Abstract
Objective
To achieve clinical recovery in canine atopic dermatitis affected pet dogs via alteration of the gut microbiome, following a meat and egg exclusion diet for 60 days.
Methods
24 atopic dermatitis-affected pet dogs, all fed poultry meat and egg, and another 48 apparently healthy controls fed both poultry meat and egg (n = 24) or vegetable diet (24) were included in the study. The study was undertaken in the Bhubaneswar Smart City, Odisha, India, from July to December 2023. Fecal samples were collected at 2 points for DNA analysis, ie, on day 0 and day 60 of the change from a meat/egg-based diet to a vegetable-based diet. Extracted DNA samples were pooled category-wise and subjected to the gut microbiome analysis in the Nanopore sequencer targeting the 16S rRNA gene. Burrows-Wheeler Transform, Ferragina-Manzini index, and Krona charts were used for taxonomical classification and visualization of relative abundances of bacterial species within the metagenome. Alpha- and beta-diversity analyses were performed.
Results
Atopic pets at day 0 showed elevation in the gut microbiome population with an adequate concentration of pathogens like Escherichia coli and Clostridiodes difficile with lower amounts of the beneficial bacteria like Lactobacillus sp, while the pets at 60 days after dietary intervention showed a significant decline in bacterial species like E coli and C difficile with higher amount of Lactobacillus sp. Both control groups showed variations of microbiome between them as well as from the atopic pets.
Conclusions
We found a close association of poultry meat/egg diet with gut microbiome population and atopic symptoms as well in dogs, and elimination of such diet could be helpful in clinical recovery.
Clinical Relevance
Dietary intervention with the exclusion of potential allergens from poultry meat and egg sources can be an effective approach for the management of canine atopic dermatitis.
Dogs possess distinct microbial communities in various body parts or regions, namely, skin, ear canal, conjunctiva, respiratory tract, genitourinary tract, and gastrointestinal tract.1 The gut microbiota, a system of interconnected host cells and resident microorganisms in the gastrointestinal tract, is a complex ecosystem of beneficial or pathogenic viruses, bacteria, fungi, and protozoa.2 Their genetic material acknowledged as a metabolically active organ has an intrinsic relationship with pet health.1,3,4 A healthy, balanced intestinal ecology of microbiota is like a crucial organ that boosts and stimulates the immune system, fights against intestinal pathogens, and enhances the provision of nutritional benefits like vitamins and nutrients to the host.2 Any disruption in the homeostasis of gut microbiota, termed dysbiosis, has an impact on the microbial transcriptome, proteome, and metabolome and triggers gastrointestinal and metabolic diseases including atopic dermatitis.4,5
Atopic dermatitis is a multifactorial, chronic, relapsing skin disease that affects dogs with a global prevalence rate of up to 15%.6 It is believed to occur through modifications of the T-cells via gut microbiome.7 Gut dysbiosis disrupts metabolites like long-chained and short-chained fatty acids, amino acids, and vitamins, hampering the proliferation and induction of regulatory T-cells and T-helper (Th) cells like Th17-type cytokines and imbalance between Th1 and Th2 cells, leading to loss of immune tolerance, which intensifies allergy and inflammatory reactions and augments the likelihood of occurrence of atopic like symptoms.7,8 It has been stated that atopy occurs when the particular gene that is causing disruption of the epithelial barrier and defense function and exacerbating atopic inflammation (via production of variety of cytokines IL-2, IL-4, IL-8, IL-22, and IL-31 and noncytokines like calcium-binding protein S100A8, serum amyloid A, and various protease inhibitors) gets triggered by allergens such as highly processed meals/meat-based diets, dust, pollutants, pollen, excessive exposure to dust-mite-infested indoor surroundings, and reduced exposure to outdoor farm environments for dogs.9,10 It is a genetically heterogeneous disease, but its phenotypic manifestations along with its prevalence may be better explained, in part, by the diet hypothesis.11–13 Food with less fiber may result in modifications to the gut microbiome and a subsequent decrease in the production of immunomodulatory products, particularly short-chain fatty acids, which have anti-inflammatory properties and support the preservation of epithelial barrier function.
