Female Worms Research Articles

Survival of worm masses of Onchocerca ochengi in gerbils and hamsters: implications for the development of an in vivo macrofilaricide screening model.

Onchocerciasis, the second leading infectious cause of blindness, afflicts approximately 21 million people globally. Its control is limited to the use of the microfilaricidal drugs, ivermectin and moxidectin. Both drugs are unable to kill the adult worms which can survive for up to 15 years in patients, justifying the urgent need for potent and novel macrofilaricides that kill adult worms. The development of such drugs has been hindered by the lack of an appropriate small laboratory animal model to evaluate potential drug candidates in vivo. This study assessed the survival of O. ochengi female worms and their embryos over time in two laboratory rodents: gerbils and hamsters and tested using "proof-of-concept" studies, whether known macrofilaricidal drugs can kill these worms. Animals were surgically implanted with mechanical or collagenase-liberated O. ochengi worm masses, and necropsied at various time points to test for survival. Recovered worm masses were assessed for viability by biochemical analysis (MTT/formazan assay) or fecundity (embryogram). Flubendazole (FBZ) administered at 20 mg/kg body weight was used to validate both rodent models. By day 26 post-implantation of 15 worm masses, a median of 7.00 (4.00-10.00) was recovered from hamsters, and 2.50 (2.00-4.00) from gerbils. Worm masses recovered from gerbils were mostly disintegrated or fragmented, with significantly higher fragmentation observed with collagenase-liberated worm masses. FBZ had no significant effect on the number of worm masses recovered, but enhanced embryo degradation in gerbils and reduced worm mass viability in hamsters. This exploratory study has revealed the gerbil and hamster as permissible rodents to adult female worms of O. ochengi. The hamsters appeared to maintain the worms longer, compared to gerbils.

Long non-coding RNAs are essential for Schistosoma mansoni pairing-dependent adult worm homeostasis and fertility

The trematode parasite Schistosoma mansoni causes schistosomiasis, which affects over 200 million people worldwide. Schistosomes are dioecious, with egg laying depending on the females' obligatory pairing with males. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein-coding potential that have been involved in other species with reproduction, stem cell maintenance, and drug resistance. In S. mansoni, we recently showed that the knockdown of one lncRNA affects the pairing status of these parasites. Here, we re-analyzed public RNA-Seq data from paired and unpaired adult male and female worms and their gonads, obtained from mixed-sex or single-sex cercariae infections, and found thousands of differentially expressed pairing-dependent lncRNAs among the 23 biological samples that were compared. The expression levels of selected lncRNAs were validated by RT-qPCR using an in vitro unpairing model. In addition, the in vitro silencing of three selected lncRNAs showed that knockdown of these pairing-dependent lncRNAs reduced cell proliferation in adult worms and their gonads, and are essential for female vitellaria maintenance, reproduction, and/or egg development. Remarkably, in vivo silencing of each of the three selected lncRNAs significantly reduced worm burden in infected mice by 26 to 35%. Whole mount in situ hybridization experiments showed that these pairing-dependent lncRNAs are expressed in reproductive tissues. These results show that lncRNAs are key components intervening in S. mansoni adult worm homeostasis, which affects pairing status and survival in the mammalian host, thus presenting great potential as new therapeutic target candidates.

Functional characterization of differentially expressed proteins coming from unisexual and bisexual infected Schistosoma japonicum female worms

Schistosomiasis is an important zoonotic disease affecting up to 40 kinds of animals and is responsible for ∼250 million human cases per year. Due to the extensive use of praziquantel for the treatment of parasitic diseases, drug resistance has been reported. Consequently, novel drugs and effective vaccines are urgently needed for sustained control of schistosomiasis. Targeting reproductive development of Schistosoma japonicum could contribute to the control of schistosomiasis. In this study, five highly expressed proteins (S. japonicum large subunit ribosomal protein L7e, S. japonicum glutathione S-transferase class-mu 26 kDa isozyme, S. japonicum UDP-galactose-4-epimerase and two hypothetical proteins SjCAX70849 and SjCAX72486) in 18, 21, 23, and 25-day mature female worms compared to single-sex infected female worms were selected based on our previous proteomic analysis. Quantitative real-time polymerase chain reaction analysis and long-term interference with small interfering RNA were performed to identify the biological functions of these five proteins. The transcriptional profiles suggested that all five proteins participated in the maturation of S. japonicum. RNA interference against these proteins resulted in morphological changes to S. japonicum. The results of an immunoprotection assay revealed that immunization of mice with recombinant SjUL-30 and SjCAX72486 upregulated production of immunoglobulin G-specific antibodies. Collectively, the results demonstrated that these five differentially expressed proteins were vital to reproduction of S. japonicum and, thus, are potential candidate antigens for immune protection against schistosomiasis.

