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Gastrointestinal nematode (GIN) infections are ubiquitous and often cause morbidity and reduced performance in livestock. Emerging anthelmintic resistance and increasing change in climate patterns require evaluation of alternatives to traditional treatment and management practices. Mathematical models of parasite transmission between hosts and the environment have contributed towards the design of appropriate control strategies in ruminants, but have yet to account for relationships between climate, infection pressure, immunity, resources, and growth. Here, we develop a new epidemiological model of GIN transmission in a herd of grazing cattle, including host tolerance (body weight and feed intake), parasite burden and acquisition of immunity, together with weather-dependent development of parasite free-living stages, and the influence of grass availability on parasite transmission. Dynamic host, parasite and environmental factors drive a variable rate of transmission. Using literature sources, the model was parametrised for Ostertagia ostertagi, the prevailing pathogenic GIN in grazing cattle populations in temperate climates. Model outputs were validated on published empirical studies from first season grazing cattle in northern Europe. These results show satisfactory qualitative and quantitative performance of the model; they also indicate the model may approximate the dynamics of grazing systems under co-infection by O. ostertagi and Cooperia oncophora, a second GIN species common in cattle. In addition, model behaviour was explored under illustrative anthelmintic treatment strategies, considering impacts on parasitological and performance variables. The model has potential for extension to explore altered infection dynamics as a result of management and climate change, and to optimise treatment strategies accordingly. As the first known mechanistic model to combine parasitic and free-living stages of GIN with host feed-intake and growth, it is well suited to predict complex system responses under non-stationary conditions. We discuss the implications, limitations and extensions of the model, and its potential to assist in the development of sustainable parasite control strategies.

The identification of gastrointestinal helminth infections of humans and livestock almost exclusively relies on the detection of eggs or larvae in faeces, followed by manual counting and morphological char-acterisation to differentiate species using microscopy-based techniques. However, molecular approaches based on the detection and quantification of parasite DNA are becoming more prevalent, increasing the sensitivity, specificity and throughput of diagnostic assays. High-throughput sequencing, from single PCR targets through to the analysis of whole genomes, offers significant promise towards providing information-rich data that may add value beyond traditional and conventional molecular approaches; however, thus far, its utility has not been fully explored to detect helminths in faecal samples. In this study, low-depth whole genome sequencing, i.e. genome skimming, has been applied to detect and char-acterise helminth diversity in a set of helminth-infected human and livestock faecal material. The strengths and limitations of this approach are evaluated using three methods to characterise and differ-entiate metagenomic sequencing data based on (i) mapping to whole mitochondrial genomes, (ii) whole genome assemblies, and (iii) a comprehensive internal transcribed spacer 2 (ITS2) database, together with validation using quantitative PCR (qPCR). Our analyses suggest that genome skimming can success-fully identify most single and multi-species infections reported by qPCR and can provide sufficient cov-erage within some samples to resolve consensus mitochondrial genomes, thus facilitating phylogenetic analyses of selected genera, e.g. Ascaris spp. Key to this approach is both the availability and integrity of helminth reference genomes, some of which are currently contaminated with bacterial and host sequences. The success of genome skimming of faecal DNA is dependent on the availability of vouchered sequences of helminths spanning both taxonomic and geographic diversity, together with methods to detect or amplify minute quantities of parasite nucleic acids in mixed samples.(c) 2023 The Author(s). Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Pianosa is a 10 km(2) Italian island in the Tyrrhenian Sea which is part of the Tuscan Archipelago National Park. In this island lives a brown hare population which, according to the literature, belongs to the ancestral taxon Lepus europaeus meridei that offers a unique opportunity to observe how the parasite biocoenosis shapes in condition of isolation, limited space availability and high population density. The aim of this work is to describe the helminth component community of a non-managed, isolated, and dense hare population, evaluating host-parasite relationship and parasite community structure. All 26 analyzed hares (13 males and 13 females) were in good physical conditions, and all of them harboured exclusively the nematode Protostrongylus oryctolagi only. This is the first report of this lungworm species in Italy. The estimated overall abundance was 48.15 worms per examined hare (range 3-258, median 50) and the parasites were unevenly distributed across host population, with few hosts having most parasites (aggregated or overdispersed distribution). No significant relationship was detected between the number of isolated parasites and hare sex and weigh. The effect of the isolation of Pianosa's hare population seems to have acted reducing parasite richness, while the high host density is probably the cause of the high prevalence and abundance of the single helminth species collected. In conclusion, despite the low impact of parasites confirmed also by the overdispersed parasite distribution, the low diversity of the studied parasite community sounds a warning for the management of the hare population and the whole Pianosa's ecosystem.

Helminths have co-evolved alongside their hosts for millions of years and haveadapted eloquent mechanisms that allow them to reside in the host withoutcausing significant pathology, or elimination. The ability of these parasites tomanipulate their specialised host is reflected by their continued persistence asa global health concern, with ~1 billion people infected with soil transmittedhelminths (STHs). Helminth infections have long been associated with reducedallergic and autoimmune diseases leading to the hypothesis that helminthsuppress the host immune system, and this has been confirmed in both animalmodels and controlled human infection studies. Many studies have shown thatmany suppressive effects of infection on the host immune system can beattributed to helminth excreted/secreted products (ES). A small but growing listof individual molecules from helminth ES have been characterised, and themechanism of action elucidated. For example, multiple helminth species havebeen identified to secrete TGFβ mimic proteins that can bind host TGFβreceptor and induce T-regulatory (Treg) immunosuppressive cells. However,the full repertoire of helminth secreted molecules that modulate the host ishypothesised to be far from complete. Our lab discovered the presence of extracellular vesicles (EVs) within ES fromthe mouse infective helminth Heligmosomoides polygyrus bakeri (H. bakeri).EVs are lipid bilayer enclosed nanoparticles that carry proteins, lipids andnucleic acids and are released ubiquitously by all cells and organisms studiedto date. In mammalian systems EVs provide a mechanism of communicationbetween near or distal cells. In the context of host-pathogen dynamics it isproposed that EVs could play a role in enabling parasites to condition theirenvironment during infection. There is mounting evidence of host-parasite EVmediated modulation occurrence between plants and colonising fungal cells,bacteria and mammalian host cells, and several parasites and theirmammalian hosts including several helminth species. During H. bakeri infection host immune suppression is thought to primarilyoccur during the adult stage of infection when the parasite resides in the lumenof the duodenum in close proximity to the intestinal epithelium. The intestinalepithelium plays an integral role both in helminth detection, and in mediatingparasite clearance. Therefore, I hypothesised that adult H. bakeri EVs targetthe intestinal epithelium and directly modulate this tissue. The goal of thisthesis was to determine the role of helminth EVs in infection dynamics andhost modulation in the intestinal epithelium. I aimed to address whether H.bakeri EVs enter the intestinal epithelium, whether uptake is targeted to aspecific cell type and how these effects the function of this tissue usingintestinal organoid models. To address these aims development and refinement of methods for high purityEV preparations and EV labelling was required in order to directly implicate H.bakeri EVs as the causative agent in host responses. In Chapter 3, I comparedvarious combinational approaches to EV isolation and improved the purity ofour EV and EV depleted HES preparations. I then assessed EV preparationsusing cryoEM which furthered our understanding of the morphology anddiversity of H bakeri secreted EVs. I trialled multiple labelling methodologiesand found a low-background labelling method that allowed high confidenceidentification of uptake for subsequent chapters. However, I later discoveredthat the majority of labelling techniques trialled had variable labelling efficiencywith low proportions of EVs labelled; this is a caveat to consider wheninterpreting results using labelled EVs. To understand how H. bakeri EVs interact with the intestinal epithelium Ideveloped methods to grow small intestinal 2-D organoids (enteroids) whichare in vitro cultures that reconstitute the intestinal epithelium (Chapter 4). 2-Denteroids have greater cellular complexity as compared to a homogenous cellline and allow us to address the question of cell type specificity for uptake. 2-D enteroid cultures maintained cellular polarisation and differentiated into 6-7major cell types of the intestinal epithelium. In Chapter 5, I demonstrated by using fluorescently labelled EVs that H. bakeriEVs enter organoid cells, however at a lower proportion than I see side-by-side for our cell line cultures. This led to the hypothesis that H. bakeri EVscould target specific cellular populations within the intestinal epithelium. Toidentify whether uptake of H. bakeri EVs occurs in a targeted fashion byspecific cell types I performed microscopy experiments aiming to co-localiseEVs with certain cell types. Microscopy approaches did not provide a definitiveanswer to the question of whether uptake is cell type specific. Next, I modifiedthe cellular proportion of our 2-D enteroids to identify whether this altered theproportion of EV uptake. Goblet and tuft cells are specialised cells of theepithelium that are strongly induced during helminth infection and mediatehelminth clearance; I reasoned that H. bakeri EVs may specifically enter andmodulate these cell types. Organoid cultures that were enriched in goblet andtuft cells showed no enhanced ability to take up EVs, suggesting that neithergoblet nor tuft cells are specifically targeted over other cell types; however, thisdata does not rule out that H. bakeri EVs can enter these cell types andmodulate them. Whether cell type specificity exists for the uptake of H. bakeriEVs within the intestinal epithelium remains unclear and is still an active areaof investigation. To understand how EV treatment of 2-D enteroids altered host geneexpression in Chapter 6, I performed RNA sequencing (RNA seq) andcharacterised the transcriptional changes within 2-D enteroids to H. bakeri EVsor EV depleted ES after 24 h. Genes critical for maintenance of stem cells, cellcycle and antimicrobial defence were downregulated by H. bakeri EVs. Withinthe intestinal epithelium only a proportion of the cells are mitotic, thereforechanges in cell cycle suggest a modulation of either stem cells of Transit-amplifying (TA) cells. I also identified several changes in cell type restricted genes expressed specifically by stem cells, Paneth cells, TA cells orEnteroendocrine cells (EECs). I now hypothesise EVs specifically modify thesecell types. To define the cell type specific responses after EV or EV depletedES treatment I performed single cell RNA seq, unfortunately the quality of ourcontrol sample made interpreting these results difficult. However, these dataserve as conformation of the cellular composition of our 2-D enteroids model. In addition, I also utilised our 2-D organoid model to perform novel co-culturedexperiments with live adult or larval stage 4 (L4) H. bakeri and performedtranscriptional analysis of the host epithelium under these conditions. Thesedata allow us to uncouple the impact of infection with whole parasites on theintestinal epithelium from any immune driven changes in the epithelium thatoccur in vivo. These data also serve as a comparison between host effectsattributed specifically to H. bakeri EVs, and changes induced by the wholeparasite. In summary, this thesis contributes new knowledge to our understanding of H.bakeri interactions with the intestinal epithelium in the absence of host immunedriven responses and distinguishes the role of secreted H. bakeri EVs inmodulating this tissue. I determined that H. bakeri EVs enter host epithelialcells in 2-D enteroids, but whether this is specifically targeted to certainsubpopulations remains elusive. I characterised the host gene expressionchanges upon H. bakeri EV treatment in 2-D enteroids, these findings furtherour understanding as a field of which host genes and pathways are targetedby H. bakeri. In the future, this thesis along with continued research, couldhave important implications for helminth eradication. Conversely, where H.bakeri EVs suppress specific genes or pathways involved in diseases of theintestinal epithelium such as ulcerative colitis, Crohn's disease, oradenocarcinoma, they could provide novel strategies for therapeutics.

