Massive Infection of Lungs with Exo-Erythrocytic Meronts in European Robin Erithacus rubecula during Natural Haemoproteus attenuatus Haemoproteosis

Haemoproteus species are widespread avian blood parasites belonging to Haemoproteidae (Haemosporida). Blood stages of these pathogens have been relatively well-investigated, though exo-erythrocytic (tissue) stages remain unidentified for the majority of species. However, recent histopathological studies show that haemoproteins markedly affect bird organs during tissue merogony. This study investigated the exo-erythrocytic development of Haemoproteus (Parahaemoproteus) attenuatus (lineage hROBIN1), the common parasite of flycatchers (Muscicapidae). Naturally infected European robins Erithacus rubecula were examined. Parasite species and lineage were identified using microscopic examination of blood stages and DNA sequence analysis. Parasitaemia intensity varied between 0.8 and 26.5% in seven host individuals. Organs of infected birds were collected and processed for histological examination. Tissues stages (meronts) were seen in six birds and were present only in the lungs. The parasites were usually located in groups and were at different stages of maturation, indicating asynchronous exo-erythrocytic development. In most parasitized individuals, 100 meronts were observed in 1 cm2 section of lungs. The largest meronts reached 108 µm in length. Mature meronts contained numerous roundish merozoites of approximately 0.8 µm in diameter. Megalomeronts were not observed. Massive merogony and resulting damage of lungs is a characteristic feature during H. attenuatus infections and might occur in related parasite lineages, causing haemoproteosis.

Genetic evidence substantiates transmission of Trichinella spiralis from one swine farm to another

Background Trichinella spiralis ranks seventh in the risk posed by foodborne parasites. It causes most human cases of trichinellosis and is the most frequent cause of Trichinella outbreaks on pig farms and in wild boar, worldwide. Veterinary inspectors seek the source of outbreaks in hopes of limiting the spread. Established molecular tools are inadequate for distinguishing among potential T. spiralis infection sources because genetic variability in these zoonotic pathogens is limited in Europe. Microsatellite markers proved successful in tracing an outbreak of T. britovi, a related parasite harboring much more genetic variation. Here, we successfully employed microsatellite markers to determine the genetic structure of T. spiralis isolates from two pig outbreaks, discovering notable uniformity among parasites within each farm and discovering an epidemiological link between these two outbreaks. Methods The individual larvae from five isolates of T. spiralis from two pig farms and from ten wild boars were genotyped using nine microsatellite markers to examine their genetic structure. Results Notably uniform parasite populations constituted each farm outbreak, and the parasites from the first and second outbreaks resembled each other to a notable degree, indicating an epidemiological link between them. Wild boar harbored more genetically variable larval cohorts, distinguishing them from parasites isolated from domestic pigs. Conclusions Microsatellite markers succeeded in distinguishing isolates of the highly homogeneous T. spiralis, aiding efforts to track transmission. Each outbreak was composed of a homogenous group of parasites, suggesting a point source of contamination. Graphical abstract

Identification of oligo-adenylated small RNAs in the parasite Entamoeba and a potential role for small RNA control

Background The RNA interference (RNAi) pathway is a gene regulation mechanism that utilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27 nt antisense sRNA populations which associate with EhAgo2–2 protein. However, there is lack of understanding about the sRNAs that are bound to two other EhAgos (EhAgo2–1 and 2–3), and the mechanism of sRNA regulation itself is unclear in this parasite. Therefore, identification of the entire pool of sRNA species and their sub-populations that associate with each individual EhAgo protein would be a major step forward. Results In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31 nt sRNAs that results from the addition of a non-templated 3–4 adenosine nucleotides at the 3′-end of the 27 nt sRNAs, indicating a non-templated RNA-tailing event in the parasite. The relative abundance of these two sRNA populations is linked to the efficacy of gene silencing for the target gene when parasites are transfected with an RNAi-trigger construct, indicating that non-templated sRNA-tailing likely play a role in sRNA regulation in this parasite. We found that both sRNA populations (27 nt and 31 nt) are present in the related parasite Entamoeba invadens, and are unchanged during the development. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27 nt sRNAs with 5′-polyphosphate (5′-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31 nt sRNAs associate with EhAgo2–2 but not with its mutant protein (C-terminal deletion), nor other two EhAgos, indicating a specific EhAgo site may be required for sRNA modification process in the parasite. Conclusion We identified a new population of sRNA with non-templated oligo-adenylation modification, which is the first such observation amongst single celled protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.

Sequence of Trypanosoma cruzi reference strain SC43 nuclear genome and kinetoplast maxicircle confirms a strong genetic structure among closely related parasite discrete typing units

Chagas disease is a zoonotic, parasitic, vector-borne neglected tropical disease that affects the lives of over 6 million people throughout the Americas. Trypanosoma cruzi, the causative agent, presents extensive genetic diversity. Here we report the genome sequence of reference strain SC43cl1, a hybrid strain belonging to the TcV discrete typing unit (DTU). The assembled diploid genome was 79 Mbp in size, divided into 1236 contigs with an average coverage reaching 180×. There was extensive synteny of SC43cl1 genome with closely related TcV and TcVI genomes, with limited sequence rearrangements. TcVI genomes included several expansions not present in TcV strains. Comparative analysis of both nuclear and kinetoplast sequences clearly separated TcV from TcVI strains, which strongly supports the current DTU classification.

