The proof is in the poo-ding: Benefits of the longitudinal molecular surveillance of drug resistance demonstrated in a New South Wales cattle herd.

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019-2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes.

A mixed amplicon metabarcoding and sequencing approach for surveillance of drug resistance to levamisole and benzimidazole in Haemonchus spp.

Anthelmintic-resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We describe a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV)-resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ)-resistant SNPs within ß-tubulin isotype-1 and the internal transcribed spacer-2 (ITS-2) nemabiome. This constitutes the first known multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing (WGS) of LEV-resistant Haemonchus contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an effective anthelmintic class for small ruminants in New South Wales, Australia. The mixed amplicon metabarcoding approach described herein paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector.

Refugia or reservoir? Feral goats and their role in the maintenance and circulation of benzimidazole-resistant gastrointestinal nematodes on shared pastures.

Gastrointestinal nematodes threaten the productivity of grazing livestock and anthelmintic resistance has emerged globally. It is broadly understood that wild ruminants living in sympatry with livestock act as a positive source of refugia for anthelmintic-susceptible nematodes. However, they might also act as reservoirs of anthelmintic-resistant nematodes, contributing to the spread of anthelmintic resistance at a regional scale. Here, we sampled managed sheep and cattle together with feral goats within the same property in New South Wales, Australia. Internal transcribed spacer 2 (ITS-2) nemabiome metabarcoding identified 12 gastrointestinal nematodes (Cooperia oncophora, Cooperia punctata, Haemonchus contortus, Haemonchus placei, Nematodirus spathiger, Ostertagia ostertagi, Teladorsagia circumcincta, Oesophagostomum radiatum, Oesophagostomum venulosum, Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus rugatus). Isotype-1 ß-tubulin metabarcoding targeting benzimidazole resistance polymorphisms identified 6 of these nematode species (C. oncophora, C. punctata, H. contortus, H. placei, O. ostertagi and T. circumcincta), with the remaining 3 genera unable to be identified to the species level (Nematodirus, Oesophagostomum, Trichostrongylus). Both ITS-2 and ß-tubulin metabarcoding showed the presence of a cryptic species of T. circumcincta, known from domestic goats in France. Of the gastrointestinal nematodes detected via ß-tubulin metabarcoding, H. contortus, T. circumcincta, Nematodirus and Trichostrongylus exhibited the presence of at least one resistance genotype. We found that generalist gastrointestinal nematodes in untreated feral goats had a similarly high frequency of the benzimidazole-resistant F200Y polymorphism as those nematodes in sheep and cattle. This suggests cross-transmission and maintenance of the resistant genotype within the wild ruminant population, affirming that wild ruminants should be considered potential reservoirs of anthelmintic resistance.

In vitro effect of burned pasture soil on eggs and free-living larvae of ruminant gastrointestinal nematodes.

Gastrointestinal nematodes are the most expensive agent of disease currently facing the livestock production industry. Spending the beginning of their life cycle as eggs and free-living larvae, nematodes are vulnerable to a multitude of external environmental factors. Fire is a naturally occurring force of nature that has both destructive and reconstructive effects on soil characteristics which nematode stages rely on for survival. The aim of this project was to evaluate in vitro the effect of burned pasture soil (200 °C and 500 °C) on the free-living stages of ruminant nematodes. We tested the effect of burned soil on the ability of eggs to hatch and produce infective larvae, and then tested survival of infective larvae within burned soil. Adding burned soil (500 °C) to larval cultures improved larval yield compared to larval cultures containing raw soil or soil burned at a lower heat (200 °C), and raw soil improved longer term survival of infective larvae. We were able to recover significantly more larvae from samples with low soil content either as raw or soil burned at 200 °C, when compared with samples with soil burned at 500 °C. This study has shown that the survival of gastrointestinal nematodes at the L3 stage is negatively impacted by the addition of soil burned at 500 °C. Although this temperature is closest to that of a medium intensity wildfire, which is a typically destructive process in agriculture, it reduces the number of infective GIN larvae available for animals to ingest. These experiments enable us to address in vitro if post-fire soil conditions alter the number of infective larvae available on pasture, and thus the infectivity of the pasture to livestock.

Post-acute long-term care COVID-19 medication optimization survey: Informing medication management initiatives.

