Amit Kumar Sinha - Biofloc Article

AQUACULTURE HEALTH I N T E R N A T I O N A L ISSUE 13

JUNE 2008

NEW CALEDONIA BREAKS

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SHRIMP INBREEDING

ALIEN FISH INDICATE AQUATIC HEALTH BIOFLOC HAS POTENTIAL TO FIGHT INFECTION

CONTENTS 3

ISSUE 13, JUNE 2008

EDITORIAL Finding the key to good biosecurity

4

RESEARCH FOCUS Breaking inbreeding in domesticated shrimp (Litopenaeus stylirostris) in New Caledonia

7

NEWS FEATURE Venezuela passes new law in fisheries and aquaculture

8

RESEARCH FOCUS Horizon scanning: the potential use of biofloc as an anti-infective strategy in aquaculture – an overview

11

NEW PUBLICATION Pearl Oyster Health Management manual on line

12

4

RESEARCH FOCUS The BC Pacific Salmon Forum, Nanaimo, Canada

15

AQUAFEED A news roundup courtesy of the Aquafeed website www.aquafeed.com

16

NEWS Updates from around the globe

22

RESEARCH FOCUS Selective breeding can reduce salmon lice problems

23

EVENTS Forthcoming fish and shellfish events

24

COMMERCIAL FOCUS Cytogenix announces agreement to develop DNA vaccines

28

LABORATORY FOCUS The Central Fish-Health Laboratory in Israel

32

RESEARCH FOCUS What’s new in pancreas diseases research?

34

BOOK REVIEW Aquaculture biosecurity: prevention, control and eradication of aquatic animal disease

22 36

ORNAMENTALS FOCUS A basic overview of multicellular parasitic infections

38

AQUACULTURE HEALTH I N T E R N A T I O N A L ISSUE 13 JUNE 2008

NEW CALEDO NIA BREAKS S

HRIMP INBREE DING

VACCINE STATISTICS Monthly Norwegian vaccine statistics provided by Pharmaq

40

RESEARCH FOCUS Spread and colonisation of alien fish species in open waters: a reliable indicator of aquatic health

36

ON THE COVER A farmed shrimp L stylirosis in New Caledonia is tagged with coloured silicone. .

ALIEN FISH IND AQUATIC HEA ICATE LTH BIOFLOC HA S TO FIGHT INF POTENTIAL ECTION

MAIN PHOTO COURTESY J Patrois, Ifremer INSET PHOTO COURTESY Y Harache, Ifremer

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An informative journal for the aquaculture health professional Published by: VIP PUBLICATIONS LTD 4 Prince Regent Drive Half Moon Bay, Auckland 1706 New Zealand Ph +64 9 533 4336, Fax +64 9 533 4337 Email [email protected] www.aquaculturehealth.com

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GENERAL: Reproduction of articles and materials published in Aquaculture Health International in whole or part, is permitted, provided the source and author(s) are acknowledged. However, all photographic material is copyright and written permission to reproduce in any shape or form is required. Contributions of a nature relevant to the aquaculture industry are welcomed and industry participants are especially encouraged to contribute. Articles and information printed in Aquaculture Health International do not necessarily reflect the opinions or formal position or the publishers unless otherwise indicated. All material published in Aquaculture Health International is done so with all due care as regards to accuracy and factual content, however, the publishers cannot accept responsibility for any errors and omissions which may occur. Aquaculture Health International is produced quarterly.

2 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

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EDITORIAL

FINDING THE KEY TO GOOD BIOSECURITY SCOTT PEDDIE, EDITORIAL DIRECTOR

T

he team at Aquaculture Health International is delighted to bring you the thirteenth issue of the magazine, celebrating our third full year of production. As always, we have endeavoured to bring you a diverse range of articles, and so we have included contributions from Israel, Ireland, Norway, India, New Caledonia, France, Venezuela, Belgium, the United States of America and Canada. This issue of Aquaculture Health International also contains a review on David Scarfe, Cheng-Sheng Lee and Patricia O’Bryen’s excellent book entitled Aquaculture Biosecurity. Although this book was first published in 2006, it is still the most authoritative and comprehensive publication on this subject that I’ve come across to date. Clearly, biosecurity is one of the most important issues for policy makers, regulators, fish health professionals and aquaculturists alike. You only have to follow the unfolding saga of the current ISA outbreak in Chile on the news wires to get a sense of the centrality of this issue to the aquaculture industry. There is, however, one area that we tend to miss when discussing biosecurity. Decision makers quite rightly have a tendency to focus almost exclusively on setting and enforcing norms and standards in this arena, whether it be on an international, national or regional platform. Where we often fall short, and this is an area that is touched on in Scarfe, Lee and O’Bryen’s book, is the issue of the mindset of those at the “coal face” in the industry, namely the staff who work on the farms. I’ve visited farms where the biosecurity procedures have been exemplary on paper but have been half-heartedly adhered to, not because the staff are unaware of company policy, but rather because

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they are “de-motivated” by the environment they are working in. This is perhaps a more serious issue than many fish health professionals are aware of, and it’s to the credit of Scarfe, Lee and O’Bryen that they have included a short chapter on this very topic. I once again find myself calling for a more holistic approach, as I often do in these columns, although this time on a different aspect of aquaculture health management. It seems to me that there is a tendency to compartmentalise the myriad functions involved in managing an aquaculture unit so that training in biosecurity and developing a highly motivated team are seen as having only a tenuous link at best. The bottom line is that a properly motivated and valued workforce is much more likely to take on ownership of the biosecurity process, and this is surely to the advantage of all concerned. After all, suboptimal biosecurity has the potential to seriously impact on public perception of the industry, in addition to a plethora of other negative impacts of the biological and economic variety. So I find myself musing on a number of issues. For example, how can the latest advances in business communication theory be utilised and developed to inculcate a sense of biosecurity awareness? What are the motivational models that can best be applied to the aquaculture environment to enable staff at all levels in the enterprise to promote and foster “good practice?” It seems to me that these issues need to be investigated more thoroughly at both a theoretical and practical level. We need all the tools we can to foster and promote optimal biosecurity in whichever ■ sector of the industry we work in.

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JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 3

BY E GOYARD, C GOARANT, D ANSQUER, F BROUTOI, P BRUN, S DE DECKER, R DUFOUR, C GALINIE, J-R MAILLEZ, JM PEIGNON, D PHAM, E VOUREY, J PATROIS AND Y HARACHE IFREMER, DEPARTEMENT AQUACULTURE EN CALÉDONIE, NOUMÉA CEDEX, NEW CALEDONIA

STYLIROSTRIS

I

n 1993, when temperatures dropped rapidly at the beginning of the cold water season, significant mortalities occurred among the farmed shrimp (Litopenaeus stylirostris) population in New Caledonia. The mortalities, due to Vibrio penaeicida, were locally referred to as “1993 syndrome” and within a few months they had spread to all the farms on the island. Survival rates (pl’s to commercial size shrimps) declined repetitively down to 30-35 percent during the winter, making winter production uneconomical. The industry concentrated on seasonal production during the warm months, with good results, although such an approach created bottlenecks for market availability and peak demand of post-larvae over short periods. At the end of 1997, another sanitary crisis appeared during the warm season in the same farm initially struck with V penaeicida. This was due to a septicaemic summer vibriosis caused by Vibrio nigripulchritudo, which has occurred every year since on the contaminated site. V nigripulchritudo (both non-pathogenic and highly pathogenic strains) was also isolated in several other sites, but was not always associated with mortalities. While V penaeicida isolates show a low genetic variability, the strains of V nigripuchritudo appear highly variable, with striking differences in their pathogenicity level. As New Caledonia’s production certified procedure totally prohibits the use of antibiotics in the pondgrowing phase, the presence of this pathogen was considered a major potential threat to industry expansion. This motivated a specific monitoring policy, undertaken by Ifremer in collaboration with DAVAR, the Nouvelle-Calédonie Veterinary Service. Significant summer mortalities, due to the same pathogen, appeared in 2003 and 2005 in two other farms. However, production continued to increase slowly, reaching 2430 tonnes in 4 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

PHOTO CREDIT: Y HARACHE – IFREMER

SODACAL, A 133HA INDUSTRIAL FARM CREATED IN 1983. PRODUCTION RANGES FROM 320 TONNES TO 490 TONNES

PHOTO CREDIT: Y HARACHE – IFREMER

RESEARCH FOCUS

BREAKING INBREEDING IN DOMESTICATED SHRIMP (LITOPENAEUS STYLIROSTRIS) IN NEW CALEDONIA

2006, with particularly strong exports markets in France, Japan and Australia. The domesticated strain, introduced into the country in the late 1970s and reproduced in captivity since, shows good growth performance profiles and an excellent virus status profile (resistant to IHHNV and free of all other known viruses). However, we were convinced that the low number of founders had induced a high level of inbreeding with an important loss of genetic variability, possibly hampering its adaptability to modifications of the rearing conditions, including resistance to new pathogens. The results of the multi-disciplinary Ifremer DéSans programme pointed out that the outbreaks of seasonal diseases were linked to the instability of the pond ecosystem (sediment and water column) during intensified production cycles, the virulence of two specific pathogens (Vibrio nigripulchritudo or V penaeicida, respectively) and the shrimps’ capacity to resist the stress induced by the interaction of these critical factors. We assumed there was a genetic component to this global resistance capacity. The necessity to re-introduce “new blood” into the broodstock line, even through a costly, complex and risky procedure, appeared unavoidable. This provided the impetus for a range of disease challenge studies using stocks of different genetic origins.

STOCKS TESTED A domesticated SPF strain from Hawaii (High Health Aquaculture Ltd) was purchased by the New Caledonian Industry (UPRAC association), allowing it to test three discrete stocks for their performance: • CC. New Caledonian stock from Ifremer and private hatcheries. A newly constituted CC control line (to reproduce every year) was

created with 16 bi-parental families and reproduced twice in 2005 and 2006. • HH. Hawaiian stock introduced through a quarantine procedure (UPRAC) was reproduced at Ifremer, constituting a new HHG9 and HH-G10 generations structured each in at least 16 biparental families from known genealogy, reducing inbreeding to a minimum. From this G9, batches of juveniles for experimental evaluation of the Hawaii stock were constituted in 2005 and 2006. • Hybrid-F1. HH and CC animals were crossed each year to obtain two first-generation hybrid stocks named F1-2005 and F1-2006 for evaluation of performance, using several families for each combination sex/origin.

EVALUATION OF THE COMPARATIVE PERFORMANCES OF THE DIFFERENT POPULATIONS: PRELIMINARY RESULTS Winter tests in earthen ponds (Ifremer 2006 and 2007) The different groups arising from 2005 (CC-2005, HH-2005 and F1-2005) and 2006 reproduction scenarios (CC-2006, HH-2006 and F1-2006) were reared separately in earthen ponds at the Ifremer station. They were then branded with silicone injections (2.9g to 11g mean weight) and re-mixed in two 500m2 ponds seeded with 10,000 shrimp and reared for 130 days using normal production techniques. The ponds were harvested after the beginning of the cold season, with identification, counting and size sampling of all groups. Under these “competitive” conditions, the average results showed, for both years, a higher survival rate for Hybrid F1 (respectively 60.5 and 53.5 percent) compared to HH (60 and 32.5 percent) and CC (51 and 34.5 percent). Although no moribund shrimp could be observed, V penaeicida was not isolated, but the presence of a few

PHOTO CREDIT: Y HARACHE – IFREMER

COLOUR SILICONE TAGGING OF HAWAII SHRIMP FAMILIES

EXPERIMENTAL FLOATING CAGES ON SITE AT THE AIGUE-MARINE FARM AFFECTED BY OUTBREAKS OF SUMMER VIBRIOSIS

PHOTO CREDIT: J PATROIS – IFREMER

PHOTO CREDIT: Y HARACHE – IFREMER

HAWAII SHRIMPS IN QUARANTINE AT UPRAC

PHOTO CREDIT: J PATROIS – IFREMER

THE IFREMER RESEARCH STATION AT SAINT VINCEN

dead shrimp found following rapidly dropping temperatures was typical of syndrome 93, suggesting a higher resistance by Hybrids-F1 to V penaeicida. The final weight, and the subsequent growth rate, were significantly higher for Hybrid F1 (0.22g and 0.24g/day) than for both parent HH and CC lines (0.15g to 0.17g/day). Summer tests in floating cages (Private farm 2007) Because no summer mortalities caused by V nigripulchritudo had ever been observed in the Ifremer ponds, it was decided, in coordination with the Farmers Association, to compare the results of the three populations in a farm showing typical outbreaks of V nigripulchritudo since 2003. This test was conducted in small floating cages (4m2/3.2m3) installed in a production pond affected by summer mortalities. Shrimps were stocked either as “pure line” cages or under “competition” (mixed population) during the recurrent outbreak period. The final survival after 77 days appeared significantly higher in Hybrids F1 (51 percent) than the parent lines, which expressed no significant difference between CC and HH (41 and 44 percent) with no significant difference between the conditions of testing (“pure line” cages vs “competition” cages). Haemolymph samples taken at day 29 showed a V nigripulchritudo prevalence of 90 to 100 percent with no difference between groups, but a higher number of vibrios in CC samples (50 percent with more than 300 vibrios in 10µl) while 50 percent of HH and F1 bore fewer than 50 pathogens. Further samples did not indicate significant trends in prevalence between groups, but confirmed a higher number of V nigripulchritudo CFUs found in CC haemolymph samples. The average growth rates observed in Hybrids F1 were 40 percent higher than that of the parent lines (0.28g v 0.19g/day), JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 5

EXPERIMENTAL STRUCTURES AND PROGRAM FUNDED THOUGH A 5 YEARS “CONTRAT DE DÉVELOPPEMENT” ESTABLISHED BETWEEN THE STATE OF FRANCE AND THE PROVINCES OF NEW-CALEDONIA

PHOTO CREDIT: J PATROIS – IFREMER

RESEARCH FOCUS

DISEASE CHALLENGE STUDIES OF INBRED AND OUTBRED SHRIMP IN NEW CALEDONIA

THE EXPERIMENTAL INFECTION ROOM FOR STANDARDISED TEST RESISTANCE TO VIBRIO IFREMER STAFF AT SAINT VINCENT, MAY 2007 (PERMANENT STAFF CONTRIBUTING FROM 2002 TO 2006) D ANSQUER, P BOISARD, O BOUISSOU, F BROUTOI, P BRUN, L CHIM, D COATANEA, L DELLA-PATRONA, R DUFOUR, E GOYARD, C GOARANT, L GOURMELEN, Y HARACHE, A HERBLAND, J HERLIN, JS LAM, C LAMBERT, P LEMAIRE, H LEMONNIER, JR MAILLIEZ, AL MARTEAU, A MATEAU, H MICHAUT, C MUGNIER, J PATROIS, JM PEIGNON, J PICHON, D PHAM, E PITA, JM RANOUIL †, E SAULNIER, BG SERY, SOULARD, N WABETE, N WAHMETRUA, B WAPOTRO AND K WASSAUMII

IFREMER SHRIMP PONDS

TEMPORARY STAFF: M CASTEX, C JUSTOU, A LEGRAND, R LUCAS, S DE DECKER, F IMBERT, E VOUREY AND E ZIPPER. INPUTS FROM OTHER IFREMER LABORATORIES OR EXTERNAL PARTNERS: E BACHÈRE, C COURTIES, C CAHU, G CUZON, J DE LORGERYL, C GALINIÉ, F LEROUX, AG MARTIN, JL MARTIN, JC MASSABUAU, D SAULNIER, S VIRLY. ESTABLISHED PARTNERSHIPS WITH IRD, INSTITUT PASTEUR, UNIVERSITÉ DE NOUVELLE-CALÉDONIE, DAVAR (VETERINARY SERVICES OF NOUVELLCALÉDONIE, UNIVERSITÉ DE BORDEAUX ET PARIS VI-BANYULS)

FIGURE 1: Relative performances of Hybrid-F1 population (A) and of the Hawaiian population (B) compared with the Caledonian population Production of Biomass in ponds - year 1

Production of Biomass in ponds - year 2

Production of Biomass in cages

Growth in ponds - year 1

Growth in ponds - year 2

Growth in cages

Survival in ponds - year 1

Survival in ponds - year 2

Survival in cages

with a much better food conversion ratio than the CC line in the testing conditions, though this remains to be confirmed in other conditions.

240%

A

220% 200% 180%

160% 140% 120% 100% 30%

35%

40%

45%

50%

55%

Biomass production in ponds - year 1

Biomass production in ponds - year 2

Biomass production in cages

Growth rate in ponds - year 1

Growth rate in ponds - year 2

Growth rate in cages

Survival in ponds - year 1

Survival in ponds - year 2

Survival in cages

60%

140%

B

120%

Experimental infections with Vibrio penaeicida (Ifremer 2007) Six experimental infection tests were undertaken, three each in 2006 and 2007, in a bio-safe infection room at Ifremer. The experimental population comprising the three tagged populations (CC, HH and Hybrid F1) was reared in an outdoor pond for three weeks. Groups of each population were then introduced in the experimental facility in 16 or 32 x 200-litre tanks. After acclimatisation for one week, the animals were infected by balneation or injection with a reference strain of V penaeicida, following a standardised technique. Natural contamination in 2007 with V nigripulchritudo prior to the V penaeicida experimental infections made the expression of results and the characterisation of a specific resistance to V penaeicida difficult. However, in all experiments, the final survival of hybrids F1 was higher than that of CC and HH, with a significant difference for 2006 experiments (57 percent v 50 and 47 percent respectively).

