A mixed amplicon metabarcoding and sequencing approach for surveillance of drug resistance to levamisole and benzimidazole in Haemonchus spp.

Anthelmintic-resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We describe a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV)-resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ)-resistant SNPs within ß-tubulin isotype-1 and the internal transcribed spacer-2 (ITS-2) nemabiome. This constitutes the first known multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing (WGS) of LEV-resistant Haemonchus contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an effective anthelmintic class for small ruminants in New South Wales, Australia. The mixed amplicon metabarcoding approach described herein paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector.

Which larvae are they? Use of single larva for the molecular confirmation of Cooperia pectinata and Cooperia punctata in Australian cattle.

In Australia, Cooperia spp. are often overshadowed by parasites believed to be more pathogenic production-limiting nematodes. A rise in anthelmintic resistance and reports of reduced growth rates attributed to infection with Cooperia spp. in Europe increases the need to be able to monitor the presence of C. pectinata, C. punctata and C. oncophora in Australian cattle. Here, we present the first molecular confirmation of C. pectinata and C. punctata in Australian cattle using ITS2 rDNA and COXII mtDNA. Cultured larvae were morphologically differentiated to the genus level with the aid of iodine solution and their DNA was screened using a cattle nematode MT-PCR panel. By isolating individual iodine stained and morphologically identified nematode larvae, we demonstrated the presence of C. pectinata and C. punctata using a generic ITS2 rDNA qPCR assay following DNA amplicon sequencing. A novel suite of COXII mtDNA species/genus-specific PCR assays for Cooperia speciation from complex nematode samples enabled us to detect all three species (C. oncophora, C. pectinata, C. punctata) in Australia cattle samples. Our approach, utilising traditional techniques coupled with the manipulation of individual nematode larvae, provides a foundation for the inclusion of Cooperia spp. into existing high throughput molecular diagnostic panels for cattle nematode surveillance.

Efficacy of imidacloprid 10%/moxidectin 1% spot-on formulation (Advocate®) in the prevention and treatment of feline aelurostrongylosis.

BACKGROUND: In three randomized, controlled laboratory efficacy studies, the efficacy in the prevention of patent infections of a topical combination of imidacloprid 10%/moxidectin 1% (Advocate® spot-on formulation for cats, Bayer Animal Health GmbH) against larval stages and immature adults of Aelurostrongylus abstrusus, as well as the treatment efficacy of a single or three monthly treatments against adult A. abstrusus, were evaluated. METHODS: Cats were experimentally inoculated with 300-800 third-stage larvae (L3). Each group comprised 8 animals and the treatment dose was 10 mg/kg bodyweight (bw) imidacloprid and 1 mg/kg bw moxidectin in each study. Prevention of the establishment of patent infections was evaluated by two treatments at a monthly interval at three different time points before and after challenge infection. Curative efficacy was tested by one or three treatments after the onset of patency. Worm counts at necropsy were used for efficacy calculations. RESULTS: In Study 1, the control group had a geometric mean (GM) of 28.8 adult nematodes and the single treatment group had a GM of 3.4 (efficacy 88.3%). In Study 2, the control group had a GM of 14.3, the prevention group had a GM of 0 (efficacy 100%), while the treatment group had a GM of 0.1 (efficacy 99.4%). In Study 3, the GM worm burden in the control group was 32.6 compared to 0 in all three prevention groups (efficacy 100% for all of those groups). CONCLUSIONS: The monthly administration of Advocate® reliably eliminated early larval stages and thereby prevented lung damage from and patent infections with A. abstrusus in cats. Regarding treatment, a single application of Advocate® reduced the worm burden, but it did not sufficiently clear the infection. In contrast, three monthly treatments were safe and highly efficacious against A. abstrusus.

Minimal modulation of macrocyclic lactone susceptibility in Caenorhabditis elegans following inhibition of cytochrome P450 monooxygenase activity.

