BACKGROUND
micrornas  are a class of endogenous non-coding small rna molecules, typically  <dig> nucleotides in length, which bind primarily to the 3'utr of target mrnas to repress translation and/or accelerate the decay  <cit>  of up to 30 % of all expressed transcripts  <cit> .
numerous studies have found roles for mirnas in regulation of gene expression for important biological processes including cellular proliferation and differentiation, tissue development, and immune response.
of interest for our study, mirnas help regulate the development of immune cells and modulate the innate and adaptive immune responses  <cit> .
some specific examples include the role of mir- <dig> in inhibiting synthesis of the transcription factor c-myb to help regulate b-cell differentiation  <cit> , and mir- <dig> targeting of insulin regulatory subunit- <dig> transcripts to positively control the fate of b-cells  <cit> .
similarly, mir-146a functions as a negative regulator of tnf receptor-associated factor  <dig> and interleukin- <dig> receptor-associated kinase  <dig> transcripts during and/or after the innate immune system responds to a bacterial infection  <cit> .
these examples demonstrate how micrornas can exert some regulatory control of the immune system within a cell.
more recent studies have shown that extracellular mirnas stably present in body fluids through encapsulation into exosomes or microvesicles; may also play a significant role in intercellular communication during an immune response .
therefore, the presence of specific mirnas in exosomes may be indicative of various pathological conditions and provide biomarkers for detection of certain disease conditions  <cit> .
despite the recognized importance of mirnas in host immune response to disease in humans and mice, their comparative roles in regulating the immune response of livestock are still in preliminary stages of study.
expression surveys of bovine mirnas have been reported for embryos  <cit> , muscle  <cit> , adipose  <cit> , mammary gland  <cit> , testicular and ovarian tissues , and various tissues involved in immune response  <cit> ; but none of these reports have interrogated mirna expression changes in response to infection.
more recently, lawless and colleagues reported two studies surveying mirna expression changes in response to bacterial infection of a bovine cell line  <cit>  and circulating monocytes from blood and milk  <cit> .
in the latter report, bovine specific mirnas were identified as amplifiers of monocyte inflammatory response networks and repressors of several metabolic pathways in response to staphylococcus uberis  infection of the mammary gland.
based on these findings and the hypothesis that exosomes may contain specific mirnas indicative of infection means that characterizing the mirna profile of bovine milk exosomes could provide further understanding of host immune response signaling during lactation.
it is well known that human milk is the most essential nutritional source for infant health during postnatal development  <cit> , containing nutrients that closely match infant requirements for brain development, growth, and a healthy immune system  <cit> .
in the past decade, many immune-related substances like secretory immunoglobulins, leukocytes, and antimicrobial factors such as lysozyme, lactoferrin, and oligosaccharides have been detected in milk  <cit> .
more specific analyses of the exosome component of human milk found abundant immune-related proteins  <cit>  as well as mirnas  <cit> .
the presence of mirnas has also been confirmed from cursory surveys of bovine  and porcine milk  <cit> .
taken together, the immune-related factors in milk exosomes could impart some pathogen resistance to the newborns lacking a fully developed immune system  <cit> .
some mirnas may play a role in these effects  <cit> , since there is evidence that exosome mirnas are readily absorbed within the neonate digestive tract after ingestion of milk  <cit> .
thus, the goal of our study was to comprehensively survey by deep sequencing the mirna content of bovine milk exosomes derived from both healthy and infected mammary glands, and then attempt to identify if host response to infection significantly changes the content of specific mirnas in exosomes.
we chose to harvest exosomes from lactating holstein cows challenged with staphylococcus aureus ; because holsteins are the primary source of milk-related products consumed by humans globally, and s. aureus is a leading causative agent of bovine mastitis.
furthermore, identification of differentially expressed exosome mirnas in this infection model serve as potential molecular targets for development of biomarkers assays to provide early detection of sub-clinical mastitis.
RESULTS
sequence analysis of milk exosome mirnas
next generation sequence analysis of the eight small rna libraries derived from milk exosomes prior to  and 48 h post s. aureus infection  yielded more than  <dig>  million 36 nt sequences.
after filtering, approximately  <dig>  and  <dig>  million high-quality sequence reads from the control and infected libraries were available for reference genome alignment, respectively .
the unbalanced read results between treatments was due to an intentional increase of sequencing coverage of the infection sample  <dig> library, which showed the rate of unique sequence discovery was reduced with additional depth of sequencing but still comparable to the other libraries with fewer sequence reads .
in total, all reads represented about  <dig>  million unique sequences, and soap alignment  <cit>  to the bostau <dig> reference genome assembly  <cit>  resulted filtered this set of unique sequences down to  <dig>  bovine specific sequences corresponding to about  <dig>  million total sequences  for further analyses.
consistent with a previous report  <cit> , the relatively high sequence depth  and multiple, unpooled samples  provided enough replication for bona fide discovery of potentially novel mirnas expressed from the bovine genome.
sequence identities were assigned to the  <dig>  unique sequences by alignments to rfam and bovine-specific sequences within mirbase, repeat and reference mrna databases.
