scholarly journals Development of high throughput method for the analysis of anthelmintic resistance allele frequencies in field populations of gastrointestinal nematodes

2019 ◽  
Author(s):  
Neil D. Sargison ◽  
Madison MacLeay ◽  
Alison A. Morrison ◽  
David J. Bartley ◽  
Mike Evans ◽  
...  
Keyword(s):  
Anthelmintic Resistance ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Allele Frequencies ◽  
Management Tool ◽  
Resistance Allele ◽  
Resistance Mutations ◽  
Field Samples ◽  
The Impact ◽  
Β Tubulin

AbstractDrug resistant helminths have become a major cause of poor health and production in sheep and goats, and there is a need for diagnostic markers and tools to determine the frequency of resistance alleles in field parasite populations. Gastrointestinal nematode resistance to benzimidazole drugs is caused by a mutation in one of three positions on the isotype 1 β-tubulin locus, and in the absence of markers for resistance to other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field gastrointestinal nematode populations can be impractical using conventional molecular methods, which may be error prone or lack sensitivity at low levels of resistance. Here, we report the development of a novel method based on an Illumina Mi-seq deep amplicon sequencing platform; to sequence the isotype 1 β-tubulin locus of the small ruminant gastrointestinal nematode,Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias in the isotype 1 β-tubulin locus, comparing the results of Illumina Mi-seq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible L3. Finally, we applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic drug resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.

scholarly journals A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 β-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA

2021 ◽  
Author(s):  
Livio M. Costa-Junior ◽  
Umer N. Chaudhry ◽  
Philip J. Skuce ◽  
Seamus Stack ◽  
Neil D. Sargison
Keyword(s):  
Genetic Susceptibility ◽  
Management Practices ◽  
Anthelmintic Resistance ◽  
Lamp Assay ◽  
Gastrointestinal Nematode ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Snp Typing ◽  
Field Samples ◽  
Β Tubulin

AbstractDevelopment of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples.

Multi-species Grazing for Control of Gastrointestinal Nematodes – a Review

2021 ◽  
Vol 99 (Supplement_2) ◽  
pp. 36-37
Author(s):  
Joan M Burke ◽  
James E Miller
Keyword(s):  
Anthelmintic Resistance ◽  
Small Ruminants ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Copper Tolerance ◽  
Resistant Species ◽  
Complex Interactions ◽  
Teladorsagia Circumcincta ◽  
Management Scheme ◽  
Time Required

Abstract Multi-species grazing, alternate grazing of cattle with sheep or goats, offers benefits to gastrointestinal nematode (GIN) control as GIN species differ between hosts. In the Southeastern United States, common GIN species of cattle include Cooperia spp., Ostertagia ostertagi, Haemonchus spp., whereas species of sheep and goats are Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp., Cooperia curticei, and Oesophagostomum spp. Anthelmintic resistance is widely prevalent in small ruminants, and is an increasing problem in cattle, but remains less prevalent. Thus, it is recommended to farmers to use a multi-faceted approach of GIN control, including multi-species grazing. Because management differs between cattle and small ruminants in regard to copper tolerance in minerals, predator control and fencing, alternate grazing of species has most commonly been used. Other considerations include age or susceptibility to GIN, as young calves may be vulnerable to GIN, but mature cows often have negligible GIN infection. Similarly, weanling lambs and kids are susceptible, and depending on breed, adults range from highly resistant to susceptible. Research indicates that GIN burden of more resistant animals is less influenced by multi-species grazing, and highly susceptible animals will likely need additional measures of control. The more resistant species used in the multi-species grazing may lend more control to the more susceptible. Some research showed reductions in O. ostertagi, but not C. oncophora in cattle during extended grazing of cattle and sheep, and reductions of H. contortus and T. colubriformis in sheep. However, other research reported similar worm burdens. Weight gains were improved in lambs grazed alternately with cattle in some studies, and most studies showed little benefit to cattle. Little has been published on alternate grazing with cattle and goats. The complex interactions, larger plot sizes needed, and time required to complete experiments limits published studies of this important management scheme.

scholarly journals Epidemiology and Multidrug Resistance of Strongyle Nematodes in Ordos Merino Sheep

2020 ◽  
Author(s):  
Ledege Eye ◽  
Jiya Wuen ◽  
Xiuling He ◽  
Buhe Buyin ◽  
Ying Hai ◽  
...  
Keyword(s):  
Anthelmintic Resistance ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Infection Rates ◽  
Merino Sheep ◽  
Predominant Species ◽  
Anthelmintic Efficacy ◽  
Nematode Eggs ◽  
Repeated Use

