Gastrointestinal parasite community structure in horses after the introduction of selective anthelmintic treatment strategies.

A relatively new method to study the species richness and diversity of nematode parasites in grazing animals is to perform deep sequencing on composite samples containing a mixture of parasites. In this work, we compared species composition of strongyles in two groups of horses as a function of egg count and age, based on a DNA barcoding approach. Faecal egg counts and larval cultures were obtained from nearly 300 horses, i.e., domestic horses (n = 167) and trotters (n = 130) sampled nationwide. The second internal transcribed spacer region (ITS2) of strongyle nematodes in the larval cultures was first amplified using barcoded universal primers and then sequenced on the PacBio platform. Subsequently, bioinformatic sequence analysis was performed using SCATA to assign operational taxonomic units (OTU). Finally, species occurrence and composition were assessed using R. ITS2 sequences were found in the majority (89%) of larval samples. Sequencing yielded an average of 140 (26 to 503) reads per sample. The OTUs were assigned to 28 different taxa, of which all but three could be identified as species. The average relative abundance of the seven most abundant species (all Cyathostominae) accounted for 87% of the combined data set. The three species with the highest prevalence in both horse groups were Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus calicatus, and they were frequently found in different combinations with other species regardless of horse group. Interestingly, this result is largely consistent with a previous Swedish study based on morphological analysis of adult worms. In addition, two migratory strongylids (Strongylus vulgaris and S. edentatus) occurred in few domestic horses and trotters. Except for C. minutus and C. nassatus, which decreased with age, and C. catinatum and S. vulgaris, which increased, no specific trends were observed with respect to horse age. Taken together, these results are broadly consistent with data obtained before the introduction of selective targeted treatment in Sweden in 2007. All in all, our results suggest that this treatment strategy has not led to a significant change in strongyle nematode community structure in Swedish horses. The study also confirms that nemabiome analysis in combination with diversity index analysis is an objective method to study strongyle communities in horses.

Benzimidazole-resistance associated mutation in Haemonchus contortus in Norwegian sheep, as detected by droplet digital PCR.

The aim of this study was to investigate the occurrence of benzimidazole-resistant Haemonchus contortus in Norwegian sheep flocks. Screening was based on detection of one of the resistance-conferring mutations in the ß tubulin isotype 1 gene (F200Y, TAC) in larvae (L3) cultivated from H. contortus eggs from naturally infected sheep. Faecal samples were collected in 2021/2022 from flocks in the northern (n = 34), central (n = 7), eastern (n = 40), southern (n = 1), and western (n = 87) areas of Norway. In total, samples were taken from 169 flocks (spring-ewes samples: 167, autumn-lambs samples: 134). Individual faecal samples were collected from 10 randomly selected ewes (spring) and 10 randomly selected lambs (autumn) in each flock. Faecal samples collected from each flock on each occasion were pooled (lamb and ewe samples pooled separately) and cultured for L3 development. After harvest of larvae (Baermann method), DNA was extracted and then analysed using droplet digital PCR with primer/probe sets targeting the BZ-associated F200Y (TAC) mutation. Haemonchus was found in 60% (80/134) of samples from lambs, and in 63% (106/167) from ewes. Among these, the F200Y mutation was detected in 73% (58/80) of larval samples from lambs and 69% (73/106) of larval samples from ewes, respectively. Although regional differences were evident, the mutation was detected in all areas indicating a widespread distribution and a strong potential for an increasing problem with treatment-resistant haemonchosis in Norwegian sheep flocks.

Distribution of large lungworms (Nematoda: Dictyocaulidae) in free-roaming populations of red deer Cervus elaphus (L.) with the description of Dictyocaulus skrjabini n. sp.

