APPLICATIONS OF BIOTECHNOLOGY IN AQUACULTURE

John A. Howard

ProdiGene

101 Gateway Boulevard

College Station, TX 77845

Jhoward@prodigene.com

ABSTRACT

The opportunity to custom design feed ingredients for the aquaculture industry now exists. This can include a better balance of amino acids, low phytic acid, neutraceuticals, and the oral delivery of vaccines. Examples of transgenic corn are used to illustrate how the oral delivery method can work in mice and swine. Extrapolation of these results to fish leads to the question, what are the most important targets for the aquaculture industry with this technology?

KEY WORDS: Transgenic proteins, oral vaccines, maize

INTRODUCTION

Advances in plant biotechnology have led to the realization of over one hundred million acres of crops today in production with input traits such as pest resistance. This represents the growing utility of this technology for traits that growers can appreciate. The technology, however, is not limited to input traits. The same technology can be used to design output traits for various applications, including aquaculture. This could include better nutritionally balanced feed, nutraceuticals, as well as the possibility of edible vaccines. While there has been very little effort toward engineering plant traits for aquaculture; there are a growing number of laboratories researching these new traits for other animals that may be applicable.

One example to date has been the engineering of maize to produce high lysine corn. The objective is to increase the lysine content in corn such that it can be used directly in feed without supplements. This approach has shown great promise by increasing lysine content in corn by 400% [1]. This has potential application in aquatic feed, as well. In addition, it paves the way to the introduction of a variety of proteins that can be introduced to give aquatic feed additional functional properties, including the potential for edible vaccines.

While the ability to engineer traits for nutrient value would seem relatively straightforward in terms of application, efficacy of pharmaceuticals by oral consumption appears more problematic. The first difficulty encountered is the ability of the protein to survive in the gut and, in some cases, reach the mid gut for absorption. The second problem would be to get the desired physiological response. There has recently been research on mice and now swine that could answer some of these basic questions.

RESULTS AND CONCLUSIONS

In an attempt to answer the question, can proteins survive in the gut of an animal, genetically engineered corn with the protein, avidin, was fed to mice. As can be seen in Figure 1, the avidin corn was able to survive the mid gut and appear in fecal material. No avidin was found in non-transgenic corn, and more importantly, no avidin was found in mice that had been given avidin orally at the same dose but not engineered in the corn [2]. This indicates that the avidin is broken down much more rapidly when not in corn material. The stability in corn may be due to the bioencapsulation of the avidin in the cell wall, which allows for the slow release. Similar results were also found when the antigen for Traveler’s Diarrhea (Lt-B) was fed to mice as well. (Data not shown).

With the ability to deliver proteins to the mid gut, the potential for a physiological response is possible. This was tested for several proteins in mice, as well as the antigen for Traveler’s Diarrhea (Lt-B). The data in Figure 2 illustrates the antibody response in mice when fed transgenic corn with the antigen Lt-B. Alternatively, purified Lt-B could be given orally, and while it still illicits a response, a much lower response than Lt-B in transgenic corn. This illustrates the benefits of bioencapsulated material [3].

Attempts were made to answer a more critical question, can this immune response actually lead to protection. In the case of Lt-B, the mice can have symptoms similar to that in humans while not as pronounced. In this case, the animals were fed transgenic corn and examined for protection against the toxin. The results shown in Figure 3 illustrate that the mice were protected when given the transgenic corn prior to administering the toxins.

Another example of oral delivery can be seen with swine. Transmissible Gastroenteritis Virus (TGEV) is a disease that can be fatal in young pigs and cause a variety of symptoms including diarrhea. In this case, the pigs were fed transgenic corn with a viral coat protein (TGEV-S) from the virus to try and induce an immune response. The results demonstrated that pigs are capable of eliciting an immune response when fed TGEV-S corn. (Data not shown).

Pigs were also challenged with the virus to see if the oral dose of vaccine in corn would give protection. The pigs that were fed the TGEV-S corn showed much fewer systems than the control (Figure 4). This protection was comparable to that of the commercial product, which calls for injected material [3].

CONCLUSIONS

In conclusion, there is growing evidence that corn, a major component of animal feed, can be used effectively to deliver a variety of feed additives, including nutraceuticals, and even orally delivered vaccines. Examples in mice and swing demonstrate that these transgenic proteins can provide physiological responses. This suggests that there should be an opportunity for this approach in fish.

RECOMMENDATIONS

Short-term

Currently the biotechnology industry is designing genetic material with great potential for feed additives with little understanding of aquaculture. The most immediate need is for the aquaculture industry to define the various opportunities and identify where this technology would have the greatest benefit. This would result in a list of potential applications based on economic potential. This would be followed by studying the technical feasibility of these various applications suitable to this technology. This could include exploratory projects to demonstrate proof of principle. Possibilities could include a list of diseases where edible vaccines are needed, which species are nutrient-limited and require supplements such as amino acids, what nutraceuticals could be of benefit, or other environmental issues such as reduction of phytic acid in feed or by the addition of phytase in feed. This will require funding from granting agreements.

Mid-term

After proof of principle is accomplished, development programs need to be put in place. This may require some funding from granting agencies however, with proof of principle, the industry may be prepared to pay for the bulk of this work. Also, the impact that this technology will have on the regulatory requirements, production practices, and public perception needs to be addressed.

Long-term

The long-term recommendations will be to look at synergistic traits, as well as potentially antagonistic effects from combining many traits together. This should also include, not only traits engineered in corn, but also those engineered in fish.

REFERENCES

1. Beach, L.R., Ph.D., Pioneer Hi-Bred International, Inc. (personal communication.)
2. Bailey, M.R. A Model System for Edible Vaccination Using Recombinant Avidin Produced in Corn Seed. (December 2000 Thesis, Texas A&M University.)
3. Streatfield, S.J., Jilka, J.M., Hood, E.E., Turner, D.D., Bailey, M.R., Mayor, J.M., Woodard, S.L., Beifuss, K.K., Horn, M.E., Delaney, D.E., Tizard, I.R., Howard, J.A. Plant-based Vaccines: Unique Advantages. (in press)

Figure 2. Antibody response of mice fed Lt-B transgenic

Corn. Mice were given either control corn, LT-S transgenic

Corn or purified Lt-B from E. coli. The mice were tested

On day 28 for the Lt-B specific antibody.

Figure 3. Protective immune responses of mice fed transgenic

With 95% confidence levels, and the sample size is given.

Figure 4. Protection against TGEV of piglets fed transgenic

corn expressing the S protein or modified live vaccine

(MLV) TGEV. Percent morbidity incidence.