Abstract Background: Regular whole-flock treatments and long-term repeated use of similar anthelmintics has led to the development of anthelmintic resistance and widespread epidemic of sheep nematodiasis. The current work was carried out to understand the prevalence of gastrointestinal nematodes in Ordos merino sheep and to evaluate the efficacy of currently used anthelmintics. Results: A total of 4014 fresh fecal samples were collected from sheep from March 2017 to April 2019 and fecal nematode eggs were qualitatively and quantitatively analyzed for understanding the prevalence of sheep nematodiasis. The anthelmintic efficacy of currently used drugs was evaluated in naturally infected sheep. The gastrointestinal nematode infection was serious in Ordos merino sheep. The infection rates of nematodes in three consecutive years were 84.3%, 36.9%, and 42.3%, respectively. The Hemonchus contortus and Nematodirus sp. were the dominantly infected species with the infection rates of 84.3% and 65.6% in 2017. Moreover, these species were acquired highly resistance to the Ivermectin, Doramectin, Albendazole and Levamisole. The percentages of FERC were 6.9%, 1.7%, 3.5% and 79.0%, respectively, following single administration. However, Nitroxynil and Closantel showed a strong anthelmintic efficacy on popular dominant species Haemonchus contortus and some other nematodes, while almost no effect on Nematodirus sp. Conclusions: The prevalence of gastrointestinal nematodes in Ordos merino sheep and their resistance to commonly used anthelmintics were comprehensively understood, and the high efficacy drugs on predominant species were screened out. The results will pay a good foundation for developing of reasonable drug use.

scholarly journals PSII-37 Determining the possible anthelmintic properties of allicin and pumpkin seed oil for mitigation of anthelmintic resistance found in gastrointestinal nematodes infecting ruminants

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 217-218
Author(s):  
Collin Hayes ◽  
Elizabeth Benavides ◽  
Malorie Diamond ◽  
Trixie L Wetmore ◽  
Ken Mix
Keyword(s):  
Seed Oil ◽  
Anthelmintic Resistance ◽  
Small Ruminants ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Current Treatment ◽  
Economic Losses ◽  
Ovicidal Activity ◽  
Pumpkin Seed ◽  
Pumpkin Seed Oil

Abstract Anthelmintics are a widely utilized treatment option for gastrointestinal nematodes infecting ruminants. Recently, these parasites have exhibited increasing resistance towards these drugs. Haemonchus controtus, a gastrointestinal nematode commonly found in small ruminants, has developed resistance towards multiple classes of anthelmintics, leading to huge economic losses worldwide. There are few novel drugs currently being developed, but certain plant-derived products have demonstrated anthelmintic properties when administered in various forms to ruminants. By utilizing these plant products, producers have the opportunity to decrease the rate of development of anthelmintic resistance, and prevent eggs from hatching when excreted by infected livestock. Therefore, it is hypothesized that specific plant-derived products will act as ovicidal agents in H. contortus eggs. The objective of this study was to determine the ovicidal activity of allicin (from Allium sativum) and pumpkin seed oil (PSO). H. contortus eggs were extracted from a pooled fecal sample of Boer and Spanish goats naturally infected with H. contortus. The eggs were then exposed to allicin or PSO at different concentrations. Experiments were replicated four times per concentration. To determine ovicidal activity, samples were observed for unhatched eggs and hatched worms for 24 hours. The 1:100 allicin dilution treatment tended (P = 0.053) to inhibit hatching more than the control. Furthermore, any worms that hatched in allicin treatments exhibited limited signs of motility. Pumpkin seed oil does not appear to possess the same ovicidal potency as allicin, but may still have potential as a larvicidal agent. Based on the current results, allicin is a potential ovicidal agent that may be used independently or incorporated with current treatment methods for H. contortus control.

scholarly journals Phenotypic and genotypic approaches for detection of anthelmintic resistant sheep gastrointestinal nematodes from Brazilian northeast

2021 ◽  
Vol 30 (2) ◽  
Author(s):  
José Vilemar de Araújo-Filho ◽  
Wesley Lyeverton Correia Ribeiro ◽  
Weibson Paz Pinheiro André ◽  
Géssica Soares Cavalcante ◽  
Jéssica Maria Leite dos Santos ◽  
...  
Keyword(s):  
Anthelmintic Resistance ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Northeastern Brazil ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Egg Hatch ◽  
Fecal Egg Count ◽  
Phenotypic Tests ◽  
Brazilian Northeast

