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Abstract Onchocerca lupi is a zoonotic filarioid parasite of dogs and cats with widespread distribution. A specific non-invasive diagnostic assay for the detection of O. lupi infections remains unavailable. This study aimed to assess the accuracy, specificity, and sensitivity of an ELISA test designed using nine peptides from two O. lupi proteins. Sera (n = 54) collected from O. lupi infected dogs from endemic areas (Portugal and USA), alongside sera from dogs positive for Dirofilaria immitis, D. repens, Cercopithifilaria bainae, and Acanthocheilonema reconditum (n = 53) from a non-endemic area for O. lupi, as well as from helminth-free dogs (n = 60), were tested. The checkerboard titration method was applied for the optimization of peptide concentrations and conjugate anti-dog dilutions. Sensitivity, specificity, and optimal cut-off values were calculated using ROC curve analysis. All peptides reacted against sera of O. lupi, with no correlation between optic density (OD) values and microfilariae (mfs) loads. Sensitivity and specificity values ranging from 85.45 to 100%, and 88.89% to 100%, respectively, were recorded for all peptides examined, with 100% specificity and sensitivity observed for peptides 40_3, 40_5, 130_3, 120_3 and 40_1, 130_5, respectively. The maximum cut-off value was observed for peptides 40_5 (0.765) and 40_3 (0.708). Testing of sera from dogs positive for other filarioids resulted in lower OD values (up to 1.565) for peptides 40_3 and 40_5 when compared with O. lupi (up to 2.929). The availability of this assay will be of value in epidemiological studies of canine O. lupi infection in both endemic and non-endemic areas, and in assessing the risk for zoonotic transmission.

Abstract Background The microbiome is known to play key roles in health and disease, including host susceptibility to parasite infections. The freshwater snail Galba truncatula is the intermediate host for many trematode species, including the liver and rumen flukes Fasciola hepatica and Calicophoron daubneyi, respectively. The snail-parasite system has previously been investigated. However, the specific interaction between the snail-associated microbiota and intra-snail developmental stages of trematodes has yet to be explored. Methods Galba truncatula snails were collected from farms in Northern Ireland and trematode infection was diagnosed using PCR. High-throughput sequencing analysis of the bacterial 16S ribosomal DNA V3-V4 hypervariable regions was subsequently applied to characterise the microbiota of both uninfected and infected snails. Results We first showed that the snail harboured microbiota that was distinct for its environment. The microbiota of infected snails was found to differ significantly from that of uninfected snails. In particular, the bacterial genera Mycoplasma and Methylotenera were significantly more abundant in infected snails, while genera Sphingomonas and Nocardioides were predominantly associated with uninfected snails. Conclusion These findings pave the way to future studies on the functional roles of bacteria in host-parasite relationships. Graphical Abstract

Toxoplasma gondii is a zoonotic parasite able of infecting all warm-blooded animals. Toxoplasmosis is one of the major foodborne diseases globally. The consumption of wild boar (Sus scrofa) meat from recreational hunting has been linked to outbreaks of human toxoplasmosis. The island of Sardinia (Italy) contains a large wild boar population, thus providing an opportunity to assess the distribution of Toxoplasma in this species and the associated risks of transmission to humans. A total of 562 wild boars were screened: heart and meat juice samples were tested for T. gondii DNA via nested-PCR and IgG anti-Toxoplasma by commercial ELISA. Anti-Toxoplasma IgG were detected in 24.6% (138/562) of animals, while 37.2% (209/562) of the heart samples were PCR positive. The prevalence of T. gondii antibodies and DNA highlights the potential role of wild boar as an important reservoir for this parasite. The study suggests that wild boar could play a significant role in spreading the parasite to humans. As wild boar numbers are increasing throughout their range, their potential role in transmitting toxoplasmosis should be communicated to stakeholders, and the impact of different population control methods on disease transmission should be thoroughly assessed to mitigate potential threats effectively.

