Biology subjects

Ali, Q.

Publications and source records attributed to Ali, Q..

13 recordsLinked to original sources

bioRxiv2025

Development of a qPCR assay and tremabiome deep amplicon sequencing method for differentiation of fluke species in livestock

BackgroundTrematode parasites, or flukes, are a significant economic threat to ruminant production worldwide. Traditional diagnostic methods rely on egg sedimentation from faeces, a time-consuming methodology lacking sensitivity and specificity. This study aimed to develop and validate two diagnostic methods: firstly, qPCR for accurate identification of Fasciola spp, and secondly, tremabiome, deep amplicon sequencing technique for identifying fluke species using faecal egg DNA. MethodologyTo detect fluke infection primers targeting mitochondrial DNA were repurposed to develop a SYBR Green qPCR diagnostic. For the identification of fluke species, a tremabiome approach was developed. A reference sequence library and taxonomy file were generated for 21 fluke species, enabling species sequence read separation and extracting amplicon sequence variants (ASVs). To validate the qPCR and tremabiome approach, 402 faecal samples were collected from cattle and sheep across the UK. Fluke eggs were isolated by sedimentation, detected by microscopy and qPCR, and tremabiome used to identify fluke eggs to species level. ResultsqPCR demonstrated high analytical sensitivity, detecting Fasciola hepatica DNA down to 19.2fg and F. gigantica down to 6.4fg, with no cross-amplification of other flukes. Tremabiome was able to detect as few as five F. hepatica and Calicophoron daubneyi eggs and identify mixed infections. High levels of co-infection (14.4%) of F. hepatica and C. daubneyi were observed in faecal samples, followed by single infections with C. daubneyi (12.6%) and F. hepatica (3.2%). Notably, tremabiome detected F. hepatica in 20 samples missed by qPCR. Data analysis identified 55 and 32 ASVs for F. hepatica and C. daubneyi, respectively, with phylogenetic clustering within their respective clades. ConclusionThis study developed qPCR assay for Fasciola detection and validated a tremabiome deep amplicon sequencing for fluke species differentiation. These approaches have improved capacity to identify fluke species compared to microscopy and are valuable tools for enhancing fasciolosis surveillance and control. Author SummaryFlukes are flatworm parasites that cause disease domestic and wild animals and humans. The main species infecting cattle and sheep globally are the liver flukes F. hepatica and F. gigantica, with other species including the rumen fluke Calicophoron daubneyi locally important or emerging. Infections result in serious economic losses. The traditional method of diagnosing fluke infection involves observation of eggs in faecal samples under the microscope, but this can be time-consuming and error prone, since the eggs of different species often look similar. In this study, we developed and validated two methods to improve detection: qPCR, a sensitive DNA-based test to identify Fasciola infections, and tremabiome, a DNA sequencing technique that can accurately differentiate between different fluke species. We tested these methods using faecal samples collected from cattle and sheep across the UK. The qPCR could detect small amounts of Fasciola DNA, while tremabiome was more sensitive, identifying different fluke species from as few as five eggs. Our study found that co-infections of F. hepatica and C. daubneyi are common in the UK. The approaches we have developed could be valuable tools for to improve fluke diagnosis and enable better control of this important parasitic disease.

molecular biology↗

bioRxiv2024

Liver-specific Mettl14 deletion induces nuclear heterotypia and dysregulates RNA export machinery

Modification of RNA with N6-methyladenosine (m6A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m6A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m6A writers Mettl3 and Mettl14, nor the m6A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro. To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling.

molecular biology↗

bioRxiv2022

Genetic characterisation of the Theileria annulata cytochrome b locus and its impact on buparvaquone resistance in ruminants

