Anthelmintic resistant gastrointestinal helminths have become a major cause of poor health in sheep and goats. Sensitive and specific molecular markers are needed to monitor the genotypic frequency of resistance in field parasite populations. Gastrointestinal nematode resistance to benzimidazole is caused by a mutation in one of three positions within the isotype 1 β-tubulin gene. In the absence of markers for resistance to the other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field nematode populations can be impractical using conventional molecular methods to examine individual parasites; which can be laborious and lack sensitivity in determining low levels of resistance in parasite populations. Here, we report the development of a novel method based on an Illumina MiSeq 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, comparing the results of Illumina MiSeq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible larvae. 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 resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.
Early life performance traits in dairy cattle can have important influences on lifetime productivity. Poor health and fertility are of great economical and animal welfare concern. Circulating miRNAs have been linked to several livestock traits, including resistance to infection, fertility, and muscle development. This study aimed to identify circulating miRNAs associated with early life performance traits and aging in dairy cattle. Plasma samples from female calves (n = 12) identified retrospectively as differing in health, growth, and fertility outcomes prior to first calving were analyzed using PCR arrays detecting 378 miRNAs. Levels of 6 miRNAs differed significantly in calves with poor growth/fertility relative to controls (t-test: P<0.05). Additionally, general(ized) (non)linear mixed models identified 1 miRNA associated with average daily gain until weaning, 22 with live bodyweight at one year of age, 47 with age at first service, and 19 with number of infections before first calving. Out of 85 distinct miRNAs that were associated with at least one animal trait, 9 miRNAs were validated by RT-qPCR in a larger cohort (n = 91 animals), which included longitudinal plasma samples (calf, heifer, first lactation cow). Significant associations (P<0.05) involving individual miRNAs or ratios between miRNAs and early-life performance traits were identified, but did not retain significance after multiple testing adjustment. However, levels of 8 plasma miRNAs (miR-126-3p, miR-127, miR-142-5p, miR-154b, miR-27b, miR-30c-5p, miR-34a, miR-363) changed significantly with age, most prominently during the calf-to-heifer transition. Comparative RT-qPCR analyses of these miRNAs across 19 calf tissues showed that most were ubiquitously expressed. Online database mining identified several pathways involved in metabolism and cell signaling as putative biological targets of these miRNAs. These results suggest that miR-126-3p, miR-127, miR-142-5p, miR-154b, miR-27b, miR-30c-5p, miR-34a, miR-363 are involved in regulating growth and development from birth to first lactation (~2 years old) and could provide useful biomarkers of aging in cattle.
Drug 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.