Lungworms of the genus Dictyocaulus are causative agents of parasitic bronchitis in domestic and wild ungulates. This study investigates the distribution, morphology and genetic diversity of D. cervi and a new lungworm species, Dictyocaulus skrjabini n. sp. infecting red deer Cervus elaphus, fallow deer Dama dama and moose Alces alces in Poland and Sweden. The study was conducted on 167 red deer from Poland and on the DNA of lungworms derived from 7 fallow deer, 4 red deer and 2 moose collected in Sweden. The prevalence of D. cervi and D. skrjabini n. sp. in dissected red deer in Poland was 31.1% and 7.2%, respectively. Moreover, D. skrjabini n. sp. was confirmed molecularly in 7 isolates of fallow deer lungworms and 1 isolate of red deer lungworms from Sweden. Dictyocaulus skrjabini n. sp. was established based on combination of their distinct molecular and morphological features; these included the length of cephalic vesicle, buccal capsule (BC), buccal capsule wall (BCW), distance from anterior extremity to the nerve ring, the width of head, oesophagus, cephalic vesicle, BC and BCW, as well as the dimensions of reproductive organs of male and female. Additionally, molecular analyses revealed 0.9% nucleotide sequence divergence for 1,605 bp SSU rDNA, and 16.5­17.3% nucleotide sequence divergence for 642 bp mitochondrial cytB between D. skrjabini n. sp. and D. cervi, respectively, and 18.7­19% between D. skrjabini n. sp. and D. eckerti, which translates into 18.2­18.7% amino acid sequence divergence between D. skrjabini n. sp. and both lungworms.

Ascaridia galli - An old problem that requires new solutions.

Reports of Ascaridia galli in laying hens in Europe have increased since the ban on conventional battery cages in 2012. As this parasite is transmitted directly via the faecal-oral route by parasite eggs containing a larva, it is reasonable to assume that the escalating problem is related to the increased exposure now occurring in modern welfare-friendly cage-free housing systems. On many farms, A. galli reappears in subsequent flocks, even though the birds have no access to the outdoors, biosecurity is high and empty houses are cleaned and disinfected during downtime. Since the egg production cycle lasts only ≈80 weeks and recombinant antigen production for helminth vaccines has not yet been solved, the development of a vaccine seems to be an unrealistic option. Therefore, disrupting the life cycle of the parasite by other means, including the strategic use of dewormers, appears to be the key to controlling infection. Of concern is that only one class of anthelmintics is licenced for poultry in Europe and that are usually administered indiscriminately through the birds' drinking water and often too late when the parasite is already established. If current calendar-based parasite control strategies are not changed, there is a risk that resistance to anthelmintics may develop, as has already been demonstrated with nematodes in livestock. We insist that treatments can be more effective and the risk of developing drug resistance can be mitigated if we invest in a better understanding of A. galli responses to more prudent and judicious use of anthelmintics. This review identifies knowledge gaps and highlights aspects of sustainable parasite control that require further research to support commercial egg producers.

Review and Evaluation of Ostertagia ostertagi Antibody ELISA for Application on Serum Samples in First Season Grazing Calves.

The O. ostertagi-Ab ELISA assay is widely used as a diagnostic tool for monitoring gastrointestinal (GI) nematodes using milk samples from adult dairy cows. This assay is potentially also useful to analyse serum samples from first-season grazing (FSG) calves, providing a more cost-effective and robust diagnostic technique than the current serum pepsinogen assay. However, a comprehensive evaluation of its use in serum samples from FSG calves has not yet been conducted. In this study, we first reviewed the available scientific literature in which the O. ostertagi-Ab ELISA was applied to serum samples from FSG calves. Then, a field study was conducted to compare results from the O. ostertagi-Ab ELISA assay with a serum pepsinogen assay on a set of 230 serum samples from 11 commercial dairy herds (seven in Belgium and four in Sweden). The literature review showed an increase in mean antibody levels, expressed as optical density ratio (ODR) values, from <0.4 (early grazing season) to values of 0.7-1.1 (late grazing season). Three out of five studies found a negative correlation between O. ostertagi antibody levels measured during the late grazing season and weight gain, while the other two studies found no correlation between the two variables. Our field studies showed a good correlation between O. ostertagi antibody levels and the results from the pepsinogen assay. Both indicators were negatively related to average daily weight gain in the Belgian herds, but not in the Swedish herds. Overall, the results suggest that the O. ostertagi-Ab ELISA test can be a useful tool in FSG calves and could replace the use of the serum pepsinogen assay at the end of the grazing season for general monitoring purposes.