the average rna content of milk exosomes based on alignments of mapped, annotated reads was approximately: 1) 58 % other non-coding rna , 2) 13 % known bovine mirna, 3) 23 % repetitive sequences, 4) 1 % mrna , and 5) 4 % non-annotated sequence .
comparing the ratio of total sequence reads to unique sequences for each identity category revealed mirna associated sequences were the least diverse class of rna present in milk exosomes with mrna and other non-coding rna associated sequences having 500- and 50-fold more observed diversity, respectively.
because the purpose of this study was to characterize mirna-associated sequences and attempts to identify bovine snorna and pirna identities were hampered by lack of annotation for the bovine reference genome, none of the other categories of sequence were analyzed further.
however, non-mirna sequence data is available for future analyses through geo accession gse <dig>
in order to examine the potential mirna content of milk exosomes more deeply, the  <dig> mirna-associated unique sequences  were clustered into homolog groups based on annotation of best match to mirbase.
using soap and mirdeep <dig>  <cit>  methods, identities of unique sequences could be assigned to  <dig> mirna homolog groups corresponding to  <dig> mirna loci and  <dig> mirna homologs corresponding to  <dig> mirna loci, respectively .
the union of these two results yielded  <dig> known mirnas corresponding to over 53 % of known bovine mirnas in mirbase release  <dig>
the reduction of  <dig> unique sequences to a set of  <dig> homologs is most likely attributable to the presence of isomirs, which are heterogeneous variants of canonical mirna species  <cit> .
this finding was supported by variation in sequence read length  for some unique sequence alignments within a homolog groups .
however, mirna length distribution analysis  showed the highest count of mirna-associated reads was 22 nt in length , and the majority of unique sequences reads ranged within the expected length of 21–23 nt.
interestingly, a significant portion of unique reads was also present in the length ranging from 18–20 nt.
this result requires further investigation, but the length variation may be attributed to enzyme modifications and imprecise processing of primary or precursor mirnas by drosha and dicer enzymes, such as rna editing in mirna-mediated gene silencing  <cit> , 3'-editing  <cit> , and degradation of micrornas by a family of exonucleases  <cit> .fig.
<dig> distribution by length of mapped sequence reads.
the distribution of mapped sequence reads from milk exosomes matching known bovine mirnas found in mirbase binned by length  and shown by percentage of unique  and total  sequence read counts
looking at diversity within the  <dig> mirna homolog groups, the number of total sequences representing isomirs ranged from one to hundreds.
as expected, the most highly expressed isomir usually corresponded to the bovine reference sequence in mirbase .
a total of  <dig> mirnas were found to have at least one isomir expressed at a level of ≥ <dig> sequence reads and more than  <dig> different isomirs were identified in total.
the group designated bta-mir- <dig> had the highest diversity of isomirs at a level of ≥ <dig> sequence reads with counts of  <dig> variants at the 5' end and  <dig> variants at the 3' end.
the presence of such mirnas variants was previously suggested to be cell type specific, have functional differences, and vary in their response to biological stimuli  <cit> .
in our dataset, about 38 % of the isomirs were variant at the 5' end, which should change the seed sequence , and thereby effect changes in target mrna and possibly mirna function  <cit> .
overall, these results fully illustrated that mirna diversity in exosomes is greatly extended through the presence of multiple isomirs, which is a result that cannot be elucidated by real-time pcr or microarray expression analyses.
after accounting for isomirs, we evaluated the overall distribution of mirna diversity based on homolog groupings.
the  <dig> most prevalent mirnas accounted for between 76-88 % of the total normalized sequence reads depending on analysis method, while the members of this list were nearly conserved between methods .
furthermore, eight of these mirnas  shared common ranking as top  <dig> expressed mirnas when parsing the sequence data based on treatment , suggesting these mirnas could be important nutritional components of milk.
recently, similar mirnas were found by sequencing of lactating caprine mammary gland  <cit> , bovine mammary epithelial cells  <cit> , and porcine  <cit>  and human  <cit>  milk.
in the latter two studies; mir-30a, −148a, − <dig> and -27b were also represented as the top  <dig> mirnas.
previous mirna expression studies using bovine mammary tissue  <cit>  or milk  <cit>  also found mir-148a and -181a as significantly elevated during lactation, respectively.
however, most of the mirnas reported as elevated in mammary tissue during ovine and bovine lactation do not match the most prevalent mirnas from exosomes  <cit> .
some of these differences could be because previous studies only profiled expression for a pre-selected subset of mirnas, which did not overlap with our top  <dig> list.
however, mir-148a-3p, which accounted for ~ <dig>  % of the total mirna associated sequence reads, has previously been suggested as a nutritional biomarker corresponding to the protein content of various bovine-derived milk products  <cit> .
interestingly, feeding studies that have shown exogenous plant  <cit>  or milk  <cit>  mirnas can be found in the sera and tissues and influence regulation of target genes in recipient animals.
