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1.  Recurrent bottlenecks in the malaria life cycle obscure signals of positive selection 
Parasitology  2014;142(0 1):S98-S107.
Detecting signals of selection in the genome of malaria parasites is a key to identify targets for drug and vaccine development. Malaria parasites have a unique life cycle alternating between vector and host organism with a population bottleneck at each transition. These recurrent bottlenecks could influence the patterns of genetic diversity and the power of existing population genetic tools to identify sites under positive selection. We therefore simulated the site-frequency spectrum of a beneficial mutant allele through time under the malaria life cycle. We investigated the power of current population genetic methods to detect positive selection based on the site-frequency spectrum as well as temporal changes in allele frequency. We found that a within-host selective advantage is difficult to detect using these methods. Although a between-host transmission advantage could be detected, the power is decreased when compared with the classical Wright-Fisher population model. Using an adjusted null site-frequency spectrum that takes the malaria life cycle into account, the power of tests based on the site-frequency spectrum to detect positive selection is greatly improved. Our study demonstrates the importance of considering the life cycle in genetic analysis, especially in parasites with complex life cycles.
PMCID: PMC4139472  PMID: 24560397
Plasmodium; life history; temporal; site-frequency spectrum
2.  Proteomics on the rims; insights into the biology of the nuclear envelope and flagellar pocket of trypanosomes 
Parasitology  2012;139(9):1158-1167.
Trypanosomatids represent the causative agents of major diseases in humans, livestock and plants, with inevitable suffering and economic hardship as a result. They are also evolutionarily highly divergent organisms, and the many unique aspects of trypanosome biology provide opportunities in terms of identification of drug targets, the challenge of exploiting these putative targets, and at the same time significant scope for exploration of novel and divergent cell biology. We can estimate from genome sequences that the degree of divergence of trypanosomes from animals and fungi is extreme, with perhaps one third to one half of predicted trypanosome proteins having no known function based on homology or recognizable protein domains/architecture. Two highly important aspects of trypanosome biology are the flagellar pocket and the nuclear envelope, where in silico analysis clearly suggests great potential divergence in the proteome. The flagellar pocket is the sole site of endo- and exocytosis in trypanosomes and plays important roles in immune evasion via variant surface glycoprotein (VSG) trafficking and providing a location for sequestration of various invariant receptors. The trypanosome nuclear envelope has been largely unexplored, but by analogy with higher eukaryotes, roles in the regulation of chromatin and most significantly, in controlling VSG gene expression are expected. Here we discuss recent successful proteomics-based approaches towards characterization of the nuclear envelope and the endocytic apparatus, the identification of conserved and novel trypanosomatid-specific features, and the implications of these findings.
PMCID: PMC4241632  PMID: 22309600
Proteomics; nuclear pore complex; flagellar pocket; systems biology; molecular evolution; protein complex; protein interactions
3.  Epichromatin is conserved in Toxoplasma gondii and labels the exterior parasite chromatin throughout the cell cycle 
Parasitology  2013;140(9):1104-1110.
Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed.
PMCID: PMC4189827  PMID: 23701822
Toxoplasma gondii; epichromatin; nuclear envelope; cell cycle; replication
4.  Toxoplasma gondii Hsp90: potential roles in essential cellular processes of the parasite 
Parasitology  2014;141(9):1138-1147.
Hsp90 is a widely distributed and highly conserved molecular chaperone that is ubiquitously expressed throughout nature, being one of the most abundant proteins within non-stressed cells. This chaperone is up-regulated following stressful events and has been involved in many cellular processes. In Toxoplasma gondii, Hsp90 could be linked with many essential processes of the parasite such as host cell invasion, replication and tachyzoite-bradyzoite interconversion. A Protein-Protein Interaction (PPI) network approach of TgHsp90 has allowed inferring how these processes may be altered. In addition, data mining of T. gondii phosphoproteome and acetylome has allowed the generation of the phosphorylation and acetylation map of TgHsp90. This review focuses on the potential roles of TgHsp90 in parasite biology and the analysis of experimental data in comparison with its counterparts in yeast and humans.
PMCID: PMC4181529  PMID: 24560345
Toxoplasma gondii; Hsp90; cell cycle; post-translational modification; development
5.  Vertical transmission of Toxoplasma gondii in Australian marsupials 
Parasitology  2009;136(9):939-944.
