Clinical efficacy of spray‐based heat‐treated lactobacilli in canine atopic dermatitis: a preliminary, open‐label, uncontrolled study

Background – Canine atopic dermatitis (cAD) is a common inflammatory and pruritic skin disease, with various treatment options. The use of topical products containing natural ingredients has proven increasingly popular. Objective – To evaluate the effects of a spray solution containing heat‐killed Lactobacillus rhamnosus and L. reuteri, on the clinical signs and cutaneous microbiota of atopic dogs. Conclusions and clinical importance – There was a significant and rapid decrease in the clinical signs associated with cAD after use of the spray. Future larger, randomized, controlled studies are needed to confirm these results and to assess the effects on the cutaneous immunity and microflora of atopic dogs.

Abstract

Background

Canine atopic dermatitis (cAD) is a common inflammatory and pruritic skin disease, with various treatment options. The use of topical products containing natural ingredients has proven increasingly popular.

Objective

To evaluate the effects of a spray solution containing heat‐killed Lactobacillus rhamnosus and L. reuteri, on the clinical signs and cutaneous microbiota of atopic dogs.

Animals

Ten privately owned, mildly affected, nonseasonally atopic dogs.

Methods and materials

The spray was applied to the ventrum every 24 h for 28 days. Clinical scores, skin barrier function and owner assessment were evaluated on day (D)0, D14, D28 and D42. The cutaneous microbiota was analysed on D0 and D28.

Results

A reduction in the total clinical score was seen at each time point (D14, P = 0.03; D28, P = 0.04; D42, P = 0.001). A reduction in the regional clinical scores was seen after D28 (P = 0.01) and D42 (P = 0.003). A significant reduction in the pruritus score was seen on D42 (P = 0.01). A lower hydration value was seen on D28 (P = 0.02) and D42 (P = 0.02) on the pinnae. A good‐to‐excellent response and an easy‐to‐use administration was reported by owners. There were no significant changes in the cutaneous microbiota after 28 days.

Conclusions and clinical importance

There was a significant and rapid decrease in the clinical signs associated with cAD after use of the spray. Future larger, randomized, controlled studies are needed to confirm these results and to assess the effects on the cutaneous immunity and microflora of atopic dogs.

Introduction

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases in dogs. Genetic and immune system alterations along with defects of the skin barrier and modifications of the cutaneous microflora have been reported in atopic dogs. ¹ , ² , ³ , ⁴ Multiple treatment options are available; ⁵ , ⁶ there is a growing interest in the use of alternative products based on so‐called natural ingredients. ⁷

In human AD there are reports of the potential positive effects of topical applications of heat‐killed beneficial bacteria. ⁸ , ⁹ , ¹⁰ In particular, the use of topical products containing heat‐killed bacteria (Vitreoscilla filiformis, Lactobacillus johnsonii and L. reuteri) has been reported to show a significant decrease in clinical signs and pruritus in both atopic people and mice. ⁸ , ⁹ , ¹⁰ , ¹¹ There was an overall reduction in the Staphylococcus aureus population reported in one study. ¹⁰ Some studies report that the use of rapidly killed beneficial bacteria (e.g. lactobacilli) can ameliorate the clinical signs of human AD and positively stimulate the local immunity, to the point of influencing the local microbiome. ¹² , ¹³ , ¹⁴

The objective of this preliminary, noncontrolled, prospective clinical trial was to assess the effects of a spray version of a veterinary product containing heat‐killed lactobacilli (L. rhamnosus and L. reuteri) marketed as an adjuvant therapy for allergic dogs (LinkSkin spray, DRN Inc.; Cremona, Italy) on the clinical signs of canine (c)AD (primary outcome), the skin barrier and the cutaneous microbiota (secondary outcomes).

Methods and materials

Animal welfare documentation

All dogs were recruited at the authors’ institution with previous approval from the University of Florida and a signed owner consent form.

Canine population

Naturally affected, privately owned dogs with mild‐to‐moderate [Canine Atopic Dermatitis Extent and Severity Index, 4th iteration CADESI‐04 with scores of 10 to 59] ¹⁵ nonseasonal AD were enrolled in the study. Diagnosis of cAD was based on published guidelines. ¹⁶ All dogs received monthly flea prevention.

