Utilizing Immunoassay Techniques to Evaluate the Prevalence of Ergovaline Toxins Extracted from Neotyphodium coenophialum in Fescue

This study evaluated the tall fescue pasture land in Craig County for the endophytic fungus, Neotyphodium coenophialum and for the alkaloid, ergovaline, commonly produced by the fungus. Ergovaline produces toxins that cause many serious side effects in livestock. However, not all endophytic fescue produces ergovaline, thus rendering the fescue harmless to livestock. This project evaluated the Immunoblot and ELISA techniques in the identification of the endophytic fungus in fescue and in the extraction of ergovaline toxins associated with the endophytic fungus. It is hypothesized that all sites testing positive for the endophytic fungus will also test positive for ergovaline toxins. Immunoassay procedures were followed to identify the fungus and ergovaline. The results of the Immunoblot test indicate that of the thirty-six fescue pastures tested, 91.70% were infected with Neotyphodium coenophialum. A positive identification of the fungus was indicated by a dark pink color on the nitrocellulose membrane. Negative results where recorded if a salmon color was present . The Immunoblot test only tests for the fungus and isn’t conclusive about whether the fungus is involved in producing toxic substances; therefore, an ELISA assay was performed to test for the toxins. The results of the ELISA assay indicate that 72% of the sites tested positive for the alkaloid, ergovaline. Positive results for the ELISA assay where identified by clear wells. Negative identification was recorded if a green color was present in a well. Of the fescue sites that tested positive for Neotyphodium coenophialum, 79% produced ergovaline. Future studies might focus on analyzing emerging fescue seed for the production of ergovaline toxins.

Tall fescue (Festuca arundinacea Schreb.) is a cool-season, perennial bunchgrass that came to North America from Europe in the late 1800’s. Since the discovery of a field of fescue in eastern Kentucky in 1931 and the subsequent release of the Kentucky31 variety in 1943, tall fescue has become the dominant cool-season perennial grass in the southeastern United States (Redmon, Pratt, and Woods 1). For decades Kentucky31 tall fescue was planted because it was easy to establish, widely adaptable, resistant to insects, disease and weed competition, and had a long growing season. However, with its increased use in pastures after World War II, disturbing reports of poor animal performance and visible disorders appeared in the 1950’s. This was puzzling since everything suggested that well-managed fescue should result in good animal performance (Cooper and Johnson 2). The Plant Materials Program states in its article, “Tall Fescue”, that it is now recognized that the presence of an endophytic fungus has contributed to both the tough nature of the grass and the poor performance of grazing animals (3).

The endophytic fungus lives between the plant tissue layers where its entire life cycle takes place. The relationship between the grass and the endophyte is symbiotic; that is, it benefits both. The fungus gives fescue its hardy characteristics and the fescue provides the fungus with a place to live and grow. The fungus grows as the seed germinates, invading the seedling shortly after germination. Within two days of germination, the fungus can be observed in the first emerging shoot. Infection of the first and subsequent leaves does not occur until sheath differentiation occurs (Freeman , Pratt, and Woods 3). Only seed transmits the fungus, so a plant cannot become infected from its neighbors. A stand of a endophyte-free variety will remain endophyte free. However, endophytic fescue tends to dominate over endophyte-free fescue because the fungus makes it hardier.

Although the endophyte does not harm the grass, it produces toxins that are harmful to livestock. The endophytic fungus either produces a chemical or causes the fescue to produce a chemical, which scientists believe to be an alkaloid toxin. Three groups of alkaloids, diasiphernanthrene, pyrrolizidine, and ergot, are found in endophytic fescue. Tall fescue toxicity is caused by the toxin ergovaline (ergot). The alkaloid ergovaline is found in all portions of the plant with the highest concentration in the developing seed head and the lowest concentration in expanded leaf blades.

The effects of this toxicity on livestock include hypothermia, lower feed intake and weight loss, lower pregnancy rates, and decreased milk production. These clinical signs, although more apparent during hot weather, can occur at any time of the year.

