Assessment of the nasal microbiota in dogs with fungal rhinitis before and after cure and in dogs with chronic idiopathic rhinitis.

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Emilie Vangrinsven, Aline Fastrès, Bernard Taminiau, Frédéric Billen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1703144/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Apr, 2023 Read the published version in BMC Microbiology → Version 1 posted 12 You are reading this latest preprint version Abstract Background Pathogenesis of canine fungal rhinitis is still not fully understood. Treatment remains challenging, after cure intranasal remodeling may be associated with clinical disease mimicking chronic idiopathic rhinitis and recurrence can occur. Alterations of the nasal microbiota have been demonstrated in dogs with chronic idiopathic rhinitis and nasal neoplasia, although whether they play a role in the pathogenesis or are a consequence of the disease is still unknown. The objectives of the present study were (1) to describe nasal microbiota alterations associated with fungal rhinitis in dogs, compared with chronic idiopathic rhinitis and controls, (2) to characterize the nasal microbiota modifications associated with successful treatment of fungal rhinitis. Forty dogs diagnosed with fungal rhinitis, 14 dogs with chronic idiopathic rhinitis and 29 healthy control dogs were included. Nine of the fungal rhinitis dogs were resampled after successful treatment with enilconazole infusion. Results Only disease status influenced the nasal microbiota variance. The relative abundance of the genus Moraxella was decreased in the fungal rhinitis (5.4 ± 18%) and chronic idiopathic rhinitis (4.6 ± 8.7%) groups compared to controls (51.8 ± 39.7%). Fungal rhinitis and chronic idiopathic rhinitis groups also showed an increased richness and α-diversity at species level compared with controls. Increase in unique families were associated with fungal rhinitis (Staphyloccaceae, Porphyromonadaceae, Enterobacteriaceae and Neisseriaceae) and chronic idiopathic rhinitis (Pasteurellaceae and Lactobacillaceae). In dogs with fungal rhinitis at cure, only 1 dog recovered a high relative abundance of Moraxellaceae. Conclusions Results confirm major alterations of the nasal microbiota in dogs affected with fungal rhinitis and chronic idiopathic rhinitis, consisting mainly in a decrease of Moraxella . Besides, a specific dysbiotic profile further differentiated fungal rhinitis from chronic idiopathic rhinitis. In dogs with fungal rhinitis, whether the NM returns to its pre-infection state or progresses toward chronic idiopathic rhinitis or fungal rhinitis recurrence warrants further investigation. Nasal cavity dog nasal microbiota aspergillosis fungal rhinitis mycotic rhinitis chronic idiopathic rhinitis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background With advances in culture-independent technologies, the role of the upper respiratory tract microbiota in health and disease has become an intense area of research in human medicine 1 – 7 and to a much lesser extend in canine medicine 5,8−12 . Fungal rhinitis secondary to infection with Aspergillus fumigatus is a common cause of nasal disease in dogs while it is uncommon in humans. Why young otherwise healthy dolichocephalic dogs have their nasal cavity and sinus invaded by the fungus is not yet totally understood, despite extensive investigation 13 – 15 . Local dysimmunity is suspected, which associates a host response, consisting in Th1 and inflammatory response 16 , 17 with an in-host adaptive genetic and phenotypic response of the fungus itself, enabling growth of the pathogen and contributing to the suppression of the host immune response 15 , 18 . In human beings, reports over the past five years suggest that the microbiota can modulate the host immune response to invading fungal micro-organisms 19 – 21 . The microbiota is expected to influence immune homeostasis through host-to-microbe and microbe-to-microbe interactions 4,22−24 . It has been shown, by correlating changes in metabolite profiles with microbiota metagenomic composition, that certain bacterial species contribute to host-fungal symbiosis and mucosal homeostasis in humans with Aspergillus -related lung disease 25 . Formation of sinonasal bacterial biofilms of multiple species (such as Staphylococcus aureus , Staphylococcus epidermidis and Pseudomonas aeruginosa ) have been reported to damage epithelia sufficiently for the establishment of fungal biofilms 26 while the release of extracellular molecules by P. aeruginosa has been shown to stimulate the growth of A. fumigatus 27 . It can therefore be suspected that, in the nasal cavities of dogs with fungal rhinitis, the microbiota is able to influence the immunological response to fungi, the clinical fungal disease severity, as well as the response to treatment. Chronic idiopathic rhinitis is a common heterogeneous disease characterized by lymphoplasmacytic to mixed inflammation of the sinonasal cavities without any identifiable cause 28 . In human medicine, chronic rhinosinusitis represents a significant health problem, the causes underlying the onset of the disease are multiple and not well defined 29 . Disruption of the microbiota homeostasis has been described as being the primary driver or at least exacerbating factor for nasal chronic inflammatory diseases such as chronic idiopathic rhinitis and allergic rhinitis 2,6,24,30−33 . In the pathophysiology of chronic idiopathic rhinitis in humans, two other interesting notions are the importance of bacterial biofilms 34 as well as the concept of keystone species maintaining a stable and healthy state by providing resistance to colonization by pathogens 32 . Altogether, this suggests that specific bacterial strains, as well as factors influencing the microbial composition and/or modulating microbial disturbances may be an untapped source of therapeutics to mitigate the severity of upper respiratory tract infections and/or inflammation. However, the role of nasal microbiota (NM) alterations in the pathophysiology of canine chronic nasal diseases has been very little studied since only one single study describes alterations of the NM in dogs with nasal neoplasia or chronic rhinitis 10 . So far, data relative to the NM in dogs with fungal nasal disease are not available. Therefore, whether bacterial dysbiosis exist in fungal rhinitis, and whether it may be one of the factors able to either initiate or entertain the local fungal development, or allow recurrence, is unknown. Likewise, all therapeutic protocols for fungal rhinitis described are based on attempts to eliminate the fungus 13 , 35 , 36 instead of targeting the relationship between the host and the fungus, and a possible approach based on NM modulation has not been considered yet. For all these reasons, further knowledge concerning the alterations of the NM in canine chronic nasal diseases such as fungal rhinitis and chronic idiopathic rhinitis is warranted. Improved understanding of the relationships between the microbiota, host responses and non-native microorganisms would help to develop future therapeutic approaches acting to prevent the invasion of pathogenic microorganisms. A first step is to characterize any specific dysbiosis associated with both chronic idiopathic rhinitis and fungal rhinitis. Therefore, the aim of this study was to describe and compare the NM in dolichocephalic dogs with fungal rhinitis (at diagnosis and cure in a subpopulation of dogs) and in dogs with chronic idiopathic rhinitis versus a control population of healthy dogs. Results Study population Eighty-three client-owned dogs were recruited and divided into 3 groups: 29 in the control group, 40 dogs in the fungal rhinitis (FR) group and 14 in the chronic idiopathic rhinitis (CR) group (Table 1 ). A first batch (7 healthy, 9 FR and 8 CR dogs) was collected and sequenced in 2017 37 , a second batch (22 healthy dogs and 17 dogs with FR) in 2018 and finally a last group was analyzed in 2020 (6 dogs with CR and 14 dogs with FR among which 9 dogs were sampled twice: at diagnosis and at cure). The data of the three sequencing sets were gathered in one table and processed together. Table 1 Characteristics of the groups according to the disease status Control group CR group FR group Number 29 14 40 Age (years) 7 (0.8–11.3)* 9 (1-14.3)* 6.4 (1.2–14.3)* Gender 12 males, 17 females 6 males, 8 females 27 males, 14 females Weight (kg) 30 (14.8–47.8)* 24 (6–36)* 30.2 (3.7–55)* Antibiotic treatment / 1 (topical) 11 (systemic) Antifungal treatment / / 10 *Median (min-max) FR: fungal rhinitis CR: chronic idiopathic rhinitis In the control group, breeds included were Labrador retriever (n = 4), Belgian shepherd (n = 4), Border collie (n = 4), Australian shepherd (n = 4), Beauceron (n = 3), Golden retriever (n = 3), Alaskan malamute (n = 2), German shepherd (n = 1), Doberman (n = 1), Dalmatian (n = 1), White Swiss shepherd (n = 1), and mixed-breed (n = 1). Included dogs had a normal general examination and bloodwork, and were not receiving any treatment within one month before sampling. Breeds included in the FR group were Border collie (n = 5), rottweiler (n = 3), American Staffordshire terrier (n = 3), mixed-breed (n = 3), Labrador (n = 2), Bull Terrier (n = 1), Greater Swiss Mountain dog (n = 1), Golden retriever (n = 11), Cocker spaniel (n = 1), Great Dane (n = 1), Australian shepherd (n = 1), giant poodle (n = 1), Rhodesian ridgeback (n = 1), Beauceron (n = 1), Siberian husky (n = 1), German shepherd (n = 1), Dobermann (n = 1), Jack Russel (n = 1) and Dachshund (n = 1). At the time of sampling, 11 dogs were receiving systemic antimicrobials (Table 1 ), 10 dogs were treated with oral antifungal drugs, 4 with non-steroidal anti-inflammatory drugs and 2 with steroids, within the 2 previous weeks. Breeds included in the CR group were mixed-breed (n = 3), Siberian husky (n = 2), Jack Russel terrier (n = 2), Dalmatian (n = 1), American Staffordshire terrier (n = 1), poodle (n = 1), Bernese mountain dog (n = 1), Barzoï (n = 1), Dachshund (n = 1) and Border collie (n = 1). One dog was treated with topical antimicrobial therapy (thiamphenicol) and saline at the time of sampling (Table 1 ). All the other dogs did not receive anti-inflammatory or antimicrobial treatment for at least two weeks before sampling albeit this was not an exclusion criterion. Nasal microbiota analysis At the finest taxonomic level 4,887 operational taxonomic units (OTUs) were present throughout all samples. The Good’s coverage of all samples was higher than 96% with median 99.3% (96.6%-99.9%) indicating that the sequencing depth was sufficient for reliable analysis of these nasal microbial community samples. The distribution of age, sex and bodyweight according to disease status is reported in Table 1 . Age did not differ significantly between groups. Healthy dogs. The most common taxa at phylum level were Proteobacteria (mean relative percentage 54.1%, min 1.0%-max 99.9%), Firmicutes (15.5%, 0.1–96.8%), Tenericutes (8.7%, 0.0-81.5%) and Actinobacteria (7.8%, 0.0-83.7%), representing 97% of the bacterial population in this group (Table 2 ). Beside the family Moraxellaceae, three dogs had a high relative abundance (> 50%) of Cardiobacteriaceae (phylum Proteobacteria) and two dogs a high relative abundance of an unclassified family of the Mollicutes class (phylum Tenericutes). Among the phylum Proteobacteria, the genus Moraxella represented the most abundant taxa with a mean relative percentage at 51.8%. Table 2: Bacterial groups at >1% mean relative abundance among the control, FR and CR groups at phylum, family and genus level. Taxon Control group (n=29) CR group (n=14) FR group (n=40) Phylum Family Genus Mean rel. freq. (%) SD (%) Tukey's multiple comparisons test (corrected p<0.05) Detected in n dogs Mean rel. freq. (%) SD (%) Tukey's multiple comparisons test (corrected p<0.05) Detected in n dogs Mean rel. freq. (%) SD (%) Tukey's multiple comparisons test (corrected p<0.05) Detected in n dogs Proteobacteria Moraxellaceae Moraxella Neisseriaceae Conchiformibius Neisseria Enterobacteriaceae Escherichia_Shigella Proteus Pasteurellaceae Pasteurella Pasteurellaceae_ge Cardiobacteriaceae Suttonella Pseudomonadaceae Pseudomonas 71.6% 52.0% 51.8% 0.4% 0.3% 0.0% 0.9% 0.3% 0.0% 0.1% 0.0% 0.1% 9.8% 9.8% 6.1% 6.1% 29.3 39.5 39.7 1.4 1.4 0.1 2.8 1.1 0.0 0.4 0.0 0.4 23.9 23.9 12.5 12.5 A A A A A A NS NS NS A*** A** A** A* A* NS NS 29 29 29 12 8 3 15 11 1 11 1 6 19 19 26 25 30.3% 5.4% 4.6% 7.1% 4.4% 2.6% 3.6% 1.7% 1.4% 10.5% 6.0% 3.9% 0.1% 0.0% 0.6% 0.6% 22.2 9.6 8.7 9.9 8.7 6.2 6.1 2.8 5.4 14.5 13.4 7.5 0.2 0.0 1.1 1.1 B*** B*** B*** AB AB B* NS NS NS B B B AB AB NS NS 14 13 11 13 12 9 13 13 2 13 11 9 6 2 13 13 34.2% 6.3% 5.4% 7.7% 6.7% 1.0% 9.2% 7.0% 0.8% 1.6% 0.1% 1.2% 0.4% 0.4% 6.2% 6% 29.8 18.0 18.0 11.6 11.3 2.3 21.9 18.1 5.1 2.5 0.3 2.3 1.6 1.6 20.4 20.4 B*** B*** B*** B* B* AB NS NS NS A*** A** A* B B NS NS 42 36 32 34 28 25 36 35 7 29 19 26 15 7 31 30 Firmicutes Peptostreptococcaceae Lactobacillaceae Lactobacillus Lachnospiraceae Bacillales_Family_XI Gemella Streptococcaceae Lactococcus Streptococcus Staphylococcaceae Staphylococcus 10.3% 0.0% 1.6% 1.6% 0.1% 0.1% 0.1% 4.0% 0.4% 3.6% 2.1% 2.1% 17.3 0.1 5.3 5.3 0.2 0.7 0.7 12.4 1.3 12.7 4.4 4.4 A NS A A A A*** A*** A A NS NS NS 29 5 10 10 8 15 1 22 6 20 20 19 39.1% 0.6% 19.6% 19.6% 0.8% 1.3% 1.3% 7.7% 5.3% 2.4% 4.4% 4.4% 27.2 0.9 23.9 23.9 1.3 1.6 1.6 6.0 6.3 2.7 7.6 7.6 B** NS B* B* AB B B B** B** NS NS NS 14 10 14 14 11 11 9 14 14 12 11 11 35.1% 1.0% 9.4% 9.4% 1.1% 0.3% 0.3% 5.1% 2.5% 2.6% 14.1% 13.9% 31.9 2.5 18.4 18.4 1.7 0.5 0.5 6.3 4.4 4.3 29.4 29.3 B** NS AB AB B* A*** A*** AB AB NS NS NS 41 31 28 28 32 28 14 35 27 34 39 39 Bacteroidetes Porphyromonadaceae Porphyromonas Bacteroidaceae Bacteroides Flavobacteriaceae Capnocytophaga Flavobacterium Prevotellaceae Weeksellaceae Elizabethkingia 2.0% 0.7% 0.7% 0.0% 0.0% 0.1% 0.0% 0.0% 0.0% 1% 0.0% 5.6 3.5 3.5 0.1 0.1 0.4 0.2 0.2 0.1 3.0 0.0 A A A NS NS A NS NS NS NS NS 26 5 5 4 5 10 3 9 8 6 1 13.4% 4.0% 4.0% 0.4% 0.4% 4.7% 3.2% 1.4% 0.5% 3.3% 2.3% 11.7 7.3 7.3 0.5 0.5 11.3 7.5 4.6 0.8 5.7 4.7 B* AB AB NS NS B* NS NS NS NS NS 14 10 10 8 8 10 9 8 9 8 6 13.4% 6.5% 6.5% 1.3% 1.3% 1.1% 0.2% 0.8% 1.0% 2.2% 1.1% 14.8 12.5 12.5 3.9 3.9 1.8 0.4 1.5 2.7 4.0 2.6 B*** B* B* NS NS AB NS NS NS NS NS 41 35 35 30 30 29 22 22 28 19 16 Actinobacteria Corynebacteriaceae Corynebacterium_1 Microbacteriaceae Leucobacter Micrococcaceae Micrococcus Propionibacteriaceae Cutibacterium Actinomycetaceae Actinomyces 5.4% 0.5% 0.2% 2.3% 2.0% 1.6% 1.4% 0.3% 0.3% 0.1% 0.1% 9.7 1.1 0.4 4.6 4.4 7.5 7.3 0.8 0.8 0.5 0.5 NS NS NS NS NS NS NS NS NS A NS 29 21 13 21 19 19 