DFID Applied studies on epizootic ulcerative syndrome Final Report February 2001 Project R6979 of the Aquaculture Research Programme of the Department for International Development of the United Kingdom This document is an output from a project funded by the UK Department for International Development (DFID) for the benefit of developing countries. The views expressed are not necessarily those of DFID. Hard copies of this report can be obtained from: ARP Manager Institute of Aquaculture University of Stirling Stirling FK9 4LA Scotland, UK Institute of Aquaculture University of Stirling, Scotland Aquatic Animal Health Research Institute Department of Fisheries, Thailand PROJECT COMPLETION SUMMARY SHEET Sheet Completed 28 February 2001 TITLE OF PROJECT: Applied studies on EUS - The ecology, immunogenicity and treatment of Aphanomyces invadans R NUMBER: R6979 PROGRAMME: Aquaculture Research Programme (ARP) PROGRAMME MANAGER (INSTITUTION): Prof J.F. Muir PROGRAMME PURPOSE: Productive benefits of aquatic resources for poor people generated through improved knowledge of aquaculture processes and their management PRODUCTION SYSTEM: Land/Water Interface BENEFICIARIES: Asian freshwater fish farmers TARGET INSTITUTIONS: AAHRI, FRI, BAU, CARE-LIFE, CIFA, BFAR, FDD, FRTI, RIA1 GEOGRAPHIC FOCUS: Southeast and South Asia Planned Actual START DATE: 1 July 1997 1 July 1997 FINISH DATE: 31 December 2000 28 February 2001 TOTAL COST: £250,613 £248,613 1. Project purpose: To generate the information needed for the formulation of strategies to contain EUS; and to develop and introduce improved prophylactic and therapeutic treatments to provide fish farmers with a means of reducing losses due to EUS. 2. Outputs: Some modifications were made to the original logical framework in January 1999. This was partly due to problems that were encountered in developing an environmental DNA probe for Aphanomyces invadans, but the change meant that the project research could be redirected towards having a greater immediate impact for small-scale fish farmers. The studies that aimed to develop the DNA probe for environmental testing were scaled down and greater emphasis was placed on field studies, particularly pond and farm trials, and epidemiological surveys in Bangladesh and Nepal. The outputs stated below in inverted commas are those listed in the January 1999 logframe, along some other outputs given in the original July 1997 logframe. 2.1 DIAGNOSIS OF EUS "Improved diagnostic procedures. Development of immunological tools for diagnosis of EUS " Jan 99. A DNA probe procedure was developed that enables identification of cultures of A. invadans. A detailed description of the molecular characterisation of A. invadans was prepared and submitted for publication. A variety of histological stains were compared and the fluorescent stain, Uvitex, was considered the most practical and useful for the diagnosis of EUS. Further procedures were tested using the A. invadans-specific DNA sequence (e.g. in situ hybridisation, PCR of DNA extracted from paraffin embedded tissues) and monoclonal antibodies (e.g. immunohistochemistry, immunocytochemistry) but these did not prove robust enough techniques for the diagnosis of EUS. - i - 2.2 ECOLOGY & GEOGRAPHICAL DISTRIBUTION OF A. INVADANS "Better understanding of the geographical distribution of A. invadans and identification of areas at risk." Jan 99 An effective environmental probe could not be developed using the available DNA sequence. However, through contact with other researchers, a better understanding of the distribution of EUS was achieved. Reports of outbreaks of EUS in the region were monitored, and samples were obtained and processed at AAHRI. The project also identified an isolate of A. invadans from USA, confirming that the EUS fungus is involved in outbreaks of “ulcerative mycosis” in the USA. Surveys showed that A. invadans is endemic in natural water bodies in Bangladesh. A technique for identifying the presence of A. invadans by studying the serum of snakehead fish was tested in the Philippines and Thailand. 2.3 ZOOSPORE PHYSIOLOGY "Based on in vitro studies, an understanding of developmental morphology of zoospores, optimal and lethal conditions for zoospore production and motility" July 97 Zoospore physiology studies provided information that enabled management recommendations to be made on the exclusion of A. invadans from fish farms. 2.4 TANK STUDIES "A challenge system tested and available for further studies, including fungal pathogenicity in relation to other disease agents." July 97 Challenge systems were developed in which snakehead developed EUS after abrasion or being placed in pH5 water for 30 mins, and then being bathed in A. invadans zoospores. The systems were then used in pond trials to assess the ability of various treatments to prevent EUS in snakehead and mrigal. Some challenge work was undertaken using bacterial pathogens in Bangladesh and India. Challenge studies also investigated the susceptibility of various non-Asian fish species to A. invadans infection. 