SNP-based quantitative deconvolution of biological mixtures: application to the detection of cows with subclinical mastitis by whole-genome sequencing of tank milk.

Biological products of importance in food (e.g., milk) and medical (e.g., donor blood-derived products) sciences often correspond to mixtures of samples contributed by multiple individuals. Identifying which individuals contributed to the mixture and in what proportions may be of interest in several circumstances. We herein present a method that allows to do this by shallow whole-genome sequencing of the DNA in mixed samples from hundreds of donors. We show the efficacy of the approach for the detection of cows with subclinical mastitis by analysis of farms' tank mixtures containing milk from as many as 500 cows.

Mammary epithelial cell-derived exosomal miR-155-inhibitor played a key role in the treatment of mastitis via down-regulation of TLRs/NF-κB signaling pathway to inhibit inflammatory response.

Mastitis is a common disorder in women capable of altering the normal physiological function of the mammary gland. It has been reported that mammary epithelial cells (MECs) could be involved in treating mastitis by regulating the inflammatory response and miR-155 might participate in this process. However, the effects of MECs-derived exosomal miR-155-inhibitor in treating mastitis and the regarding mechanism are still unknown. In our study, mouse mammary epithelial cells (HC11) were applied to study the role of MECs-derived exosomal miR-155-inhibitor in the treatment of mastitis and explore the mechanism. Results in our study showed that specific markers including CD63 and Apo-A1 were expressed in blank exosomes and exosomes containing miR-155-inhibitor isolated from transfected HC11 cells. Results of immunofluorescence showed that the blank exosomes and exosomes (containing miR-155-inhibitor) labeled with PKH26 were absorbed in HC11 cells. The level of miR-155 was decreased obviously in Engineered exosomes with miR-155-inhibitor and HC11 cells Transfected with exosome containing miR-155-inhibitor. The level of miR-155 was increased and cell apoptosis was promoted obviously in HC11 cells induced by LPS, however, they were decreased obviously after transfecting with an exosome containing miR-155-inhibitor. The level of TLR2, TLR4, TLR6, NF-κB, TNF-α, and IL-1ß was increased obviously in LPS-induced HC11 cells, however, they were decreased obviously after transfecting with an exosome containing miR-155-inhibitor. The change in IL-10 level is opposite to the above genes. Taken together, exosomal miR-155-inhibitor could decrease the apoptosis of MECs and inhibit the inflammatory response to treat mastitis by down-regulation in the TLRs/NF-κB signaling pathway, which might be a new therapeutic target for mastitis.

Genomic clues of association between clinical mastitis and SNPs identified by ddRAD sequencing in Murrah buffaloes.

The total milk production of India is 209.96 MT out of which 45% is contributed by the indigenous buffalo and due to their high producing virtue, the prevalence of mastitis is 5-20%. Despite the increasing level of technological advancement, mastitis is still an issue of concern for dairy industry in India as well as across the world. Therefore, the present study aimed to identify the SNPs and associate them with the incidence of clinical mastitis in Murrah buffalo using the ddRAD sequencing approach taking mastitis incidence data of 96 Murrah buffaloes. A total of 246 million quality controlled reads were obtained with an average alignment rate of 99.01% and at a read depth of 10, quality controlled SNPs obtained were 18,056. The logistic regression model was used and a total of seven SNPs were found significantly associated (p < 0.001) with mastitis incidence and seven genes were identified viz., NCBP1, FOXN3, TPK1, XYLT2, CPXM2, HERC1, and OPCML. The majority of them were having tumor suppressing action, related to immunogenetics or glycolytic and energy production. Conclusively, the SNPs identified in this study may be useful for future studies on mastitis incidence in Murrah buffalo and the SNP associations can be further validated.

Allelic diversity at BoLA DRB3 locus and association with predisposition to clinical mastitis in indicus and crossbred cattle.

