Conference Agenda

Advances second and third-generation sequencing technologies over the last decade have revolutionised the analysis of microbial communities and are now being increasingly applied in helminthology research. There are many opportunities, as well as some specific challenges, when applying these approaches to helminths. Long-read and short-read shotgun sequencing are generating many more, and higher quality, reference helminth genomes which will be crucial to apply genome-wide approaches to helminth population genetic studies. However, metabarcoding and targeted deep sequencing have an important roles and are particularly powerful when dealing with large sample numbers, low amounts and/or low quality template DNA (for example in stool samples), and complex and/or poorly defined helminth communities. This talk will focus on current resources and future directions in both short-read and long-read metabarcoding (nemabiome metabarcoding) and targeted deep sequencing and their application to study anthelmintic drug resistance, molecular epidemiology and molecular diagnostics of parasitic nematodes in both animals and humans. Examples will include recent work on the widespread emergence of anthelmintic resistance in canine hookworm Ancylostoma caninum across north America as well as the role of metabarcoding in the discovery of a new species of human Trichuris, T. hominis, which is refractory to albendazole-ivermectin combination therapy.

Tick-borne diseases (TBDs) present challenges due to complex interactions among hosts, tick toxins, salivary proteins, and microbes. Limited understanding of these interactions leads to undetected cases despite clinical guidelines. The rise of omics technologies offers a promising solution by shifting from pathogen-centric to host-centric studies, which can elucidate the complexity of TBDs. The skin, the primary site of tick bites, has been largely overlooked. We hypothesise that studying interactions at the skin can provide insights into disease progression. Using the NanoString GeoMx platform, we performed spatial transcriptomics on skin tissues from tick-bitten patients, comparing the tick-bite epicentre within 72 hours post-bite and contralateral ‘healthy’ skin. Our data reveal perturbations in several pathways. Spatial analysis identified tissue signatures distinguishing acute from chronic tick bites, highlighting differentially expressed genes. Our study shows that skin signatures can correlate with biomarkers from other omics data, improving diagnostic outcomes and informing treatment options. This approach enhances understanding of tick-associated pathogenesis, aiding in developing better management strategies for TBDs.

Scabies is a neglected tropical disease caused by the obligate parasitic mite Sarcoptes scabiei. With an estimated 400 million cases annually, mainly in the tropics, scabies is one of the most common dermatological diseases globally. The burrowing of mites within the epidermis combined with mite excretory proteins interfering with the host immune system causes secondary bacterial infections. Epidemiology has demonstrated a correlation between scabies and disease caused by Staphylococcus aureus and Streptococcus pyogenes, though there is little molecular evidence to underpin this link. We undertook a collaborative multi-national study collecting skin scrapings from scabies patients in three countries. Microbial DNA was extracted and 16s full length rRNA and ITS^1-4 sequencing were performed using the PacBio sequel. Using an established bioinformatics pipeline, a total of 22,678 amplicon sequence variants were identified from 751 samples. Community composition and microbial abundance was analysed using the programming language R. Our results demonstrate a significant decrease in microbial diversity and an increase of pathogenic bacteria in scabies infected lesions (P<0.05). This study is the first to identify and quantify the scabies associated microbiome at the molecular level to provide a basis to improve treatment approaches for this disease complex.

Arthropod ectoparasites play a crucial role in disease transmission worldwide, with ticks being particularly significant due to their ability to carry and transmit a diverse array of pathogens. Understanding the population structure of ticks is essential for predicting their movement and spread of associated pathogens. This study employs ddRADseq, a cost-effective population genetic tool that provides high-resolution SNP data. This was used to assess the population structure of Amblyomma triguttatum (ornate kangaroo tick) in the Swan Coastal Plain of Western Australia. One hundred ninety-two A. triguttatum specimens were collected and analysed, most of which fell within a 330km range. Results indicate that specimens collected in closer geographic proximity are more genetically related. Urbanisation (e.g., cities and suburbs) and natural barriers (e.g., the Swan River) contribute to genetic segregation. In contrast, within non-disrupted areas, genetic connectivity was more homogeneous, although genetic distance still increased with spatial separation. These findings provide valuable insights into previously unknown genetic structure and movement patterns of A. triguttatum. This information is crucial for future research on tick-borne diseases affecting humans, companion animals, and livestock, as it helps identify potential transmission corridors for current and emerging pathogens.

Ixodes holocyclus, the Australian eastern paralysis tick, is an obligate hematophagous ectoparasite known for causing flaccid paralysis in animals and humans due to its potent neurotoxin, holocyclotoxins. Treatment typically involves administering antiserum once symptoms develop. However, a lack of genomic, transcriptomic, and proteomic resources hampers understanding of I. holocyclus biology, including toxin production, host preference, and survival mechanisms. To address this resource gap, we generated the first high-quality chromosome-scale genome assembly of I. holocyclus. This integrated approach, combining Oxford Nanopore long-read sequencing, Illumina short-read sequencing, Hi-C chromatin interaction maps, and long- and short-read RNA sequencing, facilitated the construction of comprehensive gene models. The resulting chromosomal-scale draft genome spans 1.9GB and includes approximately 35,000 predicted protein-coding genes, alternative splice isoforms, and transposable elements. Given I. holocyclus's limited distribution along Australia's eastern coast, we generated ~100 I. holocyclus genomes from this region and intent to perform comparative analyses. This aimed to understand genetic complexity, link genomic variation with geographic distribution, ecological adaptation, vector capacity, and potential drug resistance emergence. Our study provides the first genetic resource for I. holocyclus, a potential paralysis-inducing hematophagous ectoparasite. These insights can inform future interventions against this tick and tick-transmitted infections.

RNAi technology has emerged as a potential biocontrol strategy against various pests, including Lucilia cuprina, an ectoparasite that causes significant harm to sheep. Blowfly strike is the second most costly parasitic disease of sheep in New Zealand and costs the New Zealand industry more than $37 million per year. In this study, we explored the effects of dsRNA on the blowfly. Initially, six dsRNA constructs were assessed via microinjection, focusing on their impact on RNA expression, hatching, and mortality. Building on these insights, we evaluated two novel materials systems: Bentonite combined with either Polyethylenimine or Poly (dimethylaminoethyl methacrylate), referred to as BenPol. These systems were assessed for cytotoxicity, stability in midgut juice, and their protective effects on dsRNA. Initial findings showed that the BenPol composites exhibited low cytotoxicity. Further investigations focused on the loading and pH-dependent release of dsRNA from these nanoparticles. The BenPol systems demonstrated effective protection of dsRNA within the harsh gastric environment of L. cuprina for over 24 hours. Subsequent experiments involved loading RNAi targets onto the BenPol systems through oral feeding. Results from these bioassays indicated that both BenPol systems (PEI and pDMAMAE) significantly enhanced RNAi efficacy while safeguarding the integrity of dsRNA.

Trichomonas vaginalis (TV), the most common non-viral sexually transmitted human pathogen, feeds on the vaginal microbiota including protective Lactobacillus spp. (LB) through phagocytosis. Subsequent destruction occurs in lysosomes through degradative hydrolases. Secretion of protein factors by TV may play a fundamental role in TV-LB interaction prior to phagocytosis. However, a comprehensive profile of the secretome upon interaction remains elusive.

Here we co-cultivated TV and LB for multiple time frames to investigate their interactions. Phagocytosis was observed and quantified by microscopy and Fluorescence-activated cell sorting. The dynamics of protein secretion during TV-LB co-incubation was analyzed in the cell-free supernatant by label-free quantitative mass spectrometry. Gene expression levels of differentially secreted proteins were examined through real-time quantitative PCR. LB significantly stimulated the secretion of 24 TV proteins including a variety of proteases and surface proteins such as Lysozyme-like, Cathepsin-L like, and GP63-like peptidases, and Trichomonas beta-sandwich repeat proteins, a group of heterogenous surface transmembrane proteins of unknown function. Overexpression of selected proteins in TV confirmed their effect on LB viability.

Our analysis provides a comprehensive set of secreted proteins that may be involved in parasite-LB interactions and potentially contribute to the decolonization of LB during trichomoniasis and the development of vaginal dysbiosis.

Infection with Trichomonas vaginalis is associated with an imbalance in the bacteria that normally reside in the female reproductive tract. T. vaginalis is increasingly detected in women that have reduced amounts or a complete absence of Lactobacillus species. This is alarming, as Lactobacillus crispatus promotes better pregnancy outcomes and protects against urogenital infection. We hypothesize that T. vaginalis has antibacterial activity against L. crispatus. Using confocal microscopy, we observed that T. vaginalis adheres to L. crispatus as soon as both microbes are exposed to one another. Furthermore, T. vaginalis exerts an antibacterial effect against L. crispatus leading to a statistically significant reduction in L. crispatus colony forming units as early as 30 minutes, and even greater microbicidal activity is observed at 3 and 6 hours of co-incubation. Additionally, T. vaginalis displayed this antibacterial activity in nutrient-rich and nutrient-limiting conditions, whereas the viability of T. vaginalis was unaffected by exposure to L. crispatus. The molecular mechanisms contributing to this microbe-microbe interaction are under study. Our work will yield critical insight as to how T. vaginalis infection leads to changes in the cervicovaginal microbiome and potentially reveal a therapeutic need to ameliorate the microbiological insults inflicted by T. vaginalis.

Lactobacillus iners is the most common bacteria found in the cervicovaginal microbiome. This bacterial community is most prone to shift towards a microbial community found in women with bacterial vaginosis. Infection with Trichomonas vaginalis is also associated with bacterial vaginosis. To our knowledge, nothing is known about T. vaginalis-L. iners interactions. We hypothesize that T. vaginalis exerts antibacterial effects on L. iners. We first optimized the conditions to maximally quantify L. iners colony formation on agar plates. Upon co-incubation with T. vaginalis, we found that at a ratio of 155 L. iners to 1 T. vaginalis, there was a statistically significant 96% reduction in L. iners colony formation. At a ratio of 0.02 bacteria to 1 parasite, L. iners growth was promoted by 1.7 fold in the presence of T. vaginalis. Interestingly, in 12 biologically independent experiments, T. vaginalis viability was unaffected by the presence of L.iners. Lastly, using transmission electron microscopy, we have observed L. iners uptake by T. vaginalis. Delineating the factors that influence the balance between coexistence vs. antibacterial killing of L. iners by T. vaginalis can potentially lead to the identification of novel therapeutic targets to ease the burden of T. vaginalis infections.

The human vaginal microbiome is a complex ecosystem primarily governed by lactobacilli, which create an acidic environment and defend against pathogens. However, when lactobacilli decrease, anaerobic bacteria such as, Gardnerella vaginalis among others may flourish as a polymicrobial condition known as bacterial vaginosis (BV). Coincidently, the vaginal infection by the protozoan Trichomonas vaginalis (i.e., trichomoniasis) is accompanied by this dysbiotic BV-like microbiome including G. vaginalis. Therefore, the interactions between this bacterium and the protozoan, despite poorly understood, might be crucial for disease development. Because G. vaginalis is the initiator of a pathogenic biofilm, here we aimed to understand how these two microorganisms may help disperse each other. We employed complementary approaches to evaluate biofilm growth and dispersion, and the adhesion of T. vaginalis to this biofilm. We also examined whether extracellular vesicles (EVs) produced by lactobacilli and G. vaginalis would influence the dispersion of T. vaginalis on agar-based assays. We show that T. vaginalis avidly adheres to the G. vaginalis biofilm and enhances bacterial dispersion in a strain-independent manner while bacterial EVs affect T. vaginalis dispersion. Further experiments are necessary to quantify these phenotypes and to reveal the molecular mechanisms underlying the synergistic interactions between G. vaginalis and T. vaginalis.

Vaginal trichomoniasis, resulting from Trichomonas vaginalis infection, is characterized by a dysbiotic microbiome including anaerobic bacterial species associated with bacterial vaginosis (BV). Recent research by us and others highlights important disease-related synergism between BV-bacteria and T. vaginalis. BV-bacteria are known to metabolically collaborate among themselves. Therefore, our study aimed to investigate whether BV-bacteria and T. vaginalis would also engage in metabolic cooperation. Here we demonstrate that, unlike host-protective lactobacilli, BV-bacteria and T. vaginalis mutually support each other's growth, even in depleted conditions where neither organism can grow alone. Furthermore, metabolomics analyses using gas-chromatography coupled with mass-spectrometry revealed alterations in metabolite profiles, indicating a metabolic shunt towards amino acid catabolism, notably through upregulation of the arginine dihydrolase (ADH) pathway. This metabolic pattern mirrors previous reports of cooperation between T. vaginalis and its mycoplasma endosymbionts. Gene expression analysis further confirmed upregulation of ADH enzymes, particularly carbamate kinase. In conclusion, our findings unveil a previously unrecognized metabolic cooperation between T. vaginalis and the associated dysbiotic microbiome, providing insights into the synergistic relationships underlying vaginal trichomoniasis.

Trichomonas species live at various mucosal surfaces characterised by complex microbiota amongst a range of animal hosts. Trichomonas infections associate with significant changes in microbiota taxonomic composition, commonly referred to as dysbiosis. Trichomonas vaginalis and Trichomonas gallinae (a bird parasite) infections result in the depletion of the mutualist bacteria Lactobacillus spp., known to play important roles in female urogenital defence against pathogens. However, the molecular and cellular basis of interactions between Trichomonas and the microbiota are poorly understood.

We exploited Trichomonas gallinae - Escherichia coli co-cultures as a model system alongside comparative genomics and transcriptomics (RNAseq) to identify candidate enzymes and peptides targeting bacteria. A number of genes encoding homologues of bacterial cell wall targeting enzymes, including candidate lysozymes, and anti-microbial peptides, which are conserved throughout Trichomonas spp., were identified and with a number of them being significantly upregulated within the co-culture system. We also observed changes in bacterial and parasite behaviour and morphology.

Integrating comparative genomics between species, transcriptomics within our model and known interactions between Trichomonas and bacteria, for example the peptidoglycan targeting NlpC/P60 endopeptidases, illustrates a number of mechanisms for the parasites to potentially out-compete bacteria, with a likely important role of deconstructing bacterial cell wall peptidoglycans.

Gastrointestinal nematodes (GIN) are a challenge for goat producers worldwide, causing high morbidity and mortality rates, and data pertaining to current GIN distribution in Australian goats is sparse. The objective of this study was to characterise the distribution of GIN in Queensland and New South Wales meat goats and explore influential factors. Between April 2022 and March 2024, 2,615 individual faecal egg counts (FEC) were conducted across 35 goat farms. All goats had not received anthelmintic treatment for at least 8 weeks prior to sampling. A number of effects were explored including rainfall, system intensity, supplementary feeding, breed and age. A high prevalence (96.7%) of GIN infection was observed, with 46% of goats responsible for 80% of total FEC output. The primary GIN species present were Haemonchus contortus and Trichostrongylus colubriformis. Significant differences (p < 0.05) were found in the FEC of goats across differing rainfall categories, system intensities, breeds, age classes and those that received supplementary feeding. The findings from this study indicate that GIN infection is a widespread issue for meat goat producers, and infection levels exist at magnitudes that indicate potential production losses.

Small strongyles (cyathostomin) infection in equines can cause clinical symptoms such as lethargy, sudden weight loss, debilitation, and diarrhoea. The encysted stages of cyathostomin can enter a dormancy stage called hypobiosis, which are not detected with a standard faecal egg count (FEC). Although a commercialised IgG(T) sera test identifying encysted larvae is available, the requirement for blood samples impedes its wide scale adoption. The aim of the current study was to investigate whether saliva can be used as an alternative to blood to detect antibodies against encysted larvae.

Saliva blood and faeces were collected at three timepoints (Spring, Autumn, and Winter) from horses (n=27) in regional Victoria, Australia. Blood was processed for IgG(T) antibodies against encysted stage larvae, the Mini-FLOTAC method was used to calculate FEC, and an IgA/IgG(T) ELISA was optimised for saliva. A positive correlation between sera and saliva IgG(T) in Winter was found (r=0.49, p=0.022). No correlations were observed with FEC for either blood or saliva. These results demonstrate antibodies to encysted stage larvae antigens used in the commercial test are also present in saliva and may have potential as an alternative test to detect encysted stage of cyathostomin infection in equines.

The pig industry contributes approximately AUD 5.2 billion annually to the Australian economy and provides around 36,000 full-time jobs across various sectors. Gastrointestinal parasites in pigs can lead to poor feed conversion and reduced weight gain, contributing to economic losses. Despite the recognised impact gastrointestinal parasites can have on pigs, little is known about their prevalence in Australia. This study aims to assess the prevalence of internal parasites across different pig production systems in Victoria through a cross-sectional coprological survey. A total of 50 commercial and backyard piggeries will be selected for the study. Approximately 30 faecal samples will be collected from each farm and tested using a modified McMaster technique. Selected parasite species will be characterised using morphological and genetic methods. Preliminary testing of samples from 29 farms shows a farm-level prevalence of 17% for Trichuris suis and 28% for coccidia (Cystoisospora suis/Eimeria spp.) in Victorian pigs. The findings of this project will generate baseline data and enhance our understanding of the prevalence of various internal parasites among pigs of different age groups and farming systems, thereby facilitating the development of effective parasite control strategies for Victorian pigs.

Ascaris suum is a significant internal parasite of pigs and is transmitted via faecal-oral route. It can result in reduced growth rate, poor intestinal absorption and contamination of livers with migrating larvae, leading to economic losses. Larval migration through the liver results in lesions (milk spots) and condemnation of livers at slaughter. The most commonly used diagnostic technique for detecting A. suum in live animals is the faecal egg count (FEC). However, this method cannot detect A. suum at the larval stages. This study investigates the effectiveness of enzyme-linked immunosorbent assay (ELISA) in diagnosing A. suum infection in pigs, by detecting antibodies against A. suum in pigs’ serum. Faecal, blood and liver samples will be collected from the pigs at the abattoir. A comparison of FEC results and liver milk spots scores to those of ELISA will help to identify early infection at larval stages. Preliminary screening of Victorian piggeries has identified 7% (2/29) of farms positive for A. suum infection using the FEC technique. The findings of the study will help to select an improved diagnostic technique for A. suum infection in pigs, resulting in timely detection of the parasite, thereby reducing economic losses.

Gastrointestinal nematode (GIN) infections cause economic losses in livestock and remains a global challenge for farmers. Haemonchus contortus is one of the most pathogenic GIN of small ruminants, often leading to death. In Victoria (Australia), outbreaks of H. contortus infections have been sporadic and linked to years with high rainfall. However, changing climatic conditions suggest infections may become more endemic and rapid and specific diagnosis is critical for effective treatment. Currently, Faecal Egg Counts (FEC) and larval cultures are the gold standard commercial diagnostic method which are carried out largely in regional, low technology laboratories. FEC are rapid but are limited by confirming patent infections, not GIN species. Conversely, larval culture allow species identification but are slow taking 1-2 weeks. There are many molecular diagnostic techniques used routinely in research allowing specific and rapid identification of GIN species including polymerase chain reaction (PCR), Loop-mediated isothermal amplification (LAMP) and sequencing of the ITS-2 rDNA region (nemabiome). This project will examine the commercial feasibility of such biomolecular tests in partnership with regional diagnostic laboratories to enable rapid diagnostics to confirm species presence. This will assist in the surveillance of H. contortus in Victoria throughout changing climatic conditions.

Managing parasites with fewer anthelmintic inputs requires a better understanding of parasite epidemiology, especially in situations where some species exhibit high levels of resistance and other species remain highly susceptible to anthelmintics. The seasonality of parasite species abundance has been difficult to establish because visual speciation of infective stage larvae is difficult. Now, relatively new DNA techniques have made this easier.

Nine farms distributed around New Zealand were enrolled in the project and tasked with sending in 10 fresh faecal samples from each of 3 stock classes of sheep (mixed age ewes, lambs and 2^nd year (2Tooth) ewes) on their property. Faecal nematode egg counts were performed on each sample and the remaining faeces was cultured by stock class. The resulting 3^rd-stage larvae (L3) were extracted, counted, and aliquoted for nemabiome sequencing.

As expected, initial results indicate differences in the species composition between age classes of sheep and regional variations, probably associated with climatic differences. Interestingly the seasonality of the species revealed by the data, raises some questions about the best time of year to carry out Faecal egg count reduction tests. As well as emphasising the need to truly know what species you have present when making treatment decisions.