combining the findings of these reports with the pleiotropic roles of the  <dig> most prevalent mirnas in our study suggests the enrichment of specific mirnas derived from mammary secretory cells may also influence development of the immune system of neonates.
specifically, the mir-30a-5p and -30d  are known to be involved in regulation of autophagy in cancer progression and treatment by suppressing the expression of beclin  <dig>  <cit>  and also cellular invasion and immunosuppression by targeting galnac transferase galnt <dig> to increase synthesis of the immunosuppressive cytokine interleukin- <dig>  <cit> .
also widely studied for possible involvement in tumor progression, mir-148-3p has been shown to directly target the tgif <dig> gene in ovarian cancer  <cit> , the drug metabolizing pxr gene in human cancer metastasis  <cit> , and cand <dig> expression in human prostate cancer to promote prostate cell growth  <cit> .
in the case of mir-27b, mrna stability of ppargamma is destabilized, which is often associated with chronic inflammatory diseases provoked by an immune response  <cit> .
mir-27b has also been found to be degraded by a viral transcript in lytic murine cytomegalovirus  infection  <cit> , further highlighting its role in immunity.
another prevalent milk mirna, mir-181a, regulates t-cell selection by altering sensitivity to peptide antigens, which is partly achieved through the down regulation of multiple phosphatases that act as negative regulators of t cell receptor signaling  <cit> .
additional supporting evidence includes the roles of plasma mir- <dig> as a biomarker for colon cancers  <cit> , mir- <dig> as a tumor-suppressor for the development and progression of lung adenocarcinoma  <cit> , mir-26a in mirna biogenesis to target lin28b and zcchc <dig> as a suppression mechanism for tumor growth and metastasis  <cit> , and mir- <dig> targeting of estrogen receptor α mrna to inhibit estrogen signaling associated with some forms of breast cancer  <cit> .
finally, mir- <dig>  a well characterized oncogenesis-related mirna, is a biomarker of colorectal cancer  <cit> , primary effusion lymphoma  <cit> , and hepatocellular carcinoma  <cit> .
even though there is compelling evidence that the most prevalent mirnas in our study could potentially exert an influence on immune response, the specific functional roles of each mirna needs further detailed investigations to obtain a thorough understanding of the specific targets and mechanistic effects of consumption of mirna-loaded bovine milk exosomes by a recipient animal.
this is especially relevant considering that effects of microrna consumption are still not well validated  <cit> .
novel mirna discovery
after assignment of sequence identities by various alignments , nearly 38 % of all unique sequences had no identity or were considered non-annotated.
a mireap analysis  <cit>  of these sequences identified  <dig> potentially novel bovine mirnas corresponding to  <dig> genomic loci .
interestingly, the total read counts for each of these potential mirnas were much lower on average than for those matching known bovine mirs already present in mirbase.
for instance, there were only  <dig> unique sequences with a total read count > <dig>  and many of our initial novel mirnas  had read counts < <dig>  because known mirnas use as little as 6– <dig> nucleotides on the 5' end to recognize target mrnas  <cit> , the sequence of this seed region for the potentially novel mirnas was compared to those seed regions found among  <dig>  mirnas in mirbase.
a total  <dig>  % of novel mirnas had a seed sequence identical to those reported for known mature mirnas, and this sequence conservation supports functionality for many of the mirnas predicted by mireap analysis of non-annotated reads.
after removing the  <dig> unique sequences with < <dig> total reads counts, the remaining  <dig> unique sequences were also aligned to mirbase using blast.
only three unique sequences had 100 % identity with known mirnas found exclusively in other species .
in addition,  <dig> and  <dig> unique non-annotated sequences had significant homology with the bta-mir- <dig> and bta-mir- <dig> families, respectively .
these results were consistent with a previous report that mir- <dig> and − <dig> families had possibly over  <dig> members spanning the entire bovine genome  <cit> .
although we are confident these analyses identified previously undiscovered bovine mirnas, none of these sequences were used for the differential expression analyses between exosomes from control and infected milk.
differentially expressed mirnas in response to s. aureus infection
compared to previous ngs studies investigating the milk mirnas in bovine  <cit> , porcine  <cit>  and human  <cit> , we generated sequence reads from unpooled mirna libraries corresponding to individual animal replicates under different treatment .
this allowed exploration of statistically significant changes in mirna content of milk exosomes in early response to s. aureus infection of the mammary gland.
a separation of control  from infected samples was confirmed by principal component analysis  of the normalized sequence reads representing the  <dig> mirna homolog clusters .
pc <dig> explained  <dig>  % of the overall mirna expression variability, whereas pc <dig> explained  <dig>  % of the variability.fig.
<dig> principal component analysis of mapped reads by animal.
principal component analysis of the mapped sequence reads matching known bovine mirnas from the eight individual libraries.
control represents sequences from non-infected milk exosome libraries and infection represents those from infected quarters of the same animals 48 h post-infection
the primary differential expression  analysis was done using edger and compared expression values of the unique mirnas as determined by mirdeep <dig> normalization .
this approach detected a total of  <dig> de mirnas .
heatmap analysis of these  <dig> de micrornas shows the clustering of infected from control samples , ageing with the infection challenge statistics of these animals .table  <dig> known bovine mirnas with significant changes in exosome content in response to infection
p valuea
ap value determined by edger analysis of upper quartile normalized sequence reads corresponding to bovine mirna homolog groups
bbta-mir-296-5p was not present in infection samples and bta-mir- <dig> was not present in control samples
calso significant by soap analysis
fig.
