, 2009). Because of this sensitivity, Selleck Dasatinib it was noticed that is important to keep good control by removing all possible agents that can negatively affect the

regular life cycle of trichostrongylids. Regarding water solubility, although desirable, it might not be required in some cases. For instance, tests in dogs using carbon tetrachloride against hookworms showed that the efficacy of the tested compounds increased as solubility in water decreased (Bennet-Jenkins and Bryant, 1996). Another point of difficulty found in in vitro tests was the contamination. Álvarez-Sánchez et al. (2005) used the LFIA to detect anthelmintic resistance to ivermectin and levamisole and reported that such assay offers the advantage of simplicity and rapidity in

comparison to the LDA. LDA takes a long time to be performed and problems related to contaminations frequently occur. These problems reflected in fewer laboratories using the assay as the primary screen for activity in vitro ( Jackson and Hoste, 2010). In our work we dealt with both bacterial and fungal contamination by using sterile techniques as much as possible because development stages could not stand higher quantities of antibiotics than found in nutritive medium ( Hubert and Kerboeuf, 1992). In addition, instead of seven days of incubation at 23 °C ( Bizimenyera et al., 2006), we increase temperature to 27 °C and decreased incubation time to five days. This modification allowed larval development 3-Methyladenine manufacturer at higher rates and more PAK6 reproducible results. It also decreased fungal and bacterial contamination in the plates. Different LC50 values found between assays can be attributed to the sensitivity of each stage. Eggs are more resistant than L1 due to its hard and resistant shell. On the other hand, L3 were more resilient due to their double sheath. L1 was the most sensitive stage due to its pharynx that is more sensitive to the paralysis caused by drugs than the axial muscles (Molan et al., 2002).

These facts lead to higher or lesser volumes of active compound to achieve LC50 for each test. Várady et al. (2007) used the criterion of LC99 concentration to evaluate the sensitivity to thiabendazole under field screening. Those authors found in egg hatch and larval development assay the comparison of LC50 values was not significantly different, however the LC99 concentration is able to differentiate susceptible group, susceptible heterozygote group and resistant group in test for resistance diagnosis. Our LC99 results in EHA, LDA and LEA showed the same pattern of activity found in LC50 values. For trichostrongylids, the L3 exsheathment is a key process in the life cycle because it is the transition step between the free living and the parasitic stages (Hertzberg et al., 2002).