Animal & Veterinary
CVM Scientists Develop PCR Test to Determine Source of Animal Products in Feed, Pet Food
by Michael Myers, Ph.D., Research Pharmacologist, Office of Research
FDA Veterinarian Newsletter January/February 2004 Volume XIX, No 1
A test, derived to determine source of pentobarbital in pet food, is so sensitive it can identify the species of origin for animal products on a scale of 7 lbs. per 500 tons. Tests find no cat or dog DNA in pet food.
Anecdotal reports from veterinarians during the 1990s suggested pentobarbital appeared to be losing its effectiveness as an anesthetizing agent in dogs. These reports led to speculation that pentobarbital was present as a contaminant in dog food, and that this was altering the physiological response to pentobarbital-induced anesthesia in some dogs.
Pentobarbital is used to humanely euthanize unwanted animals (including dogs and cats in animal shelters) as well as animals in situations of severe pain and suffering. Pentobarbital is a member of the barbiturate family of drugs, which are proven inducers of drug metabolism. Agents that induced drug metabolism enhance the metabolism and elimination of drugs from the body, which has a net effect of decreasing the effectiveness of those drugs. While pentobarbital is a weak inducer of drug metabolism, there was speculation that the dogs had been exposed somehow to pentobarbital, which increased the dogs' metabolism to the drug to cause reduced effectiveness. Until recently, carcasses from pentobarbital-euthanized animals were disposed of by rendering. Pentobarbital is known to survive the rendering process and partition equally into protein and fat 1 .
Scientists working at the Food and Drug Administration's Center for Veterinary Medicine developed an analytical method for the determination of pentobarbital residues in dog food, and in a limited survey found that several lots of commercial dog food contained confirmable levels (10-60 parts per billion) of pentobarbital 2 . These results confirmed an earlier study that also found detectable levels of pentobarbital in dry dog food samples 3 . Thus, one of the two central premises for why pentobarbital could be losing effectiveness—the presence of pentobarbital in dry dog food—was demonstrated.
However, the second premise, namely auto-induction of drug metabolism, could not be demonstrated. In a toxicological study designed to answer this very question, FDA scientists demonstrated that pentobarbital could affect the drug metabolizing system of dogs, but only at greatly elevated levels of pentobarbital 4 ; i.e., at levels well above those possible by prolonged consumption of dog food containing the highest levels of pentobarbital.
These studies did not address the central question of the source of the pentobarbital in dog food. It has been presumed that pentobarbital was present in these dog food samples because euthanized animals, such as dogs, cats, and horses, might be included with other animal-byproducts used in preparing dog food. However, this presumption was difficult to test due to the limitations of existing analytical methods. Therefore, CVM scientists developed a polymerase chain reaction (PCR)-based approach to identify species-specific products that might be present in dog food.
The approach the scientists used was a modification of the PCR-based method validated for detecting bovine-derived materials in complete animal feed. 5 The polymerase chain reaction is an in vitro, test-tube diagnostic approach using DNA precursor molecules, two distinct oligonucleotides (termed “primers”), magnesium ions, the template or target DNA, and DNA polymerase, the enzyme that catalyzes the process.
The PCR primers are short, synthetic stretches of DNA that bind to the template DNA. The primers are designed to bind to only one particular section of DNA, such that with repeated cycles of amplification, logarithmic increases in the DNA sequence between where the primers bind are produced. This results in an easily detectable level of product.
Because of the specificity of the primers, it is possible to detect not only one genetic sequence in an organism's entire genome, but to determine from which species of several closely related species a particular DNA sequence was derived.
The underlying principle of the method we developed is the amplification of a mitochondria-specific DNA sequence (mtDNA) using PCR primer pairs that permit species-specific amplification.
Mitochondria are small organelles found in eukaryotes which are responsible for directing cellular respiration. Mitochondria are more commonly called “the cells' powerhouse” because of their unique role in generating the energy sources used by cells. Mitochondria have their own DNA called mitochondrial DNA (mtDNA) which is distinct from genomic DNA. The genes encoded by mtDNA are used to control cell respiration. Genomic DNA is equally inherited from both parents, while mtDNA comes only from the mother.
The use of mtDNA sequences increases the number of targets available for amplification relative to genomic DNA, which increases the sensitivity of the method. This approach is possible because there are roughly 50-100 mitochondria in each cell, all having a copy of mtDNA.
