Nadine P. Randle

Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae.

How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S) in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr) to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs.

A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

BackgroundA single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation.MethodsA Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique.Results and DiscussionThe HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous.ConclusionThe method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.

The genomic basis of parasitism in the Strongyloides clade of nematodes

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families—families encoding astacin-like and SCP/TAPS proteins—is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

Occurrence and Diversity of Giardia duodenalis Assemblages in Livestock in the UK

Summary Giardia duodenalis is a common intestinal parasite in humans and a wide range of livestock species. It is a genetically heterogeneous parasite that has been characterized in seven distinct genetic assemblages or cryptic species, and molecular markers can be used to differentiate both animal-specific and potentially zoonotic genotypes. Little is known about G. duodenalis and the range of assemblages occurring in domestic livestock species in the UK. Here, we present data on the occurrence and molecular diversity of G. duodenalis detected in the faeces or large intestinal contents of cattle, sheep, pigs, goats and camelids from farms in the north-west of England. Both healthy and clinically diseased animals were included in the survey. The presence of Giardia spp. and assemblages was determined by sequencing of the small-subunit ribosomal RNA gene. The potential association of infection with various clinical and epidemiological parameters was studied in cattle using both univariate and multivariate analyses. Giardia spp. were detected in 127 (34.3%) of the 370 animals tested. G. duodenalis assemblage E was found to be predominant in cattle and sheep, followed by assemblage A. Mixed infections with assemblages A and E were also detected. Interestingly, some cattle, sheep and pigs were found to be infected with more unexpected assemblages (C, D, F). Pre-weaned calves were more likely to test positive than adult animals, but no association between the occurrence of overt intestinal disease and G. duodenalis infection was detected. The common occurrence of assemblage A and the finding of unusual assemblages in atypical hosts suggest that in future, a multilocus analysis should be used to confirm the actual diversity of G. duodenalis in livestock and the presence of potentially zoonotic genotypes. These data also suggest that there is a need to re-evaluate the clinical significance of G. duodenalis infection in livestock.

Identification of differentially expressed proteins in sulfadiazine resistant and sensitive strains of Toxoplasma gondii using difference-gel electrophoresis (DIGE).

Graphical abstract Highlights ► Treatment failure in toxoplasmosis implies the possibility of drug resistance. ► In vitro resistance to sulfadiazine detected in three strains of Toxoplasma gondii. ► Comparison of sensitive and sulfadiazine resistant strains by DIGE. ► Thirty one proteins differentially expressed between sensitive and resistant strains.

The Yin and Yang of linkage disequilibrium: mapping of genes and nucleotides conferring insecticide resistance in insect disease vectors.

Genetic technologies developed in the last 20 years have lead to novel and exciting methods to identify genes and specific nucleotides within genes that control phenotypes in field collected organisms. In this review we define and explain two of these methods: linkage disequilibrium (LD) mapping and quantitative trait nucleotide (QTN) mapping. The power to detect valid genotype-phenotype associations with LD or QTN mapping depends critically on the extent to which segregating sites in a genome assort independently. LD mapping depends on markers being in disequilibrium with the genes that condition expression of the phenotype. In contrast, QTN mapping depends critically upon most proximal loci being at equilibrium. We show that both patterns actually exist in the genome of Anapheles gambiae, the most important malaria vector in sub-Saharan Africa while segregating sites appear to be largely in equilibrium throughout the genome of Aedes aegypti, the vector of Dengue and Yellow fever flaviviruses. We discuss additional approaches that will be needed to identify genes and nucleotides that control phenotypes in field collected organisms, focusing specifically on ongoing studies of genes conferring resistance to insecticides.

Toxoplasma gondii and Neospora caninum induce different host cell responses at proteome-wide phosphorylation events; a step forward for uncovering the biological differences between these closely related parasites

Toxoplasma gondii and Neospora caninum are closely related intracellular protozoan parasites and tissue cyst-forming Coccidia of the phylum Apicomplexa. There are remarkable similarities between the morphology, genomes and transcriptomes of both parasites. Toxoplasma is zoonotic, with a wide host range and is mainly transmitted horizontally between its definitive host, the cat, and its intermediate hosts. Neospora causes disease within a narrow host range and with reduced virulence potential to the hosts. The dog is the definitive host of Neospora and its epidemiology in cattle mainly depends on vertical transmission. What causes these biological differences is not well understood. Since these parasites secrete an array of secretory proteins, including kinases, during infection to manipulate host cell responses. Host-parasite interactions due to phosphorylation of host cell proteins by T. gondii kinases enhance virulence and maintenance of infection. In this study, proteome-wide phosphorylation events of host cell proteins were investigated in response to infection with T. gondii and N. caninum using phosphoproteomic analyses, followed by pathway analysis on host signalling pathways. A few interesting differences in host responses at both the qualitative and quantitative levels were identified between the two infections; about one third of the phosphoproteomes, approximately 21% of the phospho-motifs and several pathways such as glycolysis/gluconeogenesis and mTOR pathways of the host cell were found differentially enriched between infection with these parasites. Identifying the differences in host-parasite interactions represents a promising step forward for uncovering the biological dissimilarities between both parasites.

Developing a 3D intestinal epithelium model for livestock species

The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells’ ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore, livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated, suspended in Matrigel, cultured, cryopreserved and resuscitated. ‘Rounding’ of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with Toxoplasma gondii or Salmonella typhimurium. This 3D organoid model offers a long-term, renewable resource for investigating species-specific intestinal infections with a variety of pathogens.

Integrative transcriptome and proteome analyses define marked differences between Neospora caninum isolates throughout the tachyzoite lytic cycle

Neospora caninum is one of the main causes of transmissible abortion in cattle. Intraspecific variations in virulence have been widely shown among N. caninum isolates. However, the molecular basis governing such variability have not been elucidated to date. In this study label free LC-MS/MS was used to investigate proteome differences between the high virulence isolate Nc-Spain7 and the low virulence isolate Nc-Spain1H throughout the tachyzoite lytic cycle. The results showed greater differences in the abundance of proteins at invasion and egress with 77 and 62 proteins, respectively. During parasite replication, only 19 proteins were differentially abundant between isolates. The microneme protein repertoire involved in parasite invasion and egress was more abundant in the Nc-Spain1H isolate, which displays a lower invasion rate. Rhoptry and dense granule proteins, proteins related to metabolism and stress responses also showed differential abundances between isolates. Comparative RNA-Seq analyses during tachyzoite egress were also performed, revealing an expression profile of genes associated with the bradyzoite stage in the low virulence Nc-Spain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum. SIGNIFICANCE The molecular basis that governs biological variability in N. caninum and the pathogenesis of neosporosis has not been well-established yet. This is the first study in which high throughput technology of LC-MS/MS and RNA-Seq is used to investigate differences in the proteome and transcriptome between two well-characterized isolates. Both isolates displayed different proteomes throughout the lytic cycle and the transcriptomes also showed marked variations but were inconsistent with the proteome results. However, both datasets identified a pre-bradyzoite status of the low virulence isolate Nc-Spain1H. This study reveals interesting insights into likely mechanisms involved in virulence in N. caninum and shed light on a subset of proteins that are potentially involved in the pathogenesis of this parasite.