Characterization of the bovine salivary gland transcriptome associated with Mycobacterium avium subsp. paratuberculosis experimental challenge

Abstract

Background: Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of Johne's disease is spread between cattle via the fecal-oral route, yet the functional changes in the salivary gland associated with infection remain uncharacterized. In this study, we hypothesized that experimental challenge with MAP would induce stable changes in gene expression patterns in the salivary gland that may shed light on the mucosal immune response as well as the regional variation in immune capacity of this extensive gland. Holstein-Friesian cattle were euthanized 33 months' post oral challenge with MAP strain CIT003 and both the parotid and mandibular salivary glands were collected from healthy control (n = 5) and MAP exposed cattle (n = 5) for histopathological and transcriptomic analysis.

Results: A total of 205, 21, 61, and 135 genes were significantly differentially expressed between control and MAP exposed cattle in dorsal mandibular (M1), ventral mandibular (M2), dorsal parotid (P1) and ventral parotid salivary glands (P2), respectively. Expression profiles varied between the structurally divergent parotid and mandibular gland sections which was also reflected in the enriched biological pathways identified. Changes in gene expression associated with MAP exposure were detected with significantly elevated expression of BoLA DR-ALPHA, BOLA-DRB3 and complement factors in MAP exposed cattle. In contrast, reduced expression of genes such as polymeric immunoglobin receptor (PIGR), TNFSF13, and the antimicrobial genes lactoferrin (LF) and lactoperoxidase (LPO) was detected in MAP exposed animals.

Conclusions: This first analysis of the transcriptomic profile of salivary glands in cattle adds an important layer to our understanding of salivary gland immune function. Transcriptomic changes associated with MAP exposure have been identified including reduced LF and LPO. These critical antimicrobial and immunoregulatory proteins are known to be secreted into saliva and their downregulation may contribute to disease susceptibility. Future work will focus on the validation of their expression levels in saliva from additional cattle of known infection status as a potential strategy to augment disease diagnosis.

Keywords: Biomarkers; Cattle; Johne’s disease; RNA-Seq; Saliva; Salivary glands.

Fig. 1

Principal Component Analysis (PCA) plot of the DEG dataset in Dorsal mandibular salivary gland extremity (M1); Ventral mandibular salivary gland extremity (M2); Dorsal parotid salivary gland extremity (P1) and Ventral parotid salivary gland extremity (P2) from control and MAP exposed cattle. The control (red) and MAP exposed (blue) samples are plotted along the first two principal component axes (PC1 and PC2)

Fig. 2

a Salivary glands sampling. After euthanasia, the head was positioned upside down and the skin between jaws was incised using sterile disposable scalpel. Then, diagonal incision was made from the ear to join the first incision and the skin was removed from one side to expose the adjacent tissues. Fatty tissue was incised at the site of targeted salivary glands. Parotid and mandibular glands were located at one side and two samples were collected at dorsal and ventral anatomical sections from each gland. b: a: Parotid gland; Pure serous acini consisting of rectangular granular cells with central nuclei. Central lumen hardly visible (yellow arrow). Striated duct with columnar cells with central nuclei and basal-striated appearance (red arrow). b Mandibular gland; Pure serous acini consisting of triangular granular cells with their base directed outwards and basal nuclei (yellow arrow). Mixed seromucous acini with crescents of Giannuzzi (red arrow). Bar length 20 um

Fig. 3

a Venn diagram comparing the number of DEGs identified in M1 and M2 salivary gland regions along with the intersection indicating the number of common DEGs. up = upregulated or down = downregulated in corresponding salivary gland group. b Venn diagram comparing the number of DEGs identified in P1 and P2 salivary gland along with the intersection indicating the number of common DEGs. up = upregulated or down = downregulated in corresponding salivary gland group

Fig. 4

Volcano plot of differential expression (−log10 p-value vs log2fold change) in dorsal mandibular salivary gland (M1) (a), ventral mandibular salivary gland extremity (M2) (b), dorsal parotid salivary gland (P1) (c) and ventral parotid salivary gland extremity (P2) (d), respectively. Genes with an FDR < 0.05 are highlighted in black with top 30 of them labeled by their names

Fig. 5

a Expression of Polymeric Immunoglobulin Receptor (PIGR) in salivary glands (salivary gland group in the paranthesis). The expression was downregulated in MAP infected animals in all the salivary gland groups; b Expression of lactoperoxidase (LPO) in M1 and M2 salivary gland groups (salivary gland group in the paranthesis). LPO expression was downregulated in MAP-infected animals in M1 and M2 salivary gland groups; c Expression of lactoferrin (LF) in P1 salivary gland group (salivary gland group in the paranthesis). LF expression was downregulated in MAP-infected animals in P1 salivary gland group

Fig. 6

Biological processes enriched among DEGs in dorsal mandibular salivary gland extremity (M1) (a), ventral mandibular salivary gland extremity (M2) (b), dorsal parotid salivary gland extremity (P1) (c) and ventral parotid salivary gland extremity (P2) (d)