CONCLUSION

100% 80% 60% 40% 20% 0% 30%

35%

40%

45%

50%

55%

Average values for survival, growth rate and biomass, obtained in ponds and cages, is expressed in % of the values observed in Caledonian shrimps (CC) for the same criteria. (Goyard et al., 2008)

6 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

60%

All the tests indicate that F1 hybrids are characterised by faster growth, independently of the testing conditions: in earthen ponds, in cages, under competition with other strains or alone; while the slower-growing pure strains CC and HH do not show significant differences. Though the present results do not allow a conclusion to be drawn with respect to a specific resistance of the hybrids to the pathogenic Vibrios, it was shown in all experiments that they provide higher survival than any parent population in all cases (pond or cage rearing, winter or summer period and global survival to experimental handling) and infections. Moreover, indications of a lower pathogen

SOURCE: DA CONROY PHD, CBIOL, FIBIOL, EMERITUS PROFESSOR OF FISH PATHOLOGY, FACULTY OF VETERINARY SCIENCES, CENTRAL UNIVERSITY OF VENEZUELA, MARACAY, VENEZUELA

T

▲

he government of Venezuela passed a new Fisheries and Aquaculture Law on March 14 which will have a considerable impact on the future development of its fisheries and aquaculture. The new law, known as Ley de Pesca y Acuicultura, published as Decree Nº 5930 in the Official Gazette, or Gaceta Oficial de la República Bolivariana de Venezuela (GORBV), Nº 5877 (extraordinario), on March 14, is very extensive in its coverage and attributions, as much with reference to “fisheries” as to “aquaculture”. It specifically replaces and leaves without any legal effects the previous laws on fisheries and aquaculture, published in the GORBV Nº 37323 of November 13, 2001, and in the GORBV Nº 37726 of May 22, 2003, respectively. The National Institute of Fisheries and Aquaculture (Instituto Nacional de Pesca y Acuicultura, or INAPESCA), opportunely created by these previous two laws as a dependency of the Ministry of Agriculture and Lands, or MAT, is now officially designated as the Socialist Institute of Fisheries and Aquaculture (Instituto Socialista de Pesca y Acuicultura, or INSOPESCA), which continues to be a dependency of the MAT. The printed version of this new law comprises some 120 pages of text, and its contents should be read very carefully by all those with an interest in aquaculture in Venezuela (preferably with some professional input from the interested parties’ financial, legal and technical advisers, where convenient and/or necessary). The law provides a series of quite clear definitions of the various types of aquaculture activities to which it refers, eg • freshwater and marine • extensive, semi-intensive and intensive, including ponds, tanks,

floating cages and pens • algae, crustaceans, molluscs and finfish, and their larval stages where appropriate • aspects of aquatic animal health, including quarantine matters and genetics and the levels of these activities (eg, artesanal, small-scale operations, larger commercial operations). Tariffs are established for issuing various types of permits, certificates, authorisations and other such “documentation” on a sliding scale that favours artesanal producers and becomes successively more “onerous” for the larger producers. In compliance with the terms of article 47 of Title VI (Communal Responsibility) of the law, for example, aquaculture producers at all levels are now obliged to freely donate, directly and gratuitously to government and local community organisations a minimum of five percent of their production for distribution by those agencies to needy sectors of the population. This article also states quite clearly that the competent authorities can increase this free contribution of five percent as and when deemed necessary. In accordance with the general guidelines as detailed in the text of this new Fisheries and Aquaculture Law, the future development of aquaculture in Venezuela is to be oriented above all else toward improving the “social welfare of the less economically favoured communities” within the country. The contents of this law should be carefully read and studied by any interested parties. Potential investors, in particular, would be strongly recommended to adopt a "caveat emptor" approach before ■ reaching any important decisions in principle.

CONTINUED FROM PAGE 30

of Microbiological Methods 67. pp25-27 load in haemolymph during asymptomatic infections than any of the parent lines were obtained. Although full-scale testing in commercial ponds is essential to reach a final conclusion, the combined average improvements in growth and survival inferred from all experiments would provide a biomass gain ranging from 60 to 130 percent in the historic Caledonian strain, while no difference would occur between parent lines.

SELECTED BIBLIOGRAPHY Goarant C, Ansquer D, Herlin J, Domalain D, Imbert F and De Decker S 2006. “Summer syndrome” in Litopenaeus stylirostris in New Caledonia: Pathology and epidemiology of the etiological agent, Vibrio nigripulchritudo. Aquaculture 253. pp105-113

Goyard E, Goarant C, Ansquer D, Brun P, de Decker S, Dufour R, Galinié C, Peignon JM, Pham D, Vourey E, Harache Y and Patrois J 2008. P. Cross-breeding of different domesticated lines as a simple way for genetic improvement in small aquaculture industries: Heterosis and inbreeding effects on growth and survival rates of the Pacific blue shrimp Penaeus (Litopenaeus) stylirostris. Aquaculture 275. pp43-50 Harache Y, Herbland A 2004. Le programme DéSanS (Défi Santé Stylirostris) appliqué à la filière crevette calédonienne. Trente ans de cerevetticulture en Nouvelle-Calédonie. Nouméa-Koné. June 2-6, 2003. (ed) Ifremer, Actes Colloq, 38, pp280. Saulnier D, Haffner P, Goarant C, Levy P, Ansquer D 2000. Experimental infection models for shrimp vibriosis studies: a review. Aquaculture 191. pp133-144

Goarant C, Reynaud Y, Ansquer D, de Decker S, Saulnier D and Le Roux F 2006. Virulence and molecular epidemiology of Vibrio nigripulchritudo, a pathogen for cultured penaeid shrimp (Litopenaeus stylirostris) in New Caledonia. Systematic and Applied Microbiology 29. pp570-580

Patrois J, Goarant C, Goyard E, Harache Y, Primot P and Bador R 2007b. Blue shrimp quarantined in New Caledonia; genetic variability programme. Global Aquaculture Advocate 10 (5). pp90-92

Goarant C, Merien F 2006. Quantification of Vibrio penaeicida, the etiological agent of Syndrome 93 in New Caledonian shrimp farming, by real-time PCR using SYBR Green I chemistry. Journal

Ifremer, Departement Aquaculture en Calédonie, BP 2059, 98846 Nouméa Cedex, New Caledonia. Contacts: Emmanuel.Goyard@ ifremer.fr or [email protected] ■

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JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 7

NEWS FEATURE

VENEZUELA PASSES NEW LAW IN FISHERIES AND AQUACULTURE

RESEARCH FOCUS

HORIZON SCANNING: THE POTENTIAL USE OF BIOFLOC AS AN ANTI-INFECTIVE STRATEGY IN AQUACULTURE – AN OVERVIEW BY AMIT KUMAR SINHA, KARTIK BARUAH AND PETER BOSSIER LABORATORY OF AQUACULTURE AND ARTEMIA REFERENCE CENTRE, DEPARTMENT OF ANIMAL PRODUCTION, GHENT UNIVERSITY, GHENT, BELGIUM

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ntensification of the aquaculture industry has in some cases been accompanied by environmental pollution (high nitrogen toxicity) and increasing severity of disease outbreaks. The increased level of intensification requires more supplementary protein feed and in turn contributes to nitrogen pollution in the culture system. Decomposition of dead algae, uneaten feed, fish excreta and other organic matter releases excess nitrogen in the form of ammonia and nitrite and amplifies the nitrogen toxicity level. Elevated concentrations of ammonia affect growth, moulting (in shellfish), oxygen consumption and even ammonia excretion, and can eventually cause mortality of fish/shellfish. Increased ambient nitrite concentration negatively affects the growth performance and survival of fish/shellfish (Mallasen and Valenti 2006). Hitherto, many techniques such as photo-autotrophic algae-based systems (green-water systems), frequent water exchange and the use of biofilters have been used to remove excessive nitrogen. However, none of these options has proved to be technically feasible and economically viable. Thus, the quest to develop an economical, eco-friendly and user-friendly strategy to remove or optimise the level of such toxic compounds in the pond water has become a priority.

The breakdown of PHA inside the gastrointestinal tract can be carried out via enzymatic and chemical hydrolysis One such strategy for removing ammonium nitrogen from water is through its assimilation into microbial proteins by adding carbonaceous material into the system. Nitrogenous compounds present in the water combine covalently with carbon and produce flocs of protein molecules. These small protein molecules flocculated together form a floating mass called bioflocs; these flocs are dominated by bacteria. An important aspect of this process is the potential utilisation of microbial protein as a source of protein for fish and shrimps. The success of this technique depends upon optimising the amount of carbonaceous material to be added. Recently, it was observed that excess addition of the carbon source enhances the development of polyhydroxyalkanoate (PHA) accumulating micro-organisms. The properties of PHAs are very similar to those of organic acid and have been proved to be effective bio-control agents, given that they beneficially affect the host’s microbial balance in the gut. 8 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

PRINCIPLE Bacteria and other micro-organisms require carbohydrates for their growth. As protein is the major component of new cell material, nitrogen is also required as an essential element. Thus, microbial utilisation of carbohydrate or any other low nitrogen feed is accompanied by the immobilisation of inorganic nitrogen. This is the basic process carried out by the microbial community.

BIOFLOC AND WATER QUALITY IMPROVEMENT Nitrogen control is stimulated by feeding bacteria with carbohydrates, and through the subsequent uptake of nitrogen from the water, by the synthesis of microbial protein. The relationship between adding carbohydrates, reducing ammonium and producing microbial proteins depends on the microbial conversion coefficient, the C/N ratio in the microbial biomass and the carbon contents of the added material (Avnimelech 1999). The C/N ratio has been widely used as an index of the rate at which organic matter decomposes. If the organic matter is low in nitrogen content (ie a high C/N ratio), some of the nitrogen for microbial growth will be obtained from the water column and will become immobilised as microbial protein. Previous studies have shown that the immobilisation of inorganic nitrogen only occurs when the C/N ratio of the organic matter is higher than 10 (Lancelot and Billen 1985). However, Avnimelech (1999) verified that to eliminate inorganic nitrogen, the C/N ratio in pond should be 10.75, rising to 15.78 when fish are introduced into the holding pond.

BIOFLOC AS FISH NUTRIENTS The bacterial protein and new cells (single-cell protein) synthesised by the heterotrophic bacterial population are utilised directly as a food source by the cultured organisms (carp, tilapia, shrimp), thus lowering the demand for supplemental feed protein (Avnimelech 1999). Hari et al (2004) reported that Penaeus monodon could effectively utilise the additional protein derived from the increased bacterial biomass as a result of carbohydrate addition. Burford et al (2004) suggested that “flocculated particles” rich in bacteria and phytoplankton could contribute substantially to the nutrition of the Litopenaeus vannamei in intensive shrimp ponds.

BIOFLOC AS AN ANTI-MICROBIAL AGENT It was observed that the regular addition of carbon to the culture is known to select for polyhydroxyalkanoate (PHA) accumulating bacteria (Salehizadeh and Von Loosdrecht 2004) such as Alcaligenes eutrophus, Azotobacter vinelandii, Pseudomonas oleovorans and others that synthesise PHA granules.

THE DEVELOPMENT OF MICROBIAL FLOCS AFTER THE ADDITION OF CARBON SOURCE

Such granules are synthesised under conditions of nutrient stress, that is, when an essential nutrient like nitrogen is limited in the presence of an excess carbon source. (Avnimelech 1999). These PHAs are polymers of β-hydroxy short chain fatty acids and if degraded in the gut, they could have antibacterial activity similar to short chain fatty acids (SCFAs) or organic acids. The breakdown of PHA inside the gastrointestinal tract can be carried out via enzymatic and chemical hydrolysis (Yu et al 2005). Enzyme hydrolysis is generally carried out by PHA depolymerases produced by various micro-organisms such as Aspergillus fumigatus, Pseudomonas fluorescens, Comamonas sp and others (Khanna and Srivastava 2004). Chemical hydrolysis can be carried out by treating the PHA with NaOH (Yu et al 2005). Pre-treatment of PHA polymer with NaOH can significantly accelerate the digestibility of PHA polymer. For example, an increase of up to 85 percent was demonstrated in pigs (Forni et al 1999). The working mechanism of SCFA with respect to their antibacterial activity is not well understood. It could, however, be related to the reduction of pH, as well as their ability to dissociate, or the pH of the surrounding milieu (Ricke et al 2003). The antibacterial activity increases with decreasing pH value. SCFA in undissociated form is able to pass through the cell membrane of bacteria (Cherrington et al 1991) and once internalised into the neutral pH of the bacterial cytoplasm, it dissociates into anions and protons. These ions influence the stability of functional macro-molecules and inhibit the action of important enzymes. Bacteria need to pump out excess protons to maintain the neutral pH of the cytoplasm. Removal of protons requires consumption of cellular ATP and may deplete the cellular energy and thus growth is inhibited. Another possibility is that SCFA may interfere with membrane structure and membrane protein in such a way that electron transport is uncoupled and ATP production is diminished. Russel (1992) speculated that anion accumulation is the primary reason for the antimicrobial effect of organic acid. Moreover, less direct antimicrobial activities have also been attributed to SCFA, which include interference with nutrient transport, cytoplasmic membrane damage resulting in leakage, disruption of outer membrane permeability and hindrance in macromolecule synthesis (Ricke 2003). Apart from inhibiting the growth of pathogenic bacteria by lowering the pH of surrounding milieu, SCFA have also been shown to specifically down-regulate virulence factor expression and positively influence the gut health of animals (Teitelbaum and Walker 2002). Moreover, these compounds are capable of

exhibiting bacteriostatic and/or bacteriocidal properties, depending on the physiological status of the host and the physiochemical characteristics of the external environment (Ricke 2003). These indicate that biofloc can be a novel strategy for disease management on a long-term basis, in contrast to conventional approaches such as antibiotic, probiotic and prebiotic application, especially since in many cases the efficacy of the latter two remains to be demonstrated. Short chain fatty acids have been known for a long time to

JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 9

inhibit the growth of pathogenic bacteria and they also have been used in commercial diets of terrestrial animals to control pathogens such as Salmonella and E coli (Van Immerseel et al 2005) but their use in aquafeed is very limited (on a commercial scale). Recently, Defoirdt et al (2006) reported increased survival of Artemia nauplii when fed formic, acetic, propionic, butyric and valeric acid and challenged with a luminescence pathogenic Vibrio campbellii strain. In another study, the same author (Defoirdt et al 2007) reported that commercial polyhydroxy butyrate (PHB) particles or PHB accumulating bacteria offered a preventive and curative protection to Artemia against luminescent vibriosis. They observed complete protection at a level of 1000mg/l of commercial PHB or 107 cells/ml of PHB accumulating bacteria. Although there are no reports of direct use of biofloc (PHA or SCFA) as antimicrobial neutraceuticals in fish feed, many projects have and are focussing on the characterisation and optimalisation of PHA production by these flocs and analysing their bio-control efficacy in different host-microbe systems.

P and Verstraete W 2007. The bacterial storage compound polyb-hydroxybutyrate protects Artemia franciscana from pathogenic Vibrio campbellii. Environmental Microbiology 9. pp445-452

CONCLUSION

Mallasen M and Valenti WC 2006. Effect of nitrite on larval development of the giant river prawn, Macrobrachium rosenbergii. Aquaculture 261. pp1292-1298

In essence, biofloc technology could be useful in improving the sustainability of fish/shellfish farming in both extensive and modified extensive culture systems. There is clearly scope for further improvement of this management strategy, not only by optimising the quantity of carbohydrate addition at various intensities of culture, but also by comparing the potential of other carbohydrate sources. Additional research is required with respect to feed composition planning and feeding rate determination.

REFERENCES Avnimelech Y 1999. C/N ratio as a control element in aquaculture systems. Aquaculture 176. pp227-235 Burford MA, Thompson PJ, McIntosh PR, Bauman RH and Pearson DC 2004. The contribution of flocculated material to shrimp, Litopenaeus vannamei nutrition in a high-intensity, zero-exchange system. Aquaculture 232. pp525-537 Cherrington CA, Hinton M, Pearson GR and Chopra I 1991. Short chain organic acids at pH5 kills Escherichia coli and Salmonella spp without causing membrane perturbation. Journal of Applied Bacteriology 70. pp161-165 Defoirdt T, Halet D, Sorgeloos P, Bossier P and Verstraete W 2006. Short-chain fatty acids protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii. Aquaculture 261. pp804-808 Defoirdt T, Halet D, Vervaeren H, Boon N, Wiele T, Sorgeloos P, Bossier

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Forni D, Bee G, Kreuzer M and Wenk C 1999. Novel biodegradable plastics in sheep nutrition 2: Effect of NaOH pre-treatment of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) on in-vivo digestibility and in-vitro disappearance (Rusitec). Journal of Animal Physiology and Animal Nutrition 81. pp41-50 Hari B, Kurup BM, Varghese JT, Schrama JH and Verdegem MCJ 2004. Effects of carbohydrate addition on production in extensive shrimp culture system. Aquaculture 241. pp179-194 Khanna S and Srivastava AK 2004. Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry 40. pp607-619 Lancelot C and Billen G 1985. Carbon-nitrogen relationships in nutrient metabolism of coastal marine ecosystems. In: Jannasch HW and Williams JJL (eds). Advances in Aquatic Microbiology, vol. 3. Academic Press, New York, USA. pp263-321

Ricke SC 2003. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Science 82. pp632-639 Russel JB 1992. Another explanation for the toxicity of fermentation acids at low pH: anion accumulation versus uncoupling. Journal of Applied Bacteriology 73. pp363-370 Salehizadeh H and Van Loosdrecht MCM 2004. Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance. Biotechnology Advances 22. pp261279 Teitelbaum JE and Walker WA 2002. Nutritional impact of pre and pro-biotics as protective gastrointestinal organisms. Annual Review on Nutrition 22. pp107-138 Van Immerseel F, Boyen F, Gantois I, Timbermont L, Bohez L, Pasmans F, Haesebrouck F and Ducatelle R 2005. Supplementation of coated butyric acid in the feed reduces colonisation and shedding of Salmonella in poultry. Poultry Science 84. pp1851-1856 Yu J, Plackett D and Chen LXL 2005. Kinetics and mechanism of the monomeric products from abiotic hydrolysis of poly [(R) -3hydroxybutyrate] under acidic and alkaline conditions. Polymer Degradation and Stability 89. pp289-299

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he FAO Fisheries and Aquaculture Department publication Pearl Oyster Health Management: a manual, is now available online. The pearl oyster industry is a growing multi-billion dollar sector of molluscan aquaculture. As pearl production relies on the health of the oyster, this manual, which serves as a guide on the management of pearl oyster health, also includes an overview of the cultured marine pearl industry, a general review of mortalities and disease problems to help reduce risks from disease, and reports from Australia, the Cook Islands, Japan, French Polynesia, the Philippines, China and other countries that have vast experience in the health management of cultured pearl oysters. Pearl Oyster Health Management: a manual (FAO Fisheries Technical Paper No. 503. Rome, FAO. 2007. pp120) was written by: • Melba G Bondad-Reantaso, Fishery Resources Officer (Aquaculture), Aquaculture Management and Conservation Service Fisheries and Aquaculture Management Division, FAO Fisheries and Aquaculture Department Rome, Italy • Sharon E McGladdery, Aquatic Animal Health Division, Canadian Food Inspection Agency Ottawa, Canada, and • Franck CJ Berthe, Animal Health and Welfare Panel, European Food Safety Authority Parma, Italy. The pearl oyster industry is a growing multi-billion dollar sector of mollusc aquaculture. Pearl farming occurs throughout Australasia, the Middle East and South America. Few species of molluscs possess the ability to produce pearls of gem quality. The South Sea pearl oyster is one of them. Pearl production in the wild is an unpredictable and uncontrolled event which human intervention, through pearl culture, has progressively overcome by improving culture practices. Farming mother-of-pearls shares commonalties with edible mollusc aquaculture. However, the end product, pearl production, is unique to this sector. In aquatic production, health issues are of utmost importance; pearl production is based entirely upon health. The pearl itself is a product of the oyster's immune defences as a response to soft-tissue irritation. Exploited stocks receive frequent handling stresses that often predispose farmed animals to infection and diseases. The importance of health management for pearl oysters is therefore paramount. Today, most disease problems are caused by opportunistic pathogens taking advantage of oysters weakened by the stress of handling, including pearl surgery and sub-optimal growing conditions. Except for the mass mortalities experienced in Japan, the pearl oyster industry has not yet faced the types of epizootics that have impacted mollusc culture elsewhere in the world. Development of the industry will inevitably lead to increased risk of disease introduction, spread or emergence. Health management is the critical defence line against such an unwanted future. This technical paper has two objectives. The first is to review pearl oyster mortalities and disease problems in order to help design programmes aimed at reducing the risks from diseases. The other objective is to provide technical guidance to pearl oyster farmers and the industry on managing pearl oyster health so that sector development will be sustainable, not only in providing huge employment to communities

where pearl farms are located but also contributing to maintaining environmental integrity. Pearl oyster farming can serve as environmental sentinels, recognising the fact that pearl oysters thrive only in a pristine environment. This publication contains three parts. Part one consists of pearl oyster health – the current interest in it and an overview of the cultured marine pearl industry. Part two, pearl oyster health management, consists of seven sections: • introduction • general information on husbandry and handling, hatchery production, introduction and transfers; • disease diagnostic protocols dealing with field collections of samples, gross external examination, gross internal examination and laboratory protocols • health zonation • disease outbreak protocols • national strategies on aquatic animal health, and • references. Certain countries in the pearl oyster producing regions have acquired a great deal of experience in health management of cultured species. Experiences from Australia, the Cook Islands, Japan, French Polynesia, the Philippines, China, the Persian Gulf and the Red Sea are included in part three, which also contains a general review of pearl oyster mortalities and disease problems. The manual can be downloaded in full or in part via the FAO Fisheries ■ and Aquaculture Department website: http://www.fao.org/fishery

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THE BC PACIFIC SALMON FORUM, NANAIMO, CANADA Collaborative Research Initiative in British Columbia Deepens Understanding of the Broughton Archipelago and Throws Light on Salmon and Sea Lice Issues

I

n 2007 the BC Pacific Salmon Forum, an independent citizen body using science and stakeholder dialogue to advance sustainable governance of BC Pacific salmon, launched a two-year collaborative research programme to improve our understanding of the Broughton Archipelago ecosystem. The programme engages over 30 scientists and many others, with a variety of disciplines and perspectives, in some 15 research projects. They include defining oceanographic dynamics, monitoring salmon, stickleback and sea lice population dynamics, investigating the impacts of lice on salmon, and performing surveys of wild fish health. A number of key findings have been identified from individual projects in 2007, and although the data is preliminary and study will continue in 2008, some key points of interest arose. The following is an overview of the preliminary findings from the 2007 research period. (See end of article for contact details.)