Ticks and tick-borne diseases (TTBDs) are significant endemic and priority health and production constraints to the Australian cattle industry. Between November 2023 and May 2024, we conducted the first Australia-wide online survey to investigate cattle farmers' knowledge, attitudes, and practices regarding TTBDs. The questionnaire comprised 66 questions (16 close-ended and 50 open-ended) about farm demography, knowledge and perceptions of TTBDs and control. Interim results revealed that while most respondents in Queensland (91%: 51/56) and New South Wales (89.1%, 41/46) had observed ticks on their cattle, 59.4% (19/32) of Victorian respondents were uncertain although their animals had bovine theileriosis (75%:24/32). Interestingly, 49.2% (66/134) of the respondents strongly considered wild animals in the paddocks as a source of ticks to cattle, 25.4% (34/134) were unsure if all ticks seen on cattle could transmit diseases. Most of the respondents (88.8%:119/134) used acaricides as their primary tick control method. However, 32.8% (44/134) of the respondents noted that ticks usually reappeared on cattle within a month of treatment, indicating potential acaricidal resistance. This study demonstrates knowledge gaps in risk perception and integrated parasite management for controlling TTBDs among Australian cattle farmers. This highlights the need for increased awareness and targeted interventions to manage TTBDs effectively.

Andrewartha and Birch (1954) regarded the abundance and the geographic distribution of species to be two sides of the same coin. Shelford (1917) too, regarded the abundance and the distribution of species to depend on the extent to which conditions deviate from the ecological optimum (of the species). Previously, such ecological studies on the geographic distribution and abundance of species were mostly observational, correlational, and theoretical. With advancements in technologies however, sophisticated methodologies and tools to project the distributions of species and to account for their abundances have been increasingly available. Armed with these principles and these tools, I studied the effects of climate, and how variations in the weather accounted for the geographic distributions and the abundances of several species of ticks. Herein, I discuss some of my findings, and how such tools can help, and have furthered our understanding of the distribution and the abundances of ticks. I will also discuss how these projections may aid biosecurity and biosafety with a consideration of a changing climate, and the limitations of such studies.

The prevalence and distribution of mosquito-borne diseases (MBD) consistently change over time and space. Such changes result from variations in landscape, host biodiversity, and climate, which are highly influenced by anthropogenic change. This complexity makes studying MBD prevalence and distribution challenging but essential as these factors are highly variable globally.

Fortunately, mosquitoes are convenient and non-invasive tools for disease surveillance due to their haematophagous behaviours. Government Departments of Health use mosquitoes for MBD surveillance. However, these departments primarily focused on detecting common human MBD. Less prevalent or animal-specific MBD are, therefore, likely not reported.

This study has collected and identified 4,323 mosquitoes from Perth’s urban and peri-urban areas across the 4 traditional and 6 Noongar seasons. Population data has determined trends in mosquito biodiversity over seasons and between different levels of urbanisation. Collected mosquitoes are pooled according to location, date, and species, and are screened for mosquito-borne parasites, including Dirofilaria, Plasmodium, and Haemoproteus. Preliminary data has identified 11 Avian Plasmodium and 1 Haemoproteus species. Parasites were predominantly identified within mosquito of the genus Culex. Although this data does not determine the mosquito's capacity to transmit the parasites, these results can aid future mosquito control programs.

Oxythiamine, an antiplasmodial thiamine analogue, kills P. falciparum via a mechanism that likely involves its conversion by thiamine pyrophosphokinase (TPK) into the antimetabolite oxythiamine pyrophosphate (OxPP). To deepen our understanding, we used in vitro evolution to generate oxythiamine-resistant parasites. Whole-genome sequencing identified a point mutation in PfTPK at position 284. Homology modelling suggested that the mutation may decrease ATP/AMP binding and consequently reduce PfTPK activity. Consistent with this, oxythiamine-resistant parasites accumulated five times less [³H]thiamine/[³H]TPP than wild-type parasites, and the activity of oxythiamine against parasites expressing a GFP-tagged version of mutated or wild-type PfTPK was altered. To investigate the role of TPK throughout the parasite's life cycle, we generated P. berghei parasites lacking TPK (PbTPK-KO). No defect was observed in PbTPK-KO during their blood stage, but they were resistant (5-fold) to oxythiamine in vivo. Although PbTPK-KO produce a similar number of oocysts compared to wild-type parasites, PbTPK-KO oocyst failed to mature and did not produce sporozoites. Transmission electron microscopy (TEM) of mosquito midguts infected with PbTPK-KO parasites revealed small oocysts containing large empty spaces and clustered membrane whorls. In conclusion, we reveal a crucial role for TPK in the antiplasmodial activity of oxythiamine and the development of P. berghei oocysts.

Pantothenate, a precursor of the enzyme cofactor coenzyme A (CoA), is an indispensable nutrient for Plasmodium falciparum. Pantothenate kinase (PanK) catalyses the first step of CoA biosynthesis. P. falciparum expresses two PanKs, PfPanK1 and PfPanK2. To date, all PanKs with a solved structure function as homodimers. In contrast, we have shown that P. falciparum and T. gondii possess a novel heteromeric PanK complex. We performed site-directed mutagenesis of key residues in PfPanK1 and PfPanK2 predicted to be involved in active site stabilisation. Parasites expressing each mutant protein were generated, from which the complex was then purified and analysed. Heterologous expression of the PfPanK complex was attempted by using the insect cell protein expression system, with all components of the complex being expressed within the same cells. Although some of the expressed components aggregated, enough remained soluble, naturally formed the complex in situ and, crucially, when purified, the complex was functional. In addition, we generated PfPanK1 or PfPanK2 inducible knockdown parasite lines and show that knockdown of either PfPanK1 or PfPanK2 is detrimental to parasite proliferation. This research advances our knowledge of a novel P. falciparum protein complex and may facilitate the identification of new drugs targeting P. falciparum pantothenate utilisation.

Ascaris infection, a prevalent neglected tropical disease, affects 819 million people worldwide and causes significant economic losses in the livestock industry. Despite available anthelmintics, reinfection and emerging drug resistance are major concerns. Targeting the early stages of infection, specifically hepatopulmonary migration (HPM), where larvae migrate from the intestine to the liver and lungs, probably guided by chemosensation, presents a promising intervention strategy. However, our understanding of chemosensation in parasitic nematodes is limited. We explored the role of chemosensation in guiding larval Ascaris suum during infection. We conducted tissue-specific transcriptional studies, identifying a chemosensory pathway specifically present in the head and amphidal tissues of Ascaris. Subsequent larval migration assays demonstrated the chemotactic responses to pig liver and lung homogenates, as well as extracted metabolites. Transcriptional profiling of the stimulated larvae highlighted the molecular pathways involved in Ascaris chemotaxis. Additionally, we developed and characterized a polydimethylsiloxane (PDMS) microfluidic device to further investigate the chemosensory behaviour of Ascaris larvae. Our findings in microfluidics revealed distinct behavioural responses of larvae to linear concentration gradients and confirmed chemoattraction to pig liver homogenates, demonstrated by increased forward speed and reduced turning. Future studies will aim to identify target-receptor interactions to potentially block chemotaxis and disrupt HPM.

Inflammatory bowel diseases (IBD), encompassing ulcerative colitis and Crohn’s disease, are chronic inflammatory conditions affecting millions globally, with no current cure. Pediatric IBD often manifests more aggressively than adult-onset IBD, accounting for 25% of total cases. These heterogeneous diseases are prevalent in Western countries, where high levels of sanitation have reduced exposure to organisms that influence immune development and response, such as gastrointestinal hookworms. These helminths secrete diverse bioactive molecules with immunomodulatory and anti-inflammatory properties. We expressed individual hookworm-derived proteins in our lab to evaluate their therapeutic potential in inflammatory conditions. One recombinant protein significantly reduced disease severity and markers of inflammation in a mouse model of experimental colitis induced before sexual maturity. Our current research focuses on elucidating the mechanism of action of this protein by identifying its target cells, assessing its impact on intestinal barrier integrity, and examining its influence on intestinal immune processes.

Leishmaniases are caused by at least 21 species of Leishmania and significantly affect human and animal health in tropical and subtropical regions. Understanding their complex epidemiology has been hindered by traditional techniques’ inability to capture the full diversity of Leishmania species. We developed a metabarcoding tool targeting the heat shock protein 70 gene using a portable Oxford Nanopore Technologies (ONT) MinION device to establish the diversity of Leishmania species infecting vertebrate hosts and vectors. We also created a new database and used it alongside the NanoCLUST analysis pipeline. This tool allowed the accurate classification of all 14 Leishmania species tested. Using this method, all Leishmania species were separated from each other, including those within the same complex, with percentage identity scores between 99.99 - 100% when compared to reference sequences. The species tested represented four Leishmania subgenera: L. tropica, L. aethiopica, L. major, L. gerbilli, L. infantum, L. mexicana, and L. amazonensis (subgenus Leishmania); L. guyanensis and L. panamensis (subgenus Viannia); L. orientalis (subgenus Mundinia); L. tarentolae (subgenus Sauroleishmania). This nanopore sequencing method provides a useful tool for the differentiation of Leishmania species to support epidemiological studies and the prevention and control of this disease in humans and animals.

Trichomonas vaginalis (Tv) is a human parasite, causing urogenital tract infections and is commonly treated with metronidazole. Rising resistance rates have become an issue in patient treatment.

The mechanisms of resistance are not yet fully understood, but it was shown previously that strain T_V C1 displayed a similar pattern of up- and downregulated proteins when depleted of iron as its highly metronidazole-resistant cell line Tv C1 res. Proteomic analysis was performed to fully determine the differences in protein expression between wildtype, iron depleted, and resistant Tv C1, to thereby identify proteins specifically up- or downregulated in the context of metronidazole resistance. To this end, C1 cells were cultured with 2,2-bipyridine for iron depletion, alongside metronidazole resistant and wildtype C1 cell lines. Protein extraction was performed with TCA/acetone, followed by enzymatic digestion using Trypsin/ LysC and 3 kDa FASP filter units. Peptides were separated using a nano-HPLC Ultimate 3000 RSLC system directly coupled to a high-resolution Q-Exactive HF Orbitrap mass spectrometer via ESI interface. Using a quantitative proteomics approach, we were able to highlight proteomic differences between resistant, non-resistant, and iron depleted parasitic cells, to gain further understanding in the formation of metronidazole resistance in Tv.

Human trichomoniasis, a globally prevalent sexually transmitted infection caused by Trichomonas vaginalis, affects approximately 278 million people each year. It presents a challenge due to resistance to the current treatment, Metronidazole (MTZ), which is also associated with side effects. Cannabis sativa, with more than 100 phytocannabinoids and numerous studies for therapeutic applications, including parasitic infections, has undergone a significant shift in acceptance worldwide, highlighted by legalizations and substantial revenue projections. In this context, the present study delves into the effects of cannabinoids, specifically WIN 55,212-2, Cannabivarin (CBV), CBD-rich oil, and THC-rich oil, showcasing their anti-parasitic actions that influence the growth and morphology of T. vaginalis. The analysis extends to encompass the pharmacokinetic properties of these cannabinoids. Among the analyzed cannabinoids, CBV stands out for adhering to Lipinski's rules, indicating its potential suitability for oral drug delivery. They also demonstrated inhibitory effects on the growth of T. vaginalis trophozoites and a reduction in the parasite's adhesion to host cells. Several morphological alterations were observed, such as membrane projections, blebbing, autophagosomes and damaged hydrogenosomes. These results highlight the need for further research to explore the therapeutic potential of cannabinoids and understand their mechanisms of action in T. vaginalis.

Giardia duodenalis is a gastrointestinal parasite causing ~200 million symptomatic infections annually, disproportionately in lower socioeconomic tiers and children. Chemotherapeutic interventions are limited to nitroheterocyclic antibiotics such as metronidazole. However, high doses are toxic and drug-resistant treatment failures occur in up to 20% of cases, highlighting the urgency of novel and safer chemotherapeutics.

We previously identified a drug-like kinase inhibitor (IC₅₀=114nM). To identify the kinase target(s) in Giardia, we immobilise this compound to azide-agarose and -magnetic supports through “Click” chemistry, to “pulldown” its high affinity target(s). Pilot pulldown experiments showed different enrichment and non-specific binding profiles for either sets of beads. To minimise non-specific binding, we use “blank” beads and competitive binding as negative controls, with “baited” beads for the samples.

Out of the 4900 proteins in the annotated G. duodenalis proteome, DIA library-free searches identified 2065 and 931 proteins from these respective pulldowns. Relative to “blank” agarose and magnetic beads, we pulled down 819 and 132 significant proteins respectively. When further cross-referenced to secondary controls, we identify eight significantly enriched proteins from the baited agarose, and ten significantly enriched proteins from the baited magnetic beads; two kinases were identified from the baited agarose and were selected for downstream validation.

Screening for drug efficacy and safety for disease agents has historically been a costly and labour-intensive process, particularly for parasites which can have complex in vitro needs, despite efforts to improve the process.

We developed a custom robotics platform with the potential to concurrently screen for compound efficacy for parasite targets, off target effects and toxicity. A Tecan Fluent robotics platform was integrated with a gas-controlled incubator (Cytomat) and SparkCyto, capable of automated tissue culture maintenance and the capacity to perform whole cell assays using fluorescence, absorbance, bioluminescent and microscopy, followed by dose-titration on selected hits.

The platform was validated for Giardia duodenalis inhibition and compound specificity testing. From a small library of compounds 7% had potent selective inhibition of G. duodenalis, 12 of which had IC₅₀’s between 0.1 – 4.9 µM.

In conclusion, we describe a unique, high throughput platform for anti-parasite drug discovery, with the potential to initiate 18,000 assays per day. The platform could be adapted to accommodate a vast range of assays, from helminth motility assays to intracellular anti-parasite efficacy or enzyme target assays, greatly expanding the scope and improving the efficiency of anti-parasite drug discovery programs.

Drug resistance hampers the treatment of giardiasis, one of the world’s most common gastro-intestinal parasitic diseases. On an annual basis ~200 million people develop giardiasis, a disease that impacts child development and the long-term health of many adults. However, there is no vaccine for giardiasis and treatment options are failing due to multiple factors including drug resistant parasites. Moreover, current treatment strategies are monotherapies that do little to combat the development of drug resistance. To improve this position combination therapies that include new compounds with unique mechanisms of action are needed. However, little has been done to identify best practice combination therapies for giardiasis. Our team has identified new compounds with potent and selective activity against Giardia parasites. Our lead drug candidates are well-tolerated, have in vivo activity, do not impact the microbiome of mice, and have in vitro synergistic activity with currently used benzimidazole treatments. Importantly, one of the most promising candidates identified has cleared infections in mice. Preliminary data suggest that our new compounds have a different mode of action to currently used drugs that is linked to the unique cytoskeleton of Giardia parasites. Further studies with our lead candidates are now under way and will be discussed.

Giardia duodenalis is a common gastrointestinal parasite that causes ~280 million cases of giardiasis each year. While giardiasis is a ubiquitous disease that causes high rates of morbidity, Giardia is a neglected parasite with limited treatment options that are failing at an increasing rate. To improve this position, new treatment strategies and drugs with novel modes of action are desperately needed. This includes the identification and recommendation of drug combination strategies to combat treatment refractory cases. Recent work in our laboratories have identified two novel compound series with potent activity against G. duodenalis that have demonstrated tolerability and activity in mice. To further understand the clinical potential of these new anti-Giardia compounds, I have been further characterizing their in vitro interactions and biological activities. These data will be presented and discussed.

Rhipicephalus australis (Australian cattle tick) was introduced to Australia with Brahman cattle and belongs to a cryptic species group with R. annulatus and R. microplus. The tick microbiome is a target for control strategies, but few studies have investigated discrete tissues of R. microplus and no studies have utilised an un-biased metagenome approach.

Gut and salivary gland (SG) tissues were dissected from 60 semi-engorged adult female ticks, 3 replicates for each tissue. DNA was sequenced using the PromethION 24 (ONT) long read sequencer with adaptive sampling to deplete the tick genome. Tick and bovine reads were removed by mapping against relevant genomes (Minimap2). Assembled reads were classified using Kraken2 and species abundance was determined with Bracken2.9.

There was no statistical difference in alpha diversity between gut and SG tissues (p = 0.4), but ordination analysis revealed differences in species composition between the tissues. While all samples were dominated by Escherichia coli, Clostridium botulinum and Babesia bigemina were confined to the gut and Coxiella-like endosymbiont (CLEAA) sequences were identified only in SG. Species of the genus Borrelia were also identified, warranting further investigation. The identification of endosymbiotic species provides a potential new target for cattle tick control.

Ticks and tick-borne diseases (TTBDs) pose a global challenge with (re)emerging diseases affecting humans and animals. To promote animal welfare and advance future research on TTBDs, artificial tick feeding system (ATFS) is becoming an important tool. Herein, we have systematically reviewed the current knowledge on ATFS used for soft and hard ticks. A critical appraisal of 196 articles published between 1912 and 2024 revealed that capillary feeding, membrane feeding systems and semi-automated membrane feeding systems have been used to study tick biology, complete life cycle of several tick species, tick-pathogen interactions, and discover drugs and anti-tick vaccine targets under controlled environment. The majority of studies (n = 123) have used a membrane feeding system as it resembles the tick feeding on animals. It appears that there is a need for (i) finding improved attachment stimuli to enhance the success rate of tick feeding, (ii) optimising conditions for different ticks and their life stages, and (iii) developing effective ways to control entomopathogenic fungal growth during prolonged blood feeding periods. Therefore, international collaborative efforts could be made to improve the utility of ATFS in studying TTBDs and promoting animal welfare.

Microinjecting double stranded RNA (RNAi) into insect embryos can target and degrade specific messenger RNA transcripts and therefore restrict protein production. Here, we use RNAi on a sheep blowfly ectoparasite, Lucilia cuprina, to identify novel targets for pest control applications. Candidate genes were identified using transcriptomic and proteomic analysis, generated from comparisons between larvae feeding directly on sheep with larvae feeding on control agar-based media. Eighty highly expressed genes on sheep-fed groups were identified, and these genes were further filtered using lethality and phenotypic data inferred from the model organism Drosophila melanogaster. RNA injection and silencing of two of these selected genes caused high embryo lethality or developmental arrest and death at the first instar larval stage (mortality rate >90%). Quantitative PCR confirmed RNAi lowered the expression of the targeted messenger RNA levels. Gene silencing was further confirmed using three independent double stranded RNA constructs targeting the same gene. These essential, insect-specific proteins could be used for developing new insecticides and /or provide the foundation for developing environmental-friendly RNAi biopesticides against this agricultural pest.

The Australian sheep blowfly, Lucilia cuprina dorsalis, is a major sheep ectoparasite causing subcutaneous myiasis (flystrike) which can lead to reduced livestock productivity and in severe instances, death of the affected animals. Additionally, this species serves as a primary coloniser of carrion, an efficient pollinator, and is used in maggot debridement therapy and forensic investigations. The identification of closely related species within the genus Lucilia is challenging using classical morphological methods, complicating the resolution of their population structure. In this study, we report the complete mitochondrial (mt) genome of L. c. dorsalis from the Northern Territory (NT), Australia, where sheep are prohibited animals unlike the rest of Australia. The mt genome is 15,943 bp in length, comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. Phylogenetic analyses of 56 species/taxa of dipterans indicated that L. c. dorsalis and L. sericata are the closest among all sibling species of the genus Lucilia, which helps to explain species evolution within the family Luciliinae. This study provides the first complete mt genome sequence for L. c. dorsalis derived from the NT, Australia to facilitate species identification and examination of the evolutionary history of these blowflies.

The Australian sheep blowfly, Lucilia cuprina dorsalis, is a facultative ectoparasite whose larvae feed on the living tissues of domestic sheep in agricultural regions. In contrast, the closely related subspecies, Lucilia cuprina cuprina, is primarily found in urban areas and exhibits limited parasitic behavior. This study investigates the genetic and behavioral factors underlying the trophic adaptations of these two subspecies, aiming to identify genes and genomic regions associated with their physiological diversification and feeding habits. Behavioral assays were conducted to assess larval feeding preferences by measuring responses to different diets (rotten and fresh meat) at two temperatures (33ºC and 25ºC). These behavioral experiments, combined with comparative genomic analyses, aim to reveal potential differences in feeding behaviors between the subspecies. Our findings will enhance the understanding of the genetic mechanisms driving trophic specialization and the evolutionary processes contributing to the speciation of Lucilia cuprina.