Abstract The aim of this study was to characterize the anthelmintic resistance (AR) of a sheep gastrointestinal nematode population, named Caucaia, from northeastern Brazil. Phenotypic tests performed were: egg hatch (EHT), larval development (LDT) and fecal egg count reduction (FECRT). Benzimidazoles (BZs) genotypic evaluation was by frequency of single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A, and for levamisole (LEV), by frequency of resistance alleles of Hco-acr-8 gene. The primers were designed specifically for Haemonchus contortus. Effective concentrations 50% (EC50) for BZs (EHT), and for macrocyclic lactones (MLs) and LEV (LDT) were 1.02 µg/mL, 1.81 ng/mL and 0.04 µg/mL, respectively. Resistance ratios for MLs and LEV were 0.91 and 3.07, respectively. FECRT efficacies of BZs, MLs, monepantel (MPTL) and LEV were 52.4; 87.0; 94.5 and 99.6%, respectively. qPCR for BZs demonstrated resistance allele frequencies of 0%, 26.24% and 69.08% for SNPs E198A, F200Y and F167Y, respectively. For LEV, 54.37% of resistance alleles were found. There was agreement between EHT, FECRT and qPCR for BZs, and agreement between LDT and qPCR for LEV. Thus, based on higher sensitivity of qPCR, and phenotypic evaluation, the Caucaia population was considered resistant to BZs, MLs, LEV and suspect for MPTL.

scholarly journals Control measures recommended for goat gastrointestinal nematode infections after analysis of infection dynamics in the semiarid region of Brazil

2020 ◽  
Vol 50 (11) ◽  
Author(s):  
Vanessa Diniz Vieira ◽  
Franklin Riet Correa ◽  
Vinícius Longo Ribeiro Vilela ◽  
Márcia Alves de Medeiros ◽  
Jouberdan Aurino Batista ◽  
...  
Keyword(s):  
Rainy Season ◽  
Anthelmintic Resistance ◽  
Gastrointestinal Nematode ◽  
Parasite Load ◽  
Gastrointestinal Nematodes ◽  
Control Measures ◽  
Annual Rainfall ◽  
Semiarid Region ◽  
Gastrointestinal Helminths ◽  
Lactating Goats

ABSTRACT: This study aimed to determine control measures for gastrointestinal nematodes in goats in the northeastern semiarid after analyzing the dynamics of gastrointestinal helminths during the drought, the evolution of the parasitic load after the first rains and the differences in susceptibility between goats of different categories and ages. Five farms were studied from March 2013 to January 2015. Feces were collected from all goats every month, for fecal egg counts (FECs). No treatment was required on any farm during the dry period. In 2013, with annual rainfall of 265-533 mm, treatments were not necessary during the rainy season. However, in 2014, with rainfall of 604-778 mm, treatments were necessary 60-90 days, after the first rains. On three farms, gastrointestinal nematodes showed multiple anthelmintic resistance. The FECs from lactating goats were significantly higher than from dry and young goats. In conclusion, in the Brazilian semi-arid region (Caatinga biome), it is generally unnecessary to treat grazing goats during the dry season. In the rainy season, the parasite load increases 2-3 months after the first rains. In both, the dry and the rainy season, farmers should monitor their herds by means of FEC or another criterion (anemia or submandibular edema), to determine the need to treat.

scholarly journals The threat of reduced efficacy of anthelmintics against gastrointestinal nematodes in sheep from an area considered anthelmintic resistance-free

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Antonio Bosco ◽  
Jan Kießler ◽  
Alessandra Amadesi ◽  
Marian Varady ◽  
Barbara Hinney ◽  
...  
Keyword(s):  
Southern Italy ◽  
Anthelmintic Resistance ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Minor Role ◽  
Macrocyclic Lactones ◽  
On Farm ◽  
Pooled Samples

Abstract Background The worldwide increased difficulty to combat gastrointestinal nematode (GIN) infection in sheep, due to progressing anthelmintic resistance (AR), calls for an enhanced and standardized implementation of early detection of AR. This study provides a snapshot of the current AR status against benzimidazoles and macrocyclic lactones in southern Italy, generated with standardized techniques. Methods On 10 sheep farms, the efficacy of albendazole (ALB) and either eprinomectin (EPR) or ivermectin (IVM) was evaluated based on the faecal egg count reduction test (FECRT) performed with the Mini-FLOTAC. For each tested drug, 40 sheep were rectally sampled at D0 and sampled again 14 days after the treatment (D14). The FECRT was calculated from individual samples and pooled samples which consist of 5 individual samples. Efficacy was classified as ‘reduced, ‘suspected’ and ‘normal’. Coprocultures were set for D0 and D14 faecal samples of each group. From farms with FECR < 95%, an in vitro egg hatch test (EHT) and a follow-up FECRT using fenbendazole (FBZ) were conducted. Results Based on the FECR, high efficacy (from 95.7% to 100%) was observed for ALB and IVM in eight farms (Farms 3–10). On Farm 1 and Farm 2, the efficacy for the macrocyclic lactones was classified as ‘normal’, but ‘reduced’ efficacy was observed for ALB on Farm 1 (FECR = 75%) and ‘suspected’ efficacy on Farm 2 (FECR = 93.3%) with the predominant GIN genus Trichostrongylus followed by Haemonchus at D14. The FEC results of pooled samples strongly correlated with those of individual samples, for FEC at D0 (rs = 0.984; P < 0.0001) and at D14 (rs = 0.913; P < 0.0001). The classifications of efficacy in Farm 1 (FECR = 86.0%) and Farm 2 (FECR = 93.0%) in the follow-up FECRT with FBZ coincide with the main FECRT trial. The in vitro EHT confirmed AR in both farms (Farm 1: 89%; Farm 2: 74%). Conclusions In regions like southern Italy, where the negative impacts from AR have played a minor role, efficient monitoring of AR is important in order to evaluate potential risks and being able to promptly respond with countermeasures.