In addition, each mitochondria may have up to 100 copies of mtDNA, for a potential of 2,500-10,000 copies of mtDNA per cell, compared with a single copy of a given genomic DNA sequence.
Accordingly, PCR primers specific for canine, equine, or feline mtDNA sequences were developed and used to test for the possible presence of rendered materials from these species in dog food.
In addition, other species-specific PCR primers were used to assess the accuracy of the label claims by comparing the PCR results with the ingredient statements from the package label.
Looking for Canine DNA
A PCR primer set specific for a canine mtDNA sequence was deduced, and subsequently shown to amplify only that mtDNA derived from dogs, but not mtDNA derived from cattle, swine, sheep, goat, pig, cat, deer, elk, poultry, turkey, rabbit, or horse blood (Fig 1).
When the PCR process is applied to a sample, researchers look for the process to yield PCR products called amplicons that are specific both to the animal species and gene sequence the researchers are looking for.
Thirty-one dog food samples previously analyzed for the presence of pentobarbital 2 were then subjected to the DNA extraction process and tested for the presence of canine DNA. The results demonstrated the complete absence of canine DNA in all 31 samples (Fig 2) at a level exceeding 0.0007% (w/w). In other words, at this level of detection, we can say that if there is any canine material in the dog food, it is present at a rate of less than 7 lb. per 500 tons.
Cats and horses are also euthanized with pentobarbital and thus might be the source of this drug in dog food. PCR primer sets that are specific for either feline or equine mtDNA were also developed to test the same dog food samples for presence of mtDNA that might have been derived from cats or horses. The results from these analyses demonstrated the complete absence of PCR products, the amplicons, specific for either cat or horse mtDNA in all 31 dog food samples. This analysis was carried out under conditions that achieved 0.007% sensitivity.
Because the results so far were negative, it was important to demonstrate that mtDNA from these dog food samples could be amplified to increase the sensitivity of the test. Therefore, the mtDNA from these samples were subjected to PCR amplification using a set of PCR primers (termed “universal” primers) shown to amplify only mtDNA from cow, deer, elk, sheep, goat, horse and pig. These particular animal species were expected to be present in the samples due to the ingredient statements of the dog food labels.
The results demonstrated that most, but not all, samples had a PCR amplicon, indicating that one or more of these species (cow, deer, elk, sheep, goat, horse or pig) were present in these dog food samples. Interestingly, two samples that were positive for pentobarbital did not produce a PCR amplicon when the universal primers were used, suggesting a complete absence of mammalian-derived mtDNA from species that are typically euthanized with pentobarbital.
Using PCR primer sets specific for bovine, swine, or sheep mtDNA, we were able to demonstrate the presence of rendered material derived from one or more of these species. As expected, samples that did not produce a PCR amplicon using the universal primers failed to produce amplicons when the species-specific primers were used.
For the most part, the PCR results confirmed the ingredients as listed on the package label. Unexpectedly, there were four samples that had PCR results inconsistent with the package label.
Two dog food samples listed lamb in the label, yet both samples also had a PCR amplicon specific for bovine mtDNA. One of the samples labeled as containing lamb proteins produced no amplicon specific for sheep (lamb). While the remaining two samples listed only poultry on the label, one sample had a PCR amplicon specific for bovine mtDNA, whereas the other sample had a PCR amplicon specific for swine mtDNA. Pentobarbital was present in these latter two samples.
No Dog or Cat mtDNA
It is widely presumed that the principal source of pentobarbital in pet food is the rendered remains of animals euthanized at animal shelters. However, the absence of detectable feline or canine mtDNA in the samples indicates that, within the context of this limited survey, rendered proteins from euthanized dogs and cats were not present in these dog food samples. The detection limit of the method as used in this study is, at a minimum, 0.0007% on a w/w basis, or 7 lbs. of rendered protein in 500 tons of dog food. While one can argue that there may be the rendered remains of dogs or cats below this level, this amount of rendered meat and bone meal (less than 7 lbs.) is insufficient to produce the levels of pentobarbital detected in some of these dog food samples.
Horses are the other animal species euthanized with pentobarbital in relatively large numbers. Due to their large size, and the amount of drug needed for euthanasia, one horse would represent a significant portion of a large batch of meat and bone meal. However, none of the 31 dog food samples examined in this study tested positive for equine proteins.
Also, none of the samples was positive for feline proteins.