Horses are especially prone to developing serious reproductive problems, which include abortions, difficult births, and foal deaths. In a recent SeedQuestā news release, Pennington Seed Inc. remarked that these problems are presently costing livestock producers more than $1 billion each year in reduced weight gains and lower conception rates.

Removing the fungus from the fescue and replanting with endophyte-free seed has not been successful. Fungus-free fescue has not been shown to survive more than three to five years under current grazing and climatic conditions. Researchers at the University of Georgia and AgResearch of New Zealand in conjunction with Pennington hope to market a new fescue seed in the summer of 2000 that maintains the drought and pest resistance of the wild variety without producing the harmful toxins (1). This toxin-free endophytic fungus has been found sporadically in pastureland but the research team hope to supply livestock farmers with a laboratory produced endophyte seed.

The goal of this project is to use immunoblotting techniques to identify the fungus, Neotyphodium coenophialum in fescue and to use ELISA (Enzyme-Linked Immunosorbent Assay) techniques to identify ergovaline in the Neotyphodium coenophialum. According to the laboratory manual, Immunology, immunoblotting uses antibodies to detect the presence of small amounts of antigens in complex mixtures (Myers 71). An antigen is any molecule that can bind to an antibody (proteins, viruses, bacteria, cells, etc.) Antibodies are proteins that circulate in the blood stream and are also found in various secretions and on the surface of different immune cells. The function of antibodies is to recognize the antigens that bind to them and decide which should be targeted for destruction (Sims 5).

In the text, Biology, the authors explain that prior to 1975, the only source of antibodies for research and clinical applications was the blood of immunized animals Campbell, Reece, and Mitchell 856). Such antibodies are called polyclonol antibodies because they arise from many different B cell clones, each specific for a particular epitope on the immunizing antigen. A B cell is a type of lymphocyte that develops in the bone marrow and later produces antibodies, while an epitope is a small, accessible portion of an antigen that interacts with an antibody. Indeed, all immune responses are polyclonal. While polyclonal antibodies are useful in laboratory applications, the mixture often includes antibodies of undesired specificities.

Another limitation of this practice is that the source of the antibody, the immunized animal, has a limited lifespan. In 1975, George Kohler and Cesar Milstein developed a method that provides an unlimited source of monoclonal antibodies (857). Monoclonal antibodies are specifically designed to help identify cells or molecules present in relatively small amounts, or to help eradicate cells bearing specific antigens (BioOncology Online 88).

Current methods used for detection of ergot alkaloids, such as high pressure liquid chromatography or thin layer chromatography, are too laborious and time consuming to be useful for routine analyses of large numbers of samples. Immunoassays (tests that use antibodies produced in mammals against a particular compound to later detect that same compound) for other mycotoxins have been successfully developed and incorporated into routine screening procedures (Reddick et al. 1). ELISA systems can detect antibodies or soluble antigens. Often this method is used to screen for antigens because it provides a rapid and sensitive assay with few steps.

This project evaluates Immunoblot and ELISA techniques in the identification of the endophytic fungus in fescue and in the extraction of ergovaline toxins associated with the endophytic fungus. It is hypothesized that all sites testing positive for the endophytic fungus will also test positive for ergovaline toxins.

Materials

General Supplies

Fescue samples collected at 36 sites in Craig County

Map of Craig County

Tweezers

Pipette

Aluminum foil

Graduated cylinders in various sizes

Distilled water

Glass or plastic tubes

Refrigerator

Incubator

Two 4 x 6 inch plastic container

Cellulose sponge (must fit inside plastic container)

Plastic squirt bottle

Micropipettor and tips

Ziplock Bags

Razor blade

Two pieces of blotting paper

Procedures

1. Locate 36 fescue sites throughout Craig County. Record section, township, and range of each sample site and mark its location on the map.