Anthelmintic Treatment of Sheep and the Role of Parasites Refugia in a Local Context.

Gastrointestinal nematodes in grazing livestock are ubiquitous and can cause severe damage, leading to substantial losses in agricultural yields. It is undeniable that the integrated use of anthelmintics is often an essential component of successful intensive livestock management. However, anthelmintic resistance has been a major challenge for several decades, especially in pasture-based lamb production. Measures are therefore needed to reduce the risk and prevent further spread. In many countries with more extensive lamb production and pronounced resistance problems than in Sweden, the importance of keeping parasites in refugia is emphasised. To ensure that treatment is necessary, the Swedish model is based on deworming certain groups of ewes based on the parasitological results of a faecal examination and then releasing them with their lambs to safe pastures. This is intended to reduce the risk of infection, which ultimately reduces the number of subsequent treatments. Whether this preventive strategy in turn means an increased risk of resistance is debatable. In this review, we explain the importance of parasites in refugia and how they can help delay the development of resistance to anthelmintics. We also discuss how likely it is that our model contributes to an increase in resistance risk and whether there is reason to question whether it is a sustainable strategy in the long term.

Digital PCR: modern solution to parasite diagnostics and population trait genetics.

The use of polymerase chain reaction (PCR)-based diagnostic approaches has steadily increased in the field of parasitology in recent decades. The most recent large-scale technological modification of the PCR formula, also known as third-generation PCR, came in the form of digital PCR (dPCR). Currently, the most common form of dPCR on the market is digital droplet PCR (ddPCR). Unlike quantitative real-time PCR (qPCR), the digital format allows for highly sensitive, absolute quantification of nucleic acid targets and does not require external standards to be included in the developed assays. Dividing each sample into thousands of compartments and using statistical models also eliminates the need for technical replicates. With unprecedented sensitivity and enforcement of binary endpoint reactions, ddPCR not only allows the use of tiny sample volumes (especially important when working with limited amounts of DNA) but also minimises the impact of variations in amplification efficiency and the presence of inhibitors. As ddPCR is characterised by excellent features such as high throughput, sensitivity and robust quantification, it is widely used as a diagnostic tool in clinical microbiology. Due to recent advances, both the theoretical background and the practical, current applications related to the quantification of nucleic acids of eukaryotic parasites need to be updated. In this review, we present the basics of this technology (particularly useful for new users) and consolidate recent advances in the field with a focus on applications to the study of helminths and protozoan parasites.

The effect of weaning age on animal performance in lambs exposed to naturally acquired nematode infections.

The effects of mixed gastrointestinal nematode (GIN) infections on animal growth and post-weaning activity patterns were investigated in grazing intact ram lambs when naturally exposed to two different infection levels and weaned at different ages. Ewes and their twin-born lambs were turned-out to graze in two permanent pasture enclosures naturally contaminated with GIN the previous year. Ewes and lambs in the low parasite exposure group (LP) were drenched before turn-out and at weaning, respectively, with 0.2 mg ivermectin per kg body weight, whereas those in the high parasite exposure group (HP) were left untreated. Two weaning ages were applied, early weaning (EW) (10 weeks) and late weaning (LW) (14 weeks), respectively. The lambs were then allocated to one out of four groups based on parasite exposure level and weaning age (EW-HP, n = 12; LW-HP, n = 11; EW-LP, n = 13; LW-LP, n = 13). Body weight gain (BWG) and faecal egg counts (FEC) were monitored, in all groups, from the day of early weaning and every four weeks, for 10 weeks. In addition, nematode composition was determined using droplet digital PCR. Activity patterns measured as Motion Index (MI; the absolute value of the 3D acceleration) and lying time were monitored continuously from the day of weaning until four weeks post-weaning using IceQube® sensors. Statistical analyses were performed in RStudio, using mixed models with repeated measures. BWG was 11% lower in EW-HP compared with EW-LP (P = 0.0079) and 12% lower compared with LW-HP (P = 0.018), respectively. In contrast, no difference in BWG was observed between LW-HP and LW-LP (P = 0.97). The average EPG was higher in EW-HP compared with EW-LP (P < 0.001), as well as in EW-HP compared with LW-HP (P = 0.021), and LW-HP compared with LW-LP (P = 0.0022). The molecular investigation showed that animals in LW-HP had a higher proportion of Haemonchus contortus compared with EW-HP. MI was 19% lower in EW-HP compared with EW-LP (P = 0.0004). Daily lying time was 15% shorter in EW-HP compared with EW-LP (P = 0.0070). In contrast, no difference in MI (P = 0.13) and lying time (P = 0.99) between LW-HP and LW-LP was observed. The results suggest that a delayed weaning age may reduce the adverse effects of GIN infection on BWG. Contrarily, an earlier weaning age may reduce the risk of H. contortus infection in lambs. Moreover, the results demonstrates a potential use of automated behaviour recordings as a diagnostic tool for the detection of nematode infections in sheep.