8 14 12 5 5 12.5% 9.1% 2.2% 0.6% 0.4% 0.7% 0.3% 0.6% 0.5% 0.8% 0.8% 23.8 24.2 5.0 0.9 0.9 1.5 1.0 0.8 0.6 1.5 1.5 NS NS NS NS NS NS NS NS NS AB NS 14 12 8 10 8 11 6 12 9 10 10 8.5% 1.5% 1.1% 0.9% 0.4% 1.1% 0.1% 1.9% 1.7% 1.0% 1.0% 9.9 3.2 3.0 1.4 1.1 2.4 0.4 3.5 3.4 1.8 1.8 NS NS NS NS NS NS NS NS NS B* NS 36 25 22 27 15 26 14 27 25 26 26 Tenericutes Mollicutes_fa Mollicutes_ge 9.7% 9.5% 9.5% 21.3 20.9 20.9 NS A A 18 15 15 2% 1.8% 1.8% 5.4 4.8 4.8 NS AB AB 10 8 8 1.9% 1.0% 1.0% 6.4 6.2 6.2 NS B* B* 29 5 3 Fusobacteria Fusobacteriaceae Fusobacterium 0.1% 0.0% 0.0% 0.3 0.2 0.2 NS NS NS 6 6 6 1.6% 1.2% 1.2% 2.5 1.9 1.9 NS NS NS 11 11 11 4.2% 4.0% 4.0% 12.5 12.5 12.5 NS NS NS 34 32 32 Dogs with fungal rhinitis at diagnosis and cure. At diagnosis, the most common taxa at phylum level were Firmicutes (mean relative percentage 35.1%, min 0.0%-max 99.8%) followed by Proteobacteria (34.2%, 0.1–98.6%) and Bacteroidetes (13.4, 0.0-60.4). Out of the 40 dogs with FR, 9 were resampled at the time of cure. Median time to achieve cure in this subpopulation was 4.4 weeks (2.9–14). Six, 2 (dogs 5 and 6) and 1 (dog 2) dogs achieved cure after 1, 2 and 3 infusion protocols (Fig. 1 ). The most common taxa at phylum level at the time of cure was equally distributed compared to diagnosis with Firmicutes (42.4%, 0.4–99.7%), Proteobacteria (27.0%, 0.1–99.3%) and Bacteroidetes (16.1%, 0.1–96.8%). Dogs with chronic idiopathic rhinitis. At phylum level the most common taxa were Firmicutes (mean relative percentage 39.1%, min 0.1%-max 92.3%), Proteobacteria (30.3%, 0.7–75.4%), Bacteroidetes (13.4%, 0.3–40.7%) and Actinobacteria (12.5%, 0.0-97.8%). Comparison between healthy dogs and dogs with chronic nasal diseases The bacterial load quantified by 16S rRNA gene quantitative polymerase-chain reaction (qPCR) did not differ between the three groups. Constrained ordination. Redundancy analysis (RDA) showed that only disease status (p = 0.002; Adjusted R 2 0.142) contributed significantly to the variability of the microbiota (explaining 14.5% of the variance, Fig. 2 ). Intrinsic diversity values and β-diversity. Good’s coverage, species richness and α-diversity were significantly different between healthy dogs and dogs with chronic nasal diseases (Fig. 3 ). There was no difference in evenness. The non-metric multidimensional scaling graph of the β-diversity shows a clustering for the group of healthy dogs separating them from the diseased dogs (Fig. 4 ). Differences in relative abundances: FR group versus control group. Mean relative abundances at phylum and family level are represented in Figs. 5 and 6 . Table 2 shows the mean relative abundances of most abundant OTU, annotated to the levels of phylum, family and genus. The relative abundance in the Proteobacteria phylum was significantly lower in the FR group, compared with control dogs. This lower abundance in Proteobacteria was associated with a major and significant lower abundance in Moraxella (family Moraxellaceae) and Suttonella (family Cardiobacteriaceae) together with an increase of Conchiformibius (family Neisseriaceae). Other significant differences in the FR group compared with healthy dogs included an increase in the Firmicutes phylum with associated family Lachnospiraceae, an increase in the Bacteroidetes phylum with associated family Porphyromonas , an increase in Actinomycetaceae (phylum Actinobacteria), and finally a decrease in an unclassified genus of the Mollicutes class (phylum Tenericutes). Differences in relative abundances: CR group versus control group. Similar alterations were also observed between the CR and control groups especially the decrease in Proteobacteria due to the decrease in Moraxella in contrast to an increase in the phyla Firmicutes and Bacteroidetes. Specific changes were also noted such as an increase in the genera Lactobacillus (family Lactobaciliaceae) and Lactococcus (family Streptococcaceae) among the Firmicutes phylum. An increase in the genus Neisseria (family Neisseriaceae, phylum Proteobacteria) and the family Flavobacteriaceae (phylum Bacteroidetes) was also noted. Differences in relative abundances: FR group versus CR group. Between the FR and CR groups specifically, three significant differences were present: a higher relative abundance of Pasteurella and unclassified genus of the Pasteurellaceae family (both family Pasteurellaceae) as well as Gemella (family Bacillales_Family_XI) in the CR group compared to the FR group. Linear discriminant analysis effect size scores. In Fig. 7 , LEfSe scores indicate bacterial taxa that were mainly present in the different groups of the study population and shows that the highest number of specific taxa are found in the FR group, followed by the CR and the control group. Dogs with fungal rhinitis Comparison between diagnosis and cure. The microbial composition of the 9 dogs at diagnosis and cure at family level are represented in Fig. 1 . Only 1 dog (dog 2) recovered a high relative abundance of Moraxellaceae at cure. In 2 dogs (dogs 8 and 9) the NM was very similar to the one observed at diagnosis and in 5 dogs the microbiota was dominated (> 50%) by a single family: Porphyromonadaceae (dog 1), Spirochaetaceae (dog 3), Staphylococcaceae (dogs 4 and 6) and Enterobacteriaceae (dog 5). In the remaining dog (dog 7), a more heterogeneous composition was observed which was very different from its composition at diagnosis. No difference in bacterial load was observed between the two timepoints (Fig. 8 ). Among the intrinsic diversity values, only species evenness differed and was found to be lower at cure compared with the time of diagnosis (Fig. 8 ). No significant differences in relative abundances were found. NMDS plot did not show a specific pattern. Based on analysis of molecular variance (AMOVA; p = 0.202) and analysis of molecular variance homogeneity (HOMOVA; p = 0.905) beta-diversity and beta-dispersion were not different either. Effect of treatment on the NM. Among dogs with in the FR group, 11 were treated with systemic antimicrobials at the time of sampling while 29 dogs had not been receiving antimicrobials within at least the 2 previous weeks. Treated dogs were receiving amoxycillin clavulanic acid (n = 7), marbofloxacin (n = 1), marbofloxacin associated with azithromycin (n = 1), doxycycline (n = 1) or metronidazole (n = 1). Ten dogs were receiving an oral antifungal treatment (itraconazole, n = 8; ketoconazole, n = 1; or fluconazole, n = 1) at the time of sampling. For these two types of treatments, there was no significant effect on the variance (redundancy analysis), there were no differences at the level of the intrinsic diversity values, β-diversity or relative abundances at family, genus or species level. There was also no difference in bacterial load between the dogs receiving and not receiving antimicrobial or antifungal treatment. Discussion The present study is the first to describe the NM in dogs with fungal rhinitis using next generation sequencing methods. Our data showed that both fungal rhinitis and chronic idiopathic rhinitis were associated with common major alterations of the NM. These alterations were characterized by a significant lower abundance in Proteobacteria, mainly due to a lower abondance in Moraxella while more minor differences were specific either to fungal rhinitis or chronic idiopathic rhinitis. In most dogs with cured fungal rhinitis, the NM was still different from what we consider a healthy profile. Neither antimicrobial nor antifungal treatment appears to have a significant effect on the NM in dogs with fungal rhinitis. We showed that the NM in the healthy group was mostly dominated by the phylum Proteobacteria. This is in agreement with results of previous publications showing that the phyla Proteobacteria represents around 50 to 80% of the total bacterial population independently from age, breed or environment 8 – 10 , 38 . Other common phyla detected in healthy dogs in this study included Firmicutes, Tenericutes, Actinobacteria, and Bacteroidetes. This is also similar to previous publications although their frequency order may vary according to the study 8 – 10 , 38 . Like in previous studies, the Proteobacteria population was dominated by the family Moraxellaceae, and the genus Moraxella , followed by several other bacterial families at considerably lower levels 8 – 10 , 38 . However, not all healthy dogs had a microbial profile dominated by Moraxellaceae. Profiles dominated by Cardiobacteriaceae (phylum Proteobacteria), although in a much smaller amount, were also present. Cardiobacteriaceae was also one of the most frequently identified families in healthy dogs in previous studies 10 . Finally, some healthy dogs presented a more heterogenous profile, which was also the case in previous studies 10 . It can be hypothesized that, as it has been described in humans 1 , 39 , different healthy profiles exist, some of them being dominated by a particular bacterial taxon (e.g. Moraxellaceae, Cardiobacteriaceae) and others being more heterogeneous. Such a high inter-individual variability reflects the fact that the microbiota constantly undergoes changes of resident and transient micro-organisms in response to internal and external factors 4 , 12 . These factors may include the host and its local immune system, the inhaled particle-laden air, as well as atmospheric physical and chemical parameters. This is particularly true for the NM in dogs that interacts closely with the external environment which complicates the study of the NM in dogs. Based on the results of the present study and a previous study of the same group 12 , facial conformation (particularly brachycephalic breeds) and disease status are two factors able to significantly influence the NM in dogs. Both fungal rhinitis and chronic idiopathic rhinitis were associated with common major alterations of the resident nasal microbiota compared with healthy dogs. The most noticeable modification in both diseases was the marked lower relative abundance of the phylum Proteobacteria (+/- 50% reduction) and the associated family Moraxellaceae and genus Moraxella (+/- 90% reduction). Such a low relative abundance of Moraxella in dogs with chronic nasal diseases had already been described in the study by Tress and others 10 who compared the NM in healthy dogs to dogs with nasal neoplasia and chronic rhinitis. In children, nasopharyngeal Moraxella -dominated profiles have been described to be more stable and associated with a lower frequency of upper respiratory tract infections 39 . Altogether, these findings might propose Moraxella as a guarantor of nasal health. In dogs with chronic nasal disease, establishment of opportunistic species or overgrowth of some strains of the resident flora could overwhelm Moraxella , leading to a dysbiotic profile. It should be pointed out that a lower amount of Moraxella has also been observed in healthy brachycephalic dogs compared to other breed types 12 , although to a much lesser degree compared to the current data in dogs with nasal disease. This suggests that the relative abondance of Moraxella is at least partly dependent on facial conformation and/or air distribution strategy, rather than being exclusively associated with disease. The most noticeable alterations specific to dogs with fungal rhinitis included the higher abundance of genera such as Staphylococcus, Conchiformibius , Escherichia_Shigella , Porphyromonas and Fusobacteria , some taxa frequently reaching abundances of > 50% in individuals with fungal rhinitis. In healthy dogs, the same genera were also present but in small abundances, suggesting that fungal infection allows their particular development. In dogs with CR, the most noticeable alterations were different, in particular with a higher abundance of Pasteurella and Lactobacillus , underlining the fact that FR and CR are two distinct diseases each causing unique alterations of the NM. Whether these types of dysbiosis are the consequences of the alterations associated with FR and CR or if they play an active role in the development of the disease remains to be determined. In human beings, bacterial co-infections have been suggested to influence the development and persistence of clinical symptoms in patients with paranasal sinus A. fumigatus fungal balls 40 , 41 . In a sheep model of sinusitis 26 , inoculation of A. fumigatus resulted in the formation of a fungal biofilm only when co-inoculated with certain bacterial strains ( S. aureus, S. epidermidis, P. aeruginosa ). In the current study, in dogs with FR at diagnosis, genera such as Staphylococcus (n = 5), Pseudomonas (n = 2), Porphyromonas (n = 3), Escherichia_Shigella (n = 3), Conchiformibius (n = 4) and Lactobacillus (n = 3) represented the major part of the bacterial population (> 50%) in half (21/40) of the dogs, and might play an active role in the establishment, persistence and recurrence of fungal infection, either by causing epithelial inflammation and injury, or by metabolite cross-talk, and/or by modifying the immune response of the host to the fungus. A longer follow-up in dogs with FR would allow to verify the association between these specific taxa and either resolution or recurrence of the fungal infection. In dogs with fungal rhinitis at the time of cure, the NM was globally highly unpredictable. Amongst these 9 dogs, only one dog recovered a microbial composition with a high prevalence of Moraxellaceae. This dog needed 3 infusion protocols to reach cure, meaning he was the dog with the longest timeframe (3 months) between the collection of the two swabs. This may suggest that the NM needs more time to return to his “healthy state”. Another possibility would be that in some individuals the NM returns to his pre-infection state while in others it continues to shift toward a new and different bacterial community, a scenario that has already been described in humans with chronic rhinosinusitis after sinus surgery 42 . The Pasteurellaceae and Lactobacillaceae were much more prominent in dogs with chronic idiopathic rhinitis in this study. An increase in Pasteurellaceae was earlier reported in dogs with chronic rhinitis and nasal neoplasia 10 . In the current study, 2 dogs were colonized with a high amount of Pasteurella multocida , which was absent in the nose of healthy dogs in the current study. This species is considered a primary pathogen in swine 43 but is also described as an opportunistic pathogen in human and veterinary medicine. The role of P. multocida as a primary or opportunistic pathogen in dogs with chronic idiopathic rhinitis is currently unknown but deserves to be considered. Lactobacilli are commensals of the gastrointestinal and female genital tract 44 , 45 also