Although not all, some dogs with atopic dermatitis may experience food hypersensitivity. This sensitivity may exist on its own or in combination with immunoglobulin E, which is specific to other aeroallergens. In fact, in a recent international prospective trial of 743 dogs with atopic dermatitis who underwent a dietary restriction-provocation regimen, 172 dogs (23%) were diagnosed with food-induced atopic dermatitis flares.14 In some individuals with atopic dermatitis, skin lesions have been attributed to dietary allergies that cause gut microbiota to induce changes in gene expression in the patients.15 Modulation of the composition of the gut microbiota has been a potential option suggested and tested in people with atopic dermatitis.16 Atopic eczema has been linked to both a decrease and an increase in microbial diversity depending on the severity.17
Treatment options for dogs with atopic dermatitis include glucocorticoids like prednisolone and Janus kinase 1 inhibitors, such as oclacitinib (Apoquel), which targets allergic cytokines. However, their therapeutic effects are only for the short term, as there are chances of rebound of atopy after the medication dose is reduced or stopped abruptly.18 Oclacitinib (Apoquel) has been also observed to be associated with gastrointestinal adverse effects although less than cyclosporine.10,19 Dogs receiving cyclosporine experience gastrointestinal adverse effects,20 and it took a longer period to show beneficial effects. Injectables that target IL-31 are current emerging drugs, but the medication may not work for every patient.10,21 Use of other medications like antibiotics and antifungals are prescribed against secondary overlapping infections, but its prolonged use may upset the balance of the gut microbiota, allowing typically beneficial or commensal microorganisms to proliferate and become pathogenic.15 It may also develop resistant infections and a rise in antimicrobial resistance.
It is proper to mention here that the Government of Odisha promoted various animal husbandry practices including poultry farming as an engine of growth of the rural economy, providing income and livelihood opportunities to the rural people. As a result, many poultry farms (broiler, layer, and backward) were established across the state during the past 2 decades with easy availability of eggs and chicken at a cheaper rate compared to mutton and chevon. Consumption of other sources of meat as a food item is rare and illegal as well. The inclusion of chicken in the regular diet has become a common practice. Further, veterinary health records reflected an increased incidence of chronic dermatitis cases in dogs with variable degrees of therapeutic efficacy.
Moreover, there are no treatment protocols that can be followed universally for the management of canine atopic dermatitis. Individually, the management needs to be tailored based on response to therapies, adverse effects, owner compliance, and cost of medication.19 With the above facts in the forefront, an investigation was undertaken to study the dysbiosis of the gut microbiome following dietary management and its clinical response in atopic dermatitis dogs.
Methods
Pet dogs and sampling
Pet dogs with various dermatological issues registered in the University Veterinary Hospital from July 2023 to December 2023 (n = 1,326). A questionnaire was prepared to collect information with respect to the pet's breed; age; sex; living area (indoor/outdoor); duration and location of lesions; presence of macroscopic ectoparasites such as tick, lice, and fleas; last medication; and poultry meat or chicken (hereafter referred as meat) or egg diet or vegetable-based diet. Dogs with commercial food were excluded from the study. Skin scrapings, ear swabs, tape impressions, and cultural examination were performed following standard laboratory practice for differential diagnosis. Pets with macro- and microscopic ectoparasites, bacteria, and yeast infections were excluded from the study. Based on the signs of pruritus, type, position, recurrence, and chronicity of the lesions, the initial population was narrowed down to subsequent plans.
Twenty-four adult atopic pets of either sex, with either meat- or egg-based diets or both for a minimum of 1 year, were selected for this study. Atopic pets solely managed with a vegetable-based diet were not observed during the period of our study. For control, clinically healthy adult pet dogs of either sex, exclusively on meat/egg-based (n = 24) and vegetable-based (24) diets, were included in this study. All the dogs were regularly vaccinated and administered anthelmintics, and no additional medications like antibiotics or antifungals were given 6 months before this study. The experimental design and its possible benefits were explained to pet owners included in the field trial. The selected dogs were restricted from consuming cooked eggs and meat during the entire trial period. No topical preparations were applied during this trial.
Fecal samples were collected twice into sterile tubes from these 24 short-listed atopic dogs, ie, once on day 0 and another on day 60 of the trial. Fecal samples from the healthy controls with meat-based and vegetable-based diets were collected once in sterile tubes. Each fecal sample was collected in sterile vials uncontaminated using cotton swabs when freshly passed and brought to the laboratory for further analysis.
