Single-cell T-cell receptor repertoire profiling in dogs.

Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.

Characterization of the urinary microbiome in healthy dogs.

The urinary bladder in healthy dogs has dogmatically been considered free of bacteria. This study used culture independent techniques to characterize the healthy canine urinary microbiota. Urine samples collected by antepubic cystocentesis from dogs without urinary infection were used for DNA extraction. Genital tract and rectal samples were collected simultaneously from the same dogs. The V4 hypervariable region of the 16S rRNA bacterial gene was amplified and compared against Greengenes database for OTU assignment and relative abundance for urine, genital, and rectal samples. After excluding 4 dogs with cultivable bacteria, samples from 10 male (M; 1 intact) and 10 female (F) spayed dogs remained. All samples provided adequate genetic material for analysis. Four taxa (Pseudomonas sp., Acinetobacter sp., Sphingobium sp. and Bradyrhizobiaceae) dominated the urinary microbiota in all dogs of both sexes. These taxa were also detected in the genital swabs of both sexes, while the rectal microbiota differed substantially from the other sample sites. Principal component (PC) analysis of PC1 through PC3 showed overlap of urinary and genital microbiota and a clear separation of rectal swabs from the other sample sites along PC1, which explained 44.94% variation. Surprisingly, the urinary microbiota (mean # OTU 92.6 F, 90.2 M) was significantly richer than the genital (67.8 F, 66.6 M) or rectal microbiota (68.3 F, 71.2 M) (p < 0.0001), with no difference between sexes at any sample site. The canine urinary bladder is not a sterile environment and possesses its own unique and diverse microbiota compared to the rectal and genital microbiota. There was no difference between the sexes at any microbiota sample site (urine, genital, and rectal). The predominant bacterial genus for either sex in the urine and genital tracts was Pseudomonas sp.