used as probiotic strains 46 , 47 or feed additives 48 , 49 in dogs. However, it seems unlikely that Lactobacillus play a role in the pathogenesis of CR in dogs since they are uncommonly depicted as an opportunistic pathogen 50 , and were not reported to be elevated in dogs with CR in the study by Tress and others 10 or in humans with chronic rhinosinusitis. Results of the present study showed that systemic antimicrobials do not seem to significantly influence the NM in dogs with fungal rhinitis. In human beings with chronic rhinosinusitis, contradicting results have been published with variable effect on the diversity, evenness and bacterial burden 42,51−54 . Another study in dogs with nasal neoplasia also showed that pretreatment with antibiotics did not significantly altered NM 10 . The lack of effect could be due to a small concentration of drug reaching the nasal mucosa or a high resilience 42 of the NM to short-term antibiotic treatments and makes the use systemic antimicrobial questionable in canine chronic nasal diseases. The influence of age and bodyweight on the NM is unclear in dogs, based on previous studies this influence seems weak or absent 9 , 10 , 12 . Facial conformation however has been associated with significant changes of the NM in healthy dogs 12 . These changes were mostly present in dogs of brachycephalic breeds compared to other breed types, but minor variations were also observed between dolichocephalic and terrier breeds. Antibiotic pretreatment has also been reported to influence NM at varying degrees in humans and dogs 10,51−54 and the possible influence of antifungal treatment is unknown. For these reasons we decided to take age, bodyweight, breed type (meso-/dolichocephalic or terrier breed) and treatment status (antibiotic and antifungal) into account along with disease status (FR, CR or control group) for the RDA, as we believe these individual factors were the most likely to influence the variance in microbiota community composition. Sex and living environment (rural versus industrial regions) were considered unlikely to influence the NM 9 , 10 , 12 . The present study is essentially descriptive. We did not measure local microenvironmental parameters such as intranasal pH, humidity or temperature. Neither did we determine viral or fungal populations and the host immune response, preventing interpretation of the NM in light of these parameters. Another limitation concerns the size of the group, essentially the dogs with chronic idiopathic rhinitis, which is moreover a heterogeneous disease, of unclear and possibly variable etiology. A long-term follow-up was not performed in dogs with fungal rhinitis to evaluate the evolution of the NM in the presence or absence of relapse or recurrence of the disease. And finally, the present study was not designed to assess the effect of antimicrobial treatment: the molecules and duration of treatment were not standardized and the number of dogs in the treated group was small. This could possibly explain why we failed to show statistical differences between treated and non-treated groups. In conclusion, in dogs with chronic nasal diseases such as FR and CR, major alterations are present compared to healthy dogs while more subtle but significant differences might distinguish both diseases. Most dogs with fungal rhinitis probably did not recover their core microbiota at cure. The NM in dogs with fungal rhinitis at cure was unpredictable and a longer follow-up is needed to draw a conclusion. The present study lays the first groundwork to the realization and comprehension of the complex interactions between the nasal microbiota and nasal Aspergillus fumigatus infection in dogs. Further studies are warranted to discover if modulation of the nasal microbiota might be an interesting perspective for the treatment of this disease. Methods Study sample Client-owned dogs with a diagnosis of fungal rhinitis (FR group) or chronic idiopathic rhinitis (CR group) were prospectively recruited. A control group of healthy dolichocephalic dogs, age and breed matched with the FR group, was also recruited. Part of the dogs with fungal rhinitis that were treated and cured were examined at checkup (cured FR group). Diagnosis of fungal rhinitis was based on the presence of compatible clinical signs and per-endoscopic identification of fungal plaques with turbinate destruction. Additional diagnostic procedures consisted of computed tomography of the head, histopathology and fungal culture or polymerase chain reaction (PCR). All dogs were treated with endoscopic debridement of the fungal plaques followed by a 15-minutes enilconazole infusion protocol 55 . Control rhinoscopy was performed 3 to 6 weeks after treatment. Cure was based on resolution of clinical signs and absence of fungal plaques. As a non-negligible amount of the dogs included in the FR group were treated with antimicrobial and/or antifungal treatment at the time of sampling, the potential effect of these treatments on the NM was also investigated. Diagnosis of chronic idiopathic rhinitis was based on compatible clinical signs, endoscopic and/or histopathologic lesions. Other nasal diseases such as fungal rhinitis, neoplasia, oronasal defect or foreign body during endoscopy, computed tomography of the head, histopathological, culture or PCR results. All healthy dogs were exempt of clinical signs and had a normal clinical examination and blood work. In all dogs, questions were asked concerning ongoing local or systemic medical treatment. Except for dogs in the control group, the presence of antimicrobial or anti-inflammatory treatment before or at the time of sampling was not an exclusion criterion. Sample collection This study was approved by the ethical committee of xxx (approval number: 1854) and all samples were obtained with owner consent. For sample collection, dogs were premedicated with a combination of butorphanol (Butomidor®, Richter Pharma) and medetomidine (Medetor®, CP-Pharma) intravenously. Propofol (Propovet®, Zoetis) on demand was used for induction. Under general anesthesia, to prevent sample contamination, a sterile speculum was inserted into the nare to allow the passage of a sterile swab (Copan → , FLOQSwabs™, 553C, Brescia, Italy) up to the distal third of the nasal cavity. Sample collection was performed either by EV, FB or CC. In case of unilateral fungal rhinitis, the affected nasal cavity was sampled. In diseased dogs, sample collection was performed before rhinoscopy. The nasal mucosa was brushed using three careful circular movements before withdrawal of the swab through the speculum. The tip of the saw was cut and stored in a sterile cryotube and banked at -80°C until further analyses. DNA extraction and high throughput sequencing Based on the manufacturer’s instructions, total bacterial DNA was extracted from the nasal swabs with the DNEasy Blood and Tissue kit (QIAGEN Benelux BV; Antwerp, Belgium). Spectrophotometry (NanoDrop ND-1000, Isogen, De Meern, The Netherlands) was used for total DNA concentration measurement and purity evaluation. After DNA extraction from samples, quantification of the bacterial load was performed with a quantitative real-time PCR targeting the V2-V3 region of the 16S rRNA gene with the following primers: forward (5’-ACTCCTACGGGAGGCAGCAG-3’) and reverse (5’-ATTACCGCGGCTGCTGG-3’) as previously described 56 . The standard curve was based upon 10-fold dilution of a quantified PCR product. This PCR product was purified (Wizard® SV Gel and PCR Clean-Up System, Promega, Leiden, The Netherlands), quantified with PicoGreen targeting double-stranded DNA (Promega). For bacterial identification, bacterial 16S rRNA gene amplicons were generated via amplification of the V1-V3 hypervariable regions of the 16S rRNA gene using the following primers: forward (5’-GAGAGTTTGATYMTGGCTCAG-3’) and reverse (5’-ACCGCGGCTGCTGGCAC-3’) and Illumina overhand adapters. The DNA was purified with the Agencourt AMPure XP beads kit (Beckman Coulter; Pasadena, CA, USA) and submitted to a second PCR round for indexing, using the Nextera XT index primers 1 and 2. A final quantification, performed by quantitative PCR, of each sample in the library was performed using the KAPA SYBR" FAST qPCR Kit (KapaBiosystems; Wilmington, MA, USA) before normalization, pooling and sequencing on a MiSeq sequencer using V3 reagents (Illumina; San Diego, CA, USA). Positive control using DNA from 20 defined bacterial species and a negative control (from the PCR step) were included in the sequencing run. Amplicon profiling analysis Alignment and clustering were done with MOTHUR software package (v1.41.0) with an OTU clustering distance of 0.03 and based on the SILVA database (V1.32) of full-length 16S rRNA gene sequences. Vsearch algorithm was used for chimera detection 57 . After the chimera removal, reads corresponding to chloroplastic and mitochondrial 16S rRNA genes and reads whose taxonomic assignation fall outside the bacterial kingdom are removed during the cleaning process. From 16,220,278 raw reads, we obtained 14,714,808 reads after cleaning (length and sequence quality). Finally, we retained 6000 reads (median 5999 reads per sample) to adjust for uneven sequencing depth across samples. All biosample raw reads were deposited at the National Center for Biotechnology Information (NCBI) and are available under de Bioproject ID PRJNA841569. Alpha- and beta diversity. Subsample data sets including bacterial richness, evenness and α-diversity were obtained with MOTHUR at species level using the Chao1 index, Simpson index-based measure and the inverse Simpson’s index respectively. Beta-diversity at species level was assessed with MOTHUR using a dissimilarity matrix of Bray-Curtis. Non-metric multidimensional scaling plots for visual assessment were performed based on a Bray-Curtis dissimilarity matrix at species level with Rstudio (R v1.2.5033 package vegan v2.5-6 and ggplot2 v3.3.0) to represent the β-diversity between groups (FR versus CR versus control group, diagnosis versus cure within the FR group, treated versus non-treated dogs within the FR group). Statistical analysis Redundancy analysis. A RDA on values at genus level was performed to evaluate the relationships between the NM and the different potential explanatory variables (age, bodyweight, breed type, disease status and, among the FR group, antimicrobial or antifungal treatment status) that could influence/shape it. Forward selection was conducted to select significant variables using the “ordiR2step” function (with adjusted R2 coefficient) from the vegan package 58 . Relative abundances of taxa. A Kruskal-Wallis test with Benjamini-Hotchberg FDR correction followed by Tukey’s multiple comparison test in STAMP (v2.1.3) were used to identify differences in relative abundance at phylum, family, genus and species level (FR versus CR versus control group and treated versus non-treated dogs within the FR group). Alpha- and beta diversity. Bacterial richness, evenness, α-diversity, good’s coverage index and bacterial load were compared between the three groups (FR versus CR versus control group and treated versus non-treated dogs within the FR group) using a Kruskal-Wallis test and Dunn post-hoc test with Bonferroni correction or a Wilcoxon rank test for paired samples (diagnosis versus cure within the FR group). These analyses were performed using XLstat (2020.5.1, Addinsoft, Paris, France). Differences were considered significant for a p-value < 0.05. Beta-diversity was estimated with AMOVA (analysis of molecular variance; 10,000 iterations) and beta-dispersion was assessed with HOMOVA (analysis of molecular variance homogeneity; 10,000 iterations). Linear discriminant analysis (LDA) effect size (LEfSe) score. LEfSe was performed to detect differences in bacterial composition between groups (FR versus CR versus control group) at phylum, family, genus and species level with MOTHUR (significant for an LDA score > 3.0 59 ). Abbreviations NM nasal microbiota FR fungal rhinosinusitis CR chronic idiopathic rhinitis OTU operational taxonomic unit rRNA ribosomal ribonucleic acid DNA deoxyribonucleic acid rDNA ribosomal deoxyribonucleic acid PCR polymerase chain reaction qPCR quantitative polymerase chain reaction RDA redundancy analysis LEFSe Linear discriminant analysis effect size NMDS non-metric multidimensional scaling AMOVA analysis of molecular variance HOMOVA homogeneity of molecular variance LDA linear discriminant analysis Declarations -Ethics approval and consent to participate: Samples from healthy domestic dogs were collected according to a protocol approved by the Ethical Committee of the University of Liège (protocol #1854) and with the informed consent of the owners. All methods were carried out and reported in accordance with relevant guidelines and regulations (including ARRIVE guidelines). -Consent for publication: Not applicable -Availability of data and materials: All sample raw reads associated with this study have been deposited at the National Center for Biotechnology Information (NCBI) under the accession number PRJNA841569. -Competing interests: The authors declare that they have no competing interests -Funding: The study was conducted with personal funding. -Authors' contributions: EV and CC conceived and designed the study. EV, AF, FB, BT, GD and CC participated in data acquisition for the study. EV, AF and BT analyzed the data. EV, AF and BT performed the statistical analyses. EV and CC interpreted the results. EV and CC wrote the manuscript and all the authors read and approved the final version of the manuscript. -Acknowledgements: The authors would like to thank Albert Belinda, Phan Kim-Thu and Romijn Sylvain for their help in samples collection and storage. -Authors' information (optional): Vangrinsven Emilie, DVM Dipl. ECVIM-CA Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Liège, Quartier Vallée 2, Avenue de Cureghem 3, 4000 Liège, Belgium. References Bassis CM, Tang AL, Young VB, Pynnonen MA. The nasal cavity microbiota of healthy adults. Microbiome . 2014;2:27. Published 2014 Aug 11. doi:10.1186/2049-2618-2-27 Lal D, Keim P, Delisle J, et al. Mapping and comparing bacterial microbiota in the sinonasal cavity of healthy, allergic rhinitis, and chronic rhinosinusitis subjects. Int Forum Allergy Rhinol . 2017;7(6):561-569. doi:10.1002/alr.21934 Salzano FA, Marino L, Salzano G, et al. 