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.

Comparison of constitutive and thiabendazole-induced expression of five cytochrome P450 genes in fourth-stage larvae of Haemonchus contortus isolates with different drug susceptibility identifies one gene with high constitutive expression in a multi-resistant isolate.

Benzimidazoles (BZs) remain amongst the most widely used anthelmintic drug classes against gastro-intestinal nematode infections, although their efficacy is increasingly compromised by resistance. The primary underlying mechanisms for BZ resistance are single-nucleotide polymorphisms (SNPs) in the isotype 1 ß-tubulin gene causing the substitutions F167Y, E198A or F200Y. However, resistance is believed to be multi-genic and previous studies have shown that isolates carrying 90-100% F200Y can vary considerably in their resistance level in the egg hatch assay (EHA). Cytochrome P450 monooxygenases (CYPs) are associated with drug resistance in mammals and arthropods and have been considered as mediators of anthelmintic resistance. In Caenorhabditis elegans, several members of the CYP34/35 and CYP31 families are BZ and/or xenobiotic inducible and thiabendazole (TBZ) is metabolised by CYP35D1. Here, expression of all 5 CYPs closely related to the C. elegans CYP34/35 and CYP31 families was investigated in fourth-stage larvae of two susceptible and three BZ-resistant Haemonchus contortus isolates following in vitro exposure to TBZ for 3 and 6 h using real-time RT-PCR. The resistance status of all isolates was determined using EHAs and quantification of resistance-associated ß-tubulin SNPs using pyrosequencing. While none of the CYPs was TBZ inducible, constitutive expression of CYP34/35 family member HCOI100383400 was significantly 2.4-3.7-fold higher in the multi-drug resistant WR isolate with the strongest BZ resistance phenotype compared to susceptible and intermediate-level BZ-resistant isolates. Although this increase is only moderate, HCOI100383400 might still be involved in high-level BZ resistance by further decreasing susceptibility in isolates already carrying 100% of a ß-tubulin SNP causing BZ resistance. Lower transcript levels were observed for all CYPs in the intermediately resistant IRE isolate in comparison to the susceptible HcH isolate, which, except for CYP HCOI01579500, were statistically non-significant. This suggests that none of the investigated CYPs may contribute to protection against TBZ in this particular isolate.

Shared evolutionary origin of major histocompatibility complex polymorphism in sympatric lemurs.

Genes of the major histocompatibility complex (MHC) play a central role in adaptive immune responses of vertebrates. They exhibit remarkable polymorphism, often crossing species boundaries with similar alleles or allelic motifs shared across species. This pattern may reflect parallel parasite-mediated selective pressures, either favouring the long maintenance of ancestral MHC allelic lineages across successive speciation events by balancing selection ("trans-species polymorphism"), or alternatively favouring the independent emergence of functionally similar alleles post-speciation via convergent evolution. Here, we investigate the origins of MHC similarity across several species of dwarf and mouse lemurs (Cheirogaleidae). We examined MHC class II variation in two highly polymorphic loci (DRB, DQB) and evaluated the overlap of gut-parasite communities in four sympatric lemurs. We tested for parasite-MHC associations across species to determine whether similar parasite pressures may select for similar MHC alleles in different species. Next, we integrated our MHC data with those previously obtained from other Cheirogaleidae to investigate the relative contribution of convergent evolution and co-ancestry to shared MHC polymorphism by contrasting patterns of codon usage at functional vs. neutral sites. Our results indicate that parasites shared across species may select for functionally similar MHC alleles, implying that the dynamics of MHC-parasite co-evolution should be envisaged at the community level. We further show that balancing selection maintaining trans-species polymorphism, rather than convergent evolution, is the primary mechanism explaining shared MHC sequence motifs between species that diverged up to 30 million years ago.

Reduced efficacy of albendazole against Ascaris lumbricoides in Rwandan schoolchildren.

