Identification of Potential mRNA Biomarkers in Milk Small Extracellular Vesicles of Enzootic Bovine Leukosis Cattle

Abstract

Enzootic bovine leukosis (EBL) is a disease caused by bovine leukemia virus (BLV); only a small percentage of BLV-infected cattle develop EBL and present with B-cell lymphosarcoma. There is no vaccine against BLV, treatment for EBL, or method for predicting the possibility of EBL onset, thus making EBL control difficult. Herein, to explore biomarkers for EBL in milk, we examined the mRNA profiles of small extracellular vesicles (sEVs) in milk from four BLV-uninfected and four EBL cattle by microarray analysis. It was revealed that 14 mRNAs were encapsulated in significantly higher quantities, and these mRNAs were therefore selected as biomarker candidates. Primers for these mRNAs were designed, and nine primer sets were available for quantitative real-time PCR. Nine mRNAs were evaluated for their availability as biomarkers for EBL using sEVs from newly-collected milk of 7 uninfected and 10 EBL cattle. The quantities of eight mRNAs (TMEM156, SRGN, CXCL8, DEFB4A, FABP5, LAPTM5, LGALS1, and VIM) were significantly higher in milk sEVs of EBL cattle than in those of uninfected cattle. Therefore, our findings indicate that these eight mRNAs in milk sEVs can be used as potential EBL biomarkers with combination use, although single mRNA use is not enough. Consequently, cattle at risk of EBL onset can be identified by monitoring the fluctuation in quantities of these mRNAs in milk before they develop EBL.

Upregulated host genes during the disease progression of bovine leukemia virus independent on overexpression of viral transcriptional regulators in vitro

“…There have been several previous studies on comprehensive transcriptome or proteome analyses related to BLV infection using microarray analysis and next-generation sequencing technology: EBL cattle vs. healthy cattle [9,10], BLV-infected cattle vs. healthy cattle [11,12], and time-dependent samples from cattle experimentally infected with BLV [13]. However, an investigation reporting transcriptome pro les by comparing three different states of BLV infection is limited, which may support the new insight provided in our experiments.…”

“…Comprehensive analyses, such as microarray and next-generation sequencing technology, have promoted the identi cation of differentially expressed genes (DEGs) during the progression of BLV infection [9][10][11][12][13]. Some reports indicated up-or downregulated genes in samples from EBL cattle or BLV-infected cattle without lymphoma when compared with those from healthy cattle, while other reports indicated expression changes in samples from experimentally infected cattle with BLV at several time points.…”

Milk-Derived Extracellular Vesicles: Biomedical Applications, Current Challenges, and Future Perspectives

“…72 Hiraoka and her colleagues in a new study investigated the mRNA profiles of miEVs from 4 healthy and 4 EBL cattle to discover biomarkers. 24 Their study revealed that eight mRNAs (TMEM156, SRGN, CXCL8, DEFB4A, FABP5, LAPTM5, LGALS1, and VIM) may be potential EBL biomarkers because their expression was remarkably higher in miEVs from infected cattle. Matiur Rahman et al in a more recent study, assessed the expression of mRNAs in miEVs from BLV-infected cattle (with high or low proviral load) and uninfected cattle.…”

“…Milk is an important complex food source in mammals involved in the growth, development, and health of infants and is known as the most widely used natural drink. , This biofluid is dynamic and apart from the amino acids, fats, cytokines, vitamins, and minerals that change during the lactation stages, it contains high amounts of EVs originating from different cell populations in the mammary glands (e.g., adipocytes, epithelial cells, stem cells, and immune cells) and playing important roles in interorganismal and cross-species communication. ,, Because milk is considered a scalable, economical, and safe source of EVs, milk-derived EVs (miEVs) have recently been introduced as novel drug carriers. ,, Furthermore, miEVs can improve health and also reflect metabolic status, so they can be used as therapeutic agents or diagnostic biomarkers, respectively. , To date, the potential of miEVs for therapeutic, diagnostic, and drug delivery applications has been proven in several studies. ,− …”

Extracellular Vesicles from Animal Milk: Great Potentialities and Critical Issues

“…Indeed, the mRNA and protein expression profiles of mEVs were found to be modified by BLV infections; their monitoring can be useful in evaluating the clinical stages of the infection [ 237 , 238 ]. In particular, the concentrations of eight mRNAs were increased in the mEVs of infected cattle by Hiraoka and collaborators, which suggested a combined evaluation of the expression profiles of these genes to identify early BLV infections [ 239 ]. On the contrary, miR-424-5p was found to be significantly upregulated in the mEVs of infected animals relative to healthy ones; this was also validated in the re-qPCR of a cohort of animals selected ad hoc, thus identifying it as a valuable biomarker to identify high-risk animals for BLV transmission [ 240 ].…”