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Published 2011 Jun 24. doi:10.1186/gb-2011-12-6-r60 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 15 Apr, 2023 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Major revision 07 Dec, 2022 Reviews received at journal 31 Oct, 2022 Reviewers agreed at journal 31 Oct, 2022 Reviews received at journal 17 Sep, 2022 Reviewers agreed at journal 07 Sep, 2022 Reviews received at journal 16 Jun, 2022 Reviewers agreed at journal 06 Jun, 2022 Reviewers invited by journal 04 Jun, 2022 Editor assigned by journal 04 Jun, 2022 Editor invited by journal 01 Jun, 2022 Submission checks completed at journal 01 Jun, 2022 First submitted to journal 28 May, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1703144","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":110282655,"identity":"41aff215-d89b-410f-87be-c96e6166bb22","order_by":0,"name":"Emilie Vangrinsven","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYFACxsYDCUCKXwLMk5AhRksDWIvkDCALqIWHKHsOgAiDG2AtDIS18IsdbjjwoOJOtPHt5uOPbtRY8DCwHz66AZ8WydmJQIedeZa77c6xxOacY0CH8aSl3cCnxeA2UEti2+HcbTdyDJtz2IBaJHjM8GqxB2v5dzh38wyQln9EaDGQBmlpOJy7QQKoJbeNCC0SIFsSjj3LnXEjLXF2bp8EDxshv/DPTn/48EfNndz+GckHPud8q5PjZz98DK8WKDiAYLIRoRxNyygYBaNgFIwCdAAARVZTgGoXI/0AAAAASUVORK5CYII=","orcid":"","institution":"University of Liège","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Emilie","middleName":"","lastName":"Vangrinsven","suffix":""},{"id":110282656,"identity":"123b2505-9843-491e-bac6-7650ee63946e","order_by":1,"name":"Aline Fastrès","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aline","middleName":"","lastName":"Fastrès","suffix":""},{"id":110282657,"identity":"dd88e571-5276-4e3a-912e-684cf0202a78","order_by":2,"name":"Bernard Taminiau","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bernard","middleName":"","lastName":"Taminiau","suffix":""},{"id":110282658,"identity":"467cd55e-ce79-457d-8793-06fbc97c8a0b","order_by":3,"name":"Frédéric Billen","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Frédéric","middleName":"","lastName":"Billen","suffix":""},{"id":110282659,"identity":"3b98b466-559f-43ac-844e-dab905a574c0","order_by":4,"name":"Georges Daube","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Georges","middleName":"","lastName":"Daube","suffix":""},{"id":110282660,"identity":"8a21f099-136a-4a27-9f33-054fb49946cd","order_by":5,"name":"Cécile Clercx","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cécile","middleName":"","lastName":"Clercx","suffix":""}],"badges":[],"createdAt":"2022-05-28 15:29:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1703144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1703144/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-023-02828-7","type":"published","date":"2023-04-15T20:28:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":22415398,"identity":"dc1c6260-5c65-44e3-bc8d-ab2cd0119a2d","added_by":"auto","created_at":"2022-06-08 15:31:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":601593,"visible":true,"origin":"","legend":"\u003cp\u003eComposition of nasal microbiota at family level in dogs with fungal rhinitis at diagnosis (first bar) and cure (second bar) (other: mean relative frequency \u0026lt;1%).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/7848bef7650412efb371387f.png"},{"id":22415401,"identity":"7a532dd1-31c0-4e7d-8bcd-b40725057662","added_by":"auto","created_at":"2022-06-08 15:31:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40895,"visible":true,"origin":"","legend":"\u003cp\u003eRedundancy analysis at genus level for microbiota composition in dogs based on disease status.\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/dc75276248a4f1b8bb4cb723.png"},{"id":22415399,"identity":"42885633-6f4b-454a-aea4-9a6243ec2b39","added_by":"auto","created_at":"2022-06-08 15:31:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28519,"visible":true,"origin":"","legend":"\u003cp\u003eIntrinsic diversity values comparing healthy dogs and dogs with chronic nasal diseases\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/b63539bedefbcd4cbebdc1dc.png"},{"id":22414649,"identity":"272840ea-af2d-473a-ae61-0e43642d669c","added_by":"auto","created_at":"2022-06-08 15:26:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":127595,"visible":true,"origin":"","legend":"\u003cp\u003eNon-metric multidimensional scaling (NMDS) ordination of nasal microbiota communities using Bray-Curtis. \u003c/p\u003e\u003cp\u003eComparison between fungal rhinitis (FR) group (green), chronic idiopathic rhinitis (CR) group (black) and control group (red). CR group versus FR group p=0.054, CR group versus control group p\u0026lt;0.001, FR group versus control group p\u0026lt;0.001.\u003c/p\u003e\u003cp\u003eStress value=0.09800267\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/96d77c47073d061740470e7e.png"},{"id":22415400,"identity":"27c1aeb9-ee24-4098-a3c0-dde4caa10776","added_by":"auto","created_at":"2022-06-08 15:31:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":432330,"visible":true,"origin":"","legend":"\u003cp\u003eComposition of nasal microbiota at phylum level in the study population\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/db43c423595871b9307778da.png"},{"id":22415667,"identity":"18d17e9b-14b5-4047-a0bd-689bfdcf7f4f","added_by":"auto","created_at":"2022-06-08 15:36:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":172103,"visible":true,"origin":"","legend":"\u003cp\u003eComposition of nasal microbiota at family level in the study population.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/49c9c69377a583577e39a123.png"},{"id":22416137,"identity":"220a3725-850c-438f-9a01-3853d86c501f","added_by":"auto","created_at":"2022-06-08 15:41:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":16866,"visible":true,"origin":"","legend":"\u003cp\u003eLinear discriminant analysis (LDA) effect size (LEfSe) of Illumina sequencing datasets based on 16S rRNA gene sequences\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/e1b32a6e3552e1a15c327e33.png"},{"id":22414651,"identity":"319032c5-98b7-472e-a518-61778cd8483d","added_by":"auto","created_at":"2022-06-08 15:26:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":25162,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial load and intrinsic diversity values in dogs with fungal rhinitis at diagnosis and cure.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/3d6962ba2ff8d1df6c78282a.png"},{"id":44724848,"identity":"cb6586cd-2e1a-466b-9060-d89609fe146e","added_by":"auto","created_at":"2023-10-16 20:35:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1853577,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1703144/v1/542b0bc0-0246-45db-b449-3117a2f17ef5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of the nasal microbiota in dogs with fungal rhinitis before and after cure and in dogs with chronic idiopathic rhinitis.","fulltext":[{"header":"Background","content":"\u003cp\u003eWith advances in culture-independent technologies, the role of the upper respiratory tract microbiota in health and disease has become an intense area of research in human medicine\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and to a much lesser extend in canine medicine\u003csup\u003e5,8\u0026minus;12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFungal rhinitis secondary to infection with \u003cem\u003eAspergillus fumigatus\u003c/em\u003e is a common cause of nasal disease in dogs while it is uncommon in humans. Why young otherwise healthy dolichocephalic dogs have their nasal cavity and sinus invaded by the fungus is not yet totally understood, despite extensive investigation\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Local dysimmunity is suspected, which associates a host response, consisting in Th1 and inflammatory response\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e with an in-host adaptive genetic and phenotypic response of the fungus itself, enabling growth of the pathogen and contributing to the suppression of the host immune response\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn human beings, reports over the past five years suggest that the microbiota can modulate the host immune response to invading fungal micro-organisms\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The microbiota is expected to influence immune homeostasis through host-to-microbe and microbe-to-microbe interactions\u003csup\u003e4,22\u0026minus;24\u003c/sup\u003e. It has been shown, by correlating changes in metabolite profiles with microbiota metagenomic composition, that certain bacterial species contribute to host-fungal symbiosis and mucosal homeostasis in humans with \u003cem\u003eAspergillus\u003c/em\u003e-related lung disease\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Formation of sinonasal bacterial biofilms of multiple species (such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e) have been reported to damage epithelia sufficiently for the establishment of fungal biofilms\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e while the release of extracellular molecules by \u003cem\u003eP. aeruginosa\u003c/em\u003e has been shown to stimulate the growth of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. It can therefore be suspected that, in the nasal cavities of dogs with fungal rhinitis, the microbiota is able to influence the immunological response to fungi, the clinical fungal disease severity, as well as the response to treatment.\u003c/p\u003e \u003cp\u003eChronic idiopathic rhinitis is a common heterogeneous disease characterized by lymphoplasmacytic to mixed inflammation of the sinonasal cavities without any identifiable cause\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In human medicine, chronic rhinosinusitis represents a significant health problem, the causes underlying the onset of the disease are multiple and not well defined\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Disruption of the microbiota homeostasis has been described as being the primary driver or at least exacerbating factor for nasal chronic inflammatory diseases such as chronic idiopathic rhinitis and allergic rhinitis\u003csup\u003e2,6,24,30\u0026minus;33\u003c/sup\u003e. In the pathophysiology of chronic idiopathic rhinitis in humans, two other interesting notions are the importance of bacterial biofilms\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e as well as the concept of keystone species maintaining a stable and healthy state by providing resistance to colonization by pathogens\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAltogether, this suggests that specific bacterial strains, as well as factors influencing the microbial composition and/or modulating microbial disturbances may be an untapped source of therapeutics to mitigate the severity of upper respiratory tract infections and/or inflammation. However, the role of nasal microbiota (NM) alterations in the pathophysiology of canine chronic nasal diseases has been very little studied since only one single study describes alterations of the NM in dogs with nasal neoplasia or chronic rhinitis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSo far, data relative to the NM in dogs with fungal nasal disease are not available. Therefore, whether bacterial dysbiosis exist in fungal rhinitis, and whether it may be one of the factors able to either initiate or entertain the local fungal development, or allow recurrence, is unknown. Likewise, all therapeutic protocols for fungal rhinitis described are based on attempts to eliminate the fungus\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e instead of targeting the relationship between the host and the fungus, and a possible approach based on NM modulation has not been considered yet.\u003c/p\u003e \u003cp\u003eFor all these reasons, further knowledge concerning the alterations of the NM in canine chronic nasal diseases such as fungal rhinitis and chronic idiopathic rhinitis is warranted. Improved understanding of the relationships between the microbiota, host responses and non-native microorganisms would help to develop future therapeutic approaches acting to prevent the invasion of pathogenic microorganisms. A first step is to characterize any specific dysbiosis associated with both chronic idiopathic rhinitis and fungal rhinitis. Therefore, the aim of this study was to describe and compare the NM in dolichocephalic dogs with fungal rhinitis (at diagnosis and cure in a subpopulation of dogs) and in dogs with chronic idiopathic rhinitis versus a control population of healthy dogs.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eStudy population\u003c/h2\u003e\n \u003cp\u003eEighty-three client-owned dogs were recruited and divided into 3 groups: 29 in the control group, 40 dogs in the fungal rhinitis (FR) group and 14 in the chronic idiopathic rhinitis (CR) group (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A first batch (7 healthy, 9 FR and 8 CR dogs) was collected and sequenced in 2017\u003csup\u003e37\u003c/sup\u003e, a second batch (22 healthy dogs and 17 dogs with FR) in 2018 and finally a last group was analyzed in 2020 (6 dogs with CR and 14 dogs with FR among which 9 dogs were sampled twice: at diagnosis and at cure). The data of the three sequencing sets were gathered in one table and processed together.\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCharacteristics of the groups according to the disease status\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCR group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFR group\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7 (0.8\u0026ndash;11.3)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (1-14.3)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.4 (1.2\u0026ndash;14.3)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 males, 17 females\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6 males, 8 females\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27 males, 14 females\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeight (kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30 (14.8\u0026ndash;47.8)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24 (6\u0026ndash;36)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.2 (3.7\u0026ndash;55)*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntibiotic treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (topical)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 (systemic)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntifungal treatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e/\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e*Median (min-max)\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eFR: fungal rhinitis\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003eCR: chronic idiopathic rhinitis\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eIn the control group, breeds included were Labrador retriever (n\u0026thinsp;=\u0026thinsp;4), Belgian shepherd (n\u0026thinsp;=\u0026thinsp;4), Border collie (n\u0026thinsp;=\u0026thinsp;4), Australian shepherd (n\u0026thinsp;=\u0026thinsp;4), Beauceron (n\u0026thinsp;=\u0026thinsp;3), Golden retriever (n\u0026thinsp;=\u0026thinsp;3), Alaskan malamute (n\u0026thinsp;=\u0026thinsp;2), German shepherd (n\u0026thinsp;=\u0026thinsp;1), Doberman (n\u0026thinsp;=\u0026thinsp;1), Dalmatian (n\u0026thinsp;=\u0026thinsp;1), White Swiss shepherd (n\u0026thinsp;=\u0026thinsp;1), and mixed-breed (n\u0026thinsp;=\u0026thinsp;1). Included dogs had a normal general examination and bloodwork, and were not receiving any treatment within one month before sampling.