Control of human soil-transmitted helminths (STHs) relies on preventive chemotherapy of schoolchildren applying the benzimidazoles (BZ) albendazole or mebendazole. Anthelmintic resistance (AR) is a common problem in nematodes of veterinary importance but for human STHs, information on drug efficacy is limited and routine monitoring is rarely implemented. Herein, the efficacy of single dose albendazole (400 mg) was evaluated in 12 schools in the Huye district of Rwanda where Ascaris is the predominant STH. Ascaris eggs were detected by wet mount microscopy and the Mini-FLOTAC method to assess cure rate (CR) and faecal egg count reduction (FECR). Blood and faecal samples were analysed for co-infections with Plasmodium sp. and Giardia duodenalis, respectively. Ascaris positive samples collected before and after treatment were analysed for putatively BZ-resistance associated ß-tubulin gene single nucleotide polymorphisms. The overall CR was 69.9% by Mini-FLOTAC and 88.6% by wet mount microscopy. The FECR was 75.4% and the 95% calculated confidence intervals were 50.4-87.8% using sample variance, 55.4-88.8% by bootstrapping, and 75.0-75.7% applying a Markov Chain Monte Carlo Bayesian approach. FECR varied widely between 0 and 96.8% for individual schools. No putative BZ-resistance associated polymorphisms were found in the four Ascaris ß-tubulin isotype genes examined. Since FECRs <95% indicate reduced efficacy, these findings raise the suspicion of BZ resistance. In the absence of respective molecular evidence, heritable AR in the local Ascaris populations cannot be formally proven. However, since FECRs <95% indicate reduced efficacy, BZ resistance may be suspected which would be alarming and calls for further analyses and routine monitoring in preventive chemotherapy programs.

Economic assessment of Ostertagia ostertagi and Fasciola hepatica infections in dairy cattle herds in Germany using Paracalc®.

The aim of the current study was to estimate economic costs of Ostertagia ostertagi and Fasciola hepatica infections in dairy cattle herds in Germany using the online calculation programme Paracalc®. Following a questionnaire, survey data were available from 464 farms in 14 federal states. On those farms bulk tank milk (BTM) samples and additionally up to six serum samples collected from first season grazing calves were analysed, using a commercially available ELISA (Boehringer Ingelheim SVANOVA Biotech AB, Uppsala, Sweden), an in-house ELISA (F. hepatica) and an in-house serum pepsinogen test. In total, samples obtained from 344 farms were included in the analysis since those were the only farms with complete questionnaires. Median costs per farm and year were estimated for gastrointestinal (GI) nematode infections (€721.38) and F. hepatica infection (€565.61). Decreases in milk yield in multiparous cows were the major reason for annual production losses due to GI nematodes (€13.33 per cow) and F. hepatica infections (€7.95 per cow), which was followed by annual costs for anthelmintic treatment against GI nematode infections in adult cows (€10.00 per cow) and F. hepatica infection associated annual costs due to repeated artificial insemination (€10.13 per cow) and prolonged calving intervals (€9.40 per cow). The study demonstrated that if all required information is provided, the Paracalc® tool can assist to identify productions losses in dairy cattle herds due to helminth infections and to optimise farm economics in Germany.

A multi-country study to assess the effect of a treatment with moxidectin pour-on during the dry period on milk production in dairy cows.

A randomized clinical study was conducted in a total of 45 commercial dairy farms in France (14 farms), Germany (28 farms) and the UK (3 farms) to evaluate the effect of an anthelmintic treatment on milk yield in the subsequent lactation. A total of 1287 animals with suspected exposure to Ostertagia ostertagi were included in the study. Animals were treated during the dry period (7-77days before parturition) with moxidectin pour-on (Cydectin® 0.5% Pour-On, Zoetis; 638 animals) or left untreated (649 animals) according to a randomized block design. Animals were either heifers (n=296) or multiparous cows (n=991). The milk production was monitored at regular intervals after treatment up to 335days after lactation, and analysed using a general linear mixed model with the milk production as outcome variable and several random effects. The effect on milk yield after anthelmintic treatment over the whole subsequent lactation varied from no effect (-0.43kg/day; P=0.35) to an increase of milk yield with 2.35kg/day (P=0.01), depending on the study region and parity of the cows. Lactation curve analysis suggested that the treatment effect was mainly caused by a slower decay of the milk production in the treated animals compared to untreated animals. The present study highlights the beneficial effect of a topical treatment with moxidectin before parturition on milk yield in the subsequent lactation, as well as the importance of a careful evaluation of nematode exposure risk based on local grazing management practices to guide and target production-based anthelmintic treatment decisions.