Control of tropical theileriosis depends on the use of a single drug, buparvaquone, the efficacy of which is compromised by the emergence of resistance. The present study was undertaken to improve understanding of the role of mutations conferring buparvaquone resistance in Theileria annulata, and the effects of selection pressures on their emergence and spread. First, we investigated genetic characteristics of the cytochrome b locus associated with buparvaquone resistance in 10 susceptible and 7 resistant T. annulata isolates. The 129G (GGC) mutation was found in the Q01 binding pocket and 253S (TCT) and 262S (TCA) mutations were identified within the Q02 binding pocket. Next, we examined field isolates and identified cytochrome b mutations 129G (GGC), 253S (TCT) and 262S (TCA) in 21/75 buffalo-derived and 19/119 cattle-derived T. annulata isolates, providing evidence of positive selection pressure. Both hard and soft selective sweeps were identified, with striking differences between isolates. For example, 19 buffalo-derived and 7 cattle-derived isolates contained 129G (GGC) and 253S (TCT) resistance haplotypes at a high frequency, implying the emergence of resistance by a single mutation. Two buffalo-derived and 11 cattle-derived isolates contained equally high frequencies of 129G (GGC), 253S (TCT), 129G (GGC)/253S (TCT) and 262S (TCA) resistance haplotypes, implying the emergence of resistance by pre-existing and or recurrent mutations. Phylogenetic analysis further revealed that 9 and 21 unique haplotypes in buffalo and cattle-derived isolates were present in a single lineage, suggesting a single origin. We propose that animal migration between farms is an important factor in the spread of buparvaquone resistance in endemic regions of Pakistan. The overall outcomes will be useful in understanding how drug resistance emerges and spreads, and this information will help design strategies to optimise the use and lifespan of the single most drug use to control tropical theileriosis.

microbiology↗

bioRxiv2022

Genomic signatures of selection associated with benzimidazole drug treatments in Haemonchus contortus field populations

Genome-wide methods offer a powerful approach to detect signatures of drug selection in parasite populations in the field. However, their application to parasitic nematodes has been limited because of both a lack of suitable reference genomes and the difficulty of obtaining field populations with sufficiently well-defined drug selection histories. Consequently, there is little information on the genomic signatures of drug selection for parasitic nematodes in the field and on how best to detect them. This study was designed to address these knowledge gaps using field populations of Haemonchus contortus with well-defined and contrasting benzimidazole-selection histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 ddRADseq markers and used this resource to genotype 20 individual H. contortus adult worms from each of four H. contortus populations: two from closed sheep flocks that had an approximately 20-year history of frequent treatment exclusively with benzimidazole drugs, and two populations with a history of little or no drug treatment. The populations were chosen from the same geographical region to limit population structure in order to maximize the sensitivity of the approach. A clear signature of selection was detected on the left arm of chromosome I centered on the isotype-1 {beta}-tubulin gene in the benzimidazole-selected but not the unselected populations. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I and one near the isotype-2 {beta}-tubulin locus on chromosome II. We examined genetic differentiation between populations, and nucleotide diversity and linkage disequilibrium within populations to define these two additional regions as encompassing five genes and a single gene. We also compared the relative power of using pooled versus individual worm sequence data to detect genomic selection signatures and how sensitivity is impacted by sequencing depth, worm number, and population structure. In summary, this study used H. contortus field populations with well-defined drug selection histories to provide the first direct genome-wide evidence for any parasitic nematode that the isotype-1 {beta}-tubulin gene is the quantitatively most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole-resistance loci of secondary importance. Finally, this study provides an experimental framework to maximize the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes. AUTHOR SUMMARYBenzimidazoles are important anthelmintic drugs for human and animal parasitic nematode control with [~]0.5 billion children at risk of infection treated annually worldwide. Drug resistance is common in livestock parasites and a growing concern in humans. Haemonchus contortus is the most important model parasite system used to study anthelmintic resistance and a significant livestock pathogen. It is also one of the few parasitic nematodes with a chromosomal-scale genome assembly. We have undertaken genome-wide scans using a dense RADseq marker panel on worms from natural field populations under differing levels of benzimidazole selection. We show that there is a single predominant genomic signature of selection in H. contortus associated with benzimidazole selection centred on the isotype-1 {beta}-tubulin locus. We also identify two weaker signatures of selection indicative of secondary drug resistance loci. Additionally, we assess the minimum data requirements for parameters including worm number, sequence depth, marker density needed to detect the signatures of selection and compare individual to Poolseq analysis. This work is the first genome-wide study in a parasitic nematode to provide direct evidence of the isotype-1 {beta}-tubulin locus being the single predominant benzimidazole resistance locus and provides an experimental framework for future population genomic studies on anthelmintic resistance.

