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Transcriptome sequencing analysis for the identification of stable lncRNAs associated with bovine Staphylococcus aureus mastitis.

ABSTRACT: Staphylococcus aureus (S. aureus) mastitis is one of the most difficult diseases to treat in lactating dairy cows worldwide. S. aureus with different lineages leads to different host immune responses. Long non-coding RNAs (lncRNAs) are reported to be widely involved in the progress of inflammation. However, no research has identified stable lncRNAs among different S. aureus strain infections. In addition, folic acid (FA) can effectively reduce inflammation, and whether the inflammatory response caused by S. aureus can be reduced by FA remains to be explored. lncRNA transcripts were identified from Holstein mammary gland tissues infected with different concentrations of S. aureus (in vivo) and mammary alveolar cells (Mac-T cells, in vitro) challenged with different S. aureus strains. Differentially expressed (DE) lncRNAs were evaluated, and stable DE lncRNAs were identified in vivo and in vitro. On the basis of the gene sequence conservation and function conservation across species, key lncRNAs with the function of potentially immune regulation were retained for further analysis. The function of FA on inflammation induced by S. aureus challenge was also investigated. Then, the association analysis between these keys lncRNA transcripts and hematological parameters (HPs) was carried out. Lastly, the knockdown and overexpression of the important lncRNA were performed to validate the gene function on the regulation of cell immune response. Linear regression analysis showed a significant correlation between the expression levels of lncRNA shared by mammary tissue and Mac-T cells (P < 0.001, R2 = 0.3517). lncRNAs PRANCR and TNK2-AS1 could be regarded as stable markers associated with bovine S. aureus mastitis. Several HPs could be influenced by SNPs around lncRNAs PRANCR and TNK2-AS1. The results of gene function validation showed PRANCR regulates the mRNA expression of SELPLG and ITGB2 within the S. aureus infection pathway and the Mac-T cells apoptosis. In addition, FA regulated the expression change of DE lncRNA involved in toxin metabolism and inflammation to fight against S. aureus infection. The remarkable association between SNPs around these two lncRNAs and partial HP indicates the potentially important role of PRANCR and TNK2-AS1 in immune regulation. Stable DE lncRNAs PRANCR and TNK2-AS1 can be regarded as potential targets for the prevention of bovine S. aureus mastitis. FA supplementation can reduce the negative effect of S. aureus challenge by regulating the expression of lncRNAs.

SUBMITTER: Mi S

PROVIDER: S-EPMC8667444 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Many efforts have been made to understand the pathogenesis of bovine mastitis to reduce losses and promote animal welfare. Staphylococcus aureus may cause bovine clinical mastitis, but it is mainly associated with subclinical infection, which is usually persistent and can easily reoccur. Here, we conducted a comparative genomic analysis between strains of S. aureus causing subclinical infection (Sau170, 302, 1269, 1364), previously sequenced by our group, and two well-characterized strains causing clinical mastitis (N305 and RF122) to find differences that could be linked to mastitis outcome. A total of 146 virulence-associated genes were compared and no appreciable differences were found between the bacteria. However, several nonsynonymous single nucleotide polymorphisms (SNPs) were identified in genes present in the subclinical strains when compared to RF122 and N305, especially in genes encoding host immune evasion and surface proteins. The secreted and surface proteins predicted by in silico tools were compared through multidimensional scaling analysis (MDS), revealing a high degree of similarity among the strains. The comparison of orthologous genes by OrthoMCL identified a membrane transporter and a lipoprotein as exclusive of bacteria belonging to the subclinical and clinical groups, respectively. No hit was found in RF122 and N305 for the membrane transporter using BLAST algorithm. For the lipoprotein, sequences of Sau170, 302, 1269, and 1364 with identities between 68-73% were found in the MDS dataset. A conserved region found only in the lipoprotein genes of RF122 and N305 was used for primer design. Although the polymerase chain reaction (PCR) on field isolates of S. aureus did not validate the findings for the transporter, the lipoprotein was able to separate the clinical from the subclinical isolates. These results show that sequence variation among bovine S. aureus, and not only the presence/absence of virulence factors, is an important aspect to consider when comparing isolates causing different mastitis outcomes.

Project description:Staphylococcus aureus is one of the most important contagious bacteria causing subclinical bovine mastitis. This bacterial infection is commonly identified by determine the pathogen in bovine milk samples through conventional technique including coagulase test. However, this test has several disadvantages as low sensitivity, risk of biohazard, cost expensive, and limited preparation especially in local area.Aim of this study was to compare and assess the screening method, Mannitol fermentation test (Mannitol salt agar [MSA]), and deoxyribonuclease (DNase) test, for S. aureus identification in milk samples.A total of 224 subclinical bovine mastitis milk samples were collected from four provinces of Thailand and determined S. aureus using conventional method and also subjected to the screening test, MSA and DNase test. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) among both tests were analyzed and compared to the tube coagulase test (TCT), as reference method. Immunological test by latex agglutination and molecular assay by determined spa gene were also used to identify and differentiate S. aureus.A total of 130 staphylococci were isolated by selective media, Gram-stain, and catalase test. The number of S. aureus which identified using TCT, MSA and DNase test were 32, 102, and 74 isolates, respectively. All TCT results were correlated to results of latex agglutination and spa gene which were 32 S. aureus. MSA showed 100% sensitivity, 28.57% specificity, 31.37% PPV, and 100% NPV, whereas DNase showed 53.13% sensitivity, 41.84% specificity, 22.97% PPV, and 73.21% NPV. DNase test showed higher specificity value than MSA but the test presented 26.79% false negative results whereas no false-negative result from MSA when comparing to TCT.MSA had a tendency to be a good preference for screening S. aureus because of its high sensitivity and NPV. The result from this study will improve a choice to use a screening test to diagnose S. aureus of veterinary field for prompt disease controlling and effective treatment.

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Transcriptome sequencing analysis for the identification of stable lncRNAs associated with bovine Staphylococcus aureus mastitis.

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