Small rodents as paratenic or intermediate hosts of carnivore parasites in Berlin, Germany.

Rodents are important intermediate and paratenic hosts for carnivore parasites, including the important zoonotic agents Toxoplasma, Echinococcus and Toxocara. Monitoring of such parasites in rodents can be used to detect increasing risks for human and veterinary public health. Rodents were trapped at four sites in Berlin, two near the city center, two at the periphery. PCRs were conducted to detect Coccidia (target ITS-1) and specifically Toxoplasma gondii (repetitive element) in brain and ascarids (ITS-2) in muscle or brain tissue. During necropsies, metacestodes were collected and identified using ITS-2 and 12S rRNA PCRs. An ELISA to detect antibodies against Toxocara canis ES antigens was performed. Within the 257 examined rodents, the most frequently observed parasite was Frenkelia glareoli predominantly found in Myodes glareolus. T. gondii was only detected in 12 rodents and Microtus spp. (although strongly underrepresented) had a significantly increased chance of being positive. Neither Echinococcus nor typical Taenia parasites of dogs and cats were found but Mesocestoides litteratus and Taenia martis metacestodes were identified which can cause severe peritoneal or ocular cysticercosis in dogs, primates and humans. Using PCR, the ascarids T. canis (n = 8), Toxocara cati (4) and Parascaris sp. (1) were detected predominantly in muscles. Seroprevalence of T. canis was 14.2% and ELISA was thus more sensitive than PCR to detect infection with this parasite. Non-parametric multidimensional scaling and cluster analysis revealed that parasite communities could be grouped into an urban and a peri-urban cluster with high frequency of ascarid-positive rodents in urban and high frequency of F. glareoli in peri-urban sites. Prevalence rates of parasites in rodents with potential impact for human or veterinary public health are considerable and the monitoring of transmission cycles of carnivore parasites in intermediate rodent hosts is recommended to estimate the health risks arising from wild and domesticated carnivores.

Benzimidazole resistance survey for Haemonchus, Teladorsagia and Trichostrongylus in three European countries using pyrosequencing including the development of new assays for Trichostrongylus.

Resistance to benzimidazoles (BZs) in trichostrongyloid nematodes is a worldwide problem for livestock production, particularly regarding small ruminants. Sensitive and reliable methods are required to assess anthelmintic resistance status. Currently available methods for BZ resistance detection can be divided into three main groups, in vivo (e.g. faecal egg count reduction test), in vitro (e.g. egg hatch assay) and molecular tests. Three single nucleotide polymorphisms (SNPs) in the isotype-1 ß-tubulin gene of various nematode species correlate with BZ resistance. While PCR-based methods have been reported for the three most economically important nematodes of sheep, namely, Trichostrongylus, Haemonchus and Teladorsagia, pyrosequencing assays are so far only available for the latter two. Here, the design and evaluation of pyrosequencing assays for isotype-1 and isotype-2 ß-tubulin genes of Trichostrongylus colubriformis are described. PCR fragments carrying the susceptible and corresponding resistant genotype were combined in defined ratios to evaluate assay sensitivity and linearity. The correlation between the given and the measured allele frequencies of the respective SNPs (codons F167Y, E198A and F200Y) was very high. Pyrosequencing assays for Haemonchus, Teladorsagia and Trichostrongylus were subsequently used for a BZ resistance survey, carried out in the three European countries, namely Ireland, Italy and Switzerland. Larval cultures obtained from field survey samples in 2012 and 2013 were used for pyrosequencing. The test was applied when the target species represented at least 10% of the sample. Trichostrongylus and Teladorsagia were detected in all countries' samples whereas Haemonchus was not detected in samples from Ireland. SNPs in isotype-1 associated with resistance were detected for all three species, with frequencies at codon F200Y far exceeding those at codons F167Y and E198A. Elevated SNP frequencies in isotype-2 of Trichostrongylus were only rarely detected. Farms with BZ resistance-associated SNP frequencies above 10% were most often found in Switzerland followed by Ireland and Italy.

