A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper.

The Malaria in Melbourne 2021 conference was held online in October. This conference aims to provide a platform for students and early career researchers to share their research and develop new collaborative networks. The program covered a broad range of topics including antimalarial drug development, epidemiology, immunology, molecular and cellular biology, and other emerging technologies. This article summarises recent advances in Plasmodium research presented at the Malaria in Melbourne 2021 conference.

Acanthamoeba castellanii is a protist pathogen that can cause sight-threatening keratitis and a fatal infection of the central nervous system, known as granulomatous amoebic encephalitis. In this study, effects of five malonic acid and salicylic acid-based deep eutectic solvents (DES) on A. castellanii were investigated. These are salicylic acid-trioctylphosphine (DES 1), salicylic acid- trihexylamine (DES 2), salicylic acid-trioctylamine (DES 3), malonic acid-trioctylphosphine (DES 4) and malonic acid-trihexylamine (DES 5). The experiments were done by performing amoebicidal, encystment, excystment, cytopathogenicity, and cytotoxicity assays. At micromolar dosage, the solvents DES 2 and DES 3 displayed significant amoebicidal effects (P < 0.05), inhibited encystment and excystment, undermined the cell-mediated cytopathogenicity of A. castellanii, and also displayed minimal cytotoxicity to human cells. Conversely, the chemical components of these solvents: salicylic acid, trihexylamine, and trioctylamine showed minimal effects when tested individually. These results are very promising and to the best of our knowledge, are reported for the first time on the effects of deep eutectic solvents on amoebae. These results can be applied in the development of new formulations of novel contact lens disinfectants against Acanthamoeba castellanii.

TriTryp diseases (Leishmaniasis, Human African Trypanosomiasis (HAT), and Chagas disease) are devastating parasitic neglected tropical diseases (NTDs) that affect billions of people in developing countries, cause high mortality in humans, and impose a large socio-economic burden. The current treatment options against tritryp diseases are suboptimal and challenging due to the emergence of resistance against available tritryp drugs. Hence, designing and developing effective anti-tritryp drugs with novel targets are required. Aminoacyl-tRNA synthetases (AARSs) involved in specific aminoacylation of transfer RNAs (tRNAs), interrupt protein synthesis through inhibitors, and retard the parasite growth. AaRSs have long been studied as therapeutic targets in bacteria, and three aaRS inhibitors, mupirocin (against IleRS), tavaborole AN2690 (against LeuRS), and halofuginone (against ProRS), are already in clinical practice. The structural differences between tritryp and human aaRSs and the presence of unique sequences (N-terminal domain/C-terminal domain/catalytic domain) make them potential target for developing selective inhibitors. Drugs based on a single aaRS target developed by high-throughput screening (HTS) are less effective due to the emergence of resistance. However, designing multi-targeted drugs may be a better strategy for resistance development. In this perspective, we discuss the characteristics of tritryp aaRSs, sequence conservation in their orthologs and their peculiarities, recent advancements towards the single-target and multi-target aaRS inhibitors developed through rational design.

Glutathione transferases (GSTs) are major detoxification enzymes vital for the survival and reproduction of schistosomes during infection in humans. Schistosoma encode two GST isoenzymes, the 26- and 28-kDa isoforms, that show different substrate specificities and cellular localisations. Bromosulfophthalein (BSP) has been identified and characterised as a potent 26-kDa Schistosoma japonicum GST (Sj26GST) inhibitor with an anthelmintic potential. This study describes the structure, function, and ligandin properties of the 28-kDa Schistosoma japonicum GST (Sj28GST) towards BSP. Enzyme kinetics show that BSP is a potent enzyme inhibitor, with a specific activity decreases from 60.4 µmol/min/mg to 0.0742 µmol/min/mg and an IC50 in the micromolar range of 0.74 µM. Far-UV circular dichroism confirmed that purified Sj28GST follows a typical GST fold, which is predominantly alpha-helical. Fluorescence spectroscopy suggests that BSP binding occurs at a site distinct from the glutathione-binding site (G-site); however, the binding does not alter the local G-site environment. Isothermal titration calorimetry studies show that the binding of BSP to Sj28GST is exergonic (∆G°= −33 kJ/mol) and enthalpically-driven, with a stoichiometry of one BSP per dimer. The stability of Sj28GST (∆G(H2O) = 4.7 kcal/mol) is notably lower than Sj26GST, owing to differences in the enzyme’s dimeric interfaces. We conclude that Sj28GST shares similar biophysical characteristics with Sj26GST based on its kinetic properties and susceptibility to low concentrations of BSP. The study supports the potential benefits of re-purposing BSP as a potential drug or prodrug to mitigate the scourge of schistosomiasis.

