The impact of urine collection method on canine urinary microbiota detection: a cross-sectional study.

BACKGROUND: The urinary tract harbors unique microbial communities that play important roles in urogenital health and disease. Dogs naturally suffer from several of the same urological disorders as humans (e.g., urinary tract infections, neoplasia, urolithiasis) and represent a valuable translational model for studying the role of urinary microbiota in various disease states. Urine collection technique represents a critical component of urinary microbiota research study design. However, the impact of collection method on the characterization of the canine urinary microbiota remains unknown. Therefore, the objective of this study was to determine whether urine collection technique alters the microbial populations detected in canine urine samples. Urine was collected from asymptomatic dogs by both cystocentesis and midstream voiding. Microbial DNA was isolated from each sample and submitted for amplicon sequencing of the V4 region of the bacterial 16 S rRNA gene, followed by analyses to compare microbial diversity and composition between urine collection techniques. RESULTS: Samples collected via midstream voiding exhibited significantly higher sequence read counts (P = .036) and observed richness (P = .0024) than cystocentesis urine. Bray Curtis and Unweighted UniFrac measures of beta diversity showed distinct differences in microbial composition by collection method (P = .0050, R2 = 0.06 and P = .010, R2 = 0.07, respectively). Seven taxa were identified as differentially abundant between groups. Pasteurellaceae, Haemophilus, Friedmanniella, two variants of Streptococcus, and Fusobacterium were over-represented in voided urine, while a greater abundance of Burkholderia-Caballeronia-Paraburkholderia characterized cystocentesis samples. Analyses were performed at five thresholds for minimum sequence depth and using three data normalization strategies to validate results; patterns of alpha and beta diversity remained consistent regardless of minimum read count requirements or normalization method. CONCLUSION: Microbial composition differs in canine urine samples collected via cystocentesis as compared to those collected via midstream voiding. Future researchers should select a single urine collection method based on the biological question of interest when designing canine urinary microbiota studies. Additionally, the authors suggest caution when interpreting results across studies that did not utilize identical urine collection methods.

Dietary Features Are Associated with Differences in the Urinary Microbiome in Clinically Healthy Adult Dogs.

Nutrition plays an important role in shaping the gut microbiome composition, although the impact of diet on the urinary microbiome (i.e., urobiome) remains unknown. The aim of this pilot study was to discover how nutritional features affect the diversity and composition of the urobiome in dogs. Dietary histories were obtained for 15 clinically healthy adult dogs, including limited nutrient (protein, fat, crude fiber), commercial diet brand, and dietary diversity profiles. The urine samples were collected via cystocentesis, followed by sequencing of the bacterial 16S rRNA gene. The data were analyzed to determine associations between major nutrients and dietary sources with the urobiome's composition. The protein, fat, and crude fiber contents had no statistically significant effect on the alpha or beta diversity. However, the beta diversity values differed (PERMANOVA; p = 0.017, R2 = 0.10) between dogs fed one commercial diet brand compared to dogs consuming any other brand. The beta diversity values also differed (p = 0.019, R2 = 0.10) between dogs consuming more diverse daily diets compared to those consuming less diverse diets (≥3 or <3 unique food sources, respectively). Overall, the results of this pilot study suggest that diet might impact the urobiome and support further exploration of the relationship between diet and the urobiome's composition in dogs.

A Novel CARMIL2 Immunodeficiency Identified in a Subset of Cavalier King Charles Spaniels with Pneumocystis and Bordetella Pneumonia.

Pet dogs are a valuable natural animal model for studying relationships between primary immunodeficiencies and susceptibility to Pneumocystis and other opportunistic respiratory pathogens. Certain breeds, such as the Cavalier King Charles Spaniel, are over-represented for Pneumocystis pneumonia (PCP), suggesting the presence of a primary immunodeficiency in the breed. Here, we report the discovery of a CARMIL2 nonsense variant in three Cavalier King Charles Spaniel dogs with either PCP (n = 2) or refractory Bordetella pneumonia (n = 1). CARMIL2 encodes a protein that plays critical roles in T-cell activation and other aspects of immune function. Deleterious CARMIL2 variants have recently been reported in human patients with PCP and other recurrent pneumonias. In addition to opportunistic respiratory infection, the affected dogs also exhibited other clinical manifestations of CARMIL2 deficiencies that have been reported in humans, including early-onset gastrointestinal disease, allergic skin disease, mucocutaneous lesions, abscesses, autoimmune disorders, and gastrointestinal parasitism. This discovery highlights the potential utility of a natural canine model in identifying and studying primary immunodeficiencies in patients affected by PCP.

A scoping review of autoantibodies as biomarkers for canine autoimmune disease.