2. From each site, cut 1 tiller close to the ground from 20-30 randomly selected crowns of fescue. Place in plastic bag, label, and freeze.

Part 2

1. Place a 3.5 x 5.0 x 0.75 inch cellulose sponge on the bottom of a 4.0 x 6.0 inch plastic container. Dilute the extraction solution (EB) to 200 mL with distilled water. Add EB solution to the container. Place one piece of blotting paper on top of the sponge.

2. Using forceps or tweezers, placer the nitrocellulose membrane on top of the blotting paper.

3. Horizontally cut tillers with a single-edge razor blade 1/8” above the base of the harvested tillers. Discard this first cut. Make two more cross sections 1/16 to 1/8 in. thick. Place the cross sections on the nitrocellulose membrane. Place the cover on the container and refrigerate overnight.

4. Peel the nitrocellulose from the blotting paper and place it onto a dry piece of blotting paper. Remove the tiller pieces from the nitrocellulose membrane with a lab brush.

5. Dry the membrane in the incubator at 70^oC for 15 min. or at room temp. for one hour.

6. Place the membrane into the bottom of the reaction vessel. Dissolve the BWW reagents in 300 mL of distilled water to create a BWW solution. Add 20mL of the solution, place cover of vessel, and place on an orbital shaker for 30 min.

8. Add 10 mL of BWW solution to the MAB tube and mix by shaking. Pour MAB solution over the membrane in the reaction vessel. Rinse the MAB tube with 10 mL of BWW solution and pour over the membrane in the vessel. Place onto shaker for 1 hour.

9. Remove reaction vessel from shaker and gently pour off the antibody solution.

10. Add 20 mL of BWW solution and return reaction vessel to the shaker for 6 min. Pour off the BWW solution. Repeat this step one more time.

11. Add 10 mL of BWW solution to the RAM tube and mix by shaking. Pour RAM solution over the membrane in the reaction vessel. Rinse the RAM tube with 10 mL of BWW solution and pour over the membrane in the vessel. Place onto shaker for 1 hour.

12. Remove the vessel from the shaker and gently pour off the solution and repeat step 10.

13. Add 10mL of BWW solution to the PA tube and mix by shaking. Pour PA solution over the membrane and rinse the PA tube with 10mL of BWW solution. Place onto the shaker for 30 min.

14. Remove the vessel from the shaker and gently pour off solution and repeat step 10.

15. Dissolve TRIS with 20 mL of distilled water. Add N and shake to dissolve. Add FR and shake to dissolve and activate the chromogen. Add the chromogen solution to the reaction vessel, return vessel to the shaker, and cover with aluminum foil. Color usually develops within 20 to 30 min. After color has developed, pour off chromogen solution and stop the reaction by rinsing the membrane twice with 20 mL of distilled water. Score membrane for positive Neotyphodium tillers by viewing the dark pink color where tillers were in contact with the membrane.

ELISA Procedures

1. Cut tillers as previously done in preparation for the Immunoblot and put each tiller in the bottom of a well on the extraction microtiter plate.

2. Dilute the extraction buffer to 25 mL total volume using distilled water. Add 120 uL of the extraction buffer to each well and incubate at room temp. for 3 hours.

3. Using a pipette, gently withdraw 50 uL of extraction solution from the wells containing the tillers and place it into a corresponding well on the assay microtiter plate.

4. Dilute the monoclonal antibody solution with the buffer ABB. Add 50 uL of the monoclonal antibody solution to each well containing tiller extract.

5. Place the assay microtiter plate onto a rotary shaker revolving at 60 rpm for 2 hours at 21^OC.

6. Dilute ELISA wash to 500 mL with distilled water. Empty the contents of the microtiter wells by inverting the plate. Using a plastic squirt bottle, gently fill the microtiter wells with ELISA wash. Invert the plate to remove the solution. Repeat this step twice (total of 3 washes). Invert the plate and firmly tap it on a paper towel on the lab table to remove all wash solution.

