Dermatitis in dogs is a prevalent skin condition that affects companion animals and deteriorates the quality of life for both the pet and the owner (Outerbridge and Jordan, 2021). Dogs can present with numerous clinical signs, including itch, inflammation, hair loss, ear infections in dermatitis (Hensel et al., 2015). Environmental and dietary allergens can trigger these hypersensitivities causing dermatitis. Allergens can include dust mites, pollens, fungi, certain insects and even food proteins (Tiffany et al., 2019; Gupta et al., 2026). This dermatitis can result from a combination of a disrupted skin barrier, immunological disorders and environmental factors (Drechsler et al., 2024; Yue et al., 2024). Upon discovery of the allergens, therapies can be deployed, including allergen immunotherapy. Allergic dermatitis is diagnosed based on history, clinical signs and allergen testing (Gedon and Mueller, 2018). Environmental allergens are most commonly identified using in vivo testing, such as intradermal allergen skin testing (IDST) and skin prick test (Carmona-Gil et al., 2019; Di Tommaso et al., 2021). Complications can arise from the need for sedation and hair removal, expertise and the dog skin and drug history (Olivry et al., 2013). Skin prick test is easier compared to the intradermal test and requires less sedation. The use of in vitro testing has become more popular in recent years, as IDST can be quite capital- and human resource-intensive (Olivry et al., 2013). These in vitro tests, such as IgE ELISA, can be well tolerated and less complicated and more number of allergies can be checked at a time. Attention should be given to the sensitivity and specificity of these tests, as well as to the dogs symptoms. The reliability of both in vivo and in vitro testing for diagnosing canine allergic dermatitis is of utmost importance (Chermprapai et al., 2020). Exclusion of other conditions is also important before heading towards allergen testing. These tests should be thoroughly compared based on their diagnostic efficacy and their scientific merits. Understanding the benefits and drawbacks of both procedures will help veterinarians select appropriate testing strategies to improve therapeutic outcomes. Furthermore, comparative investigations may help generate standardised diagnostics. Limited data is available regarding region-specific allergen sensitisation patterns in dogs with allergic dermatitis from India. Thus, the current study aims to describe various skin-related conditions and to compare the efficacy of in vivo and in vitro allergen testing in the analysis of canine allergic dermatitis.

Ethics
 
The research work was approved by the Institute Animal Ethics Committee (IAEC) of Veterinary College, Anand, vide no. 434/VCC/2024. Informed consent was obtained from the owner for the participation of the animals in the present study.
 
Study period, location and animal selection
 
The study was conducted at the Veterinary Clinical Complex, Anand (22.56°N, 72.93°E), Gujarat, from January 2024 to December 2025. The temperature and relative humidity ranged from 11-42°C and 50-90%, respectively. The study comprised dogs who presented to VCC, Anand, with dermatitis as their main complaint. Throughout the study period, 1914 dogs with dermatitis were registered. Of these cases, 351 were checked for various dermatological problems by skin scraping, trichography and impression smear examination and treated accordingly (Bajwa, 2017). 24 dogs were repeatedly seen for recurrent or unresponsive cases. These 24 dogs were selected for both in vitro and in vivo (skin prick test) investigations. Ectoparasites, flea allergy, bacterial pyoderma, Malassezia dermatitis, dermatophytosis and endocrinopathies were excluded before allergen testing.
 
Skin scraping examination
 
All dogs with dermatitis were examined by deep skin scraping as described earlier (Bajwa, 2017). Hairs from the affected area were clipped, followed by the application of a drop of mineral oil to the lesions. Multiple scrapings were performed in the direction of the hair growth until capillary bleeding occurred. Skin was squeezed during or between scrapings to extrude the mites from the deep follicles to the surface. Debris was then transferred to a slide, mixed with mineral oil and examined under the microscope at low power (10x) with a coverslip.
 
Trichography
 
The trichography technique was used in difficult-to-scrape areas, such as the periocular and interdigital regions, to detect mites or fungal spores, as mentioned by earlier Saridomichelakis et al., (2007). Hairs from affected skin were plucked with forceps in the direction of the hair growth. They were placed in a drop of mineral oil on a slide and examined under low-power magnification (10x) after application of a coverslip.
 
Impression smear
 
All dermatitic dogs were examined and impression smears were prepared for diagnosing pyoderma and skin conditions associated with Malassezia. Smears were prepared by direct impression of the lesions, heat-fixed, stained with methylene blue and examined under oil immersion (100x) (Bajwa, 2017).
 
