salar FED A DIET SUPPLEMENTED WITH MICROBIAL BASED NUCLEOTIDES AND IMMUNOSTIMULANTS WORLD AQUACULTURE 2009, VERACRUZ, MÉXICO

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1 REDUCING Caligus rogercresseyi INFESTATION IN Salmo salar FED A DIET SUPPLEMENTED WITH MICROBIAL BASED NUCLEOTIDES AND IMMUNOSTIMULANTS Javier González, PhD and José Troncoso, MSc WORLD AQUACULTURE 2009, VERACRUZ, MÉXICO

2 Introduction Sea lice (Caligus rogercresseyi) infestation is of main concern for the sustainability of the Chilean salmon farming industry. Estimated costs of lice infestation: Chile, year 2007 = USD 222 millions (Bustos, 2007) Norway, year 2007 = USD 23,5 Caligus infestation significantly reduces fish performance, impairs its osmoregulation and the parasite is a possible vector for fish pathogens like Infectious Salmon Anemia orthomyxovirus, Piscirickettsia salmonis, A. salmonicida, PDv, Microsporidia sp. Moreover, sea lice represent an environmental challenge for wild fish and the benthonic fauna. 2

3 Introduction After the efficacy of the antiparasite emamectin benzoate was reduced due to increase of resistance, other anti-sealice products were authorized by the Chilean authorities (deltamethrin and diflubenzuron). However, in the long term, the control of sealice can not depend upon antiparasitic treatment. Therefore, the current situation requires the implementation of new non-terapeutical alternatives for aid in the control of sea lice infestation and reduce the development of resistance to atiparasite drugs. 3

4 Nucleotides Nitrogen base, 5-carbon sugar, phosphate group Nitrogen base Adenine, Guanine P P P purines 5-C sugar BASE Thymine, Uracil, Cytosine Nucleotides building blocks for DNA & RNA pyrimidines Essential for cell growth and physiological function Most cells in the body can synthesize nucleotides, being liver the main production organ. But some specialised cells CANNOT, as Gut mucosal cells & immune cells Takes cell 6-8 hours to replicate Time reduced 50% with addition of extra nucleotides 4

5 Introduction PGE 2 A combination of bath treatments with pyrethroids and a feed strategy (pre and post bath) with nucleotides (EWOS Boost ) can remove until 50% of the remaining attached stages and reduce the risk of resistance for Lepeophtheirus salmonis (Wadsworth et al., 1999). 5 Figure 1. Feeding EWOS Boost (nucleotides) reduced the levels of attached sea lice (L. salmonis) by 50% when combined with EXCIS pyrethroid treatments.

6 Immunostimulants Definition: - Chemicals, drugs or natural compounds that improve the specific and non-specific immune response of fish to control pathogens. Origin Chemically synthesized Type of immunostimulant Levamisole, FK-565 and muramyl dipeptide (MDP) Extracted from microbes β-glucans, lipopolysaccharides, peptidoglycans, bacterins, Freud adyuvant (FCA) and EF203. Nutritional factors Animal and plant extracts Vitamin C and Vitamin E Alginates and polysaccharides Polysaccharides Hormones Chitosan, Chitin, lentins and oligosaccharides Lactoferrin (LF), growth hormone and citoquins 6

7 How does EWOS IE (Sanictum ) works? Acquired immunity IL-1 T helper cells IL-2 Proliferation and differentiation of T and B cells Citoquins TNFα Secreted by different types of cells Inflammation Neutrophils Macrophages Lisozimes O 2- /H 2 O 2 /OH quemotaxis Opsonization, phagocytosis and cell lysis Innate immunity IE activated Complement (c1 c9) Complement activated Macrófago Bacteria 7 Macrophage action over bacteria

8 Objective of the Study To determine if the efficacy of antiparasites treatments against C. rogercresseyi could be improved by the use of a combination of nucleotides, prebiotics (PB) and immunostimulants (IE) in the diet of Atlantic salmon (S. salar) treated simultaneously with emamectin bonzoate and pyrethroids. 8

9 Material and Methods Fish rearing conditions and feed tested. - Two dietary treatments were applied in a total of six net cages with fish per cage over a period of 94 days from the 17 th of November, 2007 to the 19 th of February, 2008 at Colaco sea site, EWOS Innovation, Chile. The fish initial and final average weights were 317 g and 775 g, respectively. - Three cages were fed a commercial diet (EWOS Omega ) without additives and the others three cages were fed a diet supplemented with EWOS nucleotides (Optimun ), immunostimulant (Sanictum ) and prebiotics (Prebiosal ) in the so called EWOS boost feed. 9

10 Material and Methods Periods of treatments - The study was divided in six time periods depending on the antisealice treatment. During these periods, the fish were fed either the control diet or EWOS boost as explained before. Table 1. Treatments applied in the trail on S. salar against sea lice infestation, Colaco Experimental Station. Date Anti-sealice treatment Period Start Finish Deltamethrin Emamectin bonzoate NO YES YES NO NO NO YES YES NO YES YES YES 10

11 Material and Methods Sampling - Fish were examined for sea lice infestation 14 times during the experiment. The assessment of lice infestation was undertaken before, during and after the anti-sealice treatments. - At each sampling time, 10 or 20 fish per cage were examined to count the number of lice. The parasites were counted according to the stage of development in juveniles, mobile adults, gravid females and total lice. 11

12 Statistical Analyses The data obtained were analyzed statistically with R language and its corresponding packages. The statistical modelling was conducted by generalised linear models using the Poisson family and the log link. Because of the multilevel hierarchical structure of the data a multilevel modelling was applied for the analysis, using the cage and sampling date as grouping factores. The questions to be answered in this trial were: - Does the dietary supplementation with nucleotides, IE and PB (EWOS boost ) reduce the overall sea lice infestation? - Are there any differential effects of EWOS boost on: - the infestation during six subperiods within the study based on various additional sea lice treatments applied? - the different life stages of the sea lice overall or by periods? 12

13 Results Figure 1. Posterior probability distributions of boost effects on different life stages of C. rogercresseyi. The graph shows that overall the supplementation of an IE, nucleotides and PB reduced the infestation rate of C. rogercresseyi in Atlantic salmon (S. salar) in 7%, 9%, 20% and 12% for the juveniles, gravid females, mobile adults and total lice, respectively.

14 Discussion and Conclusions - The combine addition of an IE, nucleotides and PB reduced the infestation of C. rogercresseyi in Atlantic salmon (S. salar) between 7 and 20% when treated with anti-sealice drugs. - The direct mechanism of the supplements over sea-lice infestation was not evaluated in this trial, however, the accumulated knowledge on the immunomodulatory action of nucleotides and IE suggests an effect mediated by a better immune response of the fish against lice. - These results indicate that the efficacy of anti-sealice drugs can be improved with the use of a combination of IE, nucleotides and PB. Therefore, this dietary strategy can be a useful tool to reduce the development of resistance of lice to some antiparasitic drugs. 14