Durrington Walls was a large Neolithic settlement in Britain dating around 2500 BCE, located very close to Stonehenge and likely to be the campsite where its builders lived during its main stage of construction. Nineteen coprolites recovered from a midden and associated pits at Durrington Walls were analysed for intestinal parasite eggs using digital light microscopy. Five (26%) contained helminth eggs, 1 with those of fish tapeworm (likely Dibothriocephalus dendriticus) and 4 with those of capillariid nematodes. Analyses of bile acid and sterol from these 5 coprolites show 1 to be of likely human origin and the other 4 to likely derive from dogs. The presence of fish tapeworm reveals that the Neolithic people who gathered to feast at Durrington Walls were at risk of infection from eating raw or undercooked freshwater fish. When the eggs of capillariids are found in the feces of humans or dogs it normally indicates that the internal organs (liver, lung or intestines) of animals with capillariasis have been eaten, and eggs passed through the gut without causing disease. Their presence in multiple coprolites provides new evidence that internal organs of animals were consumed. These novel findings improve our understanding of both parasitic infection and dietary habits associated with this key Neolithic ceremonial site.

Wastewater treatment is a widely used health protection measure that can be applied to control the transmission of wastewater-related infectious diseases to communities exposed to wastewater. This study determined the efficiency of three full-scale constructed wetlands (CWs) in removing pathogenic parasites and fecal coliform (FC) bacteria from wastewater. Wastewater samples were collected from three CW systems located in the Dar es Salaam and Iringa regions of Tanzania. The modified Bailenger and modified Ziehl-Neelsen stain techniques were used to detect and quantify parasites. The membrane filtration method was used to detect and quantify FC bacteria. Data were analysed using IBM SPSS version 20. Helminth (Ascaris lumbricoides, hookworm, and Taenia spp.) eggs were completely removed by two CW systems. In all the systems, the removal of protozoa ranged from 99.8% to 100%. The mean concentrations of FCs in effluents ranged from 5 to 6 log units/100 mL. Effluents of all CW systems met the recommended parasitological quality requirements of the World Health Organization for the safe reuse of wastewater. FC effluents concentrations did not meet the local discharge standards of the Tanzania Bureau of Standards. Therefore, improvement to the CWs’ design, operation, and maintenance are required for the efficient removal of bacteria. Keywords: helminth, protozoa, fecal coliform, wastewater treatment, constructed wetland

Background Schistosomiasis is a major neglected tropical disease that affects up to 250 million individuals worldwide. The diagnosis of human schistosomiasis is mainly based on the microscopic detection of the parasite’s eggs in the feces (i.e., for Schistosoma mansoni or Schistosoma japonicum) or urine (i.e., for Schistosoma haematobium) samples. However, these techniques have limited sensitivity, and microscopic expertise is waning outside endemic areas. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become the gold standard diagnostic method for the identification of bacteria and fungi in many microbiological laboratories. Preliminary studies have recently shown promising results for parasite identification using this method. The aims of this study were to develop and validate a species-specific database for adult Schistosoma identification, and to evaluate the effects of different storage solutions (ethanol and RNAlater) on spectra profiles. Methods Adult worms (males and females) of S. mansoni and S. japonicum were obtained from experimentally infected mice. Species identification was carried out morphologically and by cytochrome oxidase 1 gene sequencing. Reference protein spectra for the creation of an in-house MALDI-TOF MS database were generated, and the database evaluated using new samples. We employed unsupervised (principal component analysis) and supervised (support vector machine, k-nearest neighbor, Random Forest, and partial least squares discriminant analysis) machine learning algorithms for the identification and differentiation of the Schistosoma species. Results All the spectra were correctly identified by internal validation. For external validation, 58 new Schistosoma samples were analyzed, of which 100% (58/58) were correctly identified to genus level (log score values ≥ 1.7) and 81% (47/58) were reliably identified to species level (log score values ≥ 2). The spectra profiles showed some differences depending on the storage solution used. All the machine learning algorithms classified the samples correctly. Conclusions MALDI-TOF MS can reliably distinguish adult S. mansoni from S. japonicum. Graphical Abstract

Currently, the incidence of helminthiasis among children is still gaining high indicators. The frequent spread of helminthiasis among children is often a sign of weak immunity, as well as frequent morbidity. The spread of helminthiasis among the child population is a problem for the field of medicine. According to the World Health Organization, helminthiasis is an annual disease-causing damage to the population. In addition, the importance of this disease in pediatric practice is its rapid prevalence as well as its significant impact on the child population. most often, helminthiasis is masked under other diseases since the child has an allergic reaction. Parasitic diseases are directly related to household infotainment and the behaviour of residents, sanitary and hygienic and natural conditions. The most common type of helminthiasis is enterobiasis. The mechanism of spread of this helminthiasis in most cases depends on the violation of sanitary and hygienic conditions, as well as on areas located in low epidemiological conditions. On the one hand, an indicator for assessing the incidence of enterobiasis at a high level indicates the incidence of helminthiasis in this population. According to the conclusions of our review, the problem indicates that it is worth reviewing the prevention of helminthiasis, В настоящее время частота встречаемости гельминтоза среди детей остается высокой. Частое распространение гельминтов среди детей часто является признаком слабого иммунитета, а также частой заболеваемости. Распространение гельминтозов среди детского населения является проблемой для сферы медицины. По данным Всемирной Организации здравоохранения ежегодно среди болезней наносящий ущерб населению является гельминтозы. Кроме того, важность этого заболевания в педиатрической практике является быстрая распространенность а также значительное влияние на детское население. Чаще всего гельминтоз маскируются под другими заболеваниями, так как у ребенка появляется аллергическая реакция. Паразитарные заболевания это напрямую связано с бытовым инфразвуком и поведением населения, санитарно-гигиеническими и природными условиями. Наиболее распространенной формой среди гельминтозов является энтеробиоз. Механизм распространения этого гельминтоза во многом зависит от нарушения санитарно-гигиенических условий, а также от районов, расположенных в эпидемиологически низких условиях. С одной стороны, высокий уровень энтеробиоза является индикатором оценки заболеваемости что свидетельствует о заболеваемости населения гельминтозом. По выводам нашего обзора проблема указывает на то, что стоит пересмотреть профилактику гельминтозов.