scholarly journals Anthelmintic resistance in gastrointestinal nematodes in sheep raised under mountain farming conditions in Northern Italy

2019 ◽  
Vol 6 (1) ◽  
pp. e000332 ◽  
Author(s):  
Christian Lambertz ◽  
Ioanna Poulopoulou ◽  
Kunlayaphat Wuthijaree ◽  
Matthias Gauly
Keyword(s):  
Macrocyclic Lactone ◽  
Anthelmintic Resistance ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Northern Italy ◽  
Controlled Conditions ◽  
Oral Formulations ◽  
Wide Range ◽  
High Prevalence ◽  
Faecal Egg Count

Anthelmintic resistance (AR) in sheep raised under mountain farming conditions in South Tyrol (Northern Italy) was assessed on eight farms (n=99 animals). A faecal egg count reduction (FECR) test was done after routine anthelmintic treatments. Furthermore, on 27 farms (n=306 animals), a FECR test was conducted after oral formulations of a macrocyclic lactone (ML), benzimidazole (BZ) (partly in combination with salicylanilide (SA)) or a combination of imidazothiazole and SA were applied under controlled conditions on the same farm. Following routine treatments, three of five ML-treated flocks showed an adequate efficacy, while the other two reached a FECR of only around 75 per cent. A wide range of gastrointestinal nematode genera were identified in one flock following the treatment. From the three BZ-treated flocks, only one showed an adequate FECR, both other farms reached 68 per cent and 84 per cent, respectively. Under controlled conditions, FECR ranged between 77 per cent and 81 per cent indicating AR for all the applied anthelmintics. Trichostrongylus species, Teladorsagia species and Haemonchus species were identified after ML treatment, Teladorsagia species after BZ treatment and Trichostrongylus species and Haemonchus species after combined BZ and SA application. Taking into consideration that underdosing might have affected results of the routine treatments, a high prevalence of AR was found in sheep under mountain farming conditions.

scholarly journals High Predatory Capacity of a Novel Arthrobotrys oligospora Variety on the Ovine Gastrointestinal Nematode Haemonchus contortus (Rhabditomorpha: Trichostrongylidae)

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 815
Author(s):  
Fabián Arroyo-Balán ◽  
Fidel Landeros-Jaime ◽  
Roberto González-Garduño ◽  
Cristiana Cazapal-Monteiro ◽  
Maria Sol Arias-Vázquez ◽  
...  
Keyword(s):  
Haemonchus Contortus ◽  
Anthelmintic Resistance ◽  
Three Dimensional ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Control Group ◽  
Arthrobotrys Oligospora ◽  
Aerial Hyphae ◽  
Conidia Production ◽  
Predatory Capacity

With the worldwide development of anthelmintic resistance, new alternative approaches for controlling gastrointestinal nematodes in sheep are urgently required. In this work, we identified and characterized native nematode-trapping fungi. We collected seven isolates of fungi with the capacity to form adhesive, three-dimensional networks as the main mechanism to capture, kill, and consume nematodes. The nematode-trapping fungi were classified into two groups; the first group includes the R2-13 strain, showing faster growth, abundant aerial hyphae, scarce conidia production, bigger conidia, and it formed a clade with Arthrobotrys oligospora sensu stricto. The second comprises the A6, A12, A13, R2-1, R2-6, and R2-14 strains, showing a growth adhering to the culture medium, forming little aerial hyphae, smaller conidia, and these formed a sister clade to A. oligospora. Except for the R2-6 strain, conidia production was induced by light. In all the strains, the predatory capacity against the sheep gastrointestinal nematode Haemonchus contortus was greater than 58% compared with the control group. The A6 and A13 strains were the most active against the infective H. contortus third instar (L3) larvae, with an average capture capacity of 91%. Altogether, our results support evidence for a novel A. oligospora variety with high nematode-trapping activity and promissory in helminthic control.

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