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U.S. Department of Health and Human Services

Animal & Veterinary

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NEW METHOD AVAILABLE FOR DETECTION OF BOVINE MATERIAL IN FEED

by Michael J. Myers, Ph.D.
FDA Veterinarian Newsletter March/April 1998 Volume XIII, No II

Bovine spongiform encephalopathy is a member of the transmissible spongiform encephalopathy (TSE) disease family. Other TSE diseases include Kuru, Creutzfeldt-Jakob Disease (CJD) which are human diseases, chronic wasting syndrome (elk) and scrapie, a disease found in sheep. These conditions cause a progressive degeneration of the central nervous system, which is always fatal.

Bovine spongiform encephalopathy (BSE) was first detected in the United Kingdom in 1986. As a consequence of epidemiological surveys, an association developed between the outbreak of BSE and the use of cattle feed containing protein derived from sheep infected with scrapie. In addition, the practice of feeding cattle protein derived in part from other cattle infected with BSE served to amplify this disease and permit further dissemination.

On June 5, 1997, the Food and Drug Administration published the final regulation prohibiting the use of mammalian protein in the manufacture of animal feeds destined to be given to ruminant animals, including cattle, sheep, and goats ( Ref.1). Products believed to pose minimal risk, including blood, blood products, gelatin, milk and milk products, protein derived solely from swine or equine sources, and inspected meat products offered for human consumption were exempted from the ban.

In the course of preparing the final regulation, several existing methods for detecting the presence of bovine derived proteins were examined, including the feed microscopic method (Ref. 2) and the ELISA method developed by the British government (Ref. 3). However, the microscopic method was determined to be very technically demanding and lacking in sensitivity. Likewise, the ELISA method, despite being used by the British government for regulatory purposes, was also determined to be inadequate, principally because of a propensity for false positive responses. In addition, this method was also found to require a high degree of technical competence which might not readily transfer to field situations.

Recently, the Center for Veterinary Medicine (CVM) became aware of another method developed by Dr. Marco Tartaglia at the Instituto Superiore di Sanita in Rome, Italy. This method is a polymerase chain reaction-based (PCR) method (Ref. 4) capable of detecting bovine DNA isolated from complete feed. The method relies on the capacity of the PCR reaction to enzymatically amplify a short segment of DNA, resulting in a method that allows the detection and amplification of a bovine-specific mitochondrial sequence from complete feedstuffs.

This method permits the detection of bovine mitochondrial DNA in feed containing as little as 0.125 percent bovine meat and bone meal. Using the bovine specific PCR primers, this method will only detect material of bovine origin. It will not detect materials of either ovine, swine, equine, avian (turkey and chicken), or lapine origin. The specificity of the PCR reaction is determined by the oligonucleotide sequence of the two PCR primers used for the polymerase chain reaction. These two regions show species specificities for all animals examined to date. No cross reactivity between PCR primers has been observed (Ref. 4). That is, a set of primers specific for swine will only detect material of swine origin, bovine-specific primers only detect material of bovine origin, etc. In addition, the method is relatively rapid, with completion in approximately 24 hours.

Species specificity/identity can also be confirmed using the oligonucleotide product generated by the PCR reaction. Within the PCR product generated from DNA of bovine origin are DNA endonuclease restriction sites for two enzymes, called SspI and DpnII. DNA endonucleases are enzymes that will cleave, or "cut" DNA at internal sites. Each endonuclease recognizes a unique set, or arrangement, of DNA base pairs.

The PCR product generated from bovine DNA is 271 base pairs in length. Endonuclease cleavage of this product by DpnII produces two fragments 57 and 217 base pairs in length. SspI produces three fragments 69, 84 and 118 base pairs in length. This cleavage pattern is unique to the PCR product derived from bovine DNA. DNA derived from ovine, swine, equine, avian, or lapine origin will not produce this cleavage pattern.

The procedure solubilizes 0.5 grams of feed in 4.5 mls of extraction buffer (5M guanidine thiocyanate; 0.05M Tris-Cl, pH 6.4; 0.02M EDTA, pH 8.0, 1.3 percent Triton X-100) with an overnight incubation at 60 C (about 15-16 hours). The following morning, the mixture is admixed, incubated for an additional 30 minutes, then centrifuged at 3000 x g for 10 min. to precipitate the gelatin. The supernatant is removed; 0.5 ml is used for DNA extraction, the remainder is stored at -20°C. The solubilized feed is mixed with an equal portion of extraction buffer (0.5 ml) and 0.040 ml of a silica suspension. This mixture is mixed, then allowed to incubate for 10 minutes at room temperature. During the incubation, the mixture is frequently admixed to keep the silica in suspension. At the end of the incubation period, the silica is removed from suspension by centrifugation. The supernatant is then discarded. The silica is washed two times with wash buffer (extraction buffer without EDTA or Triton X-100), followed by two washes with 70 percent ethanol and one final wash with acetone. The washing serves to remove any protein that precipitated with the silica. The silica pellet is allowed to dry. The DNA is extracted from the silica using 0.050 ml TE buffer (10 mM Tris-CL, pH 8.0; 0.1 M EDTA, pH 8.0).

The PCR reactions performed using 0.005 ml of the extracted DNA sample. The reaction conditions use 12.5 pmole of each primer, 50 µM of each dNTP, 1.5 mM MgCl2, 2.5 units of Taq, and Perkin-Elmer reaction buffer, all in a final volume of 50 µl. The mixture is subjected to 30 cycles at 94°C for 60 sec, 58°C for 60 sec, and 72°C for 60 sec. Upon completion of the PCR process, 20 µl of the PCR reaction is subjected to agarose gel electrophoresis using a 2 percent agarose gel. The results are visualized using ethidium bromide.

This method is currently being evaluated at CVM's Office of Research. Laboratories at FDA's National Center for Toxicological Research and Office of Regulatory Affairs are also participating in this effort. Part of this effort will include a method validation trial to determine the performance characteristics of this method for feedstuffs typically used in this country. In addition, the laboratories at the Office of Research and the National Center for Toxicological Research will be participating in an international method validation trial that is being coordinated by the Joint Research Institute, Environment Institute, Food and Drug Analysis at Ispra, Italy. This effort is tentatively planned to begin in the fall of 1998.

References

  1. Substances Prohibited From Use in Animal Food or Feed; Animal Proteins Prohibited in Ruminant Feed. Federal Register 62 (108):30935.
  2. Microscopy of Animal Feed. In: Official Methods of Analysis of the AOAC. Ed: Kenneth Helrich. AOAC, Arlington VA. 1990, pp 88-90.
  3. Ansfeld, M. 1994. Production of a sensitive immunoassay for detection of ruminant proteins in rendered animal material heated to >130°C. Food and Agricultural Immunology 6:419-433.
  4. Tartaglia M, Saule E, Pestalozza S, Morelli L, Antonucci G, Battaglia PA. A procedure for bovine mitochondrial DNA detection in animal feeds: a molecular approach to check for the presence of bovine-derived materials in ruminant feedstuffs. J Food Protection (in press).

The author was CVM's 1997 nominee for the FDA Excellence in Laboratory Science Award.