The Medication Management Guide (MMG) provides guidance on strategies to optimize medication management in PA-LTC and simplify administration to reduce the transmission risk of COVID-19. The objectives of this study were to evaluate the utility of the MMG, determine the barriers and facilitators of the MMG implementation in PALTC sites to help inform future research and initiatives. Individuals who accessed MMG during the pandemic (April 2020-March 2021) were contacted to elicit feedback on this tool via an online survey. The survey response rate was 7.7% (156/2,018) after three rounds of emails. Respondents consisted of 31% (n=49) pharmacists, 27% (n=42) physicians, 11% (n=18) nurses, and 12% (n=19) nurse practitioners. The "Other" respondents (11%, n=17) included dieticians (n=4), physician assistants (n=3), pharmacy technicians (n=3), students (n=1), consultants (n=1), and educators (n=2). From these respondents, 77% (n=122) took tactics to optimize medications at their facilities during COVID-19.

A new diagnostic approach to fast-track and increase the accessibility of gastrointestinal nematode identification from faeces: FECPAKG2 egg nemabiome metabarcoding.

Effective gastrointestinal nematode management in livestock industries is becoming increasingly difficult due to the rise of anthelmintic resistance and changes in the temporal and geographical distribution of major gastrointestinal nematodes. Underpinning the response to these challenges is the need for a fast-tracked diagnostic identification technique, making it easier for livestock producers to make informed gastrointestinal nematode management decisions. The traditional 'gold-standard' approach, larval culture followed by morphological differentiation, is laborious and potentially inaccurate. We developed a new diagnostic approach to identify gastrointestinal nematodes that integrates a remote-location digital faecal egg count platform, FECPAKG2, with internal transcribed spacer 2 (ITS2) nemabiome metabarcoding. The technique involves a quick and simple protocol to harvest concentrated strongyle eggs from the FECPAKG2 cassette utilising a repurposed pipette tip, followed by DNA isolation and Illumina next generation amplicon sequencing. The gastrointestinal nematode compositions and alpha diversity generated by our FECPAKG2 egg nemabiome metabarcoding approach was not significantly different to traditional morphological larval differentiation and nemabiome metabarcoding of larval and faecal samples. We demonstrated that storing FECPAKG2 harvested eggs in either DNA isolation lysis buffer or 80% ethanol (v/v) had no impact on gastrointestinal nematode identification outcomes for at least 60 days; enabling the transport of biological samples from their remote origins to a molecular diagnostic facility for nemabiome metabarcoding, in the absence of a cold chain. We discovered that sustained gastrointestinal nematode egg embryonation in the lysis buffer storage solution lead to higher yields of DNA compared with ethanol-stored gastrointestinal nematode eggs or faeces with gastrointestinal nematode eggs. Taking advantage of an already well-established platform such as FECPAKG2, and providing the livestock producers that use it with the option to identify gastrointestinal nematodesin their samples and contribute to large-scale gastrointestinal nematode distribution and/or anthelmintic resistance surveys, is an important future direction for the FECPAKG2 egg nemabiome metabarcoding approach.

What the fox? Cryptic Eucoleus [Capillaria] sp. in the respiratory tract of a cat from Australia.

Eucoleus aerophilus (syn. Capillaria aerophila) is a zoonotic trichuroid nematode parasite of dogs, cats and wild carnivores with a global distribution. The main reservoir species in Europe is the red fox, where it has been detected in up to 97% of animals surveyed. Despite the burgeoning feral cat and fox population in Australia, there is a paucity of information about the occurrence and molecular identity of E. aerophilus in these species. The occurrence of a gravid capillariid nematode in the bronchoalveolar lavage of a 12-week-old kitten from central New South Wales (NSW), with a history of lower respiratory signs that had been non-responsive to treatment with metronidazole or amoxicillin-clavulanic acid, prompted a detailed morphological and molecular investigation into the identity of the parasite including the examination of opportunistically-collected red fox tracheas from the region. A combination of PCR and next-generation sequencing yielded the first complete mitochondrial genome of E. aerophilus, collected from the red foxes in Australia, and revealed the presence of a cryptic Eucoleus [Capillaria] sp. in the kitten from central NSW. The protein-coding genes were 14-23% and 5-30% different (pairwise distance) at the nucleotide and amino acid sequences, respectively, which suggests the occurrence of a genetically distinct Eucoleus sp. lineage in Australia. The phylogenetic analysis using both Bayesian and the maximum likelihood methods demonstrated monophyly of the Trichuridae plus Capillariidae using amino acid sequences encoded by mitochondrial DNA. Analysis based on complete SSU rDNA sequences of Eucoleus [Capillaria] sp. and E. aerophilus placed them within Eucoleus spp. from the respiratory tract of their hosts. While Eucoleus spp. may not currently pose a significant threat to companion animals in Australia, their status as a recently emerged pathogen in Europe suggest that greater efforts should be made to understand the distribution and epidemiology of these parasites.