genomics↗

bioRxiv2020

Genetic models suggest single and multiple origins of dihydrofolate reductase mutations in Plasmodium vivax

Pyrimethamine was first introduced for the treatment of malaria in Asia and Africa during the early 1980s, replacing chloroquine, and has become the first line of drugs in many countries. In recent years, development of pyrimethamine resistance in Plasmodium vivax has become a barrier to effective malaria control strategies. Here, we describe the use of meta-barcoded deep amplicon sequencing technology to assess the evolutionary origin of pyrimethamine resistance by analysing the flanking region of dihydrofolate reductase (dhfr) locus. The genetic modelling suggests that 58R and 173L single mutants and 58R/117N double mutants are present on a single lineage; suggesting a single origin of these mutations. The triple mutants (57L/58R/117N, 58R/61M/117N and 58R/117N/173L) share the lineage of 58R/117N, suggesting a common origin. In contrast, the 117N mutant is present on two separate lineages suggesting that there are multiple origins of this mutation. We characterised the allele frequency of the P. vivax dhfr locus. Our results support the view that the single mutation of 117N and double mutations of 58R/117N arise commonly, whereas the single mutation of 173L and triple mutations of 57L/58R/117N, 58R/61M/117N and 58R/117N/173L are less common. Our work will help to inform mitigation strategies for pyrimethamine resistance in P. vivax.

genetics↗

bioRxiv2020

Molecular characterization of Fasciola gigantica in Punjab, Pakistan to infer the dispersal route among the neighbouring countries of the Indian subcontinent

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in millions of dollars production losses to the livestock industry. To understand the dispersal origin and the spread patterns of F. gigantica is important for preventing the disease. A total of 53 Fasciola flukes collected from buffalo and goat in the Punjab province of Pakistan, were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference between F. gigantica from buffalo and goats in Pakistan was indicated by the genetic analysis of two distinct mitochondrial markers [NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1)]. Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes were clustered in haplogroup A. Fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, or possibly come from neighbouring Middle East countries. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica regarding the development of sustainable parasite control strategies.

genetics↗

bioRxiv2020

Contrasting population genetics of cattle- and buffalo- derived Theileria annulata causing tropical theileriosis

The present study was designed to improve understanding of Theileria annulata in sympatric water buffalo and cattle in the Punjab province of Pakistan. The prevalence of tropical theileriosis is high, buparvaquone resistance is widespread, and vaccine protection is poor in the field. Better understanding is, therefore, needed of the factors that influence the genetics of T. annulata populations both within its hosts and in its overall populations. Here we utilise a panel of six satellites and a mitochondrial cytochrome b marker to explore the multiplicity of T. annulata infection and patterns of emergence and spread of different parasite genotypes. Parasite materials were collected from infected animals in defined regions, where water buffalo and cattle are kept together. Our results show that T. annulata is genetically more diverse in cattle- than in water buffalo-derived populations (the mean numbers of unique satellite alleles were 13.3 and 1.8 and numbers of unique cytochrome b locus alleles were 65 and 27 in cattle- and water buffalo-derived populations, respectively). The data show a high level of genetic diversity among the individual host-derived populations (the overall heterozygosity (He) indices were 0.912 and 0.931 in cattle, and 0.874 and 0.861 in buffalo, based on satellite and cytochrome b loci, respectively). When considered in the context of high parasite transmission rates and frequent animal movements between different regions, the predominance of multiple T. annulata genotypes, with multiple introductions of infection in the hosts from which the parasite populations were derived, may have practical implications for the spread of parasite genetic adaptations; such as those conferring vaccine cross-protection against different strains affecting cattle and buffalo, or resistance to antiprotozoal drugs.

