Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes

AbstractThis study assessed the anthelmintic activity of plant-derived compounds against gastrointestinal nematodes of goats using the egg hatch and larval motility assays. The compounds tested were saponins (digitonin and aescin) and their respective sapogenins (aglycones), hecogenin acetate and flavonoids (catechin, hesperidin, isocordoin and a mixture of isocordoin and cordoin). Additionally, cytotoxicity of active substances was analysed on Vero cell through 3-4,5-dimethylthiazol-2-yl,2,5diphenyltetrazolium bromide (MTT) and propidium iodide (PI) tests. Significant reduction on the egg hatching (P < 0.05) was seen only in the treatments with aescin (99%/EC50 = 0.67 mg mL−1) and digitonin (45%). The compounds that reduced the larval motility (P < 0.05) were digitonin (EC50 = 0.03 mg mL−1 and EC90 = 0.49 mg mL−1) and the hecogenin acetate (75%). The other sapogenins showed low anthelmintic activity. All the flavonoids showed low ovicidal (4–12%) and larvicidal (10–19%) effects. The aescin and digitonin showed low toxicity in PI test (viable cells >90%). Nevertheless, higher cytotoxicity was observed in the MTT assay, with IC50 of 0.20 mg mL−1 (aescin) and 0.0074 mg mL−1 (digitonin). Aescin and digitonin have a pronounced in vitro anthelmintic effect and the glycone portion of these saponins plays an important role in this activity.

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Assessment of the F200Y mutation frequency in the β tubulin gene of Haemonchus contortus following the exposure to a discriminating concentration of thiabendazole in the egg hatch test

Experimental Parasitology ◽

10.1016/j.exppara.2020.107957 ◽

2020 ◽

Vol 217 ◽

pp. 107957 ◽

Cited By ~ 1

Author(s):

Paulius Baltrušis ◽

Michaela Komáromyová ◽

Marián Várady ◽

Georg von Samson-Himmelstjerna ◽

Johan Höglund

Keyword(s):

Haemonchus Contortus ◽

Mutation Frequency ◽

Tubulin Gene ◽

Egg Hatch ◽

Egg Hatch Test ◽

Β Tubulin

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Evaluation of the Egg Hatch Assay and the Larval Migration Inhibition Assay to detect anthelmintic resistance in cattle parasitic nematodes on farms

Parasitology International ◽

10.1016/j.parint.2012.06.003 ◽

2012 ◽

Vol 61 (4) ◽

pp. 614-618 ◽

Cited By ~ 28

Author(s):

Janina Demeler ◽

Nina Kleinschmidt ◽

Ursula Küttler ◽

Regine Koopmann ◽

Georg von Samson-Himmelstjerna

Keyword(s):

Anthelmintic Resistance ◽

Parasitic Nematodes ◽

Inhibition Assay ◽

Migration Inhibition ◽

Egg Hatch ◽

Larval Migration ◽

Egg Hatch Assay

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Variability of the egg hatch assay to survey benzimidazole resistance in nematodes of small ruminants under field conditions

Veterinary Parasitology ◽

10.1016/j.vetpar.2014.03.002 ◽

2014 ◽

Vol 203 (1-2) ◽

pp. 102-113 ◽

Cited By ~ 9

Author(s):

C. Calvete ◽

L.M. Ferrer ◽

D. Lacasta ◽

R. Calavia ◽

J.J. Ramos ◽

...

Keyword(s):

Small Ruminants ◽

Field Conditions ◽

Egg Hatch ◽

Benzimidazole Resistance ◽

Egg Hatch Assay

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Comparative anthelmintic efficacy of Arundo donax, Areca catechu, and Ferula assa-foetida against Haemonchus contortus

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612021028 ◽

2021 ◽

Vol 30 (2) ◽

Author(s):

Syed Nadeem Badar ◽

Zafar Iqbal ◽

Muhammad Sohail Sajid ◽

Hafiz Muhammad Rizwan ◽

Muhammad Shareef ◽

...