A single point mutation in the Plasmodium falciparum ftsh1 metalloprotease confers actinonin resistance

The antibiotic actinonin kills malaria parasites (Plasmodium falciparum) by interfering with apicoplast function. Early evidence suggested that actinonin inhibited prokaryote-like post-translational modification in the apicoplast; mimicking its activity against bacteria. However, Amberg Johnson et al. (2017) identified the metalloprotease TgFtsH1 as the target of actinonin in the related parasite Toxoplasma gondii and implicated actinonin in the inhibition of P. falciparum growth. The authors were not, however, able to recover actinonin resistant malaria parasites, leaving the specific target of actinonin uncertain. We generated actinonin resistant P. falciparum by in vitro selection and identified a specific sequence change in PfftsH1 associated with resistance. Re-Introduction of this point mutation using CRISPr-Cas9 allelic replacement was sufficient to confer actinonin resistance in P. falciparum. Our data unequivocally identifies PfFtsH1 as the target of actinonin and suggests that actinonin should not be included in the highly valuable collection of “irresistible” drugs for combatting malaria.

Evolution of resistance in vitro reveals mechanisms of artemisinin activity inToxoplasma gondii

Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance inPlasmodium falciparum, including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability ofToxoplasma gondii, a related parasite that shows moderate sensitivity to artemisinin. Resistant populations ofT. gondiiwere selected by culture in increasing concentrations and whole-genome sequencing identified several nonconservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acid changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild-type parasites in the presence of drug, despite not changing EC50values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondria genome, including genes encoding cytochromeband cytochromecoxidase I. Prior studies in yeast and mammalian tumor cells implicate the mitochondrion as a target of artemisinins, and treatment of wild-type parasites with high concentrations of drug decreased mitochondrial membrane potential, a phenotype that was stably altered in the resistant parasites. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition of resistance inT. gondii.

Advances in the Molecular and Cellular Biology of Strongyloides spp.

Purpose of Review This paper constitutes an update of recent studies on the general biology, molecular genetics, and cellular biology of Strongyloides spp. and related parasitic nematodes. Recent Findings Increasingly, human strongyloidiasis is considered the most neglected of neglected tropical diseases. Despite this, the last 5 years has seen remarkable advances in the molecular biology of Strongyloides spp. Genome sequences for S. stercoralis, S. ratti, S. venezuelensis, S. papillosus, and the related parasite Parastrongyloides trichosuri were created, annotated, and analyzed. These genomic resources, along with a practical transgenesis platform for Strongyloides spp., aided a major achievement, the advent of targeted mutagenesis via CRISPR/Cas9 in S. stercoralis and S. ratti. The genome sequences have also enabled significant molecular epidemiologic and phylogenetic findings on human strongyloidiasis, including the first genetic evidence of zoonotic transmission of S. stercoralis between dogs and humans. Studies of molecular signaling pathways identified the nuclear receptor Ss-DAF-12 as one that can be manipulated in the parasite by exogenous application of its steroid ligands. The chemotherapeutic implications of this were unscored by a study in which a Ss-DAF-12 ligand suppressed autoinfection by S. stercoralis in a new murine model of human strongyloidiasis. Summary Seminal advances in genomics of Strongyloides spp. have transformed research into strongyloidiasis, facilitating fundamental phylogenetic and epidemiologic studies and aiding the deployment of CRISPR/Cas9 gene disruption and editing as functional genomic tools in Strongyloides spp. Studies of Ss-DAF-12 signaling in S. stercoralis demonstrated the potential of this pathway as a novel chemotherapeutic target in parasitic nematodes.

Host Preference and Seedborne Transmission of Ditylenchus weischeri and D. dipsaci on Select Pulse and Non-Pulse Crops Grown in the Canadian Prairies

The stem nematode Ditylenchus weischeri was recently reported on creeping thistle (Cirsium arvense) in Canada. Two greenhouse studies examined host suitability of crops commonly grown in the Canadian Prairies for D. weischeri and the closely related parasite of many crops, D. dipsaci. In the first study, common pulse crops (yellow pea, chickpea, common bean, and lentil), spring wheat, canola, creeping thistle, and garlic were evaluated. Plant biomass and reproductive factor (Rf = nematode recovered/inoculated) 8 weeks postinoculation were used to determine host suitability. Creeping thistle biomass was reduced by D. weischeri whereas D. dipsaci reduced biomass of four of five pea and two of three bean varieties. Two pea varieties were weak hosts for D. weischeri, with Rf slightly >1. D. weischeri aggressively reproduced on creeping thistle (Rf = 5.4). D. dipsaci reproduced aggressively on garlic (Rf = 6.4; a known host), moderately on pea varieties (Rf > 2), and weakly on chickpea and bean (Rf > 1). In the second study, using creeping thistle and yellow pea, D. weischeri was recovered from aboveground parts of the plants and seed of the former and D. dipsaci from the later. The results show that D. weischeri parasitizes creeping thistle but not other crops and that D. weischeri host preference is different from that of D. dipsaci.