Schistosoma mansoni Adult Worm Protective and Diagnostic Proteins in n-Butanol Extracts Revealed by Proteomic Analysis

The S. mansoni adult worm n-butanol extract (Sm-AWBE) has been previously shown to contain specific S. mansoni antigens that have been used for immunodiagnosis of schistosomiasis in solid phase alkaline phosphatase immunoassay (APIA) and western blot (WB) analyses. Sm-AWBE was also used in immunoprotection studies against a fatal live-cercariae challenge in experimental mouse vaccination (~43% protection). The Sm-AWBE fraction was prepared by mixing adult worm membranous suspensions with aqueous-saturated n-butanol, centrifuging and recovering n-butanol-resistant proteins in the aqueous phase. Here we report a preliminary identification of Sm-AWBE protein components as revealed from a qualitative proteomic study after processing Sm-AWBE by 1D-gel electrophoresis, in-gel and in-solution tryptic digestions, and mass spectrometry analyses. We identified 33 proteins in Sm-AWBE, all previously known S. mansoni proteins and antigens; among them, immunomodulatory proteins and proteins mostly involved in host–parasite interactions. About 81.8% of the identified Sm-AWBE proteins are antigenic. STRING analysis showed a set of Sm-AWBE proteins configuring a small network of interactive proteins and a group of proteins without interactions. Functional groups of proteins included muscle contraction, antioxidant, GPI-anchored phosphoesterases, regulatory 14-3-3, various enzymes and stress proteins. The results widen the possibilities to design novel antigen combinations for better diagnostic and immunoprotective strategies for schistosomiasis control.

Bioinformatic comparison of Kunitz protease inhibitors in Echinococcus granulosus sensu stricto and E. multilocularis and the genes expressed in different developmental stages of E. granulosus s.s.

Background Cystic and alveolar echinococcosis caused by the tapeworms Echinococcus granulosus sensu stricto (s.s.) and E. multilocularis, respectively, are important zoonotic diseases. Protease inhibitors are crucial for the survival of both Echinococcus spp. Kunitz-type inhibitors play a regulatory role in the control of protease activity. In this study,we identified Kunitz-type domain protease inhibitors(KDPIs) present in the genomes of these two tapeworms and analyzed the gene sequences using computational, structural bioinformatics and phylogenetic approaches to evaluate the evolutionary relationships of these genes. Hi-seq transcriptome analysis showed that E. granulosuss.s. KDPIs were differentially expressed in the different developmental stages. We validated some of the genes expressed in adult worm, protoscolex and cyst germinal membrane of E. granulosuss.s. and E. multilocularis by quantitative PCR. Results A total of 19 genes from E. multilocularis and 23 genes from E. granulosuss.s. were predicted to be KDPIs with the most containing a single Kunitz-domain. A maximum likelihood method phylogenetic tree indicated that the E. granulosuss.s. and E. multilocularis Kunitz domain peptides were divided into three branches containing 9 clusters. The ratio of positively charged residues and neutral residues are different between E. multilocularis and E. granulosuss.s. KDPIs. We also found that E. multilocularis had higher percentage of sequences containing signal peptides (17/19, 89.47%) than that of E. granulosuss.s. (14/23, 60.87%). Transcript analysis showed all the E. granulosuss.s. KDPI genes were expressed differentially in four developmental stages of the worm. Transcription analysis showed that 9 KDPIs (including EG_07244,EGR_08716 and EGR_10096) were highly upregulated in adult worm, and 2 KDPIs (EG_09268 and EG_09490) were highly expressed in the cyst germinal membrane. Quantitative gene expression analysis(qPCR) of four genes confirmed the expression of these genes. EGR_08716 and its homologous gene (EmuJ_001137000) were highly and specifically expressed in adult worms of the two worms. Conclusions A total 19 and 23 KDPIs were identified in the genomes of E. multilocularis and E. granulosus s.s. , respectively. The differential expression of these KDPIs in different stages may indicate their different roles in the different hosts. The difference in characterization of KDPIs may be associated with the different pathology of metacestode stage of these two parasites.