Lactobacillus strains exhibit preferable properties that make them attractive candidates for vaccine delivery systems because of their ability to regulate intestinal mucosal immunity in the body. To date, live Lactobacillus delivery vaccines reported for the defense against Eimeria tenella have been inducer-dependent systems whose applications are significantly limited due to their unattainable induction conditions in vivo. Here, a constitutive expression of Lactobacillus plantarum NC8 surface display system was constructed. Then, this system was used to prepare a live oral vaccine to constitutively express the E. tenella U6L5H2 (EtU6) protein on the NC8 surface and to evaluate its protective efficacy against E. tenella challenge in chickens. The results showed that the heterologous protein (EGFP or EtU6) was successfully expressed on the surface of L. plantarum NC8 without any inducer. The immunoprotection of EtU6 with constitutive expression in L. plantarum NC8 system (NC8/Pc-EtU6) was significantly stronger than that of EtU6 with induced expression of L. plantarum NC8 system (NC8/Pi-EtU6) (ACI: 168.28 vs. 152.74) as evidenced by increased body weight, decreased oocyst output and lesion scores. Furthermore, the constitutive system NC8/Pc-EtU6 produced higher levels of specific cecal SIgA, serum IgG, transcription of cytokines IFN-γ and IL-2, and lymphocyte proliferation than the induced system NC8/Pi-EtU6. These results indicate that, compared to the inducible system, the constitutive surface display system of L. plantarum has the advantages of continuously expressing antigens in vivo and stimulating the host immune system. It could be an ideal platform for vaccine expression. The live vector vaccine for coccidiosis constructed by this constitutive system greatly improves the application potential in chicken production and provides a novel platform for the prevention of coccidiosis in chickens.

We developed microsatellite markers to use in studying the population genetics of the trematode Alloglossidium renale, a fluke with a precocious life cycle where sexual maturation occurs in a grass shrimp. Among 21 tested loci in a Mississippi population sample, 14 were polymorphic, 12 of which significantly deviated from Hardy-Weinberg Equilibrium (HWE). We estimated identity disequilibrium (ID) to confirm whether the deviations from HWE were due to significant amounts of selfing or due to technical factors. The selfing rate derived from FIS was 86.6%, whereas the selfing rate obtained by ID was 83.9%, indicating that the deviation in HWE was due to a high amount of selfing within the population. These markers will be useful for ecological and evolutionary studies of A. renale especially in relation to the interplay of hermaphroditic mating systems, inbreeding depression, and transmission dynamics.

Diclazuril is a classic anticoccidial drug. The key molecules of diclazuril in anticoccidial action allows target screening for the development of anticoccidial drugs. Cyclin-dependent kinases (CDK) are prominent target proteins in apicomplexan parasites. In this study, a diclazuril anticoccidiosis animal model was established, and the transcription and translation levels of the CDK-related kinase 2 of Eimeria tenella (EtCRK2) were detected. mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localised in the cytoplasm of the merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group. The anticoccidial drug diclazuril against E.tenella affects the expression pattern of EtCRK2 molecule, and EtCRK2 is a potential target for new drug development.

Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; GFPHAT2 or GFPHDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.

Acanthamoeba is opportunistic pathogens that cause vision-threatening Acanthamoeba keratitis (AK). Previous studies proposed the use of chloroquine (CQ) and 5-fluorouracil (5FU) as anti-Acanthamoeba agents. The objective of this study was to determine the benefit of using 5FU and CQ nanoparticles (NP) formulations against A. castellanii that belonging to the T4 genotype and evaluate their anti-Acanthamoebic characteristic. Triplicate batches of 5FU nanoparticles (5FU-NP) were synthesized by using a modified nanoprecipitation method, while CQ nanoparticles (CQ-NP) synthesized using a modified double emulsion method. The synthesized nanoparticles were subjected to biological assays to investigate their amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects against A. castellanii, as well as cell cytotoxicity. Cytotoxicity assays were performed using human keratinocyte cells (HaCaT) to determine the effect of CQ and 5FU nanoformulations on host cells. 5FU-NP with a concentration of 60 µM showed significant inhibition to amoeba binding into human cell lines and remarkable prevention mainly during the encystation stage. Moreover, 5FU-NP resulted in less cytotoxicity and pathogenicity when compared with the free 5FU. On the other hand, CQ and CQ-NP, at the same concentration, showed poor inhibition to amoeba binding into human cells and insignificant prevention to encystation stage. Moderate human cells damage was resulted following their treatment with CQ and CQ-NP. In conclusion, 5FU may have the potential as an antiamoebic agent against Acanthamoeba spp. preferably as a nanoformulation to enhance its activity and reduce its cytoxicity.