Anthelmintic and in particular macrocyclic lactone (ML) resistance is a widespread problem in trichostrongyloid parasitic nematodes, yet mechanisms of ML resistance are still poorly understood. In the absence of target-site changes in resistant parasite field populations, increased drug extrusion and xenobiotic metabolism have been implicated in modification of susceptibility to MLs. In addition to P-glycoproteins, cytochrome P450 monooxygenases (CYPs) were considered to be involved in ML resistance. CYPs are highly divergent in nematodes with about 80 genes in the model organism Caenorhabditis elegans. Using larval development assays in the C. elegans model, piperonyl butoxide (PBO) and a temperature-sensitive variant of the emb-8 cytochrome reductase were used for chemical and genetic ablation of CYP activity. Additionally, a loss-of-function variant of cyp-14A5 was characterized to determine whether increased expression of this CYP in an ivermectin (IVM)-tolerant C. elegans line might be related to the phenotype. In a preliminary experiment with PBO, susceptibility to 5 nM IVM was synergistically increased by PBO. However, effects of genetic ablation of CYP activity on the EC50 values were small (1.5-fold decrease) for IVM and not significant for moxidectin (MOX). However, due to the steep concentration-response-curves, there were again strong differences between the wild-type and the CYP deficient genotype at individual IVM but not MOX concentrations. Although these results suggest small but significant effects on the susceptibility level of C. elegans to IVM, the cyp14A5 gene proposed by a previous study as candidate was ruled out since it was neither IVM/MOX inducible nor did a strain with a loss-of-function allele show increased susceptibility to either drug. In conclusion, the effect of the CYP system on IVM susceptibility in C. elegans is at best low while effects on MOX susceptibility were not detected. The previously suggested candidate cyp14A5 could be excluded to be involved in ML metabolism.

Dermacentor reticulatus in Berlin/Brandenburg (Germany): Activity patterns and associated pathogens.

Dermacentor reticulatus is one of the most important European tick species. However, its spatial distribution, seasonality and regional vector role are not well known. This study aimed to gather information about abundance patterns of questing ticks and associated pathogens in unfed female adult D. reticulatus in the Berlin/Brandenburg area. Using the flagging method, questing ticks were collected at four sites in 2010-2012 and 2000 D. reticulatus were analysed regarding infection with Rickettsia, Babesia, Borrelia and Anaplasmataceae by conventional or real-time PCR. Dermacentor reticulatus showed a bimodal activity pattern: highest numbers of adult ticks were recorded between March and end of May (mean 50 ticks/h) and from mid-August until end of November (mean 102 ticks/h). During summer, almost complete inactivity was observed (mean 0.4 ticks/h). Sporadic samplings from December to February revealed tick activity also during winter (mean 47 ticks/h), which was characterised by large fluctuations. Using negative binomial regression analysis, significant influences of the variables sampling site, season and temperature on the abundance of questing D. reticulatus were determined. The parameters relative humidity and year were not of significant importance. PCR analyses showed an average prevalence of 64% for Rickettsia sp. Large differences in pathogen frequencies were observed between sampling sites (31.4-78.3%). Regression analysis demonstrated a significant influence of the sampling site but not of season and year. Examinations regarding other pathogen groups indicated prevalences of 0.25% (Borrelia sp.) and 0.05% (Anaplasmataceae) but absence of Babesia sp. Sequencing of positive samples revealed infections with Rickettsia raoultii, Borrelia miyamotoi, Borrelia afzelii and Anaplasma phagocytophilum. The study shows stable populations of D. reticulatus in Berlin/Brandenburg. People should be aware of ticks throughout the year since Ixodes ricinus is co-endemic and active in spring, summer and autumn while adult D. reticulatus are active throughout the year and even in winter during periods of frost as long as it is warming up during the day. Prevalence of R. raoultii in the present study is among the highest described for D. reticulatus. Borrelia miyamotoi was detected for the first time in D. reticulatus, illustrating the importance of screening studies to evaluate the pathogen structure in D. reticulatus populations.

Factors associated with diversity, quantity and zoonotic potential of ectoparasites on urban mice and voles.

