Relative expression of genes associated with adhesion to bovine mammary epithelial cells by Streptococcus uberis
Introduction
Bovine mastitis, the inflammation of the mammary gland, is considered the most prevalent disease in dairy cattle since it affects dairy herds worldwide (Bogni et al., 2011). It causes a decline in milk production and quality, with high treatment costs or early culling of animals, and is therefore responsible for significant losses in dairy farms (Petrovski et al., 2006). Streptococcus uberis is one of the most prevalent environmental pathogens responsible for a significant proportion of clinical and subclinical bovine intramammary infections (IMI) in lactating and non-lactating cows (Zadoks, 2007). Many species of streptococci that colonize mammals exist naturally within communities of bacteria growing as biofilms (Gomes et al., 2016). They vary in their propensities to form biofilm, but in all cases, this ability depends first upon the adherence of cells to a surface (Nobbs et al., 2009). Previously, we demonstrated that S. uberis strains isolated from cows with mastitis from the central dairy region of Argentina were able to produce biofilm (Dieser et al., 2017). The ability to adhere to mammary gland epithelia is an important strategy for many bovine pathogens, including S. uberis, because it might provide an advantage to overcome other bacteria and colonize the lactating mammary gland despite the flow of milk (Leigh, 1999; Tassi et al., 2015). Several authors have demonstrated that multiple adhesins are involved in the binding of S. uberis to the surface of host cells and extracellular matrix components (Almeida et al., 2006; Leigh et al., 2010; Kerro Dego et al., 2011). Research conducted in our laboratory has shown that the adherence genes acdA, lmb, scpA, sua, fbp and lbp are highly prevalent in S. uberis isolates from clinical and subclinical bovine mastitis from the central dairy region of Argentina (Fessia et al., 2019). In addition, we found that their nucleotide and aminoacidic sequences are conserved among field strains in spite of the wide clonal heterogeneity detected. There are currently no studies characterizing gene expression in S. uberis from bovine mastitis in the presence of host cells. We hypothesized that upon contact with bovine mammary epithelial cells, S. uberis upregulates some adherence-associated genes that express factors which enable it to adhere to the host and colonize mammary glands. Hence, the objectives of this study were (a) to select S. uberis strains regarding their ability to form biofilm (b) to determine their ability to adhere to and internalize into MAC-T cells, and (c) to investigate the expression of adherence-associated genes in these strains during early bacterial interactions with the host.
Section snippets
Bacterial strains and identification
Streptococcus uberis strains (n = 34) analyzed in this study were isolated from the milk of Holstein cows with clinical and subclinical mastitis from 17 herds located in the central dairy region of Argentina. Mastitis were characterized as clinical (presence of sign in milk as clots, and/or in the udder as swelling) or subclinical (milk somatic cell count of SCC > 250,000 cells/ml, without clinical sign in the milk or the udder). Bacteria were presumably identified as S. uberis according to
In vitro biofilm formation
In the present study, 34 clinical and subclinical bovine isolates of S. uberis were screened for their ability to produce biofilm in THY medium. Results showed that most of the isolates were able to form biofilm in vitro in this medium. We observed that out of the total S. uberis isolates, three (8.82%), twenty-six (76.48%) and five (14.70%) of them were qualitatively classified as strong, moderate, and weak biofilm producers, respectively. Meanwhile, five (14.70%) isolates were sorted as
Discussion
Adherence to and internalization into the epithelium of the mammary gland are two important events in early S. uberis pathogenesis and have been extensively investigated in in vitro studies by several authors (Almeida et al., 1999; Tamilselvam et al., 2006; Patel et al., 2009; Chen et al., 2011). Although biofilm forming properties have been well demonstrated for S. uberis strains in varying degrees, in all cases biofilm formation depends chiefly upon the adherence of bacterial cells to a
Conclusion
In conclusion, 2 to 6 genes were significantly up-regulated in 6 strains and down-regulated in 5 strains, irrespectively of the clinical or subclinical origin, after the first contact between bacteria and MAC-T cells. Findings found about the relative expression of the lmb and fbp genes could indicate that initial contact between S. uberis and MAC-T cells promotes gene induction, suggesting that these genes have a potential role during early bacteria-host cells interactions. In short, our
Declaration of Competing Interest
None of the authors of this paper has any financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.
Acknowledgements
This work was supported by Argentine National Agency for the Promotion of Science and Technology (ANPCyT) (PICT 2336/2012). A. Fessia is a doctoral fellow from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
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