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Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycle
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Staffan Svärd)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-7392-1746
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Experimental infections were performed of Atlantic salmon (Salmo salar) from the Baltic Sea region with the Diplomonad fish parasite Spironucleus salmonicida in order to define the infection cycle, specifically the time-line and putative routes of transmission. An oral infection protocol using axenic parasites was developed, as were new diagnostic tools using PCR and specific antibodies. We also produced firefly luciferase expressing S. salmonicida parasites that could be identified in the infected fish using in vivo and ex vivo imaging. The new tools made it possible to follow the S. salmonicida infection cycle in detail. Three different stages of the infection were identified: one initial intestinal stage, followed by a blood stage and a final tissue stage. Parasites intubated into the intestine attached to the intestinal surface and were identified in the blood after 1-3 weeks. Skin lesions and infections of the muscles, internal organs and eyes were seen 4-10 weeks after initiation of infection. Several morphologically different forms of S. salmonicida cells were detected in ex vivo cell-cultures of biopsies from skin lesions. By this infection trial we have been able to show that S. salmonicida may use several alternative routes of transmission. One alternative is the fecal-oral route, similar to other Diplomonad parasites but the parasites can also be excreted directly into the surrounding water from the mucous layer of the skin or from an ulcerated skin lesion. This information can be used to prevent the transmission of the parasite in fish farms.

Keywords [en]
Diplomonads, Spironucleus salmonis, Salmo salar, fish infection, pathology
National Category
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-378925OAI: oai:DiVA.org:uu-378925DiVA, id: diva2:1295066
Funder
Swedish Research Council Formas, 2016-00539Available from: 2019-03-09 Created: 2019-03-09 Last updated: 2019-03-19
In thesis
1. Pathogenesis and Cell Biology of the Salmon Parasite Spironucleus salmonicida
Open this publication in new window or tab >>Pathogenesis and Cell Biology of the Salmon Parasite Spironucleus salmonicida
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Spironucleus species are classified as diplomonad organisms, diverse eukaryotic flagellates found in oxygen-deprived environments. Members of Spironucleus are parasitic and can infect a variety of hosts, such as mice and birds, while the majority are found to infect fish. Massive outbreaks of severe systemic infection caused by a Spironucleus member, Spironucleus salmonicida (salmonicida = salmon killer), have been reported in farmed salmonids resulting in large economic impacts for aquaculture.

In this thesis, the S. salmonicida genome was sequenced and compared to the genome of its diplomonad relative, the mammalian pathogen G. intestinalis (Paper I). Our analyses revealed large genomic differences between the two parasites that collectively suggests that S. salmonicida is more capable of adapting to different environments. As S. salmonicida can infiltrate different host tissues, we provide molecular evidence for how the parasite can tolerate oxygenated environments and suggest oxygen as a potential regulator of virulence factors (Paper III). To further investigate the molecular responses of the parasite and in addition, its host, during infection we set up an interaction system of S. salmonicida and ASK (Atlantic salmon kidney) cells (Paper VI).

To study the cell biology in S. salmonicida we optimized an enzymatic proximity labeling method using ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) (Paper IV). As the system is robust and versatile, we showed the localization and performed ultrastructural characterization of numerous proteins in S. salmonicida and G. intestinalis. We furthermore utilized the APEX system to study the annexin protein family in S. salmonicida (Paper II). Super resolution microscopy and TEM were applied to show that the annexins are mostly associated with cytoskeletal and membranous structures. In addition, we performed phylogenetic analyses concluding that the annexin gene family is expanded in diplomonads.

We performed experimental infection in Atlantic salmon and derived a potential model for the route of infection (Paper V). The results suggested multiple routes of transmission between hosts for the parasite.

To conclude, the comprehensive work in this thesis has provided valuable insights into the pathogenesis and cell biology of the highly adaptable diplomonad parasite S. salmonicida.      

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 70
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1785
Keywords
Diplomonads, Spironucleus, Giardia, genome, annexin, oxygen stress, APEX, pathology
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-379671 (URN)978-91-513-0604-9 (ISBN)
Public defence
2019-05-10, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Research Council Formas
Available from: 2019-04-17 Created: 2019-03-19 Last updated: 2019-06-18

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