<dig> heatmap of differentially expressed mirna.
heatmap of  <dig> differentially expressed mirnas  between infected and non-infected as determined by mirdeep <dig> analysis of normalized sequence reads corresponding to known bovine mirnas.
note: in = infected sample; c = control or non-infected sample
as a comparison to the edger analysis, a differential expression analysis was also done by soap , and six common mirnas  with differential presence in milk exosomes between uninfected and infected animals were identified .
comparing our de mirna results from milk to previously reported changes in bovine mammary-derived mirna expression revealed observations in support of our findings.
for example, we detected increases of bta-mir-142-5p and − <dig> in milk exosomes 48 h post-infection, while both lactating mammary epithelium  <cit>  and bovine monocytes  <cit>  under infection challenge with s. uberis also were found to increase levels of mir- <dig>  other reports surveying bovine milk  <cit>  found higher levels of mir- <dig> in colostrum possibly in support of increased immunity for early neonates or the mammary gland during a period of higher susceptibility  to bacterial infection.
lawless and colleagues also found significant increases in mir-142-5p in mammary monocytes post-infection  <cit> .
the cellular source of increases in our results, mammary epithelium or monocytes or both, remains to be determined; however, the lower levels of relative expression in comparison to the most prevalent mirnas in exosomes suggests only a subset of cells responding to infection are contributing these rnas to milk.
the decreases in -15b, and -193a-5p found only using soap analysis are supported by similar findings in mammary epithelium  <cit>  and monocytes  <cit> , respectively.
in contrast, we did not find any expression of bta-mir- <dig>  which was previously found to be upregulated in response to bacterial infection of mammary gland, where a potential role as a modulator of immune response was supported through differential regulation of a2m transcript binding  <cit> .
regarding the roles of these mirnas in immune response, a previous study demonstrated elevated mir- <dig> in systemic lupus erythematosus patients compared to healthy controls  <cit> .
mir- <dig> has also been shown to be abundant in t cells  <cit> , which implied its role as an immune-relevant mirna while its function remains unclear.
on the other hand, mir- <dig> has a potential role in balancing metabolism and immune response during infection  <cit> , as previous bioinformatic analysis provides support for its importance in down-regulating lipid metabolism  <cit> ; a process very important during milk production.
beyond its role in response to mammary infection in cattle, mir- <dig> was also recently reported to regulate granulopoiesis  <cit> .
in spite of the limited knowledge of milk enriched mirnas in response to bacterial infection, our results present significant information about highly expressed mirnas in bovine milk exosomes from healthy and infected animals, and provides specific mirnas that can be targeted for biomarker development as a potential means for early diagnostic detection of sub-clinical mastitis.
prediction and analysis of target genes
a total of  <dig> unique genes were predicted by rnahybrid to be targeted based by the six de mirnas found in common between both analysis methods .
of these,  <dig> genes were predicted to be targeted by up-regulated mirnas;  <dig> were targeted by down-regulated mirnas; and  <dig> were targeted by both up and down-regulated mirnas .
because many of these target mrnas may not actually be expressed during lactation, we filtered our candidate list of  <dig> genes for mammary specific genes by only selecting those encoding proteins identified in previous reports  <cit> .
from this reduced list of  <dig> genes encoding mammary derived protein found in exosomes, a total of  <dig> overlapping target genes were identified .
david analysis results showed  <dig> biological processes go terms for  <dig> genes, which were significantly related to host immune processes and inflammation .
we therefore hypothesized that differentially expressed mirnas can target sequences in these  <dig> genes to regulate the immune responses with s. aureus infection.
this new knowledge of milk mirna expression between healthy cows and cows with mastitis will provide information important for the immune function in the mammary gland.
CONCLUSIONS
in conclusion, we have comprehensively analyzed the rna content of milk exosomes from infected and uninfected animals using next generation sequencing to identify  <dig> known bovine mirnas and  <dig> novel candidate mirnas.
expression analyses identified six mirnas that were significantly differentially present in exosomes in response to bacterial infection of the mammary gland, and provided two promising targets in bta-mir-142a and − <dig> for biomarker development of bacterial infection.
overall, this study expands the repertoire of bovine mirnas and provides some specificity for the most prevalent mirnas in milk, which may have effects on downstream gene expression through ingestion.