To date, little is known about the dynamics of vertical transmission of Toxoplasma gondii in Australian marsupials. Studies in mice demonstrate that vertical transmission of T. gondii is common and that chronically infected mice can transmit T. gondii to successive generations. In this study, PCR and immunohistochemistry were used to detect T. gondii in chronically infected marsupial dams and their offspring. T. gondii was detected in the unfurred pouch young of 2 out of 10 chronically infected western grey kangaroos (Macropus fuliginosus) and in the unfurred pouch young of a brush-tailed bettong (Bettongia penicillata). Results of the study suggest vertical transmission of T. gondii can occur in chronically infected Australian marsupials.
PMCID: PMC4029946  PMID: 19549348
Toxoplasma gondii; Marsupial; Vertical transmission; Australia; Congenital; Toxoplasmosis
6.  Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones 
Parasitology  2013;140(4):499-508.
Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulateCon-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.
PMCID: PMC3690601  PMID: 23279803
Cooperia oncophora; ivermectin resistance; ABC transporters; PGP; HAF; MRP
7.  Successful vaccines for naturally occurring protozoal diseases of animals should guide human vaccine research. A review of protozoal vaccines and their designs 
Parasitology  2014;141(5):624-640.
Effective vaccines are available for many protozoal diseases of animals, including vaccines for zoonotic pathogens and for several species of vector-transmitted apicomplexan haemoparasites. In comparison with human diseases, vaccine development for animals has practical advantages such as the ability to perform experiments in the natural host, the option to manufacture some vaccines in vivo, and lower safety requirements. Although it is proper for human vaccines to be held to higher standards, the enduring lack of vaccines for human protozoal diseases is difficult to reconcile with the comparatively immense amount of research funding. Common tactical problems of human protozoal vaccine research include reliance upon adapted rather than natural animal disease models, and an overwhelming emphasis on novel approaches that are usually attempted in replacement of rather than for improvement upon the types of designs used in effective veterinary vaccines. Currently, all effective protozoal vaccines for animals are predicated upon the ability to grow protozoal organisms. Because human protozoal vaccines need to be as effective as animal vaccines, researchers should benefit from a comparison of existing veterinary products and leading experimental vaccine designs. With this in mind, protozoal vaccines are here reviewed.
PMCID: PMC3961066  PMID: 24476952
Protozoal diseases; attenuation; vaccination; efficacy; review; animal models of human disease; malaria; one medicine; apicomplexa
8.  Anti-Wolbachia drug discovery and development: safe macrofilaricides for onchocerciasis and lymphatic filariasis 
Parasitology  2013;141(1):119-127.
Anti-Wolbachia therapy delivers safe macrofilaricidal activity with superior therapeutic outcomes compared to all standard anti-filarial treatments, with the added benefit of substantial improvements in clinical pathology. These outcomes can be achieved, in principle, with existing registered drugs, e.g. doxycycline, that are affordable, available to endemic communities and have well known, albeit population-limiting, safety profiles. The key barriers to using doxycycline as an mass drug administration (MDA) strategy for widespread community-based control are the logistics of a relatively lengthy course of treatment (4–6 weeks) and contraindications in children under eight years and pregnancy. Therefore, the primary goal of the anti-Wolbachia (A·WOL) consortium is to find drugs and regimens that reduce the period of treatment from weeks to days (7 days or less), and to find drugs which would be safe in excluded target populations (pregnancy and children). A secondary goal is to refine regimens of existing antibiotics suitable for a more restricted use, prior to the availability of a regimen that is compatible with MDA usage. For example, for use in the event of the emergence of drug-resistance, in individuals with high loiasis co-infection and at risk of severe adverse events (SAE) to ivermectin, or in post-MDA ‘endgame scenarios’, where test and treat strategies become more cost effective and deliverable.
PMCID: PMC3884836  PMID: 23866958
Wolbachia; onchocerciasis; lymphatic filariasis; drug discovery; macrofilaricide
9.  The effect of spatial heterogenity on the aggregation of ticks on white-footed mice 
Parasitology  2012;139(7):10.1017/S003118201200008X.