THE BROUGHTON ARCHIPELAGO

ECOSYSTEM DYNAMICS Oceanographic factors play a significant role in the dynamics of any ecosystem, and in particular, in the distribution of organisms (both fish and potential pathogens such as sea lice) within that system. The Broughton Archipelago is a complicated system and to better understand it, and to provide insights into the distributions of fish and lice, several research groups have gathered oceanographic data. The data indicates a considerable degree of wind-driven surface water circulation that may result in surface transport in directions counter to tidal currents in the Broughton Archipelago. Although currents near the surface move predominantly in a seaward direction, winds can drive surface water (and potentially any particulates, including sea lice, contained in these surface waters) up inlets contrary to the current flow. Oceanographic fluctuations have significant impacts on organisms living within an ecosystem. A delicate balance exists between a fish, the environment it lives in and potential pathogens. It is an accepted fact that any change (environmental, nutritional or physiological) that pushes an organism beyond its ability to cope results in stress, which can upset homeostasis and lead to increased pathogen susceptibility. A comparison of data between 2003 and 2006 demonstrated that surface water salinity and surface water temperatures were greater in 2004 compared to other years, and this corresponded with an increased abundance of sea lice. Surface water temperatures increased (from 8.5˚C to 12.5˚C) and surface salinity decreased (from 26ppt to 19ppt) between April and June 2007.

SEA LICE STUDIES Plankton tows were performed to identify the distribution patterns of planktonic lice, both near to and distant from salmon farms. The majority of early samples (February and March) contained no lice, but 12 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

MAP OF THE BROUGHTON ARCHIPELAGO

although larvae were relatively rare, some patterns did emerge. Caligus copepodids were found in higher abundance in side inlets and low salinity areas, while Lepeophtheirus copepodids were most abundant near active farms. Plankton tows in April were hampered by dense plankton blooms that congested nets and precluded further sampling. In 2008, new equipment will allow the examination of vertical distributions of sea lice larvae in the water column. Their salinity and temperature preferences and their diel (ie daily) migration patterns will also be investigated. Several projects involved collecting wild juvenile pink and chum salmon and evaluating their infestation rates. Although there was some degree of variation between research groups with respect to the prevalence and intensity of lice observed on wild pink salmon captured in 2007, lice levels were low early in the season (March and April) and increased over time (April to June). Overall, the prevalence of lice on juvenile pink salmon in 2007 was described as being low, relative to previous years. In one study approximately 80 percent of all juvenile pink and chum salmon captured had no lice (ie the prevalence was approximately 20 percent). This represented the overall infection rate (prevalence) observed for

ABOVE: SALMON SMOLTS IN A SEA CAGE RIGHT: SAMPLING

all pink and chum salmon caught between March and June, including fish captured close to and distant from salmon farms. Juvenile chum salmon appeared to be more heavily infected than other species in all the years examined. Concerns have been raised in the past that different identification methodologies may preclude the ability to compare data; a comparison of sampling methods for fish and lice identification and quantification was performed by two different investigation teams that resulted in almost complete agreement in findings. Two further studies found similarly low lice prevalence (<25 percent to 31 percent), and a fourth study that collected juvenile pink and chum salmon for survival studies described an abundance of lice on wildcaught fish as generally being less than one louse per fish, and chalimus and copepodids as declining, with motiles rarely exceeding an overall ratio of 0.4 lice/fish over 20 days. Sampling of lice prevalence on wild fish was carried out by all research teams both near to and distant from salmon farms, but a statistical analysis of transmission estimates has not yet been completed. Lice levels in the Broughton Archipelago were found to differ compared with another geographical region on the mid-BC coast. Although this area encompasses different environmental conditions from those in the archipelago, the prevalence of sea lice on juvenile pink and chum salmon in the Bella Bella region averaged 4.2 percent, with lice slightly more prevalent on chum than pink salmon in this area. Statistical analysis of the differences in lice prevalence has yet to be undertaken.

IMPACTS OF SEA LICE ON JUVENILE SALMON There has been significant debate on the impacts of sea lice on juvenile salmon and there is not yet complete agreement on the sufficiency of current data to allow firm conclusions. It is common in nature to find fish with parasites, yet these organisms must maintain a fine balance lest they overwhelm their host. Similarly, the host, if in a sufficient state of fitness and health, can maintain homeostasis in the face of pressures exerted by a reasonable number of parasites. What defines “balance”, “fitness”, “health” and “reasonable” with respect to these species is not yet completely known, as these specific research questions are relatively recent. Part of the research

SAMPLING JUVENILES EXAMINING JUVENILES

being undertaken by the BC Pacific Salmon Forum is designed to address these questions. A health survey conducted in the region reported that both the weight and the length of juvenile pink salmon increased between April and June. The condition factor (one measure of the physical condition of fish that describes the relationship between body weight and body length) was significantly associated with the sampling month, while the presence or absence of sea lice and sea lice density (lice/g of fish) was not significantly associated with the condition factor in any general linear model tested. Skin lesions were found to be associated with the presence of sea lice, as were lower liver glycogen stores (energetic reserves). No other health parameters (ie the condition factor, histopathological findings, viral or bacterial infections) were apparently associated with lice. Histological studies were performed on saltwater entry juveniles and, based on the findings, fish first entering the marine environment appeared healthy. A portion of those fish sampled one month later displayed liver lesions consistent with toxicants. There was a positive correlation between some liver lesions and a myxosporean parasite (25 percent of fish were infected with the myxosporean) but in no cases were liver lesions or myxosporean infections correlated to lice infestation. Additional sampling is scheduled in 2008 to provide a stronger statistical basis for analysing these relationships. Wild pink salmon have been successfully transferred to and raised in a laboratory setting displaying growth patterns similar to those observed in the wild. Two studies that collected and held naturally infected wild fish found that lice disappeared over approximately two weeks. Repeatable methods of measuring swimming performance were developed and, when fish with one louse attached were compared JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 13

RESEARCH FOCUS

THE BC PACIFIC SALMON FORUM, NANAIMO, CANADA

ABOVE LEFT: LICE COUNTS ABOVE CENTRE: JUVENILE SALMON UNDERGO A FITNESS TRIAL ABOVE RIGHT: SAMPLING FOR JUVENILE SALMON RIGHT: A SEA LOUSE IS VISIBLE ON THIS INDIVIDUAL FAR RIGHT: JUVENILE SALMON

with fish having no lice, there were no significant differences in their maximum swimming performance. Louse stage (at one louse per fish) had no apparent effect on maximum swimming performance. Copepodids artificially raised from egg strings were successfully used to infect juvenile pink salmon in the laboratory, and while the duration of survival of juvenile salmon did decrease with increasing lice loads, this effect was not statistically supported unless lice loads were 10 times greater than levels reported in nature. Interestingly, mortalities of pink salmon spiked when the lice entered a moulting stage of their development. An examination of the impact of sea lice on the schooling behaviour of juvenile pink salmon in tanks in the field was carried out, and preliminary findings suggest that predation may occur selectively on lice-infected fish. This data has not yet been fully analysed, and modelling is necessary to substantiate the results and evaluate the implications more thoroughly. Salmon populations can and do fluctuate for a great number of reasons, many of them environmental. Variations in the freshwater environment can impact early rearing stages of fry, environmental conditions on marine entry can impact what is known to be a physiologically challenging period, oceanic productivity can impact prey availability and parasite populations, and the high seas are a black box into which we have yet to accurately peer. The overall catch of juvenile salmon in the Broughton Archipelago, for sampling purposes in 2007, was described as being the lowest over the previous five-year period of monitoring (despite an increase in the number of sampling sites) and there was greater variation in the size of fish than previously observed. Freshwater counts from a variety of systems and the migratory patterns of adult and juvenile salmon were tracked through the installation of video equipment, an acoustic counter, aerial surveys and fry traps. The 2007 pink adult salmon returns (escapement), as a whole, in the mainland inlets of the Broughton Archipelago system are described as being similar or slightly improved, relative to the brood return in 2005. The out-migration from the Glendale River spawning channel was estimated at 7.7 million pink fry and 34,000 chum salmon fry during 2007, but fry appear to have migrated out later in 2007 than in past years. The three spine stickleback fish has been of interest for several years, following numerous reports of them being heavily infested with lice. 14 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

In some cases sticklebacks have been described as being, “feathery with lice”. There is no evidence to date that lice are capable of carrying out their entire life cycle on the stickleback, and the stickleback’s heavy armour is thought to protect it. In 2007 sticklebacks were captured in large numbers during juvenile salmon fry sampling and numbers were high relative to previous sampling years. In many cases sticklebacks were reportedly more heavily infested by lice than juvenile pink and chum salmon captured in the same sets. Interestingly, in a captive study, sticklebacks were observed cropping egg strings from gravid females on juvenile salmon and actively removing adult lice in some instances. It is not known what, if any, role sticklebacks play in the transmission dynamics of lice in the Broughton.

CONCLUSION The 2007 research period has ended and much of the data is still being analysed. It is difficult, if not impossible, to make well-founded statements about the dynamics of organisms in a region without accurate assessments of populations and the environment over time. For this reason, the majority of the projects being funded by the Forum are being repeated for a second year to provide more data. The 2008 research programme is now underway, and new data emerging over the coming year will add to our expanding knowledge of this complex system. The Broughton Archipelago is very intricate, and its currents, winds, geography and ecology are very complicated. The Forum has stated that, “Since it is clear we are dealing with a dynamic ecosystem that includes many factors, not simply sea lice, funding is being allocated in 2008 to engage a range of researchers to develop an analytical framework that will incorporate all possible ecosystem factors in order to interpret the data that is emerging from this research programme.” This analysis would be a first attempt to undertake a whole system evaluation of many of the key environmental factors that affect salmon in the Broughton Archipelago. While research will not unravel all of the ecological mysteries of lice and salmon in the archipelago, perhaps some of the more pressing questions can be, at least in part, addressed through this scientific process. ■ See www.pacificsalmonforum.ca/research/index.php

AQUAFEED

NEWS ROUNDUP BY SUZI FRASER DOMINY

This news roundup appears courtesy of the Aquafeed website. See www.aquafeed.com

SALMON GET TRAINING TIPS FROM FOOTBALL What do juvenile salmon and the Spanish football team Barcelona have in common? Both use intensive training to improve their form. The head coach is a Nofima scientist and the training is healthy for fish, too. In order to make salmon more robust for transfer to sea water, an interdisciplinary research group involving Nofima, the Norwegian Institute for Water Research (NIVA), the Norwegian School of Veterinary Science, NTNU, Thelma AS and Aakvik Settefisk AS decided to strengthen the salmon’s heart capacity and health through intensive training. Benefits to humans through training include strengthening of the heart, muscles, skeleton and immune system and reduction of stress. “We were concerned that the fish would develop lifestyle diseases,” says senior scientist Harald Takle of Nofima Marin (formerly Akvaforsk), who headed the research group. The scientists have found hearts in farmed salmon that differ from the heart form of wild salmon, so the thought that the fish needed training wasn’t far off the mark. It was just a matter of starting to train 50g salmon. The scientists increased the tank water velocity to get the fish to swim faster. One of the challenges was to get tiny heart rate monitors made. These were inserted into the fish’s stomach with sensors attached to the heart. The fish in the trial were divided into three groups: • a control group that lived the normal life of juvenile salmon • one that received increased tank water velocity around the clock, and • one also received continual “jogging”, a daily spell of high-intensity training. The findings showed that the fish that trained grew considerably quicker, but that it did not influence feed utilisation. “We are now conducting tests at the VESO Vikan fish farm to see if the fish with headbands and heart rate monitors have greater powers of resistance to a deadly virus. The findings are extremely promising. Moreover, the fish tackle stress better when they are in better form. Less stress means the fish have greater energy reserves to tackle the challenges of everyday life,” says Takle. The new knowledge about juvenile salmon could lead to salmon farmers putting more jets in fish tanks and regulating the tank water velocity. “In the long term, we believe that this can make the fish even more robust. It’s just like with us humans, healthier fish thrive better, and this will in turn increase profitability for the salmon farmers,” Takle said.

generation shrimp feed additive, IMS. SemBioSys is developing a second-generation shrimp feed additive product in safflower, called ImmunoSphere, which it says could significantly reduce the manufacturing costs of the feed additive. “As a result of the decision, SemBioSys Genetics intended to evaluate the impact it had on ImmunoSphere,” said the president and chief executive, Andrew Baum. ImmunoSphereTM offered a significant reduction to the cost of producing immuneostimulatory feed additives, Baum said. “We intend to evaluate all the available options prior to committing to the next steps with this product, which targets a large potential market that has a significant unmet need.” The global shrimp industry represents a US$18 billion market. One of the major challenges facing commercial shrimp producers is the outbreak of diseases, which cost the shrimp aquaculture industry more than US$3 billion per year. Immuno-stimulatory feed additives, like ImmunoSphere, are designed to provide prophylactic protection to shrimp production.

PROSOL CREATES NEW PRODUCTS BASED ON NUCLEOTIDES The Italian company Prosol, which is a private producer of natural nucleotides for dietary food applications, has launched a new range of nucleotide-based products for aquaculture. Based on well-known independent scientific literature, these products provide a specific source of Free 5' nucleotides (F5N). It has been proved that only nucleotides in free form enhance the immune system’s response to stress factors.

The new knowledge about juvenile salmon could lead to salmon farmers putting more jets in fish tanks and regulating the tank water velocity

AQUA BOUNTY SCALES BACK COMMITMENT SemBioSys Genetics Inc., a biotechnology company developing a portfolio of therapeutic proteins for metabolic and cardiovascular diseases, says its joint development partner on the ImmunoSphere shrimp feed additive programme, Aqua Bounty Technologies Inc, intends to scale back marketing and registration efforts on its first-

Nucleo 5 Prime, the latest in the Prosol range, is developed from the company's deep knowledge in nucleotide extraction technology. In response to feed producers’ requirements, Nucleo 5 Prime is said to offer: • expertise in production • a constant ratio among free 5' nucleotides • a precise amount of free Single nucleotides • determination of free 5' nucleotides in each batch • GMP+ certification, and • optimal ratio quality/price. Nucleo 5 Prime is obtained in its free form by enzymatic hydrolysis of ribonucleic acid in yeast. Nucleo 5 Prime is then spray dried and provided in two different concentrations: six percent F5N and 40 ■ percent F5N content. JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 15

new

INTERNATIONAL Skretting tests marine zooplankton against lice

Skretting has entered into a contract with Calanus AS to develop and document a product based on the marine zooplankton Calanus finmarchicus to counteract sea lice infestations in salmon and trout. Commercial-scale field trials by selected farmers in Chile and Norway are now starting. The pelagic stages of Calanus finmarchicus and the infective stage of salmon lice are derived from a common ancestor and have many similarities. Therefore they may share what researchers call immunological structures. The trials will test whether it is possible to achieve higher protection from salmon lice by stimulating the immune system of the salmon with this new zooplankton product. It is known that the immune system of mammals can be stimulated to improve protection against external parasites. It has also been documented that the immune system of salmon can be improved to repel lice attacks. “The development of new, alternative salmon lice treatments is important. Although the outcome of this project is unknown, we believe the idea is too good not to try it out,” said David Knudsen, Skretting’s international product manager, raw materials. Sea lice infection is a major problem for the fish-farming industry in Chile, Canada, the United Kingdom and Norway. If the test results are sufficiently positive, the product will be launched for commercial use. “We are very pleased with the agreement and that Skretting is willing to invest significant resources to test our product on a commercial scale in an attempt to help fish farmers solve a serious problem,” said the managing director of Calanus AS, Gunnar Rørstad.

