Vibrio aestuarianus, a pathogen of oyster Crassostrea gigas? Epidemiology study and bacterial characterization

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1 Vibrio aestuarianus, a pathogen of oyster Crassostrea gigas? Epidemiology study and bacterial characterization IFREMER PFOM-UMR-PE2M Brest Matthieu GARNIER - Jean Louis NICOLAS- Céline GARCIA (LGP La Tremblade)

2 Epidemiological study : MOREST Bay of Veys Argenton Morbihan Bouin Quiberon Fouras La Tremblade Arcachon Hatcheries Growing out Experimental sites Sète : Analysis Dying oysters: 53 ( hatchery) and 54 (growout sites) Healty oysters: 190 (growout sites) Hemolymph sampling Plating on marine agar: concentration and diversity of bacteria Genotyping of dominant strains ( RFLP)

3 Occurrence of bacteria in hemolymph nbre oysters S >5 S 1 B >5 B LCB Dying oysters Low conc B>10 5 Type of infection Heathy Dying Healthy oysters Low conc B> S >5 S 1 B >5 B LCB 1 dominant bact- >10 8 no dominant bacteria > dominant bact-10 6 no dominant bacteria <10 5 During summer mortalities hemolymph of dying and healthy oysters contain bacteria. Obviously more in dying bivalves.

4 splen 25% Types of bacterial infection in dying oysters pseud 8% aestu 67% Bacterial species in facilities 100% 80% 60% 40% 20% 0% dominant Facilities Low bact.conc Pure or dominant Mixture Field other 28% splend 28% splend 25% aestua 44% Bacterial species in all oysters aestua 75% Bacterial species in oysters infected by one dominant bact

5 Frequency of vibrios in hemolymph V. aestuarianus V. splendidus group Other vibrios Pseudoalteromonas 60% 50% 40% 30% 20% 10% % of mortality cases 0% V aestuarianus represented 60% of isolates in pure culture and zt high concentration in hemolymph in oysters maintained at9-22 C When isolated in mixture strains were more diversified

6 Phenotypic characterization Cells are motile and curve rods colony are less than 1mm diameter, circular white and translucent (easy to recognize) Growth Optimum : C % 35% 40% 45% 50% 55% 60% 65% 70% 75% 80% 85% 90% 95% 100% /151 V.aest 1---* /008 V.aest I /015 V.aest 1--* /308 V.aest I. I /140 V.aest 75% similarity * 2--* /064 V.aest I I /093 V.aest I I /103 V.aest I 2-* /041 V.aest I /032 V.aest * /323 V.sple I I * /083 V.sple I I 1--* /337 V.sple I I * /034 V.orie I.. I.I V.aest 02/069 V.sple. I I I /114 V.aest I * 1* /089 V.sple * I I /053 V sple I I1---* /004 V.sple I....II.I /003 V.sple Dendrogramm using 50 differential tests V aestuarianus They are phenotypically as diversified as the strains of V. splendidus group Groupe V splendidus

7 16S 20species, 1162 sites (global removal gap ) Neighbor Joining Method Jukes andcantor distance 500bootstrap replicates v_splend v_tasman v_lentus v_pomeroyi 98 v_crassostreae _aj582 v_kanaloae v_chagasii 01_140 02_103 01_031 01_308 02_ _064 03_015 02_114 02_093 01_151 01_312 ATCC _032 Genotypic characterization gyrb 19species, 1158 sites (global removal gap ) Neighbor Joining Method Jukes andcantor distance 500bootstrap replicates v_crassostreae _aj577 v_pomeroyi _aj v_kanaloae _aj v_tasmaniensis _aj v_lentus_aj v_splendidus _aj51529 v_chagasii _aj _008_ATCC _093_gyrb 02_114_gyrb 02_041_gyrb 01_140_gyrb 02_103_gyrb 03_015_gyrb 01_031_gyrb 01_064_gyrb 01_308_gyrb 01_151_gyrb gyr ToxR 35 species, 232 sites (global gap removal) Neighbor Joining Method Jukes and Cantor distance 500 bootstrap replicates On the contrary of V. splendidus group and phenotypic characters the cluster of V. splendidus is highly homogenenous ay ay ay ay ay _034_toxr 04_007_toxr 02_103_toxr 02_041_toxr 01_031_toxr_1 01_031_toxr 02_114_toxr_bis 02_114_toxr 01_064_toxr 02_103_toxr_bis 01_140_toxr_bis 01_140_toxr 01_151_toxr 01_308_toxr 03_008_toxr 02_093_toxr

8 Genotypic characterization : DNA-DNA Hybridization A species is determined at 70% of homology V aest C. gigas Reference Pathogenic Vaestuaranus 01/032 V aestuarianus ATCC / / / / / / / / / / / ATCC Only one species but the homology is below 70% with the reference strain

9 01/308 01/312 02/114 03/008 01/064 01/151 02/093 01/031 01/032 03/015 01/140 02/103 02/041 Virulence of V aestuarianus strains % de mortalités Gradient of virulence No relation between virulence and other phenotypic characters This vibrio can invade oyster and be no virulent

10 Type of infection: septicemia B w e A :Necrosis of muscular fibers (mf) ith ou ta ut ho ra ut ho riz A Hematoxylin-eosin scale bar = 10 µm at io n A to C : Necrosis of muscular fibers of the adductor muscle of Crassostrea gigas. D D o no td is se m in at C B: Image of phagocytosis (arrow) of the muscular debris (md) C: Condensation of muscular fibers D: Epithelium atrophy of digestive diverticules cell atrophy (arrow) of the digestive diverticules (dd). (mf) and clear gap (arrow)

11 Development of Infection The development of infection is slower than infection with V.splendidus Koch postulate is verified. Exposure of Oysters with 02/041 : the most % of mortality virulent V aestuarianus strain days

12 absorbance 0,3 0,25 0,2 0,15 0,1 0,05 0 Effect of Temperature The natural infection is generally triggered at 19 C. 01/032 02/041 22,5 C température ( C) absorbance température ( C) 28 C Two different optima but not related with the virulence

13 bacterial concentration in hemolymph in apparently healthy oysters E+06 1.E+05 1.E+04 2-mars 10-mars 17-mars 25-mars 14-avr. 22-avr. 29-avr. 7-mai 17-mai 25-mai 1-juin 9-juin 16-juin 24-juin 1-juil. 9-juil. 17-juil. 25-juil. 1-août 9-août 19-août 26-août 3-sept. 11-sept. 19-sept. C Cell/ml Température Conc. Bact. Mortality Spawn 1.E+03 1.E+02 The bacteria concentration reached a maximum just before mortality

14 Conclusion Vibrio aestuarianus is an ubiquist bacteria phenotypically diversified but with homogenous genotypic characters. Until now not reported as pathogenic bacterium It can be considered as pathogen/ opportunist in hatchery even the mortalities are not massive, except in spat where it provoked sudden mortality. Its role in summer mortalities remains imprecise since not virulent bacteria can invade dying and apparently healthy oysters. Condition of appearance of infection. Weakness of oyster during maturation phase 19 C was not an absolute limit but favors its growth Invasive bacteria without direct effect on hemocytes but its Extra Cellular Product attack tissues : frequently found in moribund oysters (MOREST) 1983 :Shellfish on the west coast (USA) : reference strain 2002 Sea water of turbot rearing (Spain) 2004 : Flat oyster( Spain) :