Anthelmintic resistance (AR) has thus far only rarely been reported for intestinal helminths of dogs and cats, in contrast to parasites of livestock and horses. We highlight possible reasons for this striking and important discrepancy, including ecological, biological and genetic factors and/or intervention regimens of key intestinal helminths concerning both host groups. In view of the current knowledge related to the genetics, mechanisms and principles of AR development, we point at issues which in our view contribute to a comparatively lower risk of AR development in intestinal helminths of dogs and cats. Finally, we specify research needs and provide recommendations by which, based on the available information about AR in ruminant and equine helminths, the development of AR in dog and cat helminths may best be documented, prevented or at least postponed., Graphical abstract Image 1, Highlights • Identifying causes of differences in anthelmintic resistance between livestock and dogs & cats. • Discussion of roles of AR mechanisms, genetics and husbandry in delayed AR evolution in dogs & cats. • Research needs for improved tools to monitor AR development in dogs & cats.

Introduction: This paper provides an overview of the current immunologic research findings of lymphatic filarial worms, which affect millions of people worldwide. Objectives: This paper aims to discuss the immunologic features of lymphatic filarial worms. It also aims to highlight their potential anti-inflammatory actions and the use of anti-filarial drugs against COVID-19. Methods: A literature review was performed to obtain insights on the immunologic features of lymphatic filarial worms. Results: The CD4+ (Th2) response profile is the main defence against filarial worms. Modulation of human immune responses are primarily mediated by parasite-secreted peptides. Lymphatic filarial worms have anti-inflammatory potentials. Drug repurposing of diethylcarbamazepine, doxycycline, and ivermectin can be looked upon against COVID-19. Conclusion: Lymphatic filarial worms have several immunologic effects on host immune systems, which promote chronic infection and curtail anti-inflammatory responses. Insights in this paper can serve as a guide for the understanding of immunologic aspects of lymphatic filarial worms.

This study examined the prevalence and intensity of intestinal helminth infections and their association with anemia, undernutrition, and academic performance among school children in Maksegnit, northwestern Ethiopia. A total of 1205 school children, who attended Maksegnit Number Two Elementary School between May and July 2021, participated in this study. To determine helminth infection status, two thick Kato–Katz slides were examined for each child. Hemoglobin level was measured using a HemoCue machine. Academic performance was assessed using the mean score of all subjects children have taken for the Spring 2020/2021 academic term. Out of 1205 children examined, 45.4% were infected with at least one helminth species, 7.9% were anemic, and 35.8% were undernourished. The means for hemoglobin level and z-scores of weight for age, height for age, body mass index for age, and academic scores were lower among helminth-infected children than the uninfected. Children infected with intestinal helminths showed higher odds of anemia than those uninfected with helminths. In conclusion, there was a moderate prevalence of intestinal helminth infection and undernutrition among school children in Maksegnit. Intestinal helminth infection could increase the risk of anemia, undernutrition, and poor academic performance.

Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminth-derived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum, were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)-induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS-induced levels of tumour necrosis factor (TNF-α) in a dose-dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone.

Diabetes is recognised as the world's fastest growing chronic condition globally. Helminth infections have been shown to be associated with a lower prevalence of type 2 diabetes (T2D), in part due to their ability to induce a type 2 immune response. Therefore, to understand the molecular mechanisms that underlie the development of T2D-induced insulin resistance, we treated mice fed on normal or diabetes-promoting diets with excretory/secretory products (ES) from the gastrointestinal helminth Nippostrongylus brasiliensis. We demonstrated that treatment with crude ES products from adult worms (AES) or infective third-stage larvae (L3ES) from N. brasiliensis improved glucose tolerance and attenuated body weight gain in mice fed on a high glycaemic index diet. N. brasiliensis ES administration to mice was associated with a type 2 immune response measured by increased eosinophils and IL-5 in peripheral tissues but not IL-4, and with a decrease in the level of IL-6 in adipose tissue and corresponding increase in IL-6 levels in the liver. Moreover, treatment with AES or L3ES was associated with significant changes in the community composition of the gut microbiota at the phylum and order levels. These data highlight a role for N. brasiliensis ES in modulating the immune response associated with T2D, and suggest that N. brasiliensis ES contain molecules with therapeutic potential for treating metabolic syndrome and T2D. This research was supported by a program grant from the National Health and Medical Research Council (1132975), and a strategic research initiative award from the Australian Research Council to The Australian Institute of Tropical Health and Medicine at James Cook University (SRI40200003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Many helminth life cycles, including hookworm, involve a mandatory lung phase, where myeloid and granulocyte subsets interact with the helminth and respond to infection-induced lung injury. To evaluate these innate subsets, we employ Nippostrongylus brasiliensis infection of reporter mice for myeloid cells (CX3CR1(GFP)) and granulocytes (PGRP(dsRED)). Nippostrongylus infection induces lung infiltration of reporter cells, including CX3CR1(+) myeloid cells and PGRP(+) eosinophils. Strikingly, CX3CR1(GFP/GFP) mice, which are deficient in CX3CR1, are protected from Nippostrongylus infection with reduced weight loss, lung leukocyte infiltration, and worm burden compared to CX3CR1(+/+) mice. This protective effect is specific for CX3CR1 as CCR2-deficient mice do not exhibit reduced worm burdens. Nippostrongylus co-culture with lung Ly6C(+) monocytes or CD11c(+) cells demonstrates that CX3CR1(GFP/GFP) monocytes secrete more proinflammatory cytokines, and actively bind the parasites causing reduced motility. RNA sequencing of Ly6C(+) or CD11c(+) cells shows Nippostrongylus-induced gene expression changes, particularly in monocytes, associated with inflammation, chemotaxis, and extracellular matrix remodeling pathways. We also identify cytotoxic and adhesion molecules as potential effectors against the parasite, such as Gzma and Gzmb, which are elevated in CX3CR1(GFP/GFP) monocytes. These studies validate a dual innate cell reporter for lung helminth infection and demonstrate that CX3CR1 impairs monocyte-helminth interaction.

Background: Iron deficiency is a dominant source of anaemia in many settings. To evaluate the key cause of anaemia in the study area, the prevalence of anaemia due to major public health diseases was compared with anaemia due to iron deficiency. Methods: Pregnant women were recruited from ante-natal (n=490) and HIV clinics (n=217) with their personal data docu- mented using a questionnaire. Microscopy of Giemsa-stained thick smears was used for detection of malaria parasites while helminths in stools were detected using direct smear method. Haematocrit values were determined by capillary method. Se- rum ferritin levels were determined using enzyme-linked immunosorbent assay. Data was analysed using SPSS version 22.0. Results: The mean age of the recruited women was 28.6±5.4 years old. There were 68.1% cases of anaemia of which 35.5% was due to infections only predominantly HIV and malaria, 14.9% from unknown sources while anaemia due to iron deficiency only was 7.1%. Conclusion: It can safely be inferred that malaria and HIV predispose to anaemia than iron deficiency in the study area. Although pregnant women are dewormed and given IPTp for helminths and malaria treatment respectively, there should be complementary routine malaria screening at ANC visits for those with HCT values

Graphical abstract, Highlights • Current vaccine candidates against helminth infection have shown limited success. • Helminths release extracellular vesicles (EVs) which act on host cells and are a rich source of antigens for new vaccines. • The biogenesis, release and immunomodulatory functions of helminth EVs are reviewed. • Utilisation of EVs in vaccine generation are discussed, including potential antigens and routes of delivery., The hunt for effective vaccines against the major helminth diseases of humans has yet to bear fruit despite much effort over several decades. No individual parasite antigen has proved to elicit full protective immunity, suggesting that combinatorial strategies may be required. Recently it has been discovered that extracellular vesicles released by parasitic helminths contain multiple potential immune modulators, which could together be targeted by a future vaccine. Increasing knowledge of helminth extracellular vesicle components, both enclosed by and exposed on the membrane, will open up a new field of targets for an effective vaccine. This review discusses the interactions between helminth extracellular vesicles and the immune system discovered thus far, and the advantages of targeting these lipid-bound packages with a vaccine. In addition, we also comment upon specific antigens that may be the best targets for an anti-helminth vaccine. In the future, extensive knowledge of the parasites' full arsenal in controlling their host may finally provide us with the ideal target for a fully effective vaccine.

Interactions between wildlife and livestock have increased over time with increased anthropogenic pressure on limited available natural habitats. These interactions have resulted in sharing of pathogens between the species resulting in impacting the wild animals’ fitness and reproduction and further influencing their abundance and diversity. The spatial overlap between Swamp Deer and livestock was studied at Jhilmil Jheel Conservation Reserve (JJCR), Uttarakhand and Kishanpur Wildlife Sanctuary (KWLS), Uttar Pradesh in India, having different levels of interaction with livestock. The prevalence, load and commonality of gastro-intestinal parasites in the species was studied through coprological examination. Parasitic ova of Strongyle sp., Trichostrongylus sp., Fasciola sp., and Moniezia sp. Amphistomes were encountered in swamp deer and livestock from both the sites. The parasitic species richness and prevalence however, varied between JJCR and KWLS. The study recorded significant differences between the parasitic load in Swamp Deer with the eggs per gram of 487.5±46.30 at JJCR and 363.64±49.97 at KWLS at varying levels of livestock interactions.