The Australian sheep blowfly, Lucilia cuprina dorsalis, is a serious sheep parasite causing subcutaneous myiasis (flystrike) resulting in production losses, injury or mortality. Flystrike control relies heavily on breech modification surgery (mulesing) and insecticide treatments; however, widespread and accelerating resistance to existing insecticides is occurring with a limited understanding of the emergence of resistance alleles in Australian blowfly populations. An integrated genomics methodology, combining Oxford Nanopore Technologies (ONT) and Illumina sequencing with high-throughput chromosomal conformation capture sequencing (Hi-C), was used to achieve a chromosomal-level assembly of this important pest. The comprehensive assembly will provide valuable insights into the genetic makeup, evolutionary history, and potential mechanisms underlying key biological processes in L. c. dorsalis. Furthermore, the chromosomal-level genome facilitates the study of genetic variation within blowfly populations across the country, which is crucial for monitoring the emergence and spread of resistance alleles against control measures like insecticides.

The alkyne modified thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) is a gold standard chemical probe for DNA synthesis and proliferation detection in mammalian cells. EdU is incorporated into nuclear DNA via the thymidine salvage pathway and can be detected using copper catalysed azide-alkyne cycloaddition (CuAAC) with a fluorescent azide. However, as Plasmodium malaria parasites lack the thymidine kinase enzyme that is essential for metabolism, EdU and similar probes (e.g., BrdU) cannot be utilised. While P. falciparum engineered to express thymidine kinase from Herpes simplex virus can overcome this limitation and enable DNA labelling studies using BrdU and EdU, this approach may not be feasible for analysis different Plasmodium species, multiple laboratory lines and field isolates. We have designed and synthesised new thymidine-based probes that overcome the need for an endogenous thymidine kinase enzyme. We have shown that these DNADetect™ probes robustly label replicating asexual intraerythrocytic P. falciparum parasites using CuAAC with a fluorescent azide and flow cytometry. The DNADetect™ chemical probes are synthetically accessible and thus have broad applicability as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

Simulium blackflies of the damnosum species complex, known vectors of the parasitic nematode causing human onchocerciasis (river blindness), can travel long distances of 20 to 600 km with wind assistance. Onchocerciasis is marked by severe skin lesions, blindness, and epilepsy, mainly affects remote rural areas in sub-Saharan Africa, Yemen, Brazil, and Venezuela. In Ghana, extensive small-scale mining has polluted fast-flowing rivers, the natural breeding habitats for these blackflies, prompting their migrate in search of suitable environments and raising concerns about the spread of onchocerciasis to previously unaffected areas. In June 2023, blackflies were reported in Accra, a city previously free of onchocerciasis. This prompted an investigation, where 270 female blackflies were collected from 14 communities in Ghana using human landing catches. Whole genome sequencing and principal components analysis of 138,128 SNPs, along with k-means clustering, indicated that blackflies in Accra were genetically closer to those in the Eastern region of Ghana than to those from other parts of the country. This suggests a southeastern origin. These findings highlight the need for further research into the migration pathways of these blackflies and the implications of their urban presence for the spread of onchocerciasis.

The major intermediate host for the medically significant blood fluke Schistosoma haematobium, which is endemic primarily in African, is the freshwater snail Bulinus globosus. The taxonomic status of the species that comprise the B. globosus group is still remains controversial. We examined genetic variation of B. globosus using analyses of the intron 3 region of the arginine kinase gene (AkInt3). A total 81 B. globosus snails were collected from three different localities in Kenya. The 81 B. globosus snails were classified into 44 haplotypes (H1 – H44). These haplotypes were separated into three haplogroups (I – III). In addition, heterozygosity in the AkInt3 sequence was also detected. DNA recombination haplotypes between different haplogroups were commonly found in the heterozygous samples. High levels of genetic variability and heterozygosity were shown by AkInt3 sequence analysis, supporting the potential as a genetic marker to shed light on B. globosus population genetics. Additionally, our research shows that the B. globosus populations in Kenya formed a “species complex”.

Parasites, such as the malaria parasite P. falciparum, are critically dependent on host nutrients. Interference with nutrient uptake can lead to parasite death and, therefore, serve as a successful treatment strategy. P. falciparum parasites cannot synthesise cholesterol, and instead source this lipid from the host. Here, we tested whether cholesterol uptake pathways could be ‘hijacked’ for optimal drug delivery to the intracellular parasite. We found that fluorescent cholesterol analogues were delivered from the extracellular environment to the intracellular parasite. We investigated the uptake and inhibitory effects of conjugate compounds, where proven antimalarial drugs (primaquine and artesunate) were attached to steroids that mimic the structure of cholesterol. These conjugated antimalarial drugs improved the inhibitory effects against multiple parasite lifecycle stages, multiple parasite species, and drug-resistant parasites, whilst also lowering the toxicity to human host cells. Steroids with introduced peroxides also displayed antimalarial activity. Similar activity was observed against other apicomplexan and other protozoan parasites.

These results provide a proof-of-concept that cholesterol mimics can be developed as a drug delivery system against apicomplexan parasites with the potential to improve drug efficacy, increase therapeutic index, and defeat drug resistance.

Malaria parasites have developed resistance to all currently available antimalarials, and it is now critical novel targets against which new inhibitors can be developed are identified. To aid malaria elimination, these targets should play important roles in both malaria disease and transmission. We have identified Protein Disulfide Isomerases (PDIs), a family of enzymes responsible for the formation/cleavage of disulfide bonds ensuring correct protein folding, as novel targets for antimalarial development. Importantly, a wide repertoire of proteins involved in host cell invasion (associated with malaria disease) and transmission must be properly folded in order to function. Here we have interrogated whether PDIs can be inhibited by commercially available inhibitors, with a view to repurposing these compounds as novel antimalarials. We show that commercial PDI inhibitors prevent growth of key malaria-causing species (Plasmodium falciparum and P. knowlesi) whilst also inhibiting their ability to successfully invade host cells. Furthermore, through medicinal chemistry we have enhanced the activity of one of these inhibitors and our data suggests that it specifically targets PDI-Trans, a PDI found to be critical to malaria transmission. Collectively our data suggest that PDI inhibitors could provide dual stage activity and are attractive targets for antimalarial development.

Antibiotics that inhibit translation in the Plasmodium apicoplast are useful antimalarials, but these inhibitors have a “delayed death” effect. These compounds have no immediate impact on growth after treatment, and stop parasite growth only one cycle after drug exposure. A notable exception is the prophylactic drug doxycycline, which has a delayed death effect, but also kills parasites quickly at higher concentrations. We characterised the impact of delayed death drugs on Plasmodium protein translation using quantitative proteomics, simultaneously assaying translation in the apicoplast, mitochondrion and cytosol. We find that delayed death drugs lead to a specific block of apicoplast translation, but doxycycline additionally inhibits translation in the Plasmodium mitochondrion. Seahorse metabolic assays also show that doxycycline decreases parasite oxygen consumption rate, consistent with a perturbation to mitochondrial metabolism consequent to mitochondrial translation inhibition. This provides a rational for doxycycline’s faster inhibition of parasite growth at higher doses. Importantly, these data reveal the first known inhibitor of Plasmodium mitochondrial protein synthesis, and support mitochondrial translation as a potential target for future drugs. The discovery of an additional target for a widely-used malaria prophylactic may also inform future drug combinations for malaria treatment or prevention.

Trichomonas vaginalis is a sexually transmitted parasite that colonizes the human urogenital tract. As an extracellular pathogen, adherence to host cells is an important step to colonize the human host. Host: parasite interaction is regulated by changes in gene expression, but it is still largely unknown how these changes in transcriptional profiles are controlled. Our recent works highlighted the importance of epigenetics in the regulation of transcription. We identified transcriptionally active or repressive intervals flanked by 6mA-enriched regions, which are in close spatial proximity, suggesting a role for 6mA in modulating three-dimensional (3D) chromatin structure and gene expression. We now aim to analyze the role of 6mA and chromatin during the host-parasite interaction. We compare MedIP-seq data with ATAC-seq and RNA-seq data in free vs. host cell-attached parasites. We identified 2000 active/repressive intervals flanked by 6mA in the presence and absence of host cells, suggesting a role for 6mA in modulating 3D genome architecture through the formation of chromatin loops. Interestingly, our data showed differentially accessible chromatin regions and differentially expressed genes in pathogenesis-related genes when T. vaginalis is exposed to host cells. These findings underscore the importance of chromatin structure and its role in gene regulation during parasite infection.

To establish infection, T. vaginalis expresses diverse proteins/virulence factors targeted to the trichomonad’s surface and secreted into the vaginal environment. These factors interact with human cells and bacteria, namely lactobacilli, whose population decreases during acute trichomoniasis. We found that T. vaginalis significantly increased the secretion of 19 proteins in response to Lactobacillus jensenii, six of which belong to the TBSR family. This family comprises over 200 members, sorted into two groups based on the presence of a large domain of unknown function (lDUF) and several subgroups based on the number of cadherin-like beta-sheet repeats, presence of a transmembrane domain with a cytosolic tail, and a small domain of unknown function (sDUF). AlphaFold was used to predict potential interacting partners of TBSR1. Beta-catenins appeared among the best candidates to interact with the cytosolic tail, while a surface chaperone-like protein of L. jensenii was predicted to interact with the sDUF. Using Foldseek, the lDUF was identified as part of glycan hydrolases. AutoDock Vina and Alphafold predicted branched N-acetylglucosamines in mucins to represent the best lDUF binding candidates. Altogether, our predictions highlight TBSRs as prominent membrane-associated and secreted proteins potentially involved in recognizing and engulfing lactobacilli and other interactions within the vaginal environment.

Tetraspanins (TSPANs) are a family of proteins highly conserved in all eukaryotes, and known to be involved in adhesion, migration, and intracellular signaling. TSPANS are also often regarded as a marker for extracellular vesicles. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica. Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native PAGE analysis showed that these TSPANs form several complexes of 120-250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites.

Entamoeba histolytica is an intestinal protozoan parasite and causes amebiasis, which affects 50 million people annually. Phosphatidyl inositol (PI) metabolism is essential for signaling and trafficking in general and also essential for survival, proliferation, stage conversion, and pathogenesis of this organism. Among PI and phosphorylated PIs, phosphatidylinositol 3-phosphate (PI3P) is in general involved in endocytic and autophagic pathways. PI3P is generated from PI by class III PI-3kinase (C3PI3K). As the E. histolytica genome encodes a single C3PI3K, it is conceivable this gene is expected to play a pivotal role in biology and pathogenesis. However, it remains elusive how C3PI3K regulates endocytosis and autophagy and what accessory molecules are involved in its regulation in E. histolytica. In this study, we identified two major components of C3PI3K, vps34 (Ehvps34) and p150 (Ehp150), from E. histolytica. We also identified a unique E. histolytica-specific C3PI3K component, unlike mammalian counterparts (forming a heterotetramer), by coimmunoprecipitation followed by mass spectrometric analysis. We also demonstrated that EhC3PI3K is necessary for growth. Furthermore, we showed that EhC3PI3K localizes on the cell periphery by confocal laser microscopy. We presume that EhC3PI3K is involved in the endosome maturation, and are further investigating the mechanism how EhC3PI3K regulates endocytosis and autophagy.

The treatment of malaria, a deadly infectious disease caused by Plasmodium parasites, relies on an arsenal of chemotherapies that are hampered by drug resistance. New antimalarial drugs with mechanisms of action that are different to currently used drugs are needed to combat Plasmodium drug resistance. Robenidine is a known antiparasitic drug with modest activity against P. falciparum (50% growth inhibitory concentration; IC₅₀ 0.76 µM), the most lethal human malaria parasite species. To aid in the identification of new antiplasmodial drug candidates, a library of robenidine analogues were tested for antiplasmodial activity and selectivity for parasites versus human cells (selectivity index; SI). Multiple compounds with hit and lead activities were identified including NCL146 and NCL123 which demonstrated IC₅₀ values of <100 nM against P. falciparum 3D7 parasites and a SI of 100. Further studies with NCL146 and NCL123 have demonstrated that these compounds are active against multiple multidrug resistant P. falciparum, zoonotic P. knowlesi (IC₅₀ <200 nM) and are well tolerated in vivo. Furthermore, parasites treated with NCL146 display a unique metabolic signature, suggesting a mode of action different to currently used drugs.

There is a need for compounds that can target the transmission-capable gametocytes of P. falciparum. Using a molecular and chemical biology approach we have developed a novel antimalarials with a unique mechanism of action against blood stage Plasmodium. Based on the structure of the human protein PF4, an engineered peptide termed PDIP, has potent activity against trophozoites. PDIP selectively enters parasitised erythrocytes and causes death by lysis of the digestive vacuole. We sought to determine whether PDIP was similarly active against gametocytes and other transmission stages. We found no direct gametocytocidal activity, however PDIP-fluorochrome conjugates rapidly enter gametocyte infected erythrocytes, and persist within for several days, highlighting their specificity. Novel peptide drug conjugates (PDCs) of PDIP covalently linked to known antimalarials, which use the PDIP scaffold to selectively deliver the compound to the parasite, have direct killing and inhibit further activation during gametogenesis. We are working with collaborators to determine the effects of PDIP in the full mosquito transmission infection model, and against sporozoite viability. This project reinforces the selectivity of membrane active proteins, and supports PDIP as a novel antiplasmodial compound. We also validate the PDC approach as a strategy for selective delivery of potent antimalarials.

The human malaria parasite Plasmodium falciparum scavenges key nutrients for its survival. Among these is riboflavin, the metabolites of which are used in crucial intracellular processes such as redox homeostasis. Flavin adenine dinucleotide synthetase (FADS) is an integral part of riboflavin metabolism, and consequently, a potential antimalarial target. The riboflavin analogue roseoflavin (RoF) binds competitively to FADS, disrupting riboflavin metabolism and generating toxic metabolites. Previous work identified an additional riboflavin analogue with antiplasmodial activity, namely 8-aminoriboflavin (8AF). We will employ a two-fold approach to investigating riboflavin metabolism; the characterisation of FADS and the testing of riboflavin analogue activity against transgenic parasites.

We will attempt to generate a conditional PfFADS-knockout to ascertain the enzyme’s necessity in parasite proliferation. Additionally, the apicomplexan parasite Leishmania tarentolae will be used to express PfFADS. This will enable us to characterise the enzyme in isolation. Literature has shown that the over-expression of an enzyme which metabolises an antiplasmodial compound can increase the sensitivity of the parasite to that compound. We will explore this effect in the context of RoF and 8AF by using transgenic parasites which are over-expressing FADS. This work will provide novel insights into the suitability of riboflavin metabolism as an antimalarial target.

An important biological process in the biology of Entamoeba is stage conversion, which plays a crucial role in disease propagation facilitating parasite survival outside the host and spread to new hosts. Multiple mechanisms contribute to controlling the expression of amebic stage-specific genes. Identification of early transcriptional control regulators is crucial to understand initiation of the encystation cascade. Recent results including the identification of new regulators involved in Entamoeba development will be presented. The work represents an important advance in a critical aspect of parasite biology.

The South Pacific is a global hotspot for seabird diversity, and the waters of New Zealand and southeast Australia stand out as a critical region for threatened species of seabirds, ranging from plovers to albatrosses. Various parasites such as protozoa, ticks, helminths, among others, have been shown to cause significant mortality of seabirds in this region and there is growing evidence that human factors such as climate change and invasive species may synergize and/or enhance the impacts of these parasites on the survival or fitness of their seabird hosts. However, there are important knowledge gaps that limit our ability to mitigate these impacts and ensure that these parasites will not jeopardize the conservation of their hosts. In addition, this region’s wildlife community is markedly different from that of the northern hemisphere, where most studies on seabird host-parasite dynamics were historically conducted, providing opportunities for potentially unique and understudied interactions. In this presentation, I will present case studies that exemplify the relevance of parasites to the conservation of seabirds in the South Pacific and the uniqueness of the host-parasite dynamics in this region, highlighting some significant research gaps.

Abstract: Community engagement is often pitched for specific age groups and abilities. Music and song serve as a special medium that can effortlessly connect the subject to a broad audience regardless of age and background, which can thus be leveraged as an effective outreach tool. As a scientist and artist, singer-song writer, the author has composed a number of parasite themed songs for toddlers, primary school children, high school aged and grown-ups from a non-scientific background as well as professional scientists.

Dr Rina’s songs include Hail to Parasites, Big Bugs Small Bugs Microscopic Bugs, Hook-a-Worm and Fight Against Malaria. These songs are original compositions, professionally recorded at a studio, and road-tested in over 100 workshops in the community and public events, university classes, scientific conferences and outreach activities that are enjoyed by children and adults alike, including those with intellectual disabilities. Some of the pieces are produced with engaging music videos and lyrics, Australian sign language (AUSLAN) that are ready-to-use for parasitology outreach and for leisure listening on Spotify, Apple Music and other platforms. Dr Rina will provide a taste of these pieces with a live performance including behind-the-scenes production and implementation for outreach.

Beef + Lamb New Zealand’s gastrointestinal nematode (GIN) research programme fosters genuine co-development partnerships with sheep and beef farmers. The aim is to find common ground and language between farmers and researchers to support mutual learning and practice change.

We explore key considerations for effectively delivering research-based GIN material to farmers, emphasising a strategic approach that begins with understanding farmers' circumstances and challenges. This involves active listening, trust-building, and relationship cultivation to identify farmers’ values and priorities, shaping the communication narrative.

Our discussion highlights the importance of producing clear, accessible content, acknowledging the diversity in farmers' preferences for receiving information. We also stress the significance of a dynamic feedback loop through engagement with our farmer advisory group and others, who provide practical perspectives and guidance.

We share insights and learnings to support on-farm practice change in GIN management by increasing awareness and understanding. Additionally, we discuss the evolution of our communication strategies and the importance of consolidating and adapting learnings across various research programs. B+LNZs research is driven by co-development, fostering lasting partnerships among farmers, researchers, and rural professionals to support resilient farm systems.

Immersive virtual reality (iVR) technology designed for education purposes has been heralded as the next big thing in experiential learning. Previous work has shown that iVR use in teaching parasitology is fun, useful and that users would like to see more of this technology in parasite education. Consequently, during 2022 Australian Society for Parasitology (ASP) Concepts in Parasitology Course (CIP), participating students were asked to design and pitch iVR games to teach concepts in parasitology. The winning pitch, as voted by peers, was then developed into an iVR game prototype, Parasite Camping VR, funded via the ASP education committee. The game explores the ‘One Health’ approach to parasite control, highlighting that ‘Prevention is Better than a Cure’. It is set in a natural Australian bush environment, with users required to navigate common camping activities, including taking preventive measures to avoid parasite infections. Tasks include boiling water and cooking food for safe consumption, protecting against mosquitoes, and performing parasite treatment in pets. Feedback for further development was gathered from participants at the ASP 2023 Annual Conference and the game was further designed by 2023 CIP participants. The game is now freely available on iVR headsets for use in parasite education.

Research in parasitology can be both rewarding and challenging, particularly when faced with time constraints and limited resources. Our team encountered such a dilemma with an extensive collection of fox cadavers awaiting processing for parasite data collection. With storage space dwindling and only one researcher available, we sought innovative solutions to address the backlog.

By harnessing the enthusiasm and dedication of volunteers, we recruited veterinary science students at Murdoch University. Over a five-day period, this collaborative effort allowed us to process data from over 50 urban foxes. The data collection included basic body condition scores, ectoparasite surveys, sampling of organs for internal parasites and collection of GI tract for later diet and parasite analysis. Additionally, we gathered limb and jaw morphology data for a separate project.

This presentation will explore the outcomes of our volunteer-driven initiative, share insights gained during the process, and provide practical tips for effectively engaging volunteers in research. Our experience underscores the potential of collaborative efforts in overcoming resource limitations and achieving significant research milestones.

Parasitology research aims to improve the health of people and animals, often with an emphasis on developing nations where parasitic diseases are most prevalent and detrimental. Historically, helicopter research, where researchers enter communities to collect and disseminate data without community involvement, have negatively impacted many communities around the world. Mistrust and other negative impacts associated with these practices decreases the translation of research, to the detriment of participating communities. While often underappreciated, involvement of community members in research also presents a significant burden on communities, and in many cases, there is a prolonged time course for communities to experience benefits from the research.