 micrornas are a class of endogenous non-coding small rna molecules, typically nucleotides in length, which bind primarily to the 3'utr of target mrnas to repress translation and/or accelerate the decay of up to 30 % of all expressed transcripts. numerous studies have found roles for mirnas in regulation of gene expression for important biological processes including cellular proliferation and differentiation, tissue development, and immune response. of interest for our study, mirnas help regulate the development of immune cells and modulate the innate and adaptive immune responses. some specific examples include the role of mir- in inhibiting synthesis of the transcription factor c-myb to help regulate b-cell differentiation, and mir- targeting of insulin regulatory subunit- transcripts to positively control the fate of b-cells. similarly, mir-146a functions as a negative regulator of tnf receptor-associated factor and interleukin- receptor-associated kinase transcripts during and/or after the innate immune system responds to a bacterial infection. these examples demonstrate how micrornas can exert some regulatory control of the immune system within a cell. more recent studies have shown that extracellular mirnas stably present in body fluids through encapsulation into exosomes or microvesicles; may also play a significant role in intercellular communication during an immune response. therefore, the presence of specific mirnas in exosomes may be indicative of various pathological conditions and provide biomarkers for detection of certain disease conditions. despite the recognized importance of mirnas in host immune response to disease in humans and mice, their comparative roles in regulating the immune response of livestock are still in preliminary stages of study. expression surveys of bovine mirnas have been reported for embryos, muscle, adipose, mammary gland, testicular and ovarian tissues, and various tissues involved in immune response; but none of these reports have interrogated mirna expression changes in response to infection. more recently, lawless and colleagues reported two studies surveying mirna expression changes in response to bacterial infection of a bovine cell line and circulating monocytes from blood and milk. in the latter report, bovine specific mirnas were identified as amplifiers of monocyte inflammatory response networks and repressors of several metabolic pathways in response to staphylococcus uberis infection of the mammary gland. based on these findings and the hypothesis that exosomes may contain specific mirnas indicative of infection means that characterizing the mirna profile of bovine milk exosomes could provide further understanding of host immune response signaling during lactation. it is well known that human milk is the most essential nutritional source for infant health during postnatal development, containing nutrients that closely match infant requirements for brain development, growth, and a healthy immune system. in the past decade, many immune-related substances like secretory immunoglobulins, leukocytes, and antimicrobial factors such as lysozyme, lactoferrin, and oligosaccharides have been detected in milk. more specific analyses of the exosome component of human milk found abundant immune-related proteins as well as mirnas. the presence of mirnas has also been confirmed from cursory surveys of bovine and porcine milk. taken together, the immune-related factors in milk exosomes could impart some pathogen resistance to the newborns lacking a fully developed immune system. some mirnas may play a role in these effects, since there is evidence that exosome mirnas are readily absorbed within the neonate digestive tract after ingestion of milk. thus, the goal of our study was to comprehensively survey by deep sequencing the mirna content of bovine milk exosomes derived from both healthy and infected mammary glands, and then attempt to identify if host response to infection significantly changes the content of specific mirnas in exosomes. we chose to harvest exosomes from lactating holstein cows challenged with staphylococcus aureus; because holsteins are the primary source of milk-related products consumed by humans globally, and s. aureus is a leading causative agent of bovine mastitis. furthermore, identification of differentially expressed exosome mirnas in this infection model serve as potential molecular targets for development of biomarkers assays to provide early detection of sub-clinical mastitis.  sequence analysis of milk exosome mirnas next generation sequence analysis of the eight small rna libraries derived from milk exosomes prior to and 48 h post s. aureus infection yielded more than million 36 nt sequences. after filtering, approximately and million high-quality sequence reads from the control and infected libraries were available for reference genome alignment, respectively. the unbalanced read results between treatments was due to an intentional increase of sequencing coverage of the infection sample library, which showed the rate of unique sequence discovery was reduced with additional depth of sequencing but still comparable to the other libraries with fewer sequence reads. in total, all reads represented about million unique sequences, and soap alignment to the bostau reference genome assembly resulted filtered this set of unique sequences down to bovine specific sequences corresponding to about million total sequences for further analyses. consistent with a previous report, the relatively high sequence depth and multiple, unpooled samples provided enough replication for bona fide discovery of potentially novel mirnas expressed from the bovine genome. sequence identities were assigned to the unique sequences by alignments to rfam and bovine-specific sequences within mirbase, repeat and reference mrna databases. the average rna content of milk exosomes based on alignments of mapped, annotated reads was approximately: 1) 58 % other non-coding rna, 2) 13 % known bovine mirna, 3) 23 % repetitive sequences, 4) 1 % mrna, and 5) 4 % non-annotated sequence. comparing the ratio of total sequence reads to unique sequences for each identity category revealed mirna associated sequences were the least diverse class of rna present in milk exosomes with mrna and other non-coding rna associated sequences having 500- and 50-fold more observed diversity, respectively. because the purpose of this study was to characterize mirna-associated sequences and attempts to identify bovine snorna and pirna identities were hampered by lack of annotation for the bovine reference genome, none of the other categories of sequence were