\u003c/p\u003e\n \u003cp\u003eBreeds included in the FR group were Border collie (n\u0026thinsp;=\u0026thinsp;5), rottweiler (n\u0026thinsp;=\u0026thinsp;3), American Staffordshire terrier (n\u0026thinsp;=\u0026thinsp;3), mixed-breed (n\u0026thinsp;=\u0026thinsp;3), Labrador (n\u0026thinsp;=\u0026thinsp;2), Bull Terrier (n\u0026thinsp;=\u0026thinsp;1), Greater Swiss Mountain dog (n\u0026thinsp;=\u0026thinsp;1), Golden retriever (n\u0026thinsp;=\u0026thinsp;11), Cocker spaniel (n\u0026thinsp;=\u0026thinsp;1), Great Dane (n\u0026thinsp;=\u0026thinsp;1), Australian shepherd (n\u0026thinsp;=\u0026thinsp;1), giant poodle (n\u0026thinsp;=\u0026thinsp;1), Rhodesian ridgeback (n\u0026thinsp;=\u0026thinsp;1), Beauceron (n\u0026thinsp;=\u0026thinsp;1), Siberian husky (n\u0026thinsp;=\u0026thinsp;1), German shepherd (n\u0026thinsp;=\u0026thinsp;1), Dobermann (n\u0026thinsp;=\u0026thinsp;1), Jack Russel (n\u0026thinsp;=\u0026thinsp;1) and Dachshund (n\u0026thinsp;=\u0026thinsp;1). At the time of sampling, 11 dogs were receiving systemic antimicrobials (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), 10 dogs were treated with oral antifungal drugs, 4 with non-steroidal anti-inflammatory drugs and 2 with steroids, within the 2 previous weeks.\u003c/p\u003e\n \u003cp\u003eBreeds included in the CR group were mixed-breed (n\u0026thinsp;=\u0026thinsp;3), Siberian husky (n\u0026thinsp;=\u0026thinsp;2), Jack Russel terrier (n\u0026thinsp;=\u0026thinsp;2), Dalmatian (n\u0026thinsp;=\u0026thinsp;1), American Staffordshire terrier (n\u0026thinsp;=\u0026thinsp;1), poodle (n\u0026thinsp;=\u0026thinsp;1), Bernese mountain dog (n\u0026thinsp;=\u0026thinsp;1), Barzo\u0026iuml; (n\u0026thinsp;=\u0026thinsp;1), Dachshund (n\u0026thinsp;=\u0026thinsp;1) and Border collie (n\u0026thinsp;=\u0026thinsp;1). One dog was treated with topical antimicrobial therapy (thiamphenicol) and saline at the time of sampling (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). All the other dogs did not receive anti-inflammatory or antimicrobial treatment for at least two weeks before sampling albeit this was not an exclusion criterion.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eNasal microbiota analysis\u003c/h2\u003e\n \u003cp\u003eAt the finest taxonomic level 4,887 operational taxonomic units (OTUs) were present throughout all samples. The Good\u0026rsquo;s coverage of all samples was higher than 96% with median 99.3% (96.6%-99.9%) indicating that the sequencing depth was sufficient for reliable analysis of these nasal microbial community samples. The distribution of age, sex and bodyweight according to disease status is reported in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Age did not differ significantly between groups.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy dogs.\u003c/strong\u003e The most common taxa at phylum level were Proteobacteria (mean relative percentage 54.1%, min 1.0%-max 99.9%), Firmicutes (15.5%, 0.1\u0026ndash;96.8%), Tenericutes (8.7%, 0.0-81.5%) and Actinobacteria (7.8%, 0.0-83.7%), representing 97% of the bacterial population in this group (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Beside the family Moraxellaceae, three dogs had a high relative abundance (\u0026gt;\u0026thinsp;50%) of Cardiobacteriaceae (phylum Proteobacteria) and two dogs a high relative abundance of an unclassified family of the Mollicutes class (phylum Tenericutes). Among the phylum Proteobacteria, the genus \u003cem\u003eMoraxella\u003c/em\u003e represented the most abundant taxa with a mean relative percentage at 51.8%.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Bacterial groups at \u0026gt;1% mean relative abundance among the control, FR and CR groups at phylum, family and genus level.\u003c/p\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.151515151515152%\"\u003e\n \u003cp\u003eTaxon\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" width=\"28.282828282828284%\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl group (n=29)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" width=\"28.282828282828284%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCR group (n=14)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" width=\"28.282828282828284%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFR group (n=40)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.129032258064516%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003ePhylum\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;Family\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Genus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.451612903225806%\"\u003e\n \u003cp\u003eMean rel. freq. (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.301075268817204%\"\u003e\n \u003cp\u003eSD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.67741935483871%\"\u003e\n \u003cp\u003eTukey\u0026apos;s multiple comparisons test (corrected p\u0026lt;0.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.526881720430108%\"\u003e\n \u003cp\u003eDetected in n dogs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.451612903225806%\"\u003e\n \u003cp\u003eMean rel. freq. (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.301075268817204%\"\u003e\n \u003cp\u003eSD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.67741935483871%\"\u003e\n \u003cp\u003eTukey\u0026apos;s multiple comparisons test (corrected p\u0026lt;0.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.526881720430108%\"\u003e\n \u003cp\u003eDetected in n dogs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.451612903225806%\"\u003e\n \u003cp\u003eMean rel. freq. (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.301075268817204%\"\u003e\n \u003cp\u003eSD (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.67741935483871%\"\u003e\n \u003cp\u003eTukey\u0026apos;s multiple comparisons test (corrected p\u0026lt;0.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.526881720430108%\"\u003e\n \u003cp\u003eDetected in n dogs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eProteobacteria\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Moraxellaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Moraxella\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eNeisseriaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Conchiformibius\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Neisseria\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Enterobacteriaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cem\u003eEscherichia_Shigella\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; Proteus\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Pasteurellaceae\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Pasteurella\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Pasteurellaceae_ge\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Cardiobacteriaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Suttonella\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePseudomonadaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Pseudomonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e71.6%\u003c/p\u003e\n \u003cp\u003e52.0%\u003c/p\u003e\n \u003cp\u003e51.8%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.4%\u003c/p\u003e\n \u003cp\u003e0.3%\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.9%\u003c/p\u003e\n \u003cp\u003e0.3%\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9.8%\u003c/p\u003e\n \u003cp\u003e9.8%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6.1%\u003c/p\u003e\n \u003cp\u003e6.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e29.3\u003c/p\u003e\n \u003cp\u003e39.5\u003c/p\u003e\n \u003cp\u003e39.7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e23.9\u003c/p\u003e\n \u003cp\u003e23.9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n 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\u003cp\u003e26\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eFirmicutes\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Peptostreptococcaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Lactobacillaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Lactobacillus\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Lachnospiraceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Bacillales_Family_XI\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; Gemella\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Streptococcaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Lactococcus\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Streptococcus\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Staphylococcaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Staphylococcus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e10.3%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.6%\u003c/p\u003e\n \u003cp\u003e1.6%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.0%\u003c/p\u003e\n \u003cp\u003e0.4%\u003c/p\u003e\n \u003cp\u003e3.6%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.1%\u003c/p\u003e\n \u003cp\u003e2.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e17.3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003cp\u003e5.3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e12.4\u003c/p\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003cp\u003e12.7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA***\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA***\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e39.1%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.6%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e19.6%\u003c/p\u003e\n \u003cp\u003e19.6%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.8%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.3%\u003c/p\u003e\n \u003cp\u003e1.3%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7.7%\u003c/p\u003e\n \u003cp\u003e5.3%\u003c/p\u003e\n \u003cp\u003e2.4%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.4%\u003c/p\u003e\n \u003cp\u003e4.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e27.2\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e23.9\u003c/p\u003e\n \u003cp\u003e23.9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7.6\u003c/p\u003e\n \u003cp\u003e7.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003e\u003cstrong\u003eB**\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB**\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB**\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e35.1%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9.4%\u003c/p\u003e\n \u003cp\u003e9.4%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.1%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.3%\u003c/p\u003e\n \u003cp\u003e0.3%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5.1%\u003c/p\u003e\n \u003cp\u003e2.5%\u003c/p\u003e\n \u003cp\u003e2.6%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e14.1%\u003c/p\u003e\n \u003cp\u003e13.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e31.9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e18.4\u003c/p\u003e\n \u003cp\u003e18.4\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e29.4\u003c/p\u003e\n \u003cp\u003e29.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003e\u003cstrong\u003eB**\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA***\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA***\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eBacteroidetes\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;Porphyromonadaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cem\u003ePorphyromonas\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Bacteroidaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Bacteroides\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFlavobacteriaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Capnocytophaga\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Flavobacterium\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Prevotellaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Weeksellaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Elizabethkingia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e2.0%\u003c/p\u003e\n \u003cp\u003e0.7%\u003c/p\u003e\n \u003cp\u003e0.7%\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u0026nbsp;0.0%\u003cbr\u003e\u0026nbsp;0.0%\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1%\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e5.6\u003c/p\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e13.4%\u003c/p\u003e\n \u003cp\u003e4.0%\u003c/p\u003e\n \u003cp\u003e4.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.4%\u003c/p\u003e\n \u003cp\u003e0.4%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.7%\u003c/p\u003e\n \u003cp\u003e3.2%\u003c/p\u003e\n \u003cp\u003e1.4%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.5%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3.3%\u003c/p\u003e\n \u003cp\u003e2.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e11.7\u003c/p\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003cp\u003e4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e13.4%\u003c/p\u003e\n \u003cp\u003e6.5%\u003c/p\u003e\n \u003cp\u003e6.5%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.3%\u003c/p\u003e\n \u003cp\u003e1.3%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.1%\u003c/p\u003e\n \u003cp\u003e0.2%\u003c/p\u003e\n \u003cp\u003e0.8%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.2%\u003c/p\u003e\n \u003cp\u003e1.