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Testing Two Somatic Cell Count Cutoff Values for Bovine Subclinical Mastitis Detection Based on Milk Microbiota and Peripheral Blood Leukocyte Transcriptome Profile.

ABSTRACT: Somatic cell count (SCC) is an important indicator of the health state of bovine udders. However, the exact cut-off value used for differentiating the cows with healthy quarters from the cows with subclinical mastitis remains controversial. Here, we collected composite milk (milk from four udder quarters) and peripheral blood samples from individual cows in two different dairy farms and used 16S rRNA gene sequencing combined with RNA-seq to explore the differences in the milk microbial composition and transcriptome of cows with three different SCC levels (LSCC: <100,000 cells/mL, MSCC: 100,000-200,000 cells/mL, HSCC: >200,000 cells/mL). Results showed that the milk microbial profiles and gene expression profiles of samples derived from cows in the MSCC group were indeed relatively easily discriminated from those from cows in the LSCC group. Discriminative analysis also uncovered some differentially abundant microbiota at the genus level, such as Bifidobacterium and Lachnospiraceae_AC2044_group, which were more abundant in milk samples from cows with SCC below 100,000 cells/mL. As for the transcriptome profiling, 79 differentially expressed genes (DEGs) were found to have the same direction of regulation in two sites, and functional analyses also showed that biological processes involved in inflammatory responses were more active in MSCC and HSCC cows. Overall, these results showed a similarity between the milk microbiota and gene expression profiles of MSCC and HSCC cows, which presented further evidence that 100,000 cells/ml is a more optimal cut-off value than 200,000 cells/mL for intramammary infection detection at the cow level.

SUBMITTER: Zhang J

PROVIDER: S-EPMC9264859 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSubclinical intramammary infection (IMI) represents a significant problem in maintaining dairy cows' health. Disease severity and extent depend on the interaction between the causative agent, environment, and host. To investigate the molecular mechanisms behind the host immune response, we used RNA-Seq for the milk somatic cells (SC) transcriptome profiling in healthy cows (n = 9), and cows naturally affected by subclinical IMI from Prototheca spp. (n = 11) and Streptococcus agalactiae (S. agalactiae; n = 11). Data Integration Analysis for Biomarker discovery using Latent Components (DIABLO) was used to integrate transcriptomic data and host phenotypic traits related to milk composition, SC composition, and udder health to identify hub variables for subclinical IMI detection.ResultsA total of 1,682 and 2,427 differentially expressed genes (DEGs) were identified when comparing Prototheca spp. and S. agalactiae to healthy animals, respectively. Pathogen-specific pathway analyses evidenced that Prototheca's infection upregulated antigen processing and lymphocyte proliferation pathways while S. agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle, and carbohydrate and lipid metabolism. The integrative analysis of commonly shared DEGs between the two pathogens (n = 681) referred to the core-mastitis response genes, and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells (r2 = 0.72), followed by the udder health (r2 = 0.64) and milk quality parameters (r2 = 0.64). Variables with r ≥ 0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cytohubba plug-in. The genes in common between DIABLO and cytohubba (n = 10) were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals (sensitivity > 0.89, specificity > 0.81, accuracy > 0.87, and precision > 0.69). Among these genes, CIITA could play a key role in regulating the animals' response to subclinical IMI.ConclusionsDespite some differences in the enriched pathways, the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response. The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.

Project description:Mastitis is a condition in which the mammary tissue becomes inflamed. Changes in color and the appearance of clots, as well as increases in cell counts in the milk, are all indicators of inflammation. Mastitis is a common occurrence in cows as a result of inframammary infections. The present study aimed to find out how often nontuberculous mycobacteria (NTM) mastitis occurs and how hp65 affects Interleukin (IL) 6 concentrations. The findings of the Modified Whiteside Test (MWT) on the milk samples from 70 cows, 50 sheep, and 30 goats revealed that 82.9%, 76.7%, and 46.7% of milk samples from cows, sheep, and goats were positive, respectively. This test demonstrated a range of positive milk sample MWT reactions, and the difference among the current positivity score results was statistically significant (P<0.05). The presence of NTM in analyzed milk samples from cows and sheep was confirmed by hsp65-based polymerase chain reaction (PCR) and gene sequencing, with significant differences (P<0.05) in 71.4% and 20% of milk samples from cows and sheep, respectively. The PCR detection of the NTM hsp65 gene in fecal samples from cows, sheep, and goats indicated that cows (80%) had the highest proportion of NTM hsp65 gene amplification, followed by goats (70%), while sheep fecal samples had the lowest amount (22%). The difference among the positive NTM hsp65-based PCR was statistically significant (P<0.05). The phylogenetic tree and sequence analysis of the hsp65 gene revealed two novel variant NTM hsp65 genes that were deposited in Gene Bank (GenBank acc. LC636294 and LC636295). The current examined NTM Hsp65 Mycobacterium sequences which were included in the Mycobacterium avium clade in the currently produced tree ELISA detection of IL6 concentration in cow's milk revealed that IL-6 concentration in mastitis milk was varied. The mean of IL-6 concentration in cow's mastitis milk with MWT scores (+++ve) and mean of IL6 concentration in each MWT scores (++ve), MWT scores (+ve), and -ve MWT cow's milk had a highly significant difference (P<0.001).

Dataset Information

Testing Two Somatic Cell Count Cutoff Values for Bovine Subclinical Mastitis Detection Based on Milk Microbiota and Peripheral Blood Leukocyte Transcriptome Profile.

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