ANALYSIS OF THE LUNG MICROBIOTA IN DOGS WITH BORDETELLA BRONCHISEPTICA INFECTION AND CORRELATION WITH CULTURE AND QUANTITATIVE POLYMERASE CHAIN REACTION

Purpose of the study: Infection with Bordetella bronchiseptica, one of the principal pathogens involved in canine infectious respiratory disease complex (CIRD-C), can be diagnosed using bacterial culture or quantitative polymerase chain reaction (qPCR). The lung microbiota (LM) has been described in healthy experimental dogs, while it has not yet been studied in dogs with lower respiratory infection. In the present study we aimed to analyze the LM in dogs with B. bronchiseptica infection in comparison with healthy dogs, and to assess the correlation between 16S rDNA amplicon sequencing and culture and qPCR/ and to correlate 16S rDNA amplicon sequencing with culture and qPCR results.
Methods used: Twenty dogs with B. bronchiseptica infection and 4 healthy age-matched dogs were retrospectively included. B. bronchiseptica infection was diagnosed based on either positive qPCR (n=19/19) and/or positive culture (n=11/17, including 1 dog with culture available only) in the bronchoalveolar lavage fluid (BALF). qPCR for Mycoplasma cynos were also available in 18 of the dogs with B. bronchiseptica infection and the 4 healthy dogs. qPCRs results were categorized into 6 classes based on the cycle threshold values. 16S rDNA sequences were obtained from naïve BALF after DNA extraction, PCR targeting the V1-V3 region of the 16S rDNA and sequencing. Sequences were mapped on the SILVA database with an OTU clustering distance of 0.03. Total bacterial load was calculated with a qPCR targeting the V2-V3 region of the 16S rDNA. Analysis of the LM were performed using MOTHUR v1.39 for the ecological data calculation including the β and α-diversity, the richness and the evenness. Mann-Whitney tests were used to compare the ecological data and the bacterial load. Welsh t-tests and FDR correction were used to compare the differences in relative abundances. Correlations between sequencing results at each taxonomic level and qPCRs or culture were done by Spearman tests.
Summary of the results: 16S rDNA sequencing results showed the presence of B. bronchiseptica in large amounts in all BALF samples in diseased dogs. Seven of them were co-infected with M. cynos, one dog with another species of Mycoplasma, and one with bacteria of the Pseudomonas genus. A shift in the β-diversity of the LM was observed in diseased compared with healthy dogs (P=0.002). Compared with healthy dogs, there were significantly more Burkholderiaceae at family level and Bordetella at genus level (75.92 ± 25.99 versus 2.94 ± 2.56%; P=0.058 and 75.65 ± 26.04 versus 0.11 ± 0.19%; P=0.10 respectively) in diseased dogs. The richness and the α-diversity were significantly lower (93.09 ± 76.19 versus 340.50 ± 158.68; P=0.012 and 1.54 ± 0.48 versus 17.74 ± 9.36; P=0.006 respectively) and the bacterial load higher (5.72 ± 0.38 versus 4.93 ± 0.15; P=0.004) in diseased compared with healthy dogs. B. bronchiseptica qPCR classes and positive culture results positively correlated with the relative abundance of B. bronchiseptica obtained by 16S rDNA amplicon sequencing (r= 0.56, P=0.028 and r=0.70, P=0.002 respectively). M. cynos qPCR classes also positively correlated with the relative abundance of M. cynos (r=0.84, P<0.0001).
Conclusions: CIRD-C induce a major dysbiosis of the LM, characterized by high bacterial load, low richness and diversity and increased abundance of B. bronchiseptica, in comparison with healthy dogs. Results of the LM analysis highly correlate with results obtained by specific qPCR and culture.