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e14.8\u003c/p\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3.9\u003c/p\u003e\n \u003cp\u003e3.9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003e\u003cstrong\u003eB***\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eActinobacteria\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eCorynebacteriaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Corynebacterium_1\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Microbacteriaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Leucobacter\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Micrococcaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Micrococcus\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Propionibacteriaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cutibacterium\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Actinomycetaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Actinomyces\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e5.4%\u003c/p\u003e\n \u003cp\u003e0.5%\u003c/p\u003e\n \u003cp\u003e0.2%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.3%\u003c/p\u003e\n \u003cp\u003e2.0%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.6%\u003c/p\u003e\n \u003cp\u003e1.4%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.3%\u003c/p\u003e\n \u003cp\u003e0.3%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e9.7\u003c/p\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7.5\u003c/p\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e12.5%\u003c/p\u003e\n \u003cp\u003e9.1%\u003c/p\u003e\n \u003cp\u003e2.2%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.6%\u003c/p\u003e\n \u003cp\u003e0.4%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.7%\u003c/p\u003e\n \u003cp\u003e0.3%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.6%\u003c/p\u003e\n \u003cp\u003e0.5%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.8%\u003c/p\u003e\n \u003cp\u003e0.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e23.8\u003c/p\u003e\n \u003cp\u003e24.2\u003c/p\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e8.5%\u003c/p\u003e\n \u003cp\u003e1.5%\u003c/p\u003e\n \u003cp\u003e1.1%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.9%\u003c/p\u003e\n \u003cp\u003e0.4%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.1%\u003c/p\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.9%\u003c/p\u003e\n \u003cp\u003e1.7%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.0%\u003c/p\u003e\n \u003cp\u003e1.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e9.9\u003c/p\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.129032258064516%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eTenericutes\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;Mollicutes_fa\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; Mollicutes_ge\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e9.7%\u003c/p\u003e\n \u003cp\u003e9.5%\u003c/p\u003e\n \u003cp\u003e9.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e21.3\u003c/p\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e2%\u003c/p\u003e\n \u003cp\u003e1.8%\u003c/p\u003e\n \u003cp\u003e1.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003cp\u003e4.8\u003c/p\u003e\n \u003cp\u003e4.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.451612903225806%\"\u003e\n \u003cp\u003e1.9%\u003c/p\u003e\n \u003cp\u003e1.0%\u003c/p\u003e\n \u003cp\u003e1.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.301075268817204%\"\u003e\n \u003cp\u003e6.4\u003c/p\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003cp\u003e6.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eB*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"16.129032258064516%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eFusobacteria\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;Fusobacteriaceae\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cem\u003eFusobacterium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.451612903225806%\"\u003e\n \u003cp\u003e0.1%\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.301075268817204%\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.451612903225806%\"\u003e\n \u003cp\u003e1.6%\u003c/p\u003e\n \u003cp\u003e1.2%\u003c/p\u003e\n \u003cp\u003e1.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.301075268817204%\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.451612903225806%\"\u003e\n \u003cp\u003e4.2%\u003c/p\u003e\n \u003cp\u003e4.0%\u003c/p\u003e\n \u003cp\u003e4.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.301075268817204%\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.67741935483871%\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.526881720430108%\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cstrong\u003eDogs with fungal rhinitis at diagnosis and cure.\u003c/strong\u003e At diagnosis, the most common taxa at phylum level were Firmicutes (mean relative percentage 35.1%, min 0.0%-max 99.8%) followed by Proteobacteria (34.2%, 0.1\u0026ndash;98.6%) and Bacteroidetes (13.4, 0.0-60.4).\u003c/p\u003e\n \u003cp\u003eOut of the 40 dogs with FR, 9 were resampled at the time of cure. Median time to achieve cure in this subpopulation was 4.4 weeks (2.9\u0026ndash;14). Six, 2 (dogs 5 and 6) and 1 (dog 2) dogs achieved cure after 1, 2 and 3 infusion protocols (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe most common taxa at phylum level at the time of cure was equally distributed compared to diagnosis with Firmicutes (42.4%, 0.4\u0026ndash;99.7%), Proteobacteria (27.0%, 0.1\u0026ndash;99.3%) and Bacteroidetes (16.1%, 0.1\u0026ndash;96.8%).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDogs with chronic idiopathic rhinitis.\u003c/strong\u003e At phylum level the most common taxa were Firmicutes (mean relative percentage 39.1%, min 0.1%-max 92.3%), Proteobacteria (30.3%, 0.7\u0026ndash;75.4%), Bacteroidetes (13.4%, 0.3\u0026ndash;40.7%) and Actinobacteria (12.5%, 0.0-97.8%).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eComparison between healthy dogs and dogs with chronic nasal diseases\u003c/h2\u003e\n \u003cp\u003eThe bacterial load quantified by 16S rRNA gene quantitative polymerase-chain reaction (qPCR) did not differ between the three groups.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eConstrained ordination.\u003c/strong\u003e Redundancy analysis (RDA) showed that only disease status (p\u0026thinsp;=\u0026thinsp;0.002; Adjusted R\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e 0.142) contributed significantly to the variability of the microbiota (explaining 14.5% of the variance, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIntrinsic diversity values and \u0026beta;-diversity.\u003c/strong\u003e Good\u0026rsquo;s coverage, species richness and \u0026alpha;-diversity were significantly different between healthy dogs and dogs with chronic nasal diseases (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). There was no difference in evenness. The non-metric multidimensional scaling graph of the \u0026beta;-diversity shows a clustering for the group of healthy dogs separating them from the diseased dogs (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDifferences in relative abundances: FR group versus control group.\u003c/strong\u003e Mean relative abundances at phylum and family level are represented in Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the mean relative abundances of most abundant OTU, annotated to the levels of phylum, family and genus. The relative abundance in the Proteobacteria phylum was significantly lower in the FR group, compared with control dogs. This lower abundance in Proteobacteria was associated with a major and significant lower abundance in \u003cem\u003eMoraxella\u003c/em\u003e (family Moraxellaceae) and \u003cem\u003eSuttonella\u003c/em\u003e (family Cardiobacteriaceae) together with an increase of \u003cem\u003eConchiformibius\u003c/em\u003e (family Neisseriaceae). Other significant differences in the FR group compared with healthy dogs included an increase in the Firmicutes phylum with associated family Lachnospiraceae, an increase in the Bacteroidetes phylum with associated family \u003cem\u003ePorphyromonas\u003c/em\u003e, an increase in Actinomycetaceae (phylum Actinobacteria), and finally a decrease in an unclassified genus of the Mollicutes class (phylum Tenericutes).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDifferences in relative abundances: CR group versus control group.\u003c/strong\u003e Similar alterations were also observed between the CR and control groups especially the decrease in Proteobacteria due to the decrease in \u003cem\u003eMoraxella\u003c/em\u003e in contrast to an increase in the phyla Firmicutes and Bacteroidetes. Specific changes were also noted such as an increase in the genera \u003cem\u003eLactobacillus\u003c/em\u003e (family Lactobaciliaceae) and \u003cem\u003eLactococcus\u003c/em\u003e (family Streptococcaceae) among the Firmicutes phylum. An increase in the genus \u003cem\u003eNeisseria\u003c/em\u003e (family Neisseriaceae, phylum Proteobacteria) and the family Flavobacteriaceae (phylum Bacteroidetes) was also noted.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDifferences in relative abundances: FR group versus CR group.\u003c/strong\u003e Between the FR and CR groups specifically, three significant differences were present: a higher relative abundance of \u003cem\u003ePasteurella\u003c/em\u003e and unclassified genus of the Pasteurellaceae family (both family Pasteurellaceae) as well as \u003cem\u003eGemella\u003c/em\u003e (family Bacillales_Family_XI) in the CR group compared to the FR group.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eLinear discriminant analysis effect size scores.\u003c/strong\u003e In Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, LEfSe scores indicate bacterial taxa that were mainly present in the different groups of the study population and shows that the highest number of specific taxa are found in the FR group, followed by the CR and the control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003eDogs with fungal rhinitis\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eComparison between diagnosis and cure.\u003c/strong\u003e The microbial composition of the 9 dogs at diagnosis and cure at family level are represented in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Only 1 dog (dog 2) recovered a high relative abundance of Moraxellaceae at cure. In 2 dogs (dogs 8 and 9) the NM was very similar to the one observed at diagnosis and in 5 dogs the microbiota was dominated (\u0026gt;\u0026thinsp;50%) by a single family: Porphyromonadaceae (dog 1), Spirochaetaceae (dog 3), Staphylococcaceae (dogs 4 and 6) and Enterobacteriaceae (dog 5). In the remaining dog (dog 7), a more heterogeneous composition was observed which was very different from its composition at diagnosis. No difference in bacterial load was observed between the two timepoints (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). Among the intrinsic diversity values, only species evenness differed and was found to be lower at cure compared with the time of diagnosis (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). No significant differences in relative abundances were found. NMDS plot did not show a specific pattern. Based on analysis of molecular variance (AMOVA; p\u0026thinsp;=\u0026thinsp;0.202) and analysis of molecular variance homogeneity (HOMOVA; p\u0026thinsp;=\u0026thinsp;0.905) beta-diversity and beta-dispersion were not different either.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEffect of treatment on the NM.\u003c/strong\u003e Among dogs with in the FR group, 11 were treated with systemic antimicrobials at the time of sampling while 29 dogs had not been receiving antimicrobials within at least the 2 previous weeks. Treated dogs were receiving amoxycillin clavulanic acid (n\u0026thinsp;=\u0026thinsp;7), marbofloxacin (n\u0026thinsp;=\u0026thinsp;1), marbofloxacin associated with azithromycin (n\u0026thinsp;=\u0026thinsp;1), doxycycline (n\u0026thinsp;=\u0026thinsp;1) or metronidazole (n\u0026thinsp;=\u0026thinsp;1). Ten dogs were receiving an oral antifungal treatment (itraconazole, n\u0026thinsp;=\u0026thinsp;8; ketoconazole, n\u0026thinsp;=\u0026thinsp;1; or fluconazole, n\u0026thinsp;=\u0026thinsp;1) at the time of sampling.\u003c/p\u003e\n \u003cp\u003eFor these two types of treatments, there was no significant effect on the variance (redundancy analysis), there were no differences at the level of the intrinsic diversity values, \u0026beta;-diversity or relative abundances at family, genus or species level. There was also no difference in bacterial load between the dogs receiving and not receiving antimicrobial or antifungal treatment.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study is the first to describe the NM in dogs with fungal rhinitis using next generation sequencing methods. Our data showed that both fungal rhinitis and chronic idiopathic rhinitis were associated with common major alterations of the NM. These alterations were characterized by a significant lower abundance in Proteobacteria, mainly due to a lower abondance in \u003cem\u003eMoraxella\u003c/em\u003e while more minor differences were specific either to fungal rhinitis or chronic idiopathic rhinitis. In most dogs with cured fungal rhinitis, the NM was still different from what we consider a healthy profile. Neither antimicrobial nor antifungal treatment appears to have a significant effect on the NM in dogs with fungal rhinitis.\u003c/p\u003e \u003cp\u003eWe showed that the NM in the healthy group was mostly dominated by the phylum Proteobacteria. This is in agreement with results of previous publications showing that the phyla Proteobacteria represents around 50 to 80% of the total bacterial population independently from age, breed or environment\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Other common phyla detected in healthy dogs in this study included Firmicutes, Tenericutes, Actinobacteria, and Bacteroidetes. This is also similar to previous publications although their frequency order may vary according to the study\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLike in previous studies, the Proteobacteria population was dominated by the family Moraxellaceae, and the genus \u003cem\u003eMoraxella\u003c/em\u003e, followed by several other bacterial families at considerably lower levels\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, not all healthy dogs had a microbial profile dominated by Moraxellaceae. Profiles dominated by Cardiobacteriaceae (phylum Proteobacteria), although in a much smaller amount, were also present. Cardiobacteriaceae was also one of the most frequently identified families in healthy dogs in previous studies\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Finally, some healthy dogs presented a more heterogenous profile, which was also the case in previous studies\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. It can be hypothesized that, as it has been described in humans\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, different healthy profiles exist, some of them being dominated by a particular bacterial taxon (e.g. Moraxellaceae, Cardiobacteriaceae) and others being more heterogeneous. Such a high inter-individual variability reflects the fact that the microbiota constantly undergoes changes of resident and transient micro-organisms in response to internal and external factors\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These factors may include the host and its local immune system, the inhaled particle-laden air, as well as atmospheric physical and chemical parameters. This is particularly true for the NM in dogs that interacts closely with the external environment which complicates the study of the NM in dogs. Based on the results of the present study and a previous study of the same group\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, facial conformation (particularly brachycephalic breeds) and disease status are two factors able to significantly influence the NM in dogs.