Bovine lymphocyte antigen (BoLA) DRB3 locus in healthy and mastitis affected cattle has been genotyped by a polymerase chain reaction and restriction fragment length polymorphisms (PCR-RLFP) using RsaI restriction enzyme, followed by sequencing. In 130 farm animals, 25 BoLA DRB3 alleles have been detected by PCR-RFLP. Three distinct allelic patterns significantly associated with mastitis in Karan Fries crossbred and Sahiwal indicus cattle have been identified, whereas, four other allelic patterns were significantly high in frequency among healthy animals. Sequencing of RFLP genotypes revealed 25 and 47 alleles among healthy Sahiwal and Karan Fries, respectively, while 17 and 38 patterns observed in mastitis affected Sahiwal and Karan Fries animals, respectively. From Tajima's D-test of neutrality, it was concluded that alleles associated with mastitis were expanding in the population, whereas those of healthy were under contraction. Phylogenetic analysis carried out to delineate the evolutionary relationship of the farm and field animals at DRB3 locus, differentiating allelic patterns into six different clusters. Among the phylogenetic lineages, five patterns DRB3*028:01, DRB3*011:03, DRB3*031:01, DRB3*001:01 and DRB3*043:01, were previously reported, whereas one novel allelic variant was observed in indicus and crossbred cattle. This information will help in further exploring the association between BoLA-DRB3 genetic diversity and disease resistance in distinct cattle breeds, important in designing breeding strategies for increasing the distribution of favorable alleles.

CRISPR/cas9 cassette targeting Escherichia coli blaCTX-M specific gene of mastitis cow milk origin can alter the antibiotic resistant phenotype for cefotaxime.

CTX-M beta-lactamases are one of the most important extended spectrum beta-lactamase (ESBL) resistance enzymes found in E. coli. In the present study, 59% of E. coli isolates from mastitis cow milk were reported to be positive for ESBL types. The prevalence of beta-lactam (ß-lactam) antibiotic resistance was reported to be 84%, 72.7%, 52.27%, 50%, and 45.4% for cefotaxime, cefepime, cefuroxime, oxacillin, and cephalexine, respectively. The blaCTX-M gene was found in 65% (n = 17) of the E. coli isolates when they were genotyped. Further, the use of a CRISPR/cas9 cassette to target the E. coli blaCTX-M gene revealed changes in antibiotic phenotypes for cefotaxime.

A genomic assessment of the correlation between milk production traits and claw and udder health traits in Holstein dairy cattle.

Claw diseases and mastitis represent the most important disease traits in dairy cattle with increasing incidences and a frequently mentioned connection to milk yield. Yet, many studies aimed to detect the genetic background of both trait complexes via fine-mapping of quantitative trait loci. However, little is known about genomic regions that simultaneously affect milk production and disease traits. For this purpose, several tools to detect local genetic correlations have been developed. In this study, we attempted a detailed analysis of milk production and disease traits as well as their interrelationship using a sample of 34,497 50K genotyped German Holstein cows with milk production and claw and udder disease traits records. We performed a pedigree-based quantitative genetic analysis to estimate heritabilities and genetic correlations. Additionally, we generated GWAS summary statistics, paying special attention to genomic inflation, and used these data to identify shared genomic regions, which affect various trait combinations. The heritability on the liability scale of the disease traits was low, between 0.02 for laminitis and 0.19 for interdigital hyperplasia. The heritabilities for milk production traits were higher (between 0.27 for milk energy yield and 0.48 for fat-protein ratio). Global genetic correlations indicate the shared genetic effect between milk production and disease traits on a whole genome level. Most of these estimates were not significantly different from zero, only mastitis showed a positive one to milk (0.18) and milk energy yield (0.13), as well as a negative one to fat-protein ratio (-0.07). The genomic analysis revealed significant SNPs for milk production traits that were enriched on Bos taurus autosome 5, 6, and 14. For digital dermatitis, we found significant hits, predominantly on Bos taurus autosome 5, 10, 22, and 23, whereas we did not find significantly trait-associated SNPs for the other disease traits. Our results confirm the known genetic background of disease and milk production traits. We further detected 13 regions that harbor strong concordant effects on a trait combination of milk production and disease traits. This detailed investigation of genetic correlations reveals additional knowledge about the localization of regions with shared genetic effects on these trait complexes, which in turn enables a better understanding of the underlying biological pathways and putatively the utilization for a more precise design of breeding schemes.

Relationships between novel nucleotide variants within the colony-stimulating factor 1 receptor (CSF1R) gene and mastitis indicators in sheep.

