Worldwide absence of canonical benzimidazole resistance-associated mutations within ß-tubulin genes from Ascaris.

BACKGROUND: The giant roundworm Ascaris is an intestinal nematode, causing ascariasis by infecting humans and pigs worldwide. Recent estimates suggest that Ascaris infects over half a billion people, with chronic infections leading to reduced growth and cognitive ability. Ascariasis affects innumerable pigs worldwide and is known to reduce production yields via decreased growth and condemnation of livers. The predominant anthelminthic drugs used to treat ascariasis are the benzimidazoles. Benzimidazoles interact with ß-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the ß-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the ß-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations. METHODS: To further determine the putative absence of key ß-tubulin polymorphisms, we screened two ß-tubulin isotypes of Ascaris, highly expressed in adult worms. Using adult and egg samples of Ascaris obtained from pigs and humans worldwide, we performed deep amplicon sequencing to look for canonical resistance-associated mutations in Ascaris ß-tubulins. Subsequently, we examined these data in closer detail to study the population dynamics of Ascaris and genetic diversity within the two isotypes and tested whether genotypes appeared to partition across human and pig hosts. RESULTS: In the 187 isolates, 69 genotypes were found, made up of eight haplotypes of ß-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for ß-tubulin isotype A and isotype B, respectively. No evidence of any canonical benzimidazole resistance-associated mutations was found in either human- or pig-derived Ascaris isolates. There was, however, a difference in the genetic diversity of each isotype and distribution of ß-tubulin genotypes between human- and pig-derived Ascaris. Statistical tests of population differentiation show significant differences (p < 0.001) between pig- and human-derived worms; however, more diversity was seen between worms from different populations than worms from different hosts. CONCLUSIONS: Our work suggests an absence of canonical ß-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy.

Sequences Related to Chimay Rhabdovirus Are Widely Distributed in Ixodes ricinus Ticks across England and Wales.

Ticks are the main arthropod vector of pathogens to humans and livestock in the British Isles. Despite their role as a vector of disease, many aspects of tick biology, ecology, and microbial association are poorly understood. To address this, we investigated the composition of the microbiome of adult and nymphal Ixodes ricinus ticks. The ticks were collected on a dairy farm in Southwest England and RNA extracted for whole genome sequencing. Sequences were detected from a range of microorganisms, particularly tick-associated viruses, bacteria, and nematodes. A majority of the viruses were attributed to phlebo-like and nairo-like virus groups, demonstrating a high degree of homology with the sequences present in I. ricinus from mainland Europe. A virus sharing a high sequence identity with Chimay rhabdovirus, previously identified in ticks from Belgium, was detected. Further investigations of I. ricinus ticks collected from additional sites in England and Wales also identified Chimay rhabdovirus viral RNA with varying prevalence in all tick populations. This suggests that Chimay rhabdovirus has a wide distribution and highlights the need for an extended exploration of the tick microbiome in the United Kingdom (UK).

ß-Lactam resistance genes present in UK pheasants and red-legged partridges.

BACKGROUND: Despite considerable recent reductions in antimicrobial use, the UK gamebird industry continues to struggle with production diseases during the rearing season, necessitating significant antibiotic use. This observational study investigated the presence of genes conferring resistance to ß-lactam antibiotics within industry-reared pheasants and red-legged partridges in the UK. METHODS: DNA was extracted from 60 pooled caecal samples collected from gamebirds at routine postmortem examinations during the rearing season. Genes encoding extended-spectrum ß-lactamases (ESBL) were detected by PCR and the corresponding alleles were determined. RESULTS: Over half (53%) of the samples harboured genes encoding blaTEM resistance, with blaSHV identified in 20% of samples. The blaTEM gene was more common on sites with higher antibiotic use, whereas blaSHV was predominantly found in birds younger than 5 weeks. Genotyping of the identified resistance genes revealed the presence of blaTEM-1 , blaSHV-1 and blaSHV-11 alleles. LIMITATIONS: This was a small-scale study conducted at four sites in southern England. CONCLUSION: This is the first report of the presence of ESBL genes in gamebirds, highlighting the need for further research into antimicrobial resistance in UK gamebirds.

Identification of key interactions of benzimidazole resistance-associated amino acid mutations in Ascaris ß-tubulins by molecular docking simulations.

Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to ß-tubulin proteins and destabilising microtubules. Three mutations in the ß-tubulin protein family are associated with BZ resistance. Seven shared ß-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all ß-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two ß-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris ß-tubulin isotype A clusters with helminth ß-tubulins previously shown to interact with BZ. Molecular dynamics simulations using ß-tubulin isotype A highlighted the key role of amino acid E198 in BZ-ß-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with ß-tubulins have been identified and show how mutations can lead to resistance in nematodes.

Metabolites of Cannabis Induce Cardiac Toxicity and Morphological Alterations in Cardiac Myocytes.

