Diagnostic methods for adverse food reactions in dogs

Introduction

Adverse food reactions (AFRs) include food intolerance (FI) and food hypersensitivity, with the latter subdivided into food allergy (FA), usually associated with IgE-dependent reactions, and cell-mediated inflammatory reactions to food allergens or dietary components, which are common in dogs with atopic dermatitis 1. The gold standard for diagnosing an AFR is based on the improvement of skin lesions and pruritus during the restrictive phase of an elimination diet trial (EDT) and a relapse of clinical signs after a food challenge (FC) with the original diet, as reliable commercial tests for diagnosis do not exist 2,3,4.

The practical difficulties involved with elimination diets mean that there has been a growing interest in diagnostic protocols that are easier to execute and more acceptable to owners 1. The use of allergic testing in dogs with chronic pruritus can be a useful tool in formulating a restrictive diet and developing guidelines for a provocative challenge, helping avoid unsuccessful attempts at dietary composition, as well as being beneficial in terms of time and cost demands. This article aims to review the veterinary literature on the use of tests with food extracts for evaluating AFRs in dogs.

Elimination diets 

The possibility of an AFR should be investigated in all dogs with chronic and perennial itching, after ruling out other pruritic skin diseases of infectious-parasitic origin and allergy related to reactions to arthropod saliva 5. There is generally suspicion of an AFR when itching and lesions are reduced by at least 50% after establishing an EDT, and the condition is confirmed when clinical signs recur after the FC 3.

Dietary components for an EDT are usually chosen based on the animal’s history, and preferably only use ingredients to which the animal has not been regularly exposed previously. Studies show that dogs with food allergy rarely respond to an elimination diet during the first week of feeding, but around 50% will show a significant reduction in signs after three weeks, and more than 85% improve after five weeks; this figure exceeds 95% if the dietary trial is extended to eight weeks. A few dogs may require an extension of the diet to 12 weeks 3 (Figure 1).

It has been shown that the time required for an EDT can be reduced if the initial allergic pruritus and inflammation are controlled with a short course of anti-inflammatory drugs 6. One study used either prednisolone (for two or three weeks) or oclacitinib (for three weeks), with the provocative food challenge started after four weeks, and a diagnosis could be made within six weeks. In this study, the diagnosis of food-induced reactions in dogs with AD was 100% in both groups, and the negative predictive value was 95% when using prednisolone, compared to 63% positive and 100% negative predictive values when using oclacitinib 7. Therefore, prednisolone seems to be the better option for most dogs, probably due to the more rapid onset and broader anti-inflammatory effects of glucocorticoids 6,7.

Homemade restrictive diets

Such diets consist of a source of original protein and carbohydrate, preferably to which the animal has never been regularly exposed 8. For many decades they were considered ideal for establishing a diagnosis of AFR, but these are nearly always unbalanced, particularly in young and rapidly growing dogs. In addition, associated limitations such as preparation factors, the risk of contamination, cross-allergic reactions between food components, the need for supervision by veterinary nutritionists, and the necessary owner dedication have led to increasing use of commercial foods for the diagnosis and maintenance of animals with AFRs 8.

Hydrolyzed protein (hypoallergenic) diets 

The main objective when manufacturing specialized diets by hydrolysis is to sufficiently disrupt the protein structure to remove any allergens and allergenic epitopes, thus preventing immunological recognition by patients already sensitized to the intact protein. A secondary goal may be to break down the proteins to the point where there are no antigens capable of eliciting an immune response which would lead to sensitization 9.

Hydrolyzed diets therefore consist of protein fragments with molecular weights less than 10 kDa, and provide greater digestibility, absorption, and lower antigenicity. This can be achieved by disrupting the three-dimensional structure of the protein, altering the structure of the amino acid side chains, and can be achieved via various methods, including heat treatment, pH manipulation, enzymatic hydrolysis and filtration 9. 