Parasites are often aggregated on a minority of the individuals in their host populations. Although host characteristics are commonly presumed to explain parasite aggregation on hosts, spatio-temporal aggregation of parasites during their host-seeking stages may have a dominant effect on the aggregation on hosts. We aimed to quantify, using mixed models, repeatability and autocorrelation analyses, the degree to which the aggregation of blacklegged ticks (Ixodes scapularis) on white-footed mice (Peromyscus leucopus) is influenced by spatio-temporal distributions of the host-seeking ticks and by heterogeneity among mice. Host-seeking ticks were spatially aggregated at both the larval and nymphal life-stages. However, this spatial aggregation accounted for little of the variation in larval and nymphal burdens observed on mice (3% and 0%, respectively). Conversely, mouse identity accounted for a substantial proportion of the variance in tick burdens. Mouse identity was a significant explanatory factor as the majority of ticks parasitized a consistent set of mice throughout the activity seasons. Of the characteristics associated with mouse identity investigated, only gender affected larval burdens, and body mass and home range sizes in males were correlated with nymphal burdens. These analyses suggest that aggregation of ticks on a minority of mice does not result from the distribution of host-seeking ticks but from characteristics of the hosts.
PMCID: PMC3856707  PMID: 22409977
burden; host-seeking ticks; index of dispersion; Ixodes scapularis; Pennsylvania; Peromyscus leucopus; repeatability
10.  Rates and intensity of re-infection with human helminths after treatment and the influence of individual, household, and environmental factors in a Brazilian community 
Parasitology  2011;138(11):10.1017/S0031182011001132.
This study quantifies the rate and intensity of re-infection with human hookworm and Schistosoma mansoni infection 12 months following successful treatment, and investigates the influence of socio-economic, geographical and environmental factors. A longitudinal study of 642 individuals aged over 5 years was conducted in Minas Gerais State, Brazil from June 2004 to March 2006. Risk factors were assessed using interval censored regression for the rate and negative binomial regression for intensity. The crude rate and intensity of hookworm re-infection was 0·21 per year (95% confidence interval (CI) 0·15–0·29) and 70·9 epg (95% CI 47·2–106·6). For S. mansoni the rate was 0·06 per year (95% CI 0·03–0·10) and intensity 6·51 epg (95% CI 3·82–11·11). Rate and intensity of re-infection with hookworm were highest among males and positively associated with previous infection status, absence of a toilet and house structure. Rate and intensity of S. mansoni re-infection were associated with previous infection status as well as geographical, environmental and socio-economic factors. The implications of findings for the design of anti-helminth vaccine trials are discussed.
PMCID: PMC3827741  PMID: 21819640
Necator americanus; hookworm; Schistosoma mansoni; risk factors; re-infection; negative binomial
11.  Novel amidines and analogues as promising agents against intracellular parasites: a systematic review 
Parasitology  2013;140(8):10.1017/S0031182013000292.
Parasitic protozoa comprise diverse aetiological agents responsible for important diseases in humans and animals including sleeping sickness, Chagas disease, leishmaniasis, malaria, toxoplasmosis and others. They are major causes of mortality and morbidity in tropical and subtropical countries, and are also responsible for important economic losses. However, up to now, for most of these parasitic diseases, effective vaccines are lacking and the approved chemotherapeutic compounds present high toxicity, increasing resistance, limited efficacy and require long periods of treatment. Many of these parasitic illnesses predominantly affect low-income populations of developing countries for which new pharmaceutical alternatives are urgently needed. Thus, very low research funding is available. Amidine-containing compounds such as pentamidine are DNA minor groove binders with a broad spectrum of activities against human and veterinary pathogens. Due to their promising microbicidal activity but their rather poor bioavailability and high toxicity, many analogues and derivatives, including pro-drugs, have been synthesized and screened in vitro and in vivo in order to improve their selectivity and pharmacological properties. This review summarizes the knowledge on amidines and analogues with respect to their synthesis, pharmacological profile, mechanistic and biological effects upon a range of intracellular protozoan parasites. The bulk of these data may contribute to the future design and structure optimization of new aromatic dicationic compounds as novel antiparasitic drug candidates.
PMCID: PMC3815587  PMID: 23561006
intracellular parasites; chemotherapy; aromatic amidines; arylimidamides
12.  New approaches for understanding mechanisms of drug resistance in schistosomes 
Parasitology  2013;140(12):1534-1546.