International audience; In the Mekong Delta region of Vietnam, small-scale chicken farming is common. However, high levels of disease or mortality in such flocks impair eco- nomic development and challenge the livelihoods of many rural households. We investigated 61 diseased small-scale flocks (122 chickens) for evidence of infec- tion with 5 bacteria, 4 viruses, and helminths. Sero- logical profiles (ELISA) were also determined against 6 of these pathogens. The aims of this study were the following: (1) to investigate the prevalence of different pathogens and to compare the probability of detection of bacterial pathogens using PCR and culture; (2) to investigate the relationship between detection of or- ganisms in birds’ tissues and the observed morbidity and mortality, as well as their antibody profile; and (3) to characterize risk factors for infection with specific viral or bacterial pathogens. We used PCR to test for viral (viruses causing infectious bronchitis [IB], highly pathogenic avian influenza [HPAI], Newcastle disease, and infectious bursal disease [IBD]) and bacterial pathogens (Mycoplasma gallisepticum, Pas- teurella multocida, Avibacterium paragallinarum, and Ornithobacterium rhinotracheale [ORT]). The latter two were also investigated in respiratory tissues by conventional culture. Colisepticemic Escherichia coli was investigated by liver or spleen culture. In 49 of 61 (80.3%) flocks, at least one bacterial or viral pathogen was detected, and in 29 (47.5%) flocks, more than one pathogen was detected. A. paragallinarum was detected in 62.3% flocks, followed by M. gallisepticum (26.2%), viruses causing IBD (24.6%) and IB (21.3%), septicemic E. coli (14.8%), ORT (13.1%), and HPAI viruses (4.9%). Of all flocks, 67.2% flocks were colonized by helminths. Mortality was highest among flocks infected with HPAI (100%, interquartile range [IQR]: 81.6– 100%) and lowest with flocks infected with ORT (5.3%, IQR: 1.1–9.0%). The results indicated slight agreement (kappa 0.167) between detection by PCR and culture for both A. paragallinarum and ORT, as well as be- tween the presence of cestodes and ORT infection (kappa 5 0.317). Control of A. paragallinarum, viruses causing HPAI, IBD, and IB, M. gallisepticum, and gastrointestinal helminths should be a priority in small- scale flocks.

The tapeworm Hymenolepis diminuta fails to establish in mice. Given the potential for helminth-bacteria interaction in the gut and the influence that commensal bacteria exert on host immunity, we tested if worm expulsion was related to alterations in the gut microbiota. Specific pathogen-free (SPF) mice, treated with broad-spectrum antibiotics, or germ-free wild-type mice were infected with H. diminuta, gut bacterial composition assessed by 16S rRNA gene sequencing, and worm counts, blood eosinophilia, goblet cells, splenic IL-4, -5 and -10, and colonic cytokines/chemokines mRNA were assessed. Effects of a PBS-soluble extract of adult H. diminuta on bacterial growth in vitro was tested. H. diminuta-infected mice displayed increased α and β diversity in colonic mucosa-associated and fecal bacterial communities, characterized by increased Lachnospiraceae and clostridium cluster XIVa. In vitro analysis revealed that the worm extract promoted the growth of anaerobic bacteria on M2GSC agar. H. diminuta-infection was accompanied by increased Th2 immune responses, and colon from infected mice had increased levels of IL-10, IL-25, Muc2, trefoil factor 3, and β2-defensin mRNA. SPF-mice treated with antibiotics, or germ-free mice, expelled H. diminuta with kinetics similar to control SPF mice. In both settings, measurements of Th2-immune responses were not significantly different across the groups. Thus, while infection with H. diminuta results in subtle but distinct changes to the colonic microbiota, we have no evidence to support an essential role for gut bacteria in the expulsion of the worm from the mouse host.

Neuropeptides comprise a diverse and broad group of neurotransmitters in vertebrates and invertebrates, with critical roles in neuronal signal transduction. While their role in controlling learning and memory in the brains of mammals is known, their extra-synaptic function in infection and inflammation with effects on distinct tissues and immune cells is increasingly recognized. Helminth infections especially of the central nervous system (CNS), such as neurocysticercosis, induce neuropeptide production by both host and helminth, but their role in host–parasite interplay or host inflammatory response is unclear. Here, we review the neurobiology of helminths, and discuss recent studies on neuropeptide synthesis and function in the helminth as well as the host CNS and immune system. Neuropeptides are summarized according to structure and function, and we discuss the complex enzyme processing for mature neuropeptides, focusing on helminth enzymes as potential targets for novel anthelminthics. We next describe known immunomodulatory effects of mammalian neuropeptides discovered from mouse infection models and draw functional parallels with helminth neuropeptides. Last, we discuss the anti-microbial properties of neuropeptides, and how they may be involved in host–microbiota changes in helminth infection. Overall, a better understanding of the biology of helminth neuropeptides, and whether they affect infection outcomes could provide diagnostic and therapeutic opportunities for helminth infections.

Helminths produce excretory/secretory products (E/S) which can modulate the immune responses of their hosts. Dendritic cells (DC) are essential for initiating the host T cell response and are thus potential targets for modulation by helminth E/S. Here we study immunomodulation of porcine peripheral blood DC subsets following ex vivo stimulation with E/S from Ascaris suum, a common helminth of pigs with considerable public health and economic importance. Our data showed that the relative frequencies of DC subsets in porcine blood differ, with plasmacytoid DC (pDC) being the most prominent in healthy 6-month-old pigs. pDC are an important cytokine source, and we found that A. suum E/S suppressed production of the type 1 cytokines IL-12p40 and TNF-α by this subset following toll-like receptor (TLR) ligation. In contrast, conventional DC (cDC) are more efficient antigen presenters, and the expression of CD80/86, costimulatory molecules essential for efficient antigen presentation, were modulated differentially by A. suum E/S between cDC subsets. CD80/86 expression by type 1 cDC (cDC1) following TLR ligation was greatly suppressed by the addition of A. suum E/S, while CD80/86 expression by type 2 cDC (cDC2) was upregulated by A. suum E/S. Further, we found that IFN-γ production by natural killer (NK) cells following IL-12 and IL-18 stimulation was suppressed by A. suum E/S. Finally, in the presence of E/S, IFN-γ production by CD4+ T cells co-cultured with autologous blood-derived DC was significantly impaired. Together, these data provide a coherent picture regarding the regulation of type 1 responses by A. suum E/S. Responsiveness of pDC and cDC1 to microbial ligands is reduced in the presence of E/S, effector functions of Th1 cells are impaired, and cytokine-driven IFN-γ release by NK cells is limited.

The aim of this study is to determine the species of parasites that affected the inhabitants of the city of Acre on the coast of the eastern Mediterranean during the Ottoman Period. This is the first archaeological study of parasites in the Ottoman Empire. We analysed sediment from a latrine dating to the early 1800s for the presence of helminth eggs and protozoan parasites which caused dysentery. The samples were examined using light microscopy and enzyme-linked immunosorbent assay (ELISA) kits. We found evidence for roundworm (Ascaris lumbricoides), whipworm (Trichuris trichiura), fish tapeworm (Dibothriocephalus sp.), Taenia tapeworm (Taenia sp.), lancet liver fluke (Dicrocoelium dendriticum), and the protozoa Giardia duodenalis and Entamoeba histolytica. The parasite taxa recovered demonstrate the breadth of species present in this coastal city. We consider the effect of Ottoman Period diet, culture, trade and sanitation upon risk of parasitism in this community living 200 years ago.

Inflammatory bowel disease (IBD) is a set of immunological disorders which can generate chronic pain and fatigue associated with the inflammatory symptoms. The treatment of IBD remains a significant hurdle with current therapies being only partially effective or having significant side effects, suggesting that new therapies that elicit different modes of action and delivery strategies are required. TGM1 is a TGF-β mimic that was discovered from the intestinal helminth parasite Heligmosomoides polygyrus and is thought to be produced by the parasite to suppress the intestinal inflammation response to help evade host immunity, making it an ideal candidate to be developed as a novel anti-inflammatory bio-therapeutic. Here we utilized the expression system of the edible green algae Chlamydomonas reinhardtii in order to recombinantly produce active TGM1 in a form that could be ingested. C. reinhardtii robustly expressed TGM1, and the resultant recombinant protein is biologically active as measured by regulatory T cell induction. When delivered orally to mice, the algal expressed TGM1 is able to ameliorate weight loss, lymphadenopathy, and disease symptoms in a mouse model of DSS-induced colitis, demonstrating the potential of this biologic as a novel treatment of IBD., Highlights • A novel cytokine from a parasitic worm expressed in green algae induces an anti-inflammatory response when fed to mice. • Recovery from inflammatory colitis is accelerated by the algal-expressed protein. • Oral administration offers an attractive route for the novel therapy.