BACKGROUND: Autoantibody biomarkers are valuable tools used to diagnose and manage autoimmune diseases in dogs. However, prior publications have raised concerns over a lack of standardization and sufficient validation for the use of biomarkers in veterinary medicine. OBJECTIVES: Systematically compile primary research on autoantibody biomarkers for autoimmune disease in dogs, summarize their methodological features, and evaluate their quality; synthesize data supporting their use into a resource for veterinarians and researchers. ANIMALS: Not used. METHODS: Five indices were searched to identify studies for evaluation: PubMed, CAB Abstracts, Web of Science, Agricola, and SCOPUS. Two independent reviewers (AET and ELC) screened titles and abstracts for exclusion criteria followed by full-text review of remaining articles. Relevant studies were classified based on study objectives (biomarker, epitope, technique). Data on study characteristics and outcomes were synthesized in independent data tables for each classification. RESULTS: Ninety-two studies qualified for final analysis (n = 49 biomarker, n = 9 epitope, and n = 34 technique studies). A high degree of heterogeneity in study characteristics and outcomes reporting was observed. Opportunities to strengthen future studies could include: (1) routine use of negative controls, (2) power analyses to inform sample sizes, (3) statistical analyses when appropriate, and (4) multiple detection techniques to confirm results. CONCLUSIONS: These findings provide a resource that will allow veterinary clinicians to efficiently evaluate the evidence supporting the use of autoantibody biomarkers, along with the varied methodological approaches used in their development.

Characterization of the urogenital microbiome in Miniature Schnauzers with and without calcium oxalate urolithiasis.

BACKGROUND: Calcium oxalate (CaOx) uroliths are common in dogs. Humans with CaOx urolithiasis exhibit alterations of the urinary and urogenital microbiomes that might mediate urolith formation. Detection of urogenital microbes associated with CaOx in dogs could inform disease pathophysiology. OBJECTIVE: To identify compositional differences in the urogenital microbiome of Miniature Schnauzers with and without CaOx uroliths. ANIMALS: Nineteen midstream, voided urine samples from Miniature Schnauzers with (n = 9) and without (n = 10) a history of CaOx urolithiasis. METHODS: Analytical cross-sectional study. Microbial DNA was extracted from previously frozen urine samples and sequenced for the bacterial 16S rRNA V3-V4 hypervariable regions. Diversity and composition of microbial populations were compared between urolith formers and controls. RESULTS: Alpha and beta diversity measures were similar between groups. Five individual bacterial taxa differed in abundance (indicator values >0.5 and P < .05): Acinetobacter, 2 Geobacillus variants, and Hydrogenophaga were overrepresented in the urine of urolith formers, and Sphingopyxis was overrepresented in controls. Two distinct subtypes of urine microbial composition were observed based on beta diversity measures, independent of urolith status, and other clinical variables. CONCLUSIONS AND CLINICAL IMPORTANCE: Although we did not detect a difference in the overall urogenital microbial composition between groups, observed differences in individual bacterial taxa might be clinically relevant. For example, Acinetobacter was overrepresented in urolith formers and is associated with CaOx urolithiasis in humans. Two unique clusters of the microbiome were identified, independent of urolith status, which may represent distinct urotypes present in Miniature Schnauzers.

Comparison of immediate versus delayed streak plate inoculation on urine bacterial culture and susceptibility testing in dogs and cats.

BACKGROUND: Quantitative bacterial culture and susceptibility testing is the gold standard diagnostic for determining bacterial urinary tract infection. Transport of samples to external reference laboratories is common practice in veterinary medicine. OBJECTIVE: To compare bacterial culture and susceptibility results from clinical urine samples when streak plate inoculation is performed immediately after sample collection versus after transport to a reference laboratory. To determine the clinical implications of discrepant culture results. ANIMALS: One hundred and ninety-four canine and 45 feline urine samples that were submitted for urinalysis and urine culture and susceptibility testing. METHODS: This was a prospective, cross-sectional study. Streak plate inoculations were performed on urine samples immediately after collection and also after transport to a reference laboratory. Samples were stored in plain sterile tubes and refrigerated up to 24 hours before transport. Culture results were compared, and discordant results were evaluated for clinical relevance. Signalment, comorbidities, lower urinary tract signs, and antimicrobial history were recorded. RESULTS: Kappa coefficient for agreement between plating methods was 0.884. Twenty-two (71%) of 31 discrepant results were determined to have no clinical impact. Though 35% of clean midstream samples had discrepant culture results, only 8% of these had clinical impact. Conversely, 8.6% from cystocentesis were discrepant, but 41% of these had clinical impact. CONCLUSIONS AND CLINICAL IMPORTANCE: Provided urine samples are stored and transported appropriately, the immediate preplating of urine for culture and susceptibility testing is unnecessary in the majority of cases. Despite more discrepancies in plating methods for midstream samples, the minority were of clinical importance.