Keyword(s):

Ethyl Acetate ◽

Haemonchus Contortus ◽

Anthelmintic Activity ◽

Parasitic Nematodes ◽

Egg Hatch ◽

Faecal Egg Count ◽

Areca Catechu ◽

Better Than

Abstract In the present study, anthelmintic activities of Arundo (A.) donax L., Areca (Ar.) catechu L., and Ferula (F.) assa-foetida L. were determined. Leaves of A. donax L., latex of F. assa-foetida L. and seeds of Ar. catechu L. in different solvent fractions were subjected to in vitro (egg hatch assay; EHA, and adult motility assay; AMA) and in vivo (faecal egg count reduction test; FECRT) tests of anthelmintic activity using Haemonchus contortus model. In the AMA, crude aqueous methanol extracts (CAME) and ethyl acetate fractions of F. assa-foetida at 10 hr post-treatment showed maximum mortality of H. contortus at 12.5-50 mg mL-1. In the EHA, CAME of F. assa-foetida was identified as a potent ovicide based on its low LC50 (16.9 µg mL-1), followed in order by Ar. catechu and A. donax. Results from the FECRT also showed the extract of F. assa-foetida L. to be more effective than those of Ar. catechu L. and A. donax L., against the gastrointestinal parasitic nematodes. Chloroform and ethyl acetate fractions showed better anthelmintic activities against the adult worms in vitro, while CAME of these plants were better than their crude powders in vivo. It is recommended to document and investigate indigenous knowledge of possible medicinal plants to plan scientific trials that may justify their endorsement.

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In Vitro Anthelminthic Efficacy of Aqueous Pomegranate (Punica granatum L.) Extracts against Gastrointestinal Nematodes of Sheep

Pathogens ◽

10.3390/pathogens9121063 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1063

Author(s):

Fabio Castagna ◽

Domenico Britti ◽

Manuela Oliverio ◽

Antonio Bosco ◽

Sonia Bonacci ◽

...

Keyword(s):

Gallic Acid ◽

Anthelmintic Resistance ◽

Punica Granatum ◽

In Vivo Studies ◽

Insoluble Residue ◽

Methanol Fraction ◽

Egg Hatch ◽

Anthelmintic Efficacy ◽

Egg Hatch Test

The worldwide increased difficulty to counteract gastrointestinal nematode (GIN) infection in sheep, due to progressing anthelmintic resistance, has led to the evaluation of other alternative helminth control options, mainly from plants. The anthelmintic efficacy of an aqueous Punica granatum macerate was evaluated in sheep naturally infected by GIN in southern Italy. The macerate was chemically characterized by chromatographic analysis coupled with high-resolution mass spectrometry (LC/HRMS) and an aliquot was concentrated to obtain a dry extract. A part was characterized, the remaining washed with methanol to obtain an insoluble residue and methanol phase. In the methanol fraction, the quantitatively predominant gallic acid was purified to obtain the pure molecule. The three fractions thus obtained were used for in vitro studies (i.e., egg hatch test) to verify anthelmintic efficacy. For this purpose, fecal samples were collected from sheep naturally infected by GINs. Fractions were diluted in H2O/DMSO 0.5% at 1.00, 0.5, 0.25, 0.125, 0.05, and 0.005 mg/mL concentrations. Thiabendazole (0.25 and 0.5 mg/mL) and deionized water were used as positive and negative controls, respectively. Egg hatch test results indicated that all fractions caused a significant (p < 0.05) egg hatch inhibition within 48 h of exposure highlighting a high (>82%) efficacy in vitro at all tested doses. Maximal egg hatching inhibition effect was exhibited by the methanol fraction (99.3% and 89.3% at 1 and 0.005 mg/mL concentrations), followed by the insoluble residue and gallic acid (94.7% and 85.3% and 94.0% and 82.7% at 1 and 0.005 mg/mL, respectively). The current study validated the anthelmintic potential of traditional P. granatum macerate against GIN infection in sheep, thus highlighting the role of gallic acid as principal component and justifying a need to undertake further in vivo studies on these ethno-veterinary remedies.