To determine whether small mammals living in natural settings harbor helminth infections in their mammary glands, we conducted a survey of helminths infecting rodents and soricimorphs in three widespread locations in the eastern United States: states of New York, Tennessee, and Georgia. We examined all the primary organs in all hosts, and identified all helminths. We also excised the complete mammary glands within their subcutaneous fat pads, then stained and mounted each whole mammary gland set for microscopical examination. A total of 53 individual hosts were examined, including 32 Peromyscus spp., 11 Mus musculus, 5 Sigmodon hispidus, 4 Clethrionomys gapperi, and 1 Blarina carolinensis. Helminths collected included Heligmosomoides sp., Hymenolepisdiminuta, Hymenolepis nana, Pterygodermatites peromysci, Schistosomatium douthitti, Syphacia obvelata, Syphacia sigmodontis, and Trichostrongylus sigmodontis. Four S. hispidus were infected by T. sigmodontis in the small intestine, in all four, we also found nematode larvae in lactiferous duct lumen and lactogenic tissue of the mammary glands. We were unable to identify the species of nematode larvae, but the co-occurrence with T. sigmodontis in all cases may suggest an association. Future studies should seek to identify such larvae using molecular and other methods, and to determine the role of these mammary nematode larvae in the life cycle of the identified species. No other host species harbored helminths in the mammary glands. Overall, our results suggest that mammary infections in wild small mammals are not common, but warrant inclusion in future surveys.

Spirocerca lupi is the etiological agent of spirocercosis in canids from tropics and subtropics in the world. This parasite can lead to life threatening complications, including the formation of fibro and osteosarcomas. A 5-year- old crossbreed Maltese dog from Costa Rica was presented to a veterinary clinic with two nodules in the sub- cutaneous tissues of the cervical region. One of the nodules resolved with intramuscular antibiotic and anti- inflammatory treatment. However, the other nodule persisted and became purulent. The content of the nodule was excised releasing two coiled 3-cm reddish worms. Identity of the specimens was confirmed by amplifying an 850 bp fragment of the 18S rDNA. The obtained sequence showed 99.96% similarities with S. lupi from Hungary. The present study highlights the need of veterinary clinicians' awareness towards nematode aberrant migrations, and the require of molecular studies to gain insight into the parasite's evolutionary history. Spirocerca lupi es el agente etiológico de la espirocerosis en los cánidos de los trópicos y subtrópicos del mundo. Este parásito puede provocar complicaciones mortales, como la formación de fibrosis y osteosarcomas. Un perro maltés de 5 años de edad, procedente de Costa Rica, acudió a una clínica veterinaria con dos nódulos en los tejidos subcutáneos de la región cervical. Uno de los nódulos se resolvió con un tratamiento antibiótico y antiinflamatorio intramuscular. Sin embargo, el otro nódulo persistió y se volvió purulento. El contenido del nódulo fue extirpado liberando dos gusanos rojizos enrollados de 3 cm. La identidad de los especímenes se confirmó mediante amplificación de un fragmento de 850 pb del ADNr 18S. La secuencia obtenida mostró una similitud del 99,96% con S. lupi de Hungría. El presente estudio pone de relieve la necesidad de que los clínicos veterinarios tomen conciencia de las migraciones aberrantes de los nematodos, y de que se requiera una mayor concienciación al respecto y la necesidad de realizar estudios moleculares para conocer la historia evolutiva del parásito. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

Signal Transducer and Activator of Transcription (STAT) 1 signaling is critical for IFN-γ-mediated immune responses and resistance to protozoan and viral infections. However, its role in immunoregulation during helminth parasitic infections is not fully understood. Here, we used STAT1−/− mice to investigate the role of this transcription factor during a helminth infection caused by the cestode Taenia crassiceps and show that STAT1 is a central molecule favoring susceptibility to this infection. STAT1−/− mice displayed lower parasite burdens at 8 weeks post-infection compared to STAT1+/+ mice. STAT1 mediated the recruitment of inflammatory monocytes and the development of alternatively activated macrophages (M2) at the site of infection. The absence of STAT1 prevented the recruitment of CD11b+Ly6ChiLy6G− monocytic cells and therefore their suppressive activity. This failure was associated with the defective expression of CCR2 on CD11b+Ly6ChiLy6G− cells. Importantly, CD11b+Ly6ChiLy6G− cells highly expressed PDL-1 and suppressed T-cell proliferation elicited by anti-CD3 stimulation. PDL-1+ cells were mostly absent in STAT1−/− mice. Furthermore, only STAT1+/+ mice developed M2 macrophages at 8 weeks post-infection, although macrophages from both T. crassiceps-infected STAT1+/+ and STAT1−/− mice responded to IL-4 in vitro, and both groups of mice were able to produce the Th2 cytokine IL-13. This suggests that CD11b+CCR2+Ly6ChiLy6G− cells give rise to M2 macrophages in this infection. In summary, a lack of STAT1 resulted in impaired recruitment of CD11b+CCR2+Ly6ChiLy6G− cells, failure to develop M2 macrophages, and increased resistance against T. crassiceps infection.

An inherent elevation in type 2 immunity is a feature of maternal and offspring immune systems. This has diverse implications for maternal and offspring biology including influencing success of pregnancy, offspring immune development and maternal and offspring ability to control infection and diseases such as allergies. In this review we provide a broad insight into how this immunological feature of pregnancy and early life impacts both maternal and offspring biology. We also suggest how understanding of this axis of immune influence is and may be utilised to improve maternal and offspring health.

Viral infections are often studied in model mammalian organisms under specific pathogen-free conditions. However, in nature, coinfections are common, and infection with one organism can alter host susceptibility to infection with another. Helminth parasites share a long coevolutionary history with mammalian hosts and have shaped host physiology, metabolism, immunity, and the composition of the microbiome. Published studies suggest that helminth infection can either be beneficial or detrimental during viral infection. Here, we discuss coinfection studies in mouse models and use them to define key determinants that impact outcomes, including the type of antiviral immunity, the tissue tropism of both the helminth and the virus, and the timing of viral infection in relation to the helminth lifecycle. We also explore the current mechanistic understanding of how helminth-virus coinfection impacts host immunity and viral pathogenesis. While much attention has been placed on the impact of the gut bacterial microbiome on immunity to infection, we suggest that enteric helminths, as a part of the eukaryotic macrobiome, also represent an important modulator of disease pathogenesis and severity following virus infection.

Orientador: Bruno Coraucci Filho Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil Resumo: o lodo de esgoto é um resíduo gerado no final do processo de tratamento de esgotos. É produzido em grandes quantidades e há necessidade de promover sua disposição de maneira adequada. A disposição do lodo no solo é uma alternativa que combina reuso e reciclagem de constituintes orgânicos e minerais, pois o lodo contém matéria orgânica e elementos como N e P, que são importantes para o desenvolvimento das plantas. No entanto, este lodo pode estar contaminado por patógenos, como os protozoários e helmintos, ou por metais pesados, causando problemas em relação à disposição. O objetivo desta pesquisa foi avaliar a presença destes patógenos no solo e no líquido percolado que recebeu aplicação de lodo em 3 dosagens diferentes: 2,5 ; 5,0 e 7,5 TDS/ha, com pH natural e 5,0 TDS/ha pH corrigido, além do grupo controle. Também foram testadas duas formas de desinfecção do lodo: calagem (20, 30 e 50%) e desinfecção natural utilizando a luz solar, sendo que neste experimento, além de helmintos e protozoários, foram feitas análises de coliformes totais e E. colí. Os resultados obtidos para aplicação de lodo no solo mostram que os patógenos concentram-se na camada superficial do solo (0-20cm), e quanto maior a dose de lodo aplicada maior a concentração destes organismos. No líquido percolado não foram detectados patógenos. Os testes de calagem indicam que a 50% os patógenos são eliminados em 15 dias, e o experimento de desinfecção natural também demonstra que este método pode ser utilizado, com eliminação total da E. colítambém em periodo de 15 dias Abstract: Sewage sludge is a residue trom wastewater treatment process that has been produced in large scale and must be disposed appropriatelly. Land disposal of sludge is an altemative that arrange reuse and recicling ot organic and mineral constituints like nitrogen (N) and phosporus (P). However, sludge can be contamined with pathogens, helminth and protozoan, or heavy metal, causing problems with disposal. The objective was to evaluate the pathogens in soil and inflitrated liquid in sai', that received sewage sludge application in three differents dosagens: 2,5; 5,0 e 7,5 TDS/ha in natural pH and 5,OTDS/ha in corriged pH. There were tested two forms os sludge disinfection: chemical stabilization with lime a (20, 30 and 50%) and natural solar disinfection. In the last was checkin total coliforms and E. colí. The research to land appplication in soU shows that the pathogens are concentring on superficial layer at soil (D-20cm) and the concentration increases in accordance with elevation dosage. In the infilirated liquid has not been detected pathogens. The chemical stabilization with lime (50%) shows that pathogens are eliminated within 15 days. Solar disinfection proved an alternative efficient, with destruction of E coli within 15 days too Doutorado Saneamento e Ambiente Doutor em Engenharia Civil

The translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of theC. elegansRNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5′UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5′UTR dependent manner. Our study reveals anin vivomechanism for eIF3 in governing neuronal protein levels to control activity states and offers insights into how eIF3 dysregulation contributes to neuronal disorders.