Thus, the pentobarbital found in 10 of these 31 dog food samples does not appear to be due to contamination of meat and bone meal containing the remains of euthanized dogs, horses, or cats.
In fact, the PCR results on the species of origin in the various dog food samples do not support a single point source of protein for the origin of the pentobarbital.
The only common feature of all samples containing pentobarbital is the presence of animal fat. This suggests that animal fat might be the source for pentobarbital. This hypothesis is supported by observations from the initial survey for pentobarbital in dog food. A positive relationship was noted between the ingredients listed on the package label and the likelihood a sample contained pentobarbital.
While the results of this study narrow the search for the source of pentobarbital, it does not define the source (i.e., species) responsible for the contamination.
This PCR method developed by FDA scientists can also be used in assuring the validity of label claims on feed or pet food.
Twenty-seven of the 31 samples showed agreement between the PCR results and the package label for mammalian and avian derived components. In only four samples did the PCR results not agree with the label claims. In all four cases, bovine materials were noted by the PCR results; there were no bovine protein sources listed on the labels for these samples. However, these samples all list either animal fat or beef tallow on the label, suggesting that this component might be the source of the bovine material. Residual levels of animal derived proteins contaminating the animal fat might explain these findings; whether this is the case or not cannot be determined at present.
The absence of a PCR amplicon in these samples could also be due to experimental error or sample misbranding. Based on previous results, the rate of false negatives and false positive for this method is 1.25% and 0.83%, respectively. 4 However, these samples were analyzed by two different investigators on two different occasions, with both analysts obtaining the same result, suggesting that the product is incorrectly labeled (i.e. misbranded or adulterated).
The results of this study demonstrated a lack of correlation between species identity and the presence of pentobarbital in dog food. They also provide evidence against the presumption that euthanized pets are routinely rendered and used in pet food.
In addition, the results of this study have established a methodology for identification of the types (re: species) of meat and bone meal present in dog food. This method should prove useful for analysis of dog (and cat) food for the accuracy of the label claims.
- O'Connor JJ, Stowe CM, Robinson RR. Fate of sodium pentobarbital in rendered products. Am J Vet Res 1985; 46:1721-1724.
- Heller DN, Lewis KM, Cui W. Method for determination of pentobarbital in dry dog food by gas chromatography/mass spectrometry. J Agric Food Chem 2001; 49:4597-602.
- Adam LA, Reeves VB. Procedure for detecting and confirming pentobarbital residues in dog food by gas chromatography/mass spectrometry. J Assoc Off Analyt Chem 1998; 81:359-367.
- Kawalek JK, Howard KD, Farrell DE, Derr J, Cope CV, Jackson J, Myers MJ. Effect of low doses of pentobarbital in beagles. Am J Vet Res 2003; .
- Myers MJ, Friedman SL, Farrell DE, et al. Validation of a polymerase chain reaction method for the detection of rendered bovine-derived materials in feedstuffs. J Food Protect 2001: 64:564-566.
[The following charts go with the ‘PCR Test' article]
Specificity of Canine mtDNA Primers
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Detection of canine mitochondrial DNA using PCR primers specific for Canis familiaris . PCR primers specific for Canis familiaris were used to amplify mtDNA obtained from numerous species. The PCR product was separated in a 2% agarose gel containing ethidium bromide. Lanes 1-7 (top picture) contained DNA obtained from the blood of cattle, elk, horse, goat, sheep, dog (beagle), and pig, respectively. Lanes 8-14 (bottom picture) contained DNA obtained from the blood of chicken, geese, cat, rabbit, turkey, dog (mixed breed), and deer, respectively. Only mtDNA from the two dogs produced a PCR amplicon. Similar results were obtained when either the feline-specific primers or the equine-specific primers were used. That is, these latter primers only amplified mitochondrial DNA from their respective species.
Analysis of Dog food Samples
MW 1 2 3 4 5 6 7 8 9 10 11
PCR analysis for canine mtDNA in dog food. Representative results from some of the 31 different dog food samples analyzed. The 10 samples shown here had previously been found to contain pentobarbital (2). The DNA from the dog food samples was extracted and subjected to PCR amplification using the canine specific PCR primers. The PCR product was separated in a 2% agarose gel containing ethidium bromide. MW; molecular weight standards. Lanes 1-9, dog food samples. Lane 10, negative control. Lane 11, purified canine DNA (positive control). Only the positive control sample (Lane 11) demonstrated the presence of a PCR amplicon.