Schistosomes are parasitic flatworms that cause schistosomiasis, a neglected tropical disease that affects hundreds of millions worldwide. Treatment and control of schistosomiasis relies almost entirely on the single drug praziquantel (PZQ), making the prospect of emerging drug resistance particularly worrisome. This review will survey reports of PZQ (and other drug) resistance in schistosomes and other platyhelminths, and explore mechanisms by which drug resistance might develop. Newer genomic and post-genomic strategies that offer the promise of better understanding of how drug resistance might arise in these organisms will be discussed. These approaches could also lead to insights into the mode of action of these drugs and potentially provide markers for monitoring the emergence of resistance.
PMCID: PMC3775338  PMID: 23552512
schistosomiasis; drug resistance; multidrug transporters; praziquantel
13.  Quantification of Plasmodium falciparum malaria from complex infections in the Peruvian Amazon using quantitative PCR of the merozoite surface protein 1, block 2 (PfMSP1-B2): in vitro dynamics reveal density-dependent interactions 
Parasitology  2012;139(6):701-708.
The majority of Plasmodium falciparum field isolates are defined as complex infections because they contain multiple genetically distinct clones. Studying interactions between clones in complex infections in vivo and in vitro could elucidate important phenomena in malaria infection, transmission and treatment. Using quantitative PCR (qPCR) of the P. falciparum merozoite surface protein 1, block 2 (PfMSP1-B2), we provide a sensitive and efficient genotyping method. This is important for epidemiological studies because it makes it possible to study genotype-specific growth dynamics. We compared 3 PfMSP1-B2 genotyping methods by analysing 79 field isolates from the Peruvian Amazon. In vivo observations from other studies using these techniques led to the hypothesis that clones within complex infections interact. By co-culturing clones with different PfMSP1-B2 genotypes, and measuring parasitaemia using qPCR, we found that suppression of clonal expansion was a factor of the collective density of all clones present in a culture. PfMSP1-B2 qPCR enabled us to find in vitro evidence for parasite-parasite interactions and could facilitate future investigations of growth trends in naturally occurring complex infections.
PMCID: PMC3773723  PMID: 22339946
Plasmodium falciparum; merozoite surface protein 1; block 2; quantitative PCR; density regulation
14.  Trichuris muris research revisited: a journey through time 
Parasitology  2013;140(11):1325-1339.
The mouse whipworm Trichuris muris has long been used as a tractable model of human Trichuriasis. Here we look back at the history of T. muris research; from the definition of the species and determination of its life cycle, through to the complex immune responses that we study today. We highlight the key research papers that have developed our understanding of immune responses to this parasite, and reflect on how original concepts have been transformed, as our knowledge of immunology has grown. Although we have a good understanding of host–parasite interactions in the context of the underlying cellular immunology, there are still many aspects of the biology of the Trichuris parasite that remain undefined. We predict that advances in parasite biology will be key in the future development of new and improved treatments for Trichuriasis.
PMCID: PMC3761323  PMID: 23965819
Trichuris muris; helminth; nematode; parasite; mouse
15.  Seroepidemiological survey for canine angiostrongylosis in dogs from Germany and the UK using combined detection of Angiostrongylus vasorum antigen and specific antibodies 
Parasitology  2013;140(11):1442-1450.
Dogs infected with Angiostrongylus vasorum, a potentially lethal parasite parasitizing the heart and pulmonary arteries, may present severe respiratory, haematological and neurological signs. In this first large-scale seroepidemiological survey, 4003 sera originating from Germany and 4030 from the UK were tested by an ELISA for the detection of circulating antigen of A. vasorum, and by a separate ELISA detecting specific antibodies. In Germany, where mainly western federal states were sampled, 0·3% (n = 13, CI: 0·2–0·6%) of dogs were positive in both ELISAs, whereas in total 0·5% (n = 20, CI: 0·3–0·8%) were antigen-positive and 2·25% (n = 90, CI: 1·8–2·8%) were positive for specific antibodies. Regions with antigen- and antibody-positive animals were overlapping. In the UK, where mainly the south of the country was sampled, 0·97% (n = 39, CI: 0·7–1·3%) of dogs were antigen- and antibody positive. In total, 1·32% (n = 53, CI: 1·0–1·7%) were antigen-positive, and 3·2% (n = 129, CI: 2·7–3·8%) were positive for specific antibodies, again in overlapping regions. These results confirm the occurrence of A. vasorum in a random dog population originating from large parts of the countries investigated. The use of the tests alone or in combination was considered as a function of their sensitivities and specificities, in order to guide efficient clinical and epidemiological application.