Colony-stimulating factor 1 receptor (CSF1R) plays an important role in the process of innate immunity and inflammation, thus it was hypothesized that the CSF1R gene might affect the occurrence of mammalian mastitis. The purpose of this study was to investigate the association between nucleotide variations of CSF1R gene and mastitis in Australian white sheep (AUWs). Two indel variants (Intron5-27 bp and Intron5-22 bp) within the CSF1R gene have been found in AUWs. The Chi-square test for different mastitis symptoms demonstrated that individuals without symptoms of mastitis had higher 'I' allele frequencies and 'II' genotype frequencies (p < 0.01). We found strong correlation between mastitis and lactation score through Pearson correlation analysis. Therefore, we also analyzed the relationship between the two indel loci and lactation, we found that the lactation ability of individuals with type II was stronger than that of DD genotype at the Intron5-22 bp (p < 0.05). Additionally, we found that the combined genotype of the two loci was significantly associated with mastitis (p < 0.01). These findings indicated that CSF1R mutations were significantly associated with mastitis, and could affect lactation performance, suggesting that two deletion sites could be used as the effective molecular markers against mastitis in sheep breeding.

Devastating Gynecological Infections in Women with STAT3 Deficiency.

We provide the first description of a series of 9 severe gynecological infections (mastitis and pelvic cellulitis) occurring in the French national cohort of women with STAT3 deficiency. Each episode had unique features in terms of clinical presentation, microbial documentation, location, treatment duration, and related persistent esthetic damage.

DNA methylation analysis of porcine mammary epithelial cells reveals differentially methylated loci associated with immune response against Escherichia coli challenge.

BACKGROUND: Epigenetic changes such as cytosine (CpG) DNA methylations regulate gene expression patterns in response to environmental cues including infections. Microbial infections induce DNA methylations that play a potential role in modulating host-immune response. In the present study, we sought to determine DNA methylation changes induced by the mastitis causing Escherichia coli (E. coli) in porcine mammary epithelial cells (PMEC). Two time points (3 h and 24 h) were selected based on specific transcriptomic changes during the early and late immune responses, respectively. RESULTS: DNA methylation analysis revealed 561 and 898 significant (P < 0.01) differentially methylated CpG sites at 3 h and 24 h after E. coli challenge in PMEC respectively. These CpG sites mapped to genes that have functional roles in innate and adaptive immune responses. Significantly, hypomethylated CpG sites were found in the promoter regions of immune response genes such as SDF4, SRXN1, CSF1 and CXCL14. The quantitative transcript estimation indicated higher expression associated with the DNA CpG methylation observed in these immune response genes. Further, E. coli challenge significantly reduced the expression levels of DNMT3a, a subtype of de novo DNA methylation enzyme, in PMEC indicating the probable reason for the hypomethylation observed in the immune response genes. CONCLUSIONS: Our study revealed E. coli infection induced DNA methylation loci in the porcine genome. The differentially methylated CpGs were identified in the regulatory regions of genes that play important role in immune response. These results will help to understand epigenetic mechanisms for immune regulation during coliform mastitis in pigs.

Genome-wide association study of milk and reproductive traits in dual-purpose Xinjiang Brown cattle.

BACKGROUND: Dual-purpose cattle are more adaptive to environmental challenges than single-purpose dairy or beef cattle. Balance among milk, reproductive, and mastitis resistance traits in breeding programs is therefore more critical for dual-purpose cattle to increase net income and maintain well-being. With dual-purpose Xinjiang Brown cattle adapted to the Xinjiang Region in northwestern China, we conducted genome-wide association studies (GWAS) to dissect the genetic architecture related to milk, reproductive, and mastitis resistance traits. Phenotypic data were collected for 2410 individuals measured during 1995-2017. By adding another 445 ancestors, a total of 2855 related individuals were used to derive estimated breeding values for all individuals, including the 2410 individuals with phenotypes. Among phenotyped individuals, we genotyped 403 cows with the Illumina 150 K Bovine BeadChip. RESULTS: GWAS were conducted with the FarmCPU (Fixed and random model circulating probability unification) method. We identified 12 markers significantly associated with six of the 10 traits under the threshold of 5% after a Bonferroni multiple test correction. Seven of these SNPs were in QTL regions previously identified to be associated with related traits. One identified SNP, BovineHD1600006691, was significantly associated with both age at first service and age at first calving. This SNP directly overlapped a QTL previously reported to be associated with calving ease. Within 160 Kb upstream and downstream of each significant SNP identified, we speculated candidate genes based on functionality. Four of the SNPs were located within four candidate genes, including CDH2, which is linked to milk fat percentage, and GABRG2, which is associated with milk protein yield. CONCLUSIONS: These findings are beneficial not only for breeding through marker-assisted selection, but also for genome editing underlying the related traits to enhance the overall performance of dual-purpose cattle.