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Silencing of cyp-33C9 Gene Affects the Reproduction and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus

International Journal of Molecular Sciences ◽

10.3390/ijms20184520 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4520 ◽

Cited By ~ 1

Author(s):

Xiuwen Qiu ◽

Lili Yang ◽

Jianren Ye ◽

Wei Wang ◽

Tiantian Zhao ◽

...

Keyword(s):

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Economic Losses ◽

Parasitic Nematodes ◽

Pine Wood Nematode ◽

Egg Hatch ◽

Pine Wood ◽

Large Numbers ◽

Cytochrome P450 Genes ◽

Pine Trees

Cytochrome P450 genes are very important for plant-parasitic nematodes to reproduce and to metabolize xenobiotic compounds generated by their host plants. The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes very high annual economic losses by killing large numbers of pine trees across Asia and into Europe. In this study, we used RNA interference (RNAi) to analyze the function of the cyp-33C9 gene of PWN. Our results showed that expression of the cyp-33C9 gene was suppressed successfully after soaking nematodes for 24 h in cyp-33C9 double-stranded RNA (dsRNA). The silencing of the cyp-33C9 gene significantly decreased the feeding, reproduction, oviposition and egg hatch of B. xylophilus. Meanwhile, the migration speed of B. xylophilus in Pinus thunbergii was reduced in the early stages when the cyp-33C9 gene was silenced in the nematodes. Moreover, knockdown of the cyp-33C9 gene in B. xylophilus caused a decrease in pathogenicity to pine trees. These results suggest that the cyp-33C9 gene plays an important role in the reproduction and pathogenicity of B. xylophilus. This discovery identified several functions of the cyp-33C9 gene in B. xylophilus and provided useful information for understanding the molecular mechanism behind pine wilt disease caused by PWN.

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Responses of Heterodera glycines and Meloidogyne incognita to exogenously applied neuromodulators

Journal of Helminthology ◽

10.1017/s0022149x07850243 ◽

2007 ◽

Vol 81 (4) ◽

pp. 421-427 ◽

Cited By ~ 18

Author(s):

E.P. Masler

Keyword(s):

Meloidogyne Incognita ◽

Soybean Cyst Nematode ◽

Heterodera Glycines ◽

Body Movement ◽

Parasitic Nematodes ◽

Plant Parasitic Nematodes ◽

Root Knot Nematode ◽

Egg Hatch ◽

Movement Frequency ◽

Plant Parasitic

AbstractBiogenic amines regulate important behaviours in nematodes and are associated with pharyngeal activity in plant-parasitic nematodes. A robust behavioural assay based upon nematode body movements was developed to expand the study of these and other neuroregulators in plant-parasitic nematodes. Dopamine, octopamine and serotonin each had significant but differing effects on the behaviour of soybean cyst nematode Heterodera glycines and root-knot nematode Meloidogyne incognita juveniles. Body movement frequency was increased twofold in H.glycines by 5 mM dopamine (P = 0.0001), but decreased by 50 mM dopamine in H. glycines (88%) and M. incognita (53%) (P < 0.0001). Movement frequency in both species was increased by 50–70% (P < 0.0001) by 50 mM octopamine, and 5 mM octopamine increased M. incognita movement frequency more than twofold (P < 0.0001). Movement frequency in each species was reduced by more than 90% by 5 mM serotonin (P < 0.0001). While amplitude of body movement in H. glycines was unaffected by any amine, it was significantly reduced in M.incognita by all amines (P < 0.0006). Stylet pulsing frequencies in either species were unaffected by dopamine or octopamine, but 5 mM serotonin stimulated pulsing in H. glycines by nearly 13-fold (P < 0.0001) and in M. incognita by more than 14-fold (P < 0.0001). The invertebrate neuropeptide FLRFamide (N-Phe-Leu-Arg-Phe) increased M. incognita body movement frequency 45% (P = 0.02) at 1 mM but did not affect stylet activity. Finally, H. glycines egg hatch was completely suppressed by 50 mM serotonin, and partially suppressed by 50 mM dopamine (75%; P < 0.0001) and 50 mM octopamine (55%; P < 0.0001).

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