The Occurrence of the Strongylid Nematodes, Kalicephalus viparae viparae (Nematoda: Diaphanocephalidae), in Viper Snakes, Macrovipera lebetina (Reptilia: Viperidae), Southwestern Iran.

ancylostomatid Kalicephalus spp. is the common parasitic intestinal nematode of reptiles. West-Asian blunt-nosed viper is a venomous snake found in extensive areas of Iran. From June to September 2017, two dead viper snakes were referred to a parasitology laboratory and examined for intestinal parasites. Several white elongated roundworms were collected and fixed to identify under light and scanning electron microscopes (SEM) based on morphological and molecular characteristics. For the molecular survey, some parts of the identified worms were extracted and the ITS of nuclear ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR). Five roundworms were found in one snake and three worms with similar morphological characteristics in another one. All the collected female hookworms were taxonomically identified as Kalicephalus viperae viperae. The SEM findings showed the head was small and had three dorsal, ventral, and middle circumoral papillae with a spike-like process on the median papilla of K. viperae. Moreover, the buccal capsule was bivalvular and included two lateral valves consisting of several chitonid pieces. The tail of the female worm was slim and long with a blunt end and had a terminal spike at its end. In the molecular survey, the ITS of rDNA amplified at about 850 bp was identified as K. viperae. The ITS gene rDNA phylogeny analysis of the K. viperae sequence showed that the isolated species had high similarity to Ancylostoma species from around the world and is close to Ancylostoma braziliense with 88% discrepancies in the phylogenetic tree. The morphological characteristics and a large part of K. viperea viperea rDNA nucleotide sequence were reported in viper snakes for the first time in the world and in Iran.

Inhibition of signal peptidase complex expression affects the development and survival of Schistosoma japonicum.

Schistosomiasis, the second most neglected tropical disease defined by the WHO, is a significant zoonotic parasitic disease infecting approximately 250 million people globally. This debilitating disease has seriously threatened public health, while only one drug, praziquantel, is used to control it. Because of this, it highlights the significance of identifying more satisfactory target genes for drug development. Protein translocation into the endoplasmic reticulum (ER) is vital to the subsequent localization of secretory and transmembrane proteins. The signal peptidase complex (SPC) is an essential component of the translocation machinery and functions to cleave the signal peptide sequence (SP) of secretory and membrane proteins entering the ER. Inhibiting the expression of SPC can lead to the abolishment or weaker cleavage of the signal peptide, and the accumulation of uncleaved protein in the ER would affect the survival of organisms. Despite the evident importance of SPC, in vivo studies exploring its function have yet to be reported in S. japonicum. The S. japonicum SPC consists of four proteins: SPC12, SPC18, SPC22 and SPC25. RNA interference was used to investigate the impact of SPC components on schistosome growth and development in vivo. qPCR and in situ hybridization were applied to localize the SPC25 expression. Mayer's carmalum and Fast Blue B staining were used to observe morphological changes in the reproductive organs of dsRNA-treated worms. The effect of inhibitor treatment on the worm's viability and pairing was also examined in vitro. Our results showed that RNAi-SPC delayed the worm's normal development and was even lethal for schistosomula in vivo. Among them, the expression of SPC25 was significantly higher in the developmental stages of the reproductive organs in schistosomes. Moreover, SPC25 possessed high expression in the worm tegument, testes of male worms and the ovaries and vitellarium of female worms. The SPC25 knockdown led to the degeneration of reproductive organs, such as the ovaries and vitellarium of female worms. The SPC25 exhaustion also reduced egg production while reducing the pathological damage of the eggs to the host. Additionally, the SPC-related inhibitor AEBSF or suppressing the expression of SPC25 also impacted cultured worms' pairing and viability in vitro. These data demonstrate that SPC is necessary to maintain the development and reproduction of S. japonicum. This research provides a promising anti-schistosomiasis drug target and discovers a new perspective on preventing worm fecundity and maturation.

