Altered avr-14B gene transcription patterns in ivermectin-resistant isolates of the cattle parasites, Cooperia oncophora and Ostertagia ostertagi.

Ivermectin (IVM) resistance is an emerging problem for the control of gastrointestinal nematodes of cattle such as Cooperia oncophora and Ostertagia ostertagi. Although there is still a poor understanding of the molecular basis of macrocyclic lactone (ML)-resistance, it is clear that IVM exerts its activity by binding to glutamate-gated chloride (GluCl) channels within the parasite's neuromuscular system. One of the GluCl genes (avr-14) encodes, via alternative splicing, two subunits, AVR-14A and AVR-14B; the latter is suggested to be the main target for IVM. The genomic DNA (gDNA) sequence of avr-14 in C. oncophora contains 21 exons separated by 20 introns and spans approximately 10 kb of gDNA. Intron 13 contains a sequence with high homology to a mammalian mariner transposase. The L256F polymorphism in the avr-14 gene, which was shown to be associated with IVM resistance in a UK isolate of C. oncophora, was not found in the IVM-resistant C. oncophora and O. ostertagi isolates investigated in this study. However, genetic analyses on C. oncophora indicated a loss in allelic diversity of the avr-14 gene in the resistant isolates compared with the susceptible isolate. This suggests that the avr-14 gene, or another genetically linked locus, is under selection in these Belgian C. oncophora isolates. Comparison of the full-length avr-14B coding sequence in the susceptible and resistant C. oncophora isolates did not show any polymorphisms specifically linked to IVM resistance, although a decrease in the number of avr-14B isoforms was observed in the resistant isolates compared with the susceptible one. Measuring the transcription levels of avr-14B in adult male and female C. oncophora and O. ostertagi worms showed significantly lower levels in resistant worms compared with susceptible ones. Whether the down-regulation of this IVM target actually contributes to the resistance mechanism in these worms remains unclear.

Standardization of the larval migration inhibition test for the detection of resistance to ivermectin in gastro intestinal nematodes of ruminants.

Resistance to anthelmintics is an increasing problem in sheep, goat and cattle industries worldwide. For parasite management on farms reliable methods for the detection of resistance are required and it is important that tests give comparable, reproducible and reliable results in different laboratories. The ability of the larval migration inhibition test (LMIT), to detect ivermectin resistance in cattle and sheep nematodes has been evaluated through ring testing in up to six different laboratories in Europe, supported by an EU sixth Framework Project (PARASOL). Third stage larvae of Ostertagia ostertagi, Cooperia oncophora, and Haemonchus contortus with a known resistance status were obtained from faecal cultures of experimentally infected calves and sheep. Following a series of ring tests using identical protocols, reproducible results were obtained within and between participating laboratories. In all tests dose-response curves with R(2) values >0.90 were obtained by all laboratories. Resistance ratios of 8.3 and 8.4 were found when susceptible and IVM-resistant isolates of C. oncophora and H. contortus were compared and differences in the EC(50) values were highly significant (p<0.0001). Protocols for the LMIT and the preparation of ivermectin solutions are described in a supplementary file.

Monitoring macrocyclic lactone resistance in Cooperia oncophora on a Belgian cattle farm during four consecutive years.

In this study we have used the faecal egg count reduction test (FECRT) to monitor the evolution of ivermectin resistance on a Belgian cattle farm between 2006 and 2009. The presence of ivermectin resistant Cooperia oncophora worms on this farm was first detected in 2006. During the following years, the FECRs on day 21 post-treatment decreased from 73% (95% CI: 8-99) in 2006, over 40% (95% CI: 0-89) in 2007, to 0% (95% CI: 0-41) in 2008. The ivermectin resistant C. oncophora showed side-resistance against moxidectin, indicated by a FECR of 83%, suggesting that the use of any type of ML is discouraged once ivermectin resistance has been detected. Benzimidazoles on the other hand were still fully effective on this farm. The resistant C. oncophora larvae collected in 2007 (CoIVR07) and 2008 (CoIVR08) were also used to evaluate a modified version of the larval migration inhibition assay (LMIA). The results indicated that it is possible with the LMIA to differentiate susceptible from ivermectin resistant C. oncophora isolates. The EC50 values of CoIVR07 (542 nM) and CoIVR08 (698 nM) were, respectively, 4.5- and 5.8-fold higher than the value of a susceptible isolate (120 nM). Furthermore, the results of the LMIA reflected the outcome of the FECRT, with the C. oncophora isolate collected in 2008 being more resistant than the isolate collected in 2007. However, the test should be further optimized, e.g. more isolates with different susceptibility to ivermectin and mixtures of species, in order to use the test in field conditions.

The use of a simplified faecal egg count reduction test for assessing anthelmintic efficacy on Belgian and German cattle farms.

Anthelmintic resistant cattle nematodes have been reported in different regions around the world. However, in Western Europe the assessment of the problem relies largely on case reports and no prevalence data based on wide-scale surveys are available. Therefore, we performed a survey to (1) screen for reduced anthelmintic efficacy in Belgian and German cattle farms; (2) evaluate the usefulness of a simplified faecal egg count reduction test (FECRT), where efficacies are based on the mean FECs of 10 at random collected faecal samples pre- and post-treatment per farm and (3) identify possible risk factors for reduced anthelmintic efficacy. Of 88 farms included in this study, 84 farms used macrocyclic lactones (MLs). A FECR <95% was observed on 39% of these 84 farms. However, using a Markov chain Monte Carlo simulation analysis, to correct for the used McMaster FEC technique with a detection limit of 50 epg, reduced efficacy could only be confirmed in 25% of the farms (21/84). Only Cooperia spp. were found in significant numbers in the coprocultures post-treatment. Reduced efficacy was significantly associated with farm type and with a lower efficacy in beef herds compared to dairy herds. Four farms were revisited and a standardized FECRT was performed to confirm anthelmintic resistance (AR). Surprisingly, macrocyclic lactone resistance against Cooperia oncophora was only confirmed in one of four farms. In conclusion, our results show that a reduced efficacy observed in a FECRT are not only caused by AR but that the detection limit of the FEC technique used and the (in)correct administration of the anthelmintic drugs are confounding factors of major importance.