The presence and relative frequency detection of the levamisole-resistance-associated S168T substitution in hco-acr-8 in Haemonchus contortus.

Parasitic sheep nematodes, among which Haemonchus contortus is often considered to be the most clinically important, exact a significant toll on the animals, not least because of their capacity to evolve drug resistance. Despite decades of research, our understanding of the mechanism of resistance to compounds such as levamisole is fairly limited, which therefore constrains our ability to develop sensitive and efficient molecular diagnostic tools for rapid and accurate resistance detection in field settings. Herein, we investigated the presence and frequency of the newly reported, levamisole-resistance-associated, mutation, yielding a S168T substitution in exon 4 of hco-acr-8, in six different phenotypically described isolates (three susceptible and three resistant), three Swedish field isolates and eight larvae culture samples, the latter two of which originated on farms where levamisole showed complete parasite elimination. For this purpose, we created both an allele-specific and droplet digital PCR approaches and found the mutated allele to be present only in the Kokstad isolate, whereas the other five as well as both the Swedish isolates and larvae cultures displayed only the non-mutated, serine-encoding, allele. While the finding of only the non-mutated allele in the phenotypically susceptible and Swedish isolate and larvae culture samples seemed sensible, we speculate that for the other two phenotypically resistant isolates, different (perhaps secondary) variants are responsible for conferring the resistance to levamisole phenotype, given the polygenic nature of levamisole resistance. All in all, despite the limited number of samples tested here, the mutation causing the S168T substitution in hco-acr-8 represents a plausible levamisole resistance-associated variant in, at least, some isolates of H. contortus.

Practical guide to the diagnostics of ruminant gastrointestinal nematodes, liver fluke and lungworm infection: interpretation and usability of results.

The diagnostics of ruminant parasites remains one of the cornerstones for parasite control best practices. Field veterinarians have several techniques at their disposal (fecal egg count, coproculture, FAMACHA®, plasma pepsinogen, ELISA-Ostertagia, ELISA-Fasciola, Baermann and ELISA-Lungworm) for the identification and/or quantification of gastrointestinal nematodes, lungworms and liver fluke infecting small ruminants and cattle. Each of these diagnostic tools has its own strengths and weaknesses and is more appropriate for a specific production operation and/or age of the animal (young and adults). This review focuses on the usability and interpretation of the results of these diagnostic tools. The most advanced technical information on sampling, storage, advantages and limitations of each tool for different types of production operations and animal categories is provided.

Signs of multiple anthelmintic resistance in sheep gastrointestinal nematodes in Sweden.