In terms of efficacy, one study evaluated 12 dogs with cutaneous manifestations of allergic dermatitis after exposure to chicken meat and reported that 11 improved when fed a hydrolyzed chicken diet 10. Additionally, dogs allergic to soy showed a significant increase in pruritus after oral challenge with soy, but not when fed a hydrolyzed soy diet 11. 

Studies comparing the effectiveness of home-prepared diets with hydrolyzed diets demonstrated similar results in the diagnosis of canine AFR, although dogs fed the hydrolyzed commercial diet improved more quickly, suggesting a lower antigenic potential 12. It is important to note that there is no standardization in the market for commercial diets with hydrolyzed proteins, and such foods may contain anything from simple amino acids to polypeptides of large molecular weight, depending on the degree of hydrolysis; the latter can favor cross-reactivity between food allergens and reduce the diet’s diagnostic sensitivity 8,9.

In a review of eleven studies analyzing the presence of clinical reactions to hydrolyzed or partially hydrolyzed diets in dogs, evidence of reactions to hydrolyzed diets was found in four studies, and 20-50% of dogs with food allergies showed worsening of clinical signs when fed partial-hydrolyzed diets 9. Therefore, hydrolysate-containing diets are probably best used in dogs suspected not to be hypersensitive to their individual components, rather than using them as a diagnostic tool for AFR 8,9.

Elemental diets

Hydrolysates generally contain simple amino acids and/or polypeptides of large molecular weight (depending on the degree of hydrolysis), and traces of enzymes used in the process 13. Elemental diets are produced by ultrafiltration of hydrolyzed diets, leading to products which have molecular weights below 3 kDa and which are rich in amino acids (ranging from 75-204Da), thus preventing IgE-dependent reactions and minimizing protein contamination 14. 

In a study comparing serum levels of specific chicken IgE in dogs fed either non-hydrolyzed, hydrolyzed, and ultra-hydrolyzed commercial diets, it was demonstrated that dogs fed the latter had the lowest presence of serum IgE, avoiding the triggering of an allergic food reaction 13. Additionally, a ultra-hydrolyzed poultry feather diet did not induce pruritic flares in dogs allergic to chicken, in contrast to a ultra-hydrolyzed chicken liver diet that led to flares in 40% of the dogs 15. Therefore, such diets may benefit animals with IgE-dependent reactions to food and should be used for the diagnosis of AFR, but in animals with delayed cell-mediated reactions the results are controversial 15.

In vitro tests for AFR

Serological tests

Serological tests for diagnosing adverse responses to foods present controversial results. Paired sera from dogs with AFRs, along with healthy dogs, were subjected to laboratory testing for specific IgE and IgG to foods, and no significant differences in the results were found between the groups 16. Another study that evaluated specific IgE and IgG responses to foods in dogs with allergic skin disease also showed unsatisfactory repeatability of these tests 16. In addition, a study assessing the Western blot serological test as a tool for diagnosis of AFRs concluded that it could be useful in formulating an elimination diet but not in the definitive diagnosis of food allergy 17. Thus, the usefulness of serological tests for specific IgE and IgG to foods remains limited, and does not appear to be recommended for the diagnosis of canine AFRs 8,16,17.

Lymphocyte proliferation test (LPT)

The lymphocyte proliferation response to food antigens can be useful in detecting in vitro non-IgE-mediated inflammatory reactions to food allergens in dogs, with reported positive predictive values of 100% and negative predictive values of 93% 18. In one study, out of 14 dogs that showed a positive reaction only to food in the LPT, complete medical information was available for 12 cases, and all dogs exhibited a significant reduction in itching after an EDT without any other medication 19. However, due to practical difficulties, this test is currently useful only in experimental protocols and is not available for routine clinical practice.

In vivo tests for AFR

Intradermal skin tests

In human medicine, intradermal testing (whereby the allergen is injected directly into the skin) is not recommended with food allergens extracts due to high irritability, which can lead to false positive results, and because there may be a greater risk of anaphylactic reactions. In veterinary medicine, studies have been conducted in healthy animals, but the lack of standardization of food extracts, variations in the technique and cutaneous irritability make the method very sensitive and not very specific 8.