Abstract Background Cystic echinococcosis (CE) is a widespread zoonosis and a significant economic concern and cause of morbidity in humans. A scarcity of education on the sources of CE infection and containment measures is considered to be a key factor responsible for persistent transmission within communities. Recently, edutainment approaches have captured the attention of health education (HE) professionals due to the benefits of integrating cognitive and emotional learning processes. Methods A study was carried out in Sardinia, Italy, between 2020 and 2022, amid the SARS-Covid-19 pandemic. The project, designed to involve primary school children (via remote or face-to-face learning depending on the evolving Covid-19 containment measures) consisted of four distinct phases: (i) creation of material for school children and teachers focused on cystic echinococcosis; (ii) pre-intervention evaluation of CE knowledge (i.e. True False Don’t Know [TFD] pre-intervention questionnaire based on CE-related knowledge and practices); (iii) edutainment activity (e.g. interactive lessons enhanced by the comic booklet and the “Fight the parasite” cartoon video, hands-on educational activities and drawing activities on CE); and (iv) post-intervention evaluation of CE knowledge (via TFD post-intervention questionnaire [same questionnaire as used for the pre-intervention assessment] on CE-related knowledge and practices) and on-site edutainment tour in primary schools taking part to the project. Results The percentage of correct answers increased from 65% for the questionnaire administered pre-intervention to 87.9% for the same questionnaire administered post-intervention (χ 2 = 648.12, df = 1, P

Abstract Background Over the past decade, evidence has emerged of the ability of gastrointestinal (GI) helminth parasites to alter the composition of the host gut microbiome; however, the mechanism(s) underpinning such interactions remain unclear. In the current study, we (i) undertake proteomic analyses of the excretory-secretory products (ESPs), including secreted extracellular vesicles (EVs), of the ‘brown stomach worm’ Teladorsagia circumcincta, one of the major agents causing parasite gastroenteritis in temperate areas worldwide; (ii) conduct bioinformatic analyses to identify and characterise antimicrobial peptides (AMPs) with putative antimicrobial activity; and (iii) assess the bactericidal and/or bacteriostatic properties of T. circumcincta EVs, and whole and EV-depleted ESPs, using bacterial growth inhibition assays. Methods Size-exclusion chromatography was applied to the isolation of EVs from whole T. circumcincta ESPs, followed by EV characterisation via nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of EVs and EV-depleted ESPs was conducted using liquid chromatography-tandem mass spectrometry, and prediction of putative AMPs was performed using available online tools. The antimicrobial activities of T. circumcincta EVs and of whole and EV-depleted ESPs against Escherichia coli were evaluated using bacterial growth inhibition assays. Results Several molecules with putative antimicrobial activity were identified in both EVs and EV-depleted ESPs from adult T. circumcincta. Whilst exposure of E. coli to whole ESPs resulted in a significant reduction of colony-forming units over 3 h, bacterial growth was not reduced following exposure to worm EVs or EV-depleted ESPs. Conclusions Our data points towards a bactericidal and/or bacteriostatic function of T. circumcincta ESPs, likely mediated by molecules with antimicrobial activity. Graphical Abstract

Abstract Gastrointestinal (GI) helminth infections cause significant morbidity in both humans and animals worldwide. Specific and sensitive diagnosis is central to the surveillance of such infections and to determine the effectiveness of treatment strategies used to control them. In this article, we: (i) assess the strengths and limitations of existing methods applied to the diagnosis of GI helminth infections of humans and livestock; (ii) examine high-throughput sequencing approaches, such as targeted molecular barcoding and shotgun sequencing, as tools to define the taxonomic composition of helminth infections; and (iii) discuss the current understanding of the interactions between helminths and microbiota in the host gut. Stool-based diagnostics are likely to serve as an important tool well into the future; improved diagnostics of helminths and their environment in the gut may assist the identification of biomarkers with the potential to define the health/disease status of individuals and populations, and to identify existing or emerging anthelmintic resistance. Graphical Abstract

Increasing evidence shows that the host gut microbiota might be involved in the immunological cascade that culminates with the formation of tissue granulomas underlying the pathophysiology of hepato-intestinal schistosomiasis. In this study, we investigated the impact of Schistosoma mansoni infection on the gut microbial composition and functional potential of both wild type and microbiome-humanized mice. In spite of substantial differences in microbiome composition at baseline, selected pathways were consistently affected by parasite infection. The gut microbiomes of infected mice of both lines displayed, amongst other features, enhanced capacity for tryptophan and butyrate production, which might be linked to the activation of mechanisms aimed to prevent excessive injuries caused by migrating parasite eggs. Complementing data from previous studies, our findings suggest that the host gut microbiome might play a dual role in the pathophysiology of schistosomiasis, where intestinal bacteria may contribute to egg-associated pathology while, in turn, protect the host from uncontrolled tissue damage.