Gastrointestinal nematodes in small ruminants are clinically and economically important parasites that often are controlled with anthelmintics. In this study, we compiled information on the anthelmintic efficacy collected on sheep farms according to routines established by Farm & Animal Health in Sweden. The efficacies of benzimidazoles (i.e. albendazole or fenbendazole, n = 30), ivermectin (n = 47), levamisole (n = 2) or moxidectin (n = 2) were examined between 2015 and 2021 in 81 treatment groups on 49 non-randomly selected farms in south-central Sweden. Drug efficacies were estimated with the faecal egg count reduction test. In addition, efficacy data were in most cases supplemented with data on the abundance of the three most common nematode genera in sheep by performing droplet digital (dd) PCR on coprocultures. Efficacies of <95% for benzimidazoles or ivermectin were identified in 37% and 77% of the tested groups, respectively. In addition, on 27 (55%) of the 49 farms where both benzimidazoles and ivermectin were tested, multiple resistance was found on 8 (30%). In contrast, on each of the two farms tested for levamisole and moxidectin both drugs proved to be 100% effective. However, because post-sampling was performed earlier than recommended in several susceptible groups (benzimidazoles = 15, and ivermectin = 10 groups), this could have underestimated the severity of the situation. Mainly larvae from the genus Haemonchus were detected in post-treatment coprocultures, in all groups with declared resistance, suggesting that this parasite was primarily associated with anthelmintic resistance. Unexpectedly, the DNA of larvae, which survived treatment, was also detected on farms declared as susceptible. Taken together, this indicates that the situation regarding the anthelmintic efficacy has deteriorated compared with the latest nationwide study on Swedish sheep farms conducted more than a decade ago. Unlike the previous study, the farm selection here was not strictly randomized but rather opportunistic i.e., only farms with a recognized parasite problem were included. Thus, there is a need for a truly randomized study to get an update on the extent of the situation of anthelmintic resistance at a national level, as well as to identify risk factors involved in the resistance selection. Research is also required to establish the optimal intervals for sampling post-treatment.

Parasitic strongyle nemabiome communities in wild ruminants in Sweden.

BACKGROUND: Wildlife hosts may serve as reservoirs for strongyles, which can be transmitted to domestic livestock. Therefore, studies evaluating nemabiome compositions in wildlife ruminants are of great use in assessing the possibility of transmission of important nematode pathogens to domestic sheep in Sweden. METHODS: First, fecal samples were collected from roe deer (n = 125), fallow deer (n = 106), red deer (n = 18) and mouflon (n = 13) in south central Sweden during the hunting season in 2019. Second, after fecal examination samples were cultured and the larvae were harvested, followed by DNA extractions. Third, all samples were barcoded and processed for sequence analysis on the PacBio platform. Finally, bioinformatic sequence analysis was conducted with DADA2, while species diversity and richness, as well as interactions between the different hosts, were calculated and analyzed in R. RESULTS: Nematode ITS2 sequences were found in 225 of 262 (86%) samples. In total, 31 taxa were identified, among which 26 (86%) to the species level. These were found in different combinations, among which 24 (77%) occurred in roe deer, 19 (61%) in fallow deer, 20 (65%) in red deer and 10 (32%) in mouflon. Five of the species found are known to be associated with livestock (Chabertia ovina, Haemonchus contortus, Oesophagostomum venulosum, Teladorsagia circumcincta and Trichostrongylus axei). However, in the present study the relative abundance and prevalence of most of these species were low. The most striking exception was T. axei, which was relatively abundant in all wildlife hosts. Mostly a wide range of wildlife specific nematodes such as Ostertagia leptospicularis and Spiculopteragia spp. were identified including the invasive nematode Spiculopteragia houdemeri, which was found for the first time in red deer, fallow deer, and mouflon in Sweden. The difference in the number of shared species between mouflon and all cervids (n = 6) was less than among all three cervids (n = 8). CONCLUSION: In this study, we investigated the community structure of parasitic intestinal nematodes in four wildlife hosts, and we found that the majority of the parasite species identified were wildlife specific. We also found a new, potentially invasive species not reported before. After comparing the nemabiome of the wildlife hosts in this study with a previous study in sheep from the same geographical region, we conclude that the horizontal transmission potential appears to be relatively low. Still, cross-infections of nematodes between game and sheep cannot be completely ignored.

Estimation of the impact of three different bioinformatic pipelines on sheep nemabiome analysis.