Functional evaluation of a monotreme-specific antimicrobial protein, EchAMP, against experimentally induced mastitis in transgenic mice.

EchAMP, the tenth most abundant transcript expressed in the mammary gland of echidna, has in vitro broad-spectrum antibacterial effects. However, the effects of EchAMP on mastitis, a condition where inflammation is triggered following mammary gland infection, has not been investigated. To investigate the impact of EchAMP against mastitis, EchAMP transgenic mice were generated. In antibacterial assays, the whey fractions of milk from transgenic mice significantly reduced growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa compared with whey fractions from wildtype mice. Furthermore, a mastitis model created by infecting mammary gland with these four bacterial strains displayed a significant reduction in bacterial load in transgenic mice injected with S. aureus and B. subtilis. On further confirmation, histomorphologic analysis showed absence of necrosis and cell infiltration in the mammary glands of transgenic mice. To understand the role of EchAMP against inflammation, we employed an LPS-injected mastitis mouse model. LPS is known to induce phopshorylation of NF-κB and MAPK pathways, which in turn activate downstream proinflammatory signaling mediators, to promote inflammation. In LPS-treated EchAMP transgenic mice, phosphorylation levels of NF-κB, p38 and ERK1/2 were significantly downregulated. Furthermore, in mammary gland of transgenic mice, there was a significant downregulation of mRNA levels of proinflammatory cytokines, namely TNF-α, IL-6 and IL-1ß. Taken together, these data suggest that EchAMP has an antiinflammatory response and is effective against S. aureus and B. subtilis. We suggest that EchAMP may be a potential prophylactic protein against mastitis in dairy animals by expressing this gene in their mammary gland.

A validation study of loci associated with mastitis resistance in two French dairy sheep breeds.

BACKGROUND: The identification of loci associated with resistance to mastitis or of the causative mutations may be helpful in breeding programs for dairy sheep as it is for cattle worldwide. Seven genomic regions that control milk somatic cell counts, an indirect indicator of udder infection, have already been identified in sheep (Spanish Churra, French Lacaune and Italian Sardinian-Lacaune backcross populations). In this study, we used a 960 custom-designed ovine single nucleotide polymorphism (SNP) chip in Lacaune and Manech Tête Rousse dairy sheep to validate these seven genomic regions associated with mastitis. RESULTS: The most significant SNP (rs868996547) on Ovis aries chromosome (OAR) 3 was a previously described mutation in the suppressor of cytokine signalling 2 (SOCS2) gene. An antagonist effect of this causal candidate between health and growth in Lacaune sheep was confirmed. Effects of the mutation on the infectious status of the udder, i.e. increases in milk somatic cell counts and bacteria shedding, were also identified. This SNP was not present in the data available on Manech Tête Rousse. Three other regions associated with mastitis were also confirmed on OAR16 (Manech Tête Rousse), 19 (Lacaune) and 2 (both breeds). For the OAR2 region, we validated previously detected SNPs in several other breeds (Sarda, Churra, and Chios). For significant SNPs in the four mastitis regions, the effect varied from 0.24 to 0.67 phenotypic standard deviation of the traits. Two of the mastitis quantitative trait loci (QTL) regions (OAR2 and 16) that we validated here were also associated in opposite ways with milk production traits in both populations. CONCLUSIONS: These results indicate, at least in part, a genomic basis for the trade-off between milk production and mastitis resistance. Four of the seven mastitis QTL regions that were previously identified in independent populations, were confirmed in this study, which demonstrates partial sharing of mastitis-related genetic mechanisms between different distant dairy sheep populations.

Genetic associations of lactose and its ratios to other milk solids with health traits in Austrian Fleckvieh cows.

