czech Republic

Veterinární Medicína ◽

10.17221/2935-vetmed ◽

2009 ◽

Vol 54 (No. 10) ◽

pp. 467-472 ◽

Cited By ~ 2

Author(s):

E. Vernerova ◽

R. Vondrova ◽

H. Kisova ◽

V. Svobodova ◽

A. Hera

Keyword(s):

Czech Republic ◽

Larval Development ◽

Infection Intensity ◽

Gastrointestinal Nematode ◽

Egg Hatch ◽

The Czech Republic ◽

Nematode Parasites ◽

Benzimidazole Resistance ◽

Faecal Egg Count ◽

Larval Development Test

During 2004–2007 the efficacy of benzimidazole type drugs was studied on 14 sheep farms in the Czech Republic. The study was based on the evaluation of faecal egg count reduction test (FECRT), egg hatch test (EHT), and larval development test (LDT). The prevalence of benzimidazole resistance on farms ranged from 25% to 98%; however, the infection intensity was typically low to moderate on most of the farms. Based of FECRT, resistance was identified on three farms. Resistance was suspected on one of the farms after repeated examination performed the following year.

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Piper aduncum against Haemonchus contortus isolates: cross resistance and the research of natural bioactive compounds

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612016073 ◽

2016 ◽

Vol 25 (4) ◽

pp. 383-393 ◽

Cited By ~ 7

Author(s):

Yousmel Alemán Gaínza ◽

Rafaela Regina Fantatto ◽

Francisco Celio Maia Chaves ◽

Humberto Ribeiro Bizzo ◽

Sérgio Novita Esteves ◽

...

Keyword(s):

Haemonchus Contortus ◽

Steam Distillation ◽

Resistant Isolate ◽

Cross Resistance ◽

Egg Hatch ◽

Susceptible Isolate ◽

Piper Aduncum ◽

Eggs And Larvae ◽

Larval Development Test

Abstract The anthelminthic activity of the essential oil (EO) of Piper aduncum L. was tested in vitro on eggs and larvae of resistant (Embrapa2010) and susceptible (McMaster) isolates of Haemonchus contortus. The EO was obtained by steam distillation and its components identified by chromatography. EO concentrations of 12.5 to 0.02 mg/mL were used in the egg hatch test (EHT) and concentrations of 3.12 to 0.01 mg/mL in the larval development test (LDT). Inhibition concentrations (IC) were determined by the SAS Probit procedure, and significant differences assessed by ANOVA followed by Tukey’s test. In the EHT, the IC50 for the susceptible isolate was 5.72 mg/mL. In the LDT, the IC50 and IC90 were, respectively, 0.10 mg/mL and 0.34 mg/mL for the susceptible isolate, and 0.22 mg/mL and 0.51 mg/mL for the resistant isolate. The EO (dillapiole 76.2%) was highly efficacious on phase L1. Due to the higher ICs obtained for the resistant isolate, it was raised the hypothesis that dillapiole may have a mechanism of action that resembles those of other anthelmintic compounds. We further review and discuss studies, especially those conducted in Brazil, that quantified the major constituents of P. aduncum-derived EO.

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Levels of benzimidazole resistance in Haemonchus contortus and Trichostrongylus colubriformis recorded from an egg hatch test procedure

Research in Veterinary Science ◽

10.1016/s0034-5288(18)32954-0 ◽

1978 ◽

Vol 25 (3) ◽

pp. 360-363 ◽

Cited By ~ 37

Author(s):

C.A. Hall ◽

N.J. Campbell ◽

N.J. Richardson

Keyword(s):

Haemonchus Contortus ◽

Test Procedure ◽

Trichostrongylus Colubriformis ◽

Egg Hatch ◽

Benzimidazole Resistance ◽

Egg Hatch Test

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In vitro Anthelmintic Activity of Two Aloe-derived Active Principles against Sheep Gastrointestinal Nematodes

Natural Product Communications ◽

10.1177/1934578x1701201221 ◽

2017 ◽

Vol 12 (12) ◽

pp. 1934578X1701201

Author(s):