Abstract Parasites, including viruses, bacteria, fungi, protists, helminths, and arthropods, are ubiquitous in the animal kingdom. Consequently, hosts are frequently infected with more than one parasite species simultaneously. The assessment of such co-infections is of fundamental importance for disease ecology, but relevant studies involving non-domesticated animals have remained scarce. Many amphibians are in decline, and they generally have a highly diverse parasitic fauna. Here we review the literature reporting on field surveys, veterinary case studies, and laboratory experiments on co-infections in amphibians, and we summarize what is known about within-host interactions among parasites, which environmental and intrinsic factors influence the outcomes of these interactions, and what effects co-infections have on hosts. The available literature is piecemeal, and patterns are highly diverse, so that identifying general trends that would fit most host–multiparasite systems in amphibians is difficult. Several examples of additive, antagonistic, neutral, and synergistic effects among different parasites are known, but whether members of some higher taxa usually outcompete and override the effects of others remains unclear. The arrival order of different parasites and the time lag between exposures appear in many cases to fundamentally shape competition and disease progression. The first parasite to arrive can gain a marked reproductive advantage or induce cross-reaction immunity, but by disrupting the skin and associated defences (i.e., skin secretions, skin microbiome) and by immunosuppression, it can also pave the way for subsequent infections. Although there are exceptions, detrimental effects to the host are generally aggravated with increasing numbers of co-infecting parasite species. Finally, because amphibians are ectothermic animals, temperature appears to be the most critical environmental factor that affects co-infections, partly via its influence on amphibian immune function, partly due to its direct effect on the survival and growth of parasites. Besides their importance for our understanding of ecological patterns and processes, detailed knowledge about co-infections is also crucial for the design and implementation of effective wildlife disease management, so that studies concentrating on the identified gaps in our understanding represent rewarding research avenues. Graphical Abstract

Introduction: Over 1 billion humans carry infectious helminth parasites that can lead to chronic comorbidities such as anemia and growth retardation in children. Helminths induce a T-helper type 2 (Th2) immune response in the host and can cause severe tissue damage and fibrosis if chronic. We recently reported that mice infected with the soil-transmitted helminth, Nippostrongylus brasiliensis, displayed elevated levels of endocannabinoids (eCBs) in the lung and intestine. eCBs are lipid-signaling molecules that control inflammation; however, their function in infection is not well defined. Materials and Methods: A combination of pharmacological approaches and genetic mouse models was used to investigate roles for the eCB system in inflammatory responses and lung injury in mice during parasitic infection with N. brasiliensis. Results: Hemorrhaging of lung tissue in mice infected with N. brasiliensis was exacerbated by inhibiting peripheral cannabinoid receptor subtype-1 (CB(1)Rs) with the peripherally restricted CB(1)R antagonist, AM6545. In addition, these mice exhibited an increase in nonfunctional alveolar space and prolonged airway eosinophilia compared to vehicle-treated infected mice. In contrast to mice treated with AM6545, infected cannabinoid receptor subtype-2-null mice (Cnr2(−/−)) did not display any changes in these parameters compared to wild-type mice. Conclusions: Roles for the eCB system in Th2 immune responses are not well understood; however, increases in its activity in response to infection suggest an immunomodulatory role. Moreover, these findings suggest a role for eCB signaling at CB(1)Rs but not cannabinoid receptor subtypes-2 in the resolution of Th2 inflammatory responses, which become host destructive over time.

High-throughput sequencing has become a standard tool for analyzing RNA and DNA. This method usually needs a cDNA/DNA library ligated with specific 5′ and 3′ linkers. Unlike mRNA, small RNA often contains modifications including 5′ cap or triphosphate and 2′-O-methyl, requiring additional processing steps before linker additions during cloning processes; due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. Here we present a new strategy to clone 5′ modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. The 7-h cloning process only needs ∼1 h of labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA; the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, the all-liquid-based reaction can be performed in an automated manner.

Duddingtonia flagrans is a nematode-trapping fungus that has shown promising results as a tool to combat parasitic nematode infections in livestock. The fungus interrupts the parasitic lifecycle by trapping and killing larval stages on pasture to prevent re-infection of animals. One barrier to the fungus’ commercial use is scaling up production of the fungus, and specifically of chlamydospores, which survive the digestive tract to grow in fecal pats on pasture, thus have potential as a feed through anthelmintic. The purpose of this study was to evaluate the effect of dehydration on sporulation of the fungus. Disks of Duddingtonia flagrans type strain (ATCC® 13423™) were grown on 17% cornmeal agar for 26 days at 30 °C, then split into three groups; dried quickly at 38 °C and 37% humidity over 48 h (“incubated”), dried more slowly at 24 °C and 55% humidity over 10 days (“air-dried”), or kept at 30 °C and sealed with parafilm to prevent loss of moisture as a control (“wet”). Half of each dried culture was resuspended in water, then heated to liquify and homogenized through vortexing. Spores were then counted in a Neubauer hematocytometer. Both the “air-dried” and “incubated” drying techniques yielded significantly more spores than the “wet” control (Welch’s two sample t test p values of .0359 and .0411, respectively). The difference in average chlamydospores per milliliter was insignificant between the two drying techniques, although a visual representation of the data shows less spore count variability in the “air-dried” technique.

Children in the Peruvian Amazon Basin are at risk of soil-transmitted helminths (STH) infections. This study aimed to determine the prevalence of STH infection in children from a rural Amazonian community of Peru and to elucidate epidemiological risk factors associated with its perpetuation while on a school-based deworming program with mebendazole. Stool samples of children aged 2–14 years and their mothers were analyzed through direct smear analysis, Kato–Katz, spontaneous sedimentation in tube, Baermann’s method, and agar plate culture. A questionnaire was administered to collect epidemiological information of interest. Among 124 children, 25.8% had one or more STH. Individual prevalence rates were as follows: Ascaris lumbricoides, 16.1%; Strongyloides stercoralis, 10.5%; hookworm, 1.6%; and Trichuris trichiura, (1.6%). The prevalence of common STH (A. lumbricoides, T. trichiura, and hookworm) was higher among children aged 2–5 years than older children (31.6% versus 12.8%; P = 0.01). In terms of sanitation deficits, walking barefoot was significantly associated with STH infection (OR = 3.28; CI 95% = 1.11–12.07). Furthermore, STH-infected children more frequently had a mother who was concomitantly infected by STH than the non-STH–infected counterpart (36.4% versus 14.1%, P = 0.02). In conclusion, STH infection is highly prevalent in children from this Amazonian community despite routine deworming. Institutional health policies may include hygiene and sanitation improvements and screening/deworming of mothers to limit the dissemination of STH. Further studies are needed to address the social and epidemiological mechanics perpetuating these infections.

Summary The present study proposes a new methodology for the quantification of parasite eggs in animal tissue. Quantification of parasites are important to understand epidemiology of spirorchiid infections in sea turtles, however different methodologies for quantifying Spirorchiidae eggs in turtle tissues have been used. The most representative way to quantify Spirorchiidae burdens in tissues is counting eggs / g of tissue, however, this method is very laborious. As an alternative, we propose quantifying number of Spirorchiidae eggs/area of tissue on a microscope slide. We compared this method to number of eggs / slide, a common metric of egg burden in turtle tissues. Both methods correlated well with eggs / g with eggs/mm2 of tissue having better correlation.

Internal parasitic nematodes are a global animal health issue causing drastic losses in livestock. Here, we report a H. contortus representative draft genome to serve as a genetic resource to the scientific community and support future experimental research of molecular mechanisms in related parasites. A de novo hybrid assembly was generated from PCR-free whole genome sequence data, resulting in a chromosome-level assembly that is 465 Mb in size encoding 22,341 genes. The genome sequence presented here is consistent with the genome architecture of the existing Haemonchus species and is a valuable resource for future studies regarding population genetic structures of parasitic nematodes. Additionally, comparative pan-genomics with other species of economically important parasitic nematodes have revealed highly open genomes and strong collinearities within the phylum Nematoda.

Two species of Acanthobothrium ( Onchoproteocephalidea : Onchobothriidae ) are described from the spiral intestine of Narcine entemedor Jordan & Starks, 1895, in Bahia de Acapulco, Acapulco, Guerrero, Mexico. Based on the four criteria used for the identification of species of Acanthobothrium , A. soniae sp. nov. is a Category 2 species (less than 15 mm in total length with less than 50 proglottids, less than 80 testes, and with the ovary asymmetrical in shape). Acanthobothrium vidali sp. nov. is a Category 6 species (more than 15 mm in total length with more than 50 proglottids, fewer than 80 testes, and the ovary is asymmetrical). The new species differ from similar species from the Pacific Ocean by total length, the number of proglottids, diameter of the accessory sucker, the length of the cirrus sac, the number of testes per proglottid and the measurements of hooks. With the recognition of A. soniae sp. nov. and A. vidali sp. nov., 42 species of Acanthobothrium have been reported from the Pacific coast of the Americas. This is the first report of species of Acanthobothrium from a member of Narcine from Mexico and it brings the number of species reported from elasmobranchs from the Pacific Coast of Mexico to 13.