The genetic correlations (ra) of milk lactose percentage (LP), lactose yield (LY), and ratios of LP to other milk solids with udder, metabolic, and fertility disorders have not been assessed in dairy cattle so far. To evaluate the potential role of milk lactose as indicator of cow health, 142,285 lactation records of 84,289 Austrian Fleckvieh cows were analyzed with univariate and bivariate animal models. Milk traits were on a 150-d basis and health traits were coded as binary (0/1). Other than LP and LY, 3 new phenotypes were defined and included in the present study, namely the lactose-to-fat, lactose-to-protein, and lactose-to-solids ratios. The most heritable trait was LP (0.566 ± 0.008) and heritability of LY was much lower (0.145 ± 0.005). Heritability estimates close to 0.50 were assessed for the ratios. The frequency of health disorders was higher in multiparous cows yielding milk with low LP (≤4.553%) compared with cows yielding milk with high LP (≥5.045%). Heritabilities of health traits were in the expected ranges, with the highest estimate for ovarian cysts (CYS; 0.037 ± 0.004) and the lowest for retained placenta (0.005 ± 0.001). Mastitis (MAS) genetically correlated with LY (0.518 ± 0.057); considering that the amount of synthesized lactose is the key regulator of milk volume, this result confirmed that high-producing cows are more genetically susceptible to MAS than low-producing animals. Similar to MAS, ketosis (KET) was also positively genetically associated with LY (0.420 ± 0.077) and a weak and unfavorable ra between KET and lactose-to-protein ratio was estimated (0.159 ± 0.077). The ra of LY with milk fever (MFV) and CYS were approximately 0.20. The ra of LP with MAS, KET, and MFV were negative (-0.142 on average), supporting the idea that LP is a potential health indicator. Genetic correlations between health traits ranged from zero (retained placenta with MAS and CYS) to 0.463 ± 0.090 (MAS and MFV). Results of the present study suggest that LP has potentiality to be used as indicator trait to improve udder health in Austrian Fleckvieh population.

Divergent selection on milk somatic cell count in goats improves udder health and milk quality with no effect on nematode resistance.

Milk somatic cell count (SCC) is commonly higher in goats than in cattle and sheep. Furthermore, the ability of milk SCC to predict mastitis is considered lower in goats than in cattle and sheep, and the relevance of somatic cell score (SCS)-based selection in this species has been questioned. To address this issue, we created 2 divergent lines of Alpine goats using artificially inseminated bucks with extreme estimated breeding values for SCS. A total of 287 goats, 158 in high- and 129 in low-SCS lines, were scrutinized for mastitis infections. We subjected 2,688 milk samples to conventional bacteriological analyses on agarose and bacterial counts were estimated for positive samples. The SCS, milk yield, fat content, and protein content were recorded every 3 wk. Clinical mastitis was systematically noted. A subset of 40 goats (20 from each line) was subsequently challenged with Haemonchus contortus and monitored for anemia (blood packed cell volume) and fecal egg counts to see if SCS-based selection had an indirect effect on resistance to gastrointestinal nematodes. Milk production traits, including milk quantity, fat content, and protein content, were similar in both goat lines. In contrast, the raw milk SCC almost doubled between the lines, with 1,542,000 versus 855,000 cells/mL in the high- and low-SCS lines, respectively. The difference in breeding value for SCS between lines was 1.65 genetic standard deviation equivalents. The Staphylococcus spp. most frequently isolated from milk were S. xylosus, S. caprae, S. epidermidis, and S. aureus. The frequency of positive bacteriology samples was significantly higher in the high-SCS line (49%) than in the low-SCS line (33%). The highest odds ratio was 3.49 (95% confidence interval: 11.95-6.25) for S. aureus. The distribution of bacterial species in positive samples between lines was comparable. The average quantity of bacteria in positive samples was also significantly higher in high-SCS goats (69 ± 80 growing colonies) than in low-SCS goats (38 ± 62 growing colonies). Clinical cases were rare and equally distributed between high- (n = 4; 2.5%) and low-SCS (n = 3; 2.3%) lines. Furthermore, the larger the amounts of bacteria in milk the higher the SCS level. Conversely, goats with repeatedly culture-negative udders exhibited the lowest SCC levels, with an average of below 300,000 cells/mL. We therefore confirmed that SCS is a relevant predictor of intramammary infection and hygienic quality of milk in goats and can be used for prophylactic purposes. After challenge with H. contortus, goats were anemic with high fecal egg counts but we found no difference between the genetic lines. This result provides initial evidence that resistance to mastitis or to gastrointestinal nematodes infections is under independent genetic regulation. Altogether, this monitoring of the goat lines indicated that SCS-based selection helps to improve udder health by decreasing milk cell counts and reducing the incidence of infection and related bacterial shedding in milk. Selection for low SCC should not affect a goat's ability to cope with gastrointestinal nematodes.