Research partnerships that provide direct tangible benefits to communities, ensure appropriate knowledge translation, and strengthen local capacity to address parasite management have a higher likelihood of translating outcomes for the benefit all parties involved. A model incorporating stakeholder engagement and co-design at the very start of a research project is optimal. Engaging community members as full and equal partners, on par with investigators and research that might be conducted with an industry organisation, should also form best practice. Here, we present a toolkit for parasitologists to ensure respectful research collaborations to benefit participating communities.

Scabies outbreaks cause significant disruption in aged care facilities and other institutional settings. Failure to manage scabies outbreaks may be attributable to low awareness among health care workers. A survey was distributed to several aged care facilities in South-East Queensland, Australia. The survey captured demographics, prior scabies experience and knowledge-based questions. Scabies was common in aged care facilities, with 41% of 128 respondents encountering the disease in the ten years prior. Participants demonstrated sound theoretical knowledge regarding scabies (median knowledge score 82%). The level of scabies knowledge was associated with respondent age (p = 0.017). Respondents demonstrated an inconsistent ability to identify atypical clinical presentations of scabies, showing discordance between theoretical knowledge and its practical application. The ability to identify crusted scabies was low, reflecting the high frequency of misdiagnosis of index cases in scabies outbreaks. Respondents considered scabies to be a problem and were supportive of improved management guidelines. These study outcomes will inform the design of accessible, targeted educational resources for scabies to help prevent and reduce the impact of outbreaks.

Temperature has been known to affect the distribution of parasites in their hosts, including the hibernation period. Studies have shown that parasite numbers decline during hibernation, while others are able to survive alongside the host, known as co-hibernation. Hedgehogs (Erinaceus europaeus) hibernation is influenced by temperature, but in New Zealand temperatures vary, affecting the length of time hedgehogs hibernate with hedgehogs in the north possibly not hibernating at all. The aim of this study is to see if parasite load is affected by the length of time in hibernation. We hypothesise that with increased time in hibernation there will be a decrease in the parasite load. Hedgehogs will be caught across New Zealand before hibernation with both endoparasites and ectoparasites species being identified. Tracking devices will be placed on the hedgehogs so they can be recaught and analysed once the hibernation finishes. We expect to find that the Northern end of the North Island where hibernation is either absent or minimal in New Zealand will have a higher abundance and diversity of parasites in hedgehogs. This study hopes to explore how hibernation duration influences the host-parasites dynamics in hedgehogs, potentially contributing to a broader ecological and parasitological knowledge.

The broad aim of my research is to explore the evolutionary trends that exist between a parasite which induces a high rate of mortality in its host, and its group of hosts. The relationship between the genetic variation of the parasite and the variation of the parasite’s microbiome will also be a focus. This will be done by investigating both the geographic distribution of variation in these three groups as well as the cophylogeny between the parasitoids and their hosts.

This study focuses on members of the order Mermithida, a group of parasitic nematodes that infect invertebrates, with mermithids and hosts being collected from around the South Island of New Zealand. The morphological and behavioural effects of mermithid infection on their hosts will also be studied to inform how the cophylogenetic history and geographic distribution of mermithids and their variation may be affecting the current relationship between the parasites and their hosts. This raises an opportunity to investigate whether the phenotypic effects that a parasite has on its hosts varies over a geographic distance and potentially whether it correlates with genetic or microbial variation between parasite populations.

Toxoplasma gondii, an Apicomplexa protozoan parasite, poses significant risk to wildlife and human health with an increasing prevalence in new and endemic areas. Since introduction to New Zealand, toxoplasmosis has been found to cause morbidity and mortality in a range of native New Zealand species, however, T. gondii remains poorly understood in avian species. Toxoplasmosis has been found in cherished native birds such as kiwi (Apteryx spp.), kākā (Nestor meridionalis), and kererū (Hemiphaga novaeseelandiae), necessitating further investigation into the impacts and distribution of T. gondii in New Zealand. In this talk, I will summarize previous global T. gondii research on wild birds to provide a broader perspective on infections in New Zealand. Additionally, I will outline the appropriate next steps to advancing T. gondii research in New Zealand. These include testing currently used detection methods for accuracy and precision, expanding on our current understanding of avian toxoplasmosis prevalence in New Zealand, and conducting a multifaceted inquiry into ecological and anthropogenic factors driving infection dynamics. By addressing these knowledge gaps, we aim to develop effective strategies for mitigating the impacts of T. gondii on avian populations and safeguarding both wildlife and human health in New Zealand and on a global scale.

The Tasmanian wedge-tailed eagle, Aquila audax subsp. fleayi, is an endangered species found only in Tasmanian and some of the surrounding islands. As a major predator, it plays a key role in the ecosystem and by extension, so does its parasites. Previously, only one species of parasite, Microtetrameres paraccipiter, had been reported from this subspecies and only four species had been reported from the mainland species, Strigea glandulosa, Neodiplostomum spathula, Thelazia aquilina and “Eye worms”. A helminthological survey of 137 Tasmanian wedge-tailed eagle carcasses from various locations across Tasmania found nine different species of parasite including Capillaria, Microtetrameres, Strigea, Neodiplostomum, Dispharynx and Brachylaimidae. A detailed morphological and molecular examination of the Microtetrameres sp. and Strigea sp. suggested these could be new species. For the Microtetrameres sp., histopathology and stable isotope analysis was also performed. A comprehensive analysis of parasite ecology for Aquila audax subsp. fleayi is provided, including prevalence and mean intensity of infection.

Birds play a significant role in the spread of diseases as they can migrate great distances in relatively short periods of time. Waterbirds are of particular interest to parasitology as they are exposed to a great diversity of parasites due to using both aquatic and terrestrial environments. Much like the rest of Australia’s native fauna, very little is known of the parasitic fauna of Australia’s native ducks. The current knowledge is outdated with the last significant works being completed close to 50 years ago. Since this publication the way in which parasites are studied has significantly changed with the introduction of improved morphological and molecular techniques.

This study aimed to determine the cause of proventricular lesions observed in native Australian ducks. Postmortem examination revealed the presence of Tetrameres nematodes. While Tetrameres spp. are known to cause pathology in poultry, our findings suggest low burdens may not induce significant mortality in ducks. This study provides important morphological and molecular data, emphasising the importance of accurate pathogen identification and a multidisciplinary approach for disease investigations. Future research on Tetrameres species can enhance disease control and conservation efforts in avian populations.

We describe a comparative and evolutionary analysis of the new Trichomonas vaginalis reference genome with the first genomes of two other human parasites, T. tenax and Pentatrichomonas hominis, and four avian Trichomonas species, including the closest T. vaginalis relative T. stableri [2]. Long-read assembly of six chromosome-size T. vaginalis scaffolds vastly improves on the fragmented 2007 [1] assembly, enabling accurate annotation of protein-coding and RNA genes, and highly repetitive multigene families and transposable elements (TEs). We report trichomonad genome sizes, gene content, synteny, and multigene families; resolve phylogenetic ambiguities; and find evidence to support at least two host switches in Trichomonas evolution. We assessed TE abundance across genomes, its role in expansion of human Trichomonas genomes relative to avian, its relation to TE expression, and classified >4600 long virus-like Maverick TEs that constitute >40% of the T. vaginalis genome. Our evolutionary analyses further report: 1) evidence that the increase in repeat sequences (TEs and multi-gene families) was driven by relaxed selection and genetic drift; 2) the functional roles of T. vaginalis gene families that expanded the most and convergently expanded in T. tenax; and 3) single-copy genes putatively important for the transition of the parasite from avian to human host

The protozoan parasite Histomonas meleagridis is the causative agent of histomonosis, an important poultry disease emphasized by the absence of any licensed product for prophylaxis and treatment. The interplay between the parasite and its bacterial environment highlights a unique interaction in medicine and infection biology. Clonal monoxenic cultures used to establish high-quality genome sequence data from virulent and attenuated strains, originating from the same parental lineage of H. meleagridis, enabled the link between genomic variations and attenuation. Furthermore, comparative proteomic and surfaceome studies on in vitro propagated parasites revealed molecular data associated with virulence and in vitro attenuation. An intricate relationship between H. meleagridis and bacteria was revealed, demonstrating that the parasite itself substantially influenced co-cultivated bacteria's proteome pattern. Recently, we performed an immunoproteome analysis to identify the key antigens involved in the antibody response to H. meleagridis. Immunogenic proteins were isolated by immunoprecipitation employing sera from chickens and turkeys with different infection background. Comparative analysis of immunogenic proteins using a virulent and an attenuated antigenic preparation demonstrated minor variations depending on the host. Most differential immunogenic proteins were detected with either chicken or turkey sera, underlying a specific immune reaction of each host.

Trichomonas vaginalis, a flagellated protozoan parasite, thrives predominantly in the anaerobic environment of the human genitourinary tract. Previous research has established that the genus Trichomonasvirus, a double-stranded ribonucleic acid (dsRNA) virus belonging to the Totiviridae family, can specifically infect T. vaginalis. Within the Trichomonasvirus family, there are five distinct species, referred to as Trichomonas vaginalis virus 1 through Trichomonas vaginalis virus 5. Notably, a single T. vaginalis isolate can be infected by multiple Trichomonasvirus species simultaneously. While many studies suggest that Trichomonasvirus affects the virulence, physiology or drug resistance of T. vaginalis, there are also numerous studies that hold opposing views. A consensus remains elusive due to disparate findings leading to varying conclusions. As a result, we postulate that there may exist heterogeneity in Trichomonasvirus species distribution among cells within the same T. vaginalis isolate. To investigate this phenomenon, we utilized single-cell RNA sequencing (scRNA-seq) alongside an anti-dsRNA antibody-based immunofluorescent assay on the T. vaginalis isolate, ATCC 30236, as a model to study the inconsistent distribution of Trichomonasvirus. Importantly, our scRNA-seq data not only indicate the presence of heterogeneity in virus distribution but also have the potential to reveal subpopulations within the population derived from a single isolate.

Several studies have reported that Trichomonas vaginalis, the causative agent of trichomoniasis, can cause persistent infections in the human reproductive tract. In addition to the parasite’s trophozoite stage, a nonmotile pseudocyst stage has also been described, which may be a factor in such persistent infections. Methods to induce the development of pseudocysts have been described including culturing parasites (i) in iron-depleted media, (ii) at cold temperatures, and in acidic media that is physiologically appropriate to reproductive-age vaginas. Testing all three methods, we determined that incubating at cold temperatures is not physiologically relevant, and culturing in acidic media leads to parasite death, but iron-depletion yields live, persistent pseudocysts for up to a week. To determine gene expression patterns and putative marker genes associated with pseudocysts, we generated RNA-Seq data from triplicate cultures of T. vaginalis generated under iron-depletion. The data revealed hundreds of significantly differentially expressed genes in pseudocysts that are distinct from trophozoites in each daily dataset with 189 downregulated and 214 upregulated protein coding genes found across the first four days. The functions of these genes shed light on the structural and metabolic upheaval required to form pseudocysts and provide several promising candidate marker genes.

Bovine trichomonosis, caused by Tritrichomonas foetus, is a venereal disease that impairs cattle reproduction. Traditional vaccines use whole inactivated T. foetus, requiring extensive culturing. This study explores reverse vaccinology by sequencing Australian T. foetus strains to identify putative vaccine candidates against bovine trichomonosis.

In 2020-2021, T. foetus was detected across northern Australia using qPCR, and two isolates (TfOz5 and TfOz-N36) were purified from preputial samples from Queensland and Northern Territory. Genomic DNA was extracted via cold alkaline lysis, sequenced using SQK-LSK110 kit in MinION Mk1C for 48 hours using R9.4.1 flow cells. Base calling used Guppy 6.5.7 with super-high accuracy and reads under 20 Kbp with an average Q-score under 12 were filtered out using NanoFilt. Genome assembly was conducted with Flye, and ab initio gene prediction was performed with Augustus.

The genome assembly of the TfOz5 strain resulted in 194 contigs (110 Mb, N50 of 1.49 Mb), while TfOz-N36 had 368 contigs (105 Mb, N50 of 0.48 Mb). The isolates share 99.2% similarity and represent a 10 to 100-fold improvement in contig reduction compared to previous T. foetus genomes. Augustus predicted 28,480 genes and further bioinformatic analyses will screen for vaccine candidates using reverse vaccinology approaches.

Trichomonads infect a broad range of hosts, including wild and farmed animals and humans. The vast majority of studies focus on human infecting species, and Trichomonas vaginalis in particular. Here we will illustrate, and argue for the importance of expanding, comparative studies of Trichomonads across a broad range of hosts to gain new insights into the molecular and cellular basis of the complex interactions between Trichomonads, the host microbiota and host cells. Animals hosting Trichomonads are also important as sources of food and as nodes in terrestrial ecosystems. Hence increasing our knowledge of Trichomonads is relevant in a number of contexts including medical and veterinary sciences. A selection of examples will be covered to illustrate these points and aim to stimulate: (i) collaborations across disciplines including microbial ecology and evolutionary biology and more generally (ii) a One Health approach for the Trichomonads.

Canine heartworm (Dirofilaria immitis) is a filarial parasitic nematode that causes severe cardiopulmonary disease in domestic and wild canids. D. immitis is present in tropical, subtropical, and temperate regions of the world, with infections reported on almost every continent. Despite its global prevalence, the origins and global dispersal history of D. immitis remain unknown. In addition, the recent discovery of drug-resistant D. immitis in the USA underscores the urgent need to further understand the genomic landscape of this parasite. We conducted whole-genome sequencing on 130 adult D. immitis from nine countries (Australia, Thailand, Malaysia, USA, Panama, Costa Rica, Greece, Italy, and Romania), with D. repens and D. ursi as outgroups. This dataset represents the world’s largest collection of D. immitis genomes. Preliminary analyses revealed distinct partitioning between broad geographical regions, aligning with an ancient origin and dispersal scenario. Genetic similarity between some D. immitis from Central America and those from Europe was consistent with modern transatlantic transportation of dogs. This presentation will reveal the final outcomes of this population genomics study.

Canine vector-borne pathogens (CVBP), such as Anaplasma platys, Babesia vogeli, and Dirofilaria immitis, are known to be endemic in Indigenous communities in Australia, while information on pet dogs is limited. Recent outbreaks of Ehrlichia canis, a pathogen previously exotic to Australia, highlighted the urgent need to assess the status of CVBP in pet dogs in the country. To address this knowledge gap, we screened 729 canine blood samples from temperate and non-temperate regions of Australia. In addition, we administered two independent questionnaires to veterinarians and dog owners to assess their awareness and preventative practices around CVBP. Our findings revealed an overall apparent prevalence of 3% for CVBP, with haemotropic Mycoplasma being the most common pathogen (2.1%, 95% CI 1.2-3.4%), followed by canine filarial worms (0.8%, 95% CI 0.3-1.8%). Additionally, one dog from Woree, Queensland, tested molecularly positive for Hepatozoon canis. Despite veterinarians considering CVBP less important in their practices and dog owners having low awareness, satisfactory prophylaxis recommendations and administration practices were identified. However, to maintain low CVBP prevalence and mitigate the associated biosecurity risk in Australia, we recommend educating dog owners, ensuring adequate surveillance, and implementing risk-based control measures for CVBP.

Companion dogs are a popular pet, providing physiological and psychological benefits to owners, with Australia retaining one of the highest pet ownership rates in the world with over six million dogs. Gastrointestinal (GI) parasites are a common health concern in dogs, often causing chronic or recurrent infections, with some parasites possessing zoonotic potential.

To determine the prevalence of GI parasites in pet dogs, faecal samples were collected from healthy six-week-old puppies in kennel environments, and healthy juvenile/adult dogs in public dog areas to analyse via coproscopy. Protozoans, Cystoisospora spp. and Giardia sp. were the dominant parasite genera detected in both kennels and juvenile/adult dogs, and persisted throughout almost all age groups.

This persistence of protozoans is likely linked to the common use of monthly ‘all-wormers’ which have active ingredients not effective against protozoan parasites. As both helminths and protozoa inhabit the same environmental niche, the reduction of helminths through the use of anthelmintics may allow protozoans to proliferate. Upon genotyping of microscopy-positive Giardia samples, two puppies and one juvenile/adult were detected to be carrying zoonotic Giardiaassemblages, highlighting the potential public health implications associated with Giardia, and the need for suitable anti-protozoan drugs to reduce parasite transmission to the community.

Dogs and cats are now considered integral members of families, present in nearly 50% of
households in Australia and New Zealand. However, their health can be impacted by
numerous pathogens, including helminths, some of which are transmissible to humans. In
spite of the importance of these parasites, surprisingly, there are limited data on the
epidemiology of parasitic diseases in dogs and cats in these countries. Clinical diagnosis is
sometimes challenging and diagnostic methods can be unreliable. In this study, we are
investigating the prevalence, distribution, and risk factors associated with helminths in
dogs and cats in parts of Australia and in New Zealand, and will critically assess the
sensitivity and specificity of conventional and molecular methods. We aim to achieve a
comprehensive investigation of parasitic helminths affecting dogs and cats in parts of
Australasia and of the performance of diagnostic methods.

The main aim of this project is to determine if (raw) food-borne parasites can infect cats in Australia. This project aims to answer this question by analysing the VetCompass database to identify cats fed raw food. This will be done to identify links between signs of gastrointestinal disease and parasitic infection.

This project will also involve surveying Australian cat owners. Faecal samples of cats will be requested and examined for the possible presence of food-borne parasites. It is hypothesised that cats fed raw diets will have parasites of different species and lifecycles/hosts.

Surveying of Australian cat owners will also be used to identify households that use parasite prevention products. It is hypothesised that households that (regularly) use parasite prevention products will have cats with no or very low parasite burdens. On the other hand, households that do not use parasite prevention products for their cats will likely correlate with higher parasite burdens.

Background: Heartworm (Dirofilaria immitis) disease poses a significant risk to domestic and wild canines and felines. Traditional diagnostic methods for dogs face limitations in accuracy, cost and accessibility, leading to underdiagnosis. Near-infrared spectroscopy (NIRS) combined with Artificial Intelligence (AI) presents a promising solution to overcome these limitations. NIRS utilises infrared radiation to analyse biological samples cost-effectively, while AI interprets the resultant data to predict samples' infection status.

Methods: Utilising a handheld NIRS device, we scanned the ears and blood smears of 183 dogs. Each dog was tested for D. immitis by antigen detection, microfilariae presence in blood and PCR. We developed machine learning algorithms to detect the presence/absence of infection using artificial neural networks, and we used the models obtained to predict the infection status of independent samples.

Results: We attained optimal accuracies by scanning specific regions of the ears (90.9%, n=22) and blood smears (88.9%, n=18). Both models exhibited 100% sensitivity (n=8) and 85.7% specificity (n=14) for the ears and 80% specificity (n=10) for blood smears. Additionally, we identified distinctive D. immitis NIRS spectra peaks.

Conclusion: This study demonstrates that NIRS /AI has the potential to be a rapid and effective surveillance and diagnostic tool for heartworm infection.

In Australia invasive species such as deer and pigs are now wide reaching. With six introduced species of deer and one of feral pigs now thriving it is important to understand the parasites that they have brought over with them into the Australian ecosystem. As sarcocystis along with many of its like in the Sarcocystidae family are potentially zoonotic, the impact of these parasites could be far greater than anticipated. Through PCR and histopathological analysis of organ tissue the first instance of Sarcocystis in feral deer and pigs in Australia was found in 2023. Species identification of S. miesheriana was achieved for pig samples, however, deer samples were not identifiable to a species level. All positive PCR samples were also found to have microscopic sarcocysts during histopathological analysis. Further research is now being conducted to identify species of Sarcocystis in deer and other invasive species in Australia as well as the impact they have on native wildlife.

Australian marsupials are particularly susceptible to Toxoplasma gondii, an introduced zoonotic protozoan parasite. The eastern barred bandicoot (Perameles gunnii) is a small native marsupial species classified as Critically Endangered. Although the species has previously been described as highly susceptible to infection with T. gondii, there is currently no information on the genotypes occurring in this species. This study employed qPCR for the detection of T. gondii in opportunistically obtained tissue samples from eastern barred bandicoot carcasses (n = 113) from Victoria, followed by determination of genotype using a DNA sequence-based virtual restriction fragment length polymorphism (RFLP) method. Overall, 19.5% of the samples were positive for T. gondii using qPCR. The RFLP analysis revealed the dominance of T. gondii type II while the type II-like genotype was found in two isolates. This is the first study to demonstrate the presence of T. gondii in eastern barred bandicoot tissues using molecular methods and to provide information on prevalent genotypes. Epidemiological studies of definitive and intermediate hosts, including further genotyping, are recommended to better understand T. gondii epidemiology for the successful recovery of eastern barred bandicoots in Australia.