Cannabis is one of the most commonly used recreational drugs worldwide. Rrecent epidemiology studies have linked increased cardiac complications to cannabis use. However, this literature is predominantly based on case incidents and post-mortem investigations. This study elucidates the molecular mechanism of Δ9-tetrahydrocannabinol (THC), and its primary metabolites 11-Hydroxy-Δ9-THC (THC-OH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). Treatment of cardiac myocytes with THC-OH and THC-COOH increased cell migration and proliferation (p < 0.05), with no effect on cell adhesion, with higher doses (250-100 ng/mL) resulting in increased cell death and significant deterioration in cellular architecture. Conversely, no changes in cell morphology or viability were observed in response to THC. Expression of key ECM proteins α-SMA and collagen were up-regulated in response to THC-OH and THC-COOH treatments with concomitant modulation of PI3K and MAPK signalling. Investigations in the planarian animal model Polycelis nigra demonstrated that treatments with cannabinoid metabolites resulted in increased protein deposition at transection sites while higher doses resulted in significant lethality and decline in regeneration. These results highlight that the key metabolites of cannabis elicit toxic effects independent of the parent and psychoactive compound, with implications for cardiotoxicity relating to hypertrophy and fibrogenesis.

Characterization of the ß-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance.

BACKGROUND: The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding ß-tubulin proteins, the principal targets of BZ drugs. METHODOLOGY AND PRINCIPAL FINDINGS: First, the ß-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different ß-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. SIGNIFICANCE: This study demonstrated the presence of at least seven ß-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the ß-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of ß-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Cocaine Induces Cytoskeletal Changes in Cardiac Myocytes: Implications for Cardiac Morphology.

Cocaine is one of the most widely abused illicit drugs worldwide and has long been recognised as an agent of cardiac dysfunction in numerous cases of drug overdose. Cocaine has previously been shown to up-regulate cytoskeletal rearrangements and morphological changes in numerous tissues; however, previous literature observes such changes primarily in clinical case reports and addiction studies. An investigation into the fundamental cytoskeletal parameters of migration, adhesion and proliferation were studied to determine the cytoskeletal and cytotoxic basis of cocaine in cardiac cells. Treatment of cardiac myocytes with cocaine increased cell migration and adhesion (p < 0.05), with no effect on cell proliferation, except with higher doses eliciting (1-10 µg/mL) its diminution and increase in cell death. Cocaine downregulated phosphorylation of cofilin, decreased expression of adhesion modulators (integrin-ß3) and increased expression of ezirin within three hours of 1 µg/mL treatments. These functional responses were associated with changes in cellular morphology, including alterations in membrane stability and a stellate-like phenotype with less compaction between cells. Higher dose treatments of cocaine (5-10 µg/mL) were associated with significant cardiomyocyte cell death (p < 0.05) and loss of cellular architecture. These results highlight the importance of cocaine in mediating cardiomyocyte function and cytotoxicity associated with the possible loss of intercellular contacts required to maintain normal cell viability, with implications for cardiotoxicity relating to hypertrophy and fibrogenesis.

Zoonotic transmission of intestinal helminths in southeast Asia: Implications for control and elimination.

Intestinal helminths are extremely widespread and highly prevalent infections of humans, particularly in rural and poor urban areas of low and middle-income countries. These parasites have chronic and often insidious effects on human health and child development including abdominal problems, anaemia, stunting and wasting. Certain animals play a fundamental role in the transmission of many intestinal helminths to humans. However, the contribution of zoonotic transmission to the overall burden of human intestinal helminth infection and the relative importance of different animal reservoirs remains incomplete. Moreover, control programmes and transmission models for intestinal helminths often do not consider the role of zoonotic reservoirs of infection. Such reservoirs will become increasingly important as control is scaled up and there is a move towards interruption and even elimination of parasite transmission. With a focus on southeast Asia, and the Philippines in particular, this review summarises the major zoonotic intestinal helminths, risk factors for infection and highlights knowledge gaps related to their epidemiology and transmission. Various methodologies are discussed, including parasite genomics, mathematical modelling and socio-economic analysis, that could be employed to improve understanding of intestinal helminth spread, reservoir attribution and the burden associated with infection, as well as assess effectiveness of interventions. For sustainable control and ultimately elimination of intestinal helminths, there is a need to move beyond scheduled mass deworming and to consider animal and environmental reservoirs. A One Health approach to control of intestinal helminths is proposed, integrating interventions targeting humans, animals and the environment, including improved access to water, hygiene and sanitation. This will require coordination and collaboration across different sectors to achieve best health outcomes for all.

Divergence across mitochondrial genomes of sympatric members of the Schistosoma indicum group and clues into the evolution of Schistosoma spindale.

Schistosoma spindale and Schistosoma indicum are ruminant-infecting trematodes of the Schistosoma indicum group that are widespread across Southeast Asia. Though neglected, these parasites can cause major pathology and mortality to livestock leading to significant welfare and socio-economic issues, predominantly amongst poor subsistence farmers and their families. Here we used mitogenomic analysis to determine the relationships between these two sympatric species of schistosome and to characterise S. spindale diversity in order to identify possible cryptic speciation. The mitochondrial genomes of S. spindale and S. indicum were assembled and genetic analyses revealed high levels of diversity within the S. indicum group. Evidence of functional changes in mitochondrial genes indicated adaptation to environmental change associated with speciation events in S. spindale around 2.5 million years ago. We discuss our results in terms of their theoretical and applied implications.