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

Abstract

Infection 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 < 0.001 and r = 0.70, P = 0.0022). Mc qPCR classes also correlated with the abundance of Mc (r = 0.73, P < 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.

Figure 1

Species-level composition of the lung microbiota in dogs affected withB. bronchiseptica. Bar charts showing relative abundance annotated to the taxonomic level of species of all taxa detected in the bronchoalveolar lavage fluid of 20 dogs affected with B. bronchiseptica.

Figure 2

Taxa detected in healthy dogs and dogs affected withB. bronchiseptica. Bar charts showing the relative abundance of all taxa detected in the bronchoalveolar lavage fluid of 4 healthy dogs and 7 dogs affected with B. bronchiseptica, annotated to the taxonomic level of phylum (A), family (B), genus (C) and species (D).

Figure 3

Principal component analysis representing the β-diversity between healthy dogs and dogs affected withB. bronchiseptica. Lung communities are clustered by groups (diseased (n = 7) and healthy (n = 4) dogs).

Figure 4

Ecological parameters comparison between healthy dogs and dogs affected withB. bronchiseptica. Box plot graphs representing the bacterial alpha diversity (A), richness (B) and evenness (C) in healthy (n = 4) compared with diseased dogs (n = 7). The medians are represented by the central horizontal bars. The lower and upper limits of the box are the first and third quartiles, respectively. *P = 0.012; **P = 0.006.

Figure 5

Bacterial load in healthy dogs and dogs affected withB. bronchiseptica. Box plot representing the logarithm of the number of 16S rDNA copies per microliter (bacterial load) between healthy (n = 4) and diseased dogs (n = 7). The medians are represented by the central horizontal bars. The lower and upper limits of the box are the first and third quartiles, respectively. *P = 0.004.