Introduced species play a significant role in the distribution of novel parasites and subsequent diseases. Hemidactylus frenatus is known to be the most invasive and widely dispersed lizard in the world, proven to be an aggressive displacive competitor, with the potential to transmit novel parasites and amplify endemic parasites. There is little published research of the parasites of reptiles in Australia, and especially blood protozoa, to date there are no published papers on blood parasites of Gehyra dubia.This research aims to determine the occurrence of blood parasites in two gecko species found within Australia native species Gehyra dubia and introduced species Hemidactylus frenatus. This will be done by examining 259 Gehyra dubia and 102 Hemidactylus frenatus blood smears collected in 1998 from northern Queensland. This knowledge can be useful to understand whether native geckos are susceptible and/or impacted by the parasites carried by the introduced species of gecko and in the process create a baseline dataset for future research into native geckos to examine the parasitic implications of Hemidactylus frenatus on Gehyra dubia.

Polychaetes constitute a significant portion of the biodiversity and abundance in marine sediments, playing essential roles as prey and predators to other free-living species and as hosts for various parasites. Over a two-year period in coastal British Columbia, Canada, we screened diverse groups of polychaetes for parasites and hyperparasites. As a result, we discovered more than 40 novel species of parasitic alveolates (including ciliates, dinoflagellates, and apicomplexans) and animals (e.g. orthonectids). Among these parasites, gregarine apicomplexans are particularly interesting since they are highly diverse and abundant and can harbour their own parasites (i.e., hyperparasites), namely metchnikovellids. We observed the morphology of these novel parasites and hyperparasites using light and electron microscopy. Also, we inferred single-gene phylogenetic trees based on small subunit ribosomal RNA sequences for larger taxonomic coverage and multigene phylogenetic trees using transcriptomic and genomic data for better resolution. By synthesizing results from morphological and molecular analyses, we explore phylogenetic diversity and relationships, patterns of coinfection and host specificity, and evidence for codiversification between hosts and parasites.

Animal microbiomes have been recognized to have significant impact on animal behaviour, development, and health. For parasites, the source of their microbes is likely acquired from their hosts, making host identity a potential factor to explain the variation of microbiome composition among conspecific parasites. To test this, we compared the microbiomes of trematode Maritrema poulini metacercariae (encyst larval form) across three different host hierarchical levels: parasites within the same host individual; parasites from different conspecific host individuals; and parasites from amphipod Paracalliope fluviatilis versus isopod Austridotea annectens hosts. We found there was no difference in alpha diversity for the microbiome of M. poulini metacercariae across all three different host hierarchical levels. However, there were differences in beta diversity for the parasites’ microbiomes under two hierarchy levels: (i) for parasites from amphipod vs isopod hosts; (ii) for parasites from different individual isopod hosts. Differential abundance analysis also showed differences of some bacteria at phylum level. To conclude, the study shows that host identity, such as host species or conspecific host individuals, impacts parasite microbiome composition, but has no influence on taxonomic diversity and richness within parasite microbiomes.

Cardicola forsteri and C. orientalis are economically significant aporocotylids infecting the circulatory system of ranched Southern bluefin tuna (SBT), Thunnus maccoyii, in South Australia. SBT are typically ranched for 3-6 months and without praziquantel treatment Cardicola spp. can cause substantial losses to the SBT industry. The aim of this study was to determine the Cardicola spp. infection dynamics during an extended period of SBT ranching. SBT gills and hearts were collected at 12 time points (10 to 40 weeks post praziquantel treatment) from fish farmed by a single company for a twelve-month period. Cardicola spp. infection was quantified by adult fluke counts in the heart of SBT and quantitative polymerase chain reaction (qPCR) to detect C. forsteri and C. orientalis ITS-2 DNA in the gills. C. forsteri adult prevalence and intensity peaked 25 weeks post treatment (PT) before the infection intensity significantly decreased for the remainder of the study. At 17 weeks PT C. forsteri prevalence reached and stayed at 100%. Ranching outside of the usual timeline will allow the SBT industry to provide a fresh product for a longer period and understanding the infections risks will help farmers make informed health management decisions.

The Yanomami, Indigenous inhabitants of the Amazon, confront multifaceted challenges endangering their health and cultural integrity. Of immediate concern is the surge in malaria cases within Yanomami communities, unfolding amid the controversial environmental policies of former President Bolsonaro's administration in Brazil (2018-2022). Through causal inference methods, we examined the impact of land-use changes on malaria incidence among the Yanomami, drawing on annual health data spanning 2003-2022. Further, difference-in-differences analyses were performed to confirm if the Yanomami people suffered disproportionately when illegal mining raised in the region. A remarkable ~300% increase in malaria incidence was observed from 2016 to 2022. We show that mining is the primary driver of malaria incidence among the Yanomami people, with an increase of 31% in malaria incidence for every 1% increase in mining cover. A protective effect of forest cover was found, with a decrease of 0.81% in malaria incidence for a 1% forest cover increase. We also confirmed that Yanomami suffered disproportionally more (up to 15%) from malaria than other communities as a result of increasing illegal mining. Overall, we demonstrate that the increase in illegal mining contributed to the Yanomami humanitarian crises by boosting the high malaria burden suffered by these Indigenous people.

Giardia intestinalis is a protozoal parasite regularly found in both humans and animals in New Zealand and worldwide. Several genetically distinct assemblages of G. intestinalis, A to H, have been described, alongside their tendency to associate with different host species. Work conducted so far in New Zealand has focused mostly on which assemblages are found in humans, with very little DNA typing done on isolates from animals such as dogs and cats and therefore the zoonotic potential of infections in animals is relatively unknown. A total of 95 canine and feline samples positive for Giardia by faecal floatation and centrifugation were submitted for PCR and further diagnostics to identify assemblage. Of the 95, 44% were from dogs and 56% from cats, 90% were from animals in local animal shelters and 10% were from client owned pets. PCR returned a total of 22% negative results. Canine samples returned with 33% assemblage C, and 43% assemblage D. Feline cases had a return of 36% assemblage A1 which is generally considered one of the human giardia types, and 42% assemblage F. These results highlight the potential for human associated assemblages to be common in Giardia-infected cats, more so than in dogs.

A case of peritoneal cystic echinococcosis (CE) in a domestic cat is described from Queensland state of Australia. Physical examination indicated a large, distended abdomen, that palpated as fluid filled. Ultrasonography showed numerous cysts with hyperechoic walls and anechoic contents within the cat’s abdominal cavity. Molecular identification based on mitochondrial DNA genes indicated that the causative agent was Echinococcus granulosus sensu stricto (G1 strain). Moreover, the cat was also found infected with Feline Leukemia Virus (FeLV) and FeLV induced immunosuppression could have led to the development of CE in this cat. This is the first report of CE in a FeLV infected cat in Australia.

Approximately 2/3 of the Trichomonas vaginalis genome is composed of Type II transposable elements (TEs). TEs represent a strong mutational force shaping genomic evolution, raising questions as totheir impact on T. vaginalis function and evolution. We generated the first chromosome-grade assemblies, annotation and transcriptomes offour T. vaginalis strains and three close relatives in birds: Trichmonas stableri, and two T. vaginalis-like species even more closely related to T. vaginalis. We found that the genome of T. vaginalis is massively expanded, follows a mosaic pattern of synteny, and that TEs have expanded and altered the genomic architecture. Comparison of regulatory mechanisms revealed a bias towards piRNA machinery in T. vaginalis and the least expression of TEs among all the species. We inferred the functional consequences of this TE expansion by genotyping 5,759 TE polymorphisms in 12 T. vaginalis strains and associating TE insertion with gene expression. We found 69 eQTLs (loci that explain variation in mRNA expression) in low frequency, indicating TEs likely play a deleterious role in the capacity of the parasite to regulate gene expression. This is the first genome-wide study in Trichomonas to show the evolution of TEs, their regulatory mechanisms, and their impact on transcriptional regulation.

Abstract: Spliceosomal introns, distinctive features of eukaryotic genomes, are non-coding sequences excised from pre-mRNAs by the spliceosome, contributing to genome evolution and protein diversity. Although spliceosomal introns have been characterised in several eukaryotic lineages, their origin and evolution remain unresolved. The protozoan parasite Trichomonas vaginalis is a deep-branching eukaryote with a large genome and a rich gene repertoire despite apparently having very few spliceosomal introns. We have recently uncovered a group of short introns in T. vaginalis. Here, using a reporter gene disrupted by short intron sequences, we employed an extensive series of mutagenesis to investigate their conserved features of splice signal sequences and intron length, comparing them to long introns. We found that short introns have reasonable but limited flexibility in their length, including extremely close juxtaposition of the branch and the 3’ splice sites. Additionally, their exhibit distinctive consensus sequences with a very degenerate 5’ splice site. Lastly, and surprisingly, we found that T. vaginalis is capable of trans-splicing split introns, as previously documented in Giardia lamblia, another deep-branching protozoan of the group Excavata. In conclusion, our findings expand on the diversity of introns in deep-branching eukaryotes, offering new insights into the evolution of spliceosomal introns.

Alternative splicing, a eukaryotic-originated process, involves the processing of cis-acting elements (spliceosomal introns and exons) within pre-mRNA transcripts by trans-acting elements (spliceosomal proteins and splicing factors), resulting in the generation of multiple mRNA isoforms. Alternative splicing is tightly linked to eukaryotic complexity. Spliceosomal introns have expanded despite consistent spliceosomal protein composition from yeast to humans. This raises questions about the emergence of the spliceosome during eukaryotic evolution and the proteins influencing the complexity of spliceosomal introns. Considering that protists evolved over 500 million years before yeast, studying alternative splicing in Giardia duodenalis, an early eukaryotic protist, could provide insights into the origins of the spliceosome and spliceosomal introns. Cis- and trans-factors guiding alternative splicing in Giardia are understudied. We conducted a comprehensive bioinformatic analysis of annotated intronic and proteomic data from the basal protist Giardia to complex protist Plasmodium falciparum, alongside other model eukaryotes (Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Saccharomyces cerevisiae). Our preliminary ortholog mining suggests the presence of spliceosomal proteins across established spliceosomal complex sub-categories in Giardia. This study represents one of the first attempts to comprehend the spliceosome and spliceosomal introns at the base of eukaryogenesis, shedding light on the origin of alternative splicing.

The central dogma (DNA-to-RNA-to-Protein) is key to cellular regulation, with chromatin/transcriptional and post-translational tiers achieving complexity in higher eukaryotes. Post-transcriptional regulation (PTR) by RNA-binding proteins (RBPs) is ancient and ubiquitous, expanding significantly with eukaryotic evolution, making it central to cellular life. We investigated whether novel RBPs emerged during the prokaryotic-to-eukaryotic transition. Our phylogenomic atlas across the tree of life reveals that the eukaryotic RBPome is shaped by bacterial and archaeal RBP systems, alongside the emergence of “novel” RBP families. In the early-branching eukaryote Giardia duodenalis, predating yeast by a billion years, RBPs for RNA-splicing, RNA-silencing, translation repression, and cellular fate regulation emerged. We characterized the G. duodenalis RBPome through in silico modelling, transcriptomic, proteomic profiling, and interactome capture, revealing canonical and non-canonical RBPs. Functional genetics, RNA-network capture, and phase-separation assays of key eukaryotic originated RBPs, including early Pumilio homologs (PUF, PUM) and helicases DDX3x and EIF4A, and other non-canonical RBPs like Phospho-glycerate kinase suggest Giardia RBPs exhibit complexity similar to higher eukaryotes, with roles in translational repression, biomolecular condensates, and cell differentiation. Our findings indicate that complex RBP regulation emerged early in eukaryotic evolution, potentially pivotal for the emergence and evolution of eukaryotes.

Sri Lanka is known for remarkable marine biodiversity, yet the parasite faunas of its fishes remain unexamined. For digenean trematodes, perhaps the richest of metazoan faunas parasitic in fishes, few species have been documented in Sri Lanka. Conversely, the broader South Asia region has been intensively studied, though comparative molecular data are almost entirely lacking, and the quality of taxonomic work has varied wildly such that the historical record poses a daunting mire of obscure records and complex synonyms. We are undertaking the first broad and intensive collection effort for marine fish digeneans in Sri Lanka. Utilising an integrated taxonomic approach that combines detailed morphological study with molecular genetic analyses, our research aims to identify the fauna, prospect for new taxa, and apply molecular analyses to compare the South Asia fauna with that of the broader Indo-West Pacific. The marine environments in Sri Lanka, and much of the developing tropical world, face significant and imminent threats; our opportunity to collect, document and understanding parasites faunas in these regions is diminishing, at an unknown rate. Our research will build taxonomic capacity in the region.

Polystomatid flatworms (Monogenea, Polystomatidae) are known from the Australian lungfish; all three orders of amphibians namely salamanders, frogs and caecilians; freshwater turtles; and from the eye of the common hippopotamus. Of the 30 currently known polystome genera, 19 are known from amphibians, nine from freshwater turtles, one from the lungfish and one from the hippopotamus. Parasite transmission can only occur in an aquatic environment, involving the oncomiracidium. As a result, the diversity of host species is limited to aquatic or semi-aquatic hosts. Polystomes co-evolved with their amphibious hosts and unique life cycles evolved in close synchronisation with that of the host.

Although polystomes are globally distributed, most genera are restricted to specific continents or host groups. If we include Australia in the area of interest we have Concinnocotyla from the Australian lungfish, Parapolystoma and Sundapolystoma from anuran hosts and Aussietrema, Fornixtrema, Pleurodirotrema and Uropolystomoides from chelonian hosts. No polystomes are known from New Zealand. Globally we find a direct correlation between polystome diversity and search effort. It could thus be a case that many more polystomes remain undiscovered in the Pacific.

Evaluating the host body condition index (BCI) based on relationships between host mass and length is a general and pervasive approach to assess the negative effects of parasites on host health. Although many researchers, especially fish biologists and fisheries managers, commonly utilize BCI, overall patterns among BCI – infection relationships remain unclear. Here, we systematically reviewed 973 fish BCI – infection relationships from 216 publications and investigated factors potentially affecting the relationships’ significance and direction. Trematodes (22%) were the most commonly studied parasites across all datasets, followed by Cestodes (15%), Nematodes (14%), and Copepods (9%). Surprisingly, most studies reported non-significant associations between BCI and infection (72%), while significant negative (20%) or positive (7%) relationships were rarely reported. Almost half of the studies used Fulton’s BCI (mass/length³). Simple generalized linear mixed models based on a subset of publications revealed that (1) studies using Fulton’s BCI were more likely to report significant associations compared to other BCI measures, and (2) studies focused on Acanthocephalans tended to report significant positive relationships. Our study suggests that the outcomes of fish BCI- infection relationships may vary depending on several factors such as BCI measure used and parasite taxonomic group.

Introduction: Intestinal parasitic infections are common among in travelers from tropical countries. We present a clinical case series of unusual intestinal protozoa and helminths diagnosed and treated at the University Hospital Bulovka in Prague, Czech Republic between 2017 and 2023.

Case 1: A 36-year-old female visited South America from October 2019 to March 2020. She suffered from watery diarrhea without fever since January 2020. Parasitological stool Examinatio (flotation with ZnSO₄) revealed oocysts of Sarcocystis sp. Molecular analysis confirmed Sarcocystis hominis, and treatment with co-trimoxazole was successful.

Case 2: A 29-year-old man worked in Kenya from September 2021 to May 2023. He developed chronic watery diarrhea without fever after visiting Uganda in March 2023. In June 2023, investigation revealed significant eosinophilia (3,070/μl) and presence of Trichostrongylus sp. eggs in stool (flotation with ZnSO₄). After treatment with albendazole, both diarrhea and eosinophilia resolved. Molecular analysis of samples is ongoing.

Case 3: Hookworm infection is common among travelers. A case of Ancylostoma ceylanicum was diagnosed at female traveler returning from Southeast Asia in 2017.

Conclusion: Routine multiplex PCR diagnostic kits for intestinal parasitic infections do not detect unusual parasites. Therefore, stool microscopy performed by experienced parasitologist is essential for diagnostics of these infections.

The zoonotic hookworm Ancylostoma ceylanicum is the second commonest hookworm infecting humans in the Asia Pacific. However, except from anecdotal cases, reports of this hookworm in the Americas have been limited. This is most likely due to the predominant use of microscopy techniques for diagnosis, which has prevented the identification of hookworms to the species level. Recent reports, which used molecular techniques, suggested A. ceylanicum infection in humans in South America in the early 2000’s. However, there was no recent information about zoonotic hookworm species on the continent. Recently, we conducted a survey of dogs and coyotes on the island of Grenada and Costa Rica using a molecular approach and revealed the presence of A. ceylanicum. We also reviewed the current state of knowledge of A. ceylanicum in the Americas and propose that this hookworm is much more widespread in this part of the world than previously assumed. Future epidemiological studies should assess the distribution, prevalence, transmission patterns and impact of A. ceylanicum infection in humans and animals in the Americas using molecular tools, in order to critically evaluate the need for the prevention and control of ancylostomiasis in a One Health context.

Recently, a review was conducted to establish the prevalence and distribution of dirofilariasis in India and Sri Lanka. Current evidence suggests that D. repens is prevalent in dogs (35.8%) and humans in India and Sri Lanka, but D. immitis is reported in dogs in India (9.7%), but, surprisingly, has not yet been reported in Sri Lanka. This apparent difference could be due to a lack studies in some regions, the presence/lack of vectors in geographic regions and/or the use of inaccurate methods for diagnosis. Our work also identified that there are a number of other Dirofilaria species and taxa of importance in animals, and emphasised the importance of zoonotic dirofilariasis, and the need for accurate diagnosis using molecular (DNA-based) methods. Interestingly, recent reports suggest that the Hong Kong genotype of Dirofilaria sp. might be more prevalent in the Asia-Pacific than previously assumed, and there is evidence of its potential to spread. Indeed, there is evidence of their occurrence of human dirofilariasis in non-endemic countries, such as Australia. Clearly, there is a need to investigate the epidemiology of dirofilariasis in animals and humans using genetic and genomic methods to aid in the prevention and control of zoonotic disease.

The species Giardia lamblia (syn. intestinalis, duodenalis) is a fascinating example of evolution by reduction. Its limited number and types of endomembrane compartments and corresponding molecular machinery, make it an excellent model system to investigate protein trafficking pathways in the absence of eukaryotic hallmark organelles such as the Golgi and bona fide endosomes and mitochondria. In my presentation, I shall attempt to take the audience on a journey through Giardia’s eccentric exocytic and endocytic organelles and associated molecular complements, with a strong focus on this parasite’s unique feeding organelles and their possible role as bidirectional protein trafficking hubs. Furthermore, I shall highlight how investigating Giardia’s unique cell biology, despite challenges in genetic tractability for this species, has opened up truly fascinating windows on the diversity of eukaryotic protein trafficking pathways, while also providing important puzzle pieces to the evolutionary history of well-characterized but not well-conserved trafficking machineries.

Veterinary anthelmintics excreted from treated ruminants enter the environment, can be absorbed by plants, and, upon grazing, re-enter the ruminants ¹. Such circulation of anthelmintics can influence non-target species, particularly free-living invertebrates. However, contact with traces of anthelmintics can also promote drug resistance in helminths. Our research focuses on the non-target site mechanisms of drug resistance in nematodes, which can be based on the enhanced deactivation of anthelmintics via their increased biotransformation and elimination from the nematode body. In previous studies, increased expression and activity of xenobiotic-metabolizing enzymes (XME) in drug-resistant strains of Haemonchus contortus were found. In addition, significantly faster deactivation of benzimidazole anthelmintics via oxidation or reduction and subsequent glucose conjugation was detected in resistant strains in comparison to susceptible one. We have explored the effect of sub-lethal concentrations of anthelmintics on H. contortus adults in vitro and in vivo, mimicking the real farm conditions and exploring their effect on the expression and activity of various XME². Alarmingly, even the lowest concentration of anthelmintics caused enhancement of biotransformation in H. contortus, which can potentially result in inefficient treatment of haemonchosis.