Gianluca Fichi ◽

Matteo Mattellini ◽

Elisa Meloni ◽

Guido Flamini ◽

Stefania Perrucci

Keyword(s):

Larval Development ◽

Larval Mortality ◽

Plant Secondary Metabolites ◽

Anthelmintic Activity ◽

Gastrointestinal Nematodes ◽

Egg Hatch ◽

Reference Drug ◽

Faecal Samples ◽

Aloe Emodin

The in vitro anthelmintic activity on sheep gastrointestinal strongyle (GIS) eggs and larvae of 0.5% aloin and 0.1% aloe-emodin was investigated. From fresh faecal samples collected by ewes naturally infected by Haemonchus, Trichostrongylus and Teladorsagia nematodes, GIS eggs were isolated and cultivated in Petri dishes (100 eggs/dish). For the in vitro evaluation of the anthelmintic activity of tested compounds, the Egg hatch test (EHT), the Larval development test (LDT) and the Larval mortality/paralysis test (LMT) were used. In each assay, the activity of tested compounds was compared to untreated and treated (0.1% thiabendazole, TBZ) controls. Six repetitions were made through the experiment. Obtained data were statistically elaborated using the X2 test. In EHT, 0.5% aloin gave highly significantly different (P<0.01) results from the untreated controls. In LDT, both 0.1% aloe-emodin and 0.5% aloin almost completely prevented the larval development from L1 to L3, showing no significant differences (P<0.01) when compared to TBZ. In LMT, larval mortality observed in 0.5% aloin treated plates was significantly higher (P<0.01) than that observed in TBZ treated controls. These results show the in vitro anthelmintic properties on sheep GIS of the examined plant secondary metabolites. In LDT and/or LMT, the activity of 0.5% aloin and 0.1% aloe-emodin was comparable to or higher than that of the reference drug.

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In Vitro Anthelmintic Activity of Crude Extracts of Artemisia herba-alba and Punica granatum against Haemonchus contortus

Journal of Parasitology Research ◽

10.1155/2020/4950196 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Aliyi Hassen Ahmed ◽

Mebrat Ejo ◽

Teka Feyera ◽

Dereje Regassa ◽

Bahar Mummed ◽

...

Keyword(s):

Haemonchus Contortus ◽

Anthelmintic Activity ◽

Punica Granatum ◽

Limiting Factor ◽

Egg Hatching ◽

Traditional Use ◽

Egg Hatch ◽

Inhibition Effect ◽

Crude Extracts

Gastrointestinal nematodes (GINs) are the major limiting factor for the successfulness of livestock production throughout the world. Emergence of resistance strains as well as scarcity and high cost of the currently available drugs has led to the evaluation of other alternative helminth control options, mainly from plants. The current study is aimed at investigating the in vitro anthelmintic efficacy of crude methanolic extracts of two traditionally important medicinal plants, Artemisia herba-alba and Punica granatum, against Haemonchus contortus using adult motility assay (AMA) and egg hatch inhibition assay (EHIA). Four graded concentrations of the extracts were tested for both the AMA (10, 5, 2.5, and 1.25 mg/mg) and EHIA (0.1, 0.25, 0.5, and 1 mg/mL) in replicates. Albendazole and phosphate-buffered saline (AMA) or distilled water (EHIA) were used as the positive and negative controls, respectively. The crude extracts of A. herba-alba and P. granatum exhibited a potential anthelmintic activity at all dose levels in a concentration- and time-dependent fashion. The highest concentration (10 mg/mL) of all the extracts caused a significantly (p<0.05) superior nematocidal activity compared to the negative control. Moreover, significant and concentration-dependent egg hatching inhibition effect was observed from both plant extracts. Maximal (98.67%) egg hatching inhibition effect was exhibited by the flower extract of A. herba-alba at 1 mg/mL concentration. The relative egg hatch inhibition efficacy indicated that both plants caused a significantly (p<0.05) greater egg hatch inhibition within 48 hr of exposure. The current study validated the traditional use of both plants as a natural anthelmintic against H. contortus justifying a need to undertake detail pharmacological and toxicological investigation on both plants.

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