7. Add 50 uL of polyclonal antibody solution to each well. Incubate the microtiter plate for 2 hours at 21^OC.

9. Dilute the chromogenic agent with the CB buffer. Add 50 uL of chromogenic solution to each microtiter well. Place in the dark at 21^OC for 15-30 min. Place the microtiter plate on a white piece of paper and view for color development. A clear color indicates that ergovaline was present in the tiller. Any color development indicates that ergovaline was not present in the tiller.

Conclusion

The results of the Immunoblot test indicate that of the thirty-six fescue pastures tested, 91.70% were infected with the fungus Neotyphodium coenophialum. A positive identification of the fungus was indicated by a dark pink color on the membrane. Negative results were recorded if a salmon color was present. The Immunoblot test only tests for the fungus and isn’t conclusive about whether the fungus is involved in producing toxic substances; therefore, an ELISA assay was performed to test for the toxins. The results of the ELISA assay indicate that 72% of the sites tested positive for the alkaloid, ergovaline. Positive results for the ELISA assay were identified by clear wells. Negative identification was recorded if a green color was present in a well. Of the fescue sites that tested positive for Neotyphodium coenophialum, 79% produced ergovaline. Two sites were ruled inconclusive for ergovaline due to mixed readings.

It had been hypothesized that all sites testing positive for the endophytic fungus will also test positive for ergovaline toxins. This hypothesis has been rejected since only 79% of the sites that tested positive for the endophytic fungus also tested positive for ergovaline toxins.

Possible sources of error in this experiment could include discrepancies in reading the Immunoblot color results on the membrane and in reading the color test results in the ELISA assay. Color is subjective and timing of recording the color is crucial. Future studies might focus on analyzing emerging fescue seed for the production of ergovaline toxins.

Works Cited

BioOncology Online. 1998. On-line. Onenet. 8 December 2000. http://www.biooncology.com/biooncology/orr_gloss.htm

Campbell, Neil A., Jane B. Reece and Lawrence G. Mitchell. Biology. Menlo Park, California: Addison Wesley Longman, Inc., 1999.

Cooper, Tom and Keith Johnson. “The Diagnosis of the Tall Fescue Endophyte.” O June 1998. On-line. Onenet. 8 December 1999. http://agry.purdue.edu/agronomy/ext/forages/publications/endophyte.htm.

Freeman, David, Phillip W. Pratt, and Robert L. Woods. “Fescue Toxicity and Horses.” OSU Oklahoma Cooperative Extension Service.

http://pss.ag.utk.edu/fescue/fesart10.html.

Myers, Richard. Immunology-- A Laboratory Manual. Dubuque, IA: Wm. C. Brown Publishers, 1995.

Reddick, B. B. et al. “Development of an Immunoassay for Detection of Ergovaline Alkaloids in Tall Fescue. On-line. Onenet. 9 January 2000.

Redmon, Larry A., Phillip W. Pratt, and Robert L. Woods. “Tall Fescue in Oklahoma.” OSU Extension Facts.

SeedQuestā. Pennington Seed Inc. Madison, Georgia. 6 October 1998.

Sims, Thomas. “Defense Against Disease.” Lecture Notes. On-line. Onenet. 18 December 2000. http://www.bios.niu.edu/sims/immunesystem.html.

“Tall Fescue.” Plant Materials Program. 22 December 1997. On-line. Onenet.

16 January 2000. http://ironwood.itc.nrcs.usda.gov:90/pmc/grasses/fear3.html

The following individuals were invaluable to the completion of this project:

Sally A. Fenska, MEd. and Pam Benne, NBCT, for their endless support in the experimental and production stages of the project;

Nicholas Hill, Ph.D., University of Georgia and Agrinostics Ltd. for his helpful support via e-mail and most generous donation of Immunoblot test and ELISA assay materials;

Phillip Pratt, Regional Extension Agent, for his encouragement and support in pursuing the project; and

Whitney James for help with the technology involved in representing the results of the tests.