genetics↗

bioRxiv2019

Novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

Various PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have been recently developed for the first time a novel high throughput haemoprotobiome metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species and to assess the accuracy of proportional quantification. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. Metabarcoded DNA sequencing showed better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.

genetics↗

bioRxiv2019

Genetic diversity and multiplicity of infection in Fasciola gigantica isolates of Pakistani livestock

Fasciola gigantica liver flukes are responsible for over 3 billion US dollars of production loss annually in farmed livestock and cause widespread zoonotic disease. Nevertheless, the understating of the emergence and spread of the trematode species is poor. The multiplicity of F. gigantica infection and its spread is potentially influenced by multiple factors, including the abundance of suitable intermediate hosts, climatic conditions favoring the completion of the parasites lifecycle, and translocation of infected animals or free-living parasite stages between regions. Here we describe the development of a tremabiome metabarcoding sequencing method to explore the numbers of F. gigantica genotypes per infection and patterns of parasite spread, based on genetic characteristics of the mitochondrial NADH dehydrogenase 1 (mt-ND-1) locus. We collected F. gigantica from three abattoirs in the Punjab and Balochistan provinces of Pakistan, and our results show a high level of genetic diversity in 20 F. gigantica populations derived from small and large ruminants consigned to slaughter in both provinces. This implies that F. gigantica can reproduce in its definitive hosts through meiosis involving cross- and self-breeding, as described in the closely related species, Fasciola hepatica. The genetic diversity between the 20 populations derived from different locations also illustrates the impact of animal movements on gene flow. Our results demonstrate the predominance of single haplotypes, consistent with a single introduction of F. gigantica infection in 85% of the hosts from which the parasite populations were derived. This is consistent with clonal reproduction in the intermediate snail hosts.

genetics↗

bioRxiv2019

Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

Piroplasmosis is caused by tick-borne haemoprotozoa of the genera Theileria and Babesia. These parasitic infections can cause serious impact on the health of livestock and production. Multiple piroplasm species can infect a single host, but reliable molecular diagnostic tools are needed with which to understand the composition of these complex parasite communities. Theileria and Babesia vary in their epidemiology, drug sensitivity, pathogenicity and interaction of co-infecting species, but are similar in the animals, become persistent carriers after recovery from primary infection, acting as reservoir hosts. Here, we describe for the first time the use of a deep amplicon sequencing platform to identify proportions of piroplasm species in co-infecting communities and develop the concept of a \"haemoprotobiome\". First, four phenotypically-verified species of Theileria and Babesia were used to prepare mock pools with random amounts of the parasites and amplified with four different numbers of PCR cycles to assess sequence representation of each species. Second, we evaluated the detection threshold of the deep amplicon sequencing assay for each of the four species and to assess the accuracy of proportional quantification of all four species. Finally, we applied the assay to the field samples to afford insight of the species composition of piroplasm communities in small and large ruminants in the Punjab province of Pakistan. The \"haemoprotobiome\" concept has several potential applications in veterinary and human research, including understanding of responses to drug treatment; parasite epidemiology and ecology; species interactions during mixed infections; and parasite control strategies.

microbiology↗

bioRxiv2019

Report on the spatial spread of defective interfering particles and its role in suppressing infectious particles

Defective interfering particles (DIPs) are categorized as non-infectious viruses with large deletions in their genomic material. A cell infected by a DIP require co-infection by a wild-type virus to complete its replicative lifecycle. There is an increasing interest in developing DIP based therapies in the form of molecular parasites that steal genetic resources of infectious particles. This parasitic behavior is enhanced by constructing engineering designs of DIPs to optimize their role in suppressing the virus infection within-host. Recent experimental studies characterize viral infection as a spatial process and emphasize on its spread rate and the area populated by the infectious particles (IPs). We developed a spatio-temporal model in the framework of reaction-diffusion equations to depict the functional organization of virus particles distributed over a tissue surface. Our model investigates the scenarios and figures out the aspects that can play a vital role to suppress the infection within-host. We studied the impact of initial dose of DIPs, the efficiency of DIP production and the role of cell maturation. Our results show that an engineered DIP can substantially decrease the concentration of IPs. We assert that the decrease in the rate of spatial spread of IPs requires non-deterministic settings.