Blood collection
 
A total of 4 ml of blood was collected from the cephalic vein and transferred to an EDTA vial and a clot activator vial for haematological examination. Haemoglobin (g/dL), packed cell volume (%), total erythrocyte count (×10⁶/µL), total leucocyte count (×10³/µL), lymphocyte (%), monocyte (%), neutrophils (%), eosinophils (%), basophils (%) values were estimated from the blood using the Abacus Vet 5 haematology analyser machine. Serum was separated and sent for in vitro allergen testing. The inclusion criteria for the healthy control group required the absence of dermatological lesions, pruritus, systemic illness and recent treatment with corticosteroids, antihistamines, or immunosuppressive drugs. All control dogs underwent a comprehensive physical and dermatological examination before inclusion and only dogs with unremarkable clinical findings and normal haematological values were enrolled.
 
In vitro testing
 
Serum samples were sent to Endocrine and Allergy Laboratory Pvt Ltd, Ahmedabad, Gujarat, for estimation of allergen-specific IgE using Immuno-EIA (Enzyme Immunoassay). Results were expressed as allergen-specific IgE concentrations (IU/mL) and values ≥0.35 IU/mL were considered positive, following the manufacturer’s recommended interpretative criteria and previous veterinary studies employing ELISA-based canine allergen-specific IgE assays (Mueller et al., 2016). Dogs were tested for allergens such as house dust, house dust mites, Parthenium, tomato, mango, wheat, fish, milk, egg, Aspergillus fumigatus and Candida.
 
In vivo testing
 
Skin prick testing (SPT) was performed using standardised allergen extracts (All Cure Pharma Pvt. Ltd., Haryana, India) according to the manufacturer’s instructions. The allergen extracts were glycerinated aqueous preparations and were stored at 2-8°C until use. Testing was performed on the clipped lateral thoracic wall after the skin was cleansed with 70% isopropyl alcohol and allowed to dry completely. Individual test sites were marked at 2 cm intervals to prevent overlapping wheal reactions. Histamine hydrochloride served as the positive control, while normal saline (or glycerinated diluent supplied with the kit) served as the negative control (Carmona-Gil et al., 2019). Sedation was not used because it may influence cutaneous vascular responses and interfere with test interpretation. Excess allergen solution was gently removed after testing and reactions were evaluated 15-20 minutes after allergen application. The largest wheal diameter and its perpendicular diameter were measured using a transparent millimetre ruler and the mean wheal diameter was calculated. A reaction was considered positive when the wheal diameter was at least 3 mm greater than the negative control and demonstrated an appropriate response relative to the histamine positive control.
 
Statistical analysis
 
Data on different skin conditions were analysed by descriptive statistics and presented as frequencies and percentages. Various haematological parameters were statistically analysed using an unpaired t-test in GraphPad Prism (V.9.0).

A total of 1914 dog dermatological cases were recorded at the Veterinary Clinical Complex, Anand, during the study period, of which 351 dogs were studied in detail (Table 1). Pyoderma was the most common skin condition, making up 22.51% (79/351) of all cases. Undiagnosed/various skin conditions accounted for 19.66% (69/351) of the cases, indicating a large variety of clinical presentations. Malasseziosis was the second most diagnosed condition with an frequency of 16.52% (58/351), followed by fungal dermatitis (13.11%; 46/351). Allergic flea dermatitis accounted for 10.54% (37/351) of all dermatologic cases. Demodicosis was diagnosed in 8.55% (30/351) of dogs with dermatitis and recurrent/non-responding dermatitis in 6.84% (24/351) of cases. Sarcoptic mange was the least common, with an frequency of 1.71% (6/351). The lowest was for contact dermatitis.


       
Breed-wise distribution of recurrent/non-responding dermatitis cases revealed that labrador retrievers were the most commonly affected breed, with 12 cases (50.00%), followed by Beagles with 4 cases (16.67%). Non-descript dogs accounted for 2 cases (8.33%). German Shepherd, Bully, Pug, Dalmatian, Dobermann and Shih Tzu each contributed 1 case (4.17%). 
       
