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Cycle 5 (2015 Deadline)

Enhanced research capacity and fish health infrastructure to assist Tunisian aquaculture

PI: Nadia Chérif (, Institut National des Sciences et Technologies de la Mer (INSTM)
U.S. Partner: James Winton, U.S. Geological Survey, Western Fisheries Research Center
Project Dates: December 2016-November 2019

Project Overview:

Aquaculture is the fastest growing food-production sector in the world, providing a significant supplement to aquatic organisms harvested from the wild. However, the high density of animals reared in intensive aquaculture frequently produces infectious diseases that have emerged as major constraints to the successful development of aquaculture in many areas of the world. The project will support both basic and applied research that will generate tools and knowledge needed for promoting social acceptance and good governance of Tunisian aquaculture, as well as solutions for sustainable production. The project has three objectives: (1) establishment of a Phase-1 prototype aquatic health network, an initiative towards development of a National Aquatic Health Network (NAHN) for Tunisia; (2) development of improved surveillance tools and characterize host defense mechanisms; (3) application of nodavirus RNA3 as a biosensor. Key benefits from the research include providing an enabling environment for sustainable aquaculture, protecting investments from aquatic diseases and pests, and increasing research capacity for the prevention, early detection, and response to aquatic disease threats.

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The PEER project team. Picture courtesy of Dr. Cherif

This project combines expertise in genomics, fish virology, biotechnology, and immunology to enhance technical capacities for improving aquaculture productivity and marketing in Tunisia. It also represents an exciting opportunity to use novel molecular approaches including biosensor technology to answer questions about the viral status of commercially important aquaculture species, potentially serving as an early warning system to prevent diseases that could affect fish harvests. Planned capacity building activities under the project include proficiency testing and assay validation with the aid of the laboratory of the U.S. partner. Other components of the training will include proper sampling protocols for fish, as well as recruitment and training of lab technicians across Tunisia. Another important product of this initiative will be the assessment of to a national fish health program. Other products include the results from research on the effects of immune stimulants on the health of fish in aquaculture farms in Tunisia and an expanded understanding of their impact on antiviral immunity. The results will be shared with stakeholders and will be used at the end of the validation stage.

Summary of Recent Activities: 

The aim of this project which ended in December 2019 was to support both basic and applied research that would generate tools and knowledge needed for promoting social acceptance and good governance of Tunisian aquaculture, as well as solutions for sustainable production.

The first work package activities focused on the improving of the fish health surveillance in Tunisian Aquaculture resulted in the creation of new National Zoo-sanitary Program covering a set of listed diseases affecting farmed fish species in Tunisia. The virology laboratory of the National Institute of Sea Sciences and Technologies (INSTM) was designated by the competent authority as the National Reference Laboratory to undertake diagnostic activities for certain of the listed aquatic diseases. The program enabled the veterinary services to move from a passive surveillance to an active surveillance targeting two listed viral diseases as recommended by the World Organization for Animal Health (International Organization of Epizooties; OIE). In addition to causing significant mortality and economic losses in fish farms, the presence of these two viral diseases, Viral Hemorrhagic Septicemia (VHS) and Infectious Hematopoietic Necrosis (IHN), will prevent the export of fish to market partners located in OIE-member countries such as the USA, Canada, Morocco and some European countries due to the risk of the introduction of these diseases via the movement of live fish or aquaculture products. Consequently, the determination of an official zoo-sanitary status of the Tunisian fish farming zones became mandatory.

During the project period, one farm participated in the pilot program. Two inspections and two sampling protocols were accomplished with a good collaboration between our team and the farm technical staff. Per guidelines from the OIE, virus-free status will require two successive years to perform four inspections and four samplings of all the farm cages and fish species. By the end of the survey, the farm can ask for an official zoo-sanitary status. Using their preliminary results, the farm has already received very good feedback from its economic partners and guaranteed a regular weekly export market to Canada. The success of this certification pilot program has led to the expansion of the program, starting in December 2019, to cover all the 25 active fish farming units in Tunisia including two hatcheries. A Policy Document is in preparation with the participation of the General Directorate of Veterinary Services and the National Institute of Sea Sciences and Technologies presenting guidelines for the application of the national zoo-sanitary surveillance network that will significantly improve aquatic animal health and export opportunities for Tunisian aquaculture.