Disease affects Marine Harvest results Marine Harvest says the on-going disease situation in Chile continues to impact the group, giving a negative operational EBIT of 65 million kroner in the first quarter. The situation in Chile remained a challenge, but Marine Harvest said it was still confident of a positive long-term outcome. The largest operation, Marine Harvest Norway, continued to deliver good figures in the first quarter of 2008, with higher harvest volume and lower cost of harvested fish than the same period last year. VAP Europe also continued to show positive development with an increased EBIT. “We have taken a number of operational actions to contribute to short and long-term improvements in Chile, but it takes time to achieve the envisaged improvements,” said the chief executive, Åse Aulie Michelet. “The number of new ISA sites identified has been reducing in the last months, however the overall situation remains uncertain. Still, we are optimistic about the long-term outcome for Chile as an attractive and profitable production region.” Michelet said the company had taken strong action to reduce costs in the quarter, including closing a processing plant and making 900 staff redundant. Marine Harvest Norway had shown good operational improvement compared with last year. The reduction in operational EBIT was to a large extent due to lower market prices and currency effects. “We are further pleased with the results in VAP Europe that improved its EBIT, despite a lower revenue compared to the same period last year,” said Michelet. Marine Harvest generated operating revenues of 3080 million kroner in the first quarter 2008 compared to 3795 million kroner for the same period last year. The reduction in revenues 16 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

of 715 million kroner is mainly a result of lower prices in all regions. The reduction in revenues in Chile alone amounted to 594 million kroner, although a large part of this reduction was also due to lower volumes and smaller fish harvested. EBITDA before fair value adjustment of the biomass and restructuring was 107 million kroner in the quarter, down from 740 million kroner in first quarter 2007. Operational EBIT was -65 million kroner in the first quarter of 2008 compared to positive 552 million kroner in the first quarter of 2007. The total harvest volumes in the first quarter were similar to the levels of the comparable period last year, as the reduction in Chile due to disease was compensated by a volume increase in Norway. At the end of the first quarter 2008, Marine Harvest had a net interest-bearing debt of 6314 million kroner compared to 6725 million kroner in the first quarter 2007 and 6744 million kroner at the end of 2007. The equity ratio was 53.8 percent compared to 54.7 percent in the first quarter 2007. Marine Harvest is divided into five business areas: Marine Harvest Norway, Marine Harvest Chile, Marine Harvest Canada, Marine Harvest Scotland and Marine Harvest VAP Europe (value added products). It also has operating units in Ireland, the Faroe Islands and Japan, and halibut farming in Norway. Marine Harvest Norway had operating revenues of 1228 million kroner for the first quarter. Operational EBIT was 93 million kroner. The harvest volume in the period was a record high for a first quarter at 40,353 tonnes (HOG), contributing to an EBIT/kilogram (HOG) of 3.16 kroner. EBIT levels were lower than the 2007 first quarter due to reduced market prices and currency impacts. Marine Harvest Chile had operating revenues of 568 million kroner for the first quarter. Operational EBIT was -155 million kroner. The sold volume in the period was 23,883 tonnes (HOG), giving a negative EBIT margin of EBIT/kilogram 6.48. The negative result was impacted by lower market prices, smaller fish size, lower volumes and inventory write-downs. Marine Harvest Canada had operating revenues of 318 million kroner for the first quarter. Operational EBIT was 19 million kroner. The harvest volume in the period was 11,348 tonnes (HOG), contributing to an EBIT/kilogram of 1.64 kroner. The biological situation in Canada remains good. Difficult circumstances with lower prices and the falling US dollar compared with the Canadian dollar had a negative effect on profitability in the first quarter. Marine Harvest Scotland had operating revenues of 184 million kroner for the first quarter, and operational EBIT was -35 million kroner. The harvest volume in the period was 6275 tonnes (HOG), resulting in an EBIT/kilogram negative margin of 5.54 kroner. Scotland’s underlying performance showed improvement, but performance figures were influenced by an incident that led to a product recall. The result included provision for 34 million kroner to cover the costs of the recall. Marine Harvest VAP Europe had operating revenues of 839 million kroner for the first quarter. Operational EBIT was 29 million kroner, giving an EBIT margin of 3.4 percent compared with 1.4 percent in the corresponding period last year. The increase in EBIT margin despite the reduced revenues in the quarter was satisfactory, the company said. Marine Harvest expected to harvest 319,000 tonnes of salmonids in 2008, an increase of 11,000 tonnes compared with the previous year, of which 73,000 tonnes would be harvested in the second quarter. “The situation in Chile remains uncertain. The frequency of new ISA outbreaks has been reduced over the last months. However, 2008 will be a challenging year for Marine Harvest

Chile. The restructuring of our operations in Chile has the highest priority,” Michelet said. Feed costs were a challenge for salmon farming as well as for other protein farming industries, and Marine Harvest had for the first time negotiated global agreements with its feed supplier. “This will be important for the attention we will pay to the use and composition of feed in the future.” Marine Harvest anticipated a breach of one of the covenants in the loan agreement by the end of the first quarter of 2008. However, the company had obtained a waiver from the bank syndicate of this financial covenant in relation to the first quarter of 2008. “We are in a constructive dialogue with the banks to get the current covenants amended.” The global supply of Atlantic salmon grew by five percent compared to the first quarter last year, significantly below recent growth rates. “As a result of the situation in Chile we reiterate a global supply growth of one or two percent for the year and improved market balance in the main markets.” The board expected improvement in the EBIT for the second quarter compared to the first quarter, reflecting higher prices and slightly lower costs. “However, several measures have to be taken to meet the long-term financial targets for this business.”

New team at Intervet/Schering-Plough

UNITED STATES PerOs granted licence for oral vaccine formulations PerOs has been granted an establishment licence to produce bacterins as well as an autogenous bacterin licence utilising its patented oral delivery formulation methods (Oralject). The Centre for Veterinary Biologics at the United States Department of Agriculture granted the licences in April. “The approval of our manufacturing facility and our oral

Ohio extends emergency order Ohio’s Agriculture Director, Robert Boggs, extended an emergency order on May 16 that prohibits the intrastate transportation, sale or distribution of 28 fish species susceptible to viral haemorrhagic septicemia, or VHS, out of the affected region in northern Ohio. The Ohio Department of Agriculture is responsible for protecting animals from potentially devastating diseases. “This emergency order not only protects Ohio’s fish population but also others in points beyond the state’s borders,” Boggs said. VHS is a fish disease that must be reported to the department under state law. It was introduced into the wild fish population by an invasive species. It is not harmful to humans or other animals. Tests by the ODA’s Animal Disease Diagnostic Laboratory has revealed that VHS is not present inland in Ohio. The department will continue to test and monitor for the disease. Ohio’s ban prohibits the intra-state distribution of VHSsusceptible fish or eggs out of the area in Ohio north of US Highway 6 from the Indiana border to the intersection of H6 and Interstate 90 near Fremont, continuing on I-90 to the Pennsylvania border. This includes the Sandusky River south of H6 to the Ballville Dam. Twenty-seven fish species are known to be susceptible to VHS. The prohibition is in effect in Ohio until the US Department of Agriculture’s Division of Animal and Plant Health Inspection Service releases its embargo and prohibition of the movement of live VHS-susceptible fish. The prohibition does not apply to live fish or eggs removed directly from production facilities that have tested negative for VHS. It also excludes live fish or eggs that are being

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Schering-Plough has appointed the executive team members of the company’s global animal health unit: Intervet/ScheringPlough Animal Health. These appointments define the major steps in the design process of the animal health unit. The executive team is chaired by Ruurd Stolp, senior vicepresident and president animal health. Dr Stolp reports to the chairman and chief executive of Schering-Plough, Fred Hassan. The executive team members are • Raul Kohan, senior vice-president corporate excellence, and deputy head animal health • Hugo Wahnish, vice-president global AH regions • René Aerts, vice-president animal health, research and development biologicals • KJ Varma, vice-president AH, research and development pharmaceuticals • Jochen Bader, vice-president global finance, animal health • Malte Greune, vice-president global supply chain animal health • Gráinne Higgins, vice-president HR, animal health • Mark van Heumen, law lead global animal health, and • Andreas König, quality head, global quality operations, animal health. The new combination of Intervet/Schering-Plough Animal Health strengthens the animal health portfolio in several areas and brings together complementary lines of pharmaceuticals, biologicals and innovative services, the company says. Schering-Plough completed its acquisition of Intervet last November. The American company purchased the animal health division from the Dutch chemical giant Akzo Nobel for €11 billion.

vaccine formulation platform in the United States marks a significant milestone for our company,” said the chief scientific officer, Grant Vandenberg, who invented Oralject. “We look forward to offering these capabilities to the livestock producers and opening new possibilities to the growing aquaculture market, as well as collaborating with traditional animal health companies to expand the oral enablement of the industry as a whole.” PerOs’ oral delivery platform focuses on the use of natural products to deliver safe therapeutic and prophylactic compounds. Its formulations are designed to enable the delivery of biological compounds such as vaccines, peptides and probiotics, among others, while eliminating the stress associated with handling, injection and other current delivery practices. The company says the new technology platform will enhance the value of vaccines by offering a labour-saving, practical oral delivery approach for the livestock industry and open up new possibilities to the rapidly growing aquaculture market. Oral vaccination provides animal producers with an innovative tool for disease prevention through vaccination, rather than relying on disease treatment using antibiotics. The PerOs facility, said to be the first of its kind, was made possible by the contribution of Benchmark Biolabs’ development team. Benchmark managed the regulatory interface and established the QA and QC standards, while also designing, constructing and now operating the PerOs plant. The chairman of PerOs, Ian Kott, said the work of the Benchmark team was “perfect. Using their BEAM process flow, they led our technology assets in record time toward regulatory approval.” Contact Jean-Simon Venne, PerOs Inc. Phone (514) 516 3003, email [email protected], or Tom Overbay, DVM, Benchmark Biolabs, Inc. Phone (402) 475 8104 or email [email protected]

JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 17

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transported for use by research scientists in closed research facilities with diagnostic laboratories. See www.ohioagriculture.gov

CHILE New general manager for Pharmaq Chile Dr Bernd Wrege has been appointed general manager of Pharmaq Chile Ltda, situated in Puerto Montt. He will continue his responsibilities as technical director. Dr Wrege has held key positions within the company since 2004, and has extensive experience within the aquaculture industry in Chile. Pharmaq Chile Ltda is as a subsidiary of the Norwegian company Pharmaq AS, and started its activities in Chile in 2001. The company says it has experienced a strong growth in the Chilean market and occupies the number one position in vaccines and treatment against Caligus. The aquaculture industry in Chile is facing several challenges related to diseases, and the company says it is committed to helping the industry by introducing new products and customer services.

Pharmaq introduces vaccine in a new product line Pharmaq Chile Ltda has been granted a licence by Servicio Agricola y Ganadero de Chile (SAG) for the new vaccine Alpha Ject® micro 3. This is a three-component vaccine for salmon in a micro-dose (0.05ml/dose) to protect against infectious pancreatic necrosis, vibriosis caused by Vibrio ordalii, and SRS caused by Piscirickettsia salmonis. “With all the efforts and resources put into this project we are confident that we have developed an effective and safe vaccine for our customers,” said the company’s general manager in Chile, Dr Bernd Wrege. Alpha Ject® micro 3 represents the first micro-dose SRS vaccine delivered from Pharmaq. “The documentation of this new-generation micro-dose product from Pharmaq is comprehensive. We had to develop new technologies to ensure the right concentrations of antigens for optimum protection of this product.” As well as ensuring the efficacy of fish vaccines from Pharmaq, all the products go through laboratory and field tests to establish safety documentation as well as safeguard the maximum growth of the fish. The introduction of the new vaccine follows the company’s launch one year ago of the first SRS vaccine, Alpha Ject® 4-1. Contact Bernd Wrege, general manager Pharmaq Chile Ltda. Phone (+56) 65 483091, or email [email protected]

UNITED KINGDOM Isolation of spring viraemia of carp virus Recent routine import checks have identified spring viraemia of carp, or SVC virus in goldfish imported from a supplier in Hong Kong, the Tung Hoi Aquarium Company. SVC has no implications for human health. It is nonetheless, a serious viral disease affecting common and ornamental carp, as well as tench, roach, rudd, goldfish, pike and wels catfish. The virus was found in a random sample taken directly from imported boxes of fish. The Fish Health Inspectorate at Cefas has placed movement controls on, and taken samples from, all sites receiving fish from the SVC-positive consignment. The FHI has also provided advice to other businesses that may have recently imported fish from the same source to increase awareness of any potential SVC risk. The authorities in the exporting country have been informed of the isolation of SVC and are conducting investigations to 18 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

identify the source of the infection. Importation of fish from the affected source will be prohibited pending the conclusion of these investigations. The clinical signs of SVC can include darkening of the skin, swollen eyes, abdominal swelling, pale gills, trailing faecal casts and protrusion of the anus. Infected fish may be lethargic and show areas of bleeding in the gills and skin. Anyone noting deaths in carp or any other species susceptible to SVC, with signs of disease similar to those above, should immediately contact the Cefas Fish Health Inspectorate at the Weymouth laboratory on 01305 206673/4. Anyone who imports, keeps or retails carp or other susceptible fish should take strict precautions to prevent the spread of SVC and follow the advice set out in Defra’s booklet Keep Fish Disease Out, available from the Cefas Fish Health Inspectorate or through www.efishbusiness.com

Salmon industry to invest £400k in research and development Scottish salmon farmers are to benefit from a £400,000 investment in research and development projects over the next three years to help secure the long-term socio-economic and environmental sustainability of the industry. “This project will be highly significant in developing new farming practices in the Scottish industry,” said the chief executive of the Scottish Salmon Producers’ Organisation, Sid Patten. “It is hoped that the information generated will enhance sustainability, while improving both environmental performance and reducing the cost of production. “To be successful, it must report good quality, robust scientific evidence. By working closely with stakeholders, the initiatives address many of the current challenges and they will help the industry to achieve its full potential,” Patten said. A range of initiatives has been identified, including a scientific environmental modelling project and the adoption of new technology and husbandry practices on farms. An enhanced monitoring programme has been commissioned to develop the accuracy of predicting environmental performance of marine farms. A second initiative is to look at economic modelling of production to determine optimum sustainable farming conditions. Projects addressing alternative sea lice management strategies, the application of new technology in freshwater farms and further development of acoustic devices to deter predators are under consideration. The initiative is being managed and partly funded by the producers’ organisation under the auspices of the new Demonstration Project, with financial and other support from the Scottish government, salmon farmers, industry suppliers, Highlands and Islands Enterprise, The Crown Estate, Scottish Aquaculture Research Forum and regulators.

Scientists gather for workshop Marine scientists from around the world took part in a special workshop in Weymouth on infections and diseases in marine life during May. The 27 delegates took part in a histopathology workshop at the Centre for Environment, Fisheries and Aquaculture Science Laboratory. The three-day workshop featured presentations from experts, laboratory tours and breakout sessions, and placed significant emphasis on “hands-on” practical training and the diagnosis of diseases and pathological changes. Participants studied tissue samples through microscopes and examined tissue sections under high-magnifying monitors. Species examined included salmonids, cyprinids, molluscs and crustacea. One of the organisers, pathologist John

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Bignell, said that scientists came from as far afield as Tasmania and South Africa to learn about histopathology.

Contact Global Communications, Animal Health, Boxmeer, The Netherlands Email [email protected]

Gettinby honoured with award

Professor George Gettinby from the University of Strathclyde in Scotland was honoured with the Intervet Dieter Lütticken Award at World Aquaculture 2008, held in Busan, Korea from May 19 to 23. The award, which carried a prize of €20,000, was for his innovative statistical and epidemiological research methods in sea lice treatments in salmon farming. The jury found his research methods were an PROFESSOR excellent example of commitment GEORGE to the advancement of reducing, GETTINBY refining and replacing the use of animals in research, development and production of veterinary medicines. Professor Gettinby’s research focused on applying statistical and epidemiological methods in sea lice treatments in salmon farming in order to minimise the number of animals required in test groups. The research also provides an alternative to assessing the effectiveness of veterinary medicines in fish, which supports the replacement principle. This alternative is the adoption of mathematical models, which can simulate the effects of treatments on lice populations. Dr Dieter Lütticken, who presented the award, said there had been a long and intimate relationship between life sciences and statistics. “Professor Gettinby’s innovative statistical and epidemiological research methods are an excellent example of refining and reducing the use of animals in sea lice treatments in salmon farming.” The jury panel praised Professor Gettingby’s innovating mathematical model approach for investigating the optimal use of treatments that involve the minimal use of animals. ”In addition, these methods also minimise the use of veterinary medicines in the environment.” Professor Gettinby said he was honoured to receive the Dieter Lütticken Award. “It is pleasing to know that the award has come as a result of the increasing role of statistics and mathematical modelling in the pursuit of medicines for animals and the improved health of species in aquaculture.” He acknowledged the collaboration, support and work of Marine Harvest Ltd, Scottish Quality Salmon, the UK Department for Environment, Food and Rural Affairs and the Epi-informatics research group at the University of Strathclyde. The Dieter Lütticken award, established in 2004, aims to encourage research into the use of alternative models for animal testing with significant impact on the development or production of new animal health products. It is named after Dr Dieter Lütticken, a committed researcher in microbiology and virology. He led Intervet’s research and development for more than 25 years, and retired in 2003 as vice-president and head of research and development. The award covers in-vitro models used in research and development which replace animal testing for licensing purposes, as well as studies avoiding the use of animals in efficacy, safety and quality testing in the production of biologicals and pharmaceuticals for animals. Intervet said it welcomed submissions from all life-science research institutions for the next award. This year’s deadline is November 15. Commercial organisations are excluded.

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IRELAND Improved sea lice strategy for salmon farms The Minister for Agriculture, Fisheries and Food, Mary Coughlan TD, and Minister of State John Browne TD, recently launched a new strategy to improve pest control on Irish salmon farms. The sea louse is the common enemy of both the salmon farmer and wild fisheries. Monitoring of sea lice on salmon farms was initiated in 1991 and involves inspecting and sampling at all fish farms 14 times per year. Treatment is required where sea lice levels are above target levels. The strategy intends to build on the existing monitoring and treatment regime through intensifying and revitalising the single-bay management approach and making it central to national policy for sea lice management. The new approach will incorporate “real time” management of sea lice infestations on a case-by-case basis. The strategy also calls for more work to be done to identify further treatment options, and optimising the use of available licensed salmon farm sites from a sea lice management perspective. A national implementation group comprising representatives from the Minister’s Department, the industry, BIM and the Marine Institute, is to be established to review progress made to reduce sea lice levels and to report within six months with recommendations on further steps, if any, required to redress the situation.