bioengineering↗

bioRxiv2018

Selective sweep and phylogenetic models for the emergence and spread of pyrimethamine resistance mutations in Plasmodium vivax

Pyrimethamine resistance is a major concern for the control of human haemoprotozoa, especially Plasmodium species. Currently, there is little understanding of how pyrimethamine resistance developed in Plasmodium vivax in the natural field conditions. Here, we present first time the evidence of positive selection pressure on a dihydrofolate reductase locus and its consequences on the emergence and the spread of pyrimethamine resistance in P. vivax in the Punjab province of Pakistan. First, we examined the pyrimethamine resistance locus in 38 P. vivax populations to look for evidence of positive selection pressure in human patients. The S58R (AGA)/S117N (AAC) double mutation was most common, being detected in 10/38 populations. Single mutation S117N (AAC), I173L (CTT) and S58R (AGA) SNPs were detected in 8/38, 2/38 and 1/38 populations, respectively. The F57L/I (TTA/ATA) and T61M (ATG) SNPs were not detected in any population examined. Although both soft and hard selective sweeps have occurred with striking differences between populations, there was a predominance of hard sweeps. A single resistance haplotype was present at high frequency in 9/14 populations, providing a strong evidence for the single emergence of these mutations. In contrast, 5/14 populations carried multiple resistance haplotypes at high frequencies, providing an evidence of the emergence of resistance by recurrent mutations, characteristics of soft selective sweeps. Our phylogenetic relationship analysis suggests that S58R (AGA)/S117N (AAC) and S117N (AAC) mutations arose multiple times from a single origin and spread to multiple different cities in the Punjab province through gene flow. Interestingly, the I173L (CTT) mutation was present on a single haplotype, suggesting that it arises rarely and has not spread between cities. Our work shows the need for responsible use of exiting and new antimicrobial drugs and their combinations, control the movement of infected patients and mosquito vector control strategies.

evolutionary biology↗

bioRxiv2018

Emergence and the spread of the F200Y benzimidazole resistance mutation in Haemonchus contortus and Haemonchus placei from buffalo and cattle

Benzimidazoles have been intensively used in the livestock sector, particularly in small ruminants for over 40 years. This has been led to the widespread emergence of resistance in a number of small ruminant parasite species, in particular Haemonchus contortus. In many counties benzimidazole resistance in the small ruminants H. contortus has become severely compromising its control; but there is a little information on benzimidazole resistance in H. contortus infecting buffalo and cattle. Resistance to benzimidazoles have also been reported in the large ruminant parasite, Haemonchus placei, but again there is relatively little information on its prevalence. Hence it is extremely important to understand how resistance-conferring mutations emerge and spread in both parasites in the buffalo and cattle host in order to develop the approaches for the recognition of the problem at an early stage of its development. The present study suggests that the F200Y (TAC) mutation is common in H. contortus, being detected in 5/7 populations at frequencies between 7 to 57%. Furthermore, 6/10 H. placei populations contained the F200Y (TAC) mutation, albeit at low frequencies of between 0.4 to 5%. The phylogenetic analysis suggests that the F200Y (TAC) mutation in H. contortus has emerged on multiple occasions in the region, with at least three independent emergence of resistance alleles across the populations. In contrast, the F200Y (TAC) resistance-conferring mutation in H. placei is only seen on a single haplotype. A high level of haplotype frequency of the susceptible alleles in the region, suggests that the unique resistance conferring-mutation has spread from a single emergence; likely by anthropogenic animal movement. Overall, these results provide the first clear genetic evidence for the spread of benzimidazoles resistance-conferring mutations to multiple different locations from a single emergence in H. placei; while supporting previous small ruminant-based observations of multiple emergence of resistance mutations in H. contortus.

genetics↗