Exclusion criteria

Dogs with clinical evidence or a history of malignant neoplasia, metabolic/endocrine, parasite or other allergic skin conditions (food/contact) were excluded. Dogs with superficial infections, diagnosed based on published guidelines, ¹⁷ were excluded. Owners withdrew drugs for their dog using the following periods: oral and topical antibiotics and antifungal agents (four weeks), corticosteroids (eight weeks for depo‐injectable, four weeks for oral, three weeks for topical), antihistamines (two weeks), ciclosporin (four weeks), lokivetmab (eight weeks) and oclacitinib (one week). Dogs receiving allergen‐specific immunotherapy for less than one year before enrolment were excluded from the study.

Intervention

The treatment consisted of a water‐based spray solution containing a mixture of lactobacilli (L. reuteri and L. rhamnosus) together with the culture media containing metabolites produced by the bacteria, tamarind extract and polyphenols. This solution had undergone Tyndallization, a process used to sterilize food from heat‐resistant endospores and bacteria, based on the use of heat to reach boiling point (~100°C) for 15 min for three consecutive days. This methodology, although designed to allow endospores to germinate and be killed, allows minimal degradation of the bacteria walls with minimal alteration of the immunological properties of the bacteria. ¹² , ¹³ , ¹⁴

Other topical and/or systemic treatments including antimicrobial, humectant or antipruritic, were not allowed during the study. Owners sprayed the glabrous areas (axillae, ventral thorax, inguinal area and medial thighs) every 24 h for 28 days, irrespective of the distribution of the dog’s skin disease. Licking and subsequent alteration of the cutaneous microbiota was prevented by distracting the dog and, when unsupervised, placing a T‐shirt on the dog, throughout the 28 days of the study. The use of the T‐shirt was preferred to the use of an E‐collar for ethical reasons and tolerability. Each dog was clinically evaluated by the same investigator on Day (D)0, D14, D28 and D42.

Clinical assessment

The primary outcome of this study was to assess the clinical efficacy of the spray for cAD. On D0, D14, D28 and D42, the severity of clinical signs was assessed using the CADESI‐04 score system recording 20 body sites that are commonly affected in AD (total CADESI‐04). These areas were scored on a scale between 0 and 3 (0,none;1, mild; (2, moderate; 3, severe) with a maximum score of 180. ¹⁵ A separate CADESI‐04 score (regional) was calculated adding the score of only the treated areas (axillae, ventral thorax, inguinal area and medial thighs). At each visit, the pruritus was scored by the owners using a pruritus Visual Analog Scale (pVAS). ¹⁸ , ¹⁹ The percentage of dogs with a CADESI‐04 N (<10) and with a pVAS10‐N (<2) was calculated. ²⁰ An owner global assessment of treatment efficacy (OGATE) score was recorded using a scale between 0 and 4 (0, no response; 1, poor; 2, fair; 3, good; 4, excellent). The percentage of dogs with an OGATE‐G2E (>2) was calculated in a similar way to the CADESI‐04 N. ²⁰ An “ease to administer” score by the owners was recorded, based on asking the owners if the product was not easy, easy, or very easy to use. ²¹ Parameters considered by the owners included ease‐of‐spray, skin residues, stickiness and time of absorption.

Skin barrier function evaluation

As secondary outcome, the skin barrier function was assessed in all dogs on D0, D14, D28 and D42; assessing skin hydration via corneometry and pH. These parameters were analysed on the inguinal, axillary, pedal and aural surfaces using a corneometer and expressed as microsiemens values [µS] (Corneometer CM825, Courage + Khazaka electronic GmbH; Cologne, Germany) and a pH meter (Skin‐pH‐meter PH 905, Courage + Khazaka electronic GmbH) as described previously. ²² Before each evaluation, if present, the T‐shirt was removed and dogs were allowed to acclimatise to the testing room for 30 min. Temperature and humidity settings were maintained at 25 ± 5°C and 50 ± 10% for the entire duration of the study and as recommended by the manufacturer to optimize the performance of the instruments.