CANADA Public updated on salmon migration British Columbia’s largest aquaculture company, Marine Harvest Canada, placed full-page advertisements in the North Island Gazette and a later edition of the Victoria TimesColonist on April 3 to update the public on its success in minimising the number of sea lice on its farmed fish in the Broughton Archipelago area during the current outmigration of wild juvenile salmon. The advertisement said that that four of its Broughton farms were now empty of fish. Six other farms showed sea lice levels of no more than 0.2 lice per farmed fish, well under the provincial government’s threshold limit of three lice per fish, at which time fish must be treated to eliminate the lice. The latest monitoring was completed in early March. “This data has been available on our website since 2004,” said the Director of Environmental Compliance and Community Relations, Clare Backman. “We monitor for sea lice levels diligently and manage our operations closely to ensure that our fish do not add to the many other threats that young wild salmon face.” The ad was the second placed by Marine Harvest Canada to update the public on its sea lice management plan since the out-migration began in March. “It is part of our commitment to being open with those interested in our business about what we are doing and what results we are attaining. We do not pretend to have all the answers, but we are committed to reviewing and supporting science and improving our practices so that we minimise risk to our salmon and wild stocks.” Marine Harvest Canada produces 45,000 tonnes of salmon year-round in farms off Vancouver Island and the central coast. The company employs 500 people from Duncan to Klemtu. See www.marineharvestcanada.com/documents/MHC_ ■ sealice_update_April_08. pdf

BY BJARNE GJERDE (NOFIMA, NORWAY) AND SCOTT PEDDIE (AQUACULTURE HEALTH INTERNATIONAL UK)

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ome salmon of the same species are more susceptible to lice infestation than others. Utilising this knowledge in designing selective breeding programmes not only has the potential to save millions in lost revenue, but also reduce the infestation pressure of salmon lice among wild salmonid populations. These are the findings of a recent Nofima research project, and follows on from work published by Akvaforsk researchers in 2005 (Kolstad et al 2005). This initial work recorded susceptibility to the salmon louse Lepeophtheirus salmonis in three year-classes of Atlantic salmon with 300 (year-classes 2000 and 2001) and 50 (year-class 2001) fullBJARNE GJERDE MARINE SALMON FARM sib families. The genetic correlations between body weight and lice numbers of salmon that are more resistant to lice infestation. lice were of moderate size, showing that it was possible to improve “The aquaculture industry and authorities should join forces both body weight and resistance to L salmonis simultaneously to breed a salmon with greater powers of resistance to salmon through selection. Moreover, the genetic correlation between the lice,” says Nofima (formerly Akvaforsk) senior scientist Bjarne numbers of lice recorded in a challenge test and during a natural Gjerde. “Breeding for resistance to salmon lice should be included infection was very high (See Figure 1). as a supplementary measure in the industry’s and authorities’ It was concluded in this initial study that the potential for action plan against lice, in the same way as there is today an improving resistance to sea lice in Atlantic salmon by selective order to delouse as soon as a stipulated number of lice per fish is breeding was good. In addition, as natural infection is highly variable exceeded.” in time and magnitude, the study recommended that challenge tests INFESTATION OF WILD SALMON should be used in selective breeding to increase the resistance of Although the issue is a complex one, salmon lice from farmed salmon to L salmonis. salmon is one of the threats facing wild salmonids, given that wild Recent Norwegian media coverage has indicated that salmon salmon may be infested by lice spread by coastal currents. “The lice have become resistant to anti-lice medication. However, one responsibility to take good care of wild salmonids and the risk that measure to combat this particular problem is by breeding farmed the lice develop resistance to medication means that it is important to increase the farmed salmon’s powers of resistance to salmon SEA LICE lice,” says Gjerde. “Purposeful breeding for a farmed salmon that is infested by salmon lice to a lesser extent than today will also reduce the infestation pressure on wild salmonids, as there would be fewer salmon lice along the coast.” FIGURE 1: Breeding values for the number of lice per fish of 50 Atlantic salmon full-sib families under controlled (challenge test with post smolts) and natural infection (Gjerde et al 2008, data from Kolstad et al 2005)

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Field test, no. of lice per fish

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SELECTIVE BREEDING CAN REDUCE SALMON LICE PROBLEMS

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THE 1ST INTERNATIONAL CONGRESS ON HEALTH MANAGEMENT AND DISEASES OF AQUATIC ANIMALS Tehran, Iran January 27-28, 2009 This international meeting will be hosted by the Veterinary Council, IR Iran, as organiser, with the collaboration of Contemporary Conference Organisers as the co-organiser. The congress has the support of the Iranian Veterinary Organisation, Faculty of Veterinary Medicine of University of Tehran, Iranian Fisheries Research Organisation, Iranian Fisheries Organisation, Iranian Veterinary Association, Faculty of Veterinary Medicine of Islamic Azad University and the Iranian Department of Environment. The main aim of this congress and exhibition is to create a dynamic scientific environment for presenting, transferring and exchanging the latest advanced research findings and scientific achievements in all health and nutrition management aspects of aquatic animals. There will be particular emphasis on diseases, prevention and treatment, nutrition health management, water quality management and health management in aquaculture. This will be achieved through the active participation of Iranian and overseas veterinarians, researchers, scientists and experts. See www.ichmda.com

THE 11TH AQUACULTURAL INSURANCE AND RISK MANAGEMENT CONFERENCE

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Grand Villa Argentina, Frana Supila 14, 20000 Dubrovnik, Croatia March 26-27, 2009 Aquaculture Underwriting and Risk Management Services Ltd has

announced the 11th biennial conference. It is targeted at insurance underwriters, brokers, loss adjusters, surveyors, lawyers, fish farm risk managers, university experts, investors and all those interested in insurance, risk management and loss prevention in aquaculture. Confirmed speakers to date include: • High risk, low reward: has our industry peaked or simply reached a plateau? Speakers: Tom Rutter and Martin Coull • The use of geographical information systems (GIS) in risk managing aquaculture. Speaker: Andreas Siebert • Federal trout and catfish aquaculture insurance development in the US. Speakers: Prof Keith H Coble and Assoc Prof Terry Hanson • Effects of global warming on low-lying coastal mariculture. Speaker: Ian Thomas • A review of the current disease situation in aquaculture. Speaker: Dr Scott Peddie See www.conference.aquacultureinsurance.com/index.cfm

2008 FISH HEALTH SECTION – AMERICAN FISHERIES SOCIETY MEETING Atlantic Veterinary College, The University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, Canada July 9-12, 2008 The meeting is being hosted by the Atlantic Veterinary College University of Prince Edward Island, and will mark only the second time that the Fish Health Section annual meeting has been held in Canada. We are encouraging our EAFP and other international fish health colleagues to attend. Prince Edward Island is easily accessible from Europe, Asia and South America through Halifax, Nova Scotia and Moncton, New Brunswick by air on several major airlines. ■ See http://ocs.vre.upei.ca/index.php/FHS/FHS2008

CONTINUED FROM PAGE 18

ECONOMICS Economic losses due to sea lice infestation are substantial in the salmon industries of the major producing countries. Selective breeding for increased resistance to salmon lice can potentially provide significant economic gains in two ways. Firstly, farmed salmon will require fewer treatments against salmon lice and secondly, the infestation pressure on wild salmonids will decrease. The first is of major economic value to the salmon farming industry, while the latter is of major value to society. “But it is important to emphasise that this, as with other traits selected for in breeding programmes, is a long-term measure,” says Gjerde. “It will be some years before we reap the big gains, so it is important to commence this work as quickly as possible.”

ACKNOWLEDGEMENTS The project is financed by the Research Council of Norway and Salmo Breed AS. The findings from a fresh project involving

150 families and financed by the Aquaculture Industry Research Fund (FHF) and Salmo Breed AS will be available later this (northern) summer. Contact Bjarne Gjerde, phone (+47) 93 06 15 41 or email bjarne. [email protected]

REFERENCES Kolstad K, Heuch PA, Gjerde B, Gjedrem T and Salte R (2005). Genetic variation in the resistance of Atlantic salmon (Salmo salar) to the salmon louse Lepeophtheirus salmonis. Aquaculture 247. pp145-151 Gjerde B, Sonesson A, Storset A and Rye M 2008. Selective Breeding and Genetics – Atlantic salmon. In: Aquaculture – Production of Aquatic Organisms (2000-2005). Aquaculture Research: From Cage to Consumption (eds) M Thomassen, R Gudding, B Norberg and L Jørgensen. The Research Council of Norway, ISBN 978-82-1202409-0 (pdf version). ■ JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 23

EVENTS

FORTHCOMING FISH AND SHELLFISH EVENTS

COMMERCIAL FOCUS

CYTOGENIX ANNOUNCES AGREEMENT TO DEVELOP DNA VACCINES SCOTT PEDDIE, AQUACULTURE HEALTH INTERNATIONAL, UNITED KINGDOM

T

he US-listed company CytoGenix Inc announced on March 5 that it has completed an agreement with Taiwan Cobia Inc to develop DNA vaccines against fish viruses. Under this agreement, CytoGenix will develop DNA vaccines against selected viruses, and TCI will vaccinate the fish and test immunological responses. The major diseases and target species of interest include viral nervous necrosis disease (VNN; Betanodavirus, Nodaviridae) affecting marine fishes such as parrot fish, groupers, flounders, sea bass, turbot, and striped jack. DNA vaccines against pathogenic Iridovirus in grouper (Lymphocystivirus, Megalocytivirus or Ranavirus) are also being investigated. “Fish is man’s most important single source of high-quality protein and provides ~16 percent of the animal protein consumed by the world’s population (United Nations Food and Agriculture Organization, 1997),” says the president and chief executive officer of TCI, Cory Huang. According to the FAO, aquaculture is one of the fastest growing food-producing sectors of the world, and it continues to grow more rapidly than all other animal food-producing sectors. Asia and the Pacific region contribute over 91.5 percent of the total global production and 82 percent of the value, Huang said. “However, aquaculture faces many challenges, especially when it comes to health management of fish stocks. CytoGenix’ synDNATM technology has the capability to provide the large quantities of affordable DNA vaccines necessary to address the need for simpler and more effective aquaculture vaccination regimens than currently available.” CytoGenix’s chief scientific officer, Dr Yin Chen, says DNA vaccines have already been shown to provide protection against pathogenic challenges in several animal models, including fish. “The vaccines carry only a small portion of the infective agent, and therefore carry almost no risk of inadvertent infection, particularly when manufactured using CytoGenix’s proprietary cell-free method (synDNATM),” Dr Chen said. “Additionally, the prophylactic potential of genetic vaccination in fish culture is being commercialised, due to the advantages offered by genetic vaccines over conventional preparations ie, ease of production, stability, cost and production of vaccines against organisms which are difficult or dangerous to culture in the laboratory.” The president and chief executive officer of Cytogenix, Malcolm Skolnick, says the synDNATM vaccine platform is highly adaptable. “These vaccines are based on an expression cassette that may be used against a large number of pathogens by substituting appropriate sequences to express antigenic proteins for a given pathogen. “This agreement represents a beneficial joint effort where CytoGenix develops a vaccine and our strategic partner tests it and has the ability to commercialise the results. This agreement with Taiwan Cobia further exemplifies the versatility of our technology and provides an avenue for expansion into the worldwide marketplace,” Skolnick said. 24 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

THE CYTOGENIX TECHNOLOGY The CytoGenix team has developed a method for manufacturing large amounts of high quality therapeutic nucleic acid constructs (synDNATM) which is conducive for the rapid development of compounds with new sequences of interest. The process offers significant improvements over traditional bacteria-based fermentation for plasmid DNA production and polymerase chain reaction (PCR)-based methods. It incorporates a cell-free method for producing therapeutic quality DNA for use as drug substances in both humans and animals. According to CytoGenix, such an approach offers a number of advantages, including: • Minimal hazardous contaminants: Cell-free amplification of therapeutic nucleic acid has many important benefits, beginning with a smaller size and less complex version of the plasmid. Under this system, there is no need for bacterial replication sequences or selection markers such as antibiotic resistant genes found in the plasmid DNA backbone. In most cases, this will reduce the size and weight of the therapeutic product by at least 3000 base pairs. In addition, the absence of bacteria and growth medium greatly simplifies the methodology required to purify the manufactured product. This process employs a simplified chromatography-based purification procedure developed at CytoGenix. Depending on the intended application, nucleic acids of various grades of purity can be produced ranging from 70 percent (ie, no purification) to about 95 percent pure. This reduces or eliminates the need for mechanical or chemical purification methods to remove contaminants such as bacteria cellular debris, endotoxins, RNA or genomic DNA molecules. • Robust biological activity: According to CytoGenix, the results of their experiments have shown that the biological response to this material (devoid of vector backbone) is similar to traditional plasmids. Moreover, experiments conducted in several animal models have shown that linear DNA prepared with CytoGenix synDNATM process triggers a robust response (immune or physiological, depending on the application) in treated groups compared to placebo or plasmid DNA-treated animals. • Low risk, competitive cost, universal accessibility and fast cycle time: CytoGenix scientists claim that the entire process is benchscale and requires little equipment, space or human intervention

SENIOR SCIENTIST FRÉDÉRIC KENDIRGI, PHD NEXT TO THE HPLC PURIFICATION STATION FOR SYNDNATM TECHNOLOGY

SENIOR LABORATORY DIRECTOR MS HARILYN MCMICKEN AT THE DENSITOMETRY STATION FOR SYNDNATM

in comparison to bio-process or bacterial fermentation manufacturing facilities. This process easily lends itself to liquid-handling automation, and a skilled technician can synthesise multi-gram quantities of this material within a few weeks, while working in a compact, room-sized facility. Unlike PCR, this process requires only basic laboratory equipment and is therefore accessible to many facilities around the world, especially rural settings in the field, where access to specialised equipment is limited and often prohibitive. • Improved regulatory profile: A major benefit of using this cell-free DNA manufacturing technology, again according to CytoGenix representatives, relates to the regulatory agency review and compliance perspective. CytoGenix claims that product cGMP manufacturing procedures detailing methods for cell collection, processing and cell culture conditions are no longer necessary, and therefore reduces the level of risk, the amount of documentation and the amount of required space, as well as QA/ QC and compliance costs. In terms of production, CytoGenix states that once all the production parameters are optimised for a given construct, manufacturing of gram quantities of the desired product can usually be accomplished within five to 10 working days. Quality control tests are incorporated for quality assurance and within another week, the DNA can be released for use. Additional developments may reduce the lag period between custom optimisation identification and DNA release. CytoGenix has entered into an exclusive supply agreement with GE Healthcare Bio-Sciences Corp to purchase custom kits and reagents for making large amounts of nucleic acid in accordance with our proprietary technology. The agreement gives CytoGenix the exclusive, worldwide right to purchase the necessary reagents from GE Healthcare for use in producing therapeutic synDNATM.

Prior to joining the School of Public Health, Dr Skolnick managed the Health Science Centre’s Office of Technology Management and oversaw the university's activities in protecting and licensing its patent portfolio of technology. Dr Skolnick held several clinical trials while he was at the Health Science Centre in Houston in pain management, smoking cessation and reduction of withdrawal symptoms in drug addiction. He is a registered patent attorney, a patented inventor and is licensed to practice law in the state of Texas. Dr Skolnick has been active in patent prosecution and licensing for selected clients, and has served as an expert witness in intellectual property, product liability and accident reconstruction matters.

ABOUT CTYOGENIX CYTOGENIX MANAGEMENT TEAM Malcolm Skolnick, PhD, JD. Chairman/president and chief executive officer Malcolm Skolnick received his PhD in physics from Cornell University and his JD from the University of Houston Law Centre. Prior to joining CytoGenix, Dr Skolnick held academic positions in the Graduate School of Biomedical Sciences and the School of Public Health at the University of Texas Health Science Centre at Houston. He is currently an adjunct professor in the School of Public Health, where he formerly served as Professor of Technology and Health Law.

Greg S Taylor, CPA. Vice-president of finance and administration and chief financial officer. Greg Taylor has almost 20 years of investment banking, venture capital, corporate advisory and entrepreneurial experience and

INFORMATION BOX Taiwan Cobia Inc, a private company founded in 2004, was the first company to introduce the concept of organic aqua-farming in Taiwan. The company is currently adding production sites in China and South East Asia. TCI also has collaborative agreements with research institutes and universities, including Academia Sinica, University of California at San Diego, National PengHu University, and National Taiwan University, as well as various government agencies in Asia and Africa. CytoGenix Inc is a Houston-based bio-pharmaceutical company pioneering the new field of gene-based medicine. It is focused on developing innovative vaccines and therapeutic products using its three proprietary technology platforms: • synDNATM enzymatic, non-bacterial production methodology • single stranded DNA (ssDNA) expression, and • OligogenixTM, an oligonucleotide based anti-bacterial technology. CytoGenix currently holds 13 granted patents, two allowances and 55 additional international or US patent applications claiming methods and materials in connection with these platform technologies. See www.cytogenix.com

JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 25

COMMERCIAL FOCUS

CYTOGENIX ANNOUNCES AGREEMENT TO DEVELOP DNA VACCINES

FREDERIC AND HARILYN NEXT TO TC HOOD (FUNCTIONAL ANALYSIS IN CULTURED CELLS OF THERAPEUTIC DNA PREPARED WITH SYNDNATM PLATFORM)

has completed over $2BB in transactions. He has also served in management positions as both a chief executive officer and chief financial officer. He was most recently managing director of Monterey Capital Partners, a private equity investment firm, for over 10 years. Previously, he served as a vice-president with the investment banking division of Sumitomo Trust and Banking and with Bunker Hill Associates, a merchant banking firm. Greg Taylor began his career with Ernst and Whinney, a big eight accounting firm in Houston, Texas as a certified public accountant. He has served on the board of directors of several private companies and charitable organisations. He has a Bachelor of Business Administration in accounting and finance from Baylor University and a Master of Taxation from Baylor University. Yin Chen, PhD. Chief scientific officer and vice-president of research and development Dr Chen earned his PhD in molecular biology and biochemistry from the University of Maine. Subsequently, he was a post-doctoral fellow at the Beth Israel Deaconess Medical Centre, a teaching hospital of the Harvard Medical School. He was briefly employed by InGene, Inc in Kansas City, MO as a senior research scientist. Dr Chen joined CytoGenix In 2000 as chief research scientist. He was promoted to vice-president of research and development in 2001. Dr Chen oversees the research and development team at the company and is the leading inventor of the company’s proprietary technologies. Pam Schertz, CPA, Controller Pam Schertz has been appointed as interim chief financial officer. Ms Schertz has served the company as controller since 2003. She received her BBA in accounting from the University of Houston and is a licensed CPA in the state of Texas. After establishing her own practice offering consultant services as a CPA in October 2003, Cytogenix became her first client. 26 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

THE CYGX SCIENTIFIC TEAM. FROM LEFT: CHIEF SCIENTIFIC OFFICER YIN CHEN, PHD, SENIOR LABORATORY DIRECTOR MS HARILYN MCMICKEN, BS, MT, SENIOR SCIENTIST FRÉDÉRIC KENDIRGI, PHD AND SENIOR RESEARCH SCIENTIST XIN-XING TAN, PHD

Prior to working for Cytogenix, Ms Schertz has had over 15 years of experience in accounting in various industries, including a casket manufacturer, an independent power company and an architecture firm. Management positions have included serving as controller for a high-end clothier and a civil engineering firm. Cindee Ewell, PhD, JD. Vice-president of legal affairs and secretary Dr Ewell has been with CytoGenix since 2004 and is the company’s resident corporate and patent counsel. She received her law degree from the South Texas College of Law, her bachelor’s degree in microbiology from Cornell University, and her doctorate in biochemistry and molecular biology from the University of Texas Health Science Centre at Houston. She is a registered patent attorney and is licensed to practice law in the state of Texas. Her experience includes nearly eight years of research at Baylor College of Medicine, about four years of patent prosecution and freedom to operate experience in a large law firm environment, and several years of contract and corporate law experience. Ms Ewell entered the legal field in 1999 as a biotechnology technical advisor with a focus on intellectual property. She has since attained her law degree and has established herself as a patent and corporate attorney with a specialty in biotechnology. Key CytoGenix staff Xin-Xing Tan, PhD. Senior research scientist Dr Tan earned his PhD in biochemistry from the Chinese Academy of Science, Shanghai, China. He was an NIH-sponsored postdoctoral fellow at Rice University, Houston. He has authored or coauthored 20 scientific published papers. Dr Tan joined CytoGenix in 2002 and is co-inventor of an anti-bacterial technology developed in the company’s laboratory. He leads the company’s research efforts in developing novel antimicrobial therapeutics. Frederic Kendirgi, PhD. Senior scientist

Dr Kendirgi earned his MSc in virology and immunology in 1995 from the University of Quebec/INRS-Institut Armand-Frappier, Canada, and his PhD in biochemistry and molecular biology in 2000 from the University of Calgary, Canada. Following four years of postdoctoral studies in cell biology at Washington University and Vanderbilt University, Dr Kendirgi joined the CytoGenix research and development team in October 2004. He is a co-inventor of the synDNATM technology and oversees manufacturing operations as well as the synDNATM vaccine platform. Harilyn W McMicken, BS, MT (ASCP). Senior laboratory director Harilyn McMicken graduated from Abilene Christian University and University of Texas at Galveston. She has done postgraduate work at the University of Texas School for Biomedical Sciences in Houston, Texas. Prior to her employment, Ms McMicken was senior research assistant for 23 years in the Department of Paediatrics at the Baylor College of Medicine. Her responsibilities and expertise lie in the areas of molecular biology and laboratory and personnel management.

