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.

Demonstration of reduced efficacy against cyathostomins without change in species composition after pyrantel embonate treatment in Swedish equine establishments.

Consisting of approximately 50 different species, the cyathostomin parasites are ubiquitous in grazing horses. Co-infection with several species is common, and large burdens can cause the fatal disease of larval cyathostominosis. Due to intense anthelmintic drug use, cyathostomin resistance has developed to all available anthelmintic drug groups. Resistance to the anthelmintic drug pyrantel (PYR) has been documented in over 90% of studies published over the past two decades. In Sweden, a study performed in the early 2000s only confirmed resistance in 4.5% of farms. Further, prescription-only administration of equine anthelmintic drugs was enforced in Sweden in 2007. However, it is unknown if this conservative drug use has maintained PYR efficacy in cyathostomins. The aim of the present study was to investigate the effect of PYR on cyathostomin infection in Sweden using fecal egg count reduction tests (FECRTs). Further, the effect of PYR treatment on cyathostomin species composition was studied using metabarcoding. Sixteen farms with at least six horses excreting a minimum of 100 eggs per gram feces were included. Using the current World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines, PYR resistance was demonstrated in nine of farms, with seven farms showing full susceptibility. Farms with low biosecurity measures had significantly lower efficacy of PYR treatment. The most common cyathostomin species were Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus longibursatus, Cys. calicatus, Cys. goldi, Cys. minutus, Coronocyclus coronatus and Cya. pateratum, accounting for 97% of all sequence reads prior to treatment. Of these, Cyc. nassatus and Cya. catinatum had the highest occurrence, accounting for 68% of all sequence reads prior to PYR treatment. Treatment did not significantly affect the species composition. The results highlight the importance of drug efficacy testing when using PYR to treat cyathostomin infection, even when selective anthelmintic treatment and thus low treatment intensity, is used on the farm.

Avian malaria parasite infections do not affect personality in the chestnut thrush (Turdus rubrocanus) on the Qinghai-Tibet Plateau.

Personality traits, the consistent individual behavioral differences, are currently gaining much attention in studies of natural bird populations. However, associations between personality traits and parasite infections are not often investigated. Even less attention has been given to studies of birds in the high-elevation region such as the Tibetan plateau. This research aims to examine the relationship between avian malaria parasites and two personality traits in a population of the Chestnut Thrush (Turdus rubrocanus) breed in the Tibetan plateau. Our results revealed no evidence of sex bias in malaria parasite prevalence. Furthermore, we found no effect of infection status on two personality scores: activity and boldness. Additionally, no effects on the activity level or boldness were observed for different parasite lineages of Haemoproteus, Leucocytozoon, the sex of the birds, or their interactions. Similarly, we did not find any relationship between activity level and boldness with nestling numbers, sex, or their interactions. Notably, individuals with a larger number of offspring tended to display greater boldness. Our findings indicate that blood parasite infections are common in this population but do not significantly impact the personality of the birds.

Retained efficacy of ivermectin against cyathostomins in Swedish horse establishments practicing selective anthelmintic treatment.

Cyathostominae are ubiquitous to grazing horses and regarded the most prevalent internal parasite in the horse. Unfortunately, decades of indiscriminate use of anthelmintic drugs have resulted in the development of resistance in cyathostomins to all currently available drug groups, the most recent being a documented lack of efficacy to the macrocyclic lactones (ML). In vivo determination of anthelmintic resistance in horses most often utilises the faecal egg count reduction test (FECRT). Further, a shortened egg reappearance period (ERP) can indicate a change in response to the applied treatment and suggest an upcoming reduction of efficacy. Although both true resistance as demonstrated by the FECRT and shorter ERPs after ML treatment have now been shown in cyathostomins worldwide, the efficacy of ML as regards to cyathostomins in Sweden is currently unknown. The aim of the present study was therefore to determine FECRTs and ERPs after ivermectin (IVM) treatment in Swedish horses. Sixteen equestrian establishments with a minimum of six horses excreting at least 150 eggs per gram faeces (EPG) at screening were selected. For each establishment, FECRTs and ERPs were determined by collecting faecal samples prior to and 14 days after IVM treatment (200 µg/kg), and thereafter at weekly intervals for a total of eight weeks. All participants responded to a questionnaire detailing pasture management methods and anthelmintic routines.Questionnaire results showed that the majority of establishments (69%) only treated horses with anthelmintic drugs if indicated by faecal diagnostics and all of the establishments had a mean FECRT exceeding 99.0% and ERPs ranging from six to over eight weeks. The ERP was shown to increase with age as young individuals were shown to excrete cyathostomin eggs earlier after treatment compared with older horses (R = 0.21, p = 0.015). Riding schools, stud farms and those declaring not to use separate summer and winter paddocks had significantly shorter ERPs (p <0.01).In conclusion, retained ERPs and no confirmed resistance to IVM were found in Swedish equine establishments practising selective anthelmintic treatment, and supports the use of selective deworming regimens as a means of reducing the risk of anthelmintic resistance development.