This project was supported by New Technologies for Translational Research in Pharmaceutical Sciences /NETPHARM, project ID CZ.02.01.01/00/22_008/0004607.

1 Navrátilová et al. (2021) Proof of the environmental circulation of veterinary drug albendazole in real farm conditions. Environmental Pollution 286: 117590

2 Dimunová et al. (2022) Environmental circulation of the anthelmintic drug albendazole affects expression and activity of resistance-related genes in the parasitic nematode Haemonchus contortus. Science of the Total Environment 822: 153527

Malaria causes significant morbidity and mortality, with 249 million cases and 608,000 deaths in 2022. Progress towards elimination and eradication goals are threatened by parasite drug-resistance. To combat drug-resistance, therapies that possess modes of action different to current antimalarial drugs are needed. The indoloquinolizidine alkaloid natural product alstonine was identified in a screen of compounds from the NatureBank library, Griffith University. Alstonine was shown to have a P. falciparum in vitro IC₅₀ of 0.18 µM, >1,000 selectivity for parasites over human cells, and a slow action phenotype. Alstonine was hypothesized to have a novel mode of action due to a lack of cross-resistance seen by a panel of drug-resistant P. falciparum lines and a different slow action activity to delayed-death compounds. To investigate the antiplasmodial target of alstonine, P. falciparum parasites were generated with 20-fold less sensitivity to alstonine compared to wildtype parasites. Whole genome sequencing of alstonine-resistant subclones identified a point mutation and copy number variation in a gene encoding a putative inner-mitochondrial membrane protein (PfMPV17). CRISPR editing was undertaken to induce the reported point mutation in wildtype P. falciparum parasites. PfMPV17 transgenic parasites displayed a decrease in sensitivity to alstonine, implicating PfMPV17 in the resistance mechanism to alstonine.

The emergence and spread of drug resistance stymies our efforts to control malaria. Clindamycin is a safe and inexpensive antimalarial partner drug candidate. Clindamycin kills malaria parasites by targeting the apicoplast prokaryotic translation machinery, but uncertainties surrounding the clindamycin resistance mechanism have restricted its development in combination therapies. To explore clindamycin resistance, we used two different selection regimes to generate clindamycin resistant lines in human malaria parasites (Plasmodium falciparum). Resistance under both selection regimes resulted from identical point mutations in the apicoplast 23S ribosomal RNA, mirroring the canonical resistance mechanism observed in bacteria.

Notably, the clindamycin resistant parasites exhibited reduced fitness in mosquitoes compared to drug-sensitive parasites. To quantify this transmission deficiency, we developed an amplicon sequencing technique to measure the growth of clindamycin resistant parasites in direct competition with drug-sensitive lines during the mosquito life stages. The mosquito host presents vastly different selection pressures compared to the human blood, and our new assay allows us to quantify parasite fitness during the mosquito stage for the first time. Understanding the impact of Plasmodium clindamycin resistance on transmission will inform decisions about the use of this drug in combination therapy to help curb antimalarial drug resistance.

We developed an axenic amastigote line of Leishmania donovani (MHOM/SD/75/1246/130) that retains macrophage-infecting characteristics and proliferates better than metacyclic promastigotes in vitro and in hamsters. This line was used to induce miltefosine resistance through stepwise increasing drug pressure, achieving stable high-grade resistance (62-fold) both in vitro and in vivo. Unlike previous studies on Leishmania infantum and Leishmania major promastigotes, which showed decreased or no fitness, we assessed the in vitro fitness of our resistant line. We cultured miltefosine-sensitive (Milt-S), miltefosine-resistant (Milt-R), and a 50:50 mix in various conditions, including control, DMSO control, and different concentrations of miltefosine and amphotericin B, for 18 cycles of drug pressure.

The ratio of sensitive to resistant parasites was determined using pyrosequencing of a SNP (D897N) in the miltefosine transporter gene linJ.13.1590. Fitness was defined as the ability of one clone to overtake another over time. Under 3 or 6 μM miltefosine pressure, Milt-R dominated by cycle 5. Without miltefosine or at 1 μM, Milt-S overtook by cycle 8. The fitness advantage is conditional on drug pressure, with Milt-R's advantage manifesting faster under higher miltefosine pressure compared to Milt-S in the absence of drug pressure.

Scabies is a highly contagious dermatological condition caused by Sarcoptes scabiei var. hominis. The therapeutics, ivermectin and permethrin, mainly target parasite nervous system killing only the motile stages. Therefore, they require repeat treatments. Prolonged use of these drugs and patient incompliance to repeat treatments have led to parasite resistance. A single dose treatment which targets all stages of the S. scabiei life cycle is required. Two novel scabicides (FL01 and AB02) were tested as a combination in vitro and pre-clinically. Four groups of eight infected pigs each were treated with a single application of the combination treatment for 4h or 8h or with two-doses of ivermectin or kept untreated. LT100 for the combination was 2h for mites and young eggs, and 8h for late-stage eggs. Only the combination treatment groups were cured and free from parasites from day-1 post-treatment. Clinically, combination treatment groups showed significantly lower pruritus and much improved clinical scores compared the ivermectin and control groups. Maximum absorption of FL01 in the serum and skin was at 8h and 2h and at 2h and 8h for AB02 post-treatment. Efficacy of this combination treatment is significantly higher than available scabicides, indicating that our treatment is a promising novel scabicide.

Cryptosporidium spp., an apicomplexan intestinal parasite, represents a significant global health threat, particularly affecting children. Despite its importance, cryptosporidiosis remains a neglected disease. Research on Cryptosporidium spp. has been hindered by the absence of a tractable, continuous in vitro culture of this parasite. However, our lab has achieved progress in improving several available culture models, including an axenic model. Recent advances in short-term in vitro culture of Cryptosporidium now enable high-throughput sensitivity testing of novel therapeutics targeting this pathogen. In this study, we utilized a newly optimized in vitro drug screening platform to examine, for the first time, the anti-cryptosporidial activity of newly developed synthetic peptoids with potent antimicrobial properties. Utilizing our novel culturing platforms, we have screened a library of 18 peptoids and identified the peptoid TM9 as a promising candidate, demonstrating an anti-cryptosporidial median IC50 of 21 µg/mL. TM9 was further tested for activity against various Cryptosporidium life cycle stages, including invasion and egress, and showed a phenotypic effect against the infectious sporozoite stage. Importantly, toxicity screening revealed that TM9 displayed minimal cytotoxic effects on mammalian host cells. The high efficacy and safety profile of TM9 opens new possibilities for novel treatments and preventative strategies against Cryptosporidium infections.

Antimalarials have historically played a key role in controlling malaria in endemic regions, and for nearly all available antimalarials, cellular targets are unknown. We applied solvent-induced proteome profiling in lysates and live cells with data independent acquisition mass spectrometry (DIA-MS) to identify antimalarial protein targets. We generated the solvent denaturation curve of every single protein identified in the asexual stage of the parasite proteome using increasing percentage of solvent, following which, we choose 4-5 percentages to identify the known targets of current and novel antimalarials in lysate and live infected red blood cells. The experimental workflow involves treatment of P. falciparum infected red blood cells with compounds of interest, solvent exposure, soluble protein isolation, digestion, and DIA-MS using the Orbitrap Astral MS allowing faster throughput with deep coverage and accurate quantification. Further, methodological optimisation in the live cell workflow for the analysis of this intracellular parasite was also necessary, including an enrichment step. We have also developed new approaches for the analysis of the resulting datasets, affording better discrimination of specific compound-induced stabilisation from various experimental artefacts. We successfully identified the known targets of current and novel antimalarials and extended the proteome information of solvent-induced protein precipitation for target identification.

Mitochondria are central cellular organelles that are often metabolically modified and reduced into various anaerobic types of mitochondria (ATM) in organisms adapted to endobiosis under anaerobic conditions. It is not known whether this trade is reversible as cases of reverse adaptation to a free-living lifestyle are rare. Hence, we decided to analyse ATMs of Hexamita inflata, a diplomonad with potential parasitic ancestry living in anaerobic freshwater sediments on the edge of endobiotic and free-living lifestyles. The genome of H. inflata is about ten times larger compared to genomes of its parasitic relatives. Each cell contains about 20-30 double-membrane ATMs without cristae with a diameter of about 400-600 nm. Genome homology searches identified more than 150 proteins with possible ATM localization. However, similar to ATMs in other diplomonads, the vast majority of nuclear-encoded preproteins lack the mitochondrial N-terminal targeting presequence, which hinders reliable bioinformatic predictions. Therefore, we isolated ATM-enriched fractions and subjected them to a proteomic analysis. The proteome of the organelle contains more than 400 proteins including those involved in the iron-sulphur cluster assembly, pyruvate metabolism, ATP synthesis, hydrogen production, oxygen and ROS detoxification, and amino acid metabolism, indicating the ATM of H. inflata is an ATP-producing hydrogenosome.

Mitochondria and peroxisomes are functionally interrelated oxygen-metabolizing organelles that underwent major functional and structural changes in eukaryotes adapted for life under oxygen-limited or anaerobic conditions. Mitochondria transformed into a range of anaerobic types (ATMs) that reduced the electron transport chain to varying degrees, lost oxidative phosphorylation, and gained components for anaerobic energy metabolism, typically hydrogenase (hydrogenosomes), or lost their energy metabolism completely (mitosomes). In some cases, the organelles themselves were lost. Peroxisomes were proposed to evolve from ER for compartmentalization of a hydrogen peroxide-producing enzyme involved in fatty acid metabolism, although there is a large diversity of other peroxisomal metabolic functions. Based on the absence of peroxisomes in Metamonada, an anaerobic group of protists with ATMs, it was anticipated that peroxisomes would also be lost in other anaerobic eukaryotes. However, the discovery of anaerobic peroxisomes in Archamoebae showed that the presence of peroxisomes is not conditioned by obligater aerobiosis. Through phylogenomic searches, we found that peroxisomes are conserved in some members of anaerobic ciliates, heteroloboseans, and strictly anaerobic fungi of the Neocallimastigomycota group. We attempt to reconstruct the functional losses, gains, and links between anaerobic peroxisomes and ATMs across eukaryotic lineages, providing insights into their co-evolution in anaerobic eukaryotes.

Adaptation of eukaryotes to an anaerobic lifestyle results in reduced forms of mitochondria, such as hydrogenosomes and mitosomes, while peroxisomes were believed to be lost due to the key role of oxygen in peroxisomal metabolism. However, the discovery of anaerobic peroxisomes in Archamoebae challenges this assumption, prompting further research into the evolution of anaerobic eukaryotes. This study focused on Neocallimastix lanati, a strictly anaerobic fungus from the phylum Neocallimastigomycota. Bioinformatic, enzymatic, and proteomic analyses revealed typical hydrogenosomal enzymes related to the production of hydrogen and ATP under anaerobic conditions, components of membrane biogenesis TOM/TIM complexes, iron-sulphur cluster assembly, and amino acid biosynthesis. Notably, we identified components of respiratory complex I and II, dihydroorotate dehydrogenase, and the ATP synthase complex F1. In peroxisomes, 13 common peroxins were identified, covering all functional categories of peroxisomal biogenesis. The search for peroxisomal targeting signals predicted 140 peroxisomal proteins. Functional annotation indicated the presence of multifunctional protein, a typical fatty acid beta-oxidation component, but not acyl-CoA oxidase and catalase. Additionally, enzymes related to amino acid metabolism, cofactor biosynthesis, redox balance, and ROS metabolism were detected without a clear biochemical context. In summary, anaerobic fungi represent a eukaryotic group that possesses both hydrogenosomes and anaerobic peroxisomes.

Trichomonads that possess an undulant membrane, a recurrent flagellum and the costa. It originates at the anterior region of the protozoan and projects towards the posterior region displaying a specific striated pattern produced by the alternation of electron lucent and electron dense bands. Previous cytochemical studies and its isolation with subsequent proteomic analysis showed that the costa contains several proteins. We selected some of the most abundant proteins to produce specific antibodies making it possible to confirm, using immunocytochemistry their presence in the costa. The first protein was designated as costain 1. Here we describe two new proteins who are also localized in the costa, as shown using conventional and expansion immunofluorescence microscopy and transmission electron microscopy. They were designated as costain 2 and 3. In addition, using each protein sequence information, we built models to predict the three-dimensional structure of these proteins using ProtCHOIR software, in the attempt to further elucidate their specific functions in the costa. Our findings showed that the three costa proteins have apparently very different 3-D structures, even though they share some aminoacidic sequences. The characterization of these proteins can help elucidate further the function of this intriguing cytoskeleton structure found in trichomonads.

The internal structure of Giardia flagella, resembling that of other organisms, is characterized by an extensive cytosolic segment before emerging as functional flagella. The region of flagella emergence, known as the flagellar pore, has been a subject of limited study. Here, we analyzed the ultrastructural organization of the Giardia flagella, employing high-resolution microscopy techniques: HR-scanning EM, tomography, atomic force microscopy, and freeze-fracture. Intact cells revealed a prominent surface forming a ring at the flagella exit. The internal organization of this region showed electron-dense plaques on the cytoplasmic side, at the point where the membrane curves at the flagella base. When the plasma membrane was removed, an extra-axonemal structure associated with the outer doublets of the axoneme was visualized in this area. This structure, a novel discovery, is present in all trophozoite flagella pairs and persists even when interconnections between axonemal microtubules are disrupted by detergent treatment. In conclusion, our findings not only reveal a novel structure at the externalizing point of the G. intestinalis flagella but also enhance our understanding of the arrangement of the Giardia flagellar pore. These observations offer valuable insights into the biology, survival, and pathogenesis of this parasite. Supported by CAPES, FAPERJ, CNPq, FINEP and CENABIO.

When Giardia intestinalis differentiates into a cyst, the single cell undergoes extensive rearrangement involving the transport of cyst wall proteins (CWPs) within encystation-specific vesicles (ESVs) to the cell surface. There, the proteins combine with unique β-1,3-N-acetylgalactosamine homopolymer (giardan). By analyzing the phenotypes of Giardia mutant cell lines generated via CRISPR/Cas9—either lacking CWP1 or having defective giardan synthesis—we characterized the structural roles of the protein and sugar components in the cyst wall. Cells lacking CWP1 retained cyst wall material on the surface, but the giardan filaments condensed into thicker structures, suggesting that CWP1 plays a role in fine branching of the polysaccharide. This phenotype was reversed by reintroducing the endogenous gene or by using the CWP homologue from Carpediemonas membranifera, a free-living metamonad discovered in marine sediments of the Great Barrier Reef. Conversely, disrupting giardan synthesis by deleting UDP-N-acetylglucosamine 4-epimerase (ΔUAE) resulted in "naked" cysts surrounded only by the cytoplasmic membrane. Finally, we observed dynamic behavior of a proteasomal component on the ESV surface during encystation, indicating regulated, site-specific protein degradation. In this work, we propose the origin of the cyst wall formation and its evolution in Metamonada.

Extensive studies on model organisms like Caenorhabditis elegans (elegant worm) and Drosophila melanogaster (vinegar fly) have significantly enhanced our understanding of complex biological mechanisms in multicellular organisms. By leveraging comprehensive datasets derived from genomics, transcriptomics, and proteomics, scientists have identified essential genes, crucial for life. Recent investigations have explored the possibility of predicting essential genes using advanced bioinformatics, particularly employing machine learning techniques to extract features from DNA, RNA, protein, and cellular data. This approach shows promise for predicting essential genes within and across related species, such as those within the same evolutionary clade or phylum. In the present study, we have applied this method to predict essential genes within the phylum Nematoda (clade V), focusing on the pathogenic parasitic nematode H. contortus. By cross-predicting essential genes between C. elegans and H. contortus, we prioritized potential drug targets encoded by these genes. The analysis highlighted essential genes primarily involved in critical biological processes like ribosome biogenesis, translation, RNA processing, and signaling, with high transcription levels in specific cell types. These findings suggest that this computational approach holds promise for identifying and prioritizing drug target candidates in parasitic nematodes for further experimental validation.

Haemonchosis is a disease caused by the haematophagous barber’s pole worm, Haemonchus contortus, which affects livestock animals worldwide. Despite the major significance of this parasite worldwide and its widespread resistance to current treatments, the lack of a high-quality genome for the well-defined strain of this parasite from Australia, called Haecon-5, has constrained research in a number of areas, including host-parasite interactions, drug discovery and population genetics. To enable research in these areas, we report here a chromosome-contiguous genome (~280 Mb) for Haecon-5 with high-quality models for 19,234 protein-coding genes. Comparative genomic analyses show significant genomic similarity (synteny) with a UK strain of H. contortus, called MHco3(ISE).N1 (abbreviated as “ISE”), but we also discover marked differences in genomic structure/gene arrangements, distribution of nucleotide variability (SNPs and indels) and orthology between Haecon-5 and ISE. We used the genome and extensive transcriptomic resources for Haecon-5 to predict a subset of essential single-copy genes employing a “cross-species” machine learning approach and prioritised ten of them for further characterisation. The present Haecon-5 reference genome and associated resources now underpin a broad range of fundamental investigations of H. contortus and could assist in accelerating the discovery of novel intervention targets to combat haemonchosis.

Pituitary Pars Intermedia Dysfunction (PPID) is a detrimental endocrinopathy affecting 20-25% of geriatric horses. Formation of hyperplastic tissue in the pars intermedia leads to increased hormone secretion, resulting in symptoms such as hypertrichosis, lethargy, weight loss and increased susceptibility to infections including parasitic nematodes. The immune mechanisms underlying increased infection susceptibility is largely unknown.

This research investigated the function of Peripheral Blood Mononuclear Cells (PBMC) in PPID (n-6) and non-PPID (n=6) horses exposed to a natural infection of strongyles. PBMC were stimulated in vitro with lipopolysaccharide (LPS), concanavalin A (ConA), phorbol myristate acetate (PMA) and a small strongyle antigen (GALA-cat) for 48h. Following stimulation, cell proliferation and the presence of IgG(T) antibodies were measured.

The results showed increased cell proliferation in PPID horses following stimulation with LPS (p < 0.001), ConA (p = 0.014), PMA (p = 0.045) and GALA-cat (p = 0.017). However, there were no significant differences in the concentration of total IgG(T) antibodies in the supernatant, except for the PPID horses having lower IgG(T) levels in unstimulated conditions. These results suggest that the PBMC of PPID horses can proliferate, however the rates of the proliferation are increased and IgG(T) production is reduced in unstimulated conditions.

This study assessed gastrointestinal (GI) parasite control practices used by Australian dairy goat farmers through an online questionnaire survey. The questionnaire consisted of 54 questions on farm demographics, husbandry and grazing management, knowledge of GI parasites and their significance, diagnosis, anthelmintics, and alternative control options. After a pilot survey, the questionnaire link was sent to all registered members (n = 456) of the Dairy Goat Society of Australia Ltd on 17^th April 2023. The response rate was 14% (66/456). Most respondents owned medium-sized herds (20-100 goats; 56%, 37/66) followed by small (<20 goats; 36%) and large (>100 goats; 8%) herds. Seventy-four percent (49/66) of respondents observed parasite-related illnesses in their goats and two-thirds assessed worm burden using faecal egg counts. Most respondents (97%; 183/188) perceived that GI parasites caused production losses or health impacts and ranked Haemonchus contortus as the most significant parasite. Ninety-four percent (62/66) of respondents used anthelmintics, primarily a commercial combination of four anthelmintics (levamisole, closantel, albendazole, and abamectin), benzimidazoles and macrocyclic lactones. Although targeted deworming was the most common method of controlling GI parasites, most respondents were unaware of anthelmintic resistance. These findings will pave the way to devise guidelines for GI parasite control in goats.

East Coast fever (ECF), caused by the protozoan Theileria parva, and sheep flystrike, caused by the parasitic Lucilia cuprina, pose significant challenges to livestock health in sub-Saharan Africa and tropical regions. Current control methods for ECF rely on a live-parasite vaccine, which has practical limitations associated with it. Control methods for sheep blowfly primarily involve mulesing and use of insecticides, which has increased insect resistance. Our research focuses on developing more sustainable and effective control strategies against livestock parasites like ECF and sheep blowfly by combining molecular biology with nanotechnology. The published ECF study has shown co-immunization of cattle (Bos taurus) with two nanotechnologies along with p67C antigen resulted in stimulation of both high Ab titers and CD4 T cell responses. Our work is also investigating the innovative RNA interference (RNAi) technology in combination material science to target specific genes in sheep blowflies, to develop biological control to benefit animal health and productivity.

