The respiratory microbiota and its impact on health and disease in dogs and cats: A One Health perspective.

Healthy lungs were long thought of as sterile, with presence of bacteria identified by culture representing contamination. Recent advances in metagenomics have refuted this belief by detecting rich, diverse, and complex microbial communities in the healthy lower airways of many species, albeit at low concentrations. Although research has only begun to investigate causality and potential mechanisms, alterations in these microbial communities (known as dysbiosis) have been described in association with inflammatory, infectious, and neoplastic respiratory diseases in humans. Similar studies in dogs and cats are scarce. The microbial communities in the respiratory tract are linked to distant microbial communities such as in the gut (ie, the gut-lung axis), allowing interplay of microbes and microbial products in health and disease. This review summarizes considerations for studying local microbial communities, key features of the respiratory microbiota and its role in the gut-lung axis, current understanding of the healthy respiratory microbiota, and examples of dysbiosis in selected respiratory diseases of dogs and cats.

Ophthalmic viscoelastics commonly used in cataract surgery: A microbiota investigation.

PURPOSE: To survey commonly used, sterile ophthalmic viscoelastic materials used during routine cataract surgery for the presence of bacterial DNA and/or viable bacteria and endotoxin quantification. METHODS: Samples from three different ophthalmic viscoelastic manufacturers and three different production lots per manufacturer were collected for 16 S ribosomal ribonucleic acid (rRNA) sequencing and conventional aerobic and capnophilic bacterial culture. Other samples of viscoelastic material from the same three manufacturers were collected for endotoxin quantification using a commercially available Limulus amebocyte lysate (LAL) assay. Statistical analysis was performed using Sigma Plot 14.0, and R v4.0.2.0. Differences (p ≤ .05) between sample collection sites in total DNA concentration, microbial richness, mean intra-group distances, and endotoxin quantification alongside reagent controls were evaluated. RESULTS: Culture yielded two isolates, identified as Staphylococcus epidermidis and Bacillus megaterium. 16 S rRNA sequencing revealed no differences between brands in richness or overall composition. The most common bacterial DNA detected across all brands was Staphylococcus sp., Cutibacterium sp., Flavobacterium sp., and Lactobacillus sp. A significant difference was found between the median endotoxin concentration between Anvision and Hyvisc® viscoelastic (Anvision: 0.171 EU/mL, Hyvisc®: 0.03 EU/mL; p < .001). CONCLUSIONS: No brand-specific differences in bacterial DNA were detected in the viscoelastic materials. Staphylococcus, Cutibacterium, Flavobacterium, and Lactobacillus were the dominant contributors to the bacterial DNA detected. Although Anvision viscoelastic samples contained significantly more endotoxin than Hyvisc® viscoelastic samples, endotoxin concentrations were below the FDA limit of 0.2 EU/mL for both manufacturers. These data further the understanding of inflammatory outcomes following cataract surgery.

Molecular and microbiological evidence of bacterial contamination of intraocular lenses commonly used in canine cataract surgery.

Inflammatory outcomes, including toxic anterior segment syndrome (TASS) and infectious endophthalmitis, are potentially painful, blinding complications following cataract surgery. In an in vitro pilot study, commercially available, sterile foldable intraocular lenses (IOLs) used during routine canine cataract surgery, and their packaging fluid were surveyed for the presence of bacterial DNA and/or viable (cultivable) bacteria. Swabs from IOLs and packaging fluid from three different veterinary manufacturers and three different production lots/manufacturer were collected for 16S ribosomal ribonucleic acid (rRNA) sequencing. Packaging fluid samples were collected for aerobic/capnophilic bacterial culture. Culture yielded one isolate, identified as Staphylococcus epidermidis. 16S rRNA sequencing revealed distinct brand-specific bacterial DNA profiles, conserved between IOLs and packaging fluid of all production lots within each manufacturer. The dominant taxonomy differentiating each manufacturer was annotated as Staphylococcus sp, and was a 100% match to S. epidermidis. Distinct mixtures of bacterial DNA are present and consistent in IOLs and packaging fluid depending on the manufacturer, and Staphylococcus is the dominant contributor to the bacterial DNA detected. Caralens products had a significantly lower amount of Staphylococcus spp. compared to Anvision and Dioptrix products.

Respiratory dysbiosis in cats with spontaneous allergic asthma.