Skin microbiota collection

Another secondary outcome was the assessment of the cutaneous microbiota before (D0) and after treatment (D28). Two skin swabs, one from the groin and one from both axillae, were collected using swab applicators (Isohelix DNA Buccal Swabs, Cell Projects Ltd; Harrietsham, Kent, UK) before the acclimatisation period. Each swab applicator was rubbed on the skin 20 times (10 strokes per swab side) per anatomical site, as reported previously. ²³ In addition, an extra swab agitated for 20 s in the air was submitted as a negative control, to control for environmental contaminants. All samples were stored immediately at –80°C until processed.

Library construction of microbial samples

For each extraction, 5 ng gDNA was used for 16S library construction using Quick‐16S NGS Library Prep Kit (Zymo Research Corp.; Irvine, CA, US). PCR amplification was performed using V3–V4 primers (95°C for 10 min, 20 cycles of (95°C for 30 s, 55°C for 30 s and 72°C for 3 min). The PCR products were quantified by real‐time PCR with a Bio‐Rad CFX Real‐Time PCR Detection System (Bio‐Rad Laboratories; Hercules, CA, USA) and cleaned with the Zymo enzymatic cleanup kit. Each library was barcoded with five cycles of PCR amplification and cleaned with the Select‐a‐Size MagBeads (Zymo Research Corp.). All 48 libraries were pooled together for one MiSeq 2 x 250 cycles run.

Statistical analyses

Clinical and skin barrier assessment. Data were analysed using the intention‐to‐treat analysis (ITT) with the last value carried forward. Data collection was tested for normality using the Shapiro–Wilk test (alpha level = 0.05). Then repeated measures ANOVA (skin hydration and pH) or Friedman’s test (CADESI‐04 and pVAS) followed by Dunnett’s or Dunn’s multiple comparison test were performed to evaluate the behaviour of each variable over time. A P‐value of <0.05 was considered statistically significant. All statistical analyses were performed using prism 6.09 statistical software (GraphPad Software Inc.; La Jolla, CA, USA).

Data analysis of microbial samples. Reads of 16S RNA (V3–V4 region) acquired from the Illumina MiSeq system were cleaned up with the cutadapt program (v2.8; https://cutadapt.readthedocs.io/en/stable/) ²⁴ to trim off sequencing adaptors, low‐quality bases and potential errors introduced during sequencing. Only sequences with length ≥60 bp were included in the analyses. The cleaned paired‐end reads were merged and analysed using Quantitative Insight Into Microbial Ecology (QIIME, v1.9.1; http://qiime.org/). ²⁵

The merged 16S rRNA sequences were grouped into operational taxonomic units (OTUs) (minimum OTU cluster size = 2; OTU similarity = 0.97) using both the open reference and closed reference OTU picking strategy and classified using the SILVA 16S/18S reference database. ²⁶ The potential chimeric sequences and OTUs were detected and filtered at 97% cluster identity by Vsearch (v2.13.4; https://manpages.debian.org/stretch/vsearch/vsearch.1). ²⁷ Taxonomies were summarized at multiple taxonomic levels (L2–L6) as the OTU tables with the samtools and R‐based scripts developed at the Interdisciplinary Center for Biotechnology Research, University of Florida. The sequencing coverage was evaluated by rarefaction analysis and the estimated species richness and diversity indices were calculated with the alpha‐diversity analysis. The differences of the microbial compositions between different samples were compared with the beta‐diversity analysis, including the principal coordinate analysis (PCoA) based on the Bray–Curtis distance model in R/phyloseq. In addition, the quantitative comparison of taxa at different taxonomic levels between the different groups was performed based on the raw OTU tables using the DEB application. ²⁸

Results

Dogs

Ten dogs (five males, five females) were enrolled for this study. The average age was 5.3 ± 3.6 years. The average weight was 28.2 ± 24 kg. Breeds included great Dane (2), mixed‐breed dogs (3), pit bull terrier (2), and one each of German shepherd dog, Jack Russell terrier and French bulldog. One mixed‐breed dog was withdrawn from the study after D28 owing to relocation of the owner.