PMCID: PMC3762218  PMID: 23965824
Angiostrongylus vasorum; dog; serological survey; antigen detection; antibody detection; UK; Germany
16.  New technologies for reporting real-time emergent infections 
Parasitology  2012;139(14):1843-1851.
Novel technologies have prompted a new paradigm in disease surveillance. Advances in computation, communications and materials enable new technologies such as mobile phones and microfluidic chips. In this paper we illustrate examples of new technologies that can augment disease detection. We describe technologies harnessing the internet, mobile phones, point of care diagnostic tools and methods that facilitate detection from passively collected unstructured data. We demonstrate how these can all assist in quicker detection, investigation and response to emerging infectious events. Novel technologies enable collection and dissemination of epidemic intelligence data to both public health practitioners and the general public, enabling finer temporal and spatial resolution of disease monitoring than through traditional public health processes.
PMCID: PMC3760201  PMID: 22894823
Technology; real-time; informal; crowd-sourced
17.  Identifying permethrin resistance loci in malaria vectors by genetic mapping 
Parasitology  2013;140(12):1468-1477.
Identification of the major loci responsible for insecticide resistance in malaria vectors would aid the development and implementation of effective resistance management strategies, which are urgently needed to tackle the growing threat posed by resistance to the limited insecticides available for malaria control. Genome-wide association studies in the major malaria vector, Anopheles gambiae, have been hindered by the high degree of within-population structuring and very low levels of linkage disequilibrium hence we revisited the use of quantitative trait loci (QTL) mapping to study resistance phenotypes in this vector species. Earlier work, identified two major QTL associated with pyrethroid resistance in A. gambiae s.s. from East Africa using genetic crossing of laboratory-colonized resistant and susceptible strains. In this study, we report the results from genetic mapping of pyrethroid resistance in three isofemale pedigrees established from wild-caught female A. gambiae s.s. mosquitoes from Benin. We identified two QTL on chromosomes 2L and 3R in these field populations, in similar genomic locations to the QTL identified in laboratory strains. The relative merits of two alternative study designs are discussed and suggestions made for future genetic mapping studies of insecticide resistance in mosquitoes.
PMCID: PMC3760440  PMID: 23448678
Anopheles gambiae; natural pedigrees; QTL; malaria; insecticide resistance
18.  Discrete typing units of Trypanosoma cruzi identified in rural dogs and cats in the humid Argentinean Chaco 
Parasitology  2012;140(3):303-308.
The discrete typing units (DTUs) of Trypanosoma cruzi that infect domestic dogs and cats have rarely been studied. With this purpose we conducted a cross-sectional xenodiagnostic survey of dog and cat populations residing in two infested rural villages in Pampa del Indio, in the humid Argentine Chaco. Parasites were isolated by culture from 44 dogs and 12 cats with a positive xenodiagnosis. DTUs were identified from parasite culture samples using a strategy based on multiple polymerase-chain reactions. TcVI was identified in 37 of 44 dogs and in 10 of 12 cats, whereas TcV was identified in five dogs and in two cats –a new finding for cats. No mixed infections were detected. The occurrence of two dogs infected with TcIII –classically found in armadillos– suggests a probable link with the local sylvatic transmission cycle involving Dasypus novemcinctus armadillos and a potential risk of human infection with TcIII. Our study reinforces the importance of dogs and cats as domestic reservoir hosts and sources of various DTUs infecting humans, and suggests a link between dogs and the sylvatic transmission cycle of TcIII.
PMCID: PMC3721149  PMID: 23058180
Chagas disease; Trypanosoma cruzi; Discrete Typing Units; dogs; cats; reservoir
19.  Direct molecular identification of Trypanosoma cruzi Discrete Typing Units in domestic and peridomestic Triatoma infestans and Triatoma sordida from the Argentine Chaco 
Parasitology  2012;139(12):1570-1579.