Which larvae are they? Use of single larva for the molecular confirmation of Cooperia pectinata and Cooperia punctata in Australian cattle.

In Australia, Cooperia spp. are often overshadowed by parasites believed to be more pathogenic production-limiting nematodes. A rise in anthelmintic resistance and reports of reduced growth rates attributed to infection with Cooperia spp. in Europe increases the need to be able to monitor the presence of C. pectinata, C. punctata and C. oncophora in Australian cattle. Here, we present the first molecular confirmation of C. pectinata and C. punctata in Australian cattle using ITS2 rDNA and COXII mtDNA. Cultured larvae were morphologically differentiated to the genus level with the aid of iodine solution and their DNA was screened using a cattle nematode MT-PCR panel. By isolating individual iodine stained and morphologically identified nematode larvae, we demonstrated the presence of C. pectinata and C. punctata using a generic ITS2 rDNA qPCR assay following DNA amplicon sequencing. A novel suite of COXII mtDNA species/genus-specific PCR assays for Cooperia speciation from complex nematode samples enabled us to detect all three species (C. oncophora, C. pectinata, C. punctata) in Australia cattle samples. Our approach, utilising traditional techniques coupled with the manipulation of individual nematode larvae, provides a foundation for the inclusion of Cooperia spp. into existing high throughput molecular diagnostic panels for cattle nematode surveillance.

Oritavancin Retains a High Affinity for a Vancomycin-Resistant Cell-Wall Precursor via Its Bivalent Motifs of Interaction.

Despite its potent antibacterial activities against drug-resistant Gram-positive pathogens, oritavancin remains partially understood with respect to its primary mode of hydrogen bond interaction with a cell-wall peptide regarding the role of its lipophilic 4'-chlorobiphenyl moiety. Here we report a surface plasmon resonance (SPR) study performed in two cell-wall model surfaces, each prepared by immobilization with a vancomycin-susceptible Lys-d-Ala-d-Ala or vancomycin-resistant Lys-d-Ala-d-Lac peptide. Analysis of binding kinetics performed on the peptide surface showed that oritavancin bound ∼100-1000-fold more tightly than vancomycin on each model surface. Ligand competition experiments conducted by SPR and fluorescence spectroscopy provided evidence that such affinity enhancement can be attributed to its 4'-chlorobiphenyl moiety, possibly through a hydrophobic interaction that led to a gain of free energy with a contribution from enthalpy as suggested by a variable-temperature SPR experiment. On the basis of these findings, we propose a model for the bivalent motifs of interaction of oritavancin with cell-wall peptides, by which the drug molecule can retain a strong interaction even with the vancomycin-resistant peptide. In summary, this study advances our understanding of oritavancin and offers new insight into the significance of bivalent motifs in the design of glycopeptide antibiotics.

Observations on the biology, epidemiology and economic relevance of rumen flukes (Paramphistomidae) in cattle kept in a temperate environment.

There is concern about the probable recent introduction, increased prevalence and potential economic impact of rumen fluke infection of United Kingdom cattle. A study of 339 cattle slaughtered in a Scottish red meat abattoir was undertaken with the aims of describing the prevalence and geographical distribution of rumen fluke infection, estimating its effect on production, and evaluating faecal egg counts (FECs) as a tool to diagnose infection in live animals and study the epidemiology of the disease. The overall proportion of cattle consigned to the abattoir from northern United Kingdom with rumen fluke infection in the forestomachs was 0.29. Rumen flukes were distributed predominantly in the cranial sac of the rumen and adjacent to the reticular groove. Overall, a mean of 213 and median of 44 rumen flukes was identified in the forestomachs of rumen fluke-positive cattle. The mean and median FECs of animals were 26.01 and 5.20 eggs per gram (epg), respectively. There was a significant difference between the mean FECs per rumen fluke of 0.08 and 0.13 epg during summer/autumn and winter sampling periods, respectively. The overall correlation between rumen fluke FECs and the number of flukes in the forestomach was high, albeit lower in the summer/autumn than in the winter period. The sensitivities of rumen fluke FECs for the identification of flukes in the forestomach during the summer/autumn and winter sampling periods were 0.65 and 0.85, respectively. These results will aid in the interpretation of rumen fluke FECs when monitoring cattle health and production and studying the parasite's epidemiology in a temperate environment, thereby informing rational, precise and sustainable disease control.