Praziquantel promotes protection against Schistosoma japonicum infection in mice

Praziquantel (PZQ) is the first line drug for the treatment of schistosomiasis. Several studies have confirmed that PZQ regulates host immunity, and we have recently found that pretreatment with PZQ enhances resistance against Schistosoma japonicum infection in buffaloes. We speculate that PZQ induces physiological changes in mice that prevent S. japonicum infection. To test this hypothesis and provide a practical measure to prevent S. japonicum infection, we determined the effective dose (the minimum dose), protection period and onset time of protection by comparing the worm burden, female worm burden and egg burden in PZQ-pretreated mice and blank control mice. Morphological differences between parasites were observed by measuring the total worm length, oral sucker, ventral sucker and ovary. The levels of cytokines, nitrogen monoxide (NO), 5-hydroxytryptamine (5-HT) and specific antibodies were measured using kits or soluble worm antigens. Hematological indicators on day 0 were analyzed in mice that received PZQ on days -15, -18, -19, -20, -21 and -22. The PZQ concentrations in plasma and blood cells were monitored using high performance liquid chromatography (HPLC). The effective dose was found to be two oral administrations (interval of 24 h) at 300 mg/kg body weight (BW) or one injection at 200 mg/kg BW, and the protection period of PZQ injection was 18 days. The optimal preventive effect was observed at two days post-administration, with a >92% worm reduction rate and significant worm reduction until 21 days after administration. Adult worms from PZQ-pretreated mice were runtish showing a shorter length, smaller organs and fewer eggs in the uteri of females. Detection of cytokines, NO, 5-HT and hematological indicators showed that PZQ induced immune-physiological changes, including higher levels of NO, IFN-γ and IL-2, and a lower level of TGF-β. No significant difference in the anti-S. japonicum specific antibody levels was observed. The PZQ concentrations in plasma and blood cells 8 and 15 days post-administration were lower than the detection limit. Our results confirmed that pretreatment with PZQ promotes the protection of mice against S. japonicum infection within 18 days. Although we observed some immune-physiological changes in the PZQ-pretreated mice, the exact mechanisms involved in the preventive effect require further study.

Immunization with excretory-secretory molecules of intestinal nematodes induces antigen-specific protective memory Th2 cell responses.

Parasitic nematodes infect more than 1 billion people in the global south. The development of effective antihelminthic vaccines is a crucial tool for their future elimination. Protective immune responses to nematodes depend on Gata3+ Th2 cells, which can also be induced by nematode-released products. Whether these nematode products induce antigen-specific long-lived memory T cells and thereby confer protection against a challenge infection is not known yet. Hence, we set out to characterize the formation of memory Th2 cells induced by immunization with Heligmosomoides polygyrus excretory-secretory (HES) products, infection-induced versus immunization-induced recall responses to a challenge infection, and whether HES-induced memory T cells show protective properties following adoptive transfer. Our results show that 8 weeks postimmunization, HES induces long-lived functional memory Th2 cells at the site of immunization in the peritoneal cavity. Following a H. polygyrus challenge infection, HES-immunized mice display MHC-II-dependent antigen-specific Th2 cytokine responses in the gut-draining lymph nodes, comparable to those induced by a prior natural infection. Moreover, adoptive transfer of sorted memory CD4+ T cells from HES-immunized donors reduces female worm fecundity following a challenge H. polygyrus infection in recipient mice, highlighting a protective role for immunization-induced memory T cells.

Successful Removal of Angiostrongylus cantonensis Larvae from the Central Nervous System of Rats 7- and 14-Days Post-Infection Using a Product Containing Moxidectin, Sarolaner and Pyrantel Embonate (Simparica Trio™) in Experimental Infections

Angiostrongylus cantonensis is a nematode with an indirect lifecycle, using molluscs as intermediate hosts. Rats are the definitive host. By administering a suitable anthelmintic, at an appropriate interval, the risk of clinical neuroangiostrongyliasis occurring in paratenic hosts (e.g., dogs, man) can be eliminated. We wanted to determine if infective larvae (L3) of A. cantonensis can be safely killed during their migration through the central nervous system (CNS) by oral administration of an anthelmintic combination containing moxidectin (480 µg/kg, Simparica Trio™; M-S-P), thereby preventing patent infections in rats. Eighteen rats were used: ten received oral M-S-P every four weeks; eight rats were used as controls. Rats were initially given M-S-P as a chew to eat, but an acquired food aversion meant that subsequent doses were given by orogastric lavage. All 18 rats were challenged once or twice with approximately 30 L3 A. cantonensis larvae via orogastric lavage. Infection status was determined by faecal analysis using the Baermann technique and necropsy examination of the heart, pulmonary arteries and lungs. Eight out of ten rats dosed with M-S-P had zero lungworms at necropsy; a single female worm was detected in each of the remaining two rats. No treated rats had L1 larvae in faeces. In contrast, all eight controls were infected with patent infections, with a median of 14.5 worms per rat detected at necropsy. The difference in infection rates was significant (two tailed Fishers Exact; p = 0.0011). Moxidectin given orally once every month killed migrating larvae before they reached the pulmonary arteries in 80% of treated rats, while in 20%, only a single female worm was present. Considering the short half-life of moxidectin in the rat, it is likely that the effectiveness of moxidectin is due to larvicidal action on migrating L3, L4 and L5 larvae in the brain parenchyma or subarachnoid space, either 7 days (L3/L4 in cerebrum and spinal cord) or 14 days (L4/L5 in cerebrum and subarachnoid space) after inoculation. This study is a prelude for future research to determine if monthly moxidectin administration orally as M-S-P could prevent symptomatic neuroangiostrongyliasis in dogs.