Deoxyribonucleic acid extraction and nanopore sequencing
Deoxyribonucleic acid extractions from collected fecal samples were done using DNeasy Power Soil Pro Kit (Qiagen). The isolated DNA was quantified using spectrophotometer and fluorimetric techniques with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The quality of the DNA was assessed through agarose gel electrophoresis. The DNA samples were stored at −20 °C for further analysis.
Library preparation included 2-step PCR amplification. The first PCR amplification was done using universal full-length 16S rRNA forward (5'-TTTCTGTTGGTGCTGATATTGC-3') and reverse primers (5'-ACTTGCCTGTCGCTCTATCTTC-3') with a tailed adaptor sequence, and 50 ng of DNA was taken as template for PCR amplification by using KAPA HiFi HotStart ReadyMix (Roche). The amplicon products (∼1.5 kb) were purified by 1.6X AMPure XP beads (Beckmann Coulter) and quantified by the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Twenty-five nanograms of PCR amplicons with a tailed adapter was used for PCR barcoding by using Long Amp Taq 2X master mix (New England Biolabs). The library was prepared from the pooled barcoded sample by ligating sequencing adapters (SQK-LSK114) onto double-stranded DNA fragments using NEB Quick T4 DNA Ligase (New England Biolabs). The prepared library was purified with AMPure XP beads, and quality was checked with Qubit for quantification. Sequencing was done on the Nanopore PromethION system (PromethION P24 and Data Acquisition Unit) using PromethION flow cell (FLO-PRO114M).
Statistical data analysis
Data analysis was done after the raw reads were quality checked using NanoStat.22 Removal of adapters was performed by using the Porechop tool,23 which is specifically used for finding and removing adapter sequences from Oxford Nanopore reads. The Centrifuge tool was used for taxa classification and uses an exclusive indexing strategy that merges the Burrows-Wheeler transform with the Ferragina-Manzini index, optimized specifically for the metagenomic classification.24 Subsequently, the output files were used in the Pavian tool for an interactive exploration and estimation of the taxonomic content.25
Diversity and comparative analysis
Alpha- and beta-diversity analysis and rarefaction plots were conducted utilizing the Vegan-R program.26 Alpha diversity was quantified using Shannon-diversity27 and Simpson-diversity28 metrics. The beta diversity was calculated using a dissimilarity measure called the “z” index and is presented in the form of a distance matrix containing a dissimilarity value for each pairwise comparison. The rarefaction plot represents the annotated species richness in samples. The rarefaction curve was made by using different indexes of species alpha diversity, which plots the number of species as a function of the number of reads sampled. The relative abundance (RA) of the phylum/class/order/family/genus and species was observed and compared. Relative abundance can be defined as the percentage of a particular type of microbe/organism present in a given sample, compared to the total number of microbe/organisms found in that sample. To facilitate comparative analysis, heatmaps were employed to visualize both the prominently identified taxa and the complete array of detected taxa. Heatmaps graphically represented the percentage abundance of the particular species in terms of log2 scale values. The species with RA ≥ 0.1 in the group is displayed in heatmaps. The Krona charts were used to explore RAs and confidences within the intricate hierarchies of metagenomic classifications by using the Krona tool.29 Taxonomic rank relationships and taxonomic abundance are shown using a Sankey plot.
Altogether, there were 4 different types of pooled samples, ie, apparently healthy dogs with a nonvegetarian diet (group 1a), apparently healthy dogs with a vegetarian diet (group 1b), atopic dogs on day 0 of treatment (group 2a), and atopic dogs 60 days after the elimination of egg/meat (group 2b). Comparisons on gut microbiome were made between groups 2a and 2b, 2a and 1a, and 1a and 1b.
Results
Different contrasting microbiome population was observed among 4 categories of pooled samples. Around 200,000 bacterial reads were generated in the pooled samples, in which the most abundant domain observed was bacteria, while the least common was Archaea. The identification of specific taxa for each read based on their matches was analyzed and summarized from the broad phylum level down to the detailed species level.
Gut microbiome of the atopic group on day 0 versus day 60
The alpha diversity of gut microbiome identified by the Shannon index and Simpson index of the atopic pet group at day 0 (group 2a) was 3.2 and 0.9, while for the recovered pet at day 60 (group 2b), it was 1.4 and 0.6, respectively (Figure 1). The degree of dissimilarity between the atopic group of pets and the recovered group is 0.6.