Reference: Lacasta A, Mody KT, De Goeyse I, Yu C, Zhang J, Nyagwange J, et al. Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle. J Immunol. 2021;206(4):686-99. Epub 2021/01/10. doi:10.4049/jimmunol.2000442.

Despite being logical targets, surface tegument proteins of juvenile Fasciola hepatica have not been studied as vaccines to control fasciolosis in cattle. Nine recombinant F. hepatica tegument proteins (Tetraspanins (TSP) 2 and 3; Annexins (Anx) 2, 3 and 8;Tegument proteins FhTeg1, 5, 22 and 25), as well the immunomodulator Transforming Growth Factor Beta (TGF-β)-like homologue FhTLM, were tested as vaccines, both alone or as combinations. TSP2 was also evaluated fused to the E. coli heat-labile entero-toxin B subunit LTB adjuvant using intranasal vaccination. Five antigens were assessed in combination with Glutathione S-transferase (GST).

Female Angus/Angus-cross cattle (n=6-7) were vaccinated 2 or 3 times with proteins formulated in Freund’s adjuvant or a nanoparticle adjuvant and challenged with metacercariae. Significant reductions in mean fluke numbers (38-48%) were observed in 4 groups using combinations of TSP3, Anx2, Anx3 or Anx8, as well as with FhTLM/GST. However, there was variation between trials in vaccine efficacy. Efficacy was associated (r² = 0.915) with the fraction of animals in each group showing fluke counts less than the lowest value in control groups. The data suggest that, under our experimental conditions, 90-100% of animals show relatively low fluke counts with a vaccine efficacy of only 43-48%.

Experimental and naturally acquired human helminth infections have been shown to impart varying degrees of protection against a suite of inflammatory diseases. The proclivity of helminths to regulate their host immune response and suppress inflammation is attributed to the active release of excretory/secretory proteins (ESP) into the host tissues. Experimental infection of humans with helminths presents significant complications as a therapeutic modality due to their complex lifecycles, likely poor adoption, and unavoidable side effects in some subjects. As such, there is now considerable interest in identifying bioactive ESPs and making them more drug-like. We therefore created a recombinant library of N. americanus ESPs from both the adult and larval stage secretomes and are screening the library in a range of in vitro and in vivo assays to identify proteins with potent immunoregulatory properties. Thus far, we have identified proteins that could form the basis of novel therapeutics for treating type 2 diabetes, inflammatory bowel disease and rheumatoid arthritis based on their in vitro and/or in vivo bioactivities.

Individuals infected with Plasmodium spp. parasites or other pathogens exhibit a range of disease outcomes from asymptomatic to fatal. Current understanding of molecular mechanisms underlying immune heterogeneity is limited. This study aimed to investigate immune heterogeneity in malaria to identify immune cells and molecules associated with disease outcomes following Plasmodium infection. A particular focus was on Natural Killer (NK) cells positioned at the innate-adaptive immune interface, previously implicated in parasite control. PBMCs from individuals experimentally infected with blood-stage P. falciparum were subjected to comprehensive single-cell proteomics, bulk proteomics, and single-cell sequencing to identify cells or molecules predictive of infection outcomes. Individuals were classified based on Parasite Multiplication Rate (PMR) at 48 hours post-infection as “good” (PMR<20) or “bad” (PMR>20) responders. Single-cell-RNAseq identified transcriptional signatures showing upregulation of NKG7 and GZMB and downregulation of IL-12RB across multiple NK cell clusters, suggesting immunoregulatory phenotypes in good responders are mediated through the regulation of cytokine production and activation. Single-cell proteomics further emphasised highly inflammatory NK populations in bad responders via increased CD8a expression across NK and other immune cell subsets. These findings underscore the critical role of NK cells during P. falciparum infection, and identify unique molecular signatures associated with improved infection outcomes.

Malaria parasites have complicated lifecycles involving both human and mosquito hosts. In ~8 days, a single parasite can form an oocyst in the mosquito midgut produces thousands of daughter sporozoites. These sporozoites then egress from the oocyst and undergo a complex series of translocation and invasion events to reach the mosquito salivary gland. To facilitate host cell invasion, sporozoites use specialised secretory organelles known as rhoptries. The small size of these parasites and their rhoptries, however, has made it challenging to interrogate sporozoite rhoptry biology. To overcome this, we developed mosquito-tissue ultrastructure expansion microscopy (MoTissU-ExM), a technique that physically expands parasites and their hosts ~4.5-fold. Using MoTissU-ExM, we developed a timeline for sporozoite rhoptry biogenesis in the mosquito midgut and observed the changes they undergo during salivary gland invasion. We leverage these findings to characterise the role of rhoptry neck protein (RON) 11 in sporozoites, showing that RON11 disrupted sporozoites only have half the required number of rhoptries and that therefore RON11 is the only protein known to be involved in sporozoite rhoptry biogenesis. Further, we show that while these RON11-disrupted sporozoites can anatomically enter the salivary gland, they fail to invade salivary gland epithelial cells or the secretory cavity.

Recent breakthroughs in mRNA modifications have enabled RNA therapeutics with stabilised mRNA to improve vaccine efficacy. Despite their importance in biotechnology, the impact of mRNA modifications on stability and translation are poorly understood. N⁶-methyladenosine (m⁶A) is an mRNA modification thought to either improve translation efficacy or reduce stability of transcripts. We studied this modification in Plasmodium falciparum by disrupting the methyltransferase that makes m⁶A. Sequencing of m⁶A in mRNA has only recently become possible with Oxford Nanopore Technologies direct RNA sequencing. However, it is unclear how robust and accurate this detection of m⁶A is. We therefore compared four open-source tools that have been independently developed to identify m⁶A sites in Nanopore RNA-seq from P. falciparum trophozoites. Two of these tools detect m⁶A through machine learning systems trained to artificial RNA synthesized with m⁶A, whereas two work by detecting statistical differences between transcripts that possess or lack m⁶A. We found the former were less correlated in site prediction than the latter category. For transcripts with the most m⁶A sites predicted by each tool, six highly methylated transcripts were common to three tools. Future work involves time-series analysis of P. falciparum transcriptome using updated Nanopore flow cells with proprietary m⁶A detection.

Gastrointestinal nematodes (GINs) severely affect animals, including humans, by causing serious morbidity whilst also exacerbating cycles of poverty. Commonly used diagnostics for GIN detection are often insensitive, e.g. microscopy, whilst more sensitive molecular techniques, e.g. quantitative PCR (qPCR), may only target a few species. More recently, attempts to characterise all GIN species simultaneously, i.e. the “nemabiome” have been developed using next-generation sequencing (NGS). To date, these methods have relied on short-read NGS platforms that can only detect limited genera and for some taxa cannot classify to a species level. Improving upon this, we demonstrate how a nanopore sequencing approach can accurately characterise the complete nemabiome from animals and humans. Through targeting of both internal transcribed spacer regions of the clade V nematodes and 18S ribosomal RNA gene of other key taxa in clades I, II and IV we have shown our assay can provide a comprehensive characterisation of GIN diversity. Moreover, the performance of our assay was found to be equivalent to highly sensitive and specific qPCRs for key GIN species. Overall, long-read nanopore sequencing provides unprecedented species classification accuracy and more comprehensive characterisation of GIN communities in a way previously unachievable using older technologies.

Bovine tritrichomonosis is a venereal disease that causes economic losses around the world. Over the last 100 years, the life cycle of the protozoan T. foetus in bovines has only considered venereal transmission, almost exclusively through natural mating from an infected animal to a healthy animal. We provide the first direct evidence that T. foetus can survive the passage through the gastrointestinal tract in bovines and that the parasite can be discharged by feces and contaminate the cow’s reproductive tract. Moreover, we demonstrated that T. foetus under stress conditions can increase its DNA content per parasite without concluding the cytokinesis process (endoreplication), which represents an efficient strategy for subsequent fast multiplication by multiple fissions when the context becomes favorable. Finally, we revealed the existence of novel dormant forms of resistance cyst-like structures (multinucleated or mononucleated polyploid parasites), different than the previously described pseudocysts, that are formed under stress conditions.

Considering that the parasite could be discharged by feces into the environment and their capacity to form resistance structures, we propose a novel possible dissemination form of T. foetus, mainly in extensive livestock production systems of bovines, which should be analyzed in detail in the future.

Trichomonas vaginalis (Tvag) is an extracellular protozoan parasite and the causative agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. The parasite lacks pathways for de novo purine biosynthesis and therefore must uptake nucleotides/nucleosides from outside the cell. Here, our study aimed to investigate the involvement of purinergic signaling and extracellular vesicles (EVs) in the pathogenesis of Tvag when grown under starvation (i.e. serum deprivation or glucose restriction) with the goal of identifying new therapeutic targets. Both starvation conditions increased the enzymatic activity of NTPDase and E-5’-N in all Tvag isolates tested and this effect was reversed by adenosine. ATP-induced cytotoxic of Tvag was reversed by adenosine but not by glucose. Starvation altered the expression levels of nucleoside transporters (ENTs): Serum deprivation and glucose restriction caused downregulation and upregulation of ENT expression in Tvag, respectively. Finally, we showed that starvation modulates the release of EVs from trophozoites, suggesting a potential role for nutrient sensing in mediating cellular responses. Whether EVs released from parasites under starvation have a differential impact on host-parasite interactions is currently under investigation. Our research helps to understand a critical route for the survival of Tvag, suggesting nucleoside transporters as potential targets for therapeutic interventions.

Entamoeba histolytica, the causative agent of amoebiasis, performs a process of “cell nibbling”, called trogocytosis, that kills human cells and contributes to tissue damage. We found that E. histolytica displays human membrane proteins on its own surface after performing trogocytosis. We hypothesize that the display of human proteins following trogocytosis directly inhibits host complement activation. We allowed amoebae to perform trogocytosis on human cells and then exposed them to mouse serum. Following trogocytosis of human cells, amoebae were protected from lysis by mouse serum. We exogenously expressed negative regulators of human complement activation, CD46 and CD55, in amoebae. The resulting protection from both human and mouse serum lysis indicated that protein display was sufficient for protection, and that mouse and human complement regulators are functionally interchangeable. We are now allowing amoebae to trogocytose insect Sf9 cells to determine if trogocytosis of these cells still confers protection from lysis by human serum. We anticipate that exogenous expression of human CD46 and/or CD55 in Sf9 cells will restore protection. Since other pathogens are capable of trogocytosis, these studies should improve understanding of virulence mechanisms, and may also shed light on protein trafficking during eukaryotic trogocytosis in general.

Molecular methods are a mainstream method for the detection of protozoan infections in pathology laboratories. Recently, these assays have been applied to screen for novel animal hosts. Due to the difference in microbial flora, there is the potential for cross-reactivity to occur which was previously unexpected. Concerningly, not all studies confirm positive RT-PCR results with sequencing or microscopy. Dientamoeba fragilis is one species for which multiple new hosts have recently been identified. There are large differences in reported positivity rates of D. fragilis between pathology specimens in developed countries with similar healthcare capacities. We assessed the use of two RT-PCR diagnostics previously used to identify novel hosts of Dientamoeba fragilis and clinically. Specimens were collected from cattle, cats and dogs to screen for cross-reactivity. Additionally, the positivity rate between these two assays for 254 unknown human clinical species was compared. Positive results were assessed using melt-curve analysis, cloning and sequencing of an additional locus, and Illumina MiSeq of the RT-PCR product to assess specificity of amplification. Cross-reactivity was identified in both assays when applied to the animal specimens. When these assays are applied to the unknown clinical samples, one assay had 2.5 times more positive results than the other.

Introduction: Giardiasis is one of the most common enteric infections imported from tropical regions to Europe. The number of diagnosed giardiasis cases in the Czech Republic has decreased from over 1,000 annually in the 1990s to fewer than 100 in the early 2020s. However, the number of imported cases has remained stable, except during the COVID-19 pandemic years of 2020-21.

Methods: We conducted a retrospective analysis of the epidemiological and clinical characteristics of patients with laboratory-confirmed giardiasis diagnosed at Bulovka University Hospital (Prague), from 2004 to 2023.

Results: The infection was diagnosed in 357 patients (female:male ratio 0.69). The majority of infections were imported from the Indian Subcontinent (221 cases; 61.0%), followed by Sub-Saharan Africa (44 cases; 12.3%) and Southeast Asia (31 cases; 8.7%). The most common symptoms were diarrhea and bloating. Diagnostics were performed using parasitological stool examination and PCR. Treatment response data were available for 228 patients treated with metronidazole. Of these, 85 (37.3%) were treated successfully, 80 (35.5%) experienced treatment failure, and in 83 (36.4%) no parasitological follow-up was performed. Second-line treatments included combinations of tinidazole with albendazole or paromomycin.

Conclusion: Our study revealed low effectiveness of metronidazole, particularly in patients returning from South Asia.

Anthelmintic resistance is a major problem in the control of parasitic infections in the agriculture industry in many parts of the world. Therefore, understanding the mechanism of drug resistance and the development of new drugs is at the forefront of scientific interest.

Increased anthelmintics inactivation via biotransformation belongs to a significant drug-resistance mechanism. Long-term exposure to sub-lethal doses of drugs can lead to changes in the expression of specific detoxification enzymes and drug resistance development.

Haemonchus contortus, a gastrointestinal parasite of ruminants, is a good model organism for studying drug resistance, as it has evolved resistance to all available anthelmintics over the years.

This study focuses on biotransformation enzymes from short-chain dehydrogenases/reductases (SDR) and aldo-keto reductases (AKR) family in the nematode H. contortus. The constitutive expression of 23 SDRs genes and 22 AKRs was analyzed and compared between drug-susceptible and drug-resistant strains and among various developmental stages (eggs, larvae, adults). Furthermore, the effect of sub-lethal doses of flubendazole, benzimidazole anthelmintic bearing carbonyl group, on the expression of selected genes was evaluated.

The results suggest that SDR/AKR enzymes play different roles during the parasite's life cycle and certain enzymes might be responsible for reductions of xenobiotics, including some anthelmintics.

Anthelmintic (drench) resistance and gastrointestinal nematode (GIN) management is a significant animal health and production limiting concern for New Zealand livestock farmers. Finding useful tools to support GIN management and tackle drench resistance is critical for the sector. A potential approach is targeted selective treatment (TST), aiming to slow the development of drench resistance by reducing total drench used and thus selection pressure for resistant GIN.

Beef + Lamb New Zealand recently funded three case studies to evaluate the use of an existing TST tool, the SmartWorm® application, for use in New Zealand. Three properties in the Hawkes Bay and Wairarapa during 2023 were selected and non-replacement lambs grown out for winter slaughter were used.

SmartWorm®, with an eID tag, leverages liveweight gain, pasture quality and quantity and weather data to identify animals in real time that are most likely to benefit from a dose of drench.

Results showed that across a range of GIN pasture contamination environments, SmartWorm® was able to reduced overall drench input and provided refugia to the farm.

Across the three farms, reduction in drench use of 35%, 53% and 57% with no significant impact on weight gain was recorded. Farmers also recorded overall cost savings.

The objective of this study was to evaluate the effect of anthelmintic drugs on ewe and lamb behaviour. We randomly allocated 60 pregnant ewes to one of three groups (n=20/group): 1) administered moxidectin 2-4 weeks pre-lambing, 2) administered a controlled-release capsule (abamectin and albendazole) 2-4 weeks pre-lambing, and 3) control group. The ewes were housed indoors one month pre-lambing until 1 week post-lambing. Ewe and lamb behaviour was recorded using video-cameras for up to 7 days post-lambing and birth data was recorded. There tended to be a difference between total labour length and ewe treatment (P=0.06). Control ewes were in labour for a median of 65.8 (IQR 33.2 to 139; maximum 420.4). CRC treated ewes were in labour for a median of 67.0 (IQR 55.4 to 159.21; maximum 407.3) minutes. Moxidectin treated ewes were in labour for a median of 47.4 (IQR 30.0 to 106.8; maximum 272.8) minutes. There were no differences between treatments for latencies (from birth) to perform behaviours: lamb to shake head (P=0.68), attempt to stand (P=0.57), complete stand (P=0.7), attempt to suckle (P=0.34) and complete suckle (P=0.84). Although LA drench may not have an affected lamb behaviour, they appear to affect length of labour.

This national study investigated the epidemiology, control and anthelmintic resistance of intestinal nematodes (strongylids and ascarids) in horses using both conventional and advanced molecular techniques. Eighty Thoroughbred farms were recruited for longitudinal and cross-sectional studies, and 86 faecal egg count reduction tests were conducted on 22 farms. Coproscopic and next-generation sequencing techniques were used to determine the worm egg burdens and the types of nematodes, respectively. Questionnaire surveys were distributed to horse breeders and veterinarians to assess worm control practices. Results showed a farm-level prevalence of 100% for strongylid nematodes, with higher rates in younger horses. Only 17% of adult horses were high shedders of strongylid eggs (>500 eggs per gram). Resistance to ivermectin, abamectin, moxidectin, oxfendazole and its combination with pyrantel was found in cyathostomins and Parascaris spp. The most common species identified were species of Cylicocyclus, Cylicostephanus and Coronocyclus. Interval deworming was the most common strategy (≥55%) to control worms, and <40% of breeders performed faecal egg counts. Veterinarians rarely discuss worm control strategies with their clients. The study highlights the high prevalence of nematodes, resistance to common anthelmintics, and suboptimal parasite control practices, emphasising the need for integrated parasite management to improve parasite control in horses.

Fasciola hepatica is a zoonotic parasite of international significance. In Australia, it is the 13^th most important cause of losses to the sheep industry due to its impacts on body condition score, fibre quality and yield. Resistance to the frontline drug, Triclabendazole (TBZ), was first detected in Australia in 1995 and has since spread throughout the world. TBZ is the only drug registered for use against F. hepatica infection in people. With limited chemical control options, and no vaccine available, it is essential that we preserve the efficacy of TBZ.

Recently, livestock producers from the NSW Southern Tablelands have alerted us to their concerns over suspected increases in drug-resistant parasites. We set out to evaluate the prevalence of F. hepatica on ten naturally infected sheep or cattle properties in the region to confirm/deny suspected drug failure. Prior to the commencement of the field trial, we reviewed reports of TBZ resistance in Australia in conjunction with the recent guidelines on evaluating anthelmintic efficacy in ruminants from the World Association for the Advancement of Veterinary Parasitology (WAAVP). Here, we present a critical analysis of the strengths and limitations of these guidelines in light of the practical considerations when working in the field.

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019–2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes.

Leishmaniasis, triggered by various species of the Leishmania genus, poses a significant public health challenge with its outbreak potential. Understanding the mechanisms and pathways of cell death in these parasites is complicated due to the absence of several key apoptosis regulator genes in the Leishmania genome, despite reports of apoptosis-like cell death behaviors. Our research has centered on Bax inhibitor-1 (BI-1), a protein known for its evolutionary conservation across plants, animals, and yeast, and its role in inhibiting the Bax protein, which is involved in apoptosis. In examining the Leishmania genome, we discovered a sequence similar to Bax inhibitor-1, which we have named LdBI-1. This sequence shares limited similarity with its human analogue but retains critical transmembrane domains. Utilizing the CRISPR/Cas9 technique, we created a cell line with a disrupted LdBI-1 gene. Its absence leads to increased sensitivity to cell death stimuli. We also developed a cell line overexpressing LdBI-1 to further investigate its role, particularly in relation to autophagy and overall cellular biology, including subcellular localization. Our ongoing study seeks to understand LdBI-1's evolutionary conservation and functional role in Leishmania, potentially paving the way for new antileishmanial treatments by targeting this novel gene.