The World Health Organization indicates that more than 1.5 billion people are infected with geohelminths. Soil-transmitted helminths prevail mostly in tropical and subtropical regions, in areas with inadequate hygiene and sanitation conditions, and basic health education problems. Nematode eggs are structures of resistance and infection by fecal–oral transmission. When STH eggs are ingested, they can infect the potential host, causing abdominal pain, diarrhea, anemia, malnutrition, and physical-cognitive impacts in children. Taking advantage of the increasing employment of three-dimensional models of these structured based on light microscopy images to improve the research area and education could be an alternative to improve health education and spread scientific information on transmission and prevention. The objective of this work was to produce 3D printed models from bi-dimensional images of eggs based on their real morphological and morphometric characteristics. The virtual models were reconstructed from the acquisition and selection of images obtained using light microscopy. After selecting referential images, we constructed the models based on the vectorization of the egg structures. After vectorization, 3D modeling was performed and printed in PLA. 3D models have a high potential to contribute to the advanced morphological studies and teaching of parasitological sciences, enriching the teaching-learning process applicable in presential or remote teaching of basic education, undergraduate, and post-graduation classes.

We report a case of autochthonous infection of the eye worm Thelazia callipaeda in a dog in the northeastern United States. Integrated morphologic identification and molecular diagnosis confirmed the species. Phylogenetic analysis suggested introduction from Europe. The zoonotic potential of this parasite warrants broader surveillance and increased awareness among physicians and veterinarians.

Trematode parasites of the genus Fasciola are the cause of liver fluke disease (fasciolosis) in humans and their livestock. Infection of the host involves invasion through the intestinal wall followed by migration in the liver that results in extensive damage, before the parasite settles as a mature egg-laying adult in the bile ducts. Genomic and transcriptomic studies revealed that increased metabolic stress during the rapid growth and development of F. hepatica is balanced with the up-regulation of the thiol-independent antioxidant system. In this cascade system thioredoxin/glutathione reductase (TGR) reduces thioredoxin (Trx), which then reduces and activates peroxiredoxin (Prx), whose major function is to protect cells against the damaging hydrogen peroxide free radicals. F. hepatica expresses a single TGR, three Trx and three Prx genes; however, the transcriptional expression of Trx1 and Prx1 far out-weighs (>50-fold) other members of their family, and both are major components of the parasite secretome. While Prx1 possesses a leader signal peptide that directs its secretion through the classical pathway and explains why this enzyme is found freely soluble in the secretome, Trx1 lacks a leader peptide and is secreted via an alternative pathway that packages the majority of this enzyme into extracellular vesicles (EVs). Here we propose that F. hepatica Prx1 and Trx1 do not function as part of the parasite’s stress-inducible thiol-dependant cascade, but play autonomous roles in defence against the general anti-pathogen oxidative burst by innate immune cells, in the modulation of host immune responses and regulation of inflammation.

Orientador: Adriano Luiz Tonetti Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo Resumo: Diante do enorme problema de saúde pública que são as doenças de veiculação hídrica e do grande déficit sanitário existente no Brasil, principalmente nas cidades com população inferior a cinquenta mil habitantes e nas periferias dos grandes centros urbanos, o presente trabalho teve como objetivo avaliar a eficiência de um sistema simplificado, composto por filtro anaeróbio, preenchido com anéis de bambu, associado a filtro de areia, na remoção de cistos de Giardia spp. e ovos de helmintos. O filtro anaeróbio foi operado com fluxo ascendente e tempo de detenção hidráulica de 12 horas, o efluente proveniente deste reator era aplicado sobre a superfície do leito de areia em oito cargas de 25 L m-2, totalizando uma taxa de aplicação de 200 L m-2dia-1. As amostras de efluente eram coletadas em três pontos, P01 (esgoto bruto), P02 (efluente do filtro anaeróbio) e P03 (efluente do filtro de areia), mensalmente, durante 12 meses. Nos últimos quatro meses, foram realizadas análises para detecção de cistos de Giardia spp. no lodo do filtro anaeróbio e de ovos de helmintos no leito do filtro de areia e também no lodo. O sistema também foi avaliado quanto à remoção de E. coli e quanto aos parâmetros físicos e químicos. O filtro anaeróbio foi capaz de remover 99,5 ± 28,7% da concentração estimada de cistos presentes no esgoto bruto, já o filtro de areia removeu 56,25 ± 31,6% dos cistos presentes no efluente anaeróbio, sendo a eficiência de remoção total do sistema igual 99,7%. Ovos de helmintos foram detectados em apenas três amostras de efluente anaeróbio, em uma concentração média de 1,5 ovos/L, sendo alcançada a remoção de 100% dos ovos após a passagem pelo filtro de areia. O sistema também proporcionou a redução de 4 unidades logarítmicas da concentração de E. coli presente no esgoto bruto, além do mais, o efluente produzido apresentou baixas concentrações de Demanda Química de Oxigênio (52 ± 24 mgL-1) e Sólidos Suspensos Totais (17 ± 20 mgL-1) e, baixos valores de turbidez (3 ± 4 UT), comprovando assim, a eficiência do sistema simplificado proposto tanto na remoção de patógenos quanto na adequação dos parâmetros físicos e químicos Abstract: In the face of the enormous public health¿s problem that are the waterborne diseases and the big Brazilian sanitary deficit, especially at cities with less than fifty thousand inhabitants and big cities¿ peripheries, this study aimed to evaluate the efficiency of a simplified system, composed by an upflow anaerobic filter, filled with bamboo rings, associated with a sand filter, in the removal of Giardia cysts and helminths eggs. The anaerobic filter was operated with hydraulic retention time of 12 hours, the effluent from this reactor was applied to the sand bed surface in eight loads of 25 L m-2, with a total application rate of 200 L m -2 day-1. The samples were collected at three different points, P01 (raw sewage), P02 (anaerobic filter effluent) and P03 (Sand filter effluent), monthly, during 12 months. In the last four months were performed analysis to detect Giardia spp. cysts in the anaerobic filter sludge and helminths eggs in the sand filter bed and, as well, in the sludge. The system was also evaluated relating to the E. coli removal and for physical and chemical parameters. The anaerobic filter was able to remove 99.5 ± 28.7% of the estimated concentration of cysts present in raw sewage, while the sand filter removed 31.6 ± 56.25% of the cysts present in the anaerobic effluent, making the system total efficiency¿s removal of 99.7%. Helminth eggs were detected in three anaerobic effluent samples, in an average concentration of 1.5 eggs/L, and after the effluent passes through the sand filter there was a removal of 100% eggs. The system also provided a reduction of 4 log units of E. coli concentration existing in raw sewage, in addition the effluent produced showed low concentrations of Chemical Oxygen Demand (52 ± 24 mg L - ) and Total Suspended Solids (17 ± 20 mgL -1) and lower turbidity values (3 ±4 UT), proving then, the simplified system proposed efficiency both in removing pathogens, as the adequacy of physical and chemical parameters Mestrado Saneamento e Ambiente Mestra em Engenharia Civil

Objective Visceral larva migrans (VLM) caused by Ascaris suum is a major health problem in pig farming regions. The clinical characteristics of pulmonary VLM caused by A. suum, however, are unclear. We assessed the clinico-radiologic features of this disease. Methods Medical records, including the results of chest radiography and high-resolution computed tomography (HRCT), were retrospectively reviewed from January 2000 through June 2019, at the University of Miyazaki Hospital and Kyoritsuiin Hospital in Miyazaki Prefecture, Japan. Results Seven patients with VLM caused by A. suum were identified. All seven patients had a unique habit of consuming raw foods, such as organic vegetables, chicken, turkey, wild boar, and venison. All but one patient, who had eosinophilic pneumonia with a fever and severe fatigue, had only mild or no respiratory symptoms. All 7 patients had remarkable eosinophilia (median, 1,960 /μL) and high serum IgE levels (median, 1,346 IU/mL). Chest HRCT revealed multiple nodules and multiple nodular ground-glass opacities in 57% and 29% of the patients, respectively. The pulmonary lesions were located predominantly in subpleural areas. All seven patients were treated with albendazole, which led to improvement within two to three months. Neither eggs nor parasites were detected in the feces or sputum of any patient. Conclusion Consumption of raw organic vegetables or raw meat is a possible route of A. suum infection. Infected patients exhibit mild respiratory symptoms, and multiple nodules with a halo in the subpleural area are a common finding on chest HRCT. Treatment with albendazole was effective in these cases.