Antibody response and abomasal histopathology of lambs with haemonchosis during supplementation with medicinal plants and organic selenium

We evaluated the effects of dietary supplementation with medicinal plants (Herbmix) or organic selenium (Selplex) on the immune responses and histopathology of lambs infected with Haemonchus contortus. Twenty-seven lambs were infected and reinfected with approximately 11,000 third-stage larvae of H. contortus during the experiment (on days 0, 49 and 77). Lambs were divided into two supplemented experimental groups (Herbmix and Selplex) and unsupplemented group (Control). The abomasal worm counts at necropsy on day 119 were lower for Herbmix (4230) and Selplex group (3220) than to the Control (6613) which resulted in 51.3% and 36.0% of reduction, respectively. The mean length of adult female worms was in the order Control > Herbmix > Selplex (2.1, 2.08, and 2.01 cm, respectively). The specific IgG response against adults was significantly affected by time (P < 0.001). Serum-specific and total mucus levels of IgA in the Herbmix group were highest on day 15. Mean levels of serum IgM against adults were influenced by treatment (P = 0.048) and time (P < 0.001). The Herbmix group had strong local inflammation in the abomasal tissue, with the formation of lymphoid aggregates and the infiltration of immune cells, but the tissues of the Selplex group had higher numbers of eosinophils, globule leukocytes, and plasma cells. The lymph nodes of each animal had reactive follicular hyperplasia due to the infection. Dietary nutritional supplementation with a mixture of medicinal plants or organic selenium could improve local immune responses and thus enhance the resistance of animals to this parasitic infection.

Histopathological evaluation of Onchocerca volvulus nodules by microscopy and by digital image analysis for the study of macrofilaricidal drug efficacy.

Novel drugs or drug combinations that kill or permanently sterilize adult Onchocerca volvulus worms would be very helpful for treatment and elimination of onchocerciasis. In absence of a reliable biomarker for viable adult worms, histopathological assessment of worms within onchocercal nodules is a standard method to determine macrofilaricidal activity. The goal of the present study was to determine the agreement between two independent experts in the analysis of nodule sections and to assess the value of digital imaging as a means of standardizing the analysis. Two expert microscopists independently assessed 605 nodules by direct microscopy. At least two sections with two different stains hematoxylin & eosin (H&E, APR immunostain) of paraffin-embedded, ethanol-fixed whole-nodule cross-sections were analyzed. After variables were identified prone to observer discrepancies, we performed a second study to compare consolidated results for 100 nodules obtained by the two readers by microscopy and by analysis of scanned, high resolution digital images (20x magnification). The last data set analyzed was a quality panel of 100 nodules that has been previously examined by microscopy, and included additional immunostains for Wolbachia endobacteria. These slides were digitalized, read by the two assessors and results were compared with original microscopy results. The degree of agreement between assessors varied for different parameters. Agreement for female worm counts in nodules was approximately 80%, while agreement regarding female worm viability was 98%. There were no major differences observed between results obtained by microscopy or digital images. Good agreement for important parameters was also observed for the nodules of the quality panel. Nodule analysis by experienced microscopists was reproducible with regard to important parameters such as identification of living female worms or detection of normal embryogenesis. Assessments varied more for other parameters, and we recommend continued use of two independent readers for detailed analyzes. Analysis of scanned images provided similar results to direct microscopy. This facilitates training and comparison of nodule findings by readers in different locations. Analysis of high quality digital images that can be viewed remotely should improve the quality and availability of nodule assessments that are primary endpoints for onchocerciasis clinical trials.

