What method alternative to the antibiotic treatment for bivalve larval culture?

Transcription

1 What method alternative to the antibiotic treatment for bivalve larval culture? Philippe Miner, J. L. Nicolas (Ifremer,PFOM, UMR-PE2M Brest,) Régis Lasbleiz, Benoît Sérive Tinduff hatchery

2 Introduction Generally the culture of bivalve larvae does not require antibiotic treatment except Pectinidae (example Pecten maximus, Argopecten purpuratus(chile). Placopecten magellanicus ). These species are very sensitive to bacterial infections (Vibriosis or undetermined bacteria) Antibiotic is preventively used in these cases. However they present adverse effects, mainly the increase of bacterial (plasmidic) resistant to antibiotics and residues in tissues which can induce different troubles at long term exposures (chloramphenicol aplastic anaemia in human ). If chloramphenicol is banned since 1994 some antibiotics are still authorized for aquaculture purposes including flophenicol, flumequine. However the veterinary preparations contain excipient or diluent (50% or more) such as lactose. Is it possible to replace antibiotic by probiotic bacteria or by other methods?

3 What criterions to choose a probiotic? Efficient against several targets Not toxicity for larvae Reliable Easy to produce and to distribute

4 Selection of active marine bacteria Among 887 marine bacteria the most active ones (6%) belonged to Roseobacter group or Pseudoalteromonas Phylogenetic trees constructed from sequences of 16S rdna

5 Antagonism activity of these marine probiotics Test on marine agar Diameter of inhibitory zone(cm ,5 1,5 0,5 A020 BS1 BS3 X34 X129 X6 X20 X s1 R.1588 V. anguillarum V. huître V. splendidus A515 V. pectenicida 1 VB2 V. tapetis Roseobacter galleaciensis (A020, BS1, X34, X129) and the Pseudoaltermonas X153 displayed a broad spectrum of activity against vibrio

6 l o g 1 0 c e l l m l What method and concentration to add probiotic into larval culture? Live bacteria is necessary and at least 10 6 cell ml -1 to exert an antagonism in seawater (It is complex to keep alive marine bacteria) Probiotic at 10 7 ml days control X34 X129 lo g 1 0 c e ll m l Probiotic at 10 6 ml days Antagonism against V. anguillarum control X34 X129

7 Protection of scallop larvae by the probiotic X34 The probiotic X34 cells were broken by ultrasonic treatment before addition to larval culture % of mortality Days after hatching Prob 106 Prob 107 control C+ Cmp

8 Method of production D o no td is se m in at e w ith ou ta ut ho ra ut ho riz at io n In axenic algae Association with the probiotic X34 lasted several months without contamination. X34 reached about the same concentration as T-iso cells (107 cell ml-1). In casamino-acid medium (1g L-1) to add directly to larval tank

9 Protection and stimulation of growth s iz e in µ m Pecten maximus larvae fed on a mixture of 3 algal species of which one (T-iso) was axenic and associated with X34. X34 was at 104cell ml -1 and X129 at 106 cellml -1 Growth Days X34 + cmp X34 CAX129/X34 Control + cmp Control X34+Cmp X34 CA129/X34 Control + Cmp Control CA X34 CAX34 33,3 Mortality at day 16 28, % mortality 32

10 Protection of larvae facing bad quality of sea water C. gigas larvae at day 8 X34 associated with axenic T-iso Growth Mortality AX Algae X153 X34 X34R Conv. Algae G-Skel G-pav G-Iso Conv. Algae Seawater Brest Size in µm 20 0 G-SW

11 Summary about marine probiotic Probiotic bacteria were selected from a large number of isolates. They belonged mainly to Roseobacter and Pseudoalteromonas Pseudoalteromonas (H6, X153) were more active but they were toxic to bivalve larvae. Probiotic cultured in Marine Broth, broken by ultra-sonic treatment protected efficiency scallop larvae (too expensive for hatchery) The probiotic X34 associated with axenic algae protected oyster larvae (C. gigas) and sometimes scallop (P. maximus) larvae. It increased larval growth of scallop larvae. (insufficient protection and reliability) The probiotic X129 cultured in Casamino-acid protected more efficiently scallop larvae than X34 (expensive) Combination of 2 probiotics appeared necessary to replace antibiotic in larval culture of scallop. They appear insufficiently reliable.

12 Other methods Probiotic preparations used for cattle, fishes, shrimps Clay particles to adsorb the organic matter and toxic compounds, Stimulation of filtration and digestion Improvement of growth by a better nutrition Decrease of organic matter by active charcoal Decrease the stress of rearing : Change of rearing method : continuous culture Change of tank design and aeration

13 % mortality Lactobacillus and marketed probiotic preparations Control Control Cmp Mortality to Day 15 D8 D10 D13 D15 BG3 3 strains Sorbial Heated Sorbial Distribution of lactobacillus 10 4 ml -1 at every renewal of sea water Lactobacillus or probiotic preparations based on Lactobacillus were not adapted to scallop larvae.

14 Pecten maximus larvae % mortality Control Cm Clay particles D15 D18 D20 D24 Control Kp 20mg+Cm Kp 20mg Kp 10mg+Cm Kp 10mg Clay (Montmorillonite) particles (mean size 8µm) slow down the mortalities and slightly the growth rate. However larvae are not protected during metamorphosis which is a crucial phase.

15 Elimination of dissolved organic matter 07/06/ /06/ /06/ /06/ /06/ /06/2004 Do not disseminate 13/06/2004 without author authorization 14/06/ /06/ /06/ /06/ /06/ /06/ /06/ /06/ /06/ /06/ /06/ /06/2004 concentrations (mg/l) DOM is at low level in sea water. Charcoal can retain it only when it exceeds 1.5 mg L -1. It can be usefull to eliminate some toxins from harmful algae. dates 1µ 1µ + CA No improvement neither adverse effect were observed

16 Improvement of larval fitness Lengh in µm Scallop larvae fed with different mixture of microalgae: P: Pavlova lutheri, T: T-iso, C: Chaetoceros gracilis, R: Rhodomonas salina PTCc PTCg PTCR PTC Days after fecondation A better adapted diet allows to improve the growth rate. However the scallop larvae receiving Rhodomonas salina did not survive without antibiotic treatment

17 Decrease the stress: Change of tank design? Nbre of scallop cultures Mean of duration until 50% metamorphosis First appearance of significant mortalities Cylindriconical tank (30 to 150L) with aeration days Day 11 2L glass beaker days Day15 Aeration in large tank is necessary to avoid settlement of larvae but increase the stress. In recent experiment without aeration and with another type of tank, larvae were able to metamorphose without antibiotic treatment in continuous culture system. However the output was not sufficient and the handling was important

18 Conclusion The restrictive use of antibiotic as preventive method obliges to change the method of rearing, particularly in taking account the behavior of larvae. In combining probiotic associated with algae, continuous larval culture (towards a complete recycling seawater system), change of tank design, better nutrition (Rhodomonas), we hope to solve the problem of survival in Pectenidae without antibiotic and considerable additional cost.