2.5 TREATMENT STUDIES "screening of candidate fungicides" July 97 "Pond-tested treatments and interventions that prevent, or reduce the risk of, EUS outbreaks." Jan 99 Forty-nine candidate treatments were screened in vitro for activity against A. invadans. Some of these were tested in toxicity trials using silver barbs. Several treatments that showed promise were assessed further in pond trials in Thailand, Bangladesh and India. Acceptance and uptake of the treatments by farmers in Bangladesh were assessed using farmer field trials in collaboration with the CARE-LIFE project. Tank trials testing therapeutic treatments on naturally infected snakeheads and climbing perch showed that the chemicals were not effective against the fungus once it had already infected the fish. 2.6 IMMUNOLOGY "An understanding of the potential for inducing EUS-resistance in susceptible fish. Demonstration of immunoprevalence of EUS." Jan 99 A comparison of the immune reactions of various fish species revealed differences in the effectiveness of the immune mechanisms of particular susceptible and resistant fish. In tank trials, particular immunostimulants were shown to be effective in reducing mortality of snakeheads challenged with A. invadans. A tank challenge study showed that serum from - ii - snakeheads from endemic areas conferred a degree of protective immunity on naïve snakeheads. These studies demonstrate that protective antibodies against A. invadans are produced in fish, and that there is potential for vaccine development. 2.7 EPIDEMIOLOGY "Identification of factors that increase the risk of EUS in Bangladesh" Jan 99 Cross-sectional studies in Bangladesh and Nepal successfully identified risk factors regarding pond management that significantly increase the chance of EUS outbreaks in ponds. Case-control studies in Mymensingh, Bangladesh identified pond water variables and aspects of fish biology that are risk factors for EUS. 2.8 DISSEMINATION "Dissemination of information on current understanding of EUS causation, and methods for diagnosis and control, to fisheries officers and scientists." Jan 99 An EUS manual was produced and over 800 copies distributed, mainly on request. Various workshops and seminars were conducted in Thailand, Pakistan, Bangladesh, Philippines, Nepal, India, Sri Lanka and Vietnam (see Section 6). A leaflet describing control strategies for EUS was also produced and disseminated. 3. Contribution of outputs to project goal: Various management techniques and preventative pond treatments have been identified that contribute to the goal of “reducing the impact of diseases on aquaculture production in Asia”. Some treatments were assessed by farmers participating in the CARE-LIFE EUS study. Satisfaction with the treatments and uptake in the subsequent season was high among these farmers, indicating that the treatments were suitable for the farmers. Higher- cost treatments identified by the project (e.g. immunostimulants and surfactants) may be suitable for use by commercial farmers. Dissemination activities have resulted in an increased consensus among fisheries researchers in the region about EUS causation, as demonstrated by the studies reported in regional conferences. It is hoped that this will help focus research away from the study of particular pathogens and towards work on treatments for the disease, which would have greater immediate impact on farmers. Increased acceptance of a definition for EUS, and improved diagnostic techniques, will also enable laboratories to monitor outbreaks and help prevent further spread to unaffected regions. 4 Peer reviewed publications: 1998 • Khan, M.H., Marshall, L., Thompson, K.D., Campbell, R.E., and Lilley, J.H. (1998) Susceptibility of five fish species (Nile tilapia, rosy barb, rainbow trout, stickleback and roach) to intramuscular injection with the Oomycete fish pathogen, Aphanomyces invadans. Bulletin of the European Association of Fish Pathologists 18(6), 192-197. 1999 • Blazer, V.S., Vogelbein, W.K., Densmore, C.L., May, E.B., Lilley, J.H., and Zwerner, D.E. (1999) Aphanomyces as a cause of ulcerative skin lesions of menhaden from Chesapeake Bay tributaries. Journal of Aquatic Animal Health 11, 340-349. (Nominated for American Fisheries Society “Best Paper Award”) - iii - • Thompson, K.D., Lilley, J.H., Chen, S.-C., Adams, A., and Richards, R.H. (1999) The immune response of rainbow trout (Oncorhynchus mykiss) against Aphanomyces invadans. Fish and Shellfish Immunology 9(3),