analyzed further. however, non-mirna sequence data is available for future analyses through geo accession gse in order to examine the potential mirna content of milk exosomes more deeply, the mirna-associated unique sequences were clustered into homolog groups based on annotation of best match to mirbase. using soap and mirdeep methods, identities of unique sequences could be assigned to mirna homolog groups corresponding to mirna loci and mirna homologs corresponding to mirna loci, respectively. the union of these two results yielded known mirnas corresponding to over 53 % of known bovine mirnas in mirbase release the reduction of unique sequences to a set of homologs is most likely attributable to the presence of isomirs, which are heterogeneous variants of canonical mirna species. this finding was supported by variation in sequence read length for some unique sequence alignments within a homolog groups. however, mirna length distribution analysis showed the highest count of mirna-associated reads was 22 nt in length, and the majority of unique sequences reads ranged within the expected length of 21–23 nt. interestingly, a significant portion of unique reads was also present in the length ranging from 18–20 nt. this result requires further investigation, but the length variation may be attributed to enzyme modifications and imprecise processing of primary or precursor mirnas by drosha and dicer enzymes, such as rna editing in mirna-mediated gene silencing, 3'-editing, and degradation of micrornas by a family of exonucleases.fig. distribution by length of mapped sequence reads. the distribution of mapped sequence reads from milk exosomes matching known bovine mirnas found in mirbase binned by length and shown by percentage of unique and total sequence read counts looking at diversity within the mirna homolog groups, the number of total sequences representing isomirs ranged from one to hundreds. as expected, the most highly expressed isomir usually corresponded to the bovine reference sequence in mirbase. a total of mirnas were found to have at least one isomir expressed at a level of ≥ sequence reads and more than different isomirs were identified in total. the group designated bta-mir- had the highest diversity of isomirs at a level of ≥ sequence reads with counts of variants at the 5' end and variants at the 3' end. the presence of such mirnas variants was previously suggested to be cell type specific, have functional differences, and vary in their response to biological stimuli. in our dataset, about 38 % of the isomirs were variant at the 5' end, which should change the seed sequence, and thereby effect changes in target mrna and possibly mirna function. overall, these results fully illustrated that mirna diversity in exosomes is greatly extended through the presence of multiple isomirs, which is a result that cannot be elucidated by real-time pcr or microarray expression analyses. after accounting for isomirs, we evaluated the overall distribution of mirna diversity based on homolog groupings. the most prevalent mirnas accounted for between 76-88 % of the total normalized sequence reads depending on analysis method, while the members of this list were nearly conserved between methods. furthermore, eight of these mirnas shared common ranking as top expressed mirnas when parsing the sequence data based on treatment, suggesting these mirnas could be important nutritional components of milk. recently, similar mirnas were found by sequencing of lactating caprine mammary gland, bovine mammary epithelial cells, and porcine and human milk. in the latter two studies; mir-30a, −148a, − and -27b were also represented as the top mirnas. previous mirna expression studies using bovine mammary tissue or milk also found mir-148a and -181a as significantly elevated during lactation, respectively. however, most of the mirnas reported as elevated in mammary tissue during ovine and bovine lactation do not match the most prevalent mirnas from exosomes. some of these differences could be because previous studies only profiled expression for a pre-selected subset of mirnas, which did not overlap with our top list. however, mir-148a-3p, which accounted for ~  % of the total mirna associated sequence reads, has previously been suggested as a nutritional biomarker corresponding to the protein content of various bovine-derived milk products. interestingly, feeding studies that have shown exogenous plant or milk mirnas can be found in the sera and tissues and influence regulation of target genes in recipient animals. combining the findings of these reports with the pleiotropic roles of the most prevalent mirnas in our study suggests the enrichment of specific mirnas derived from mammary secretory cells may also influence development of the immune system of neonates. specifically, the mir-30a-5p and -30d are known to be involved in regulation of autophagy in cancer progression and treatment by suppressing the expression of beclin and also cellular invasion and immunosuppression by targeting galnac transferase galnt to increase synthesis of the immunosuppressive cytokine interleukin-. also widely studied for possible involvement in tumor progression, mir-148-3p has been shown to directly target the tgif gene in ovarian cancer, the drug metabolizing pxr gene in human cancer metastasis, and cand expression in human prostate cancer to promote prostate cell growth. in the case of mir-27b, mrna stability of ppargamma is destabilized, which is often associated with chronic inflammatory diseases provoked by an immune response. mir-27b has also been found to be degraded by a viral transcript in lytic murine cytomegalovirus infection, further highlighting its role in immunity. another prevalent milk mirna, mir-181a, regulates t-cell selection by altering sensitivity to peptide antigens, which is partly achieved through the down regulation of multiple phosphatases that act as negative regulators of t cell receptor signaling. additional supporting evidence includes the roles of plasma mir- as a biomarker for colon cancers, mir- as a tumor-suppressor for the development and progression of lung adenocarcinoma, mir-26a in mirna biogenesis to target lin28b and zcchc as a suppression mechanism for tumor growth and metastasis, and mir- targeting of estrogen receptor α mrna to inhibit estrogen signaling associated with some forms of breast cancer. finally, mir- a well characterized oncogenesis-related mirna, is a biomarker of colorectal cancer, primary effusion lymphoma, and hepatocellular carcinoma. even though there is compelling evidence that the most prevalent mirnas in our study could potentially exert