| 1. |
- Baltrusis, Paulius, et al.
(författare)
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Estimation of the impact of three different bioinformatic pipelines on sheep nemabiome analysis
- 2022
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Ingår i: Parasites and Vectors. - : Springer Science and Business Media LLC. - 1756-3305. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Baltrusis, Paulius, et al.
(författare)
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Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR
- 2018
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 8, s. 411-419
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Tidskriftsartikel (refereegranskat)abstract
- 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 (T (A) under barG) in the beta 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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| 3. |
- Baltrusis, Paulius, et al.
(författare)
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Genome-wide analysis of the response to ivermectin treatment by a Swedish field population of Haemonchus contortus
- 2022
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 18, s. 12-19
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 4. |
- Baltrusis, Paulius, et al.
(författare)
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Molecular detection of two major gastrointestinal parasite genera in cattle using a novel droplet digital PCR approach
- 2019
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Ingår i: Parasitology Research. - : Springer Science and Business Media LLC. - 0932-0113 .- 1432-1955. ; 118, s. 2901-2907
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Tidskriftsartikel (refereegranskat)abstract
- 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 (TM) or HEX (TM) 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.
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| 5. |
- Baltrusis, Paulius, et al.
(författare)
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The presence and relative frequency detection of the levamisole-resistance-associated S168T substitution in hco-acr-8 in Haemonchus contortus
- 2023
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 21, s. 91-95
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Tidskriftsartikel (refereegranskat)abstract
- 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 le-vamisole 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.
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| 6. |
- Baltrusis, Paulius, et al.
(författare)
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Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus
- 2021
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 15, s. 168-176
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Baltrusis, Paulius, et al.
(författare)
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Utilization of droplet digital PCR to survey resistance associated polymorphisms in the beta tubulin gene of Haemonchus contortus in sheep flocks in Sweden
- 2020
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Ingår i: Veterinary Parasitology. - : Elsevier BV. - 0304-4017 .- 1873-2550. ; 288
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Tidskriftsartikel (refereegranskat)abstract
- 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 beta 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 beta 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 beta 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 +/- 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).
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| 8. |
- Cortazar-Chinarro, Maria, et al.
(författare)
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Sign surveys for red fox (Vulpes vulpes) censuses : evaluating different sources of variation in scat detectability
- 2019
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Ingår i: Mammal Research. - : Springer Science and Business Media LLC. - 2199-2401 .- 2199-241X. ; 64:2, s. 183-190
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Tidskriftsartikel (refereegranskat)abstract
- Wildlife monitoring is essential for assessing the size of species of conservation or management concern. Wildlife monitoring could be undertaken by selected volunteers when limited resources are available. Here, we study the sources of variation in the detectability of red fox scats and to improve the monitoring of the species by means of volunteers who were briefly trained. We selected nine transects to experimentally evaluate the differences in red fox scat detectability between observers, microsite, and scat abundance. Scats were deposited in five microsites, where we simulate low, medium, and high fox scat abundance. We tested the proposed effects by using GLMM analysis incorporating observer effect as a random factor. Our results indicated effects on scat detectability of observer identity and microsite type. We did not observe the effects of scat abundance on detectability of scats. Detectability was lower for scats located in the middle of the transect and on the rocks. We conclude that large-scale red foxes surveys might be conducted using volunteers, but due to the moderate differences detected among observers should be minimized using a training period.
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| 9. |
- Ejenstam, Jesper, et al.
(författare)
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Oxidation studies of Fe10CrAl-RE alloys exposed to Pb at 550 degrees C for 10,000 h
- 2013
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Ingår i: Journal of Nuclear Materials. - : Elsevier BV. - 0022-3115 .- 1873-4820. ; 443:1-3, s. 161-170
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Tidskriftsartikel (refereegranskat)abstract
- Five experimental FeCrAl-RE alloys have been exposed up to 10,000 h in stagnant liquid Pb at 550 C. The test matrix consisted of three 10 wt.% Cr alloys, with an Al content ranging from 4 to 8 wt.% (10Cr-4Al, 10Cr-6Al and 10Cr-8Al), one alloy without additions of reactive elements (RE) (10Cr-6Al), and one reference alloy with 21 wt.% Cr and 5 wt.% Al (21Cr-5Al). The evaluation showed a clear difference in oxidation properties, and it was possible to divide the alloys into two distinct groups. A critical Al concentration in the interval of 4-6 wt.% at the given RE content was required to form a thin protective oxide. However, the absence of RE addition in one of the two 10Cr-6Al alloys resulted in a significant reduction in oxidation resistance, comparable with 10Cr-4Al. None of the alloys were severely corroded, however Pb penetrated to a relatively large extent into the porous oxide of the low performing alloys. A 100 nm thick oxide scale, partly consisting of alumina (Al2O3), was observed for the high performing 10Cr-6Al alloy. The Fe10CrAl-RE alloys showed overall very good corrosion resistance and are hence a promising new alloy category for liquid Pb applications. (C) 2013 Elsevier B.V. All rights reserved.
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| 10. |
- Eklöv, Peter, et al.
(författare)
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The trade-off between foraging activity and predation risk for Rana temporaria in different food environments
- 2000
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Ingår i: Canadian Journal of Zoology. - : Canadian Science Publishing. - 0008-4301 .- 1480-3283. ; 78:5, s. 734-739
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Tidskriftsartikel (refereegranskat)abstract
- We evaluated the trade-off between foraging activity and predation risk for larvae of an anuran species presented with different types of food resources. In the laboratory we examined the effects of benthic and phytoplankton resources and the two types combined on the activity and mortality of larvae of the common frog, Rana temporaria, exposed to predatory larva, Dytiscus marginalis.Predation mortality of tadpoles increased with the duration of the experiment and was highest in the presence of the phytoplankton resource alone. This was explained by a decrease in prey activity in the benthic- and combined-resource treatments when the predator was present, whereas in the phytoplankton treatment, the activity of the tadpoles were similar in the presence or absence of the predator.In the presence of the predator, prey mainly used the bottom of the aquarium in the benthic- and combined-resource treatments. In contrast, in the phytoplankton treatment they used the water column more than in the other treatments. In the presence of the predator, the prey had a lower consumption rate in the phytoplankton treatment than in the other treatments.Predator activity did not change among treatments. The results suggest that the trade-off between foraging activity and predation mortality for anuran larvae is strongly affected by the types of food resources present in the environment.
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