Effects of respiratory disease on Kele piglets lung microbiome, assessed through 16S rRNA sequencing

Abstract

Background and aim: Due to the incomplete development of the immune system in immature piglets, the respiratory tract is susceptible to invasion by numerous pathogens that cause a range of potential respiratory diseases. However, few studies have reported the changes in pig lung microorganisms during respiratory infection. Therefore, we aimed to explore the differences in lung environmental microorganisms between healthy piglets and piglets with respiratory diseases.

Materials and methods: Histopathological changes in lung sections were observed in both diseased and healthy pigs. Changes in the composition and abundance of microbiomes in alveolar lavage fluid from eleven 4-week-old Chinese Kele piglets (three clinically healthy and eight diseased) were studied by IonS5™ XL sequencing of the bacterial16S rRNA genes.

Results: Histopathological sections showed that diseased pigs displayed more lung lesions than healthy pigs. Diseased piglets harbored lower bacterial operational taxonomic units, α-diversity, and bacterial community complexity in comparison to healthy piglets. Taxonomic composition analysis showed that in the diseased piglets, the majority of flora was composed of Ureaplasma, Mycoplasma, and Actinobacillus; while Actinobacillus, Sphingomonas, and Stenotrophomonas were dominant in the control group. The abundance of Ureaplasma was significantly higher in ill piglets (p<0.05), and the phylogenetic tree indicated that Ureaplasma was clustered in Ureaplasma diversum, a conditional pathogen that has the potential to affect the swine respiratory system.

Conclusion: The results of this study show that the microbial species and structure of piglets' lungs were changed during respiratory tract infection. The finding of Ureaplasma suggested that besides known pathogens such as Mycoplasma and Actinobacillus, unknown pathogens can exist in the respiratory system of diseased pigs and provide a potential basis for clinical treatment.

Keywords: 16S rRNA sequencing; Kele piglets; Ureaplasma; Ureaplasma diversum; microbial diversity; respiratory diseases.

Figure-S1

The histopathological changes in experimental pigs (a-f: diseased pigs; g-j: healthy pigs). (a) Mesenteric congestion and enlargement of intestinal lymph nodes. (b and c) Pleural effusion and pulmonary adhesion. (d) Enlargement and congestion of inguinal lymph nodes. (e-f) Foam-like substances present in trachea. (g) Histological appearances of lung, trachea, mesenteric congestion, and intestinal and inguinal lymph nodes were normal.

Figure-1

HE staining of lung tissue lesions in experimental pigs. (a and c) Lung tissue lesions of healthy and diseased pigs, respectively, at 100 µm; (b and d) lung tissue lesions of healthy and diseased pigs, respectively, at 50 µm.

Figure-S2

Statistical histogram of reads alignment for all samples.

Figure-2

Relationship among sequencing data, sample size, and species diversity. Different samples are represented by curves of different colors. (a) Dilution curve. (b) Species accumulation box chart.

Figure-3

β-diversity analysis. (a) PCoA analysis. The red symbols indicate diseased piglets and the blue symbols indicate healthy piglets. (b) UPGMA clustering tree. The left side is the UPGMA clustering tree structure, and the right side is the relative abundance distribution of the species at the phylum level. Red lines and letters indicate diseased piglets and blue lines and letters indicate healthy piglets.

Figure-4

Species annotation results of family level for different groups and t-test. (a) The abundance distribution of the top 10 species in each group. Mycoplasmataceae was the most abundant species in ill piglets. (b) t-test. Mycoplasmataceae had significantly higher abundance in ill piglets than in healthy piglets (p<0.01).

Figure-5

Circos graph of genus level for different samples and t-test. (a) The abundance distribution of the top 26 species in each sample. (b) t-test. Ureaplasma had significantly higher abundance in ill piglets than in healthy piglets (p<0.01).

Figure-S3

Species annotation results of phylum level for different groups and t-test. (a) The abundance distribution of the top 10 species in each group. Tenericutes was the most abundant phylum in ill piglets. (b) t-test. Tenericutes had significantly higher abundance in ill piglets than in healthy piglets (p<0.01).

Figure-S4

Species annotation results of class level for different groups and t-test. (a) The abundance distribution of the top 10 species in each group. Mollicutes was the most abundant class in ill piglets. (b) t-test. Mollicutes had significantly higher abundance in ill piglets than in healthy piglets (p<0.01).

Figure-S5

Species annotation results of order level for different groups and t-test. (a) The abundance distribution of the top 10 species in each group. Mycoplasmatales was the most abundant order in ill piglets. (b) t-test. Mycoplasmatales had significantly higher abundance in ill piglets than in healthy piglets (p<0.01).

Figure-6

LDA value distribution histogram and evolutionary branch diagram. The histogram for the distribution of LDA values shows species with LDA scores of log₁₀ >4. In the evolutionary branch diagram, the circle radiating from the inside to the outside represents the classification level from phylum to genus. Each small circle at the different classification levels represents a classification at that level, and the diameter of the small circle is proportional to the relative abundance. Species with no significant differences are colored yellow.

Figure-7

The analysis of microorganisms’ contribution. The vertical axis represents the genus; the horizontal axis represents the samples; the size of the circle represents the relative abundance of the species; “Contribution” is the contribution of the species to the difference observed between the two groups.

Figure-8

Molecular phylogenetic analysis based on 16S rRNA partial nucleotide sequences of species of the genus Ureaplasma.