Wild rodents are important hosts for tick larvae but co-infestations with other mites and insects are largely neglected. Small rodents were trapped at four study sites in Berlin, Germany, to quantify their ectoparasite diversity. Host-specific, spatial and temporal occurrence of ectoparasites was determined to assess their influence on direct and indirect zoonotic risk due to mice and voles in an urban agglomeration. Rodent-associated arthropods were diverse, including 63 species observed on six host species with an overall prevalence of 99%. The tick Ixodes ricinus was the most prevalent species, found on 56% of the rodents. The trapping location clearly affected the presence of different rodent species and, therefore, the occurrence of particular host-specific parasites. In Berlin, fewer temporary and periodic parasite species as well as non-parasitic species (fleas, chiggers and nidicolous Gamasina) were detected than reported from rural areas. In addition, abundance of parasites with low host-specificity (ticks, fleas and chiggers) apparently decreased with increasing landscape fragmentation associated with a gradient of urbanisation. In contrast, stationary ectoparasites, closely adapted to the rodent host, such as the fur mites Myobiidae and Listrophoridae, were most abundant at the two urban sites. A direct zoonotic risk of infection for people may only be posed by Nosopsyllus fasciatus fleas, which were prevalent even in the city centre. More importantly, peridomestic rodents clearly supported the life cycle of ticks in the city as hosts for their subadult stages. In addition to trapping location, season, host species, body condition and host sex, infestation with fleas, gamasid Laelapidae mites and prostigmatic Myobiidae mites were associated with significantly altered abundance of I. ricinus larvae on mice and voles. Whether this is caused by predation, grooming behaviour or interaction with the host immune system is unclear. The present study constitutes a basis to identify interactions and vector function of rodent-associated arthropods and their potential impact on zoonotic diseases.

Comment on "The optimal timing of post-treatment sampling for the assessment of anthelminthic drug efficacy against Ascaris infections in humans".

A recent publication by Levecke et al. (Int. J. Parasitol, 2018, 8, 67-69) provides important insights into the kinetics of worm expulsion from humans following treatment with albendazole. This is an important aspect of determining the optimal time-point for post treatment sampling to examine anthelmintic drug efficacy. The authors conclude that for the determination of drug efficacy against Ascaris, samples should be taken not before day 14 and recommend a period between days 14 and 21. Using this recommendation, they conclude that previous data (Krücken et al., 2017; Int. J. Parasitol, 7, 262-271) showing a reduction of egg shedding by 75.4% in schoolchildren in Rwanda and our conclusions from these data should be interpreted with caution. In reply to this, we would like to indicate that the very low efficacy of 0% in one school and 52-56% in three other schools, while the drug was fully efficient in other schools, cannot simply be explained by the time point of sampling. Moreover, there was no correlation between the sampling day and albendazole efficacy. We would also like to indicate that we very carefully interpreted our data and, for example, nowhere claimed that we found anthelmintic resistance. Rather, we stated that our data indicated that benzimidazole resistance may be suspected in the study population. We strongly agree that the data presented by Levecke et al. suggests that recommendations for efficacy testing of anthelmintic drugs should be revised.

[Utilization of selective anthelmintic therapy on horse farms in Germany]. / Anwendung der selektiven Entwurmung in deutschen Pferdebetrieben.

OBJECTIVE: Deworming management is important for a good herd health status in horses. The aim of this study was to present differences between farms using a regular deworming management and a selective anthelmintic therapy approach (SAT), respectively. MATERIAL AND METHODS: An online survey was conducted to identify deworming practices on horse farms. The questionnaire included questions on the number of animals kept on the farm, housing, hygiene, and deworming practices. RESULTS: In total, 283 questionnaires were analyzed. A total of 155 farms used a regular deworming management (RD) and 77 farms used a SAT approach. Farms using SAT were more often small, privately organized farms in which recently introduced horses were more thoroughly checked and horse droppings were removed more frequently from the pasture. Most farms using SAT dewormed 0 to once annually (55%), whereas this was only 3% in the case of farms using RD (p ≤ 0.001). Farms using SAT had a higher annual cost of 37.50 € per horse for sample examination and deworming. Many farms using an RD (69%) were willing to change their deworming management and 43% would be interested in introducing SAT. CONCLUSION AND CLINICAL RELEVANCE: Using selective deworming reduced the number of anthelmintic therapies required annually. However, the lower costs for anthelmintic drugs did not counterweigh the cost for sample examination. Overall, there was a low willingness to improve the pasture management. A combination of pasture and deworming management is of great importance for a successful selective deworming management.