Characterization of Membrane-Associated Progesterone Receptor Component-2 (MAPRC2) from Trichinella spiralis and Its Interaction with Progesterone and Mifepristone

Trichinellosis is a foodborne zoonotic disease caused by Trichinella spp., including Trichinella spiralis. In the present study, T. spiralis membrane-associated progesterone receptor component-2 (Ts-MAPRC2) gene was cloned and characterized using protein sequencing analysis. Furthermore, the expression, purification, immunoblot assay, binding ability with progesterone antibody, and immunofluorescence assay were performed. A direct effect of progesterone (P4) and mifepristone (RU486) on the Ts-MAPRC2 gene was determined using in vitro cell culture that showed different expression levels at all developmental stages (muscle larvae (ML), female adult worm (F-AL), male adult worm (M-AL), and newborn larvae (NBL)). Subsequently, the in vitro phenotypic effects of P4, RU486, and rTs-MAPRC2-Ab on F-AL and ML stages were measured. Later, the in vivo phenotypic effect and relative mRNA expression of mifepristone on the F-AL stage were studied. Our results revealed that the Ts-MAPRC2 gene is critical to maintaining pregnancy in the female adult worm (F-AL) of T. spiralis. The 300 ng/mL of P4 and 100 ng/mL of RU486 showed downregulation of the Ts-MAPRC2 gene in F-AL (p ≤ 0.05). This plays an important role in abortion and possibly decreases the worm burden of T. spiralis in the host. Only 30 ng/mL P4 showed significant upregulation in F-AL (p ≤ 0.05). The current study provides new insights regarding the antihormone (P4 and RU486) drug design and vaccine therapy of recombinant (rTs-MAPRC2) protein as well as their combined effects to control T. spiralis infection.

Multiomic analysis of Schistosoma mansoni reveals unique expression profiles in cercarial heads and tails

Schistosomes require both molluscan and mammalian hosts for development. The larval cercaria exits the snail host and swims to identify and invade the mammalian host. The cercaria has two macrostructures, the head and the tail. The head invades the host, where it matures into an adult worm. The tail is lost after host invasion. Translation in the cercaria differs in each macrostructure, with higher levels of translation in the cercarial tail and little to no translational activity in the cercarial head. We compared the transcriptome and proteome of the cercarial head and tail and observed stark differences between the two macrostructures. We identified unique and differentially expressed transcripts and proteins, including ribosomal components expressed in higher levels in tails than in heads, which may explain the differences in translation levels between heads and tails. We also characterized the weak correlation between transcription and translation in infectious cercarial heads and tails.

An unusual case of genital filariasis of the testicular tunics in an Italian patient

At least 27 million men present with urogenital manifestations of genital filariasis (GF). Although there is a large burden of GF in residents in endemic regions, infection in short-term travellers and in non-endemic areas is rare. We report the case of a 75-year-old Italian man referred to our institution for a testicular discomfort. Clinical examination and ultrasound detected a mass of 40×18 mm in the scrotum without signs of varicocele, hydrocele and testicular or epididymal abnormalities. After ineffective medical treatment, the patient underwent surgical excision. Histology demonstrated filarial granuloma with thick cuticles pathognomonic of an adult worm in the testicular tunics. Surgery has a fundamental role in localized cases of GF to remove nematodes from the fibrotic nodules. The present manuscript describes a singular case of GF observed in Italy in a non-immigrant patient with evidence of remnants of an adult worm in the testicular tunics. SIMILAR CASES PUBLISHED: 11

Characterization of Membrane-associated Progesterone Receptor Component-2 (MAPRC2) From Trichinella spiralis and Its Interaction With Progesterone and Mifepristone