\u003c/p\u003e \u003cp\u003eBoth fungal rhinitis and chronic idiopathic rhinitis were associated with common major alterations of the resident nasal microbiota compared with healthy dogs. The most noticeable modification in both diseases was the marked lower relative abundance of the phylum Proteobacteria (+/- 50% reduction) and the associated family Moraxellaceae and genus \u003cem\u003eMoraxella\u003c/em\u003e (+/- 90% reduction). Such a low relative abundance of \u003cem\u003eMoraxella\u003c/em\u003e in dogs with chronic nasal diseases had already been described in the study by Tress and others\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e who compared the NM in healthy dogs to dogs with nasal neoplasia and chronic rhinitis. In children, nasopharyngeal \u003cem\u003eMoraxella\u003c/em\u003e-dominated profiles have been described to be more stable and associated with a lower frequency of upper respiratory tract infections\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Altogether, these findings might propose \u003cem\u003eMoraxella\u003c/em\u003e as a guarantor of nasal health. In dogs with chronic nasal disease, establishment of opportunistic species or overgrowth of some strains of the resident flora could overwhelm \u003cem\u003eMoraxella\u003c/em\u003e, leading to a dysbiotic profile. It should be pointed out that a lower amount of \u003cem\u003eMoraxella\u003c/em\u003e has also been observed in healthy brachycephalic dogs compared to other breed types\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, although to a much lesser degree compared to the current data in dogs with nasal disease. This suggests that the relative abondance of \u003cem\u003eMoraxella\u003c/em\u003e is at least partly dependent on facial conformation and/or air distribution strategy, rather than being exclusively associated with disease.\u003c/p\u003e \u003cp\u003eThe most noticeable alterations specific to dogs with fungal rhinitis included the higher abundance of genera such as \u003cem\u003eStaphylococcus, Conchiformibius\u003c/em\u003e, \u003cem\u003eEscherichia_Shigella\u003c/em\u003e, \u003cem\u003ePorphyromonas\u003c/em\u003e and \u003cem\u003eFusobacteria\u003c/em\u003e, some taxa frequently reaching abundances of \u0026gt;\u0026thinsp;50% in individuals with fungal rhinitis. In healthy dogs, the same genera were also present but in small abundances, suggesting that fungal infection allows their particular development. In dogs with CR, the most noticeable alterations were different, in particular with a higher abundance of \u003cem\u003ePasteurella\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e, underlining the fact that FR and CR are two distinct diseases each causing unique alterations of the NM. Whether these types of dysbiosis are the consequences of the alterations associated with FR and CR or if they play an active role in the development of the disease remains to be determined.\u003c/p\u003e \u003cp\u003eIn human beings, bacterial co-infections have been suggested to influence the development and persistence of clinical symptoms in patients with paranasal sinus \u003cem\u003eA. fumigatus\u003c/em\u003e fungal balls\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In a sheep model of sinusitis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, inoculation of \u003cem\u003eA. fumigatus\u003c/em\u003e resulted in the formation of a fungal biofilm only when co-inoculated with certain bacterial strains (\u003cem\u003eS. aureus, S. epidermidis, P. aeruginosa\u003c/em\u003e). In the current study, in dogs with FR at diagnosis, genera such as \u003cem\u003eStaphylococcus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5), \u003cem\u003ePseudomonas\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003ePorphyromonas\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3), \u003cem\u003eEscherichia_Shigella\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3), \u003cem\u003eConchiformibius\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4) and \u003cem\u003eLactobacillus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3) represented the major part of the bacterial population (\u0026gt;\u0026thinsp;50%) in half (21/40) of the dogs, and might play an active role in the establishment, persistence and recurrence of fungal infection, either by causing epithelial inflammation and injury, or by metabolite cross-talk, and/or by modifying the immune response of the host to the fungus.\u003c/p\u003e \u003cp\u003eA longer follow-up in dogs with FR would allow to verify the association between these specific taxa and either resolution or recurrence of the fungal infection.\u003c/p\u003e \u003cp\u003eIn dogs with fungal rhinitis at the time of cure, the NM was globally highly unpredictable. Amongst these 9 dogs, only one dog recovered a microbial composition with a high prevalence of Moraxellaceae. This dog needed 3 infusion protocols to reach cure, meaning he was the dog with the longest timeframe (3 months) between the collection of the two swabs. This may suggest that the NM needs more time to return to his \u0026ldquo;healthy state\u0026rdquo;. Another possibility would be that in some individuals the NM returns to his pre-infection state while in others it continues to shift toward a new and different bacterial community, a scenario that has already been described in humans with chronic rhinosinusitis after sinus surgery\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Pasteurellaceae and Lactobacillaceae were much more prominent in dogs with chronic idiopathic rhinitis in this study. An increase in Pasteurellaceae was earlier reported in dogs with chronic rhinitis and nasal neoplasia\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In the current study, 2 dogs were colonized with a high amount of \u003cem\u003ePasteurella multocida\u003c/em\u003e, which was absent in the nose of healthy dogs in the current study. This species is considered a primary pathogen in swine\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e but is also described as an opportunistic pathogen in human and veterinary medicine. The role of \u003cem\u003eP. multocida\u003c/em\u003e as a primary or opportunistic pathogen in dogs with chronic idiopathic rhinitis is currently unknown but deserves to be considered.\u003c/p\u003e \u003cp\u003eLactobacilli are commensals of the gastrointestinal and female genital tract\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e also used as probiotic strains\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e or feed additives\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e in dogs. However, it seems unlikely that Lactobacillus play a role in the pathogenesis of CR in dogs since they are uncommonly depicted as an opportunistic pathogen\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, and were not reported to be elevated in dogs with CR in the study by Tress and others\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e or in humans with chronic rhinosinusitis.\u003c/p\u003e \u003cp\u003eResults of the present study showed that systemic antimicrobials do not seem to significantly influence the NM in dogs with fungal rhinitis. In human beings with chronic rhinosinusitis, contradicting results have been published with variable effect on the diversity, evenness and bacterial burden\u003csup\u003e42,51\u0026minus;54\u003c/sup\u003e. Another study in dogs with nasal neoplasia also showed that pretreatment with antibiotics did not significantly altered NM\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The lack of effect could be due to a small concentration of drug reaching the nasal mucosa or a high resilience\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e of the NM to short-term antibiotic treatments and makes the use systemic antimicrobial questionable in canine chronic nasal diseases.\u003c/p\u003e \u003cp\u003eThe influence of age and bodyweight on the NM is unclear in dogs, based on previous studies this influence seems weak or absent\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Facial conformation however has been associated with significant changes of the NM in healthy dogs\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These changes were mostly present in dogs of brachycephalic breeds compared to other breed types, but minor variations were also observed between dolichocephalic and terrier breeds. Antibiotic pretreatment has also been reported to influence NM at varying degrees in humans and dogs\u003csup\u003e10,51\u0026minus;54\u003c/sup\u003e and the possible influence of antifungal treatment is unknown. For these reasons we decided to take age, bodyweight, breed type (meso-/dolichocephalic or terrier breed) and treatment status (antibiotic and antifungal) into account along with disease status (FR, CR or control group) for the RDA, as we believe these individual factors were the most likely to influence the variance in microbiota community composition. Sex and living environment (rural versus industrial regions) were considered unlikely to influence the NM\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present study is essentially descriptive. We did not measure local microenvironmental parameters such as intranasal pH, humidity or temperature. Neither did we determine viral or fungal populations and the host immune response, preventing interpretation of the NM in light of these parameters.\u003c/p\u003e \u003cp\u003eAnother limitation concerns the size of the group, essentially the dogs with chronic idiopathic rhinitis, which is moreover a heterogeneous disease, of unclear and possibly variable etiology.\u003c/p\u003e \u003cp\u003eA long-term follow-up was not performed in dogs with fungal rhinitis to evaluate the evolution of the NM in the presence or absence of relapse or recurrence of the disease.\u003c/p\u003e \u003cp\u003eAnd finally, the present study was not designed to assess the effect of antimicrobial treatment: the molecules and duration of treatment were not standardized and the number of dogs in the treated group was small. This could possibly explain why we failed to show statistical differences between treated and non-treated groups.\u003c/p\u003e \u003cp\u003eIn conclusion, in dogs with chronic nasal diseases such as FR and CR, major alterations are present compared to healthy dogs while more subtle but significant differences might distinguish both diseases. Most dogs with fungal rhinitis probably did not recover their core microbiota at cure. The NM in dogs with fungal rhinitis at cure was unpredictable and a longer follow-up is needed to draw a conclusion. The present study lays the first groundwork to the realization and comprehension of the complex interactions between the nasal microbiota and nasal \u003cem\u003eAspergillus fumigatus\u003c/em\u003e infection in dogs. Further studies are warranted to discover if modulation of the nasal microbiota might be an interesting perspective for the treatment of this disease.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStudy sample\u003c/h2\u003e \u003cp\u003eClient-owned dogs with a diagnosis of fungal rhinitis (FR group) or chronic idiopathic rhinitis (CR group) were prospectively recruited. A control group of healthy dolichocephalic dogs, age and breed matched with the FR group, was also recruited. Part of the dogs with fungal rhinitis that were treated and cured were examined at checkup (cured FR group).\u003c/p\u003e \u003cp\u003eDiagnosis of fungal rhinitis was based on the presence of compatible clinical signs and per-endoscopic identification of fungal plaques with turbinate destruction. Additional diagnostic procedures consisted of computed tomography of the head, histopathology and fungal culture or polymerase chain reaction (PCR). All dogs were treated with endoscopic debridement of the fungal plaques followed by a 15-minutes enilconazole infusion protocol\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Control rhinoscopy was performed 3 to 6 weeks after treatment. Cure was based on resolution of clinical signs and absence of fungal plaques. As a non-negligible amount of the dogs included in the FR group were treated with antimicrobial and/or antifungal treatment at the time of sampling, the potential effect of these treatments on the NM was also investigated.\u003c/p\u003e \u003cp\u003eDiagnosis of chronic idiopathic rhinitis was based on compatible clinical signs, endoscopic and/or histopathologic lesions. Other nasal diseases such as fungal rhinitis, neoplasia, oronasal defect or foreign body during endoscopy, computed tomography of the head, histopathological, culture or PCR results.\u003c/p\u003e \u003cp\u003eAll healthy dogs were exempt of clinical signs and had a normal clinical examination and blood work.\u003c/p\u003e \u003cp\u003eIn all dogs, questions were asked concerning ongoing local or systemic medical treatment. Except for dogs in the control group, the presence of antimicrobial or anti-inflammatory treatment before or at the time of sampling was not an exclusion criterion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eThis study was approved by the ethical committee of xxx (approval number: 1854) and all samples were obtained with owner consent.