Deviations from a core airway microbiota have been associated with the development and progression of asthma as well as disease severity. Pet cats represent a large animal model for allergic asthma, as they spontaneously develop a disease similar to atopic childhood asthma. This study aimed to describe the lower airway microbiota of asthmatic pet cats and compare it to healthy cats to document respiratory dysbiosis occurring with airway inflammation. We hypothesized that asthmatic cats would have lower airway dysbiosis characterized by a decrease in richness, diversity, and alterations in microbial community composition including identification of possible pathobionts. In the current study, a significant difference in airway microbiota composition was documented between spontaneously asthmatic pet cats and healthy research cats mirroring the finding of dysbiosis in asthmatic humans. Filobacterium and Acinetobacter spp. were identified as predominant taxa in asthmatic cats without documented infection based on standard culture and could represent pathobionts in the lower airways of cats. Mycoplasma felis, a known lower airway pathogen of cats, was identified in 35% of asthmatic but not healthy cats. This article has been published alongside "Temporal changes of the respiratory microbiota as cats transition from health to experimental acute and chronic allergic asthma" (1).

Temporal changes of the respiratory microbiota as cats transition from health to experimental acute and chronic allergic asthma.

In humans, deviation from a core airway microbiota may predispose to development, exacerbation, or progression of asthma. We proposed to describe microbiota changes using 16 rRNA sequencing in samples from the upper and lower airways, and rectal swabs of 8 cats after experimental induction of asthma using Bermuda grass allergen, in acute (6 weeks) and chronic (36 weeks) stages. We hypothesized that asthma induction would decrease richness and diversity and alter microbiota composition and structure in the lower airways, without significantly impacting other sites. After asthma induction, richness decreased in rectal (p = 0.014) and lower airway (p = 0.016) samples. B diversity was significantly different between health and chronic asthma in all sites, and between all time points for lower airways. In healthy lower airways Pseudomonadaceae comprised 80.4 ± 1.3% whereas Sphingobacteriaceae and Xanthobacteraceae predominated (52.4 ± 2.2% and 33.5 ± 2.1%, respectively), and Pseudomonadaceae was absent, in 6/8 cats with chronic asthma. This study provides evidence that experimental induction of asthma leads to dysbiosis in the airways and distant sites in both the acute and chronic stages of disease. This article has been published alongside "Respiratory dysbiosis in cats with spontaneous allergic asthma" (1).

Clinicopathologic features, comorbid diseases, and prevalence of pulmonary hypertension in dogs with bronchomalacia.

BACKGROUND: Reports of clinicopathologic features of bronchomalacia (BM) differ because of inconsistent definitions and frequent prevalence of comorbid cardiopulmonary disease. Pulmonary hypertension (PH) secondary to BM is poorly described. OBJECTIVES: Dogs with BM will be older but of any somatotype, and increased expiratory effort, ≥1 comorbid disease, and PH will be more common than in dogs without BM. ANIMALS: Client-owned dogs (n = 210) evaluated for respiratory signs. METHODS: Medical records of dogs with paired inspiratory: expiratory-breath-hold computed tomography, tracheobronchoscopy, or both between January 2016 and December 2019 were retrospectively reviewed. Comparisons between dogs with and without BM using Mann-Whitney rank sum or χ2 tests (P < .05 significant were made). Because of high numbers of variables, criteria with high prevalence (>25%) were identified (n = 10) for univariate analysis (P < .005 significant). Significant variables were submitted for multivariate analysis. RESULTS: Bronchomalacia was identified in 41% of dogs of all sizes/somatotypes; 38% were >10 kg. All dogs with BM had ≥1 comorbid cardiopulmonary disorder. Dogs with BM were significantly older (P < .001), smaller (P < .001), and were more likely diagnosed with tracheal or mainstem bronchial collapse (P < .001) or bronchiectasis (P < .001). Multivariate analysis confirmed associations with age, tracheal or mainstem bronchial collapse, and bronchiectasis. In dogs with BM, PH was more prevalent. CONCLUSIONS AND CLINICAL IMPORTANCE: Although significantly more common in older, smaller dogs, BM occurs in dogs of all sizes and in all instances with comorbidities. Echocardiography should be considered in dogs with BM to identify PH.

Bacterial infection in dogs with aspiration pneumonia at 2 tertiary referral practices.

