| 1. |
- Areskog, Marlene, et al.
(författare)
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Limited efficacy of pour-on anthelmintic treatment of cattle under Swedish field conditions
- 2013
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 3, s. 129-134
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Tidskriftsartikel (refereegranskat)abstract
- A study on the effect of topical macrocyclic lactones (ML) against gastrointestinal nematodes (GIN) in Swedish first season grazing cattle (FSG) was performed during the grazing seasons of 2009 and 2010. Herds were recruited through farming press and both dairy and beef cattle farms were invited. A questionnaire revealed that 64% of participating farmers dewormed their animals in previous years, and of these 76% used topical formulations with ML. Four to six weeks after turnout, 107 (2009) and 64 (2010) farmers sent in individual faecal samples from 6-10 FSG. Faecal egg counts (FEC) were determined by the FECPAK (R)-method in 2009 and the McMaster-method in 2010, when also larvae were cultured. Average FEC of >= 100 eggs per gram faeces (EPG) was seen in 39% of the herds in 2009 and 42% in 2010 and with arithmetic means of 258 +/- 110 and 252 +/- 350 EPG, respectively. Interestingly, FSG in dairy and beef herds had similar mean FEC. In herds with mean FEC of >= 100 EPG, farmers dewormed all FSG in the tested grazing group with ivermectin (IVM) or doramectin (DOR) pour-on. In 2009, 33 (31%), and in 2010, 26 (40%) of the herds were retested 7-16 days post treatment. Mean reduction was 89% and 88%, respectively, and in only 12 (36%) and 10 (38%) herds it was >= 95%. Beef herds had mean reductions similar to those of the dairy herds. No significant difference (P = 0.66) in reduction was seen between the groups treated with three different pour-on formulations, nor was there any correlation between the previous year's usage of anthelmintics and the efficacy. Larvae from post-treatment cultures analysed in 2010 with a species-specific ITS2 qPCR showed that Cooperia oncophora was the predominant species after deworming. Four (15%) groups also harboured surviving Ostertagia ostertagi post treatment. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
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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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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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| 5. |
- 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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| 6. |
- Elmahalawy, Safaa, et al.
(författare)
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Genetic variants in dyf-7 validated by droplet digital PCR are not drivers for ivermectin resistance in Haemonchus contortus
- 2018
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 8, s. 278-286
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to ivermectin (IVM) in the nematode Haemonchus contortus in small ruminants is an increasing problem throughout the world. Access to molecular diagnostics will allow early detection of IVM resistance, which in turn can limit the spread of resistant isolates. One candidate gene which has recently been suggested as a marker for IVM resistance is that for dye-filling protein (dyf-7). In this study, we critically investigated the suitability of A141G and G153T single nucleotide polymorphisms (SNP) of dyf-7 as a marker in larval cultures collected from sheep farms in Sweden, involving several isolates for which resistance status had been characterised by the faecal egg count reduction test (FECRT). Initially, we designed dyf-7 primers from a worldwide collection of adult Haemonchus contortus DNA. With the sequence data, we created a haplotype network. We then optimised and used the same sets of primers and probes in a droplet digital PCR (ddPCR) assay for precise quantification of dyf-7 allele frequencies in pre-and post-anthelmintic treatment faecal larval cultures. The fractional abundance (FA) of the mutant SNP was within the range 7.8 and 31%. However, the FA was generally stable in samples collected from the same farms, even though they were obtained on different occasions up to 25 months apart. There was also no indication that the level of IVM resistance as measured by the faecal egg count reduction test was higher on farms with high FA. Furthermore, by comparing FA in samples from the same farms pre-and post-IVM treatment, we found no evidence of a correlation between dyf-7 and level of IVM resistance. Based on these results, dyf-7 is not a suitable marker for field testing of IVM resistance in H. contortus.
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| 7. |
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| 8. |
- Hedberg Alm, Ylva, et al.
(författare)
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Demonstration of reduced efficacy against cyathostomins without change in species composition after pyrantel embonate treatment in Swedish equine establishments
- 2023
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - 2211-3207. ; 23, s. 78-86
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Tidskriftsartikel (refereegranskat)abstract
- Consisting of approximately 50 different species, the cyathostomin parasites are ubiquitous in grazing horses. Coinfection 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.
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| 9. |
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| 10. |
- Martin, Frida, et al.
(författare)
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Exploring the beta-tubulin gene family in a benzimidazole-resistant Parascaris univalens population
- 2021
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 17, s. 84-91
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Tidskriftsartikel (refereegranskat)abstract
- 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 13-tubulin gene have been correlated to BZ resistance, but little is known about the 13-tubulin genes and their possible involvement in BZ resistance in P. univalens and other ascarids. Previously two 13-tubulin genes have been identified in P. univalens. In this study, we present five additional 13-tubulin genes as well as the phylogenetic relationship of all seven genes to 13-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 13-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 13-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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| 11. |
- Novobilsky, Adam, et al.