Expression profile of the CSF3 and LPO genes in milk from buffalo (Bubalus bubalis) with and without mastitis.

This study compared the expression profile of the candidate genes, CSF3 and LPO, by investigating the immune response mechanisms involved in the phenotype of resistance and susceptibility to mastitis of healthy and infected buffaloes. The Granulocyte Colony Stimulating Factor 3 (CSF3) and Lactoperoxidase (LPO) genes expression profiles were determined in 24 milk samples from buffaloes with (N = 12) and without (N = 12) mastitis, using the quantitative real-time PCR (qRT-PCR) technique. CSF3 and LPO expressions were 5.14 (P = 0.001) and 2.41 (P = 0.097) times higher in animals with mastitis compared to healthy animals, respectively, evidencing a trend toward different expressions of this gene in the studied groups. Our finding suggests that LPO and CSF3 genes are an important defense mechanism against mastitis in dairy buffaloes, and may be putative genes for selecting healthier animals in buffalo breeding programs.

A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model.

Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2). The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host's inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.

Detection of quantitative trait loci and putative causal variants affecting somatic cell score in dairy sheep by using a 50K SNP chip and whole-genome sequencing.

This study presents a scan of the ovine genome to identify quantitative trait loci (QTL) influencing the somatic cell score (SCS), a classical indicator of subclinical mastitis in sheep, and a subsequent high-resolution analysis of one of the identified QTL regions based on the analysis of whole-genome sequence data sets. A half-sib commercial population of Churra sheep genotyped with a 50K SNP chip was analyzed using linkage analysis (LA) and combined linkage and linkage disequilibrium analysis (LDLA). By LA, 2 5% chromosome-wide significant QTL on OAR5 and OAR25 and one 5% genome-wide significant QTL on ovine chromosome 20 (OAR20) were detected, whereas 22 significant associations were identified by LDLA. Two of the associations detected by LDLA replicated LA-detected effects (OAR20, OAR25). We compared the detected associations with previously reported QTL in sheep and cattle, and functional candidate genes were identified within the estimated confidence intervals. We then performed a high-resolution analysis of the OAR20 QTL region, the most significant QTL region identified by LA that replicated a QTL previously described in Churra sheep for SCS using microsatellite markers. For that, 2 segregating trios of 2 segregating families for the OAR20 QTL (each including the Qq sire and 2 daughters, QQ and qq) were selected for whole-genome sequencing. The bioinformatic analysis of the 6 sequenced samples performed across the genomic interval considered (14.2-41.7 Mb) identified a total of 227,030 variants commonly identified by 2 independent software packages. For the 3 different concordance tests considered, due to discrepancies regarding the QTL peak in the segregating families, the list of mutations concordant with the QTL segregating pattern was processed to identify the variants identified in immune-related genes that show a moderate/high impact on the encoded protein function. Among a list of 85 missense variants concordant with the QTL segregation pattern that were within candidate immune-related genes, 13 variants distributed across 7 genes [PKHD1, NOTCH4, AGER, ENSOARG00000009395 (HLA-C, Homo sapiens), ENSOARG00000015002 (HLA-B, H. sapiens), MOG, and ENSOARG00000018075 (BoLA, Bos taurus, orthologous to human HLA-A] were predicted to cause deleterious effects on protein function. Future studies should assess the possible associations of the candidate variants identified herein in commercial populations with indicator traits of udder inflammation (SCS, clinical mastitis).

Somatic cell count-based selection reduces susceptibility to energy shortage during early lactation in a sheep model.