BACKGROUND: In dogs, antimicrobial drugs are widely prescribed for aspiration pneumonia (AP) despite poor documentation of bacterial infection in AP (b-AP) using bronchoalveolar lavage fluid (BALF) analysis. Interpretating discordant cytology and culture results is challenging, contributing to lack of a criterion standard, and highlighting differences between veterinary and human medical criteria for b-AP. OBJECTIVES: Determine how many dogs with AP had BALF collection and differences in diagnosis of b-AP using veterinary vs human medical criteria. Report findings of noninvasive markers (e.g. fever, band neutrophilia, radiographic severity score) in dogs with and without b-AP. ANIMALS: Retrospective cohort study of client-owned dogs (n = 429) with AP at 2 university veterinary hospitals. Twenty-four dogs met enrollment criteria. METHODS: Inclusion criteria were radiographic diagnosis of AP, ≥1 risk factor, CBC findings, and BALF cytology and culture results. Veterinary medical b-AP criteria were cytology findings compatible with sepsis with or without positive culture, or cytology findings not consistent with sepsis and positive culture (≥1.7 × 103  cfu/mL). Human medical b-AP criteria required culture with ≥104  cfu/mL or > 7% cells with intracellular bacteria on cytology. RESULTS: Only 24/429 dogs met all enrollment criteria; 379/429 dogs lacked BALF collection. Diagnosis of b-AP differed using veterinary (79%) vs human (29%) medical criteria. Fever, band neutrophils and high radiographic scores were noted in dogs with and without b-AP. CONCLUSIONS AND CLINICAL IMPORTANCE: Lack of routine BALF collection hampers definitive recognition of bacterial infection in AP. Differences in dogs meeting veterinary vs human medical definitions for b-AP and usefulness of noninvasive markers warrant further study to improve understanding of the role of bacteria in AP.

Comparison of Short- versus Long-Course Antimicrobial Therapy of Uncomplicated Bacterial Pneumonia in Dogs: A Double-Blinded, Placebo-Controlled Pilot Study.

Current treatment for canine bacterial pneumonia relies on protracted courses of antimicrobials (3-6 weeks or more) with recommendations to continue for 1-2 weeks past resolution of all clinical and thoracic radiographic abnormalities. However, in humans, bacterial pneumonia is often treated with 5-10-day courses of antimicrobials, and thoracic radiographs are not considered useful to guide therapeutic duration. The primary study objective was to determine whether a short course of antimicrobials would be sufficient to treat canine bacterial pneumonia. Eight dogs with uncomplicated bacterial pneumonia were enrolled in this randomized, double-blinded, placebo-controlled study comparing clinical and radiographic resolution with differing durations of antimicrobial therapy. Dogs received a course of antimicrobials lasting 10 (A10) or 21 (A21) days. Dogs randomized to the A10 group received placebo for 11 days following antimicrobial therapy. Patients were evaluated at presentation and 10, 30 and 60 days after the initiation of antimicrobials. At 10 days, 6/8 dogs had resolution of both clinical signs and inflammatory leukogram, and 5/8 dogs had improved global radiographic scores. After 60 days, clinical and hematologic resolution of pneumonia was noted in all dogs regardless of antimicrobial therapy duration; however, 3/8 dogs had persistent radiographic lesions. Thoracic radiographs do not appear to be a reliable marker to guide antimicrobial therapy in canine bacterial pneumonia as radiographic lesions may lag or persist despite clinical cure. This pilot study suggests a 10-day course of antimicrobials may be sufficient to treat uncomplicated canine bacterial pneumonia.

Serum allergen-specific IgE reactivity: is there an association with clinical severity and airway eosinophilia in asthmatic cats?

OBJECTIVES: The aim of this study was to evaluate the results of serum allergen-specific IgE testing in cats with a clinical diagnosis of asthma and to determine if the number of allergens with positive IgE reactivity and magnitude of positive IgE responses would be associated with the severity of clinical signs or airway eosinophilia. METHODS: Medical records from 2008 to 2018 were retrospectively reviewed. Inclusion required a diagnosis of feline asthma based on consistent clinicopathologic features and bronchoalveolar lavage (BAL) cytology with >10% eosinophils; additionally, cats needed to have the results of serum allergen-specific IgE tests. RESULTS: Eighteen cases satisfied the inclusion criteria. Median age was 5 years and the most common presenting clinical sign was cough (n = 10/18). Most cats lived exclusively indoors (n = 13/18). The median percentage of BAL eosinophils was 47%. Serum allergen-specific IgE testing supported an underlying allergic etiology in 14/18 (78%) cats, with all but one having polysensitization. The severity of clinical signs and magnitude of airway eosinophilia did not correlate with the degree of positive IgE reactivity. CONCLUSIONS AND RELEVANCE: This study identified a strong association between the identification of allergen-specific IgE and cats with asthma, and the majority of these cats were polysensitized. However, larger numbers of allergens with positive IgE reactivity or magnitude of IgE reactivity were not significantly associated with clinical severity or airway eosinophilia. Knowledge of positive allergen-specific IgE results could guide allergen avoidance, regardless of the magnitude of IgE reactivity.

