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.
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.
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.
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.
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.
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. 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. 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. 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.
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. 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. 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). 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.
Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered ...drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect resistance-predicting alleles. Here we propose a novel droplet digital PCR (ddPCR) assay for rapid and precise identification of H. contortus strains as being resistant or susceptible to benzimidazole drugs based on the presence or absence of the most common resistance-conferring mutation F200Y (TAC) in the β tubulin isotype 1 gene. The newly developed ddPCR assay was first optimized and validated utilizing DNA templates from single-worm samples, which were previously sequenced using the next generation PacBio RSII Sequencing (NGS) platform. Subsequent NGS results for faecal larval cultures were then used as a reference to compare the obtained values for fractional abundances of the resistance-determining mutant allele between ddPCR and NGS techniques in each sample. Both methods managed to produce highly similar results and ddPCR proved to be a reliable tool which, when utilized at full capacity, can be used to create a powerful mutation detection and quantification assay.
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•Sequencing results show country-wide distribution of resistant H. contortus in sheep.•Two novel mutations for 198th codon in β-tubulin were found in single-worm samples.•Digital droplet PCR results correlated well with those derived from NGS sequencing.•A new digital droplet PCR assay for mutation detection in H. contortus was created.
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 β-tubulin gene have been correlated to BZ resistance, but little is known about the β-tubulin genes and their possible involvement in BZ resistance in P. univalens and other ascarids. Previously two β-tubulin genes have been identified in P. univalens. In this study, we present five additional β-tubulin genes as well as the phylogenetic relationship of all seven genes to β-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 β-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 β-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.
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•First case of fenbendazole resistance in Parascaris univalens in Europe.•The P. univalens β-tubulin family contains seven genes.•P. univalens β-tubulin genes cluster with β-tubulins from other clade V nematodes.•No resistance associated SNPs were identified in P. univalens β-tubulin genes.
Kin recognition is a critical element to kin cooperation, and in vertebrates, it is primarily based on associative learning. Recognition of socially unfamiliar kin occurs rarely, and it is reported ...only in vertebrate species where promiscuity prevents recognition of first‐order relatives. However, it is unknown whether the recognition of socially unfamiliar kin can evolve in monogamous species. Here, we investigate whether genetic relatedness modulates aggression among group members in Siberian jays (Perisoreus infaustus). This bird species is genetically and socially monogamous and lives in groups that are formed through the retention of offspring beyond independence, and the immigration of socially unfamiliar nonbreeders. Observations on feeders showed that genetic relatedness modulated aggression of breeders towards immigrants in a graded manner, in that they chased most intensely the immigrant group members that were genetically the least related. However, cross‐fostering experiments showed that breeders were equally tolerant towards their own and cross‐fostered young swapped as nestlings. Thus, breeders seem to use different mechanisms to recognize socially unfamiliar individuals and own offspring. As Siberian jays show a high degree of nepotism during foraging and predator encounters, inclusive fitness benefits may play a role for the evolution of fine‐scale kin recognition. More generally, our results suggest that fine‐graded kin recognition can evolve independently of social familiarity, highlighting the evolutionary importance of kin recognition for social species.
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.
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.
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 methods of assessing dispersal have become increasingly powerful and have superseded direct methods of studying dispersal. Although now less popular, direct methods of studying dispersal ...remain important tools for understanding the evolution of dispersal. Here, we use data from Siberian jays Perisoreus infaustus, a group-living bird species, to compare natal dispersal distances and rates using visual mark–recapture, radio-tracking and microsatellite data. Siberian jays have bimodal natal dispersal timing; socially dominant offspring remain with their parents for up to 5 years (delayed dispersers), while they force their subordinate brood mates to leave the parental territory at independence (early dispersers). Early dispersers moved about 9,000 m (visual mark–recapture, radio-tracking) before settling in a group as a non-breeder. In contrast, delayed dispersers moved about 1,250 m (visual mark–recapture only) and mainly moved to a breeding opening. Dispersal distances were greater in managed habitat compared to natural habitat for both early and delayed dispersers. Molecular estimates based on 23 microsatellite loci and geographical locations supported distance estimates from the direct methods. Our study shows that molecular methods are at least 22 times cheaper than direct methods and match estimates of dispersal distance from direct methods. However, molecular estimates do not give insight into the behavioural mechanisms behind dispersal decisions. Thus, to understand the evolution of dispersal, it is important to combine direct and indirect methods, which will give insights into the behavioural processes affecting dispersal decisions, allowing proximate dispersal decisions to be linked to the ultimate consequences thereof.
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.
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•Pre- and post-treatment populations were genetically similar despite 89% reduction.•Pairwise gene and SNP frequency differentiation identified in chr4.•Difference in nucleotide diversity found in ivermectin QTL region in chr5.
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