Ecytonucleospora hepatopenaei (EHP) is a microsporidian parasite that causes hepatopancreatic microsporidiosis in shrimp, leading to growth retardation and significant economic losses in global shrimp aquaculture. Although EHP does not typically cause high mortality, its substantial economic impact due to severe growth retardation and subclinical infection makes experimental challenge models essential for studying infection dynamics, host pathogen interactions, and mitigation strategies. However, existing EHP challenge models vary widely in methodology, reproducibility, and infection outcomes. This review critically evaluates current experimental challenge models, highlighting methodological differences, advantages, limitations, and critical knowledge gaps to improve standardization and reproducibility. A structured literature review was conducted using major scientific databases to identify studies on EHP challenge models in shrimp and synthesize current methodologies.
EHP challenge models were categorized into five groups: indirect transmission (cohabitation), enteric exposure models, environmental exposure models, artificial systemic models, and alternative hosts and vectors. Cohabitation models represent biologically relevant approaches for natural transmission between infected and na��ve shrimp, although they often introduce variability in infection timing and intensity. In most cohabitation trials, infection was typically detected between 10 and 21 days post inoculation (dpi), with prevalence increasing progressively over time (Tang et al., 2016; Salachan et al., 2017). However, cohabitation models often showed greater variability compared to direct inoculation methods due to limited control over inoculum dose and environmental factors.
Enteric exposure models, including feeding of infected hepatopancreatic (HP) tissue, fecal matter, oral gavage, and reverse gavage, provide biologically relevant routes of infection but are influenced by inoculum quality and shrimp feeding behaviour. Feeding infected HP tissue is one of the most commonly used methods, due to its simplicity and biological relevance with infection typically detected within 7-14 dpi (Salachan et al., 2017; Mai et al., 2020). Fecal string based inoculation (Aranguren Caro et al., 2021,2023) and oral gavage using purified spores (Munkongwongsiri, 2021) approaches demonstrated dose-dependent infection dynamics, although infection efficiency varied across studies. Reverse gavage studies reported higher infection consistency and sustained pathogen proliferation (Mai et al., 2020).
Environmental exposure models, including immersion and waterborne transmission (Pattarayingsakul et al., 2022), provide non-invasive approaches but often result in lower infection consistency and delayed infection onset. Artificial systemic models, such as injection into hepatopancreas, ventral sinus, or muscle, although less biologically relevant, offer controlled experimental conditions for mechanistic studies (Mai et al., 2020). In addition, several studies have explored alternative hosts and potential vectors, including polychaetes, Artemia, and other aquatic organisms, as sources of infection or transmission pathways. However, evidence for sustained replication in these organisms remains limited, and their role in challenge model development requires further validation.
Across studies, infection onset ranged from 5-30 days post challenge, and varied widely depending on inoculation method, inoculum quality, and shrimp developmental stage. Quantitative infection assessment using qPCR is inconsistently applied, and environmental parameters are insufficiently reported. These inconsistencies contribute to inter-study variability and limit reproducibility. Standardization of inoculum preparation, dose quantification, and reporting of experimental conditions is therefore essential to improve challenge model reliability and reproducibility.
This review highlights the urgent need for standardized EHP challenge protocols and proposes key methodological considerations to improve reliability, reproducibility, and comparability of future experimental studies, ultimately supporting the development of effective EHP management strategies in shrimp aquaculture.
Acknowledgment
This research was supported by Flanders Innovation and Entrepreneurship (Belgium) under the Baekeland mandates program (HBC. 2023.0147).
References:
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