Elsevier

Research in Veterinary Science

Volume 132, October 2020, Pages 33-41
Research in Veterinary Science

Relative expression of genes associated with adhesion to bovine mammary epithelial cells by Streptococcus uberis

https://doi.org/10.1016/j.rvsc.2020.05.016Get rights and content

Highlights

  • The isolates were able to form biofilm in vitro in THY medium.

  • The nine strains were able to adhere to MAC-T cells.

  • S. uberis RC29 and RC37 exhibited the highest levels of adherence.

  • All strains were internalized, although at different levels.

  • We did not find a single profile of relative expression.

Abstract

Streptococcus uberis is one of the most prevalent environmental pathogens of bovine mastitis. Biofilm growth ability by S. uberis looks to depend first upon the adherence of cells to a surface. The S. uberis ability to adhere to mammary gland epithelia might provide an advantage to colonize the lactating mammary gland. The objectives of this study were (a) to select S.uberis strains according to their ability to form biofilm, (b) to determine adherence to and internalization into MAC-T cells and (c) to investigate the expression profile adherence genes in these S. uberis strains. For the assays, the MAC-T bovine mammary epithelial cell line was used. Relative expression of genes acdA, lmb, scpA, sua, fbp and lbp was quantified by RT-qPCR. We observed that the RC38 strain from clinical bovine mastitis showed in the six genes higher values than control in both conditions. While the strain with greater ability to adhere, from clinical mastitis and biofilm producer (RC29) evidenced higher values in group 1 (G1) (bacteria after the initial contact with MAC-T cells) and decrease in group 2 (G2) (both adhered and internalized bacteria) than control. Strains with a moderate or strong capacity for biofilm production showed significantly lower relative expression values in the G2. In all adherence associated genes, strain RC19 showed relative expression values incremented in G1, while in G2 decreased expression. In conclusion, we did not find a single profile of relative expression because the relative expression levels of each gene differed depending on the strain and the co-culture stage of S. uberis cells from which RNA was obtained.

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).

References (38)

  • O. Kerro Dego et al.

    PGh9:ISS1 transpositional mutations in Streptococcus uberis UT888 causes reduced bacterial adherence to and internalization into bovine mammary epithelial cells

    Vet. Microbiol.

    (2011)
  • R.A. Almeida et al.

    Adherence and internalization of Streptococcus uberis to bovine mammary epithelial cells are mediated by host cell proteoglycans

    FEMS Microbiol. Lett.

    (1999)
  • I. Moshynskyy et al.

    Characterization of a bovine lactoferrin binding protein of Streptococcus uberis

    Microb. Pathog.

    (2003)
  • J.A. Leigh

    Streptococcus uberis: a permanent barrier to the control of bovine mastitis?

    Vet. J.

    (1999)
  • E.L. Rosey et al.

    PauA: a novel plasminogen activator from Streptococcus uberis

    FEMS Microbiol. Lett.

    (1999)
  • P.N. Ward et al.

    Identification and disruption of two discrete loci encoding hyaluronic acid capsule biosynthesis genes hasA, hasB, and hasC in Streptococcus uberis

    Infect. Immun.

    (2001)
  • Y. Terao et al.

    Novel laminin-binding protein of Streptococcus pyogenes, Lbp, is involved in adhesion to epithelial cells

    Infect. Immun.

    (2002)
  • M.W. Pfaffl et al.

    Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--excel-based tool using pair-wise correlations

    Biotechnol. Lett.

    (2004)
  • K.R. Petrovski et al.

    A review of the factors affecting the costs of bovine mastitis

    J. S. Afr. Vet. Assoc.

    (2006)
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