Method for Isolation of Myxozoan Proliferative Stages from Fish at High Yield and Purity: An Essential Prerequisite for In Vitro, In Vivo and Genomics-Based Research Developments.

Ana Born-Torrijos,Joana Pimentel-Santos,Anush Kosakyan,Sneha Patra,Brian Panicucci,Tomáš Korytář,Justin Tze Ho Chan,Astrid S Holzer

doi:10.3390/cells11030377

Abstract

Myxozoans are a diverse group of microscopic cnidarian parasites and some representatives are associated with important diseases in fish, in both marine and freshwater aquaculture systems. Research on myxozoans has been largely hampered by the inability to isolate myxozoan parasites from their host tissues. In this study, we developed and optimized a method to isolate the myxozoan proliferative stages of different size and cellularity from fish blood, using DEAE-cellulose ion exchange chromatography. We optimized several parameters and obtained 99–100% parasite purity, as well as high survival and infectivity. Using polyclonal pan-carp blood cell-specific antibodies, we further developed a rapid cytometric assay for quantification of the proliferative stages, not only in highly concentrated DEAE-C isolates but also in dilute conditions in full blood. Early developmental stages of myxozoans are key to parasite proliferation, establishment, and pathology in their hosts. The isolation of these stages not only opens new possibilities for in vivo and in vitro studies, but also for obtaining purified DNA and protein extracts for downstream analyses. Hence, we provide a long-desired tool that will advance the functional research into the mechanisms of host exploitation and immune stimulation/evasion in this group, which could contribute greatly to the development of therapeutic strategies against myxozoans.

Highlights

Myxozoa is a large and diverse group of microscopic endoparasites belonging to the phylum Cnidaria
S. molnari blood stages were collected from a long-term in vivo culture line, laboratorymaintained for 4+ years in specific pathogen-free (SPF) carp, in which BS have been transferred from fish to fish by intraperitoneal (IP) injection following established protocols [20]
Using optimized DEAE-C parameters, we were able to isolate S. molnari BS from the blood of carp, with an average of 46% and a maximum of 68% recovery of parasites initially counted in infected whole blood, allowing us to isolate up to 28,000,000 BS per ml of blood

Summary

Introduction

Myxozoa is a large and diverse group of microscopic endoparasites belonging to the phylum Cnidaria. Myxozoans are characterized by a two-host life cycle, alternating between invertebrate (mostly annelid) and vertebrate hosts (mostly fishes). They are especially known for the diseases they cause in wild and cultured fishes. Myxozoan outbreaks are of particular importance in the light of climate change, which affects myxozoan geographic distribution and disease severity, leading to new emergence scenarios [1,2,3]. It is of concern that, at present, no licensed treatments or vaccines exist against myxozoans in fish, that are effective and guarantee the safety of products for human consumption. For the development of targeted antiparasitic strategies, research methodologies that can better characterize myxozoans and their host-parasite interactions at the cellular and molecular levels, are urgently required. Since the publication of the first genome [4], myxozoan ‘omics’ has become a rapidly progressing field and primary source for an improved understanding of myxozoan host exploitation mechanisms; contamination from hosts and environmental organisms pose major obstacles for the assembly and analysis of high throughput sequencing datasets [5], which is further complicated by the extremely derived character of myxozoan genomes

Methods

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Discussion

Conclusion