The allergen sensitivity profile of dogs with recurrent dermatitis was evaluated using both in vivo and in vitro allergen testing methods (Table 2). All dogs with recurrent/non-responding dermatitis showed sensitivity to at least one allergen. Among the allergens tested, house dust mite showed the highest positivity in the in vivo test, with 18 out of 24 dogs (75.00%), followed by milk with 13 cases (54.17%) and mango with 11 cases (45.83%). Candida was positive in 9 dogs (37.50%), while wheat was positive in 8 dogs (33.33%). House dust, tomato, Parthenium, Fish, Aspergillus fumigatus and egg each exhibited positive reactions in 7 dogs (29.17%).


       
In contrast, the in vitro allergen assay showed the highest positivity for house dust and house dust mite, each detected in 21 dogs (87.50%). The tomato allergen was positive in 10 cases (41.67%), whereas the milk and Parthenium allergens were positive in 7 (29.17%) and 3 (12.50%) dogs, respectively. None of the tested samples showed in vitro positivity against mango, Candida, fish, Aspergillus fumigatus, wheat or egg allergens.
       
The haematological profile of dogs with recurrent dermatitis showed few alterations compared with apparently healthy controls (Table 3). A significant reduction in total leukocyte count and neutrophil percentage was observed in allergen sensitized dogs. However, no significant differences were observed in other haematological indices between the two groups (p>0.05). Although lymphocyte levels were comparatively higher in affected dogs, the variation remained statistically non-significant.


       
The present study demonstrated that pyoderma was the most frequently encountered dermatological condition in dogs in the Anand region, followed by Malasseziosis. Similar observations have been reported by Hensel et al., (2015) and Outerbridge and Jordan (2021), who documented that bacterial and yeast-associated dermatitis are among the most common dermatological problems encountered in small animal practice. The higher frequency of pyoderma observed in the present study may be attributed to humid climatic conditions, ectoparasitic infestations, a compromised skin barrier and secondary bacterial invasion associated with an allergic skin disorder (Tanveer et al., 2024).  
       
Among recurrent and non-responding dermatitis cases, Labrador Retrievers were the most affected breed. A breed predisposition to allergic dermatitis has previously been described in Labrador Retrievers, Beagles, German Shepherds and Pugs due to inherited defects in epidermal barrier function and altered immune responses (Gedon and Mueller, 2018; Sundararajan et al., 2026). Genetic susceptibility coupled with environmental exposure may therefore explain the higher prevalence observed in these breeds. Similar breed-associated susceptibility has also been reported by Drechsler et al., (2024), who emphasised that certain pure breeds are at risk of chronic allergic dermatitis.
       
The present investigation revealed considerable variation between in vivo and in vitro allergen testing methods. House dust mite was identified as the most predominant allergen in both testing procedures, with particularly high positivity in the in vitro assay. House dust allergens also showed significantly higher positivity in the serological assay than in the skin prick test. These findings are consistent with reports by Carmona-Gil et al. (2019) and Curin et al. (2011), who documented that environmental allergen, such as dust mites, are major sensitising agents in dogs with allergies. The high prevalence of house dust mite sensitivity in the current study may be associated with tropical climatic conditions and increased indoor humidity, favouring mite proliferation (Hart, 1998).
       
In contrast, food allergens such as milk, mango and wheat demonstrated higher positivity in in vivo testing than in the in vitro assay. Similar discrepancies between intradermal or skin prick testing and serum IgE assays have been previously reported in dogs (Kang et al., 2020; Mueller and Olivry, 2017). The greater sensitivity of in vivo testing to certain allergens may be due to direct mast cell-mediated hypersensitivity reactions in the skin (Olivry et al., 2015). In contrast, in vitro measured circulating IgE concentrations may not always correlate with clinical hypersensitivity. Moreover, allergen extract quality, antigenic variability and regional environmental exposure may contribute to inconsistent test performance (Curin et al., 2011).
       
Mango, Candida, fish, Aspergillus fumigatus, wheat and egg were negative in the in vitro test, despite positive in vivo results. Similar results were reported by Gogunskaya et al. (2020) in their evaluation of skin and ELISA tests. These results arise from a variety of allergen standardisation, cross-reactivity and the biological activities of allergenic proteins. Some allergens may cause localised skin hypersensitivity even when serum IgE levels are low or undetectable, with no increase in IgE levels (Mueller and Olivry, 2017).
       