Respiratory Dysbiosis in Canine Bacterial Pneumonia: Standard Culture vs. Microbiome Sequencing.

It is unknown how the respiratory microbiome influences and is influenced by bacterial pneumonia in dogs, as culture of lung samples and not microbial sequencing guides clinical practice. While accurate identification of pathogens are essential for treatment, not all bacteria are cultivable and the impact of respiratory dysbiosis on development of pneumonia is unclear. The study purposes were to (1) characterize the lung microbiome in canine bacterial pneumonia and compare deviations in dominant microbial populations with historical healthy controls, (2) compare bacteria identified by culture vs. 16S rDNA sequencing from bronchoalveolar lavage fluid (BALF) culture-, and (3) evaluate similarities in lung and oropharyngeal (OP) microbial communities in community-acquired and secondary bacterial pneumonia. Twenty BALF samples from 15 client-owned dogs diagnosed with bacterial pneumonia were enrolled. From a subset of dogs, OP swabs were collected. Extracted DNA underwent PCR of the 16S rRNA gene. Relative abundance of operational taxonomic units (OTUs) were determined. The relative abundance of bacterial community members found in health was decreased in dogs with pneumonia. Taxa identified via culture were not always the dominant phylotype identified with sequencing. Dogs with community-acquired pneumonia were more likely to have overgrowth of a single organism suggesting loss of dominant species associated with health. Dogs with secondary bacterial pneumonia had a greater regional continuity between the upper and lower airways. Collectively, these data suggest that dysbiosis occurs in canine bacterial pneumonia, and culture-independent techniques may provide greater depth of understanding of the changes in bacterial community composition that occur in disease.

Perspectives in veterinary medicine: Description and classification of bronchiolar disorders in cats.

This Perspectives in Veterinary Medicine article seeks to define, describe putative causes, and discuss key diagnostic tests for primary and secondary bronchiolar disorders to propose a classification scheme in cats with support from a literature review and case examples. The small airways (bronchioles with inner diameters <2 mm), located at the transitional zone between larger conducting airways and the pulmonary acinus, have been overlooked as major contributors to clinical syndromes of respiratory disease in cats. Because the trigger for many bronchiolar disorders is environmental and humans live in a shared environment with similar susceptibility, understanding these diseases in pet cats has relevance to One Health. Thoracic radiography, the major imaging modality used in the diagnostic evaluation of respiratory disease in cats, has low utility in detection of bronchiolar disease. Computed tomography (CT) with paired inspiratory and expiratory scans can detect pathology centered on small airways. In humans, treatment of bronchiolar disorders is not well established because of heterogeneous presentations and often late definitive diagnosis. A review of the human and veterinary medical literature will serve as the basis for a proposed classification scheme in cats. A case series of cats with CT or histopathologic evidence of bronchiolar lesions or both, either as a primary disorder or secondary to extension from large airway disease or interstitial lung disease, will be presented. Future multi-institutional and multidisciplinary discussions among clinicians, radiologists, and pathologists will help refine and develop this classification scheme to promote early and specific recognition and optimize treatment.

Effect of blood contamination on results of dipstick evaluation and urine protein-to-urine creatinine ratio for urine samples from dogs and cats.

OBJECTIVE To evaluate effects of blood contamination on dipstick results, specific gravity (SG), and urine protein-to-urine creatinine ratio (UPCR) for urine samples from dogs and cats. SAMPLE Urine samples collected from 279 dogs and 120 cats. PROCEDURES Urine pools were made for each species (dogs [n = 60] and cats [30]). Blood was added to an aliquot of a pool, and serial dilutions were prepared with the remaining urine. Color and dipstick variables were recorded, and SG and UPCR were measured. For cats, 1 set of pools was used; for dogs, 2 sets were used. Comparisons were made between undiluted urine and spiked urine samples for individual colors. Repeated-measures ANOVA on ranks was used to compare dipstick scores and UPCR results; χ2 tests were used to compare proteinuria categorizations (nonproteinuric, borderline, or proteinuric). RESULTS Any blood in the urine resulted in significantly increased dipstick scores for blood. In both species, scores for bilirubin and ketones, pH, and SG were affected by visible blood contamination. No significant difference for the dipstick protein reagent results was evident until a sample was visibly hematuric. The UPCR was significantly increased in dark yellow samples of both species. Proteinuria categorizations differed significantly between undiluted urine and urine of all colors, except light yellow. CONCLUSIONS AND CLINICAL RELEVANCE Any degree of blood contamination affected results of dipstick analysis. Effects depended on urine color and the variable measured. Microscopic blood contamination may affect the UPCR; thus, blood contamination may be a differential diagnosis for proteinuria in yellow urine samples.