Background: Trichinellosis is a food-borne zoonotic disease caused by nematode viz., Trichinella spiralis. Physiologically, the high progesterone (P4) doses cause new borne larvae (NBL) mortality in the parasite, while the low doses maintain pregnancy. In contrast, Mifepristone (RU486) works as an antagonist against the progesterone receptor (PR) and possesses abortifacient activities. Methods: In the present study, T. spiralis membrane-associated progesterone receptor component-2 (Ts-MAPRC2) gene was cloned and characterized by protein sequencing. Furthermore, the expression, purification, immunoblot assay, binding ability with progesterone antibody, and immunofluorescence assay were performed. A direct effect of progesterone (P4) and mifepristone (RU486) on the Ts-MAPRC2 gene was determined using in-vitro cell culture that showed different expression levels at all developmental stages [muscle larvae (ML), female adult worm (F-AL), male adult worm (M-AL) and new borne larvae (NBL)]. Subsequently, the in-vitro phenotypic effect of P4, RU486, and rTs-MAPRC2-Ab on F-AL and ML stages were measured. Later on, the in-vivo phenotypic effect and relative mRNA expression of mifepristone on the F-AL stage were studied. Results: Our results revealed that the Ts-MAPRC2 gene is critical to maintaining pregnancy in the female adult worm (F-AL) of T. spiralis. The P300 ng/mL of P4 and M100 ng/mL of RU486 showed downregulation of the Ts-MAPRC2 gene in F-AL (P ≤ 0.05). This plays an important role in abortion and possibly decreases the worm burden of T. spiralis in the host. Only P30 ng/mL showed significant upregulation in F-AL (P ≤ 0.05). Conclusions: The current study provides new insights regarding the antihormone (P4 & RU486) drug design and vaccine therapy of recombinant (rTs-MAPRC2) protein as well as their combined effects to control T. spiralis infection.

Patent Macracanthorhynchus ingens infection in a 17-month-old child, Ohio

Limited data exist on Macracanthorhynchus spp. infection and sexual maturation in humans. We report an asymptomatic 17-month-old child with Macracanthorhynchus ingens infection. Eggs in stool and passage of an adult worm indicate parasite maturation and reproduction. M. ingens may have a greater capacity to mature in humans versus M. hirudinaceus.

Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections

Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal–adult-worm killing–treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4–5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.

Chemical analyses and evaluation of the anthelmintic effects of Origanum majorana essential oil, in vitro and in vivo studies

Because of the development of resistance in helminths against major anthelmintic drugs, the search for alternatives is necessary. Medicinal plants are being studied as an alternative source of anthelmintics against gastrointestinal nematodes. The objective of this study is to analyse the chemical composition and evaluate the anthelmintic efficacy of Origanum majorana essential oil. The determination of the chemical composition by gas chromatography/mass chromatography (GC/MS) revealed that the essential oil was dominated by terpenoids, particularly carvacrol (35.65%) and terpenic hydrocarbons p-cymene (15.82%). The in vitro anthelmintic effects against Haemonchus contortus were assessed by an egg hatch assay (EHA) and an adult worm motility assay (AWMA) compared with the reference drug albendazole. The essential oil showed ovicidal activity at all the tested concentrations (1, 2, 4 and 8 mg/ml) and more than 80% egg hatching inhibition was observed at the highest dose (8 mg/ml). Exposure to 0.5 mg/ml of the essential oil for eight hours induced a 50% inhibition in the worm motility. The in vivo study was performed on H. polygyrus by measuring the egg count reduction (ECR) and adult worm count reduction (AWCR) following the treatment of the animals with different doses (2 000, 4 000 and 5 000 mg/kg) of the plant essential oil, and 22 mg/kg of albendazole as the positive control. The results showed that 5 000 mg/kg of the essential oil inhibited the egg count and adult worm count by 76.3 and 74.0%, respectively, seven days post treatment. These findings support the possible use of O. majorana essential oil to control gastrointestinal nematodes.