Coles, G. C., Bauer, C., Borgsteede, F. H. M., Geerts, S., Klei, T. R., Taylor, M. A., & Waller, P. J. (1992). World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Veterinary parasitology, 44(1-2), 35-44.

Demeler, J., Kleinschmidt, N., Küttler, U., Koopmann, R., & von Samson-Himmelstjerna, G. (2012). Evaluation of the egg hatch assay and the larval migration inhibition assay to detect anthelmintic resistance in cattle parasitic nematodes on farms. Parasitology International, 61(4), 614-618

keeping them in aerobic conditions at 25 0c for 5-7 days

Ahmed Gareh , thanks for the advice.

Before, it worked...

It's weird because it worked the first time I did it again this test on February.

With the litterature that I found and with the others PH D students who did it in our team (like routine), everybody seems to practice this experiment in 48H at 27°C without any problems. (Moreover, I initiated them and confirmed the protocole like this also (2018-2019))

Find the answer for the repeatability and the reproducibility

It is interesting if we can find the roots of this problem.

To share to people if they meet this kind of observations.

Now, I can't take time to do again.

But if I need to do this test again, I will try your proposition. Thanks.

So when you do the EHA, do you always plan that it will take more than one week to have some results at each time ? It always works and reliable ?

Thanks a lot.

Sincerely,

Setha

It must be incubated in moisture, anaerobic media at 27c for 5=7 days

Estimado Setha,

El medio líquido no es el mejor para hacer evolucionar los huevos de Haemonchus, aunque la metodología del Test asía lo requiera.

Húmedo pero no líquido es lo ideal, porque así ocurre en la naturaleza.

A veces en las muestras hay buena cantidad de huevos infértiles (probablemente no fecundados).

Suerte

Claudio

Estimado Claudio Giudici ,

gracias para tu respuesta.

Es una idea que tiene sentido. En efecto, lo mas la technica esta proxima de la naturaleza, lo mejor y lo mas pertinentes estaron los resultados.

En el efecto, quizas podemos encontrar y observar huevos infértiles pero como comparar y sobre todo distinguir los huevos fertiles de no infértiles (la presencia de morula significa que habia fecundation por ejemplo, no ?)

sincera salutacion,

Setha

Setha Ketavong,

I just happened to see your question. In the lab, when we were doing the EHA in distilled water, we noticed that keeping the eggs in the cold (4°C refrigerator) for a few hours before the manipulation inhibited the hatching of H. contortus eggs for long time (or forever maybe). The longer the refrigeration time, the less the hatching.

Even if it's only a finding (we didn't push the tests very far), I have some figures on this. If you are interested I could email them to you. So, we don't put them in the fridge anymore before do coprocultures or EHA.

See you soon at the lab,

Léa

Setha Ketavong i see your question and i want to know which concept you want to use?! IC50?

Roxana Nezami I wanted to know if some plant extracts of interest could have some biological properties on the Haemonchus contortus eggs developpement (e.g. hatching). I don't really understand the aim of your question... Could you develop please ?

edit (15.02.2022) : Indeed, I prefer the concept of IC50 for EHIA (Egg Hatch Inhibition Assay) than EC50 for EHA (Egg Hatch Assay).

The positive and the negative controls are the opposite according to the concept.

Thanks to ask to be preciser.

I initiated new trainees to this technic this month. I wanted to show them why every step of our protocol was crucial for the final aim of Egg Hatch Assay. That's why after filtration of feces, and harvest of eggs in dense solution (of sugar or salt), we did two conditions : one without eggs washing and the second (positive control for hatching) with washing. We can see the effect of salt on the inhibition egg development. => here, example of test results on Haemonchus contortus eggs with or without salt.

Léa Bordes , thanks for your answer.

Indeed, good tips.

We never put feces in fridge before doing EHA. We prefered doing it directly after direct harvest with animals donors (because of litterature also).

Now, thanks to you, we have more reasons to do it the most "freshly" possible. ;-)

By the way, could you send me the figures please ?

Thanks again for your time and your advice. :-)

eggs and larvae pictures

• filtration and harvest without washing : salt seems to inhibit eggs hatching
• filtration and harvest with washing : eggs hatching

eggs and larvae pictures

• filtration and harvest without washing : salt seems to inhibit eggs hatching
• filtration and harvest with washing : eggs hatching

*The harvest will be easier if the animals donors give at least more than >1700 - 2000 eggs per gram (OPG). (for example, we have in 2021 june, 1800/2000/3000/4000 OPG per animal)