Analysis of the lung microbiota in dogs with Bordetella bronchiseptica infection and correlation with culture and quantitative polymerase chain reaction

AbstractInfection with Bordetella bronchiseptica (Bb), a pathogen involved in canine infectious respiratory disease complex, can be confirmed using culture or qPCR. Studies about the canine lung microbiota (LM) are recent, sparse, and only one paper has been published in canine lung infection. In this study, we aimed to compare the LM between Bb infected and healthy dogs, and to correlate sequencing with culture and qPCR results. Twenty Bb infected dogs diagnosed either by qPCR and/or culture and 4 healthy dogs were included. qPCR for Mycoplasma cynos (Mc) were also available in 18 diseased and all healthy dogs. Sequencing results, obtained from bronchoalveolar lavage fluid after DNA extraction, PCR targeting the V1–V3 region of the 16S rDNA and sequencing, showed the presence of Bb in all diseased dogs, about half being co-infected with Mc. In diseased compared with healthy dogs, the β-diversity changed (P = 0.0024); bacterial richness and α-diversity were lower (P = 0.012 and 0.0061), and bacterial load higher (P = 0.004). Bb qPCR classes and culture results correlated with the abundance of Bb (r = 0.71, P &lt; 0.001 and r = 0.70, P = 0.0022). Mc qPCR classes also correlated with the abundance of Mc (r = 0.73, P &lt; 0.001). Bb infection induced lung dysbiosis, characterized by high bacterial load, low richness and diversity and increased abundance of Bb, compared with healthy dogs. Sequencing results highly correlate with qPCR and culture results showing that sequencing can be reliable to identify microorganisms involved in lung infectious diseases.</jats:p

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

Additional file 1 of Analysis of the lung microbiota in dogs with Bordetella bronchiseptica infection and correlation with culture and quantitative polymerase chain reaction

Additional file 1. Comparison between the subpopulations of diseased dogs selected or not for the comparison with healthy dogs. Results of the TCC, DCC and all LM parameters comparison between the subpopulations of diseased dogs selected (n = 7) or not (n = 13) for the comparison with healthy dogs