Background Microbial eukaryotes are found alongside bacteria and archaea in natural microbial systems, including host-associated microbiomes. While microbial eukaryotes are critical to these communities, they are challenging to study with shotgun sequencing techniques and are therefore often excluded. Results Here, we present EukDetect, a bioinformatics method to identify eukaryotes in shotgun metagenomic sequencing data. Our approach uses a database of 521,824 universal marker genes from 241 conserved gene families, which we curated from 3713 fungal, protist, non-vertebrate metazoan, and non-streptophyte archaeplastida genomes and transcriptomes. EukDetect has a broad taxonomic coverage of microbial eukaryotes, performs well on low-abundance and closely related species, and is resilient against bacterial contamination in eukaryotic genomes. Using EukDetect, we describe the spatial distribution of eukaryotes along the human gastrointestinal tract, showing that fungi and protists are present in the lumen and mucosa throughout the large intestine. We discover that there is a succession of eukaryotes that colonize the human gut during the first years of life, mirroring patterns of developmental succession observed in gut bacteria. By comparing DNA and RNA sequencing of paired samples from human stool, we find that many eukaryotes continue active transcription after passage through the gut, though some do not, suggesting they are dormant or nonviable. We analyze metagenomic data from the Baltic Sea and find that eukaryotes differ across locations and salinity gradients. Finally, we observe eukaryotes in Arabidopsis leaf samples, many of which are not identifiable from public protein databases. Conclusions EukDetect provides an automated and reliable way to characterize eukaryotes in shotgun sequencing datasets from diverse microbiomes. We demonstrate that it enables discoveries that would be missed or clouded by false positives with standard shotgun sequence analysis. EukDetect will greatly advance our understanding of how microbial eukaryotes contribute to microbiomes.

The antihelmintic drug ABZ and its metabolites belong to the chemical family of benzimidazoles (BZM) that act as potent tubulin polymerization inhibitors, suggesting a potential re-direction of BZMs for cancer therapy. Applying UV-Vis spectrometry we here demonstrate ABZ as a DNA intercalator. This insight led us to determine the primary mode of ABZ action in mammalian cells. As revealed by RNA sequencing, ABZ did neither grossly affect replication as analyzed by survival and replication stress signaling, nor the transcriptome. Actually, unbiased transcriptome analysis revealed a marked cell cycle signature in ABZ exposed cells. Indeed, short-term exposure to ABZ arrested mammalian cells in G2/M cell cycle stages associated with frequent gains and losses of chromatin. Cellular analyses revealed ABZ as a potent mammalian spindle poison for normal and malignant cells, explaining the serious chromosome segregation defects. Since chromosomal aberrations promote both cancer development and cell death, we determined if besides its general cytotoxicity, ABZ could predispose to tumor development. As measured by loss of heterozygosity (LOH) in vitro and in vivo ABZ was found as a potent inducer of LOH and accelerator of chromosomal missegregation.

Trematode parasites of the genus Fasciola are the cause of liver fluke disease (fasciolosis) in humans and their livestock. Infection of the host involves invasion through the intestinal wall followed by migration in the liver that results in extensive damage, before the parasite settles as a mature egg-laying adult in the bile ducts. Genomic and transcriptomic studies revealed that increased metabolic stress during the rapid growth and development of F. hepatica is balanced with the up-regulation of the thiol-independent antioxidant system. In this cascade system thioredoxin/glutathione reductase (TGR) reduces thioredoxin (Trx), which then reduces and activates peroxiredoxin (Prx), whose major function is to protect cells against the damaging hydrogen peroxide free radicals. F. hepatica expresses a single TGR, three Trx and three Prx genes; however, the transcriptional expression of Trx1 and Prx1 far out-weighs (>50-fold) other members of their family, and both are major components of the parasite secretome. While Prx1 possesses a leader signal peptide that directs its secretion through the classical pathway and explains why this enzyme is found freely soluble in the secretome, Trx1 lacks a leader peptide and is secreted via an alternative pathway that packages the majority of this enzyme into extracellular vesicles (EVs). Here we propose that F. hepatica Prx1 and Trx1 do not function as part of the parasite’s stress-inducible thiol-dependant cascade, but play autonomous roles in defence against the general anti-pathogen oxidative burst by innate immune cells, in the modulation of host immune responses and regulation of inflammation.

Lipidomics is a relatively recent molecular research field, and explores lipids (fats) and their biology using advanced mass spectrometry technologies. Although this field has expanded significantly in biomedical and biotechnological disciplines, it is still in its infancy in molecular parasitology. Our goal here is to review and discuss technical aspects of MS-based lipidomics and its recent applications to parasitic worms, as well as challenges and future directions for worm lipid research. In a multi-omic paradigm, we expect that the exploration of lipidomic data for parasitic worms will yield important insights into lipid-associated biological pathways and processes, including the regulation of essential signalling pathways, parasite invasion, establishment, adaptation and development.

Introduction: In developing countries, immunosuppressive patients are at greater risk of parasitic infection which may cause morbidity and mortality. Socio-economic and environmental factors including lack of health hygiene in close contact with infected reservoir animal which plays an important role. Aim: To determine the prevalence of parasitic infections and their association with socio-demographic status. Materials and Methods: This was a cross-sectional study which was carried out at tertiary care hospital located in Central East India. Total 120 stool samples were collected from the immunosuppressive patients and were processed usingdirectwet mount preparation with saline and Lugol, formalin-ether concentration and Modified Ziehl-Neelsen stain. Different socio-demographic parameters were recorded. Statistical analysis was done using Graph Pad Prism version 8 Chi-square test. The p-value ≤0.05 was considered as statisticallysignificant Results: Out of total 120 patients, 20 (16.7%) were found to be infected. Majority of the population were males (66.7%). Among the total positive samples, 75% (15) showed the presence of Protozoa in which 10 samples had Entamoeba histolytica. The presence of Helminths was found in 25% (5), in which three samples showed Ascaris lumbricoides and two had Taenia species (10%). Conclusion: The prevalence of parasitic infection among immunosuppressive patients in the present study was 16.7%. Entamoeba histolytica was the most commonly observed parasite. There was no significant association between prevalence of parasitic infections and socio-demographic data variables.

19 páginas, 4 figuras, 2 tablas., World Health Organization goals against soil-transmitted helminthiases (STH) are pointing towards seeking their elimination as a public health problem: reducing to less than 2% the proportion of moderate and heavy infections. Some regions are reaching WHO goals, but transmission could rebound if strategies are discontinued without an epidemiological evaluation. For that, sensitive diagnostic methods to detect low intensity infections and localization of ongoing transmission are crucial. In this work, we estimated and compared the STH infection as obtained by different diagnostic methods in a low intensity setting. We conducted a cross-sectional study enrolling 792 participants from a district in Mozambique. Two stool samples from two consecutive days were collected from each participant. Samples were analysed by Telemann, Kato-Katz and qPCR for STH detection. We evaluated diagnostic sensitivity using a composite reference standard. By geostatistical methods, we estimated neighbourhood prevalence of at least one STH infection for each diagnostic method. We used environmental, demographical and socioeconomical indicators to account for any existing spatial heterogeneity in infection. qPCR was the most sensitive technique compared to composite reference standard: 92% (CI: 83%– 97%) for A. lumbricoides, 95% (CI: 88%– 98%) for T. trichiura and 95% (CI: 91%– 97%) for hookworm. qPCR also estimated the highest neighbourhood prevalences for at least one STH infection in a low intensity setting. While 10% of the neighbourhoods showed a prevalence above 20% when estimating with single Kato-Katz from one stool and Telemann from one stool, 86% of the neighbourhoods had a prevalence above 20% when estimating with qPCR. In low intensity settings STH estimated prevalence of infection may be underestimated if based on Kato-Katz. qPCR diagnosis outperformed the microscopy methods. Thus, implementation of qPCR based predictive maps at STH control and elimination programmes would disclose hidden transmission and facilitate targeted interventions for transmission interruption., BGP and JM received financial support for this study from Mundo Sano Foundation (www.mundosano.org). JG was personally supported at the beginning of the work by the Ramón Areces Foundation and is now funded by the Spanish ‘Juan de la Cierva’ Programme, Ministry of Economy and Competitiveness (FJC-2018-38305). MMV is personally supported by the Spanish ‘Ramón y Cajal’ Programme, Ministry of Economy and Competitiveness (RYC-2015-18368). MCP is personally supported by Junta de Castilla y León and Fondo Social Europeo (LE-135-19). ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya. CISM is supported by the Government of Mozambique and the Spanish Agency for International Development (AECID). Prof. Dr. P.C. Flu Foundation also founded this project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Symbiont microbial communities play important roles in animal biology and are thus considered integral components of metazoan organisms, including parasitic worms (helminths). Nevertheless, the study of helminth microbiomes has thus far been largely overlooked, and symbiotic relationships between helminths and their microbiomes have been only investigated in selected parasitic worms. Over the past decade, advances in next-generation sequencing technologies, coupled with their increased affordability, have spurred investigations of helminth-associated microbial communities aiming at enhancing current understanding of their fundamental biology and physiology, as well as of host-microbe interactions. Using the blood fluke Schistosoma mansoni as a key example of parasitic worms with complex life cycles involving multiple hosts, in this chapter we (1) provide an overview of protocols for sample collection and (2) outline an example workflow to characterize worm-associated microbial communities using high-throughput sequencing technologies and bioinformatics analyses of large-scale sequence data.