Does evaluation of in vitro microfilarial motility reflect the resistance status of Dirofilaria immitis isolates to macrocyclic lactones?

BACKGROUND: Several reports have confirmed that macrocyclic lactone-resistant isolates of Dirofilaria immitis are circulating in the United States; however, the prevalence and potential impact of drug resistance is unknown. We wished to assess computer-aided measurements of motility as a method for rapidly assessing the resistance status of parasite isolates. METHODS: Blood containing microfilariae (MF) from two clinical cases with a high suspicion of resistance was fed to mosquitoes and the resultant L3 injected into dogs that were then treated with six doses of Heartgard® Plus (ivermectin + pyrantel; Merial Limited) at 30-day intervals. In both cases patent heartworm infections resulted despite the preventive treatment. Microfilariae isolated from these dogs and other isolates of known resistance status were exposed to varying concentrations of ivermectin in vitro and their motility assessed 24 h later using computer-processed high-definition video imaging. RESULTS: We produced two isolates, Yazoo-2013 and Metairie-2014, which established patent infections despite Heartgard® Plus treatments. Measurements of the motility of MF of these and other isolates (Missouri, MP3 and JYD-27) following exposure to varying concentrations of ivermectin did not distinguish between susceptible and resistant heartworm populations. There was some evidence that the method of MF isolation had an influence on the motility and drug susceptibility of the MF. CONCLUSIONS: We confirmed that drug-resistant heartworms are circulating in the southern United States, but that motility measurements in the presence of ivermectin are not a reliable method for their detection. This implies that the drug does not kill the microfilariae via paralysis.

Evaluation of the larval migration inhibition assay for detecting macrocyclic lactone resistance in Dirofilaria immitis.

Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L3). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC50 values ranged from 1.57 to 5.56µM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.

Validation of the FAMACHA© system in South American camelids.

Haemonchus contortus resistant to multiple anthelmintics threaten the viability of the small ruminant industry in areas where this parasite is prevalent. In response to this situation, the FAMACHA© system was developed and validated for use with small ruminants as a way to detect clinical anemia associated with haemonchosis. Given that H. contortus and multiple anthelmintic resistance is a similar problem in camelids, the FAMACHA© system might also provide the same benefits. To address this need, a validation study of the FAMACHA© system was conducted on 21 alpaca and llama farms over a 2-year period. H. contortus was the predominant nematode parasite on 17 of the 21 farms (10 alpaca and 7 llama farms) enrolled in the study, based on fecal culture results. The FAMACHA© card was used to score the color of the lower palpebral (lower eye lid) conjunctiva on a 1-5 scale. Packed cell volume (PCV) values were measured and compared to FAMACHA© scores using FAMACHA© score cutoffs of ≥3 or ≥4 and with anemia defined as a PCV ≤15%, ≤17%, or≤20%. PCV was significantly associated with FAMACHA© score, fecal egg count (FEC), and body condition score (BCS), regardless of the FAMACHA© cutoff score or the PCV% chosen to define clinical anemia (p<0.01 in all cases). The use of FAMACHA© scores ≥3 and PCV ≥ 15% indicating anemia provided the best sensitivity (96.4% vs 92.9% for FAMACHA© ≥4), whereas FAMACHA scores ≥ 4 and PCV ≤20% provided the best specificity (94.2% vs 69.1% for FAMACHA© ≥3). The data from this study support the FAMACHA© system as a useful tool for detecting clinical anemia in camelids suffering from haemonchosis. Parameters for making treatment decisions based on FAMACHA© score in camelids should mirror those established for small ruminants.

Development of an in vitro bioassay for measuring susceptibility to macrocyclic lactone anthelmintics in Dirofilaria immitis.

For more than 20 years, anthelmintics of the macrocyclic lactone (ML) drug class have been widely and effectively used as preventives against the canine heartworm, Dirofilaria immitis. However, in recent years an increased number of lack of efficacy (LOE) cases are being reported, in which dogs develop mature heartworm infections despite receiving monthly prophylactic doses of ML drugs. While this situation is raising concerns that heartworms may be developing resistance to MLs, compelling evidence for this is still lacking. Resolution of this dilemma requires validated biological or molecular diagnostic assays, but, unfortunately, no such tests currently exist. To address this need, we developed and optimized a larval migration inhibition assay (LMIA) for use with D. immitis third-stage larvae. The LMIA was used to measure the in vitro dose-response of two ML drugs (ivermectin and eprinomectin) on a known ML-susceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated; the mean IC50 and 95% confidence interval for IVM was 4.56 µM (1.26-16.4 µM), greater than that for EPR at 2.02 µM (1.68-2.42 µM), and this difference was significant (p = 0.0428). The R (2) value for EPR assays (0.90) was also greater than that for IVM treatment (0.71). The consistency and reproducibility of the dose-response data obtained with this assay suggests that it may be a useful technique for investigating the relative susceptibilities to ML drugs in other D. immitis populations.

Modification and further evaluation of a fluorescein-labeled peanut agglutinin test for identification of Haemonchus contortus eggs.

Gastrointestinal nematodes of the family Trichostrongylidae are the most important parasites of sheep, goats and other ruminants worldwide. Of this group, Haemonchus contortus is usually the most damaging species, particularly in warmer climates or during the summer. It is therefore useful to be able to rapidly differentiate infections with this nematode from other, less pathogenic, species. However, aside from Nematodirus spp., there are only subtle differences between the egg morphology within the trichostrongyles, making it very difficult to identify eggs to the species level. It has been shown previously that peanut agglutinin specifically binds to Haemonchus eggs and not those of other trichostrongyle species. By using this lectin conjugated to fluorescein isothiocyanate, binding to Haemonchus eggs can be visualized under ultraviolet illumination. We adapted this test using eggs purified by routine sugar centrifugation methods and evaluated 26 diagnostic samples from ruminants submitted to our laboratories in Oregon and Georgia. Very good correlations were seen between this test and larval culture (adjusted R(2)=0.72132; F(1,25)=65.7; p<0.001). There was little variability between two different diagnosticians reading the same sample, suggesting that the test is robust and not subject to reader bias. An additional benefit is that eggs can be examined following preservation; fixation of H. contortus eggs in formalin at 0.5-5% for to up to 4wk did not affect their staining.