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. 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 ,snap clamp 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. 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 . The commercial RMBD tested were obtained in California due to the proximity to the testing facility . 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 feather based 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. Until such time, rigorous quality control using protein analysis methods such as qPCR or enzymelinked immunosorbent assay remains a sensitive method to confirm such contaminants are not detected in therapeutic diets fed for the purpose of the clinical diagnosis of CAFR. This remains the industry standard for quality control of commercially produced diets, including extensively hydrolyzed diets. In conclusion, this study confirms that commercial RMBD should not be considered appropriate for selection as ED in the diagnosis of CAFR as a result of their tendency to include unlisted protein ingredients, which can differ from batch to batch. A clinician should use caution when interpreting the results of an owner-directed ED trial using RMBD to exclude CAFR as a cause of their pet’s pruritic dermatopathy,growing blueberries and veterinarian-guided elimination diet oversight is still recommended. Until further evidence is presented, an elimination diet and provocation trial with a patient-appropriate prescription-based diet subjected to applicable quality control or a home-prepared novel protein diet remain the current diagnostic standard for CAFR.In vineyard production systems, canopy management practices are usually employed to control the source-sink balance and improve the cluster microclimate leading to an improved grape composition and resultant wines . Canopy density is usually controlled during the dormant season thought the winter pruning. Additional canopy management practices may be applied during berry development. Fruit-zone leaf removal and especially, shoot thinning have been widely used in order to increase the cluster exposure to solar radiation, reduce crop load as well as decreasing the pest pressures , increasing flavonoid content and diminishing herbaceous aromas . Nevertheless, when high air temperature and excessive radiation combine, detrimental effects on berry acidity and flavonoid content have been reported in warm climate regions . Leaf removal consists of removing basal leaves around the clusters in the east or north side during grape development increasing the cluster exposure to solar radiation. It is well known that an early leaf removal increased total soluble solids, anthocyanins, and flavonols . However, some authors reported increases in titratable acidity in Sangiovese and Teran cultivars while other authors found decreases in acidity with basal leaf removal on Tempranillo . Conversely, Sivilotti et al. reported a positive effect of leaf removal applied after flowering on Merlot grapevine by improving cluster integrity by reducing incidence of Botrytis, and lower herbaceous aromas without affecting yield and cluster mass. Contrariwise, Pastore et al. reported that defoliation at veraison reduced the anthocyanin content and increased the impact of sunburn. In fact, these authors found that leaf removal induced a general delay in the transcriptional ripening program, which was particularly apparent for structural and regulatory genes involved in the anthocyanin biosynthesis.

Clearly, vineyard location, cultivar , timing of leaf removal , method , and degree of leaf removal , the growing season , among others, are all factors influencing how leaf removal affects grapevine berry composition and integrity. On the other hand, shoot thinning has been related to increased cluster and berry mass and the number of berries per cluster, with a reduction on yield . Conversely, Wang et al. observed that shoot thinning had relatively minor impacts on yield components because of a compensatory effect due to the lower cluster number with concomitant increase in cluster mass. Contrarily, shoot thinning practices on grapevine did not show a great impact on berry primary metabolism , however, secondary metabolites were affected by them . In fact, we recently reported an increase of two-fold in the flavonol content of Merlot berries when leaf or shoot removal was applied mainly by increasing the proportion of quercetin and kaempferol derivatives in detriment of the myricetin derivatives . Berry composition is dependent on a complex balance between compounds derived from primary and secondary metabolism. Between secondary metabolites, flavonoids play an important role in the quality and the antioxidant properties of grapes and are very responsive to environmental factors such as solar exposure . Anthocyanin compounds are responsive of the berry color, and flavonols act as a UV shields, contribute to the wine antioxidant capacity, color stability, and hue through copigmentation with anthocyanins . On the other hand, the methoxypyrazines are wine key odorants contributing to their herbaceous characteristics and have been related to unripe berries and poor-quality wines when these are not part of the wine typicity . Since they can be present in grape berry and wines at high levels, they may have an important sensorial impact on wine quality . Among methoxypyrazines, the 3-isobutyl-2- methoxypyrazine is considered the most relevant to wine flavor due to its correlation with the intensity of the bell pepper character of wines and its content at harvest seems to be dependent of the solar exposure . The differences found in the literature about the effect of manipulating the canopy architecture on the flavonoid and aromatic content due to different solar exposure of berries in warm climates opens an important field of research. Therefore, we aimed to find the optimal ranges of berry solar exposure estimated as percent of kaempferol for flavonoid synthesis up regulation and the thresholds for their degradation, and to evaluate how canopy management practices such as leaf removal, shoot thinning and a combination of both affect the grapevine yield components, berry composition, flavonoid profile, and herbaceous aromas.An experiment was performed in 2017 on 7-year Cabernet Sauvignon vines grafted onto 110 Richter root stock with NW-SE row orientation and a vine spacing of 2 m × 2.4 m in a commercial vineyard in Oakville, CA . Individual berries were sampled at harvest according to their position in the canopy and overexposure based on visual appearance. Each independent replicate was a sample of 75 berries collected from up to 50 plants each , these plants being potentially the same for all exposures. From each sample, 55 berries were used for must analyses and berry mass, and the remaining 20 berries were storedat −20°C for analyses of flavonoids. Thus, four observational treatments with four replicates consisted in two rows of 25 vines each were established: non-exposed berries collected from interior clusters ; exposed but free of signs of overexposure, collected from northeast exposed clusters ; exposed and with mild signs of sunburn, collected from southwest exposed clusters ; and exposed and with severe signs of sunburn with signs of damage collected from southwest exposed clusters .