an influence on immune response, the specific functional roles of each mirna needs further detailed investigations to obtain a thorough understanding of the specific targets and mechanistic effects of consumption of mirna-loaded bovine milk exosomes by a recipient animal. this is especially relevant considering that effects of microrna consumption are still not well validated. novel mirna discovery after assignment of sequence identities by various alignments, nearly 38 % of all unique sequences had no identity or were considered non-annotated. a mireap analysis of these sequences identified potentially novel bovine mirnas corresponding to genomic loci. interestingly, the total read counts for each of these potential mirnas were much lower on average than for those matching known bovine mirs already present in mirbase. for instance, there were only unique sequences with a total read count > and many of our initial novel mirnas had read counts < because known mirnas use as little as 6– nucleotides on the 5' end to recognize target mrnas, the sequence of this seed region for the potentially novel mirnas was compared to those seed regions found among mirnas in mirbase. a total  % of novel mirnas had a seed sequence identical to those reported for known mature mirnas, and this sequence conservation supports functionality for many of the mirnas predicted by mireap analysis of non-annotated reads. after removing the unique sequences with < total reads counts, the remaining unique sequences were also aligned to mirbase using blast. only three unique sequences had 100 % identity with known mirnas found exclusively in other species. in addition, and unique non-annotated sequences had significant homology with the bta-mir- and bta-mir- families, respectively. these results were consistent with a previous report that mir- and − families had possibly over members spanning the entire bovine genome. although we are confident these analyses identified previously undiscovered bovine mirnas, none of these sequences were used for the differential expression analyses between exosomes from control and infected milk. differentially expressed mirnas in response to s. aureus infection compared to previous ngs studies investigating the milk mirnas in bovine, porcine and human, we generated sequence reads from unpooled mirna libraries corresponding to individual animal replicates under different treatment. this allowed exploration of statistically significant changes in mirna content of milk exosomes in early response to s. aureus infection of the mammary gland. a separation of control from infected samples was confirmed by principal component analysis of the normalized sequence reads representing the mirna homolog clusters. pc explained  % of the overall mirna expression variability, whereas pc explained  % of the variability.fig. principal component analysis of mapped reads by animal. principal component analysis of the mapped sequence reads matching known bovine mirnas from the eight individual libraries. control represents sequences from non-infected milk exosome libraries and infection represents those from infected quarters of the same animals 48 h post-infection the primary differential expression analysis was done using edger and compared expression values of the unique mirnas as determined by mirdeep normalization. this approach detected a total of de mirnas. heatmap analysis of these de micrornas shows the clustering of infected from control samples, ageing with the infection challenge statistics of these animals.table known bovine mirnas with significant changes in exosome content in response to infection p valuea ap value determined by edger analysis of upper quartile normalized sequence reads corresponding to bovine mirna homolog groups bbta-mir-296-5p was not present in infection samples and bta-mir- was not present in control samples calso significant by soap analysis fig. heatmap of differentially expressed mirna. heatmap of differentially expressed mirnas between infected and non-infected as determined by mirdeep analysis of normalized sequence reads corresponding to known bovine mirnas. note: in = infected sample; c = control or non-infected sample as a comparison to the edger analysis, a differential expression analysis was also done by soap, and six common mirnas with differential presence in milk exosomes between uninfected and infected animals were identified. comparing our de mirna results from milk to previously reported changes in bovine mammary-derived mirna expression revealed observations in support of our findings. for example, we detected increases of bta-mir-142-5p and − in milk exosomes 48 h post-infection, while both lactating mammary epithelium and bovine monocytes under infection challenge with s. uberis also were found to increase levels of mir- other reports surveying bovine milk found higher levels of mir- in colostrum possibly in support of increased immunity for early neonates or the mammary gland during a period of higher susceptibility to bacterial infection. lawless and colleagues also found significant increases in mir-142-5p in mammary monocytes post-infection. the cellular source of increases in our results, mammary epithelium or monocytes or both, remains to be determined; however, the lower levels of relative expression in comparison to the most prevalent mirnas in exosomes suggests only a subset of cells responding to infection are contributing these rnas to milk. the decreases in -15b, and -193a-5p found only using soap analysis are supported by similar findings in mammary epithelium and monocytes, respectively. in contrast, we did not find any expression of bta-mir- which was previously found to be upregulated in response to bacterial infection of mammary gland, where a potential role as a modulator of immune response was supported through differential regulation of a2m transcript binding. regarding the roles of these mirnas in immune response, a previous study demonstrated elevated mir- in systemic lupus erythematosus patients compared to healthy controls. mir- has also been shown to be abundant in t cells, which implied its role as an immune-relevant mirna while its function remains unclear. on the other hand, mir- has a potential role in balancing metabolism and immune response during infection, as previous bioinformatic analysis provides support for its importance in down-regulating lipid metabolism; a process very important during milk production. beyond its role in response to mammary infection in cattle, mir- was also recently reported to regulate granulopoiesis. in spite of the limited knowledge of milk enriched mirnas in response