Morphometric and phylogenetic study of Haemonchus isolates in small ruminants from mountainous (Lorestan) and plain (Khuzestan) regions of Iran

Haemonchus species are among ruminants' important blood-feeding and pathogenic nematodes. This study aimed to determine morphometric and phylogenetic characteristics of Haemonchus spp. isolates from sheep and goats in two different regions of Iran. A total of 340 isolates of adult Haemonchus spp. from the animals of Khuzestan and Lorestan provinces were identified using morphometric and molecular characteristics. Differentiating functions (DF) of male Haemonchus spp. as well as vulvar morphology and flap diversity for female Haemonchus spp. were determined. The twenty-four samples of Haemonchus contortus that showed the most phenotypic differences were analyzed for the sequencing of two genes, ITS-2 and ND4. Since the DF was less than 0.63, all male isolates were H. contortus. The results showed significant differences in some morphometric characteristics of males and females of H. contortus in animals of two regions. H. contortus female worms showed smooth (2–20 %), knobbed (32–65 %), and linguiform (20–66 %) forms as the lowest vulvar morphs and most common vulvar flap types. Phylogenetic and sequence analysis of ITS2 and ND4 sequences revealed the presence of five and three genotypes for all the isolates, respectively. Morphometric and molecular findings in the present study confirmed H. contortus as the only species in small ruminants from two regions of Iran. The morphometric indices of male and female isolated worms of H. contortus were significantly different concerning the species of animal (goat and sheep) and the geographical region. This study showed that ND4 is more valuable for studying the diversity and phylogeny of H. contortus than ITS2. In this study, there was no correlation between genotypes of different isolates and morphological characteristics, indicating variations in genotypes did not appear to affect the phenotype of H. contortus.

Relationship Between Child Phbs Behavior With Enterobiasis In Rt. 09/07 Susukan Village, East Jakarta

Enterobius vermicularis known as pinworms is an intestinal parasite that is disturbing and is still a public health problem worldwide, especially in children aged 6-10 years. Migration of female worms in the perianal area, especially at night, can disrupt children's sleep hours, and affect health in general. This study aims to see the correlation of clean and healthy lifestyle behavior (PHBS) in children with the presence of E. vermicularis in pre-school and elementary school age children. The method used is quantitative with a cross sectional approach. Examination results of 59 anal swab samples, one of which was positive for enterobiasis (1.7%). Filling in the child's PHBS behavior questionnaire, obtained from parental information and researchers' observations. Enteribiasis positive samples were found in pre-school children with poor PHBS behavior. Statistical tests showed that there was a significant correlation (p &lt;0.05) in the incidence of enterobiasis with poor PHBS behavior in children.

Genetic difference between two Schistosoma japonicum isolates with contrasting cercarial shedding patterns revealed by whole genome sequencing

Schistosoma japonicum is one of the major infectious agents of human schistosomiasis, mainly endemic in China and the Philippines. We have previously reported the finding of two schistosome isolates, each with a different cercarial emergence pattern adapted to their different hosts. However, there are currently no whole-genome sequencing studies to investigate the underlining genetics of the adaptive traits. We sampled schistosomes in 2013 and 2020 from a hilly area Shitai (ST) and a marshland area Hexian (HX) of Anhui, China. Ten to 15 male or female adult worms from each site/year were sent for whole genome sequencing. Genetics were analyzed, and selection signals along genomes were detected. Gene enrichment analysis was performed for the genome regions under selection. The results revealed considerable genetic differentiation between the two isolates. The genome “windows” affected by natural selection were fewer in ST (64 windows containing 78 genes) than in HX (318 windows containing 276 genes). Twelve significantly enriched genes were identified in ST, but none in HX. These genes were mainly related to specific DNA binding and intercellular signaling transduction. Some functional region changes identified along the genome of the hilly schistosome may be related to its unique late afternoon cercarial emergence.

Prevalence and magnitude of gastrointestinal helminth infections in cage-free laying chickens in Australia.

Helminth infections have been re-emerging with the growing popularity of free-range and floor-based chicken production systems. The aim of this study was to determine the prevalence and worm burdens of intestinal helminth infection in cage-free laying chickens in Australia. In an online survey about worm prevalence, a high proportion of respondents reported the detection of Ascaridia galli (77%), followed by tapeworms (69%) and caecal worms (Heterakis gallinarum) (62%), whereas fewer respondents (23%) reported the presence of hair worms (Capillaria spp.) in their flocks. Total worm recovery from 407 laying hens on four farms found that 92.1% of hens harboured one or more helminth parasite with a prevalence of 73 to 100% across farms. Mixed infections were common with 79% of hens harbouring two or more helminth species. The prevalence of nematode species H. gallinarum, A. galli and Capillaria spp. was 87, 82 and 35% respectively. Five cestode species were found with a low individual chicken prevalence (Raillietina tetragona 4.7%, Raillietina echinobothrida 3.2%, Raillietina cesticillus 5.2%, Choanotaenia infundibulum 4.4%, and Hymenolepis cantaniana 4.4%). The hens harboured an average of 71 worms with H. gallinarum having the highest mean burden (45.5 worms/hen) followed by A. galli (22.0 worms/hen), Capillaria spp. (2.7 worms/hen) and cestodes (0.8 worms/hen). The sex ratio (female:male worms) was 1.38:1 for A. galli, and 1.77:1 for H. gallinarum. There was a strong positive correlation between A. galli female worm count and excreta egg count (EECs) (rs=0.94, P<0.0001) and also between total nematode worm count and EEC (rs=0.82, P<0.0001) in individual hens. When investigating intestinal excreta (n=10) and caecal excreta (n=10) of 16 chicken flocks the prevalence of infection with ascarid worms in intestinal and caecal excreta was 71 and 78% respectively and 27% prevalence of Capillaria spp. in intestinal excreta with mean EECs of 407, 404, and 18 eggs/g of excreta (EPG), respectively. These results suggest that most chickens kept in free-range or floor production systems are infected with one or more helminth parasite species. Heavy worm infections would likely affect the production performance and welfare of birds with adverse economic impact. Strategic or tactical anthelmintic treatment with effective anthelmintic could reduce this impact.