Alfa rarefaction curves of annotated species richness (the total number of distinct species annotation [y-axis] as a function of the number of sequences sampled [x-axis]) in fecal samples collected on day 0 from 24 client-owned dogs with atopic dermatitis and a ≥ 1-year regular diet protein of poultry meat and/or egg (group 2a; red) versus fecal samples collected from the same dogs after 60 days of being fed a meat and egg elimination diet (group 2b; blue) in a study conducted between July and December 2023 to evaluate potential clinical recovery from canine atopic dermatitis through dietary management and secondary alteration of gut microbiota.
Citation: American Journal of Veterinary Research 86, 5; 10.2460/ajvr.24.09.0274
Complete taxonomical analyses of the gut microbiome of groups 2a and 2b were done through Sankey charts (Figure 2). At the phylum level, although there was no significant difference, the RA of Firmicutes was less in atopic pets (RA = 56.5) than in the recovered pet group (RA = 99.7). A higher elevation of the phylum Fusobacteria (RA = 0.6) and genus Fusobacterium (RA = 0.6) was reflected in the atopic group of pets than in the clinically recovered pets after dietary intervention.
Sankey plots of taxa with relative abundance and connections for gut microbiota identified for the dogs described on days 0 (A; group 2a; n = 24) and 60 (B; group 2b; 24) and for 48 healthy dogs (controls) that had a ≥ 1-year regular diet and had been on poultry meat- and/or egg-based (C; group 1a; 24) or vegetable-based (D; group 1b; 24) diets. The width of each plot component is proportional to the relative abundance of the given taxonomic findings at each taxonomic rank. Each number represents the taxa abundance. D = Domain. P = Phylum. C = Class. O = Order. F = Family. G = Genus. S = Species.
Citation: American Journal of Veterinary Research 86, 5; 10.2460/ajvr.24.09.0274
The top 20 bacterial species are provided in 2 situations (Table 1), and significant variations based on the percentage abundance of the top 15 species (with RA ≥ 0.1) between groups 2a and 2b are shown by a heatmap (Figure 3). The reduced RA of genus Lactobacillus in atopic pets (RA = 31.6) was found to be improved in recovered pets (RA = 60.8) at day 60. Elevated Streptococcus genus (RA = 47.6) was seen in atopic pets, while a significant reduction was observed in recovered pets (RA = 38.3).
Relative abundance (RA) of the 20 most commonly identified bacteria species in fecal samples collected on day 0 from 24 client-owned dogs with atopic dermatitis and a ≥ 1-year regular diet protein of poultry meat or egg (group 2a; n = 24), fecal samples collected from the same dogs considered recovered after 60 days of being fed a meat and egg elimination diet (group 2b; 24), control dogs on a meat- or egg-based diet (group 1a; 24), or control dogs on a vegetable-based diet (group 1b; 24) in a study conducted from July to December 2023 to evaluate potential clinical recovery from canine atopic dermatitis through dietary management and secondary alteration of gut microbiota.