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.

The complete ITS2 barcoding region for Strongylus vulgaris and Strongylus edentatus.

Gastrointestinal nematode parasites are of major concern for horses, where Strongylus vulgaris is considered the most pathogenic among the Strongylus species. Diagnosis of S. vulgaris infections can be determined with next generation sequencing techniques, which are inherently dependent on reference sequences. The best marker for parasitic nematodes is internal transcribed spacer 2 (ITS2) and we provide the first complete ITS2 sequences from five morphologically identified S. vulgaris and additional sequences from two S. edentatus. These sequences have high similarity to already published partial sequences and amplicon sequence variants (ASV) based on next generation sequencing (NGS). The ITS2 sequences from S. vulgaris matched available partial ITS2 sequences and the full ASVs, whereas the S. edentatus sequence matched another complete sequence. We also compare Sanger sequencing and NGS methods and conclude that the ITS2 variation is better represented with NGS methods. Based on this, we recommend that further sequencing of morphologically identified specimens of various species should be performed with NGS cover the intraspecific variation in the ITS2.

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.

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.

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.

Demographic history and divergence of sibling grouse species inferred from whole genome sequencing reveal past effects of climate change.

BACKGROUND: The boreal forest is one of the largest biomes on earth, supporting thousands of species. The global climate fluctuations in the Quaternary, especially the ice ages, had a significant influence on the distribution of boreal forest, as well as the divergence and evolution of species inhabiting this biome. To understand the possible effects of on-going and future climate change it would be useful to reconstruct past population size changes and relate such to climatic events in the past. We sequenced the genomes of 32 individuals from two forest inhabiting bird species, Hazel Grouse (Tetrastes bonasia) and Chinese Grouse (T. sewerzowi) and three representatives of two outgroup species from Europe and China. RESULTS: We estimated the divergence time of Chinese Grouse and Hazel Grouse to 1.76 (0.46-3.37) MYA. The demographic history of different populations in these two sibling species was reconstructed, and showed that peaks and bottlenecks of effective population size occurred at different times for the two species. The northern Qilian population of Chinese Grouse became separated from the rest of the species residing in the south approximately 250,000 years ago and have since then showed consistently lower effective population size than the southern population. The Chinese Hazel Grouse population had a higher effective population size at the peak of the Last Glacial Period (approx. 300,000 years ago) than the European population. Both species have decreased recently and now have low effective population sizes. CONCLUSIONS: Combined with the uplift history and reconstructed climate change during the Quaternary, our results support that cold-adapted grouse species diverged in response to changes in the distribution of palaeo-boreal forest and the formation of the Loess Plateau. The combined effects of climate change and an increased human pressure impose major threats to the survival and conservation of both species.

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.

First report on reduced efficacy of ivermectin on Oesophagostomum spp. on Swedish pig farms.

Anthelmintic efficacy was investigated in nine sow herds that had been identified with high faecal egg counts in a prevalence study. Faecal samples were collected from a total of 104 individual sows, and analysed using a centrifugal flotation McMaster technique. Samples positive for strongyle eggs were cultured to third stage larvae (L3) for genus identification and then further identified to Oesophagostomum species by sequencing. Following the initial sample collection, the sows were treated with either fenbendazole (FBZ, n = 5 farms) or ivermectin (IVM, n = 4 farms) at the recommended dosing and sampled again 14 days post treatment. Faecal Egg Count Reduction (FECR) was used to determine the treatment efficacy. With respect to Ascaris suum, the anthelmintic treatment was successful (FECR >90%) on the five farms where this parasite was detected, regardless of what drug had been used. In contrast, 4/9 farms were positive for Oesophagostomum spp. post treatment, out of which three had a FECR of <90%. These three herds had all been treated with injectable IVM. Out of the six farms where treatment showed good efficacy (FECR 95-100%), five herds had used FBZ and one herd IVM. This study is the first to recognise reduced efficacy to IVM on Oesophagostomum spp. in Swedish pigs. Sequencing of the Oesophagostomum L3 showed that both O. dentatum and O. quadrispinulatum were present on 5/9 farms pre-treatment and on 2/9 farms post-treatment, unrelated to what anthelmintic had been used. Given these findings we could not correlate the reduced efficacy by the species of Oesophagostomum present in the herd. Prolonged usage of only one class of anthelmintic may predispose selection of resistance and has been suspected as a cause of treatment failure of porcine Oesophagostomum spp. in other studies. On all three farms showing reduced efficacy, IVM had been used as the sole anthelmintic drug for several years, and two of the farms also used IVM twice or more per year to control sarcoptic mange. A reduced efficacy to the available anthelmintic drugs used in the control of Oesophagostomum spp., may result in a subsequent surge of the possible negative effects caused by this parasite.

Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus.

The nematode Haemonchus contortus is one of the most prevalent and pathogenic parasites in small ruminants. Although usually controlled using anthelmintics, the development of drug resistance by the parasite has become a major issue in livestock production. While the molecular detection of benzimidazole resistance in H. contortus is well developed, the molecular tools and protocols are far less advanced for the detection of levamisole resistance. The hco-acr-8 gene encodes a critical acetylcholine susceptible subunit that confers levamisole-sensitivity to the receptor. Here, we report the development of a droplet digital PCR assay as a molecular tool to detect a 63 bp deletion in the hco-acr-8 that has been previously associated with levamisole resistance. Sanger sequencing of single adult H. contortus yielded 56 high-quality consensus sequences surrounding the region containing the deletion. Based on the sequencing data, new primers and probes were designed and validated with a novel droplet digital PCR assay for the quantification of the deletion containing "resistant" allele in genomic DNA samples. Single adult worms from six phenotypically described isolates (n = 60) and from two Swedish sheep farms (n = 30) where levamisole was effective were tested. Even though a significant difference in genotype frequencies between the resistant and susceptible reference isolates was found (p = 0.01), the homozygous "resistant" genotype was observed to be abundantly present in both the susceptible isolates as well as in some Swedish H. contortus samples. Furthermore, field larval culture samples, collected pre- (n = 7) and post- (n = 6) levamisole treatment on seven Swedish sheep farms where levamisole was fully efficacious according to Fecal Egg Count Reduction Test results, were tested to evaluate the frequency of the "resistant" allele in each. Frequencies of the deletion ranged from 35 to 80% in the pre-treatment samples, whereas no amplifiable H. contortus genomic DNA was detected in the post-treatment samples. Together, these data reveal relatively high frequencies of the 63 bp deletion in the hco-acr-8 both on individual H. contortus and field larval culture scales, and cast doubt on the utility of the deletion in the hco-acr-8 as a molecular marker for levamisole resistance detection on sheep farms.

Sheep nemabiome diversity and its response to anthelmintic treatment in Swedish sheep herds.

BACKGROUND: A novel way to study the species composition and diversity of nematode parasites in livestock is to perform deep sequencing on composite samples containing a mixture of different species. Herein we describe for the first time the nematode community structures (nemabiomes) inhabiting Swedish sheep and how these are/were affected by host age and recent anthelmintic treatments. METHODS: A total of 158 fecal samples were collected (n = 35 in 2007 and n = 123 in 2013-2016) and cultured from groups of sheep on 61 commercial farms in the south-central part of the country where most animals are grazed. Among the samples, 2 × 44 (56%) were paired collections from the same groups pre- and post-treatment with anthelmintics such as macrocyclic lactones, benzimidazoles or levamisole. Samples were analyzed for their nemabiome using the PacBio platform followed by bioinformatic sequence analysis with SCATA. Species richness and diversity were calculated and analyzed in R. RESULTS: Nematode ITS2 sequences were found in all larval culture samples except two, even though the fecal egg counts were below the McMaster threshold in 20 samples. Sequencing yielded, on average, 1008 sequences per sample. In total, 16 operational taxonomical units (OTU), all with ≥ 98 % identity to sequences in the NCBI database, were recognized. The OTUs found represented nematode species of which ten are commonly associated with sheep. Multiple species were identified in all pre-anthelmintic treatment larval culture samples. No effects on nematode diversity were found in relation to host age. On the other hand, recent anthelmintic treatment lowered species richness, especially after use of ivermectin and albendazole. Interestingly, despite zero egg counts after use of levamisole, these samples still contained nematode DNA and especially H. contortus. CONCLUSIONS: Our findings provide evidence that nemabiome analysis combined with diversity index analysis provides an objective methodology in the study of the efficacy of anthelmintic treatment as both high and low abundant species were detected.

Utilization of droplet digital PCR to survey resistance associated polymorphisms in the ß tubulin gene of Haemonchus contortus in sheep flocks in Sweden.