We assessed the distribution of Trypanosoma cruzi Discrete Typing Units (DTUs) in domestic and peridomestic Triatoma infestans and Triatoma sordida specimens collected in a well-defined rural area in Pampa del Indio, northeastern Argentina. Microscopically-positive bugs were randomly selected with a multi-level sampling design, and DTUs were identified using direct PCR strategies. TcVI predominated in 61% of 69 T. infestans and in 56% of 9 T. sordida. TcV was the secondary DTU in T. infestans (16%) and was found in one T. sordida specimen (11%). Three T. sordida (33%) were found infected with TcI, a DTU also identified in local Didelphis albiventris opossums. Mixed DTU infections occurred rarely (5%) and were detected both directly from the bugs’ rectal ampoule and parasite cultures. The identified DTUs and bug collection sites of T. infestans were significantly associated. Bugs infected with TcV were almost exclusively captured in domiciles whereas those with TcVI were found similarly in domiciles and peridomiciles. All mixed infections occurred in domiciles. TcV-infected bugs fed more often on humans than on dogs, whereas TcVI-infected bugs showed the reverse pattern. T. sordida is a probable sylvatic vector of TcI linked to D. albiventris, and could represent a secondary vector of TcVI and TcV in the domestic/peridomestic cycle.
PMCID: PMC3749237  PMID: 23036510
Trypanosoma cruzi; Discrete Typing Units; PCR; Triatoma infestans; Triatoma sordida Chagas disease
20.  Lipidomic analysis of bloodstream and procyclic form Trypanosoma brucei 
Parasitology  2010;137(9):1357-1392.
The biological membranes of Trypanosoma brucei contain a complex array of phospholipids that are synthesized de novo from precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of ~500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic form T. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains of Trypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.
PMCID: PMC3744936  PMID: 20602846
Phospholipid; Trypanosoma brucei; mass spectrometry; lipidomics
21.  Advances in the sequencing of the genome of the adenophorean nematode Trichinella spiralis 
Parasitology  2008;135(8):869-880.
The adenophorean nematodes are evolutionarily distant from other species in the phylum Nematoda. Interspecific comparisons of predicted proteins have supported such an ancient divergence. Accordingly, Trichinella spiralis represents a basal nematode representative for genome sequencing focused on gaining a deeper insight into the evolutionary biology of nematodes. In addition, molecular characteristics that are conserved across the phylum could be of great value for control strategies with broad application. In this review, we describe and summarize progress that has been made on the sequencing and analysis of the T. spiralis genome. The genome sequence was used in preliminary analyses for the investigation of specific questions relating to the biology of T. spiralis and, more generally, to parasitic nematodes. For instance, we evaluated an unusually large DNase II-like protein family, predicted proteins of prospective interest in the parasite-host muscle cell interaction, anthelmintic targets and prospective intestinal genes, the encoded proteins (potentially) linked to immunological control against other nematodes. The results are discussed in relation to characteristics that are broadly conserved among evolutionary distant nematodes. The results lead to expectations that this genome sequence will contribute to advances in research on T. spiralis and other parasitic nematodes.
PMCID: PMC3718030  PMID: 18598573
Trichinella spiralis; expressed sequence tags; ESTs; genome; sequencing; intestine; host-parasite interactions
22.  Genetic manipulation of schistosomes – progress with integration competent vectors 
Parasitology  2011;139(5):641-650.
Draft genome sequences for Schistosoma japonicum and S. mansoni are now available. The schistosome genome encodes ~13000 protein-encoding genes for which the functions of few are well understood. Nonetheless, the new genes represent potential intervention targets, and molecular tools are being developed to determine their importance. Over the past 15 years, noteworthy progress has been achieved towards development of tools for gene manipulation and transgenesis of schistosomes. A brief history of genetic manipulation is presented, along with a review of the field with emphasis on reports of integration of transgenes into schistosome chromosomes.
PMCID: PMC3714224  PMID: 21854676
Schistosomes; genetic manipulation; transgenesis; chromosome integration; germ line; retrovirus; murine leukaemia virus; pseudotyped gammaretrovirus; transposon; piggyBac
23.  Oral oocyst-induced mouse model of toxoplasmosis: Effect of infection with Toxoplasma gondii strains of different genotypes, dose, and mouse strains (transgenic, out-bred, in-bred) on pathogenesis and mortality 
Parasitology  2011;139(1):1-13.
