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

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 ß-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.

TITLE: Le test d'éclosion des œufs in vitro prédit-il l'échec du traitement au benzimidazole pour Haemonchus contortus ? ABSTRACT: Des efforts de recherche considérables ont été consacrés à l'optimisation des tests in vitro permettant de diagnostiquer la résistance des parasites aux stades préparasitaires. L'objectif de cette étude était de comparer le test de réduction du nombre d'œufs fécaux in vivo (TRNOF), le test d'éclosion des œufs in vitro (TEO) et la détermination moléculaire de la fréquence d'un allèle du codon 200 de l'isotype 1 de la ß-tubuline associé à résistance au benzimidazole au stade larvaire d'Haemonchus contortus, obtenus à partir de chèvres infectées. Les animaux ont été infectés avec des doses infectieuses composites représentant 10, 20, 30, 40, 60 et 80 % d'allèles résistants. Des échantillons de matières fécales ont été prélevés pour le TEO aux jours 28, 33 et 35 après l'infection. Les résultats de la TRNOF in vivo ont indiqué que le traitement à l'albendazole réduisait respectivement de 91,3, 78,0, 63,3, 48,4, 36,5 et 41,4 % les infections composées de doses composites de 10, 20, 30, 40, 60 et 80 % de larves de l'isolat résistant. La concentration de médicament à laquelle 50 % des œufs ont été empêchés de développer des larves qui éclosent (DE50) dans le TEO in vitro variait de 0,09 ± 0,01 à 15,63 ± 12,10 µg/mL de thiabendazole. Les résultats du TEO in vitro ont indiqué que le test pouvait correctement estimer la résistance in vivo. Le TEO a ainsi pu estimer avec précision l'efficacité in vivo du médicament et le pourcentage de l'allèle de résistance dans la population en utilisant des paramètres d'éclosion dans les doses de délimitation. Cette constatation a également été étayée par la comparaison des données du TRNOF aux pourcentages d'éclosion dans le TEO sur 30 élevages de chèvres en Slovaquie, avec des infections mixtes naturelles de parasites gastro-intestinaux.

Experimental evidence for the lack of sensitivity of in vivo faecal egg count reduction testing for the detection of early development of benzimidazole resistance.

The objective of this study was to compare the results of an in vitro egg hatch test (EHT), micro-agar larval development test (MALDT) and in vivo faecal egg count reduction test (FECRT) between worm strains obtained from goats and sheep identically infected with the gastrointestinal parasitic nematode Haemonchus contortus. Results from the in vivo and in vitro tests were compared with benzimidazole (BZ)-resistance-associated ß-tubulin allele frequencies determined using Pyrosequencing™. BZ resistance was not detected by the in vivo FECRT, where reductions of > 99% for both the resistant and the susceptible H. contortus strains were detected in both species. Discriminating doses in EHT and MALDT for the resistant strain indicated a low level (approx. 25%) of resistant individuals. Genotyping indicated that the susceptible strain had 10% BZ-resistant ß-tubulin codon 200 alleles and the resistant strain had 26% respective resistant alleles. The in vitro tests and allele-frequency distribution suggested low levels of resistance in both strains; however, the FECRT did not support the evidence of resistant individuals of either strain in either species, suggesting a potential underestimation of low-level resistance in sheep and goats when employing this test.

Anthelmintic Activity of Wormwood (Artemisia absinthium L.) and Mallow (Malva sylvestris L.) against Haemonchus contortus in Sheep.

The objective of this study is to evaluate the effect of dry wormwood and mallow on the gastrointestinal parasite of small ruminants Haemonchus contortus. Twenty-four experimentally infected lambs were randomly divided into four groups of six animals each: unsupplemented lambs, lambs supplemented with wormwood, lambs supplemented with mallow and animals supplemented with a mix of both plants. Faecal samples from the lambs were collected on day 23, 29, 36, 43, 50, 57, 64 and 75 post-infection for quantification of the number of eggs per gram (EPG). The mix of both plants contained phenolic acids (10.7 g/kg DM) and flavonoids (5.51 g/kg DM). The nematode eggs were collected and in vitro egg hatch test was performed. The aqueous extracts of both plants exhibited strong ovicidal effect on H. contortus, with ED50 and ED99 values of 1.40 and 3.76 mg/mL and 2.17 and 5.89 mg/mL, respectively, in the in vitro tests. Despite the great individual differences between the treated lambs in eggs reduction, the mean EPG of the untreated and treated groups did not differ (p > 0.05). Our results indicate that using wormwood and mallow as dietary supplements do not have a sufficient effect on lambs infected with H. contortus.