Babesia bovis, an intraerythrocytic hemoprotozoan parasite, causes the most pathogenic form of bovine babesiosis, negatively impacting the cattle industry. Comprehensive knowledge of B. bovis biology is necessary for developing control methods. In cattle, B. bovis invades the red blood cells (RBCs) and reproduces asexually. Micronemal proteins, which bind to sialic acid of host cells via their microneme adhesive repeat (MAR) domains, are believed to play a key role in host cell invasion by apicomplexan parasites. In this study, we successfully deleted the region encoding MAR domain of the BBOV_III011730 by integrating a fusion gene of enhanced green fluorescent protein-blasticidin-S-deaminase into the genome of B. bovis. The transgenic B. bovis, lacking the MAR domain of the BBOV_III011730, invaded bovine RBCs in vitro and grew at rates similar to the parental line. In conclusion, our study revealed that the MAR domain is non-essential for the intraerythrocytic development of B. bovis in vitro.

Berberine, a traditional Chinese medicine, was found to exhibit anticoccidial activity. However, its mechanism is unclear. Trace metals such as copper and zinc are extremely low (less than 0.01% of the total weight of the body) but play a vital role in organisms. In the present study, we investigated the effect of berberine on copper and zinc levels in chickens infected with Eimeria tenella. Firstly, our data confirmed that infected chickens with E. tenella exhibited classic impairment on the 8th day of post infection, such as weight loss and increased feed conversion. Further study showed that E. tenella infection decreased the contents of copper and zinc in the liver and serum of chickens. Berberine was similar to amprolium and significantly improved the pathogenic conditions. Berberine could restore copper and zinc imbalance caused by E. tenella in chickens to a large extent. Studies on the development of cecum lesions demonstrated that the protective effect of berberine on the intestinal cecum was similar to that of the Cu/Zn mixture. Additionally, the mRNA expression of several metal transport related genes of the chick small intestine, including zinc transporter 1, copper transporter 1 and divalent metal ion transporter 1, was elevated by the treatment with berberine. Taken together, we speculate that the anticoccidial activity of berberine may be related to the maintenance of certain metals (Cu/Zn) homeostasis by affecting mRNA expression of their transport genes. However, the mode of action of BBR on these vital metals in the chicks infected with E. tenella still needs to be further studied.

To reveal the genetic characteristics of one member of the Plasmodium falciparum repetitive interspersed family (rif), we sequenced the rif gene (PF3D7_1254800) in 53 field isolates collected from Ghana-imported cases into China and compared them with 350 publicly available P. falciparum rif sequences from global populations. In the Ghana-imported population, the nucleotide diversities were 0.05714 and 0.06616 for the full length and variable region of rif gene, respectively. Meanwhile, 22 and 20 haplotypes were identified for the full length and variable region of rif gene (Hd = 0.843 and 0.838, respectively). Diversity of rif gene in Ghana-imported population was higher than that observed in Cambodia, Thailand, Vietnam, Myanmar, Mali, Ghana, and Senegal populations. In this analysis, we found high genetic diversity of rif gene in global P. falciparum populations and identified 158 haplotypes. Tajima's D-test shows that there are large differences in the direction of selection between the conserved and variable region of rif gene. Tajima's D value for the variable region was 0.20074, indicating that balancing selection existed in this region. We found that the variable region was the main target of selection for positive diversification, and most mutation sites were located in this region. The population structure suggested optimized cluster values of K = 6. The five groups in Ghana-imported population included a unique subpopulation. Our results reveal the dynamics of the rif gene (PF3D7_1254800) in P. falciparum populations, which can aid in the rational design of P. falciparum rif-based vaccines.