\u003c/p\u003e \u003cp\u003eFor sample collection, dogs were premedicated with a combination of butorphanol (Butomidor\u0026reg;, Richter Pharma) and medetomidine (Medetor\u0026reg;, CP-Pharma) intravenously. Propofol (Propovet\u0026reg;, Zoetis) on demand was used for induction. Under general anesthesia, to prevent sample contamination, a sterile speculum was inserted into the nare to allow the passage of a sterile swab (Copan\u003csup\u003e\u0026rarr;\u003c/sup\u003e, FLOQSwabs\u0026trade;, 553C, Brescia, Italy) up to the distal third of the nasal cavity. Sample collection was performed either by EV, FB or CC. In case of unilateral fungal rhinitis, the affected nasal cavity was sampled. In diseased dogs, sample collection was performed before rhinoscopy. The nasal mucosa was brushed using three careful circular movements before withdrawal of the swab through the speculum. The tip of the saw was cut and stored in a sterile cryotube and banked at -80\u0026deg;C until further analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and high throughput sequencing\u003c/h2\u003e \u003cp\u003eBased on the manufacturer\u0026rsquo;s instructions, total bacterial DNA was extracted from the nasal swabs with the DNEasy Blood and Tissue kit (QIAGEN Benelux BV; Antwerp, Belgium). Spectrophotometry (NanoDrop ND-1000, Isogen, De Meern, The Netherlands) was used for total DNA concentration measurement and purity evaluation.\u003c/p\u003e \u003cp\u003eAfter DNA extraction from samples, quantification of the bacterial load was performed with a quantitative real-time PCR targeting the V2-V3 region of the 16S rRNA gene with the following primers: forward (5\u0026rsquo;-ACTCCTACGGGAGGCAGCAG-3\u0026rsquo;) and reverse (5\u0026rsquo;-ATTACCGCGGCTGCTGG-3\u0026rsquo;) as previously described\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The standard curve was based upon 10-fold dilution of a quantified PCR product. This PCR product was purified (Wizard\u0026reg; SV Gel and PCR Clean-Up System, Promega, Leiden, The Netherlands), quantified with PicoGreen targeting double-stranded DNA (Promega).\u003c/p\u003e \u003cp\u003eFor bacterial identification, bacterial 16S rRNA gene amplicons were generated via amplification of the V1-V3 hypervariable regions of the 16S rRNA gene using the following primers: forward (5\u0026rsquo;-GAGAGTTTGATYMTGGCTCAG-3\u0026rsquo;) and reverse (5\u0026rsquo;-ACCGCGGCTGCTGGCAC-3\u0026rsquo;) and Illumina overhand adapters. The DNA was purified with the Agencourt AMPure XP beads kit (Beckman Coulter; Pasadena, CA, USA) and submitted to a second PCR round for indexing, using the Nextera XT index primers 1 and 2. A final quantification, performed by quantitative PCR, of each sample in the library was performed using the KAPA SYBR\" FAST qPCR Kit (KapaBiosystems; Wilmington, MA, USA) before normalization, pooling and sequencing on a MiSeq sequencer using V3 reagents (Illumina; San Diego, CA, USA). Positive control using DNA from 20 defined bacterial species and a negative control (from the PCR step) were included in the sequencing run.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAmplicon profiling analysis\u003c/h2\u003e \u003cp\u003eAlignment and clustering were done with MOTHUR software package (v1.41.0) with an OTU clustering distance of 0.03 and based on the SILVA database (V1.32) of full-length 16S rRNA gene sequences. Vsearch algorithm was used for chimera detection\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. After the chimera removal, reads corresponding to chloroplastic and mitochondrial 16S rRNA genes and reads whose taxonomic assignation fall outside the bacterial kingdom are removed during the cleaning process. From 16,220,278 raw reads, we obtained 14,714,808 reads after cleaning (length and sequence quality). Finally, we retained 6000 reads (median 5999 reads per sample) to adjust for uneven sequencing depth across samples. All biosample raw reads were deposited at the National Center for Biotechnology Information (NCBI) and are available under de Bioproject ID PRJNA841569.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAlpha- and beta diversity.\u003c/b\u003e Subsample data sets including bacterial richness, evenness and α-diversity were obtained with MOTHUR at species level using the Chao1 index, Simpson index-based measure and the inverse Simpson\u0026rsquo;s index respectively. Beta-diversity at species level was assessed with MOTHUR using a dissimilarity matrix of Bray-Curtis. Non-metric multidimensional scaling plots for visual assessment were performed based on a Bray-Curtis dissimilarity matrix at species level with Rstudio (R v1.2.5033 package vegan v2.5-6 and ggplot2 v3.3.0) to represent the β-diversity between groups (FR versus CR versus control group, diagnosis versus cure within the FR group, treated versus non-treated dogs within the FR group).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e \u003cb\u003eRedundancy analysis.\u003c/b\u003e A RDA on values at genus level was performed to evaluate the relationships between the NM and the different potential explanatory variables (age, bodyweight, breed type, disease status and, among the FR group, antimicrobial or antifungal treatment status) that could influence/shape it. Forward selection was conducted to select significant variables using the \u0026ldquo;ordiR2step\u0026rdquo; function (with adjusted R2 coefficient) from the vegan package\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRelative abundances of taxa.\u003c/b\u003e A Kruskal-Wallis test with Benjamini-Hotchberg\u003c/p\u003e \u003cp\u003e FDR correction followed by Tukey\u0026rsquo;s multiple comparison test in STAMP (v2.1.3) were used to identify differences in relative abundance at phylum, family, genus and species level (FR versus CR versus control group and treated versus non-treated dogs within the FR group).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAlpha- and beta diversity.\u003c/b\u003e Bacterial richness, evenness, α-diversity, good\u0026rsquo;s\u003c/p\u003e \u003cp\u003ecoverage index and bacterial load were compared between the three groups (FR versus CR versus control group and treated versus non-treated dogs within the FR group) using a Kruskal-Wallis test and Dunn post-hoc test with Bonferroni correction or a Wilcoxon rank test for paired samples (diagnosis versus cure within the FR group). These analyses were performed using XLstat (2020.5.1, Addinsoft, Paris, France). Differences were considered significant for a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eBeta-diversity was estimated with AMOVA (analysis of molecular variance; 10,000 iterations) and beta-dispersion was assessed with HOMOVA (analysis of molecular variance homogeneity; 10,000 iterations).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLinear discriminant analysis (LDA) effect size (LEfSe) score.\u003c/b\u003e LEfSe was performed to detect differences in bacterial composition between groups (FR versus CR versus control group) at phylum, family, genus and species level with MOTHUR (significant for an LDA score\u0026thinsp;\u0026gt;\u0026thinsp;3.0\u003csup\u003e59\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enasal microbiota\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efungal rhinosinusitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echronic idiopathic rhinitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOTU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoperational taxonomic unit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003erRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eribosomal ribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003erDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eribosomal deoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eredundancy analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLEFSe\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLinear discriminant analysis effect size\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNMDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enon-metric multidimensional scaling\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanalysis of molecular variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHOMOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehomogeneity of molecular variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elinear discriminant analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e-Ethics approval and consent to participate: Samples from healthy domestic dogs were collected according to a protocol approved by the Ethical Committee of the University of Li\u0026egrave;ge (protocol #1854) and with the informed consent of the owners. All methods were carried out and reported in accordance with relevant guidelines and regulations (including ARRIVE guidelines).\u003c/p\u003e\n\u003cp\u003e-Consent for publication: Not applicable\u003c/p\u003e\n\u003cp\u003e-Availability of data and materials: \u0026nbsp; All sample raw reads associated with this study have been deposited at the National Center for Biotechnology Information (NCBI) under the accession number PRJNA841569.\u003c/p\u003e\n\u003cp\u003e-Competing interests: The authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e-Funding: The study was conducted with personal funding.\u003c/p\u003e\n\u003cp\u003e-Authors\u0026apos; contributions: EV and CC conceived and designed the study. EV, AF, FB, BT, GD and CC participated in data acquisition for the study. EV, AF and BT analyzed the data. EV, AF and BT performed the statistical analyses. EV and CC interpreted the results. EV and CC wrote the manuscript and all the authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e-Acknowledgements: The authors would like to thank Albert Belinda, Phan Kim-Thu and Romijn Sylvain for their help in samples collection and storage.\u003c/p\u003e\n\u003cp\u003e-Authors\u0026apos; information (optional): Vangrinsven Emilie, DVM Dipl. ECVIM-CA\u003c/p\u003e\n\u003cp\u003eDepartment of Clinical Sciences, Faculty of Veterinary Medicine, University of Li\u0026egrave;ge, Quartier Vall\u0026eacute;e 2, Avenue de Cureghem 3, 4000 Li\u0026egrave;ge, Belgium.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBassis CM, Tang AL, Young VB, Pynnonen MA. The nasal cavity microbiota of healthy adults. \u003cem\u003eMicrobiome\u003c/em\u003e. 2014;2:27. Published 2014 Aug 11. doi:10.1186/2049-2618-2-27\u003c/li\u003e\n\u003cli\u003eLal D, Keim P, Delisle J, et al. 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Published 2011 Jun 24. doi:10.1186/gb-2011-12-6-r60\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nasal cavity, dog, nasal, microbiota, aspergillosis, fungal rhinitis, mycotic rhinitis, chronic idiopathic rhinitis","lastPublishedDoi":"10.21203/rs.3.rs-1703144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1703144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePathogenesis of canine fungal rhinitis is still not fully understood. Treatment remains challenging, after cure intranasal remodeling may be associated with clinical disease mimicking chronic idiopathic rhinitis and recurrence can occur. Alterations of the nasal microbiota have been demonstrated in dogs with chronic idiopathic rhinitis and nasal neoplasia, although whether they play a role in the pathogenesis or are a consequence of the disease is still unknown. The objectives of the present study were (1) to describe nasal microbiota alterations associated with fungal rhinitis in dogs, compared with chronic idiopathic rhinitis and controls, (2) to characterize the nasal microbiota modifications associated with successful treatment of fungal rhinitis. Forty dogs diagnosed with fungal rhinitis, 14 dogs with chronic idiopathic rhinitis and 29 healthy control dogs were included. Nine of the fungal rhinitis dogs were resampled after successful treatment with enilconazole infusion.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOnly disease status influenced the nasal microbiota variance. The relative abundance of the genus \u003cem\u003eMoraxella\u003c/em\u003e was decreased in the fungal rhinitis (5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;18%) and chronic idiopathic rhinitis (4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7%) groups compared to controls (51.8\u0026thinsp;\u0026plusmn;\u0026thinsp;39.7%). Fungal rhinitis and chronic idiopathic rhinitis groups also showed an increased richness and α-diversity at species level compared with controls. Increase in unique families were associated with fungal rhinitis (Staphyloccaceae, Porphyromonadaceae, Enterobacteriaceae and Neisseriaceae) and chronic idiopathic rhinitis (Pasteurellaceae and Lactobacillaceae). In dogs with fungal rhinitis at cure, only 1 dog recovered a high relative abundance of Moraxellaceae.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eResults confirm major alterations of the nasal microbiota in dogs affected with fungal rhinitis and chronic idiopathic rhinitis, consisting mainly in a decrease of \u003cem\u003eMoraxella\u003c/em\u003e. Besides, a specific dysbiotic profile further differentiated fungal rhinitis from chronic idiopathic rhinitis. In dogs with fungal rhinitis, whether the NM returns to its pre-infection state or progresses toward chronic idiopathic rhinitis or fungal rhinitis recurrence warrants further investigation.\u003c/p\u003e","manuscriptTitle":"Assessment of the nasal microbiota in dogs with fungal rhinitis before and after cure and in dogs with chronic idiopathic rhinitis.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-08 15:26:55","doi":"10.21203/rs.3.rs-1703144/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-12-07T07:46:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-10-31T09:55:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233d03d7-c20d-46d8-b015-2f9554bc65d0","date":"2022-10-31T08:56:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-09-17T11:23:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f6e222ee-004c-4e5f-b71a-cc6ecf271ed9","date":"2022-09-07T09:35:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-17T03:15:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b81041c9-fecf-4f49-b8d5-d1704070cb45","date":"2022-06-06T06:41:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-06-04T22:45:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-06-04T22:27:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-06-01T06:01:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-06-01T05:42:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2022-05-28T15:28:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16743b5d-1cf9-4813-9c1c-56ac7114c4f5","owner":[],"postedDate":"June 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:31:57+00:00","versionOfRecord":{"articleIdentity":"rs-1703144","link":"https://doi.org/10.1186/s12866-023-02828-7","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2023-04-15 20:28:23","publishedOnDateReadable":"April 15th, 2023"},"versionCreatedAt":"2022-06-08 15:26:55","video":"","vorDoi":"10.1186/s12866-023-02828-7","vorDoiUrl":"https://doi.org/10.1186/s12866-023-02828-7","workflowStages":[]},"version":"v1","identity":"rs-1703144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1703144","identity":"rs-1703144","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}