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The PI, Dr. Nadia Cherif. Picture courtesy of PI
The second work package (WP2) of the project targeted the development of innovative tools to be used for the clinical diagnosis of infectious aquatic animal diseases. Activities during this project were focused on the development of novel biosensor chips which will offer several advantages as a label-free detection system for pathogens including rapid detection, easy manipulation and low cost by minimizing reagents and effort. Biological pathogens can be detected by their attachment to specific receptors immobilized on the surface of the biosensor chips. During the reporting period, some technical problems were faced due to the quality of some reagents, however these challenges were largely overcome. In addition, promising results were obtained using experimental materials and the testing of real samples will be accomplished in the coming period. A specific aim of WP2 was to develop and optimize a simple and ultrasensitive biosensor to detect fish nodaviruses representing all the different genotypes causing significant losses in Tunisian and Mediterranean fish farms. In order to achieve this goal, two innovative approaches were used. The first was based on the detection of the viral genome using a heteroduplex (RNA/DNA) binding assay in which an electrochemical signal is generated and evaluated using computer software. The second was based on the development of specific nanobodies for improved detection of nodavirus as well as the potential inhibition of infection.

The third work package (WP3) of the project included research trials related to the evaluation of some bioactive molecules to be used as probiotics to boost the fish immune system or to serve as inhibitors of aquatic pathogens. Microbial probiotics and the polysaccharide chitosan are widely used in human and veterinary medicine; therefore, we were interested in confirming their pharmacological properties, especially its effects on cells of the immune system using our aquatic model. The lactic bacterium E. facium was isolated from tilapia and chitosan was extracted from the Tunisian shrimp, Parapenaeus longirostris. These were tested for their ability to inhibit the in-vitro expression of nodavirus using the SNN-1 fish cell line. Promising results were obtained, but it is still unknown which stage of replication is the target of chitosan as adding chitosan to a cell culture after nodavirus adsorption was as effective as adding the chitosan solution before inoculation. We also investigated the anti-nodavirus activity of extracted chitosan from P. longirostris
crustacean in reducing viral loads using the E-11 cell line. Expression changes in a panel of innate immune genes were quantified as the ΔΔCT in quantitative polymerase chain reaction assays after treatment of SNN-1 cells with both molecules in order to evaluate the transcription of genes involved in cell-mediated cytotoxicity. Results were reproducible and the approach is ready to be tested in-vivo; however, the in-vivo evaluation of bioactive molecules against nodavirus in sea bass has been delayed due to infrastructure problems within the institute which led to the loss of the fish stock dedicated to this experiment (350 juveniles). Nevertheless, based on the results of the present study, chitosan could be a potential therapeutic agent against fish nodavirus, but further studies are needed to
understand the mechanism behind the anti-nodaviral activity.

During the CapTunHealth PEER project and throughout all the phases of the workpackages, meetings with technicians and farmers took place to give information on fish health statut in aquaculture and procedures to adopt, in view of the activation of a national surveillance health plans. The project provided training and several workshops were organized on the application of biosecurity measures at aquaculture sites, methods of aquatic viral disease diagnosis and the networking modalities in a national survey period.

Work package one activities focusing on the improving of the fish health surveillance in Tunisian Aquaculture has resulted in the elaboration of a new national zoo sanitary program within the farmed fish species. A Policy document is in progress of publication with the participation of the general direction of veterinary services and the national institute of sea sciences and technologies related to guidelines for the application of the national zoo-sanitary surveillance network. The activities during this project were focused on the development of biosensors which will offer several great advantages as a label free detection system including a rapid detection, easy manipulation and low cost by minimizing reagents and efforts.

The second approach concerns the development of specific Nanobodies for the improvement of nodavirus detection and potential inhibition of its infection capability. Based on the conclusive results of the present study, chitosan could be a potential therapeutic agent
against fish nodavirus.

The project described promising results related to the evaluation of bioactive molecules to be used as probiotic boosting of fish immune system or as inhibitors of aquatic pathogens. these trials can be adopted by the fish feed producers and farmers as a prophylactic measure preventing viral disease outbreaks.

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