| Serial No. | Atopic dogs at day 0 (group 2a) | RA (%) | Recovered dogs at day 60 (group 2b) | RA (%) | Control group with meat-based diet (group 1a) | RA (%) | Control group with vegetable diet (group 1b) | RA (%) |
|---|---|---|---|---|---|---|---|---|
| 1 | Streptococcus pasteurianus | 42.9 | Lactobacillus acidophilus | 57.5 | Lactobacillus mucosae | 21.8 | Lactobacillus acidophilus | 63.5 |
| 2 | Blautia hansenii | 8.5 | Lactobacillus mucosae | 18.3 | Blautia hansenii | 17.4 | Lactobacillus salivarius | 6.1 |
| 3 | Lactobacillus acidophilus | 6.4 | Streptococcus lutetiensis | 12.7 | Lactobacillus amylovorus | 14.2 | Lactobacillus mucosae | 6 |
| 4 | Blautia sp YL58 | 5.1 | Lactobacillus reuteri | 5 | Streptococcus pasteurianus | 11.1 | Streptococcus lutetiensis | 5.9 |
| 5 | Lactobacillus reuteri | 3.3 | Lactobacillus salivarius | 1.6 | Blautia sp YL58 | 6.7 | Streptococcus gallolyticus | 3 |
| 6 | Collinsella aerofaciens | 2.9 | Lactobacillus johnsonii | 1.3 | Lactobacillus acidophilus | 3.1 | Lactobacillus reuteri | 3 |
| 7 | Escherichia coli | 2.5 | Streptococcus infantarius | 0.4 | Mordavella sp Marseille-P3756 | 2.2 | Bifidobacterium kashiwanohense | 1.5 |
| 8 | Anaerostipes hadrus | 1.9 | Lactobacillus helveticus | 0.2 | Megamonas hypermegale | 2.1 | Bifidobacterium catenulatum | 1.5 |
| 9 | Mordavella sp Marseille-P3756 | 1.8 | Lactobacillus gallinarum | 0.2 | Anaerostipes hadrus | 1.9 | Bifidobacterium longum | 1.2 |
| 10 | Lactobacillus salivarius | 1.4 | Turicibacter sp H121 | 0.2 | Paeniclostridium sordellii | 1.6 | Lactobacillus johnsonii | 0.8 |
| 11 | Streptococcus thermophilus | 1.4 | Lactobacillus acetotolerans | 0.1 | Clostridioides difficile | 1.1 | Clostridium chauvoei | 0.6 |
| 12 | Bifidobacterium catenulatum | 1.3 | Streptococcus agalactiae | 0.1 | Clostridium cellulovorans | 1.1 | Bifidobacterium breve | 0.4 |
| 13 | Paeniclostridium sordellii | 1.1 | Streptococcus gordonii | 0.1 | Lachnoclostridium phocaeense | 0.9 | Turicibacter sp H121 | 0.4 |
| 14 | Veillonella parvula | 1 | Clostridium perfringens | 0.1 | Clostridium botulinum | 0.9 | Lactobacillus fermentum | 0.3 |
| 15 | Lachnoclostridium phocaeense | 0.8 | Lactobacillus jensenii | 0.1 | Clostridium chauvoei | 0.8 | Lactobacillus delbrueckii | 0.3 |
| 16 | Lactobacillus agilis | 0.8 | Lactobacillus amylolyticus | 0.1 | Collinsella aerofaciens | 0.8 | Lactococcus lactis | 0.2 |
| 17 | Streptococcus lutetiensis | 0.8 | Lactobacillus fermentum | 0.09 | Bifidobacterium adolescentis | 0.8 | Escherichia coli | 0.2 |
| 18 | Clostridioides difficile | 0.8 | Lactobacillus amylovorus | 0.08 | Clostridium tyrobutyricum | 0.7 | Bifidobacterium pseudocatenulatum | 0.2 |
| 19 | Bifidobacterium kashiwanohense | 0.8 | Bifidobacterium longum | 0.07 | [Clostridium] saccharolyticum | 0.7 | Clostridium perfringens | 0.2 |
| 20 | Bifidobacterium longum | 0.8 | Streptococcus dysgalactiae group | 0.06 | Clostridium perfringens | 0.6 | Clostridium cellulovorans | 0.1 |
Heat maps for the most abundant bacterial species identified in fecal samples from the dogs as described (Table 1; Figure 2), with comparisons between atopic dogs on day 0 (group 2a) versus day 60 (group 2b; A), group 2a versus control dogs on a meat- and/or egg-based diet (group 1a; B), and group 1a versus control dogs on a vegetable-based diet (group 1b; C). For each heat map, the brackets and lines represent binary tree divisions or cuts. The column and the number toward the right represent log2 scale values of the percentage abundance of the species represented by the various colors of the heat map.
Citation: American Journal of Veterinary Research 86, 5; 10.2460/ajvr.24.09.0274
Extreme reduction of beneficial bacterial species like Lactobacillus acidophilus (RA = 6.4) was observed in the atopic pet at day 0, while the elevation of the same was seen in the recovered group (RA = 57.5). The RA of Escherichia coli (RA = 2.5) and Clostridioides difficile (RA = 0.82) on day 0 in atopic pets was higher, while on day 60, the recovered pets showed a significant reduction in E coli (RA = 0.25) and C difficile (RA = 0.02). The RA of Bifidobacterium catenulatum of atopic pets was 1.3, and for recovered pets, it was 0.01. The RA of Streptococcus pasteurianus was 42.9, which was reduced in recovered pets on day 60 of the restricted diet. A visible recovery of the pets in terms of pruritus and skin coat was observed on day 60.