Field study analysis of the influences of deworming regimens and housing conditions on parasites and sperm output in 21 European boar studs.

The current study reports the parasitological results of a quality control audit in 21 European boar studs. Field investigations were performed over a 2-year period (2012-2013) during the winter and spring. From each stud, an average of 30 (range, 25-33) individual faecal samples and ejaculates from 615 randomly selected Pietrain boars were analysed. Statistical analysis revealed a significant effect (P < 0.0001) of deworming regimen (DR) × age class of boar (A) and housing condition (H) × A on the presence of parasites. A second model indicated a significant effect (P = 0.0262) of DR × H × A on the presence of parasites. Sperm output was significantly affected (P < 0.0001) by the DR. Based on this study, recommendations for deworming AI boars are proposed.

Elevated temperatures and long drought periods have a negative impact on survival and fitness of strongylid third stage larvae.

In grazing cattle, infections with gastrointestinal nematodes pose some of the most important health threats and subclinical infections result in considerable production losses. While there is little doubt that climate change will affect grazing ruminants directly, mean temperature increases of ∼ 3°C and longer drought stress periods in summer may also influence the free-living stages of parasitic nematodes. Hostile climatic conditions reduce the number of L3s on pasture and therefore the refugium, which is expected to result in a higher selection pressure, accelerating development of resistance against anthelmintic drugs. The aim of the current experiments was to investigate the effects of drought stress and different temperature/humidity ranges over time on the survival and fitness of Cooperia oncophora L3s and their distribution in grass and soil under controlled conditions using a climate chamber. Grass containers inoculated with L3s were analysed after 1-6weeks using descriptive statistics as well as linear models. A large proportion of L3s was recovered from soil where fitness was also better preserved than on grass. Numbers and fitness of recovered L3s declined with duration in the climate chamber under both temperature profiles. However, the results of the linear models confirmed that higher temperatures (20-33°C versus 17-22.6°C) significantly impaired survival, distribution and fitness of L3s. Application of drought stress, known as another important factor, had a surprisingly smaller impact than its duration or higher temperatures. The climate chamber enabled exclusion of confounding factors and therefore accurate interpretation of the investigated climatic aspects. The obtained results highlight the relative importance of those factors, and will help to design better models for the population dynamics of L3s on pasture in the future. Additionally, the outcomes of these investigations may offer explanations regarding interdependencies of development of anthelmintic resistance and the presence of hot/dry weather conditions.

Susceptibility to Ticks and Lyme Disease Spirochetes Is Not Affected in Mice Coinfected with Nematodes.

Small rodents serve as reservoir hosts for tick-borne pathogens, such as the spirochetes causing Lyme disease. Whether natural coinfections with other macroparasites alter the success of tick feeding, antitick immunity, and the host's reservoir competence for tick-borne pathogens remains to be determined. In a parasitological survey of wild mice in Berlin, Germany, approximately 40% of Ixodes ricinus-infested animals simultaneously harbored a nematode of the genus Heligmosomoides We therefore aimed to analyze the immunological impact of the nematode/tick coinfection as well as its effect on the tick-borne pathogen Borrelia afzelii Hosts experimentally coinfected with Heligmosomoides polygyrus and larval/nymphal I. ricinus ticks developed substantially stronger systemic type 2 T helper cell (Th2) responses, on the basis of the levels of GATA-3 and interleukin-13 expression, than mice infected with a single pathogen. During repeated larval infestations, however, anti-tick Th2 reactivity and an observed partial immunity to tick feeding were unaffected by concurrent nematode infections. Importantly, the strong systemic Th2 immune response in coinfected mice did not affect susceptibility to tick-borne B. afzelii An observed trend for decreased local and systemic Th1 reactivity against B. afzelii in coinfected mice did not result in a higher spirochete burden, nor did it facilitate bacterial dissemination or induce signs of immunopathology. Hence, this study indicates that strong systemic Th2 responses in nematode/tick-coinfected house mice do not affect the success of tick feeding and the control of the causative agent of Lyme disease.