KEY CYTOGENIX SCIENTIFIC PUBLICATIONS Scientific publications Kendirgi F, Yun NE, Linde N, Zacks MA, Smith J, Smith J, McMicken H, Paessler S and Chen YA. Novel DNA-based vaccine protects mice against lethal infection with H5N1 influenza virus isolate A/ Vietnam/1203/04. Human vaccine 2008 (In Press). Tan X, Knesha R, Margolin W and Chen Y. DNA enzyme generated by a novel single-stranded DNA expression vector inhibits expression of the essential bacterial cell division gene ftsZ, Biochemistry 43, 2004. pp1111-1117 McMicken H, Bates P and Chen Y. * Antiproliferative activity of G-quartet-containing oligonucleotides generated by a novel singlestranded DNA expression system. Cancer Gene Therapy 10 (12) 2003. pp867-869 Chen Y and McMicken H. Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector. Gene Therapy 10. pp1776-1780, 2003 Chen Y, Ji Y and Conrad C. Expression of single-stranded DNA in mammalian cells, Biotechniques 34, 2003. pp167-171 Chen Y. A novel, single-stranded DNA (ssDNA) expression vector (Review). Expert Opinion on Biological Therapy 2, 2002. pp735-740 Chen Y. Meeting highlights, 10th international conference on gene therapy of cancer, Expert Opinion on Biological Therapy 2, 2002. pp443-445 Datta H and Glazer P. Intracelullar generation of single-stranded DNA for chromosomal triplex formation and induced recombination, Nucleic Acid Research 29, 2001. pp5140-5147 Chen Y, Ji Y, Roxby R and Conrad C. In vivo expression of singlestranded DNA in mammalian cells with DNA enzyme sequences targeted to c-raf. Antisense and Nucleic Acid Drug Development 10, 2000. pp415-422

October 2002 A marvel of biochemical engineering means cells can produce DNA enzyme to attach cancer, New Scientist. January 2001 Conference presentations Chen Y, Ji Y and Conrad C. Novel intracellular single-stranded DNA expression system: production of c-raf RNA-cleaving DNA enzyme. Keystone Symposia, Gene Therapy 2001: a gene odyssey. January 611, 2001, Snowbird, Utah Chen Y. Novel intracellular single-stranded DNA expression system: production of c-raf RNA-cleaving DNA enzyme. Tenth International Conference on Gene Therapy of Cancer. December 13-15, 2001, San Diego, California. (Invited speaker) Chen Y and McMicken H. Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector. Fifth annual meeting of the American Society of Gene Therapy. June 5-9, 2002, Boston, Massachusetts Chen Y. Application of a novel single-stranded DNA expression vector for gene regulation. Eleventh International Conference on Gene Therapy of Cancer. December 12-14, 2002, San Diego, California Chen Y and McMicken H. Application of novel single-stranded DNA expression vector for gene regulation. Keystone Symposia, Drug Target Validation Gene Suppression, January 17-22, 2003, Tahoe City, California Chen Y. Novel Intracellular Single-Stranded DNA Expression System and its Applications. Third annual conference: RNA in Drug Development – RNA as Tool and Target, San Diego, CA. November 10-13, 2003. (Invited speaker) Chen Y et al. In vivo generation of oligodeoxynucleotides for regulating gene expression. Keystone Symposia, New Advances in

Other publications Xin-Xing Tan and Yin Chen. A novel genomic approach identifies bacterial DNA-dependent RNA polymerase as the target of an antibacterial oligodeoxynucleotide, RBL1. February 9, 2005 Chen Y and Tan X-X. Oligodeoxynucleotide intervention for the prevention and treatment of sepsis, April 2004. pp21-23 Chen Y. Novel technologies for target validation. Genetic Engineering News 23 (11) 2003. pp7-9 21st Century Medicine: Genetic therapy promises dramatic change from treating symptoms to eliminating the disease. E*Street Journal, December 2002 Chen Y. Growth of oligo-based drugs, Genomics & Proteomics. JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 27

LABORATORY FOCUS

THE CENTRAL FISH-HEALTH LABORATORY IN ISRAEL BY DR RAMA FALK

KIBBUTZ NIR-DAVID SURROUNDED BY FISH PONDS WITH THE CENTRAL FISH HEALTH LABORATORY HIGHLIGHTED

T

he Central Fish-Health Laboratory is located in the Jordan River basin, Beit-Shean Valley, within a rural area where almost 70 percent of Israeli fish farming is centred. CFHL belongs to the Department of Fisheries and Aquaculture, Ministry of Agriculture and Rural Development, Israel. It specialises in fish and shellfish animal health and environmental quality, provides disease surveillance and diagnostic services, export certifications and offers advice and guidance on health management and treatment for edible and ornamental fish growers. The Central Fish-Health Laboratory addresses the basic requirements of a functioning fish-diseases laboratory in terms of fish and water sampling methodology and expertise, and experience in parasitical, viral and bacterial diseases of aquatic animals, as well as in histopathology, molecular biology, immunology, vaccine development and pathogenicity studies. The CFHL has been the only institute in Israel specialising in aquatic animal health for many years and is involved in a wide range of research projects concerning fish health and culture. The laboratory works with individuals, companies, organisations and academic institutions to develop and provide health management services, ensuring high standards of veterinary care and attention. The CFHL was established in 1941 when aquaculture in Israel began, and its main objective was research of the toxin-producing alga Prymnesium parvum. One of its founders, Professor Moshe Shilo and his team from the Hebrew University of Jerusalem, then discovered the mode of action of P parvum ichthyotoxin and developed a bioassay for its quantification and verification. This research was done in collaboration with the supervisor of 28 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

the CFHL, Shmuel Sarig, who was one of the originators of the European fish pathology associations (COPRAQ, EAFP). CFHL’s research priorities are set by the current needs of the fish industry in Israel, and these needs have changed over time. The severe shortage of water and land in Israel has lead to intensification of fish culture from 30kg to 200kg per hectare, and a move to use relatively brackish water, thus providing suitable conditions for the occurrence of Prymnesium. These conditions raised the awareness of the importance of water quality examinations introduced by the CFHL as a routine test performed within the field as well as in the lab. Israel’s location within the main routes of the mass migration of birds between Eurasia and Africa introduced a vast number of pathogens into aquaculture. Consequently, the CFHL was involved in introducing many new treatments and nutrition programmes for Israeli aquaculture, while remaining cognisant of ecological impact in accordance with internationally accepted standards. CFHL’s research focused on parameters of stress, mostly in tilapia, the main fish raised in Israel. This research, led by the CFHL’s supervisor for 30 years, Dr Itzhak Bejerano, elucidated the relationship between farm management, water quality and pathogen occurrence. A major research priority of CFHL research was the urgent need to find a substitute treatment to malachite green, the use of which was prohibited a number of years ago, for saprolegniasis. Saprolegniasis infections cause vast economic losses to Israeli aquaculture as a consequence of the immunological stress experienced by tilapia during the winter. In the course of this study the whitening agent

blankophor-BA was found to be highly effective in preventing and treating Saprolegnia infections in both fish and fish eggs. During recent years, Israel’s ornamentals industry has expanded significantly, currently constituting eight percent of the exports of freshwater fish into the European Union, and three percent of the world ornamentals market. As a consequence, the scope of the laboratory expanded to encompass export certification and exotic disease diagnosis as well. One of the CFHL’s contributions in this field was the first characterisation of mass mortalities of koi and common carp, and in the description of the disease, now known as the koi herpes virus. This research led to the development of a vaccine for the virus (KV3) and to further research concerning viral diseases. Research in CFHL is currently focused on developing a vaccine for the pathogenic bacteria Atypical Aeromonas salmonicida, finding alternative treatments to the parasites Hexamita sp and Ichthiophthyrius sp, and further development of a substitute treatment to saprolegniasis. When new, infectious micro-organisms are detected, CFHL is involved in evaluating its significance, isolation and in further studies concerning the micro-organism. Research collaboration occurs most often with academic institutes from Israel and abroad, when CFHL participates in both competitive government and internationally funded grants or contracts.

FACILITIES AND STAFF The laboratory works under QA demands and is preparing for ISO 17025 accreditation. Facilities and hardware include a necropsy and histology preparation lab, a microbiology lab, bacterial facilities, a PCR lab and a variety of routine laboratory equipment. The facilities include a wet laboratory building as well, with recirculating support systems. All procedures, care and treatment of fish are done in accordance with the principles of humane treatment outlined by the Guide for the Care and Use of Laboratory Animals (Chief Science, the Ministry of Agriculture, Israel) and are approved by the Committee for Ethical Conduct in the Care and Use of Laboratory Animals. The Department of Fisheries and Aquaculture includes two other facilities: 1 Aquaculture Research Station Dor. Located by the Mediterranean Sea, the station includes 40 hectares of earth ponds dedicated to field trials, and a recirculation aquaculture system intended for intensive culturing research, hatcheries and quarantine, researching mainly genetics, nutrition, reproduction, health, environment and broodstock development of fish. It houses 10 staff and students, all engaged in research. 2 Ginosar Research Station for Intensive Aquaculture. Located near the Sea of Galilee, the Ginosar station is focused mainly on nutritional and intensification studies. It has an area of 10 hectares, including 100 ponds of various sizes used for experiments, with a staff of four. Full cooperation between these three facilities enables the successful resolution of emerging problems concerning fish health. The research process usually begins in the CFHL, with in-vitro susceptibility tests and development of fish-models of diseases in aquariums and small tanks, proceeds to large-scale experiments in the Ginosar Research Station in larger tanks and small concrete and earth ponds, and progresses further to controlled field trials within earth ponds at the Aquaculture Research Station Dor. Professional staff at the CFHL include: • Dr Simon Tinman, DVM, a specialist in fish disease • Margarita Smirnov, an expert in fish pathology and histology • Nir Froyman, an ornamental fish genetics specialist and laboratory supervisor • Esther Parag, a laboratory technician, and • Rama Falk, PhD, a microbiologist.

HISTOLOGY SECTION OF AN EYE OF A RED-DRUM (SCIAENOPS OCCELATUS) INFECTED WITH NODAVIRUS. NOTE THE EXTENSIVE VACUOLATION IN THE GRANULAR LAYERS OF THE RETINA. (GIEMSA)

LIGHT MICROSCOPY OF THE BULBUS ARTERIOSUS OF TILAPIA INFECTED WITH THE DIGENEAN ASCOCOTYLE SP. SECTION SHOWS THE FIBROUS CAPSULE THAT SURROUNDS THE PARASITE AND DEFENDS IT FROM HOST REACTION (H&E)

HISTLOGY SECTION SHOWING EPITHELIOCYSTIS IN PARROT FISH. THE SYMPTOMS SHOWN ARE ENLARGED CELLS IN GILL EPITHELIUM FILLED WITH GRANULES. THE HYPERTHROPHIC CELLS OF VARYING SIZES OCCUR ON THE SURFACES OF THE GILL LAMELLAE. (TOLUIDINE BLUE)

JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 29

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THE CENTRAL FISH-HEALTH LABORATORY IN ISRAEL CFHL STAFF FROM RIGHT: NIR FROYMAN, ESTHER PRAG, RAMA FALK, MARGARITA SMIRNOV AND SIMON TINMAN

SERVICES PROVIDED

CERTIFICATION AND FARM SURVEILLANCE

CFHL services are essentially dedicated to fish health, and include diagnosis of the causative agent or etiology of mortality, together with programmes for preventing or controlling aquatic diseases. The laboratory promotes the use of high quality diagnostic and analytical testing, as well as technological innovation to provide concrete actions and efficient solutions to meet growers’ needs. The current fee offered by CFHL is subsidised by government contract and provides all clients with full confidentiality.

CFHL collaborates with the Veterinary Services and Animal Health to examine fish for infectious diseases notifiable to the Office International Epizooties or OIE. CFHL staff examine lots of fish intended for export and provide export certification under authorisation by the Minister of Agriculture. In addition, the staff conducts regular surveillance for fish producers, interstate zoning and transfer support and farm management health maintenance purposes. Export certification is aimed at meeting the demands of authorities from various countries and is done under strict regulations.

DIAGNOSTIC LABORATORY SERVICES These services encompass a wide range of diagnostic laboratory testing, including bacteriology, mycology, parasitology, virology, water quality, limnology, molecular biology (PCR) and descriptive histopathology. Results and recommendations of parasite infection studies and water quality analysis are given on the spot, while bacteriology results are made available within 48 hours and PCR results within 10 days. Reference samples for both histological and molecular diagnosis of many diseases of regional significance, including an archive of bacterial pathogens, are maintained under appropriate conditions.

DIAGNOSTIC INTERPRETATION AND CONSULTATION CFHL provides interpretations of laboratory results and the significance of the diagnostic findings. In addition, CFHL staff advise veterinarians and farmers and give recommendations on management and remediation procedures concerned with water treatment, sanitation, feeding and pathogen management, all in full compliance with regulatory authority requirements. These services include visits of staff members to aquaculture sites (including highintensity production facilities) and fisheries. 30 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

RESEARCH AND DEVELOPMENT SERVICES CFHL offers various companies a screening programme to verify their product’s usefulness for aquaculture. This programme includes susceptibility tests to various fish pathogens, toxicity tests, therapeutic-efficacy examinations in fish-models and further field trials for efficacy determination.

TRAINING AND EDUCATION CFHL conducts training workshops concerning fish health and fisheries management and certifies professionals in the fish industry. Participants include staff of laboratories and fish farms from Israel and overseas. The research of new, emerging pathogens over the years was the basis for MSc and PhD projects within the CFHL, in collaboration with academic institutes. The findings of these projects have then been applied in the routine work of the laboratory.

CONTACT DETAILS Central Fish Health Laboratory, Nir-David, Israel. Phone (972) 4 658 5877, Fax (972) 4 637 4329. Email [email protected] ■ or see www.vetserv.moag.gov.il/fishery

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RESEARCH FOCUS

WHAT’S NEW IN PANCREAS DISEASES RESEARCH? BY MARIAN MCLOUGHLIN AQUATIC VETERINARY SERVICES, BELFAST, UNITED KINGDOM

REPLICATING VIRUS

P

ancreas disease (PD) remains one of the most serious and economically important infectious diseases of farmed Atlantic salmon (Salmo salar L) in Europe. Over the last few years our knowledge and understanding of the PD virus and the disease has been expanded considerably, due to the efforts and collaboration of excellent research scientists, veterinarians and the salmon industry. This effort has been coordinated and greatly assisted by the formation of the Tri Nation PD Research Group. The aim of this article is to summarise the new and important information in pancreas disease research. For a recent review article see Alphavirus infections in salmonids – a review. MF McLoughlin and DA Graham, Journal of Fish Diseases, September 2007, Volume 30 Issue 9, Special Alphavirus issue. pp509-572 TABLE 1: Geographical and Species Distribution of SAV Isolated in Europe SAV SUBTYPE

SPECIES

COUNTRY

SAV 1

Atlantic salmon

Ireland (Mainly Connemara) Scotland

SAV 2

Rainbow trout

Atlantic salmon

France England & Scotland Italy Spain Germany* presumed SAV2 Scotland

SAV 3

Atlantic salmon Rainbow trout

Norway Norway

SAV 4

Atlantic salmon

Scotland Ireland

SAV 5

Atlantic salmon

Scotland

SAV 6

Atlantic salmon

Ireland

32 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

Marine only

DISINFECTION IS EFFECTIVE AGAINST SAV

PD VIRUS PD virus is classified as a salmonid alphavirus (SAV), and molecular analysis now indicates that there are at least six known sub-types, with very interesting geographical and species distribution. See Table 1. SAV 3 is the only sub-type found to date in Norway and can cause disease in both Atlantic salmon and sea-reared rainbow trout (Oncorhynchus mykiss [Walbaum]). SAV 2 has primarily been associated with disease in freshwater rainbow trout and was only recently introduced to the United Kingdom K. SAV 2, as distinct from freshwater strains, has recently been recognised in marine Atlantic salmon in Scotland. SAV 1, 4 and 5 are present in Ireland and Scotland. Further work to compare the pathogenicity of these sub-types is planned in the near future. Interestingly, there is regional separation of sub-types in Ireland with SAV 1 in Connemara (west) and SAV 4 in Donegal (north). Given that smolts from one region have regularly been transferred to the other, it suggests that vertical transmission has not been a major source of infection between these regions (Fringuelli et al 2008 submitted).

SAV SURVIVAL A major gap in the knowledge of SAV was its survival outside the fish and response to temperature, pH and disinfection, which are very important in the overall control of the disease spread. SAV was found to be rapidly inactivated in the presence of high levels of organic matter at 60˚C at pH7.2. It is also inactivated at pH4 (acid) and pH12 (alkali) at 4˚C, suggesting that composting, ensiling and alkaline hydrolysis would all be effective at inactivating the virus in fish mortalities and waste.