Clinical assessment

Compared to D0, a significant reduction in the total CADESI‐04 was seen at each time point (P = 0.028; P = 0.036; P = 0.001, respectively) (Figure S1). In particular, on D0, a median (range) total CADESI‐04 score of 16.5 (13–32) was recorded. This score decreased to 8.5 (3–25) on D14, 9.5 (3–18) on D28 and 6.5 (0–14) on D42. A CADESI‐04 N (<10) was achieved in 50% (five of 10), 50% and 90% (nine of 10) of dogs after D14, D28 and D42, respectively. Likewise, compared to D0, a reduction of the regional CADESI‐04 was seen after 28 (P = 0.013) and 42 (P = 0.003) days (Figure 1). In particular, on D0, a median (range) regional CADESI‐04 score of 6 (0–15) was recorded. This score decreased to 3 (0–6) on D14, 3 (0–4) on D28 and 0.5 (0–3) on D42.

Figure 1.

Average of the individual skin hydration and cutaneous pH values.

Hydration values are expressed in microsiemens (µS) as conductance units. Groups were compared using repeated measurements ANOVA with Dunnett's multiple comparison tests. Bars, SE.

Compared to D0, a significant reduction in the pVAS was seen on D42 (P = 0.013) (Figure S2). On D0, the pVAS was 4.9 ± 1.9, whereas on D14 it was 5.2 ± 2.2 and decreased to 4.1 ± 2.1 on D28 and to 2.5 ± 1.1 on D42. A pVAS10‐N (<2) was achieved in 10% (one of 10), 30% (three of 10) and 30% of dogs on D14, D28 and D42, respectively.

On D14, OGATEs of a fair response by seven (70%) owners and a good response by two (20%) owners were recorded (Figure S3). On D28, there was an OGATE equivalent to a fair response by three (30%) owners and a good response by six (60%) owners (Figure S3). On D42, OGATEs of a fair response by one (10%) owner and a good response by six (60%) owners and an excellent response by two (20%) owners were recorded (Figure S3). An OGATE of a poor response was reported by one (10%) owner for the entire duration of the study. An OGATE‐G2E (>2) was achieved in (two) 20%, (six) 60% and 60% of dogs on D14, D28 and D42, respectively.

On D14, two (20%) owners reported the product to be easy to use, whereas eight (80%) owners judged the product very easy to use (Figure S3). Likewise, on D28 and D42, one (10%) owner reported the product to be easy to use, whereas nine (90%) owners judged the product very easy to use (Figure S3).

During the study, major adverse effects were not observed in any dog. Only one dog had a noninfective, papular eruption after 14 days in the study. However, the eruption was not severe enough for the dog to be dropped from the study. The eruption went away after a series of topical shampoos containing oatmeal (not allowed during the study).

Skin barrier function evaluation

A significant change in the skin hydration and pH of the inguinal, axillary and interdigital areas was not seen at any time point (Figure 1). A significant reduction in the skin hydration, not the pH, was seen on the pinnae on D28 (P = 0.02) and D42 (P = 0.047) when compared to D0 (Figure 1).

Skin microbiota evaluation

Microbiome gDNA extractions were processed by using ZymoBIOMICS DNA kits following the user guide. Bacterial and archaeal microbial communities of 40 canine skin samples were characterized by sequencing the V3–V4 regions of PCR‐amplified 16S rRNA genes. An average of 160,137 paired‐end reads for each sample and approximately 159,491 cleaned merged reads (average 440 bp) were obtained for metagenomics analysis. Rarefaction analysis and alpha diversity measures showed that the bacterial communities were sufficiently sampled and that further sequencing would have been unlikely to significantly increase the observed microbial diversity detected.

Altogether, an average of 3,322 unique OTUs, ranging from 963 to 10,850, were obtained from each sample corresponding to about 14 phyla, 28 classes, 57 orders, 95 families and 164 genera of prokaryotes (Figures S4–S12). All microbial communities analysed in both groups (before and after treatment) were dominated by a few microbial genera, including Staphylococcus (Firmicutes), Conchiformibius (Proteobacteria) and Porphyromonas (Bacteroidetes) (Figures S4–S12). The microbial community profiles of the different samples based on the OTU tables and the Bray–Curtis distance model showed some differences between the two groups (Figures S4–S12). However, these differences did not reach a statistically significant level (Figure 2).

Figure 2.

The distribution of operational taxonomic units at different taxonomic levels in the (a) axillae and (b) inguinal samples.

