larval challenge protocols: the quest for virulence and ... 5 - thursday... · –sandlund et al....
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larvi 2013
ghent university, belgium, 2-5 september 2013
6th fish & shellfish larviculture symposium
Larval challenge protocols:the quest for virulence and verification
Øivind Bergh
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Experimental challengeThe quest for virulence – and protection
Øivind BerghInstitute of Marine Research, Bergen, Norway
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Why challenge?
• Basic questions:– Is ”the bug” virulent?– How does it affect the host?– How does it enter the host?
• Applied:– How can we protect the host– How can we verify that the protection is
effective?
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Immuno-protection
Immuno-suppression
Larval ontogenesis (time)
Vaccination possibleProtection induced by vaccination
No protection
Vadstein, Mo and Bergh 2004
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Challenge experiments on yolksac larvae
•Rearing of larvae in multiwell dishes•72 independent parallel wells•One egg/larvae per well•Larvae hatches in well, lives untilend of yolk sac period•Protocol developed from variouschallenge experiments during twodecades:
–Bergh et al. 1991 J. Fish Dis.–Sandlund et al. 2010 Dis. Aquat. Org.
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Immunohistochemistry
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Immunohistochemistry – yolksac larvae
Intestinalephithelium - cod
Epidermis -halibut
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• Wild type V. splendiduscauses mortality
• Aerolysin negativemutant could not beseparated fromnegative control
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Addition of Roseobacter (Rugeria) 27-4positively affected survival
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Note: probiotics in lumen– NOT attached to epithelium
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Comparative mortality
5 different Vibrio spp3 x V. anguillarumV. splendidusV. salmonicida
3 different fish larvae: turbot, halibut, cod
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Most bacteria associated with larvae areopportunists, not able to cause mortalityaloneV. anguillarum is a ”true” pathogen
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Challenges via live feed= oral administration
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Challenge with V. logei
Intestine 24 hpost challenge
72 hpost challenge
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Gills Intestine
V. splendidus
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•Immunohistochemstry: as in fish larvae•Experimental setup: as in fish larvae
•However – 10-20 larvae/well•Mortality: read as absence of motility
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Thank you for yourattention!