to bacterial infection, our results present significant information about highly expressed mirnas in bovine milk exosomes from healthy and infected animals, and provides specific mirnas that can be targeted for biomarker development as a potential means for early diagnostic detection of sub-clinical mastitis. prediction and analysis of target genes a total of unique genes were predicted by rnahybrid to be targeted based by the six de mirnas found in common between both analysis methods. of these, genes were predicted to be targeted by up-regulated mirnas; were targeted by down-regulated mirnas; and were targeted by both up and down-regulated mirnas. because many of these target mrnas may not actually be expressed during lactation, we filtered our candidate list of genes for mammary specific genes by only selecting those encoding proteins identified in previous reports. from this reduced list of genes encoding mammary derived protein found in exosomes, a total of overlapping target genes were identified. david analysis results showed biological processes go terms for genes, which were significantly related to host immune processes and inflammation. we therefore hypothesized that differentially expressed mirnas can target sequences in these genes to regulate the immune responses with s. aureus infection. this new knowledge of milk mirna expression between healthy cows and cows with mastitis will provide information important for the immune function in the mammary gland.  in conclusion, we have comprehensively analyzed the rna content of milk exosomes from infected and uninfected animals using next generation sequencing to identify known bovine mirnas and novel candidate mirnas. expression analyses identified six mirnas that were significantly differentially present in exosomes in response to bacterial infection of the mammary gland, and provided two promising targets in bta-mir-142a and − for biomarker development of bacterial infection. overall, this study expands the repertoire of bovine mirnas and provides some specificity for the most prevalent mirnas in milk, which may have effects on downstream gene expression through ingestion. 
although known bovine mirnas were identified, mirnas represented the least diverse class of rna accounting for only 1 % of all unique sequences. the most prevalent unique sequences within this class accounted for about 90 % of the total mirna-associated reads across samples. next generation sequencing of eight, unpooled small rna libraries derived from milk exosomes produced about million high-quality, bovine-specific sequence reads for comparison of mirna expression between treatments. in contrast, the mirna content of milk exosomes may also be indicative of a lactating animal’s health; whereby, the presence or absence of specific mirnas could serve as biomarkers for early detection of bacterial infection that can lead to mastitis. milk exosomes are a rich source of micrornas that are protected from degradation. in the present study, we therefore analyzed and compared mirna expression profiles of milk exosomes from four holstein cows obtained during mid-lactation prior to and after infection of the mammary gland with staphylococcus aureus. milk exosomes, purified from control and s. aureus infected cows, were extracted for rna. our survey of mirna expression from uninfected milk exosomes and those produced in response to infection provides new and comprehensive information supporting a role for delivery into milk of specific mirnas involved in immune response. expression analyses found known bovine micrornas significantly differed in frequency between exosomes from infected and control animals. sequence identity analysis showed the mirnas make up about 13 % of the average rna content of these exosomes. in particular, bta-mir-142-5p, and − are potential biomarkers for early detection of bacterial infection of the mammary gland. additionally, mammary-expressed genes involved in regulation of host immune processes and response to inflammation were identified as potential binding targets of the differentially expressed mirnas. non-annotated, unique reads provided evidence for another previously unknown bovine mirnas.
 milk exosomes are a rich source of micrornas that are protected from degradation. ingestion of milk and subsequent absorption of mirnas into recipient cells by endocytosis may play a role in the regulation of neonatal innate and adaptive immunity. in contrast, the mirna content of milk exosomes may also be indicative of a lactating animal’s health; whereby, the presence or absence of specific mirnas could serve as biomarkers for early detection of bacterial infection that can lead to mastitis. in the present study, we therefore analyzed and compared mirna expression profiles of milk exosomes from four holstein cows obtained during mid-lactation prior to and after infection of the mammary gland with staphylococcus aureus. methods milk exosomes, purified from control and s. aureus infected cows, were extracted for rna. following preparation indexed libraries from both groups the samples were subjected to next generation sequencing.  next generation sequencing of eight, unpooled small rna libraries derived from milk exosomes produced about million high-quality, bovine-specific sequence reads for comparison of mirna expression between treatments. sequence identity analysis showed the mirnas make up about 13 % of the average rna content of these exosomes. although known bovine mirnas were identified, mirnas represented the least diverse class of rna accounting for only 1 % of all unique sequences. the most prevalent unique sequences within this class accounted for about 90 % of the total mirna-associated reads across samples. non-annotated, unique reads provided evidence for another previously unknown bovine mirnas. expression analyses found known bovine micrornas significantly differed in frequency between exosomes from infected and control animals.  our survey of mirna expression from uninfected milk exosomes and those produced in response to infection provides new and comprehensive information supporting a role for delivery into milk of specific mirnas involved in immune response. in particular, bta-mir-142-5p, and − are potential biomarkers for early detection of bacterial infection of the mammary gland. additionally, mammary-expressed genes involved in regulation of host immune processes and response to inflammation were identified as potential binding targets of the differentially expressed mirnas. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bovineexosomemilkmirnamastitisissue-copyright-statement© the author 2015 