Discovery of new Schistosoma mansoni aspartyl protease inhibitors by structure-based virtual screening.

Schistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma, with a limited treatment, mainly based on the use of praziquantel (PZQ). Currently, several aspartic proteases genes have already been identified within the genome of Schistosoma species. At least one enzyme encoded from this gene family (SmAP), named SmCD1, has been validated for the development of schistosomicidal drugs, since it has a key role in haemoglobin digestion by worms. In this work, we integrated a structure-based virtual screening campaign, enzymatic assays and adult worms ex vivo experiments aiming to discover the first classes of SmCD1 inhibitors. Initially, the 3D-structures of SmCD1, SmCD2 and SmCD3 were generated using homology modelling approach. Using these models, we prioritised 50 compounds from 20,000 compounds from ChemBridge database for further testing in adult worm aqueous extract (AWAE) and recombinant SmCD1 using enzymatic assays. Seven compounds were confirmed as hits and among them, two compounds representing new chemical scaffolds, named 5 and 19, had IC50 values against SmCD1 close to 100 μM while presenting binding efficiency indexes comparable to or even higher than pepstatin, a classical tight-binding peptide inhibitor of aspartyl proteases. Upon activity comparison against mammalian enzymes, compound 50 was selective and the most potent against the AWAE aspartic protease activity (IC50 = 77.7 μM). Combination of computational and experimental results indicate that compound 50 is a selective inhibitor of SmCD2. Compounds 5, 19 and 50 tested at low concentrations (10 uM) were neither cytotoxic against WSS-1 cells (48 h) nor could kill adult worms ex-vivo, although compounds 5 and 50 presented a slight decrease on female worms motility on late incubations times (48 or 72 h). Overall, the inhibitors identified in this work represent promising hits for further hit-to-lead optimisation.

Transcriptome profiling of male and female Ascaris lumbricoides reproductive tissues

BackgroundAscaris lumbricoides causes human ascariasis, the most prevalent helminth disease, infecting approximately 1 billion individuals globally. In 2019 the global disease burden was estimated to be 754,000 DALYs and resulted in 2090 deaths. In the absence of a vaccination strategy, treatment of ascariasis has relied on anthelminthic chemotherapy, but drug resistance is a concern. The propensity for reinfection is also a major challenge to disease control; female worms lay up to 200,000 eggs daily, which contaminate surrounding environments and remain viable for years, resulting in high transmission rates. Understanding the molecular mechanisms of reproductive processes, including control of egg production, spermatogenesis, oogenesis and embryogenesis, will drive the development of new drugs and/or vaccine targets for future ascariasis control.MethodsTranscriptome profiles of discrete reproductive and somatic tissue samples were generated from adult male and female worms using Illumina HiSeq with 2 × 150 bp paired-end sequencing. Male tissues included: testis germinal zone, testis part of vas deferens, seminal vesicle and somatic tissue. Female tissues included: ovary germinal zone, ovary part of the oviduct, uterus and somatic tissue. Differentially expressed genes (DEGs) were identified from the fragments per kilobases per million reads (FPKM) profiles. Hierarchical analysis was performed to identify tissue-specific genes. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to identify significant terms and pathways for the DEGs.ResultsDEGs involved in protein phosphorylation and adhesion molecules were indicated to play a crucial role in spermatogenesis and fertilization, respectively. Those genes associated with the G-protein-coupled receptor (GPCR) signaling pathway and small GTPase-mediated signal transduction pathway play an essential role in cytoskeleton organization during oogenesis. Additionally, DEGs associated with the SMA genes and TGF-β signaling pathway are crucial in adult female embryogenesis. Some genes associated with particular biological processes and pathways that were identified in this study have been linked to defects in germline development, embryogenesis and reproductive behavior. In the enriched KEGG pathway analysis, Hippo signaling, oxytocin signaling and tight junction pathways were identified to play a role in Ascaris male and female reproductive systems.ConclusionsThis study has provided comprehensive transcriptome profiles of discrete A. lumbricoides reproductive tissue samples, revealing the molecular basis of these functionally important tissues. The data generated from this study will provide fundamental knowledge on the reproductive biology of Ascaris and will inform future target identification for anti-ascariasis drugs and/or vaccines.Graphical