Oral Probiotics Alter Healthy Feline Respiratory Microbiota.

Probiotics have been advocated as a novel therapeutic approach to respiratory disease, but knowledge of how oral administration of probiotics influences the respiratory microbiota is needed. Using 16S rRNA amplicon sequencing of bacterial DNA our objective was to determine whether oral probiotics changed the composition of the upper and lower airway, rectal, and blood microbiota. We hypothesized that oral probiotics would modulate the respiratory microbiota in healthy cats, demonstrated by the detection and/or increased relative abundance of the probiotic bacterial species and altered composition of the microbial population in the respiratory tract. Six healthy young research cats had oropharyngeal (OP), bronchoalveolar lavage fluid (BALF), rectal, and blood samples collected at baseline and 4 weeks after receiving oral probiotics. 16S rRNA gene amplicon libraries were sequenced, and coverage, richness, and relative abundance of representative operational taxonomic units (OTUs) were determined. Hierarchical and principal component analyses (PCA) demonstrated relatedness of samples. Mean microbial richness significantly increased only in the upper and lower airways. The number of probiotic OTUs (out of 5 total) that significantly increased in relative abundance vs. baseline was 5 in OP, 3 in BAL and 2 in feces. Using hierarchical clustering, BALF and blood samples grouped together after probiotic administration, and PERMANOVA supported that these two sites underwent significant changes in microbial composition. PERMANOVA revealed that OP and rectal samples had microbial population compositions that did not significantly change. These findings were visualized via PCA, which revealed distinct microbiomes in each site; samples clustered more tightly at baseline and had more variation after probiotic administration. This is the first study describing the effect of oral probiotics on the respiratory microbiota via detection of probiotic species in the airways. Finding bacterial species present in the oral probiotics in the upper and lower airways provides pilot data suggesting that oral probiotics could serve as a tool to target dysbiosis occurring in inflammatory airway diseases such as feline asthma, a disease in which cats serve as an important comparative and translational model for humans.

Dynamic changes of the respiratory microbiota and its relationship to fecal and blood microbiota in healthy young cats.

Advances in the field of metagenomics using culture-independent methods of microbial identification have allowed characterization of rich and diverse communities of bacteria in the lungs of healthy humans, mice, dogs, sheep and pigs. These data challenge the long held belief that the lungs are sterile and microbial colonization is synonymous with pathology. Studies in humans and animals demonstrate differences in the composition of airway microbiota in health versus disease suggesting respiratory dysbiosis occurs. Using 16S rRNA amplicon sequencing of DNA extracted from rectal and oropharyngeal (OP) swabs, bronchoalveolar lavage fluid (BALF), and blood, our objective was to characterize the fecal, OP, blood, and lower airway microbiota over time in healthy cats. This work in healthy cats, a species in which a respiratory microbiota has not yet been characterized, sets the stage for future studies in feline asthma in which cats serve as a comparative and translational model for humans. Fecal, OP and BALF samples were collected from six healthy research cats at day 0, week 2, and week 10; blood was collected at week 10. DNA was extracted, amplified via PCR, and sequenced using the Illumina MiSeq platform. Representative operational taxonomic units (OTUs) were identified and microbial richness and diversity were assessed. Principal component analysis (PCA) was used to visualize relatedness of samples and PERMANOVA was used to test for significant differences in microbial community composition. Fecal and OP swabs provided abundant DNA yielding a mean±SEM of 65,653±6,145 and 20,6323±4,360 sequences per sample, respectively while BALF and blood samples had lower coverage (1,489±430 and 269±18 sequences per sample, respectively). Oropharyngeal and fecal swabs were significantly richer than BALF (mean number OTUs 93, 88 and 36, respectively; p < 0.001) with no significant difference (p = 0.180) in richness between time points. PCA revealed site-specific microbial communities in the feces, and upper and lower airways. In comparison, blood had an apparent compositional similarity with BALF with regard to a few dominant taxa, but shared more OTUs with feces. Samples clustered more by time than by individual, with OP swabs having subjectively greater variation than other samples. In summary, healthy cats have a rich and distinct lower airway microbiome with dynamic bacterial populations. The microbiome is likely to be altered by factors such as age, environmental influences, and disease states. Further data are necessary to determine how the distinct feline microbiomes from the upper and lower airways, feces and blood are established and evolve. These data are relevant for comparisons between healthy cats and cats with respiratory disease.