Infections with gastrointestinal nematodes (GINs) in small ruminants are becoming increasingly harder to treat due to the development of anthelmintic resistance. Across Swedish sheep farms, Haemonchus contortus is one of the more persistent and pathogenic species encountered. Benzimidazole drugs, such as albendazole, are still widely used to control the GIN burden in small ruminants. However, the decline in efficacy of this drug has been observed across the country. In this study, we aimed to continue to investigate the presence of single nucleotide polymorphisms in the ß tubulin gene associated with benzimidazole drug resistance in H. contortus. This was carried out for sheep flocks from 67 farms around Sweden by screening for the two most commonly encountered SNPs at codons 167 and 200 in the isotype 1 ß tubulin gene utilizing the droplet digital PCR technology. We first established a good agreement (Lin's concordance correlation coefficient = 0.987) between the previously widely used pyrosequencing assay for the detection of the SNP at codon 200 (otherwise known as mutation F200Y) and our assay, as well as developed and validated primer-probe pairs for the detection of the mutation at codon 167 (mutation F167Y) in the ß tubulin gene of H. contortus. We then screened 174 pooled larval culture samples, collected either pre- or post-treatment, for the frequencies of the mutations F167Y and F200Y. Not only did we find the latter to be present at much higher frequencies, but the overall levels of this resistance conferring mutation have stayed stable throughout the years 2014-2019 at an average value of 88.5 ±â€¯20.3% in the pre-treatment samples across the tested farms (p = 0.61, Kruskal-Wallis test). Furthermore, after establishing a mixed model and fitting our data, we found a significant (p < 0.01) difference in the average frequency of the mutation F200Y between paired, pre- and post-treatment with albendazole, samples. Although the frequency difference in samples treated with albendazole was relatively minor (88.5% in pre- and 95.6% in post-treatment), no significant (p = 0.15) change in F200Y mutation frequency was observed between the samples from the flocks treated with ivermectin (90.8% and 92.6 %, respectively).

Soil contamination by Taenia solium egg DNA in rural villages in Kongwa district, Tanzania.

The presence ofTaenia solium DNA from eggs in soils around the households in four Tanzanian villages in Kongwa district were analysed in relation to seasonal fluctuations and infection risk implications. A total of 192 pooled soil samples from five sampling points per household were examined by droplet digital Polymerase Chain Reaction (ddPCR) from 96 pig-keeping households both during the dry and rainy seasons. The pooled samples were first processed by a flotation-double sieving technique, followed by screening for worm DNA employing universal primers targeting the mitochondrial cytochrome c oxidase subunit I (cox1) gene of human taeniid species and some other helminths. All DNA positive samples were later confirmed by a specific ddPCR probe assay targeting the mitochondrial cox1 gene of T. solium. A total of 17.2% (n = 33) samples were positive with the universal ddPCR, whereas T. solium DNA was confirmed by the specific ddPCR only in 3.1% (n = 3) of the surveyed households. The detection of T. solium DNA in this study spells out a low risk of exposure to T. solium eggs from contaminated household soil. Based on our results, ddPCR seems to be a promising technology for screening T. solium eggs in soil.

Genomic analysis of demographic history and ecological niche modeling in the endangered Chinese Grouse Tetrastes sewerzowi.

BACKGROUND: The Quaternary had worldwide consequences in forming the contemporary diversity of many populations, species and communities, which is characterized by marked climatic oscillations between glacial and interglacial periods. The origin and evolution of biodiversity in mountainous areas are highly dependent on historical orogenesis and associated climatic changes. The Chinese grouse Tetrastes sewerzowi is a forest-dwelling species endemic to the mountains to the east of the Qinghai-Tibet Plateau, which has been listed as Near Threatened with a decreasing trend by the IUCN because of ongoing deforestation and fragmentation of coniferous forests. It is important to place current population status into a broader ecological and evolutionary context to understand their demographic history. RESULTS: Analyses of the Chinese Grouse genome revealed fluctuations throughout the Pleistocene in effective population size. Populations decreased during early to middle Pleistocene but showed an expansion during late Pleistocene which was then followed by a sharp decline during the last glacial maximum (LGM). Ecological niche modeling indicated that a suitable habitat shift between high altitude regions to low altitude regions was due to a changing climate. This result parallels patterns of population size change in Chinese Grouse estimated from PSMC modelling, which suggested an expansion in population size from the last interglacial period (LIG) and then a peak and a bottleneck occurring at the last glacial maximum (LGM). Furthermore, the present-day distribution of Chinese Grouse is greatly reduced and fragmented. It will likely become even more fragmented in the future since coniferous forest cover is threatened in the region of their distribution and the availability of such habitat restricts their ecological niche. CONCLUSIONS: The Chinese Grouse have experienced substantial population size changes from the beginning to the LIG and reached a peak before the LGM. A sharp decrease and bottleneck occurred during the LGM, when the coniferous forests were subjected to extensive loss. The results inferred from the whole genome sequencing and species distribution models both support historical population fluctuations. The distribution of the Chinese Grouse is strongly dependent on the coniferous forest cover. To protect the fragmented coniferous forests is an essential action to protect the Chinese Grouse.

Molecular detection of two major gastrointestinal parasite genera in cattle using a novel droplet digital PCR approach.

Cooperia sp. and Ostertagia sp. are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform-based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites' ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e., Cooperia and Ostertagia) in a single mixed DNA sample.