A number of common disinfectants are effective against SAV if used at the recommended concentration Virus survival was shown to be inversely related to temperature, ie, the higher the temperature the lower the survival rate, and to be reduced by the presence of organic matter. Conversely, at low temperatures the virus survived a relatively long period of up to 61 days in sterile sea water, suggesting that SAV could be transmitted between farms by currents and tidal flows at low water temperatures. Pump-ashore systems would also be at risk unless effective disinfection procedures are in place. Survival in fresh water is markedly less than in sea water. SAV2 is reported to have lower pathogenicity at 10˚C compared with 14˚C, which may explain why SAV infections at low temperature may be predominantly sub-clinical. (Graham et al 2007, Journal of Fish Diseases 30. pp533-543)

DISINFECTION A range of commercially available disinfectants, including Virkon S, Virex, Halamid, Buffodine and FAM 30 have been tested using a protocol based on the BS EN 14675 standard for efficacy against SAV. The peroxygen disinfectants Virex and Virkon S were both effective under a range of test conditions with efficacy maintained even at 4˚C, and when contact times were reduced to five minutes, even in the presence of organic matter. Halamid and FAM 30 were also effective, but were adversely affected by the presence of organic matter. Buffodene, an iodophor disinfectant widely used for egg disinfection, was adversely affected in this study by the presence of organic matter, as has previously been reported for other fish viruses. Both Virex and FAM 30 were shown to be effective if used in sea water, while Virkon S and Halamid produced precipitates when mixed with sea water, and as a result their efficacy against SAV was not tested in seawater. In conclusion, a number of common disinfectants are effective against SAV if used at the recommended concentration, for an adequate time and after thorough cleaning of all surfaces to

TABLE 2: Diagnostic Panel for Confirmation of PD TEST

PER ACUTE

DPI

0–7

ACUTE

SUB ACUTE

CHRONIC

CARRIER

0 + 14

14 > 21

21 > 42

42 > ?

$ Appetite

Casts

Morts

Runts

Serum, Heart

Serum, Heart

Serum, Heart

–

–

Histology

N/A

+ Pancreas + Heart

+ Pancreas + Heart

+ Heart + Muscle

?

IHC

Signs Virus

N/A

+ Pan only

–

–

–

RTPCR

+

+

+

+

+/–

Serology

–

–

–

+

+

remove inhibiting organic matter. (Graham et al 2007, Journal of Fish Diseases 30. pp269-277)

PD DIAGNOSIS The three pillars of disease diagnosis are • history • clinical signs and gross pathology, and • confirmatory tests – histopathology – serology – virus isolation/detection (PCR)

EPIDEMIOLOGY AND HISTORY Increasing factual information is being gathered from field surveillance and outbreaks in Norway, Scotland and Ireland. Taksdal et al 2007 have written a comprehensive description of PD in Atlantic salmon and rainbow trout in Norway, where the incidence and geographical spread of PD is increasing each year (Taksdal et al 2007 30. pp545-558. [See also maps 1 and 2]). Individual salmon farming companies have interrogated their historic and current data for trends and clues as to the source of infection and factors that may be contributing to the prevalence and severity of PD on their sites. In Scotland, a significant sub-clinical pattern of PD disease has been revealed, indicating that infection is much more widespread than observed disease, and that poor performance in CONTINUED ON PAGE 35

FIGURE 1. THE SPREAD OF PD FROM 2004 TO 2007 IN WESTERN NORWAY

MAP 1: DISTRIBUTION OF CONFIRMED PD OUTBREAKS IN 2004

MAP 2: DISTRIBUTION OF CONFIRMED PD OUTBREAKS UP TO 01/12/2007. SOURCE MATTILSYNET

2004

JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 33

BOOK REVIEW

AQUACULTURE BIOSECURITY: PREVENTION, CONTROL AND ERADICATION OF AQUATIC ANIMAL DISEASE EDITED BY A DAVID SCARFE, CHENG-SHENG LEE AND PATRICIA J O’BRYEN. BLACKWELL PUBLISHING, 2006. PP182. ISBN: 978-08138-0539-9

D

isease in aquaculture is responsible for widespread and often economically debilitating losses and has implications for stock welfare, international trade and consumer perception and purchasing patterns. As a consequence, aquaculturists, veterinarians, policy-makers, scientists and a range of other stakeholders have become increasingly aware of the need to develop a coherent and integrated approach to aquatic animal disease prevention and management. It is against such a backdrop that the theory and practice of biosecurity has gained increasing prominence in recent years. During the World Aquaculture Society’s annual conference in Hawaii in March 2004, the Oceanic Institute and the American Veterinary Medical Association brought together a diverse group of stakeholders for a three-day session entitled Aquaculture Biosecurity 2004. The aim of this session was to present and assess the range of approaches being taken globally, and then to identify the key steps necessary to effectively implement biosecurity programmes in the production environment. Aquaculture Biosecurity is a compilation of twelve of the 30 presentations given at the stakeholder session. Chapters one and two provide an overview of the biosecurity approaches undertaken at the international level by focussing on the World Organisation for Animal Health, the World Trade Organisation and the Codex Alimentarius Commission and their respective standards aimed at eliminating trans-boundary disease transmission. Chapter three presents an interesting and informative example of a regional approach to aquatic animal health management. In this chapter the scope and utility of programmes designed by the Network of Aquaculture Centres in AsiaPacific is presented as an example of effective regional co-operation. This is followed by a comprehensive and wellwritten account of the development and functioning of Canada’s national Aquatic Animal Health Programme, using relevant case studies to highlight the need for an overarching and allencompassing approach to biosecurity. The national dimension is continued A. DAVID SCARFE in chapter five, where the United States 34 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

Fish and Wildlife Service’s aquatic animal health policy is discussed, with particular emphasis on the innovative approaches to disease management contained therein. A detailed account is presented of the plan’s risk assessment procedures, which were developed to both standardise and optimise the decision-making process pertaining to aquatic animal relocation. The situation in the USA makes for an interesting discussion point and is explored in detail in this book. That many states have no regulations, while others address only salmonids, provides an entry point to showcase Wisconsin’s veterinary approach to fish health, the topic of chapter six. Wisconsin’s approach is an interesting one, given that it includes non-salmonids and deals with fish farm registration, certification of veterinarians, health standards for fish introduced into public waters and the issuing of fish import permits. Moreover, the programme evolved as a result of an effective working relationship between the state government, aquaculturists and veterinarians, and is therefore an important example of multi-stakeholder co-operation. Chapter seven makes a coherent and succinctly presented case for a harmonised system of accreditation to ensure diagnostic data and test result validity in aquatic animal health diagnostic systems. This chapter forcefully promotes that valid data and test results are a critical pre-requisite for effective biosecurity planning and implementation. Chapter eight provides an excellent and in-depth review of the types of disinfectants applicable to aquaculture, as well as stressing the need for disinfection as an integral component of effective biosecurity at all levels. Information on the efficacy and characteristic of commonly used disinfectants, fish pathogen classification and transmission/susceptibility characteristics of important fish pathogens is presented clearly and helpfully in tabular format and makes an excellent point of reference for the interested reader. Moreover, the “example disinfection protocols” are another informative reference point and a welcome addition to a well-rounded chapter. The ninth chapter highlights the importance of aquatic animal health surveillance in effective policy making, not only for disease control but also for quarantine and health certification. Chapter 10 deals with the key issue of biosecurity at farm level and how to create the optimum environment and “state of mind” among farm workers and managers to significantly minimise risk in aquaculture facilities. This chapter succeeds in emphasising that

effective farm-level procedures are critical in the process. However, it would have been helpful to see some of the ideas presented in this chapter developed further. For example, how can the latest advances in business communication theory be utilised and developed to inculcate a sense of biosecurity awareness? What are the motivational models that can best be applied to the aquaculture environment to enable staff at all levels in the enterprise to promote and foster “good practice?” It seems to me that a more detailed and nuanced approach can be formulated, using human resource management practices, to address this issue and to develop guidelines for farm managers. The book closes with two case studies of diseases affecting the salmon industry: infectious haematopoietic necrosis and infectious salmon anaemia. Such case studies are certainly helpful in comparing

and contrasting approaches and make the case effectively for pathogen-specific strategies where necessary. Overall, Aquaculture Biosecurity: prevention, control and eradication of aquatic animal disease, is an indispensable source of detailed information for biologists, veterinarians, policy makers and aquaculturists. The editors are to be congratulated for bringing together so effectively and succinctly a diverse range of topics presented by such a distinguished list of contributors. The result is a well-balanced mix of legislative, scientific and management information that both complements and augments the existing literature on this most important of topics.

▲

TABLE 3: SUGGESTED SAMPLES FOR DIFFERENTIAL DIAGNOSIS OF PD, HSMI & CMS

CONTINUED FROM PAGE 33

the absence of mortality may be related to chronic sub-clinical infection (Graham et al 2006, Diseases of Aquatic Organisms 72. pp193-196). Recent epidemiological results from Ireland revealed that mean PD-related mortality was 18.8 percent in 2003 and 14.8 percent in 2004, with an estimated loss of growth of 11.8 percent over the two years. The risk factors associated with PD outbreaks included positive farms in the area, a history of PD on the site, more than 250,000 fish on the site and high lice burdens (Rodger and Mitchell 2007 30. pp157-167).

PD LABORATORY TESTS Various laboratory tests have been developed and tested in the past few years and will aid the confirmation of a PD outbreak. Serology and RT-PCR have also been successfully used as surveillance tools enabling the identification of acute and chronic infections. Some companies now carry out routine serology testing in advance of the risk period(s) for PD as part of their overall health plan, and have found the early warning system where viraemia is detected in pre-clinical cases to be very useful in managing PD outbreaks. Antibodies develop two to three weeks post infection at 1214˚C and have been found to persist up to harvest and are a good retrospective test for PD infection. Real-time PCR has also proven to be a very sensitive tool for detecting viral nucleic acid, but this signal does not persist as long as the presence of antibody (Graham et al 2005, Journal of Fish Diseases 28. pp373-379). Recent work indicates that pooled sera can be used effectively for detecting circulating SAV activity as individual samples, thus making population screening more cost-effective. The work on PD survival has also indicated that transporting sera for virus isolation should be at 4˚C to prevent false negative results. Table 3 summarises the test and samples required to confirm a diagnosis of SAV infection and to differentiate them from similar pathologies.

REFERENCES Alphavirus infections in salmonids – a review MF McLoughlin and DA Graham. Journal of Fish Diseases, September 2007, Volume 30 Issue 9, Special Alphavirus issue. pp509-531 Fringuelli E, Rowley HM, Wilson JC, Hunter H and Graham DA 2008. Phylogenetic analyses of partial E2 and ns P3 gene nucleotide sequences and molecular epidemiology of European salmonid alphaviruses (SAV). Submitted Graham DA, Jewhurst VA, Rowley HM, McLoughlin MF, Rodger

Dr Scott Peddie Aquaculture Health International

■

TEST

SERUM

HEART

KIDNEY

GILL

Virus

R

R

R

R

SAV RTPCR

R

R

SAV Antibody

R R

R

Histology

R

PANCREAS

MUSCLE

R

R

Serology and RT-PCR have also been successfully used as surveillance tools enabling the identification of acute and chronic infections. H and Todd D 2005. Longitudinal serological surveys of Atlantic salmon Salmo salar L using a rapid immunoperoxidase-based neutralisation assay for salmonid alphavirus. Journal of Fish Diseases 28. pp373-379 Graham DA, Jewhurst H, McLoughlin MF, Sourd P, Rowley HM, Taylor C and Todd D 2006a. Sub-clinical infection of farmed Atlantic salmon Salmo salar L with salmonid alphavirus – a prospective longitudinal study. Diseases of Aquatic Organisms 72. pp193-199 Graham DA, Cherry K, Wilson CJ and Rowley HM 2007. Susceptibility of salmonid alphaviruses to a range of chemical disinfectants. Journal of Fish Diseases 30. pp269-277 Graham DA, Staples C, Wilson CJ, Jewhurst H, Cherry K, Gordon A and Rowley HM 2007. Biophysical properties of salmonid alphaviruses; influence of temperature and pH on virus survival. Journal of Fish Diseases 30. pp533-277 Rodger H and Mitchell S 2007. Epidemiological observations of pancreas disease of farmed Atlantic salmon Salmo salar L in Ireland. Journal of Fish Diseases 30. pp157-167 Taksdal T, Olsen AB, Bjerkås I, Hjortaas MJ, Dannevig BH, Graham DA and McLoughlin MF 2007. Pathology of pancreas disease (PD) in farmed Atlantic salmon Salmo salar L and rainbow trout Oncorhynchus mykiss W in Norway. Journal of Fish Diseases 30. pp545-558 Special note: Regular updates on pancreas disease will be posted on a new, dedicated PD section at www.aqua.intervet.com ■ JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 35

ORNAMENTALS FOCUS

A BASIC OVERVIEW OF MULTICELLULAR PARASITIC INFECTIONS BY GERALD BASSLEER, BASSLEER BIOFISH, BELGIUM

This article is based on material contained in The New illustrated Guide to Fish Diseases by Gerald Bassleer ISBN 90-807831-2-9. See www.bassleer.com

CARASSIUS AURATUS SARASSA. PARASITIC AND BACTERIAL INFECTION. SARASSA WITH NUMEROUS SKIN FLUKES AND BACTERIAL INFECTION ON SKIN AND TAIL

CARASSIUS RED ORANDA. PARASITIC INFECTION. RED ORANDA WITH SMALL, WHITE SPOTS ON THE HEAD CAUSED BY SKIN FLUKES (GYRODACTYLUS)

T

he most common multicellular parasitic infections in ornamental, tropical and pond fish are skin and gill flukes. External infections are caused by skin and gill flukes (Monogenea or monogenean trematodes) Gyrodactylus species and Dactylogyrus species are especially common in aquarium fish. However, various other species, similar to these two, also occur.

GYRODACTYLUS (SKIN FLUKE) This parasitic fluke quickly reproduces itself on the skin and because it is live-bearing, the young flukes (up to three at a time) are released from the adults on the skin. It may also be found in the gills and can readily be identified in a skin scraping under the microscope. The lower the temperature the slower skin flukes will develop and reproduce. Small numbers usually do not cause any harm, except in small or juvenile fish. In terms of clinical presentation, there is usually excess slime formation on the skin, the fish scrape over substrate or against objects, red patches or lesions can be observed, with secondary bacterial infection after damage by the skin flukes. If the parasites have also infected the gills, the opercula will be flared open and the fish will be breathing rapidly. As skin flukes are usually accompanied by bleeding patches on the skin caused by physical damage or bacterial infection, the parasitic infection is often “masked”. Moreover, morbid fish may be affected by other parasitic infections, such as Ichthyobodo or Spironucleus. Treatment with Fenbendazol (200mg/100 l) is often effective, as is Trichlorfon, Flubendazol, Formalin, Mebendazol, Praziquantel or salt. In the case of severe infections, an anti-bacterial (and perhaps another anti-parasitic) medicine will have to be used to combat secondary infections. A second treatment will also be necessary within seven days of administration of the first treatment. Note, 36 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

CARASSIUS RED ORANDA. PARASITIC INFECTION. RED ORANDA WITH A CRATER IN THE HEAD CAUSED BY SKIN FLUKE (GYRODACTYLUS) DAMAGE

however, that there are strains of skin flukes that have already become resistant to certain medications.

DACTYLOGYRUS (GILL FLUKE) This parasite mainly occurs on the gills, but it can also occur on the head and skin. Reproduction occurs by means of eggs that develop in the substrate (or in the filter), so that the entire holding pond (and its filter) will have to be treated. If the water temperature is low, the larvae do not emerge from the eggs until several weeks or months have passed. This should be taken into account in the spring when the water warms up and the gill flukes may appear. These parasites (usually 1mm) can be easily identified by examining a skin or gill scraping under the microscope. In terms of clinical presentation, the fish scrape over the substrate or against objects, they are emaciated, breathe rapidly and have flared open opercula. A small number of parasites may be present on the fish without causing any damage; relatively large numbers of them are required to cause a clinically significant infection. Infection can cause physical damage, which in turn leads to secondary bacterial infections that can kill affected fish. Praziquantel (250mg/100 l for three days) is one of the most effective medicines currently available. It is recommended that treatment be repeated for three weeks to ensure that all larvae have been killed; the filter must also be treated as it may harbour larvae. Moreover, all of the sick fish can be removed from the aquarium or pond and treated in a separate tank. Over this period the flukes in the aquarium/pond will die in the absence of a host. However, at low temperatures the larvae take a very long time to hatch (at <5˚C this may even take months), or they emerge from the eggs as soon as the temperature starts to rise. This should be taken into account when treating ponds, ■ especially in the spring.

CARASSIUS AURATUS GOLDFISH. PARASITIC AND BACTERIAL INFECTION. GOLDFISH WITH ULCER CAUSED BY SKIN FLUKE (GYRODACTYLUS) AND BACTERIAL INFECTION

CARASSIUS REDCAP ORANDA. PARASITIC AND BACTERIAL INFECTION. EMACIATED REDCAP WITH GILL FLUKES AND SMALL RED PATCHES (BEGINNING OF A BACTERIAL INFECTION)

CARASSIUS AURATUS GOLDFISH. PARASITIC INFECTION. EMACIATED GOLDFISH WITH GILL FLUKE (DACTYLOGYRUS) INFECTION

CARASSIUS BUBBLE ORANDA EYE. PARASITIC INFECTION. IRREGULAR SWIMMING BUBBLE EYE WITH RESPIRATORY PROBLEMS AND EXTENDED OPERCULA CAUSED BY GILL FLUKE INFECTION CYPRINUS CARPIO KOI. PARASITIC AND BACTERIAL INFECTION. KOI WITH SEVERE GILL FLUKE INFECTION ALSO AFFECTING THE HEAD WITH SECONDARY BACTERIAL INFECTION

CARASSIUS ORANDA LIONHEAD. PARASITIC AND BACTERIAL INFECTION. LIONHEAD WITH GILL FLUKE INFECTION AND BLEEDING UNDER SKIN CAUSED BY BACTERIAL INFECTION

CYPRINUS CARPIO KOI. KOI WITH SUNKEN EYES, EXTRA MUCOUS ON HEAD AND A DAMAGED GILL COVER. GILL FLUKE INFECTION CYPRINUS CARPIO KOI. KOI WITH EXTENDED GILL COVER CAUSED BY INFECTION WITH GILL FLUKES (DACTYLOGYRUS)

PHARMAQstatistics Fish vaccines in Norway - March 2008 Oil based vaccines for salmon and trout March 2008 Doses Share 0 0,0 % 1 000 000 7,9 % 1 040 000 8,3 % 10 545 000 83,8 % 12 585 000 12 085 000 96,0 % 500 000 4,0 % 730 000

2 diseases 3 diseases 4 diseases 5 diseases Sum Salmon Trout PD vaccines

Record breaking vaccination of salmon in March

March 2007 Last 12 months Doses Share Doses Share 110 000 1,6 % 3 590 000 1,5 % 380 000 5,5 % 3 585 000 1,5 % 380 000 5,5 % 18 810 000 7,9 % 5 980 000 87,3 % 213 295 000 89,1 % 6 850 000 239 280 000 6 450 000 94,2 % 223 775 000 93,5 % 400 000 5,8 % 15 505 000 6,5 % 420 000 23 165 000

In March 12.1 million salmon were vaccinated. In comparison to March 2007 the similar number of vaccinated salmon was 6.5 million. Over the first quarter 2008 as much as 52.5 million salmon were vaccinated. This constitutes an increase of 24 % compared to the same period last year. The rolling long-term trend over the last 12 month period now shows us that nearly 240 million salmon and trout are being vaccinated. In March 2.4 million vaccine doses were used for farmed cod. This is at the same level as in March 2007. On the other hand, this month more that 2 million cod were vaccinated with an oilbased injection vaccine. Since August 2007 there has been a nice development in the long-term trend of cod vaccination.