Negative serum allergen-specific IgE results but positive in vivo reactions were observed in several allergens, suggesting a discrepancy between the two diagnostic techniques. However, because the in vitro and in vivo assays measure different aspects of the allergic response, this inconsistency should not be considered as evidence that one is superior to the other. Serum IgE testing measures circulating allergen-specific IgE antibodies, which may not correlate with tissue-bound IgE or clinical reactivity, compared with skin prick testing, which measures the immediate cutaneous hypersensitivity reaction to direct allergen contact. Differences in allergen extract composition, test sensitivity, specificity, cut-off values and biological variability can also play a role in discrepant results.
       
The present findings therefore indicate that neither in vivo nor in vitro testing alone can be considered completely reliable for diagnosing allergic dermatitis. Instead, both methods should be interpreted alongside clinical history, dermatological examination and exclusion of other pruritic skin conditions. Hensel et al. (2015) recommended that allergic testing should primarily be utilised to identify allergens for immunotherapy rather than as a sole diagnostic tool for dermatitis.
       
Haematological evaluations of test-allergic dogs showed significantly lower leucocyte counts and neutrophil percentages than those of healthy untested dogs. These changes may be indicative of recurrent secondary infection, immune-mediated responses and chronic inflammatory processes in allergic dogs with dermatitis (Mueller et al., 2016). Chronic pruritic and persistent inflammation can induce physiological stress and immunological exhaustion, leading to altered haematological profiles. Similar inflammatory and immunological alterations have been documented in canine atopic dermatitis by Outerbridge and Jordan (2021).
       
Dogs with recurrent/non-responsive status had lower leukocyte and neutrophil counts than healthy dogs. But the present study did not explore the possible mechanisms that could explain such differences. Thus, the interpretation of these findings should be observational and not indicative of a particular pathophysiological process. Differences observed may be due to biological variability, inflammatory status, previous therapeutic interventions or other unmeasured factors. Further studies, including assessments of immunological and inflammatory biomarkers, are needed to clarify the mechanisms underlying these haematological changes.
       
The comparative analysis performed in this study emphasises the complexity of diagnosing canine allergic dermatitis, as no single diagnostic method demonstrates the sensitivity and specificity required for all allergens (Mueller and Olivry, 2017). Variability in allergen detection between in vivo and in vitro methods highlights the multifactorial nature of allergic skin disease and the influence of regional allergen exposure on sensitisation pattern (Carmona-Gil et al., 2019). Environmental allergens, particularly house dust and house dust mite, appeared to play a major role in recurrent canine dermatitis in the study region, consistent with findings of Mueller et al. (2016). The findings also suggest that skin prick testing may provide a better, easier and less costly method for detecting allergen sensitivity in dogs. 
       
Canine atopic dermatitis is diagnosed based on clinical history, characteristic clinical signs and exclusion of other pruritic diseases. Once a clinical diagnosis has been established, allergen testing is used to identify sensitising allergens for the formulation of allergen-specific immunotherapy and should not be considered a stand-alone diagnostic test. Serum allergen-specific IgE assays and intradermal skin testing have limited sensitivity and specificity for diagnosing food-related adverse reactions and should not be used as stand-alone diagnostic tests. An elimination diet followed by controlled dietary provocation remains the gold standard for confirming adverse food reactions in dogs.
       
There are some limitations of the present study that need to be considered in the interpretation of the findings. The relatively small sample size and single-centre design may limit the generalisability of the results to the wider dog population. There was no gold standard for confirming dog dermatitis or individual allergen sensitisation. As such, the findings should be interpreted in conjunction with clinical assessment rather than be considered definitive evidence of diagnostic accuracy. Detailed clinical information, including lesion distribution, pruritus severity, chronicity, seasonality, diet history, flea and tick control and previous treatment history, was not consistently available for all dogs, limiting the assessment of their potential influence on allergen test results. A greater number of allergens could be used to better understand the pattern, which was very limited in the present study.

The present study supports the concept that an integrated diagnostic approach incorporating clinical examination, exclusion of parasitic and microbial skin diseases, haematological assessment and both in vivo and in vitro allergen testing can achieve greater diagnostic accuracy for canine allergic dermatitis. These combined diagnostic strategies may enable targeted therapeutic and immunotherapeutic interventions for affected dogs and improve allergen identification.

The authors acknowledge the Principal, Veterinary College, Anand, for providing the necessary facilities for the study.
 
Research funding
 
This study received no funding from any organisation.

The authors declare no conflict of interest.