A more recent anonymous Internet-based American survey indicated that 46% of dog owners and 38% of cat owners had fed RMBDs to their pets . Additionally, sales of RMBD have increased annually by as much as 15% in recent years . Over the past decade, this feeding practice has continued to increase, and market locations have expanded to include grocery stores, mass merchandisers, pet specialty stores, and veterinary clinics . A 2019 Italian-based survey shed light on dog owners’ motivations for adopting this feeding method . About 80% of respondents reported that they abandoned feeding commercial diets due to distrust in the clarity of ingredients commercial prescription veterinary diet, veterinarians may acquiesce to such requests. However, the reliability of RMBD for this purpose has not been evaluated. The primary aim of this study was to use PCR to test commercially available RMBD for the presence of DNA of animal origin other than that declared on the labels. A secondary objective was to determine the consistency of DNA presence between different batches of the same diets. The hypothesis was that the diets would contain unlisted protein ingredients, and that these unlisted proteins would vary between batches. To the authors’ knowledge, no previous studies have examined these issues. Nine commercial canine and feline RMBD were selected for analysis. All diets were marketed as balanced for complete feeding. The selected diets included a variety of commonly available North American RMBD, some formulated with novel animal source proteins , or limited ingredients , or grain-free diets which may be potentially selected for use as an elimination diet. The diets evaluated were not specifically marketed for feeding as elimination diets, but contained ingredients which may be considered by pet owners for this feeding purpose. Two lot numbers of RMBD representing separately prepared batches of each diet were selected for analysis in order to assess for consistency of any ingredient contamination between the 2 batches. One canine and one feline veterinary prescription extensively hydrolyzed poultry feather-based diet were used as negative controls. Nine species-specific qPCR assays were designed to detect beef, chicken, duck, turkey, salmon, sheep, rabbit, kangaroo, and pig DNA .

Sequences for each species were found in the National Institute of Biotechnology Information database. Two primers and an internal hydrolysis probe , 3 end, quencher dye NFQMGB were designed using Primer Express Software for all species,30 planter pot with the exception of chicken which used a locked nucleic acid probe . A Basic Local Alignment Search Tool of the amplicons confirmed unique species detection. To ensure these assays did not cross-react with DNA of other species, a crossreactivity evaluation was performed by running all assays with control DNA from each species . A housekeeping gene, eukaryotic 18S assay , was run with each sample to confirm successful DNA extraction. All assays were validated for efficiency and sensitivity by running 10-fold standard curves in triplicate from serial dilutions of control DNA. Each assay was 90% to 100% efficient and sensitive enough to detect as few as 10 copies of the target gene. Of the 9 species of animal DNA tested, 8 species, including pork, chicken, duck, rabbit, lamb, beef, salmon, and turkey, were detected in at least 1 sample of the canine and feline RMBD tested. Only kangaroo DNA was not detected in any of the RMBD. The 2 extensively hydrolyzed poultry feather-protein based diets contained either trace amounts of chicken DNA or no detectable DNA . In the canine RMBD, DNA of 1 or more animal species not indicated on the label was identified in 9 out of 9 diets, in either 1 or both of the tested batches . Of the 18 batches tested, 89% tested positive for unlisted animal-source DNA. An average of 4.4 unlisted proteins was detected in each diet. A total of 2 batches were found to contain DNA consistent with the stated label ingredients. The diet with the greatest number of unlisted proteins was a single batch , labeled as containing turkey and sardine. It contained a total of 6 unlisted proteins . The unlisted DNA most frequently detected was lamb . Discrepancy in the unlisted DNA between batches was noted in 78% of batches. In the feline RMBD, DNA of 1 or more animal species not indicated on the label was identified in 7 of 9 diets, in either 1 or both of the tested batches . Of the 18 batches tested, 61% were positive for unlisted animal-source DNA. An average of 2.6 unlisted proteins was detected in each diet. A total of 7 batches were found to contain DNA consistent with the stated label ingredients. The diet with the highest number of unlisted proteins was a single batch , labeled as containing chicken and salmon. It contained 5 unlisted proteins .

The unlisted DNA most frequently detected was turkey . Discrepancy between batches was noted in 56% of batches. All of the canine and feline RMBD included in the analysis were found to contain the proteins listed on their labels. Contamination of one or both batches in all canine RMBD and most of the feline RMBD tested was detected in this study, which supports the hypothesis that cross-contamination would be found in many RMBD. Numerous independent studies have also demonstrated significant discrepancies between label claims and actual contents of dry or canned over-the-counter commercial diets, including those marketed for the management of CAFR . While this finding may have been expected in RMBD due to the prevalence of unlisted DNA detected in other such studies, diet purity could theoretically have been improved in RMBD as they are purported to undergo less processing before distribution, allowing less opportunity for protein contamination. An additional finding of this study was that unlisted animal source proteins varied among batches in most batches tested, including both canine and feline RMBD that were analyzed. Discrepancy among batches has not been previously studied for comparison, but these results showed that differences in unlisted ingredients were common in RMBD. Due to cost limitations restricting the analysis to only 2 batches of each RMBD, a statistically significant batch contamination rate could not be determined. However, the finding of discrepancy among batches represents yet another variable which could impact interpretation of an ED trial in a patient fed a commercially prepared RMBD. While no particular manufacturer’s diets were found to be more likely to contain unlisted proteins, brand F, the producer of feline diets 7 to 9, had the least number of contaminants, as well as the most consistent agreement between each batch. It is possible that this finding is due to the nature of the processing practices of this particular manufacturer, the production of smaller batch sizes to minimize opportunity for contamination, or more limited sourcing of ingredients to restrict the potential for supplier cross-contamination. Overall, the number of animal protein ingredients included in each diet was not a predictor of the number of unlisted proteins isolated in the analysis. Even diets restricted to single proteins were as likely to contain 1 or more sources of unlisted animal DNA as those with multiple animal proteins and batch contamination was unpredictable.