Hypoxanthine guanine phosphoribosyl transferases SmHGPRTases functional roles in Schistosoma mansoni.

Extracellular/environmental stimuli trigger cellular responses to allow Schistosoma sp. parasites adaptation and decide development and survival fate. In this context, signal transduction involving eukaryotic protein kinases (ePKs) has an essential role in regulatory mechanisms. Functional studies had shown the importance of MAPK pathway for Schistosoma mansoni development. In addition, early studies demonstrated that Smp38 MAPK regulates the expression of a large set of genes, among them the hypoxanthine-guanine phosphoribosyl transferase 1 (SmHGPRTase 1, Smp_103560), a key enzyme in the purine salvage pathway that is part of a family comprising five different proteins. First, the regulation of this gene family by the MAPKs pathways was experimentally verified using Smp38-predicted specific inhibitors. In silico analysis showed significant differences in the predicted structure and the domain sequence among the schistosomal HGPRTase family and their orthologs in humans. In order to interrogate the HGPRTases (Smp_103560, Smp_148820, Smp_168500, Smp_312580 and Smp_332640, henceforth SmHGPRTase -1, -2, -3, -4, -5) functional roles, schistosomula, sporocysts, and adult worms were knocked-down using specific dsRNAs. Our results suggest that SmHGPRTases activity has an essential role in sporocysts and schistosomula development since significant differences in viability, size, and/ or shape were observed after the in vitro knockdown. Also, the knockdown of SmHGPRTases in schistosomula influenced the ovary development and egg maturation in female adult worms during mammalian infection. We also observed alterations in the movement of female adult worms knocked-down in vitro. Most of these results were shown when all gene family members were knocked-down simultaneously, suggesting a redundant function among them. Thus, this study helps to elucidate the functional roles of the SmHGPRTase gene family in the S. mansoni life cycle and provides knowledge for future studies required for schistosomiasis treatment and control.

Comparison of pathogenicity and gene expression profiles between adult Schistosoma japonicum isolated from hilly and marshland and lake regions of Anhui Province

To compare the differences in pathogenicity and gene expression profiles between adult Schistosoma japonicum isolated from hilly and marshland and lake regions of Anhui Province, so as to provide the scientific evidence for formulating the precise schistosomiasis control strategy in different endemic foci. C57BL/6 mice were infected with cercariae of S. japonicum isolates from Shitai County (hilly regions) and Susong County (marshland and lake regions) of Anhui Province in 2021, and all mice were sacrificed 44 days post-infection and dissected. The worm burdens, number of S. japonicum eggs deposited in the liver, and the area of egg granulomas in the liver were measured to compare the difference in the pathogenicity between the two isolates. In addition, female and male adult S. japonicum worms were collected and subjected to transcriptome sequencing, and the gene expression profiles were compared between Shitai and Susong isolates of S. japonicum. The differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The total worm burdens [(14.50 ± 3.96) worms/mouse vs. (16.10 ± 3.78) worms/mouse; t = 0.877, P = 0.392], number of female and male paired worms [(4.50 ± 0.67) worms/mouse vs. (5.10 ± 1.45) worms/mouse; t = 1.129, P = 0.280], number of unpaired male worms [(5.50 ± 4.01) worms/mouse vs. (5.60 ± 1.69) worms/mouse; t = 0.069, P = 0.946], number of eggs deposited in per gram liver [(12 116.70 ± 6 508.83) eggs vs. (16 696.70 ± 4 571.56) eggs; t = 1.821, P = 0.085], and area of a single egg granuloma in the liver [(74 359.40 ± 11 766.34) µm2 vs. (74 836.90 ± 13 086.12) µm2; t = 0.081, P = 0.936] were comparable between Shitai and Susong isolates of S. japonicum. Transcriptome sequencing identified 584 DEGs between adult female worms and 1 598 DEGs between adult male worms of Shitai and Susong isolates of S. japonicum. GO enrichment analysis showed that the DEGs between female adults were predominantly enriched in biological processes of stimulus response, cytotoxicity, multiple cell biological processes, metabolic processes, cellular processes and signaling pathways, cellular components of cell, organelles and cell membranes and molecular functions of binding and catalytic ability, and KEGG enrichment analysis showed that these DEGs were significantly enriched in pathways of vascular endothelial growth factor signaling, glutathione metabolism, arginine and proline metabolism. In addition, the DEGs between male adults were predominantly enriched in biological processes of signaling transduction, multiple cell biological processes, regulation of biological processes, metabolic processes, development processes and stimulus responses, cellular components of extracellular matrix and cell junction and molecular functions of binding and catalytic ability, and these DEGs were significantly enriched in pathways of Wnt signaling, Ras signaling, natural killer cells-mediated cytotoxicity, extracellular matrix-receptor interactions and arginine biosynthesis. There is no significant difference in the pathogenicity between S. japonicum isolates from hilly and marshland and lake regions of Anhui Province; however, the gene expression profiles vary significantly between S. japonicum isolates.