Anthelmintic resistance to ivermectin and moxidectin in gastrointestinal nematodes of cattle in Europe.

Anthelmintic resistance has been increasingly reported in cattle worldwide over the last decade, although reports from Europe are more limited. The objective of the present study was to evaluate the efficacy of injectable formulations of ivermectin and moxidectin at 0.2 mg per kg bodyweight against naturally acquired gastro-intestinal nematodes in cattle. A total of 753 animals on 40 farms were enrolled in Germany (12 farms), the UK (10 farms), Italy (10 farms), and France (8 farms). Animals were selected based on pre-treatment faecal egg counts and were allocated to one of the two treatment groups. Each treatment group consisted of between 7 and 10 animals. A post-treatment faecal egg count was performed 14 days (±2 days) after treatment. The observed percentage reduction was calculated for each treatment group based on the arithmetic mean faecal egg count before and after treatment. The resistance status was evaluated based on the reduction in arithmetic mean faecal egg count and both the lower and upper 95% confidence limits. A decreased efficacy was observed in half or more of the farms in Germany, France and the UK. For moxidectin, resistance was confirmed on 3 farms in France, and on 1 farm in Germany and the UK. For ivermectin, resistance was confirmed on 3 farms in the UK, and on 1 farm in Germany and France. The remaining farms with decreased efficacy were classified as having an inconclusive resistance status based on the available data. After treatment Cooperia spp. larvae were most frequently identified, though Ostertagia ostertagi was also found, in particular within the UK and Germany. The present study reports lower than expected efficacy for ivermectin and moxidectin (based on the reduction in egg excretion after treatment) on European cattle farms, with confirmed anthelmintic resistance on 12.5% of the farms.

A quantitative evaluation of the extent of fluralaner uptake by ticks (Ixodes ricinus, Ixodes scapularis) in fluralaner (Bravecto) treated vs. untreated dogs using the parameters tick weight and coxal index.

BACKGROUND: Fluralaner is a new antiparasitic drug that was recently introduced as Bravecto chewable tablets for the treatment of tick and flea infestations in dogs. Most marketed tick products exert their effect via topical application and contact exposure to the parasite. In contrast, Bravecto delivers its acaricidal activity through systemic exposure. Tick exposure to fluralaner occurs after attachment to orally treated dogs, which induces a tick-killing effect within 12 h. The fast onset of killing lasts over the entire treatment interval (12 weeks) and suggests that only marginal uptake by ticks is required to induce efficacy. Three laboratory studies were conducted to quantify the extent of uptake by comparison of ticks' weight and coxal index obtained from Bravecto-treated and negative-control dogs. METHODS: Three studies were conducted using experimental tick infestation with either Ixodes ricinus or Ixodes scapularis after oral administration of fluralaner to dogs. All studies included a treated (Bravecto chewable tablets, MSD Animal Health) and a negative control group. Each study had a similar design for assessing vitality and weighing of ticks collected from dogs of both groups. Additionally, in one study the coxal index (I. ricinus) was calculated as a ratio of tick's ventral coxal gap and dorsal width of scutum. Tick weight data and coxal indices from Bravecto-treated and negative-control groups were compared via statistical analysis. RESULTS: Ticks collected from Bravecto-treated dogs weighed significantly less (p ≤ 0.0108) than ticks collected from negative-control dogs, and their coxal index was also significantly lower (p < 0.0001). The difference in tick weights was demonstrated irrespective of the tick species investigated (I. ricinus, I. scapularis). At some assessments the mean tick weights of Bravecto-treated dogs were significantly lower than those of unfed pre-infestation (baseline) ticks. The demonstrated tick-killing efficacy was in the range of 94.6 - 100%. CONCLUSIONS: Tick weights and coxal indices confirm that a minimal uptake results in a sufficient exposure of ticks to fluralaner (Bravecto) and consequently in a potent acaricidal effect.