In vitro larval assays for anthelmintic resistance in cattle nematodes.

Summary

Internal parasite infections constitute an important endemic disease of cattle in Australia. The mainstay of worm control in cattle production systems is chemical anthelmintics (drenches or pour ons), but increasingly, resistance of all the major cattle parasites to all the currently available chemical groups is being reported from around the world. Although there are recent reports of reduced cattle anthelmintic efficacy in Australia, the information is incomplete and highlights the need for a simple and inexpensive test with which to monitor drench efficacy. 
The Faecal Egg Count Reduction Test (FECRT) can estimate anthelmintic efficacy by comparing worm egg counts before and after treatment. It is crude, laborious and time-consuming and hence, not widely used. A number of in vitro techniques have been developed in an attempt to address these shortcomings, such as an egg-hatch assay, larval motility/migration assays, biochemical detection of resistance genes and the Larval Development Assay (LDA). The LDA is convenient in that it requires the collection of only one faecal sample and is effective in diagnosing resistance in the three major worm parasites of sheep (Barber's Pole Worm  Haemonchus contortus, Black Scour Worm Trichostrongylus colubriformis and Small Brown Stomach Worm Teladorsagia circumcincta) to the white and clear drenches. It yields inconclusive results when trying to measure resistance of T. circumcincta to the macrocyclic lactone (mectin, ML) drenches. 
The LDA has been used for nematode parasites of horses and pigs and, to a limited extent, some worms of cattle. The test needed to be further validated for the different chemical groups against parasites expressing the resistant phenotype. Through a process of isolation, infection and challenge with oral ivermectin, ML resistant isolates for three of the primary nematode parasites of cattle in sub-tropical and temperate Australia, Cooperia oncophora, Cooperia punctata/pectinata and Haemonchus placei were obtained. 
A similar process did not produce a reliably ML resistant isolate of Ostertagia ostertagi. All four species were subjected to comparative tests with susceptible isolates of the same species to determine if the LDA and larval migration assay (LMA) could be used for routine diagnostic purposes. For C. punctata/pectinata and H. placei, results for the LDA with ivermectin aglycone and the LMA with eprinomectin suggest these tests could potentially be used in a routine diagnostic to determine ML resistance status of field populations of these nematode species. For C. oncophora, only the LMA with eprinomectin showed a low level discrimination between susceptible and resistant isolates suggesting that the in vitro methods used could not be reliably used for diagnosis of ML resistance for this species. For O. ostertagi, in vitro analyses using the LDA and LMA did not discriminate between the available isolates, which were all susceptible, and no diagnostic method can be recommended at this stage.It was decided to terminate the project because no O.ostertagi isolate resistant to ML could be found.