Delivery of Passive Immunity to Animals of Mariculture
Reference number: STC-LS-0350
Inventor(s): R. Durvasula; S. Durvasula
For more information, contact: Jovan Heusser, M.B.A. (505-272-7908)
Patent(s)
Application(s) pending
Background
World aquaculture production has increased to 59.4 million metric tons (MT) in 2004, with a value of $70 billion. Of this, farmed shrimp production accounts for 2.4 million MT, representing a value of nearly $10 billion (1) Diseases caused by agents such as White Spot Syndrome Virus (WSSV) and Vibrio species have decimated shrimp farming industries in many parts of Asia and South America, and account for nearly $3 billion of economic loss annually. Unregulated use of antibiotics in farmed shrimp and fish operations has been banned and contributes to the epidemic of drug-resistant bacteria in humans. Intensive practices that involve meticulous water exchange with strict standards of hygiene have been effective in reducing transmission of infectious pathogens in farmed shrimp (2, 3) but are impractical in many lower-income settings of the world. Usually, appearance of disease is associated with loss of harvest for shrimp farmers and a staggering 30% of global production is lost annually. The economic impact of infectious diseases of mariculture is overshadowed only by their tremendous threat to global food security.
Technology
The invention discloses paratransgenic methods for prevention, amelioration or treatment of infectious diseases in aquatic animals of commercial importance. In this method, common feed organisms such as microalgae, cyanobacteria and bacterial probiotics are modified to produce molecules that are toxic to infectious agents. The feed organisms can then be fed directly to animals or via an intermediate such as Artemia to confer passive immunity in shrimp, fish or shellfish against common infectious agents such as Vibrios and viruses.
For over 10 years, our laboratory has been developing a novel approach to control of infectious disease transmission, termed paratransgenesis. In this strategy, commensal or symbiotic bacteria found at mucosal sites of pathogen transmission are isolated and genetically altered to elaborate immune peptides or engineered single chain antibody fragments (scFv) that neutralize infectious agents. The transgenic bacteria are then delivered back to mucosal sites where disease transmission occurs. We initially developed this “Trojan Horse” approach to combat transmission of the Chagas disease parasite, Trypanosoma cruzi, by reduviid bug vectors (4-8). We have since applied it to commensal bacteria of the human respiratory tree (9), sharpshooter vectors of the grape bacterial disease, Pierce’s Disease (10), and sandfly vectors of visceral leishmaniasis (11).
We recently demonstrated that a marine cyanobacterium, Synechococcus bacillarus, could be genetically transformed to express a functional recombinant antibody (12). This study demonstrated that a transgenic cyanobacterium could produce an active recombinant antibody, and serves as a model for future applications of this technology. We have also demonstrated that Artemia can be used as a delivery vehicle for modified feed organisms to the commercially important shrimp, Litopenaeus vannamei (13,14). Ongoing studies involve development of several molecules with killing activity against Vibrio harveyi which will be expressed via modified probiotic bacteria and cyanobacteria to confer protection in farmed shrimp populations.
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Applications/Advantages
We now propose to use the paratransgenic approach to control several infectious diseases of commercial mariculture. Lines of marine cyanobacteria, algae and probiotic bacteria - common components of feed for farmed shrimp and fish - can be transformed to produce recombinant antibodies as well as naturally occurring antiviral and antimicrobial proteins that will neutralize infectious pathogens such as WSSV and Vibrio species. Delivery of these feed organisms, either in slurry preparations or via a bioamplification strategy with Artemia, will result in passive immunization of the alimentary tract of farmed marine animals. This is the portal of entry for many infectious agents and the delivery of neutralizing agents would either abort the infectious process or delay it sufficiently to permit harvest.
This technology aims to provide cheap and effective tools for control of many infectious processes that affect commercial mariculture. Several of the effector molecules, which we have tested against pathogenic Vibrio species, are naturally occurring components of innate immunity in both invertebrate and vertebrate animals. Expression of recombinant versions of these peptides in the gut of animals such as farmed shrimp can be accomplished through a series of expression systems that we have developed for common aquatic feed organisms such as E. coli Nissl strain, Bacillus subtilis and Synechococcus bacillarus. Innately expressed proteins such as Anti-Microbial peptides (AMP’s) and antibody fragments are found ubiquitously in nature and pose no harm to farmed animals or humans who consume the animals.
The paratransgenic approach involves genetic modification of feed organisms only. These bacteria and algae are consumed by commercial animals such as shrimp and are degraded rapidly in the gut. Release of molecules that exert toxicity toward infectious agents such as Vibrios and WSSV occurs in the gut lumen and no transgenes enter the animal itself. Therefore, concerns about introduction of genetically modified organisms in the animal itself are minimized significantly. Furthermore, we have demonstrated in the whiteleg shrimp, L. vannamei, that delivery of recombinant molecules to the gut lumen is tightly regulated through feed, either via slurry or Artemia intermediates. Accumulation of recombinant molecules in the shrimp gut can be rapidly reversed within hours of changing feed composition, suggesting that use of such feed preparations can be tightly regulated under commercial settings.
Antibiotic use in commercial aquaculture settings has been banned around the world, largely due to evolution of pathogen resistance and introduction of drug-resistant microbes to humans. In the paratransgenic strategy, technologies for rapid modification of engineered antibodies permit timely response to evolution of pathogen resistance. Vast repertoires of engineered antibodies can be made from combinatorial libraries of genes and tested against newly evolving pathogen epitopes within weeks, thus assuring a near infinite set of possibilities for molecule design and deployment. Our research consortium has expertise in design and synthesis of anti-pathogen molecules and can respond to current and future variants of common pathogens of mariculture (15-18).