Gut microbiome between atopic pet and healthy control pets with nonvegetarian diet
As all the atopic pets were on a meat/egg-based diet, it was only compared with the control group following meat/egg-based food habits. The alpha diversity as the Shannon index and Simpson index of the control group with nonvegetarian diet (group 1a) was 2.7 and 0.8, respectively, which was lower than atopic pets (group 2a). The degree of dissimilarity between the groups is 0.4. The RA of Firmicutes in group 1a was 95.1, which was higher than group 2a, but Fusobacteria RA in group 1a was observed to be the same as group 2a. Taxonomical analysis of group 1a is shown in the Sankey chart (Figure 2), the top 20 bacteria species between 2 groups is provided (Table 1), and species level diversity based on percentage abundance of the top 20 species (with RA ≥ 0.1) between groups 2a and 1a is shown by the heatmap (Figure 3).
Elevation of genus Lactobacillus (RA = 31.0), although a reduced L acidophilus (RA = 3.1), was observed in the control group with the meat/egg diet. A higher reduction in Streptococcus genus (RA = 12.2) as well as S pasteurianus (RA = 11.1) was observed in the control group with the meat/egg diet than in the atopic pet group. The RA of E coli in the control group with the meat/egg diet was 0.3. Clostridial species were observed in abundance like C difficile (RA = 1.1), Clostridioides botulinum (RA = 0.9), and Clostridioides chauvoei (RA = 0.9). The RA of B catenulatum of the control group with the meat/egg diet was 0.003, much lower than the atopic pet group on day 0.
Gut microbiome in healthy control pets with nonvegetarian diet and vegetable diet
The gut microbiome of both control groups were compared based on their dietary habit. Significant variations in the result showed the influence of diet on shaping one's core microbiome. The alpha diversity, Shannon index, and Simpson index of the control group with vegetable diet (group 1b) were 1.7 and 0.5, respectively, which was lower than the control group with the meat/egg diet (group 1a), and dissimilarity between these 2 groups was 0.5. Taxonomical analysis of the control group with the vegetable diet is shown via Sankey chart (Figure 2), the top 20 different bacteria species are provided (Table 1), and the graphical percentage abundance of the top 20 bacteria species (with RA ≥ 0.1) is shown in the heatmap (Figure 3).
Although there was not much difference in the RA of Firmicute between 2 groups (RA = 93.1), the RA of Fusobacteria in control group 1b (RA = 0.11) was much lower than in group 1a. A higher elevation in the genus Lactobacillus (RA = 67.4), as well as species the L acidophilus (RA = 63.5), was observed in healthy pets in group 1b than 1a. A higher reduction in S pasteurianus (RA = 0.1) was observed in control group 1b than in control group 1a. The RA of E coli in the control group with the vegetable diet (group 1b) was 0.2, which was higher than the control group with the meat/egg diet, but the abundance of C difficile (RA = 0.02) was much lower than the control group with the meat/egg diet. The RA of B catenulatum was 1.5, quite higher than the control group with the meat/egg diet.
Discussion
Atopic dogs need multimodal strategies to maintain good health.30 Food management could be one of the effective approaches to address this issue. The dietary patterns of companion animals have been significantly altered almost parallel to the pet owner's lifestyle. Pets, primarily considered carnivorous, become omnivorous with consumption of cooked and commercial food Further there is evidence to suggest that dogs encountering adverse food reactions and food allergy might have an increased vulnerability to developing atopic dermatitis.31,32
A survey was taken including 2,536 dogs, grouped according to their dietary preferences into 3 groups of raw meat, conventional meat, and vegan diet where skin/coat-related issues were found to be 8%, 7%, and 6%, respectively. It signifies the positive correlation of a meat diet with skin diseases.33 Based on this, the study hypothesized that a diet consisting of meat and/or egg could act as a source of allergens influencing the gut microbiome that ultimately could trigger the manifestation of prolonged skin lesions.