BACKGROUND: Next-generation sequencing (NGS) has provided an alternative strategy to study the composition of nematode communities with increased resolution and sensitivity. However, the handling and processing of gigabytes worth of amplicon sequence data produced by an NGS platform is still a major hurdle, limiting the use and adoption of faster and more convenient analysis software. METHODS: In total 32 paired, fecal samples from Swedish sheep flocks were cultured and the larvae subsequently harvested subjected to internal transcribed spacer 2 (ITS2) amplicon sequencing using the PacBio platform. Samples were analyzed with three different bioinformatic pipelines, i.e. the DADA2, Mothur and SCATA pipelines, to determine species composition and richness. RESULTS: For the the major species tested in this study (Haemonchus contortus, Teladorsagia circumcinta and Trichostrongylus colubriformis) neither relative abundances nor species diversity differed significantly between the three pipelines, effectively showing that all three analysis pipelines, although different in their approaches, yield nearly identical outcomes. In addition, the samples analyzed here had especially high frequencies of H. contortus (90-95% across the three pipelines) both before and after sample treatment, followed by T. circumcinta (3.5-4%). This shows that H. contortus is the parasite of primary importance in contemporary Swedish sheep farms struggling with anthelmintic resistance. Finally, although on average a significant reduction in egg counts was achieved post-treatment, no significant shifts in major species relative frequencies occurred, indicating highly rigid community structures at sheep farms where anthelmintic resistance has been reported. CONCLUSIONS: The findings presented here further contribute to the development and application of NGS technology to study nemabiome compositions in sheep, in addition to expanding our understanding about the most recent changes in parasite species abundances from Swedish sheep farms struggling with anthelmintic resistance.

Anticoccidial Vaccination Is Associated with Improved Intestinal Health in Organic Chickens.

Eimeria spp. and Clostridium perfringens (CP) are pathogens associated with coccidiosis and necrotic enteritis (NE) in broiler chickens. In this study we evaluated the effect of anticoccidial vaccination on intestinal health in clinically healthy organic Ross 308 chickens. On each of two farms, one unvaccinated flock (A1 and B1) was compared to one vaccinated flock (A2 and B2) until ten weeks of age (WOA). Faecal oocysts were counted weekly, and species were identified by PCR (ITS-1 gene). Lesion scoring, CP quantification and PCR targeting the CP NetB toxin gene were performed at three, four, and six WOA and chickens were weighed. Necropsies were performed on randomly selected chickens to identify coccidiosis/NE. Oocyst shedding peaked at three WOA in all flocks. Later oocyst shedding (E. tenella/E. maxima) in unvaccinated flocks at 5-7 WOA coincided with coccidiosis/NE. Although results differed somewhat between farms, vaccination was associated with lower intestinal lesion scores, reduced caecal CP counts, lower proportions of netB-positive CP, lower body weight at three-four WOA, and similar or slightly increased body weight at six WOA. In conclusion, the intestinal health of organic broilers can benefit from anticoccidial vaccination when oocyst exposure levels are high.

Implementation of a targeted treatment strategy for the sustainable control of Ascaridia galli infections in laying hens.

Background: Ascaridia galli is a widespread problem in cage-free egg production. Sustainable control of nematode infections is a key component in this sector. This study investigates the effect of a treatment strategy against A. galli, aiming to propose a guideline for anthelmintic use on commercial poultry farms. Methods: A total of eight flocks of laying hens (a-h) from five commercial poultry farms were included in this study. Faecal samples were collected on a biweekly basis starting at 7-13 weeks post-placement (WPP) and processed using the McMaster method to calculate ascarid egg shedding. Flocks were treated after the threshold of 200 eggs per gram of faeces (EPG) was reached. Results: The highest initial faecal egg count was 6700 EPG at 11 WPP, whereas the lowest was 50 at 8 WPP. The longest delay to detect A. galli was 7 weeks. The lowest and the highest number of treatments were four and six, respectively. The shortest and longest periods between any two treatments were 5 and 22 weeks, respectively. Conclusions: These results suggest that monitoring for A. galli should start at approximately 7 WPP and should be repeated every 8 weeks until hens are 50 weeks old. Treatment should be given only if moderate to high faecal egg counts are observed. Treatments after this point may be repeated every 8 weeks without eventually performing a faecal test. These findings provide practical support to veterinarians and egg producers dealing with ascarid worm infection in laying hens in their production stage.

