The bioactivity of colostrum and milk exosomes of high, average, and low immune responder cows on human intestinal epithelial cells.

Bovine milk contains bioactive components that are nutritionally and immunologically important to calves and humans. Dairy cows classified as high (H) immune responders using the patented high immune response technology have higher concentrations of immunoglobulin and specific antibodies in sera and milk compared with average (A) and low (L) responders. MicroRNA post-transcriptionally regulate expression of milk bioactive components and are enriched in extracellular vesicles known as exosomes, which protect them from degradation. The bioactivity of colostrum and milk exosomes at the human intestinal epithelial barrier remains to be explored, particularly in the context of the high immune response technology. Therefore, the purpose of this study was to evaluate the functional role of bovine milk exosomes compared with colostrum exosomes from H, A, and L responders at the intestinal interface using human colorectal adenocarcinoma epithelial (Caco-2) cells. Exosomes were isolated by successive ultracentrifugation and confirmed by western blot analysis for the presence of common exosomal proteins (CD9, CD63, and heat shock protein 70). Fluorescent labeling of exosomes using PKH67 dye confirmed their uptake by Caco-2 cells, demonstrating their potential bioavailability. The MTT assays showed that colostrum and milk exosomes maintain Caco-2 metabolic activity and are not cytotoxic to these cells. Specifically, metabolic activity after co-incubation with colostrum and milk exosomes from H responder cows was significantly greater than after co-incubation with exosomes from L responders. Caspase 3 activity, an indicator of apoptosis, was significantly lower after co-incubation of Caco-2 cells with milk exosomes compared with colostrum exosomes, suggesting that unlike colostrum exosomes, particularly those from L responders, milk exosomes do not activate the caspase 3 pathway in Caco-2 cells. This study helps us better understand the functional importance of colostrum and milk exosomes from dairy cows and emphasizes differences in functionality among exosomes from H, A, and L immune responders.

Changes in Holstein cow milk and serum proteins during intramammary infection with three different strains of Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with S. aureus is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. A sub-population of S. aureus, known as small colony variant (SCV), displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. Therefore, it was hypothesized that the host immune response will be different for SCV than its parental or typical strains of S. aureus. In this study, the local and systemic immune protein responses to intramammary infection with three strains of S. aureus, including a naturally occurring bovine SCV strain (SCV Heba3231), were characterized. Serum and casein-depleted milk cytokine levels (interleukin-8, interferon-γ, and transforming growth factor-ß1), as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three S. aureus strains. Furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of S. aureus intramammary infection. RESULTS: Serum IL-8, IFN-γ, and TGF-ß1 responses differed in dairy cows challenged with different strains of S. aureus. Changes in overall serum haptoglobin concentrations were observed for each S. aureus challenge group, but there were no significant differences observed between groups. In casein-depleted milk, strain-specific differences in the host IFN-γ response were observed, but inducible IL-8 and TGF-ß1 concentrations were not different between groups. Proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. Notably, the protein, component-3 of the proteose peptone (CPP3), was differentially expressed between the S. aureus treatment groups, implicating it as a potential antimicrobial peptide involved in host defense against S. aureus intramammary infection. CONCLUSIONS: Intramammary infection of dairy cattle with S. aureus causes an up-regulation of serum and milk immune-related proteins, and these responses vary depending on the infecting strain.

Genetic and Epigenetic Regulation of Immune Response and Resistance to Infectious Diseases in Domestic Ruminants.

Infectious diseases are the outcome of complex interactions between the host, pathogen, and environment. After exposure to a pathogen, the host immune system uses various mechanisms to remove the pathogen. However, environmental factors and characteristics of pathogens can compromise the host immune responses and subsequently alter the outcome of infection. In this article, genetic and epigenetic factors that shape the individual variation in mounting protective responses are reviewed. Different approaches that have been used by researchers to investigate the genetic regulation of immunity in ruminants and various sources of genetic information are discussed.

Characterization of a Staphylococcus aureus small colony variant (SCV) associated with persistent bovine mastitis.

Staphylococcus aureus is a common cause of bovine mastitis and foodborne and other diseases in humans. This study tested the hypothesis that small colony variants (SCVs) of S. aureus are implicated in chronic bovine mastitis. Six S. aureus isolates from foremilk samples from 11 chronically infected cows were investigated. Five isolates had typical morphology and were hemolytic and coagulase positive; one was a heterogeneous population of typical and SCV phenotype (tiny nonhemolytic colonies). In the presence of gentamicin, three of the previously typical S. aureus developed SCVs that were confirmed as S. aureus by biochemical and genetic analyses; these SCVs reverted to the typical form on antibiotic-free medium. The SCV isolate (Heba3231) from the heterogeneous population had a slow growth rate and prolonged lag phase and did not revert during 10 h of incubation. Transcriptional analysis showed that SCV Heba3231 had reduced expression of agr, hla, and coa and increased expression of indicators of fermentation pathways compared to the parent strain. Invasion of and persistence in a primary culture of bovine aortic endothelial cells (BAEC) showed that SCV Heba3231 had minimal deleterious effects, whereas the parent strain or the Newbould 305 strain caused severe damage. Recovery of the parent strain from BAEC yielded a mixture of the parent and SCV phenotypes. This study reports for the first time the isolation of S. aureus SCV from persistent bovine mastitis and suggests that SCV may be an important contributor to the prolonged survival of S. aureus in some cases of mastitis.