In the present study, a comparison of gut microbiome results of atopic dogs before and after diet management showed clear dysbiosis. The B catenulatum was reflected in a higher proportion in atopic pets than in the recovered pets at 60 days after dietary intervention. The RA of B catenulatum of atopic pets was 1.3, while in the recovered pets, it was 0.01. The outcome of increasing this microbe in allergic developments was observed in a study, where allergic infants from 1 to 6 months of age were studied and found to have higher B catenulatum than their healthy controls in rural Japan.34 However, there are reports of no alteration in B catenulatum in atopic pets as compared to healthy ones.6 However, this Bifidobacterium along with Lactobacillus was considered a protective microbiome in humans, and its reduction was also evident in human patients with atopic dermatitis.35 These variations may be due to the fact that the count and RA of B catenulatum based on the disease state as individuals with severe atopic dermatitis showed reduced counts but higher in those experiencing mild atopic symptoms.36
Escherichia coli were abundant in the pets with atopy. There was a decrease in E coli population 60 days after dietary alteration, which implies a potential connection with atopy. A similar elevation of E coli was observed in a group of 21 infants with atopic eczema and a family history of atopic dermatitis and directly correlated with an increase in the concentration of serum immunoglobulin E for the onset of eczema and later the abundance decreases after supplementation of probiotics (Bifidobacterium strain).37 Elevated level of E coli was also found in atopic human beings.35,38 Dietary intervention as a possible and better approach for patients with atopy via gut microbiome modification was suggested.38 In this study, the RA of E coli (RA = 2.5) was significantly higher in the atopic group than in the clinically recovered group (RA = 0.25), which suggests the impact of dietary intervention on the gut microbiome.
The RA of C difficile was 0.82, while in recovered pets, it was 0.02. This elevation at day 0 result was in line with a KOALA cohort study where the infants with colonized C difficile were at higher risk of having all atopic outcomes39 and elevation in Clostridium clusters was observed in 6-month-old atopic infants.17 The clinical manifestations could be attributed to the synergistic action of C difficile and E coli as these 2 microbes were found to be elevated in pets affected with atopic dermatitis.16 These 2 microbes were absent in the top 18 microbes of dogs after diet modification.
The RA of Firmicutes was more in the atopic group at day 60 than the samples collected before diet management, ie, day 0. Such microbes break down complex carbohydrates into short-chained fatty acids that ultimately reduce inflammation.5 Similarly, Lactobacillus sp became more after dietary modifications. Higher concentrations of L acidophilus and Lactobacillus mucosae could be a reason for healthy skin.36 It could be inferred that an increased population of C difficile and E coli along with a decrease in beneficial bacteria like Lactobacillus sp may be the cause of clinical manifestation of the disease in atopic pets.39 In this study, the Fusobacterium sp population was observed in higher quantity in atopic pets than in recovered pets. However, a contradiction to this result was also observed.6,16 The phylum Fusobacteria may serve as a potential biomarker to indicate the risk of canine atopic dermatitis.16 Further study is needed to link its potential association with the atopy.
A positive correlation between the Streptococcus genus and atopic dermatitis in infants was noticed.40 Streptococcus pasteurianus was identified as the primary bacterium in the atopic group. The present literature is silent to assess its role. Further studies could surface more clarity.
There were visible clinical differences in all 24 atopic pets, like reduction in pruritus, erythema, and signing of coat condition and better sleep quality. That suggests enhancing the gut microbiota may offer a therapeutic promise in ameliorating atopic conditions affecting the skin.41,42 Based on the results of gut microbiome analysis as well as clinical recovery, we suggest the exclusion of meat and egg, the known allergenic diet, from the regular diet of dogs with atopy to alleviate the sufferings. Dietary supplements with probiotics and essential fatty acids could be of additional benefit.33,43 A recent study44 suggested a weak association of diet with atopy, but other studies45–47 along with our study found changes in diet were related to atopic symptoms. However, complete or partial replacement of meat and eggs in the diet chart and/or inclusion of commercial food need further study.
The etiology of canine atopic dermatitis is multifactorial including factors linked with diet and proceeds after adverse food reactions or food allergies.15,31,46 For a deeper understanding, we have correlated the clinical improvements with modified gut microbiomes via a poultry meat/egg exclusion diet. Therefore, dietary intervention will be helpful in preventing the emergence and spread of drug-resistant bacteria10 by reducing unnecessary use of antibiotics and antifungals that are unlikely in advanced or complicated atopy situations.
Acknowledgments
The authors are thankful to all pet owners and veterinary health service providers for their cooperation and support extended during the period of study.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
The authors have nothing to disclose.
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