BACKGROUND: Cercarial dermatitis (CD), or swimmer's itch, is a water-borne allergic skin reaction caused by the penetration of the larval stages of bird schistosomes (cercariae) into the skin. Members of the genus Trichobilharzia are the primary causative agents of CD worldwide. Due to the increasing number of cases, CD is regarded as a (re)emerging disease. Outbreaks in recreational waters can significantly impact public health and local economies. Environmental monitoring of Trichobilharzia is crucial for outbreak prediction and public health management. However, conventional methods, such as cercarial shedding and snail dissections, are labour-intensive and lack sensitivity. To overcome these limitations, we present a molecular toolkit that combines loop-mediated isothermal amplification (LAMP), quantitative polymerase chain reaction (qPCR), and multiplex PCR for rapid, sensitive, and accurate detection and identification of Trichobilharzia spp. from various biological samples. METHODS: Tricho-LAMP and Tricho-qPCR were designed and optimised for Trichobilharzia DNA detection. A multiplex PCR assay was also developed and optimised to identify the three main species causing CD in Europe (Trichobilharzia franki, T. szidati, and T. regenti). RESULTS: Tricho-LAMP specifically detected T. regenti and T. franki at 10-3 ng, and T. szidati at 10-2 ng per reaction with genomic DNA. Using gBlocks synthetic DNA, Tricho-LAMP achieved 100% amplification at 10,000 copies and 85% amplification at 1000 copies, with decreasing success at lower concentrations. Tricho-qPCR showed the highest sensitivity, detecting all species down to 10-4 ng per reaction and showing a limit of detection at 10 copies of synthetic DNA in the reaction. Multiplex PCR allowed reliable species differentiation via gel electrophoresis of the PCR products, but the assay had the lowest sensitivity. CONCLUSIONS: We provide a molecular toolkit consisting of LAMP, qPCR, and multiplex PCR. By exhibiting high sensitivity, Tricho-LAMP and Tricho-qPCR assays are potentially suitable for environmental DNA (eDNA)-based environmental monitoring of bird schistosomes, by both researchers and public health authorities. Multiplex PCR can be used for species determination without the need for further sequencing.

• Keywords
• Trichobilharzia, Bird schistosomes, Cercarial dermatitis, Detection, LAMP, Monitoring, Multiplex PCR, qPCR,
• MeSH
• Molecular Diagnostic Techniques * methods   MeSH
• DNA, Helminth genetics   MeSH
• Snails parasitology   MeSH
• Trematode Infections * diagnosis   parasitology   veterinary   MeSH
• Real-Time Polymerase Chain Reaction * methods   MeSH
• Multiplex Polymerase Chain Reaction * methods   MeSH
• Birds parasitology   MeSH
• Schistosomatidae * genetics   isolation & purification   classification   MeSH
• Sensitivity and Specificity MeSH
• Nucleic Acid Amplification Techniques * methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Helminth MeSH

BACKGROUND: Venom allergen-like proteins (VALs) are abundant in the excretory-secretory products (ESPs) of numerous parasitic helminths and have been extensively studied for over 30 years because of their potential to interact with host systems. Despite substantial research, however, the precise functions of these proteins remain largely unresolved. Schistosomes, parasites of the circulatory system, are no exception, with 29 SmVAL genes identified in the genome of Schistosoma mansoni to date. The eggs of these parasites, as primary pathogenic agents, interact directly with host tissues and release excretory-secretory products that aid their egress from the host. Although SmVALs have been detected in the egg secretome in the past, direct evidence of their secretion and functional interaction with host molecules has never been demonstrated. These findings fuel the ongoing debate as to whether egg-expressed SmVALs interact with the mammalian host or are rather miracidial proteins synthesized within the egg during larval development. RESULTS: Based on complete revision of the SmVAL family and an associated robust transcriptomic meta-analysis of gene expression across the life cycle, we show that many of SmVAL genes, including 6 newly identified genes, are expressed in the infective larvae-producing stages (eggs and sporocysts). Following localization of two "egg-specific" SmVAL9 and SmVAL29 did not prove active secretion of these molecules into surrounding tissues but were aligned with miracidial structures interfacing with the molluscan host, specifically the larval surface and penetration glands. Finally, we show the complete lack of interactions between candidate SmVAL proteins and an array of 755 human cell receptors via a state-of-the-art SAVEXIS screen. CONCLUSIONS: Overall, we conclude that these "egg" SmVALs are not involved in direct host‒parasite interactions in the mammalian host and are rather proteins employed during intermediate host invasion. Our study revisits and updates the SmVAL gene family, highlighting the limitations of in silico protein function predictions while emphasizing the need for up-to-date datasets and tools together with experimental validation in host-parasite interactions. By uncovering the diversity, expression patterns, and interaction dynamics of SmVALs, we open new avenues for understanding host manipulation and reevaluating orthologous proteins in other helminths.

• Keywords
• CAP, Egg, Miracidium, SAVEXIS, SCP, Schistosomiasis, Transcriptome, Venom allergen-like,
• MeSH
• Allergens * genetics   metabolism   MeSH
• Host-Parasite Interactions * genetics   MeSH
• Humans MeSH
• Ovum * metabolism   MeSH
• Helminth Proteins * genetics   metabolism   MeSH
• Schistosoma mansoni * genetics   metabolism   MeSH
• Gene Expression Profiling MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Allergens * MeSH
• Helminth Proteins * MeSH