Oil based vaccines for salmon and trout 100,0 % 90,0 % 80,0 %

According to our information, approx. 700.000 doses of a single PD vaccine (against pancreas disease), were administered to salmon in March. Re-vaccination with another vaccine is needed to add protection against other pathogens.

70,0 % 60,0 % 50,0 % 40,0 % 30,0 %

20,0 % 10,0 % 0,0 % March 2008 2 diseases

March 2007 3 diseases

Last 12 months

4 diseases

5 diseases

Doses of vaccines for salmon and trout - 12 months rolling

Millions

250

250

200

200

150

150 Doses

Doses

Millions

Doses of oil based vaccines for salmon and trout against different diseases - 12 months rolling

100

100

50

50

0

0

2 diseases

3 diseases

4 diseases

5 diseases

Salmon

Trout - oil based

Trout - water based

Oil and water based vaccines for cod March 2008 Doses 2 425 000

Sum

March 2007 Doses 2 340 000

Last 12 months Doses 21 335 000

Millions

Doses* of oil and water based vaccined for cod 12 months rolling

Definitions 2 diseases = furuculosis and vibriosis 3 diseases = 2 diseases + coldwater vibriosis 4 diseases = 3 diseases + winter sore 5 diseases = 4 diseases + IPN

24 22 20

Doses

VA C C I N E S T A T I S T I C S

This column, focusing on monthly Norwegian commercial fish vaccine statistics, is provided by PHARMAQ. See www.pharmaq.no for monthly updates.

18

16 14 12 10

38 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

PD vaccines = vaccines against pancreas disease. Smolt needs revaccination with another vaccine to add protection against other pathogens Last 12 months value is the sum of doses used the previous 12 months. The 12 months rolling graphs shows the trend by using the 12 months values over a period - here we follow the trend for the last two years * One dose administered to cod is defined as 0.1 ml of vaccine. It is important to be aware that the calculated number of vaccinated fish shows an over estimate because immersion vaccines are included in the calculation.

PHARMAQstatistics Fish vaccines in Norway - April 2008 Oil based vaccines for salmon and trout April 2008 Doses Share 30 000 0,6 % 300 000 5,7 % 670 000 12,8 % 4 245 000 80,9 % 5 245 000 5 215 000 99,4 % 30 000 0,6 % 715 000

2 diseases 3 diseases 4 diseases 5 diseases Sum Salmon Trout PD vaccines

The vaccination of salmon generation S1 2008 has passed total S1’s put to sea in 2007

April 2007 Last 12 months Doses Share Doses Share 0 0,0 % 3 620 000 1,5 % 75 000 2,2 % 3 810 000 1,6 % 90 000 2,6 % 19 390 000 8,0 % 3 235 000 95,1 % 214 305 000 88,9 % 3 400 000 241 125 000 3 000 000 88,2 % 227 300 000 94,3 % 400 000 11,8 % 13 825 000 5,7 % 0 23 880 000

In April 5.2 million salmon were vaccinated against 3.4 million in April 2007. If we consider vaccination of S1’s 2008 from October 2007, the number vaccinated by end of April 2008 is 119 million salmon. The total S1 generation 2007 was estimated to 115 million (ref Kontali Analysis). If part of the vaccination in September 2007 is included into the S1 2008 generation we assume that a total of 125-130 million will be put to sea as one-year smolt in 2008. It was a clear reduction in the vaccination of cod, falling from 2.6 million doses in April 2007 to 990.000 doses this month. This variation has been seen earlier, and it is too early to predict any further development for 2008.

Oil based vaccines for salmon and trout 100,0 % 90,0 %

According to our information, approx. 700.000 doses of a single PD vaccine (against pancreas disease), were administered to salmon in April. Re-vaccination with another vaccine is needed to add protection against other pathogens.

80,0 %

70,0 % 60,0 % 50,0 % 40,0 % 30,0 %

20,0 % 10,0 % 0,0 % April 2008 2 diseases

April 2007 3 diseases

Last 12 months

4 diseases

5 diseases

Doses of vaccines for salmon and trout - 12 months rolling

Millions

250

250

200

200

150

150 Doses

Doses

Millions

Doses of oil based vaccines for salmon and trout against different diseases - 12 months rolling

100

100

50

50

0

0

2 diseases

3 diseases

4 diseases

5 diseases

Salmon

Trout - oil based

Trout - water based

Oil and water based vaccines for cod April 2008 Doses 990 000

Sum

April 2007 Doses 2 605 000

Last 12 months Doses 19 720 000

Millions

Doses* of oil and water based vaccined for cod 12 months rolling

2 diseases = furuculosis and vibriosis 3 diseases = 2 diseases + coldwater vibriosis 4 diseases = 3 diseases + winter sore 5 diseases = 4 diseases + IPN

24

PD vaccines = vaccines against pancreas disease. Smolt needs revaccination with another vaccine to add protection against other pathogens

22 20

Doses

Definitions

Last 12 months value is the sum of doses used the previous 12 months.

18

The 12 months rolling graphs shows the trend by using the 12 months values over a period - here we follow the trend for the last two years

16

* One dose administered to cod is defined as 0.1 ml of vaccine. It is important to be aware that the calculated number of vaccinated fish shows an over estimate because immersion vaccines are included in the calculation.

14 12 10

© PHARMAQ AS 2008

RESEARCH FOCUS

SPREAD AND COLONISATION OF ALIEN FISH SPECIES IN OPEN WATERS: A RELIABLE INDICATOR OF AQUATIC HEALTH BY AK SINGH AND WS LAKRA, NATIONAL BUREAU OF FISH GENETIC RESOURCES, LUCKNOW, INDIA, AND ARVIND MISHRA, UP STATE FISHERIES DEPARTMENT, VARANASI, INDIA

N

ew strains and varieties of alien fish species have been introduced in several countries, including India (DAIS 2004, Sena De Silva et al 2006, Singh and Lakra 2006), to compete on the world market. However, environmental, socio-economic and biodiversity issues are important considerations for regulating the importation of alien fishes in India or in other countries. The introduction of alien fish raises questions about how best to meet food demands and preserve environmental quality. Four broad categories exist for ecological impacts of alien fish species. They are: • basic species interactions such as predation and competition (DIAS 2004, Singh and Lakra 2006) • genetic impacts (Senanan et al 2004, Mabuchi et al 2008) • disease impacts (Arthur 2005) and • habitat alteration (IUCN/SSC 2000). Alien species are fundamentally indicators of biological integrity in two aspects. Firstly, the spread of alien fish species in places other than where it was originally introduced (ie, post-introduction expansion). Secondly, alien fish species have been associated with the decline in or extirpation of native fish in a range of systems because of predation, competition and/or transmission of disease. In recent years, many alien fish species have been introduced into India illegally or otherwise (Table 1). These alien species have escaped into streams and rivers by human activity and aquaculture

diversification efforts and are likely to be susceptible to invade new environments and ecosystems. Naturally populating alien fish species have therefore been considered as an indicator of degraded stream conditions or the poor health of the aquatic ecosystem. Extensive studies have demonstrated that five to 45 percent exotic fish were present in river stretches and streams of Ganga and Yamuna in Uttar Pradesh, India. The maximum contribution of commercial catch was constituted by alien tilapia (Oreochromis niloticus) and common carp (Cyprinus carpio), while there was stray occurrence of other alien fish species like grass carp, silver carp and bighead carp or even African catfish Clarias gariepinus. Tilapia (Oreochromis niloticus) and common carp (Cyprinus carpio) have formed breeding populations and contributed a large percentage of the exploited stock in the river. These alien fish species have started competing for food and space in riverine stretches, showing diet overlap with native and endemic fish species and thus environmental changes have been perceived (Table 2). Thus it may be expected that riverine ecosystems consisting of various natural foods in different niches of the river is important in diversified zones that could be homogenised by a feral population of alien fish species. Unethical over-fishing, the use of chemicals, poisons and dynamite, a wide array of prohibited fishing methods and habitat destruction of natural spawning and breeding grounds

TABLE1: Alien food fishes of common occurrence in Indian freshwater aquaculture COMMON NAME SPECIES

YEAR

SOURCE OF INTRODUCTION

REASON OF INTRODUCTION

SPREAD INTO NATURAL WATER BODIES

Mozambique tilapia

Orechromis mossambicus

1952 1962 1985

Indonesia Bangkok Sri Lanka Bangladesh Nepal

Aquaculture

Reservoirs of southern part, lakes, wetlands of West Bengal and Assam, back waters of Kerala, river stretches of northern plain.

Nile tilapia

Oreochromis niloticus

1987

Thailand Israel

Aquaculture and sewage River stretches of Ganga and Yamuna in Uttar Pradesh fed fisheries

Common carp (Scale carp)

Cyprinus carpio communis

1939 1957

Sri Lanka Bangkok

Aquaculture (Composite Most of the river stretches, reservoirs and lakes. Fish Culture) Recently formed commercial catch in Yamuna and Ganga in UP

Mirror carp

Cyprinus carpio specularis

1939 1957

Sri Lanka, Bangkok

Aquaculture in high altitudes

Yamuna, Jhelum, Beas, Satluj

Grass carp

Ctenopharyngodon idella

1959

Japan, Hongkong

Aquaculture (Composite Fish Culture)

Reservoirs and few river stretches

Silver carp

Hypophthalmichthys molitrix

1959

Japan Hongkong

Aquaculture (Composite Reservoirs, lakes and few river stretches Fish Culture)

Bighead

Aristichthys nobils

1987

Possibly Bangladesh & Nepal

Aquaculture

Reservoirs, lakes and few river stretches

African catfish

Clarias gariepinus

Not known

Neighbouring countries

Aquaculture

Few reservoirs, Yamuna and occasionally in Godawari river

Sutch catfish

Pangasius sutchi

Not known

Neighbouring countries

Aquaculture

River stretches in West Bengal

40 AQUACULTURE HEALTH INTERNATIONAL JUNE 2008

AFRICAN CATFISH IN INDIA

TRADITIONAL RIVER FISHING

of the fish through various human activities in the river has contributed to the population decline of the freshwater fish. The recent invasion of alien species in riverine stretches has further threatened the existence of local and endemic fishes (Singh and Lakra, 2006). The eroding health of the riverine ecosystem due to the invasion of alien fish species is attributable to the following facts: 1 The ability of many introduced fish species to thrive in degraded aquatic habitats and their potential to impact on aquatic ecosystem structures and functions. Thus, introduced fish may represent both a symptom and a cause of decline in river health and the integrity of native aquatic communities. 2 The varying sensitivities of many commonly introduced fish species (Cyprinus carpio, tilapia and Aristichthys nobilis, Clarias gariepinus and Pangasius sutchi) to degraded stream conditions to become invasive because of the environmental and biological characteristics of the receiving water body, are all confounding factors that may obscure the patterns of introduced fish species distribution and abundance and therefore their reliability as indicators of river health. 3 The relationships of alien species distributions and indices of abundance and biomass with the natural environmental features, the biotic characteristics of the local native fish assemblages and indicators of anthropogenic disturbance are subject to varying intensities of human interventions on alien species introduction for aquaculture without adopting biosecurity and bio-safety measures.

OREOCHROMIS NILOTICUS FROM YAMUNA RIVER

4 Alien fish species found to be widespread in distribution and often abundance in rivers and streams could be considered to be relatively tolerant to river degradation, making them good candidate indicators of river health. 5 Potential confounding factors that may influence the likelihood of successful establishment of an alien species have the potential to disrupt the biological integrity of natural aquatic ecosystems. It is therefore important to assess the associated risks and benefits of any alien fish introduction. If found appropriate, develop and implement a plan for their responsible use. The development of codes of practice is the mechanism to assist in the responsible use of introduced species. In India, the National Bureau of Fish Genetic Resources has also developed plans and guidelines to regulate the introduction of exotic fish to safeguard fish biodiversity and biological integrity. The

TABLE 2: Environmental impact of alien fish introductions in India. FISH SPECIES

ENVIRONMENTAL IMPACT

REFERENCES

Oreochromis mossambicus

Displaced Gangatic carps, Puntius dubius and Labeo kontius and now posing threat to Etroplus suratensis in back waters of Kerala

Singh and Lakra, 2006

Aristichthys nobilis

Displacement of Catla and silver carp, hybridization with silver carp and Catla

Singh and Ponniah 2001; Mia et al 2005;Taylor 2005; Singh and Lakra, 2006

Cyprinus carpio

Displacement of local spp. Schizothorax, Osteobrama belangiri, Tor putitora etc.

Singh and Das 2006; Singh and Lakra 2006

O. niloticus

Reduced catches of indigenous fish species

Sugunan 2002 ; Singh and Lakra 2006

Clarias gariepinus

Environmental problem posing threat to biodiversity. Thakur 1998; Singh and Ponniah 2001 Singh and Risks of hybridisation with native fishes, loss to local culturable fishes. Mishra 2001; Sugunan 2002; Singh and Lakra 2006

Hypophthalmichthys molitrix

Naturalised in some reservoirs and displacement of Catla

Pandey 1997; Singh 2004

Pangasius sutchi

Evironmental problems are realised but yet to be documented.

Under study by NBFGR

JUNE 2008 AQUACULTURE HEALTH INTERNATIONAL 41

RESEARCH FOCUS

SPREAD AND COLONIZATION OF ALIEN FISH SPECIES IN OPEN WATERS HARVEST OF PANGASIUS SUTCHI

COMMON CARP

aquaculturists and farmers are advised to comply with the available regulatory mechanisms for alien fish introductions, along with strict conditions of sanitary and hygienic standards.

and McAndrew BJ 2005. Detection of hybridisation between Chinese carp species (Hypophthalmichthys molitrix and Aristichthys nobilis) in hatchery brood stock in Bangladesh, using DNA microsatellite loci. Aquaculture 247. pp267-273

REFERENCES

Pandey AC 1997. Incidence and impact of capture of exotic carps in the River Gomti near Gohania village (Distt. Sultanpur). Ind Journ Anim Science 67. pp637-638

Arthur JR 2005. An historical overview of pathogen introductions and their trans-boundary spread in Asia. Preparedness and response to aquatic animal heath emergencies in Asia. FAO Fisheries Technical paper 451, FAO Rome, Italy. pp1-71 DIAS 2004. Database on Introductions of Aquatic Species. Fisheries Global Information System. FAO Rome, Italy IUCN/SSC Invasive species Specialists group 2000. Guidelines for the prevention of biodiversity loss caused by alien invasive species. IUCN Gland, Switzerland Mabuchi K, Senou H and Nishida M 2008. Mitochondrial DNA analysis reveals cryptic large-scale invasion of non-native genotypes of common carp (Cyprinus carpio) in Japan. Molecular Ecology 17. pp796-809 Mia M, Younus, Taggart JB, Gilmour AE, Gheyas AA, Das TK, Kohinoor AHM, Rahman MA, Sattar MA, Hussain MG, Mazid MA, Penman D

Sena De Silva, Thuy TT, Nguyen Nigel, Abery W and Amerasinghe US 2006. An evaluation of the role and impacts of alien finfish in Asian Inland aquaculture. Aquaculture Research 37. pp1-17 Senanan W, Kapuscinki Anne, Uthairat Na-Nakorn R and Miller LM 2004. Genetic impacts of hybrid catfish farming (Clarias macrocephalus x Clarias gariepinus) on native catfish populations in Central Thailand. Aquaculture 235. pp167-184 Singh AK 2004. Aquaculture diversification and species enhancement: problems and perspectives. In: Zoology and Human Welfare (ed) Dr Ashok Verma. Published by Dr SP Mukherji Government College, University of Allahabad. pp252-261 Singh AK and Das P 2006. Status of common carp (Cyprinus carpio – Linnaeus, 1758) in aquaculture and its environmental impact. Aquaculture 7. pp245-257 Singh AK and Mishra A 2001. Environmental issue of exotic catfish culture in Uttar Pradesh. J Envirn Biol 22. pp205-208

OIE PUBLICATIONS 2008 Available for purchase from the Office International des Épizooties (OIE), 7 Rue de Prony, 75017 Paris, France, or for consultation on-line at www.oie.int • Aquatic Animal Health Code, 11th edition 2008. English language version. 170pp • Code sanitaire pour les animaux aquatiques, 11th edition 2008. French language version. pp160 • Código Sanitario para los Animales Acuáticos, 11th edition 2008. Spanish language version. pp160 • Manual of Diagnostic Tests for Aquatic Animals, 5th edition 2006. English language version. pp469 • Manual de Pruebas de Diagnóstico para los Animales Acuáticos. 5th edition 2008. Spanish language version. pp480 • Changing trends in managing aquatic animal emergencies (ed) Eva-Maria Bernoth. Scientific and Technical Review 27 (1): pp300 (trilingual English-French-Spanish). April 2008. (Editor’s note: This publication will be reviewed in a forthcoming issue of Aquaculture Health International.) • Risk analysis in aquatic animal health (ed) CJ Rodgers. Thematic Publications 2001. pp346

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Singh AK and Ponniah AG 2001. In: Proceedings of the national symposium on fish health management and sustainable aquaculture. College of Fisheries, GB Pant University of Agriculture and Technology, Nov 1-2, 2000 Pantnagar. (eds) Singh UP, Chauhan RS and Sharma AP. pp93-96 Sugunan VV 2002. Clarias gariepinus (African catfish) gravitates into the River Yamuna, Sutlej, Godawari. Angst comes true. Fishing Chimes 22. pp50-52 Taylor RM and Pegg MA 2005. Management and ecological note response of bighead carp to a bio-acoustic behavioural fish guidance system. Fisheries Management & Ecology 12. pp283-286 Thakur NK 1998. A biological profile of African catfish, Clarias gariepinus and the impact of its introduction into Asia. In: Fish Genetics and Biodiversity conservation (eds) Ponniah AG, Das P, and Verma SR. Natcon Publication, Muzaffarnagar (UP), India. pp275-292

CONTACT DETAILS AK Singh and WS Lakra. National Bureau of Fish Genetic Resources, Canal Ring Road, PO Dilkusha, Lucknow-226002 (UP), India. Email [email protected] Arvind Mishra. UP State Fisheries Department, Varanasi-221002, ■ Uttar Pradesh, India

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