The rapid spread of drug resistant malaria parasites has necessitated the search for novel antimalarials and chemosensitizers capable of reversing drug resistance in the parasites. A number of studies have revealed the resistance reversal activities of pregnane glycosides and the antimalarial activity of a pregnane glycoside obtained from Gongronema species. However, the pregnane (2) and pregnane glycosides (1, 3–4) isolated from Gongronema latifolium leaf have not been evaluated for these activities. This study was therefore carried out to evaluate the antiplasmodial and chloroquine resistance reversal activities of a pregnane and three pregnane glycosides isolated from G. latifolium leaf in vitro. The compounds were evaluated for their inhibitory activities against P. falciparum 3D7 (a chloroquine-sensitive strain) and P. falciparum W2 (a chloroquine-resistant clone) in vitro. The activities of chloroquine in separate combination with each of the compounds against P. falciparum W2 were also evaluated. Moreover, the interaction of the active compounds (1 and 4) with selected P. falciparum proteins (PfProteins) were evaluated in silico. The results revealed that only 1 and 4 were active against P. falciparum 3D7 and P. falciparum W2. Also, 2 and 3 did not exhibit chloroquine resistance reversal activity. Activity of chloroquine against P. falciparum W2 was potentiated by 1 by 3200% at concentrations higher than 0.625 µg/mL. Also, 1 and 4 demonstrated similar binding patterns and higher binding tendencies to the selected PfProteins compared to chloroquine. Thus, 1 (iloneoside) is an antimalarial pregnane glycoside which can potentiate the activity of chloroquine against multidrug resistant P. falciparum.

The aim of this study was to evaluate the in vitro immune modulation of two de novo peptides with hypothetical identity to the serine protease family (S28) from Haemonchus spp. Expression of mRNAs encoding these peptides was confirmed by RTqPCR in L3 and adult stage parasites. Antibodies from serum samples collected from an H. contortus-infected lamb at 60 days post infection detected both peptides, as assessed by indirect ELISA. Lamb peripheral blood mononuclear cells (PBMCs) were exposed to each peptide, as well as to the peptide mixture, and cell proliferation assays were performed at 24, 48 and 72 h. The relative expression of the IL4, IL5, IL6, IL13, CXCL8 and FCεR1A genes was quantified by RTqPCR from lamb PBMCs exposed to the peptide mixture at 24 and 48 h. With respect to immune gene expression, 15- and 3-fold upregulation at 24 h was observed with IL5 and CXCL8, respectively, and 2-fold upregulation of CXCL8 at 48 h. In contrast, downregulation of IL5 was stimulated at 48 h. These data suggest that these peptides (pep-hsp and pep-pcx), which show high identity with intestinal and excretion/secretion serine proteases, can trigger immunogenic activity, and suggest that they may be useful as potential parasite vaccines.

Glutathione transferase enzymes (GSTs) are believed to be a major detoxification system in helminth parasites and have been associated with immunomodulation of the host response. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least five different GSTs, but no Omega-class enzymes have been reported in this parasite or in any other cestode. Herein we report the identification of a new member of the GST superfamily in E. granulosus s.l., which is phylogenetically related to the Omega-class: EgrGSTO. Through mass spectrometry, we showed that the 237 amino acids protein EgrGSTO is expressed by the parasite. Moreover, we identified homologues of EgrGSTO in other eight members of the Taeniidae family, including E. canadensis, E. multilocularis, E. oligarthrus, Hydatigera taeniaeformis, Taenia asiatica, T. multiceps, T. saginata and T. solium. A manual sequence inspection and rational modification yielded eight Taeniidae’s GSTO sequences, each one encoding for a 237 aa polypeptide showing 80.2% overall identity. To the best of our knowledge, this is the first description of genes encoding for Omega-class GSTs in worms belonging to the Taeniidae family -that at least in E. granulosus s.l. is expressed as a protein- suggesting the gene encodes for a functional protein.