Skin prick test 

For the assessment of sensitization to food allergens, the prick test with food extracts is considered to have high sensitivity but low specificity. Drops of the allergen solution are applied to the skin surface before the site is pricked to allow the allergen to enter the epidermis (Figure 2). In humans, when performed with standardized extracts, the method has shown a positive predictive value (PPV) of 60-75% and a negative predictive value (NPV) of up to 95% 20. In veterinary medicine, studies are progressing. In one report a prick test was conducted in 34 dogs with chronic itching; 25 dogs then underwent an EDT using a “negative” food for 60 days before being challenged with a “positive” food identified on the prick test. Four dogs did not show worsening signs after the challenge, whilst the other 21 dogs had confirmed AFRs 20. Another study (as yet unpublished) involved 30 dogs divided into three groups (control, atopic dermatitis sensu stricto, and food allergy) that were subjected to the prick test, an elimination diet based on its results, followed by a food challenge. It revealed sensitivity, specificity, and positive and negative predictive values of 46%, 97%, 66%, and 93%, respectively, when compared to the control group.

Therefore, the prick test can be useful in selecting foods for EDTs and guiding dietary ingredients for a food challenge. However, as AFRs in dogs with atopic dermatitis have a cell-mediated component, cases where these prevail may not be identified by the test, limiting its sensitivity 20.

Patch test

Patch tests are particularly interesting to identify food allergens capable of triggering atopic reactions, given the immunopathological processes of atopic dermatitis (AD), where immediate IgE-mediated mechanisms coexist with delayed T-cell-induced hypersensitivity 21. To perform the test, food extracts are placed in small chambers (8-12 mm in diameter) which are then attached to the skin with hypoallergenic adhesive tape. To aid adhesion, the tape is then bandaged and a surgical recovery suit applied. A skin contact time of 48 hours seems to be required to elicit a positive reaction 1,4 (Figure 3), with interpretation of the results primarily based on a scoring system, as shown in Table 1 21.

One study performed patch testing using proteins (raw and cooked), carbohydrates (cooked), and dry commercial food, and the NPVs, assessed after a dietary challenge with the tested foods, were 100% for proteins, 79% for carbohydrates, and 72% for dry food 4. With the aim of recognizing immediate and delayed reactions to food, the patch test was associated with the prick test in 21 dogs with atopic dermatitis with AFR, revealing sensitivity values of 80%, specificity of 66.7%, PPV of 66.7%, and NPV of 80% 1. Thus, the patch test alone or in combination with the prick test does not allow a definitive diagnosis of AFRs in dogs with chronic itching, but it can be useful in selecting components for EDTs and guiding FC 1,4.

Table 1. Assessment of food contact test reactions in dogs with atopic dermatitis with AFR (from 21)

When a food allergen is identified by FC, it should be immediately removed from the dog’s diet, and anti-pruritic medication offered as necessary, and the ED reintroduced for at least fifteen days or until the signs regress 22. After the clinical signs settle, the FC is performed with another dietary component until all aspects of the previous offending diet are exhausted. A diagnostic algorithm for adverse skin reactions to food is shown in Figure 6.

The future

In the future molecular allergology may allow better detection of sensitization to allergenic components (purified or recombinant proteins), supporting better standardization than crude allergenic extracts. There may also be a better understanding of allergenic cross-reactivity, and IgE-mediated sensitization profiles may become available. Ultimately more sensitive and specific food allergy diagnostics, with the establishment of specific dietary protocols adapted to each patient, will contribute to a multimodal protocol that enables better control of AFR 24.

Conclusion

The diagnosis of an adverse food reaction (AFR) in dogs must currently be made with an elimination diet trial followed by a food challenge; elemental diets are better for diagnostic screening, but hydrolyzed products may be suitable as a maintenance diet; Patch test and prick test using food extracts should not be used as a diagnosis for AFR, but can help screen for components of elimination diets and provocative food challenges. Dogs with food allergy sensu stricto and with food-induced atopic dermatitis should be maintained on hydrolyzed diets with components that do not trigger inflammatory reactions.