Cerebral malaria (CM) is an often-fatal neurological complication of Plasmodium falciparum infection, with survivors frequently experiencing enduring neurologic sequelae. The disruption of the blood-brain barrier (BBB) has been identified in several studies as a key factor leading to CM progression. We utilized Plasmodium coatneyi-infected rhesus macaques as a non-human primate (NHP) model to unravel the pathogenesis of falciparum malaria, given their similar pathophysiological features with P. falciparum infection in humans. Through differential gene expression analysis, we demonstrated the effectiveness of methylene blue treatment in reversing the effects of infection on the brainstem. Comparing our brainstem dataset from P. coatneyi-infected Macaca mulatta with two other transcriptomic datasets (P. coatneyi-infected M. mulatta blood and P. falciparum-infected human blood), we identified nine genes associated with CM severity. Many of these genes are expressed by neutrophils and could serve as blood biomarkers for the diagnosis of lethal falciparum CM. The potential role of P. coatneyi as a zoonotic parasite poses significant implications for public health, suggesting the need for further investigation into its capability to infect humans and contribute to malaria transmission dynamics. This study highlights the importance of considering zoonotic malaria species in the broader context of malaria research and control strategies.

Entamoeba histolytica is an enteric tissue-invasion protozoan parasite that rapidly kills host cells within about 5 minutes of contact with host cells. In this study, Entamoeba-induced host cell death was comparatively investigated using two cell death measurement methods; LDH release assay and CellTiter Glo. The widely used LDH release assay measures cell death by detecting lactate dehydrogenase, which is released through cell membrane disruption. CellTiter Glo measures cell viability by detecting ATP in living cells. First, Entamoebae were pretreated with various inhibitors such as D-galactose (a competitive inhibitor of amebic Gal-lectin), E-64 (a cysteine protease inhibitor) and Ca²⁺ chelators (EGTA or EDTA), respectively. Pretreated Entamoeba was co-cultured with Caco-2 cells, and Entamoeba-induced cell death was measured using two methods. The results of these methods commonly explained that Entamoeba treated with D-galactose, E-64, or Ca²⁺ chelators lost their ability to induce host cell killing compared to untreated amoeba. Interestingly, CellTiter Glo was able to detect Entamoeba-induced cell death after only 5 minutes of co-incubation. These results show that CellTiter Glo is more suitable and sensitive method for analyzing rapid cell death caused by Entamoeba than LDH assay.

Acanthamoeba, a free-living amoeba, causes granulomatous amebic encephalitis (GAE) and Acanthamoeba keratitis (AK), an eye infection primarily affecting contact lens users due to extended wear, improper maintenance, and corneal trauma. In vitro AK studies, especially for therapeutic drug development, require in vivo confirmation. Previously, we established an AK mouse model by loading 2 mm contact lens fragments with 5 x 10⁴ A. castellanii cells, inserting them into scratched mouse eyes under anesthesia, and suturing the eyelids. Daily observations from 1 to 14 days post-infection showed AK lesion progression, confirmed by PCR detection of Acanthamoeba DNA. This study aimed to develop an effective cultivation method to validate PCR findings in the AK mouse model. Eyeball samples collected at 1, 3, 7, and 14 days post-inoculation were homogenized and successfully cultivated on non-nutrient agar with an Escherichia coli lawn, then scaled up in PYG medium. PCR analysis confirmed the cultivated amoeba's genetic information matched the inoculated strain (> 99%). This cultivation and PCR approach provides a robust method for confirming AK development in the AK mouse model.

Anaerobic protists, although often overlooked, play a crucial role in marine ecosystems, contributing to nutrient cycling, organic matter degradation, and overall ecosystem health. Despite their importance, the species diversity and ecological significance of anaerobic protists in Korean marine environments remain largely understudied. In this study, we applied environmental DNA (eDNA) metabarcoding as an effected tool for analyzing the diversity of protist in marine water and sediments. As the result, 20 phyla of Protist are detected including Apicomplexa, Cerozoa, Telonemia, Perkinsozoa and so on. The current state of marine protist research in Korea, focusing on species diversity and ecological roles of aerobic environments. Until now, 18 phyla over 3,000 marine protist species have been reported in Korea based on morphological description. This finding underscores the need for further research to comprehensively understand the diversity and ecological functions of anaerobic protists in Korean marine ecosystems. This work was supported by the National Marine Biodiversity Institute Research Program (2024M00200).

In recent years, the interest in molecular diagnostic methods for the detection of many pathogens has grown substantially. This escalation in interest has occurred in parallel with data indicating inaccuracy of scabies diagnosis based on currently available methods such as examination of skin samples by standard microscopy, burrow ink test and handheld dermatoscopy. Mites are very few in numbers at the early stage of the disease that makes it extremely difficult for even an experienced dermatologist to make a definitive diagnosis. Hence, scabies can be misdiagnosed as an allergic reaction or eczema. With the recent availability of the scabies genome, we developed a qPCR assay targeting a more abundant repetitive region to improve the sensitivity and specificity of a PCR-based diagnostic assay, alongside a non-invasive skin sample collection using FLOQ swabs. We have initially validated our qPCR methodology by testing samples from hospitalised cases in Darwin, NT and field collected samples from Kimberley, WA and Auckland, NZ. Recently, we have optimised our assay using the digital PCR format and have demonstrated a much higher sensitivity and specificity in detecting scabies, representing a major advance. Results of our studies showing its utility in improving clinical diagnosis of scabies will be presented.

Scabies is one of the most common skin diseases worldwide and is caused by the parasitic mite Sarcoptes scabiei. Unbearable itch is the cardinal symptom of scabies. Scratching in response to itching facilities the entrance of pathogenic bacteria into the skin and can lead to life-threatening sequelae. The mechanisms underlying the scabies itch are poorly understood, hence scabies itch targeted therapies are missing. Due to the non-responsiveness to anti-histamines, a histamine-independent pathway is proposed for scabies itch. We aimed to investigate the expression of non-histaminergic itch markers(MRGPRX2, tryptase, periostin, NK1R, epidermal nerve fiber density (ENFD) and substance P) ex-vivo over a course of scabies infection in a scabies pig model. Skin biopsies from three scabies infected pigs were collected pre-infection and 2, 4, 8, 12 and 20 weeks post-infection. Primary antibodies for above listed markers and cy-3 labelled secondary antibodies were used immunohistologically. Aperio FL slide scanner and QuPath software were used to image and quantify itch markers. We observed significant increase in typtase⁺ mast cells, MRGPRX2 positive cells, periostin, and NK1R expression, and reduced ENFD at 2 Weeks post-infection, coinciding with clinical onset of the scabies-associated itch in this model. We propose that scabies infection induces the non-histaminergic pathway.

The last formal study of scabies in New Zealand was conducted in 1978, indicating little interest has been given to this mite for more than forty years. Studies conducted in Pacific Island nations and in Aboriginal and Torres Strait Islander populations show a high prevalence of scabies, with common complications such as impetigo and bacterial skin infection. Our recent surveys of scabies in Auckland have shown an alarmingly high prevalence (36.5%) in predominantly preschool children living in sociodemographically deprived areas. Many children who were identified with scabies during the survey were treated for other diagnoses with topical steroid cream and other anti-pruritic lotions. Other work we have conducted shows a very strong statistical association between scabies and acute rheumatic fever: an important cause of ethnic inequality in health status in New Zealand children. Our work highlights the importance of improving the clinical diagnosis of scabies using molecular techniques such as the polymerase chain reaction test, and further understanding the prevalence of and improving treatment for this neglected tropical disease.

The Australasian harrier Circus approximans is native to Australia, New Zealand and the South Pacific. Although an opportunistic predator, much of its diet consists of carrion, mainly roadkill. Besides a record of a single, unnamed capillariid nematode there have been no investigations into the parasites of harriers in New Zealand, and there are no parasite records for them elsewhere in their range. A helminthological survey of 65 deceased harriers from southern New Zealand uncovered a helminth fauna comprising six species. Porrocaecum circinum (Nematoda) was previously described only from poor material from Australia. Procyrnea fraseri (Nematoda) is new to science, and includes specimens previously reported from Australia under an erroneous name. Strigea falconis, a widespread trematode, is reported for the first time in New Zealand. Cladotaenia anomala (Cestoda), a species with unusual scolex spine distribution, and Polymorphus circi (Acanthocephala) were found to be new species, and both were the first of their genus from New Zealand. An unnamed species of capillariid is mainly confined to North Island and is rare in South Island. Phylogenies are presented for each species based on DNA sequence. Potential intermediate hosts are discussed, and the origins of the helminths and their potential for pathogenicity are considered.

Many parasites have the adaptive ability to modify the phenotype of their host to complete their life cycle, a trait known as host manipulation. Here, we expect to see differences in the responses of infected animals toward certain cues, especially if a cue ultimately favours parasite transmission. In freshwater hairworms (Nematomorpha: Gordiida), mature individuals somehow “drive” their terrestrial insect hosts into water, where the hairworm exits to mate. Infected insects appear more active at night and respond to environmental cues differently than uninfected ones, however, we do not know how these differences evolve throughout hairworm development. Using Acheta domesticus crickets experimentally infected with Paragordius varius hairworms in a time series of behavioural assays, we tested multiple stimuli to determine how hairworms modulate host behaviour, and whether these behaviours correlate with hairworm development. Humidity, horizontally polarised light, and direct contact with water were tested in isolated behavioural assays. We noticed a remarkable behaviour reminiscent of apple bobbing in the water assay. Also, hairworms apparently needed to contact water to egress, otherwise they would retreat back into the host. We discuss these behaviours along with the responses of infected crickets to humidity and light, and link these to the broader sensory landscape.

Alterations to natural landscapes by human activities can change patterns of parasite transmission. The type of landscape alteration and life histories of parasites and their hosts, influence parasite burden. To investigate whether parasite burden is associated with land use change, we conducted a country-wide survey of bat and rodent parasites across Cambodia. Our results show that, agricultural land use types and areas with a mosaic of different land cover types exhibit a greater parasite burden compared to other landscapes, such as forested areas. Bats showed a lower parasite burden in fragmented habitats and urban areas, whereas fragmentation had no discernible impact on parasite burden in rodents. However, rodents had a higher parasite burden in urban areas when compared to other land use types. In cities, lower parasite burden in bats may reflect changes in resource acquisition that remove parasites from their food webs. Conversely, urban rodents have higher parasite loads, likely from higher density and improved body condition enabled by human food subsidies. This dichotomy highlights the nuanced effects human-modified landscapes can have on host-parasite associations, offering insights into disease ecology in rapidly changing ecosystems.

Trichomonas vaginalis is a protozoan parasite responsible for the most common non-viral sexually transmitted infection, trichomoniasis. T. vaginalis has striking motility driven by its 5 flagella. We hypothesize that T. vaginalis motility plays a key role in mediating infection. To investigate this, we performed the first characterization of kinesin-2 proteins in T. vaginalis. Kinesin-2 proteins are motor proteins that mediate anterograde intraflagellar transport that is necessary for flagellar assembly and maintenance. To identify putative flagellar-localized kinesin-2 proteins, we performed bioinformatic analysis and identified five putative kinesin-2 genes with similarity to kinesins found in the flagella of other single cell protozoan parasites and the flagellar model organism Chlamydomonas reinhardtii. Utilizing NEB HiFi DNA assembly, fusion proteins of the candidate genes were generated with two C-terminal HA tags and mNeonGreen, a green fluorescent protein. Using confocal microscopy, we found that two putative kinesin-2 fusion proteins indeed localize to the flagella. We are in the process of optimizing CRISPR/Cas9 methodology to knockout these genes, and an update on these research efforts will be presented. Our work will generate novel knowledge about the cellular biology of T. vaginalis flagella and the contribution of T. vaginalis motility towards pathogenesis.

The sexually transmitted parasite Trichomonas vaginalis (Tv) secretes extracellular vesicles (TvEVs) which are internalized by host cell and mediate immune responses. To identify the underlying mechanisms driving TvEV induced responses, we analyzed the effect of TvEVs on host gene expression. RNA-seq analyses revealed that TvEVs have a suppressive effect on host cell innate immune gene expression. Interestingly, interferon epsilon (IFNε), a cytokine which is constitutively expressed by vaginal epithelial cells, except in peri and post-menopausal women where it is undetectable, is strongly downregulated. Epidemiological studies showing that Tv positivity is unusually high in older women suggest that physiological and immunological changes make the female reproductive tract of older women more conducive to infection. Thus, we tested the effect of pretreatment of host cells with IFNε on Tv infection. We found that pretreatment is protective against Tv killing of host cells, which may, at least in part, explain why older women are more susceptible to Tv infection. We also showed that TvEVs block IFNε-mediated nuclear translocation of transcription factors pSTAT1 and pSTAT2 and increase host cell killing and parasite burden in vitro. Further studies will elucidate how IFNε protects against Tv infection and how TvEVs downregulate IFNε to promote infection.

The parasite plays critical roles in the training and development of major components of the host’s innate and adaptive immune system. Stimulation with Trichomonas vaginalis-derived secretory products (TvSP), which contains LTB₄, induces NOX2 activation and ROS generation in human mast cells through BLT1-mediated signaling. NOX2 activity is known to be regulated by the proteasomal degradation of NOX2. Here, we investigated whether proteasome-ubiquitin pathway is associated with maturation of NOX2 and its trafficking to the cell surface in TvSP- or LTB₄-stimulated human mast cells. Stimulation with TvSP or LTB₄ for 30 min increased matured form of NOX2 protein and its surface trafficking, which was prevented by pretreatment of cells with BLT1 antagonist. When HMC-1 cells were stimulated with TvSP or LTB₄ for up to 30 min, ubiquitinated proteins and proteasome activity were significantly reduced compared with results for cells incubated with medium alone. Furthermore, treatment of quiescent cells with proteasome inhibitor alone augmented expression of NOX2 and its migration to the cell surface. In IP assay, stimulation with TvSP or LTB₄ diminished amount of ubiquitinated NOX2 compared with control. These results suggest that signaling talk between human mast cells and T. vaginalis provides example of BLT1-mediated maturation mechanism of NOX2.

The host immune response to Trichomonas vaginalis involves the chemokine IL-8 production at the site of infection. Previously, we reported that LTB₄ contained in T. vaginalis-derived secretory products (TvSP) play an essential role in IL-8 production in human mast cell via LTB₄ receptor BLT1 or BLT2. Dynamin, a GTPase, is involved in endocytosis of surface receptors for signaling of production of cytokine or chemokines. In this study, we investigated the role of dynamin in the BLT-dependent IL-8 production induced by T. vaginalis-secreted LTB_4. Pretreatment of HMC-1 cells with a dynamin inhibitor reduced BLT-dependent IL-8 production and NF-kB phosphorylation induced by TvSP or LTB₄. In addition, the exposure of HMC-1 cells with TvSP or LTB₄ for up to 60 min, BLT1 translocated from intracellular compartments to the plasma membrane at 30 minutes. At 60 min after stimulation with TvSP, BLT1 migrated from the cell surface to the intracellular areas. In dynamin-2 siRNA-knockdown cells, TvSP-induced BLT1 internalization was nicely prevented. In co-IP experiment, we also found that dynamin-2 was physically interacted with BLT1 at 60 min after stimulation with TvSP or LTB₄. These results suggest that T. vaginalis-secreted LTB₄ induces IL-8 production via dynamin-mediated endocytosis of BLT1 in HMC-1 cells.

Lucilia cuprina dorsalis is a parasitic blowfly that causes flystrike (myiasis) in many countries worldwide. In Australasia alone, flystrike costs in excess of $320 million each year resulting from production losses and costs of control that rely heavily on the use of insecticides. As a result, resistance in blowfly to almost all insecticides in current use will require the development of further intervention strategies. In contrast, the closely related subspecies, Lucilia cuprina cuprina, is primarily found in urban areas and exhibits limited parasitic behavior. In the present study, the nuclear genomes of Lucilia cuprina dorsalis (Australia) and Lucilia cuprina cuprina (USA) were sequenced and annotated. Comparative genomic analyses revealed important differences in blowfly biology, host interactions and insecticide resistance between the two subspecies. This comparative data will form the basis for downstream analyses investigating novel blowfly specific gene targets for the design of new methods for improved control of flystrike.

Haemaphysalis longicornis is an important vector of several bacterial, protozoal and viral pathogens, and is distributed in Australasia, eastern Asia and the USA. We systematically reviewed the literature on the microbiome of H. longicornis using PRISMA guidelines, with an inclusion criterion focused on studies employing next-generation sequencing techniques. We identified 46 studies from four databases (Web of Science, PubMed, Scopus and CAB Direct) published from six countries. These studies primarily characterised the bacterial components of the tick microbiome, detecting potentially pathogenic bacteria such as Anaplasma, Borrelia, Bartonella, Coxiella, Ehrlichia, Francisella, and Rickettsia as well as endosymbionts, including Arsenophonus-like, Coxiella-like and Rickettsia-like organisms. The virome studies revealed a diverse array of novel viruses, enhancing our understanding of the viral diversity within this tick species. Moreover, few studies have reported significant changes in the microbiome composition due to tick life stage, feeding status and infection. Our findings suggest that the microbiome of H. longicornis plays a potential role in the tick’s life cycle and its vectorial capability. Future investigations should encompass the “non-pathogen” microbiome components of H. longicornis to understand their role in tick biology and the transmission of pathogens to develop sustainable strategies for controlling ticks and tick-borne diseases.

Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). While fluralaner is a promising new treatment, the ‘spot-on’ formulation of fluralaner can limit in-situ treatment delivery. Dilution to a volume for ‘pour-on’ delivery is one solution. This study investigated the acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. In vitro assays determined the lethal concentration (LC₅₀) and survival time of the mites when exposed to test solutions. All compounds were highly effective at killing mites in vitro. The LC₅₀ values of Bravecto, Orange Power, limonene and citral at 1 hour were 14.61 mg/mL, 4.50%, 26.53% and 0.76%, respectively. A pilot survival assay of mites collected from a mange affected wombat showed survival times of <10 minutes when exposed to Bravecto and Orange Power, and 20 minutes when exposed to moxidectin. These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. Further research evaluating the physiochemical properties and modes of action of Orange Power constituents would be of value.

Native brown kiwi (Apteryx mantelli) chicks in conservation captive rearing programmes commonly suffer from coccidiosis, a parasitic disease that can cause significant morbidity and mortality in young birds. Disease is currently managed in captivity through a combination of preventative husbandry and medication (coccidiocides). However, recent evidence suggests that the efficacy of the primary drug used to treat coccidia (toltrazuril) is declining (Taylor, 2019) and there is a lack of information about safe and effective alternative therapeutics.

The aim of this study was to try and identify some suitable alternate options for treating coccidiosis in kiwi. During the 2023-24 breeding season we trialled one of the potential alternate treatments, diclazuril, as a prophylactic treatment for Operation Nest Egg (ONE) brown kiwi chicks in captive rearing facilities. In addition to standard oocyst reduction counts, we used a recently developed molecular tool (to assess species composition) and sporulation tests (to test oocyst viability).

The results of this efficacy study will be discussed, as well as some of the unique aspects of working with wildlife in a controlled clinical study design, such as limited sample size and need to modify protocols to gather accurate data whilst maintaining health and welfare of this rare species.

Intestinal parasites are one of the main threats that affect the health of the host and its ability to produce. Falcons are considered an innate wealth of high value in various parts of the world and in Saudi Arabia in particular. This study aimed to detect the prevalence of intestinal parasites in falcons, where 125 stool samples from different types of falcons were examined by direct fecal smear method. The examination revealed that 87.2% were infected with intestinal parasites. The overall parasite prevalence was Serratospiculum seurati 47.2%, Caryospora spp. 46.4%, Strigea falconis 6.4%, Capillaria columbae 1.6%, tapeworms 5.6%, Ascaridia spp. 0.8%, and Giardia 0.8% in the infected falcons. Infection samples were re-examined by direct fecal stain and centrifugal floatation to compare the best way for the fecal examination which appeared that the direct fecal smear is one of the most effective methods for the detection of parasites. Overall, Preserving the safety of the environment and the food provided to falcons is essential for their health. Falcons are highly sensitive to environmental changes and can be easily affected by parasites, which can cause serious health issues and even death in some cases.

Two mortality events in a colony of Tarāpuka (black-billed gulls, Chroicocephalus bulleri), an 'at risk-declining' and endemic species in Aotearoa occurred in 2023 and 2024. In a colony of 500 adult and 200 juvenile birds, nesting near the Māngere Bridge in Auckland, an estimated 160 juveniles died within a period of a few days. A collaborative effort from various wildlife agencies across Aotearoa identified the disease agent as a schistosome trematode, Austrobilharzia terrigalensis. In this talk, I'll chat about what we know about this parasite, how it got to New Zealand, into our gulls and what it means for their conservation and the surrounding ecosystem.