Farmers' perception on the control of gastrointestinal parasites in organic and conventional sheep production in Sweden.

A questionnaire was sent to sheep owners in Sweden to get information about anthelmintic drug use. The survey also investigated how respondents experienced problems with gastrointestinal nematode infections (GIN) focusing on Haemonchus contortus. The response rate was 31% and included both conventional and organic farms. The use of anthelmintics was low (45%), among which a majority (76%) drenched ewes on a single occasion, mostly with ivermectin (59%) followed by albendazole (19%). Other drugs were used rarely, however, unawareness of GIN risk was high (19%), especially among respondents with few animals. Anthelmintic dose calculations were done after visual appraisal by 63% and 22% calibrated the equipment before drug delivery, which is worrying since underdosing is a risk factor for the development of anthelmintic resistance. Like with anthelmintics, the perceived risk for GIN increased with herd size both by conventional and organic farmers. Faecal examination for the presence of GIN was done by 65% of the respondents and, among their sheep, H. contortus was or had been diagnosed in 41% of the herds. Irrespective of new stock had been imported from other countries or not, common problems were reported by 5% and 7% of the organic and conventional producers, respectively. Land use and grazing management strategies differed more in relation to herd size than by production form, with a majority (47%) having their sheep grazed in several paddocks, or at least the lambs were moved when separated from the ewes at weaning (25%). In contrast set stocked grazing was mainly reported on smaller farms. Co-grazing with cattle and horses were also frequently reported irrespective of production form, but with cattle to a somewhat greater degree on larger organic farms. Wild cervids, especially roe deer, were frequently observed on sheep pastures (87%). The veterinary involvement was higher on organic (65%) than on conventional farms (53%), and only 5% considered advice unimportant. Still, some conventional and organic producers treated sheeps routinely without a prior diagnosis, against the national regulations. 46% of the respondents drenched new and replacement stock. In conclusion, although some differences were observed between conventional and organic producers, the divergences were mainly due to herd size categories. Furthermore, despite a high veterinary involvement, we identified factors which can contribute to anthelmintic use, such as poor quarantine procedures, and deworming routines that can contribute to anthelmintic resistance in H. contortus.

Assessment of three DNA extraction kits for the absolute quantification of strongyle nematode eggs in faecal samples.

BACKGROUND: Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Methods employing detection through DNA amplification, such as droplet digital polymerase chain reaction (ddPCR), offer an advantageous approach to the diagnosis of H. contortus. However, DNA extraction protocols need to be constantly updated for the optimal retrieval of diagnostically usable template. Here, we describe the evaluation of three genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA from faecal samples, using droplet digital PCR. RESULTS: DNA samples, extracted from faecal material with the Nucleospin DNA Stool kit, produced the highest amounts of ITS2 amplicon copies and had the lowest coefficient of variation across different dilutions and sample types (fresh or frozen) out of the tested kits (Nucleospin DNA Stool, E.Z.N.A.® Stool DNA Kit and QIAamp Fast DNA Stool Mini Kit). Furthermore, the protocol of this kit has the fewest number of steps and the price of DNA extraction per sample is reasonable (2.77 €). CONCLUSIONS: The Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting.

Genome-wide analysis of the response to ivermectin treatment by a Swedish field population of Haemonchus contortus.

Haemonchus contortus is a pathogenic gastrointestinal nematode of small ruminants and, in part due to its capacity to develop resistance to drugs, contributes to significant losses in the animal production sector worldwide. Despite decades of research, comparatively little is known about the specific mechanism(s) driving resistance to drugs such as ivermectin in this species. Here we describe a genome-wide approach to detect evidence of selection by ivermectin treatment in a field population of H. contortus from Sweden, using parasites sampled from the same animals before and seven days after ivermectin exposure followed by whole-genome sequencing. Despite an 89% reduction in parasites recovered after treatment measured by the fecal egg count reduction test, the surviving population was highly genetically similar to the population before treatment, suggesting that resistance has likely evolved over time and that resistance alleles are present on diverse haplotypes. Pairwise gene and SNP frequency comparisons indicated the highest degree of differentiation was found at the terminal end of chromosome 4, whereas the most striking difference in nucleotide diversity was observed in a region on chromosome 5 previously reported to harbor a major quantitative trait locus involved in ivermectin resistance. These data provide novel insight into the genome-wide effect of ivermectin selection in a field population as well as confirm the importance of the previously established quantitative trait locus in the development of resistance to ivermectin.