During the transition from late gestation to early lactation ruminants experience a negative energy balance (NEB), which is considered to increase susceptibility to mammary infections. Our previous study in 2 divergent lines of sheep selected for high and low somatic cell score (SCS) suggested an association between the response to NEB and genetic susceptibility to mastitis. Forty-eight early-lactation primiparous dairy ewes from the 2 SCS genetic lines were allocated to 2 homogeneous subgroups-an NEB group, which was energy restricted and received 60% of the energy requirements for 15 d, and a control-fed group-to obtain 4 balanced groups of 12 ewes: high-SCS positive energy balance, low-SCS positive energy balance, high-SCS NEB, and low-SCS NEB. High-SCS ewes showed greater weight loss and increased plasmatic concentrations of ß-hydroxybutyrate and nonesterified fatty acids than low-SCS ewes when confronted with an induced NEB. The aim of this study was to further characterize this interaction by combining transcriptomic and phenotypic data with a generalized partial least squares discriminant analysis using mixOmics package framework. A preliminary analysis using 3 blocks of phenotypes (fatty acids, weight and production, blood metabolites) revealed a high correlation between fat-to-protein ratio, ß-hydroxybutyrate, and nonesterified fatty acids concentrations with milk long-chain fatty acid yields. These phenotypes allowed good discrimination of the energy-restricted high-SCS ewes and confirmed a high level of adipose tissue mobilization in this group. A second analysis, which included RNA-seq data, revealed high correlations between the long-chain fatty acid yields in milk and PDK4, CPT1A, SLC25A20, KLF10, and KLF11 expression, highlighting the relationship between mobilization of body reserves and enhanced fatty acids utilization for energy production in blood cells. Finally, analysis of milk composition measured in 1,025 ewes from the 2 genetic lines over 10 yr confirmed significant higher fat-to-protein ratio in high-SCS ewes in early lactation. Altogether, our results strongly confirmed a genetic link between susceptibility to mastitis and metabolic adaptation to energy shortage. Improving genetic resistance to mastitis using SCS should be accompanied by a favorable effect on the response to metabolic stress, especially in highly stressful early lactation. Moreover, this study suggests that the fat-to-protein ratio could be used as a low-cost tool for monitoring energy balance and ketosis during this critical phase of lactation.

Analysis of human breast milk cells: gene expression profiles during pregnancy, lactation, involution, and mastitic infection.

The molecular processes underlying human milk production and the effects of mastitic infection are largely unknown because of limitations in obtaining tissue samples. Determination of gene expression in normal lactating women would be a significant step toward understanding why some women display poor lactation outcomes. Here, we demonstrate the utility of RNA obtained directly from human milk cells to detect mammary epithelial cell (MEC)-specific gene expression. Milk cell RNA was collected from five time points (24 h prepartum during the colostrum period, midlactation, two involutions, and during a bout of mastitis) in addition to an involution series comprising three time points. Gene expression profiles were determined by use of human Affymetrix arrays. Milk cells collected during milk production showed that the most highly expressed genes were involved in milk synthesis (e.g., CEL, OLAH, FOLR1, BTN1A1, and ARG2), while milk cells collected during involution showed a significant downregulation of milk synthesis genes and activation of involution associated genes (e.g., STAT3, NF-kB, IRF5, and IRF7). Milk cells collected during mastitic infection revealed regulation of a unique set of genes specific to this disease state, while maintaining regulation of milk synthesis genes. Use of conventional epithelial cell markers was used to determine the population of MECs within each sample. This paper is the first to describe the milk cell transcriptome across the human lactation cycle and during mastitic infection, providing valuable insight into gene expression of the human mammary gland.

Assessment of genetic variation for pathogen-specific mastitis resistance in Valle del Belice dairy sheep.

BACKGROUND: Mastitis resistance is a complex and multifactorial trait, and its expression depends on both genetic and environmental factors, including infection pressure. The objective of this research was to determine the genetic basis of mastitis resistance to specific pathogens using a repeatability threshold probit animal model. RESULTS: The most prevalent isolated pathogens were coagulase-negative staphylococci (CNS); 39 % of records and 77 % of the animals infected at least one time in the whole period of study. There was significant genetic variation only for Streptococci (STR). In addition, there was a positive genetic correlation between STR and all pathogens together (ALL) (0.36 ± 0.22), and CNS and ALL (0.92 ± 0.04). CONCLUSION: The results of our study support the presence of significant genetic variation for mastitis caused by Streptococci and suggest the importance of discriminating between different pathogens causing mastitis due to the fact that they most likely influence different genetic traits. Low heritabilities for pathogen specific-mastitis resistance may be considered when including bacteriological status as a measure of mastitis presence to implement breeding strategies for improving udder health in dairy ewes.