Previous studies have shown that in rare cases,plastic growers pots ingredients listed on product packaging were found to be missing from the analysis . Our study showed that no animal DNA was missing from that declared on the packaging of any RMBD included in the analysis. Due to the target DNA of the qPCR assay, the study was unable to validate the presence of sardine or goose to confirm the inclusion of these ingredients. This was again due to cost limitations precluding the addition of these proteins in the analysis. Some diets also contained animal fat sources such as salmon oil, cod oil, or sardine oil. Through purification processes, fish oils undergo refinement to remove proteins from the oil to render them free of proteins . While cod and sardine were not included in the analysis, our study did evaluate for salmon DNA. Both canine diets 1 and 3 contained salmon oil and tested negative for salmon DNA. Feline diet 5 contained salmon oil, but also contained salmon meat, and tested positive for salmon DNA as would be expected. If extrapolating from the finding that salmon DNA was not found in the diets containing salmon oil, it may be expected that the other diets containing fish sources of oil did not contain cod or sardine DNA as potential allergens. In general, plant- and animal-based oils are not considered allergenic when highly purified . Of additional note was the finding that no diet contained the DNA of kangaroo. Since kangaroo meat is not used by any of the manufacturers in their RMBD, this finding serves as an additional negative control for this study to validate that no DNA of kangaroo origin was detected in the analysis, as would be expected. Analysis of the 2 extensively hydrolyzed poultry featherbased diets revealed no detection of unlisted animal DNA. This is consistent with previous reports that contaminants are detected less commonly and in lower numbers in hydrolyzed diets . The extensive hydrolysis of poultry feather proteins into component amino acids or very short oligopeptides is intended to avoid inducing IgE-mediated mast cell activation that can occur with proteins 10 kDa in size or greater . Extensive hydrolysis to reduce poultry allergenicity has been validated in both serum IgE and feeding trials to show the clinical benefits for CAFR . These negative control diets were selected because of the rigorous quality control methods undertaken by the manufacturer to ensure cross-contamination does not occur before market release . While the canine diet did test positive for chicken DNA, the manufacturer does list the feathers of chicken, turkey, and duck as their sourced raw materials . The target gene of the analysis for chicken DNA, transforming growth factor beta 3, is a protein expressed in chicken feathers . Additionally, the manufacturer is aware that cross-contamination needs to be avoided in a therapeutic diet and has developed and clinically validated calibration curves to prevent contamination . These calibration curves correspond to a known DNA level that was clinically tolerated based on Global Skin Scores in feeding trials in order to set a tolerance level for ancillary proteins known as the NPPI , which is strictly monitored in each diet prior to allowing market release . Based on the manufacturer’s quality control data, 72.3% of these extensively hydrolyzed diets contain DNA below the limit of detection , and 25.7% may have DNA above the LOD but below a safety threshold of 1.2 g/g . However, no diet released to market will exceed the established cut-offs of the NPPI based on the pre-established calibration curves . Therefore, trace copies of chicken DNA may be expected on PCR in some of the diets released to market, as was found in our study. The DNA in the feline extensively hydrolyzed diet in this study was below the LOD, and no animal DNA was detected in the assay. Based on the sensitivity of qPCR, it can be argued that these assays, being sensitive enough to detect as few as 10 copies of the target gene, are of greater sensitivity than that required to detect clinically meaningful contamination that would trigger CAFR. There is no established maximum tolerable level of a contaminating protein that may elicit a pruritic reaction in a food sensitized pet. In humans, soy protein concentration as low as 10 ppm may evoke a reaction in a soy-sensitized individual . Additionally, dose distribution has been demonstrated to vary between different food allergens in sensitized humans, showing that a tolerance range may exist for different food antigens themselves . The additional concern for CAFR pets is that, as opposed to most humans, pets are often fed a specific commercial diet with daily regularity, increasing their risk of chronic re-exposure to a food antigen contained therein. As a result, even small amounts of unknown allergens may lead to a cumulative reaction in a CAFR-affected pet and skew the clinical impression of their response to a particular ED. These reactions may even be sporadic if there is significant variation of the protein constituents of the diet between batches. The need to validate food allergic threshold distributions in canine and feline CAFR is an important area for future research.