Juglone: "A novel immunomodulatory, antifibrotic, and schistosomicidal agent to ameliorate liver damage in murine schistosomiasis mansoni".

Schistosomiasis remains one of the world's leading health concerns, affecting millions. The granulomatous reaction is the most significant immunopathological change associated with Schistosoma mansoni infection, resulting in significant mortality. Recent progress has been made in the search for new natural compounds to reduce schistosomiasis and its immunopathology. Walnuts contain the phenolic compound Juglone (5-hydroxy-1,4-naphthoquinone), which has antiparasitic, anti-inflammatory, immunoregulatory, and antioxidant properties. There were three groups of infected mice: untreated (IU), Juglone-treated (JUG T), and praziquantel-treated (PZ). In mice treated at 8mg Juglone /kg body weight, a reduction of 63.1% and 52.1% were observed in the number of male and female worms, respectively. In addition, the number of eggs/g tissue was reduced by 65.7% in the liver, 58.58% in the intestine, and 62.31% in the liver and intestine combined. In addition, Juglone decreased hepatic granuloma size by 55.1% and collagen fiber deposition by 23.4% compared to PZQ (41.18% and 11.2%, respectively). Interestingly, the JUG T group had significantly lower levels of IL-4, IL-13, IL-37, TNF-α, TGF-β, and IFN-γ than PZ mice (p<0.05). While IL-10 and IL-17 levels rose (p<0.01), Juglone could restore hepatic ALT, AST, GGT, and LDH activities following infection. In addition, it increased catalase, SOD, GSH, and GST while decreasing NO and LPO in comparison to the infected group. Moreover, anti-SWAP, SEA, and CAP IgG levels increased significantly. IgE levels did not change significantly, however. Juglone could be used as an antifibrotic, immunomodulatory, and schistosomicidal agent; thus, it could be used in place of PZQ.

A metabotropic glutamate receptor affects the growth and development of Schistosoma japonicum.

Schistosomiasis is a zoonotic parasitic disease caused by schistosome infection that severely threatens human health. Therapy relies mainly on single drug treatment with praziquantel. Therefore, there is an urgent need to develop alternative medicines. The glutamate neurotransmitter in helminths is involved in many physiological functions by interacting with various cell-surface receptors. However, the roles and detailed regulatory mechanisms of the metabotropic glutamate receptor (mGluR) in the growth and development of Schistosoma japonicum remain poorly understood. In this study, we identified two putative mGluRs in S. japonicum and named them SjGRM7 (Sjc_001309, similar to GRM7) and SjGRM (Sjc_001163, similar to mGluR). Further validation using a calcium mobilization assay showed that SjGRM7 and SjGRM are glutamate-specific. The results of in situ hybridization showed that SjGRM is mainly located in the nerves of both males and gonads of females, and SjGRM7 is principally found in the nerves and gonads of males and females. In a RNA interference experiment, the results showed that SjGRM7 knockdown by double-stranded RNA (dsRNA) in S. japonicum caused edema, chassis detachment, and separation of paired worms in vitro. Furthermore, dsRNA interference of SjGRM7 could significantly affect the development and egg production of male and female worms in vivo and alleviate the host liver granulomas and fibrosis. Finally, we examined the molecular mechanisms underlying the regulatory function of mGluR using RNA sequencing. The data suggest that SjGRM7 propagates its signals through the G protein-coupled receptor signaling pathway to promote nervous system development in S. japonicum. In conclusion, SjGRM7 is a potential target for anti-schistosomiasis. This study enables future research on the mechanisms of action of Schistosomiasis japonica drugs.