Humans and other hosts acquire Toxoplasma gondii infection by ingesting tissue cysts in undercooked meat, or by food or drink contaminated with oocysts. Currently, there is no vaccine to prevent clinical disease due this parasite in humans, although, various T. gondii vaccine candidates are being developed. Mice are generally used to test the protective efficacy of vaccines because they are susceptible, reagents are available to measure immune parameters, in mice, and they are easily managed in the laboratory. In the present study, pathogenesis of toxoplasmosis was studied in mice of different strains, including Human leukocyte antigen(HLA) transgenic mice infected with different doses of T. gondii strains of different genotypes derived from several countries. Based on many experiments, the decreasing order of infectivity and pathogenicity of oocysts was: interferon gamma gene knock out (KO), HLA 3.11, HLA 2.1, HLA B7, Swiss Webster, C57/black, and BALB/c. Mice fed as few as 1 oocyst of Type I and several atypical strains died of acute toxoplasmosis within 21 days p.i. Type II, and III strains were less virulent. The model developed herein should prove to be extremely useful for testing vaccines because it is possible to accurately quantitate a challenge inoculum, test response to different strains of T. gondii using the same preparations of oocysts which are stable for up to a year, and to have highly reproducible responses to the infection.
PMCID: PMC3683600  PMID: 22078010
Toxoplasma gondii; oocysts; different genotypes; pathogenicity; different mouse strains including interferon gamma knockout and HLA transgenic mice
24.  In vitro manipulation of gene expression in larval Schistosoma: a model for postgenomic approaches in Trematoda 
Parasitology  2009;137(3):463-483.
With rapid developments in DNA and protein sequencing technologies, combined with powerful bioinformatics tools, a continued acceleration of gene identification in parasitic helminths is predicted, potentially leading to discovery of new drug and vaccine targets, enhanced diagnostics and insights into the complex biology underlying host-parasite interactions. For the schistosome blood flukes, with the recent completion of genome sequencing and comprehensive transcriptomic datasets, there has accumulated massive amounts of gene sequence data, for which, in the vast majority of cases, little is known about actual functions within the intact organism. In this review we attempt to bring together traditional in vitro cultivation approaches and recent emergent technologies of molecular genomics, transcriptomics and genetic manipulation to illustrate the considerable progress made in our understanding of trematode gene expression and function during development of the intramolluscan larval stages. Using several prominent trematode families (Schistosomatidae, Fasciolidae, Echinostomatidae), we have focused on the current status of in vitro larval isolation/cultivation as a source of valuable raw material supporting gene discovery efforts in model digeneans that include whole genome sequencing, transcript and protein expression profiling during larval development, and progress made in the in vitro manipulation of genes and their expression in larval trematodes using transgenic and RNA interference (RNAi) approaches.
PMCID: PMC3682473  PMID: 19961646
Trematoda; larval stages; Schistosoma; in vitro culture; genome; transcriptome; gene manipulation; transgenesis; RNA interference
25.  When they don't bite, we smell money: understanding malaria bednet misuse 
Parasitology  2013;140(5):580-586.
Insecticide-treated nets (ITNs) are a major tool to control malaria. Over recent years increased ITN coverage has been associated with decreased malaria transmission. However, ITN ‘misuse’ has been increasingly reported and whether this emergent behaviour poses a threat to successful malaria control and elimination is an open question. Here, we use a game theory mathematical model to understand the possible roles of poverty and malaria infection protection by individual and emerging ‘community effects’ on the ‘misuse’ of malaria bednets. We compare model predictions with data from our studies in Lake Victoria Islands (LVI), Kenya and Aneityum, Vanuatu. Our model shows that alternative ITN use is likely to emerge in impoverished populations and could be exacerbated if ITNs become ineffective or when large ‘community effects’ emerge. Our model predicted patterns of ITN use similar to the observed in LVI, where ‘misuse’ is common and the high ITN use in Aneityum, more than 20 years after malaria elimination in 1990. We think that observed differences in ITN use may be shaped by different degrees of economic and social development, and educational components of the Aneityum elimination, where traditional cooperative attitudes were strengthened with the malaria elimination intervention and post-elimination surveillance.
PMCID: PMC3596778  PMID: 23343880
Plasmodium; poverty; Pareto equilibrium; Nash equilibrium; insecticide treated nets; Kenya; Vanuatu

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