If normal male meiosis occurs, it would be expected that 50 % of sperm lack an X chromosome (nullo X) and hence upon fertilisation, result in male progeny. However, for sexual reproduction within the free-living stages of Strongyloides spp. male offspring are absent. We had shown earlier by quantitative whole genome sequencing that within Strongyloides spp., nullo-X sperm are either absent (S. papillosus) or underrepresented (S. ratti) among mature sperm. To investigate how and when this elimination of male-determining sperm occurs, we characterised spermatogenesis and the dynamic localisation of important molecular players such as tubulin, actin and major sperm protein by DIC microscopy, immunohistochemistry, and fluorescent in situ hybridization (FISH) in S. ratti, S. papillosus and Parastrongyloides trichosuri. We found that meiotic divisions in these parasites proceeded as expected for organisms with XO males, resulting in four equally sized spermatocytes, two with and two without an X chromosome. However, mature sperm were found to almost always contain an X chromosome. We also observed structures that contained protein constituents of sperm, such as actin and major sperm protein (MSP) but no DNA. These structures resemble C. elegans residual bodies in appearance and may assume their function. We hypothesize that spermatocytes without an X-chromosome undergo some form of programmed cell death and transform into these residual body-like structures. As in C. elegans, MSP is found in fibrous body-membranous organelles (FB-MOs). Knocking down MSP by RNAi showed that MSP is essential for fertility in S. ratti, as it is in C. elegans.

BackgroundMalaria infection can result in distinct clinical outcomes from asymptomatic to severe. The association between patho-physiological changes and molecular changes in the host, and their correlation with severity of malaria progression is not fully understood.MethodsIn this study, we addressed mass spectrometry-based temporal profiling of serum metabolite levels from mice infected with Plasmodium berhgei (strain ANKA).ResultsWe show global perturbations and identify changes in specific metabolites in correlation with disease progression. While metabolome-wide changes were apparent in late-stage malaria, a subset of metabolites exhibited highly correlated changes with disease progression. These metabolites changed early on following infection and either continued or maintained the change as mice developed severe disease. Some of these have the potential to be sentinel metabolites for severe malaria. Moreover, glycolytic metabolites, purine nucleotide precursors, tryptophan and its bioactive derivatives were many fold decreased in late-stage disease. Interestingly, uric acid, a metabolic waste reported to be elevated in severe human malaria, increased with disease progression, and subsequently appears to be detoxified into allantoin. This detoxification mechanism is absent in humans as they lack the enzyme uricase.ConclusionsWe have identified candidate marker metabolites that may be of relevance in the context of human malaria.

The parasite responsible for causing malaria infection, Plasmodium, is known to exhibit resistance to a number of already available treatments. This has prompted the continue search for new antimalarial drugs ranging from medicinal plant parts to synthetic compounds. In lieu of this, the mitigative action of the bioactive compound, eugenol towards P. berghei-induced anaemia and oxidative organ damage was investigated following a demonstration of in vitro and in vivo antiplasmodial effects. Mice were infected with chloroquine-sensitive strain of P. berghei and thereafter treated with eugenol at doses of 10 and 20 mg/kg body weight (BW) for seven days. The packed cell volume and redox sensitive biomarkers in the liver, brain and spleen were measured. Our result demonstrated that eugenol significantly (p < 0.05) ameliorated the P. berghei-associated anaemia at a dose of 10 mg/kg BW. In addition, the compound, at a dose of 10 mg/kg BW, significantly (p < 0.05) alleviated the P. berghei-induced organ damage. This evidently confirmed that eugenol plays an ameliorative role towards P. berghei-related pathological alterations. Hence, the study opens up a new therapeutic use of eugenol against plasmodium parasite.

Despite their economic significance in agricultural cropping systems, a lack of suitable molecular tools for manipulating gene expression has hindered progress in the functional genomics of plant parasitic nematodes (PPN). Obligate sexual reproduction and the obligate nature of PPN-host interactions further complicate the development of in vivo gene delivery and expression systems in these pests. Methods such as microinjection and microprojectile bombardment have been developed for introducing gene constructs into the free-living nematode, Caenorhabditis elegans. However, these procedures can be laborious and inefficient. Electroporation has been used extensively to introduce macromolecules, including single-stranded RNAs, into eukaryotic and prokaryotic cells. The technique has also been used for the delivery of DNA and double-stranded RNA constructs into nematodes by whole-animal electroporation. Here, we describe methods for the expression of a nematode-optimized NanoLuc luciferase mRNA in the form of in vitro transcripts following whole-animal electroporation of Heterodera glycines, Meloidogyne incognita, and C. elegans. The ability to transiently express single-stranded RNA constructs in economically important PPN provides a rapid means to evaluate nematode and/or foreign genes for their biological significance and potential role in nematode management.