(författare)
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Assessment of flukicide efficacy against Fasciola hepatica in sheep in Sweden in the absence of a standardised test
- 2016
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 6, s. 141-147
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Tidskriftsartikel (refereegranskat)abstract
- Anthelmintic resistance (AR) toFasciola hepaticais emerging worldwide. Recently, AR to the adulticide compound albendazole (ABZ) was shown in Argentina and Spain. In Sweden, ABZ treatment failure againstF.hepaticawas first reported in sheep in 2012. The present study tested the efficacy of ABZ and triclabendazole (TCBZ) in sheep naturally infected withF.hepaticausing a combination of three different diagnostic methods: faecal egg counts (FEC), coproantigen ELISA (cELISA) andFasciolaegg hatch test (FEHT). Two deworming trials, in November 2014 and January 2015, were performed on two sheep farms (farms A and B) in south-western Sweden. Except ABZ in November, treatment with ABZ or TCBZ achieved sufficient efficacy (97–100%) against adultF.hepaticaon farm A. In contrast, ABZ treatment failed in the sheep flock on farm B, despite low initial faecal egg output. On farm B, ABZ efficacy based on FEC was 67% (95% CI: 35–84) and four of eight ewes tested were coproantigen-positive 21 days post-treatment. Ovicidal activity of ABZ againstFasciolaeggs in isolates from both farms and one additional bovine isolate were tested by FEHT to exclude the presence of juvenile flukes and other factors such as dosing failure and poor quality of drug product. Irrespective of drug trial, data from FEHT showed significantly lower ovicidal activity of ABZ for the ovine farm B isolate than for the isolate from farm A. This confirms that the low efficacy of ABZ in sheep flock B was associated with ABZ resistance. Overall, the usefulness of three complementary methods for detection of ABZ resistance in the field was demonstrated.
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| 12. |
- Novobilsky, Adam, et al.
(författare)
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First report of closantel treatment failure against Fasciola hepatica in cattle
- 2015
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Ingår i: International Journal for Parasitology: Drugs and Drug Resistance. - : Elsevier BV. - 2211-3207. ; 5, s. 172-177
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Tidskriftsartikel (refereegranskat)abstract
- Control of Fasciola hepatica infection in livestock is based on annual treatment using flukicides such as triclabendazole, albendazole and closantel. However, triclabendazole resistant F. hepatica populations are emerging worldwide and resistance is emerging to albendazole, whereas it has until now never been described for closantel. In Sweden, a topical formulation containing a combination of closantel and ivermectin (Closamectin Pour On) has been registered for use in cattle only since 2011. This study evaluated the efficacy of closantel against F. hepatica in naturally infected beef cattle using both coproantigen and faecal egg count reduction tests.Faecal egg counts (FEC) and coproantigen ELISA examinations were conducted in February 2014 in three beef cattle herds (A, B, C) in south-western Sweden. On each farm, 10 F. hepatica coproantigen-positive and F. hepatica egg-positive animals were allocated after 12-16 weeks of housing into groups and treated topically with a minimum of 20 mg closantel per kg body weight. Faecal samples were collected from selected animals on 0, 7 and 21 day post-treatment (PT).Based on FEC, closantel efficacy 21 days PT was 72% (95% CI: 65-77%) and 97% (95% CI: 95-98%) on farms A and B, respectively. No FEC reduction at all was observed on farm C. In total, 4, 1 and 6 animals remained coproantigen-positive at 21 days PT on farms A, B and C, respectively.Closantel treatment failure was confirmed on two of the farms. As the animals were housed 12 -16 weeks before treatment and thereafter during the entire study, failure due to the presence of juvenile flukes was excluded. Although the cause of closantel failure currently remains unclear, development of resistance or/and absorption failure of topical administration should be considered. To our knowledge, this is the first report of closantel treatment failure against F. hepatica in cattle. (C) 2015 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.
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| 13. |
- Su, Qiao, et al.
(författare)
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Transcriptomic analysis of albendazole resistance in human diarrheal parasite Giardia duodenalis
- 2023
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Ingår i: International Journal for Parasitology. - : Elsevier BV. - 2211-3207. ; 22, s. 9-19
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Tidskriftsartikel (refereegranskat)abstract
- Benzimidazole-2-carbamates (BZ, e.g., albendazole; ALB), which bind 13-tubulin to disrupt microtubule polymerization, are one of two primary compound classes used to treat giardiasis. In most parasitic nematodes and fungi, BZ-resistance is caused by 13-tubulin mutations and its molecular mode of action (MOA) is well studied. In contrast, in Giardia duodenalis BZ MOA or resistance is less well understood, may involve target-specific and broader impacts including cellular damage and oxidative stress, and its underlying cause is not clearly determined. Previously, we identified acquisition of a single nucleotide polymorphism, E198K, in 13-tubulin in ALBresistant (ALB-R) G. duodenalis WB-1B relative to ALB-sensitive (ALB-S) parental controls. E198K is linked to BZ-resistance in fungi and its allelic frequency correlated with the magnitude of BZ-resistance in G. duodenalis WB-1B. Here, we undertook detailed transcriptomic comparisons of these ALB-S and ALB-R G. duodenalis WB-1B cultures. The primary transcriptional changes with ALB-R in G. duodenalis WB-1B indicated increased protein degradation and turnover, and up-regulation of tubulin, and related genes, associated with the adhesive disc and basal bodies. These findings are consistent with previous observations noting focused disintegration of the disc and associated structures in Giardia duodenalis upon ALB exposure. We also saw transcriptional changes with ALB-R in G. duodenalis WB-1B consistent with prior observations of a shift from glycolysis to arginine metabolism for ATP production and possible changes to aspects of the vesicular trafficking system that require further investigation. Finally, we saw mixed transcriptional changes associated with DNA repair and oxidative stress responses in the G. duodenalis WB-1B line. These changes may be indicative of a role for H2O2 degradation in ALB-R, as has been observed in other G. duodenalis cell cultures. However, they were below the transcriptional fold-change threshold (log2FC > 1) typically employed in transcriptomic analyses and appear to be contradicted in ALB-R G. duodenalis WB-1B by down-regulation of the NAD scavenging and conversion pathways required to support these stress pathways and up-regulation of many highly oxidation sensitive iron-sulphur (FeS) cluster based metabolic enzymes.
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