Effects of lambing season on nematode faecal egg output in ewes.

In this study, we assessed the occurrence of a periparturient rise (PPR) in winter and spring lambing ewes in Sweden and where nematode egg excretion patterns were investigated mainly for diagnostic purposes. Gastrointestinal nematodes and Haemonchus contortus presence were monitored in parallel in all animals in each experimental group on four farms in samples that were collected mainly when the animals were stabled. Faecal examinations of the same animals were conducted on four sampling occasions between January and June 2018 Each group consisted of 12 crossbreed ewes of similar genotype. One group's peak lambing was in January or February (early), and the other in March or early May (late). The first (S1) and third samples (S3) were from approximately one to two weeks before parturition in the early (winter lambing) and late (spring lambing) group respectively, whereas the second (S2) and fourth samples (S4) were collected approximately three to five weeks post-parturition in the same groups. During the course of the study, there was a significant rise in faecal egg counts (FEC) in both groups on all farms. On three farms with a substantial amount of Haemonchus, we observed a difference in the egg excretion patterns between the two groups of ewes as revealed by a significant interaction between the sampling point and lambing period. Also, when samples corresponding to each other in relation to the number of weeks that had elapsed post-parturition (three to five weeks post-parturition, S2 for early and S4 for late) were compared, FEC were significantly lower in the early group lambing in winter than the late group lambing in spring. This indicates that besides lambing, the rise in nematode egg count is also influenced by other factors unrelated to the lambing period, such as the increased daylight in spring. Due to study limitations, we cannot provide a more detailed explanation for this, but only state that the rise appeared to be more closely linked to season than physiology as measured by day relative to parturition. Still, our results suggests that when turned out, winter lambing ewes contribute to pasture contamination to a lesser extent than those lambing in spring. These results will be used in stipulating evidence-based advice to farmers in their flock management to reduce use of anthelmintics, and at the same time efficiently produce prime lambs.

Exploring the ß-tubulin gene family in a benzimidazole-resistant Parascaris univalens population.

Benzimidazole (BZ) drugs are frequently used to treat infections with the equine ascarid Parascaris univalens due to increasing resistance to macrocyclic lactones and pyrantel. Benzimidazole resistance is rare in ascarids in contrast to strongyle parasites where this resistance is widespread. In strongyles, single nucleotide polymorphisms (SNPs) at codons 167, 198 and 200 in a ß-tubulin gene have been correlated to BZ resistance, but little is known about the ß-tubulin genes and their possible involvement in BZ resistance in P. univalens and other ascarids. Previously two ß-tubulin genes have been identified in P. univalens. In this study, we present five additional ß-tubulin genes as well as the phylogenetic relationship of all seven genes to ß-tubulins of other clade III and V nematodes. In addition, the efficacy of fenbendazole for treatment of P. univalens on a Swedish stud farm was studied in 2019 and 2020 using faecal egg count reduction test. Reductions varied from 73% to 88%, indicating the presence of a resistant P. univalens population on the farm. The emergence of BZ resistance emphasizes the need for development of molecular markers for rapid and more sensitive detection of resistant populations. We therefore investigated whether possible SNPs at positions 167, 198 or 200 in any of the ß-tubulin genes could be used to distinguish between resistant and susceptible P. univalens populations. Amplicon sequencing covering the mutation sites 167, 198 and 200 in all seven ß-tubulin genes revealed an absence of SNPs in both resistant and susceptible populations, suggesting that the mechanism behind BZ resistance in ascarids is different from that in strongyle nematodes and the search for a molecular marker for BZ resistance in P. univalens needs to continue.