Discussion

To the best of the authors’ knowledge, this is the first study to report a potentially beneficial effect from the topical use of heat‐treated bacteria in the management of cAD. A significant reduction in the clinical score and pruritus was observed for two weeks after the spray was stopped, showing a residual beneficial effect. In addition, most of the owners deemed the product to be easy to very‐easy to use and effectively to manage cAD. An overall improvement of the clinical signs was seen in areas not directly treated by the product (e.g. feet and head). Even so, no changes in the cutaneous microbiota were seen after 28 days of daily application of the compound.

In the present study, the daily application of the spray significantly and relatively rapidly decreased the clinical signs of dogs with mild‐to‐moderate AD without the use of any other therapeutic intervention. The only allowed “intervention” was the use of a T‐shirt when the dog was unsupervised. However, this was not considered sufficient to influence the results because the T‐shirt was not constantly worn and because the dogs were still able to scratch and rub. The amelioration of the disease was not limited only to the treated areas, but also to the rest of the body, suggesting a beneficial effect of the product on the systemic, not only local, immune response. These results are in agreement with previous studies showing an amelioration of the clinical signs of AD in people after oral and topical administration of heat‐killed lactobacilli. ¹⁰ , ²⁹ , ³⁰ The present study also is in agreement with a previous human clinical trial that used a topical product containing Vetrosciella filiformis extract in atopic patients. ⁸ In that study, there was a a significant improvement reported in clinical signs, pruritus and quality of sleep, after only 28 days of daily application of heat‐killed bacteria. ⁸ Altogether, these results suggest that a short‐term daily application of heat‐killed beneficial bacteria can be used as adjuvant therapy in AD.

Given that no significant changes in the microbiota were detected, it is possible to hypothesize that heat‐treated lactobacilli do not directly interfere with the cutaneous microbiota. Rather, they may have a profound effect on the local and systemic immune response in atopic dogs. Several studies have shown the beneficial direct effect of heat‐killed and treated (by Tyndallization) bacteria on the cutaneous and oral immune response without the potential drawbacks of administering live micro‐organisms (e.g. overgrowth or passage of multidrug‐resistant genes). ¹² , ¹³ , ¹⁴

This study had several limitations including the small number of dogs enrolled, wide variety of breeds and ages, as well as the short treatment period. A small number of dogs was chosen because the study was designed to collect preliminary data for future studies. The choice was arbitrary, although based on a hypothesized over‐time success rate of 75% of the product against a placebo effect of 30%. Based on these criteria a minimum number of eight dogs would have been necessary to see a significant clinical difference, and consequently 10 dogs were selected. The wide variation in breed and ages could explain the lack of significant changes in the cutaneous microbiota after treatment; furthermore, it is well‐recognised that the cutaneous microbiota can be highly variable between individuals. Thus, a reduction in interindividual variability would have been more beneficial for the microbiota evaluation. Furthermore, only two microbiological samples were taken (D0 and D28) during the study; it is unknown if microbiota changes would have been evident by D42 when a more significant clinical amelioration was seen. In addition, along with the metagenomic approach, it could have been interesting to simultaneously culture the skin microflora to evaluate for changes in the bacteria population.

Given the open‐label nature of the study and the small number of dogs enrolled, a larger double‐blinded, placebo‐controlled, randomized clinical trial is desirable to confirm the clinical benefits showed in this study and to better evaluate the effects of the LinkSkin spray on the cutaneous microbiota.

Supporting information

Figure S1. Median values for total and regional CADESI‐04 scores and pVAS scores.

Figure S2 . Median values for pVAS scores

Figure S3 . OGATE and ease of administration of lactobacilli‐based spray

Figure S4. The distribution of the top 20 operational taxonomic units at genus level in the axillae samples

Figure S5. PCoA at the genus level in the axillae samples based on Bray–Curtis dissimilarity matrices

Figure S6 . The distribution of the top 20 OTUs at the family level in the axillae samples

Figure S7. PCoA at family level in the axillae samples based on Bray–Curtis dissimilarity matrices

Figure S8 . The distribution of the top 20 OTUs at the genus level in the inguinal samples

Figure S9. PCoA at the genus level in the inguinal samples based on Bray–Curtis dissimilarity matrices

Figure S10. The distribution of the top 20 OTUs at the family level in the inguinal samples

Figure S11 . Figure S11 PCoA at the family level in the inguinal samples based on Bray–Curtis dissimilarity matrices