Bovine mastitis: frontiers in immunogenetics.

Mastitis is one of the most prevalent and costly diseases in the dairy industry with losses attributable to reduced milk production, discarded milk, early culling, veterinary services, and labor costs. Typically, mastitis is an inflammation of the mammary gland most often, but not limited to, bacterial infection, and is characterized by the movement of leukocytes and serum proteins from the blood to the site of infection. It contributes to compromised milk quality and the potential spread of antimicrobial resistance if antibiotic treatment is not astutely applied. Despite the implementation of management practises and genetic selection approaches, bovine mastitis control continues to be inadequate. However, some novel genetic strategies have recently been demonstrated to reduce mastitis incidence by taking advantage of a cow's natural ability to make appropriate immune responses against invading pathogens. Specifically, dairy cattle with enhanced and balanced immune responses have a lower occurrence of disease, including mastitis, and they can be identified and selected for using the high immune response (HIR) technology. Enhanced immune responsiveness is also associated with improved response to vaccination, increased milk, and colostrum quality. Since immunity is an important fitness trait, beneficial associations with longevity and reproduction are also often noted. This review highlights the genetic regulation of the bovine immune system and its vital contributions to disease resistance. Genetic selection approaches currently used in the dairy industry to reduce the incidence of disease are reviewed, including the HIR technology, genomics to improve disease resistance or immune response, as well as the Immunity(+)™ sire line. Improving the overall immune responsiveness of cattle is expected to provide superior disease resistance, increasing animal welfare and food quality while maintaining favorable production levels to feed a growing population.

Staphylococcus aureus small colony variants (SCVs) and their role in disease.

Persistent or difficult-to-treat Staphylococcus aureus infections in animals and humans may be related to small colony variants (SCVs) that can hide inside host cells and modulate host defenses. S. aureus SCVs have gained much attention in human medicine but have been underestimated and overlooked in veterinary medicine. Recently, an SCV isolated from a dairy cow with a history of chronic mastitis was shown to possess similar phenotypic and transcriptomic properties to those of human SCVs. SCVs form small, colorless, non-hemolytic colonies after 48 h, are only slowly coagulase positive, fail to ferment mannitol, and can revert to the parental phenotype. The phenotype of SCVs is mostly related to alterations in hemin and/or menadione biosynthesis or to thymidine deficiency. Transcriptomic analysis of SCVs shows up-regulation of genes involved in glycolytic and arginine-deiminase pathways, capsular biosynthesis; increased sigma B activity; and down-regulation of genes for α-hemolysin, coagulase and effector molecule RNA III of the global virulence regulator Agr. Similar results are reported at the protein level. SCVs are less virulent but successful persisters in infection models. SCVs persist longer and at higher numbers within non-phagocytes than do their parents. SCVs survive within spacious vacuoles up to 24 h within cultured bovine mammary epithelial cells, likely due to up-regulation of protective mechanisms that counteract the lethal acidic environment of the phagolysosome. Persistence of SCVs within host cells may explain failures in antimicrobial therapy and vaccinations.

Persistence of a Staphylococcus aureus small colony variants (S. aureus SCV) within bovine mammary epithelial cells.

Persistent bovine Staphylococcus aureus mastitis is attributable to the versatility of this pathogen within the mammary gland environment and to the formation of small colony variants (SCVs) that can survive within host cells. Previous studies had shown that S. aureus SCV Heba3231, isolated from a cow with chronic mastitis, had invaded and persisted in primary bovine aortic endothelial cells but caused minimal deleterious effects. The objective of this study was to investigate the interaction of SCV Heba3231 with bovine mammary epithelial cells (MAC-T cells) compared to its parent strain 3231 and to prototype strain Newbould 305. Monolayer cells were infected with each strain at various multiplicity of infections (MOIs) for 1 and 3.5h, followed by 20 min incubation with lysostaphin. Recovery of the SCV was significantly higher (P<0.05) after 3.8h with MOI of 100 compared to recovery of strains 3231 and Newbould 305. Upon further incubation, viable SCV were detected up to 96 h while 3231 were not isolated at 24h or later. Transmission electron microscopy demonstrated SCV uptake by MAC-T cells following a series of events similar to those for strain 3231. At 24h, multiple SCV were seen within enclosed vacuoles, while the 3231 parent strain was released extracellularly and the monolayer cells were damaged. The ability of SCV Heba3231 to survive inside vacuoles could be related to up-regulation of protective mechanisms. These findings highlight the potential role of bovine mammary epithelial cells and S. aureus SCV in persistent bovine mastitis.

Antibody and cell-mediated immune responses to Staphylococcus aureus small colony variants and their parental strains associated with bovine mastitis.

Persistence of bovine Staphylococcus aureus mastitis may be associated with the small colony variant (SCV) form that is adapted to intracellular life and resists elimination by the immune system. This study evaluated antibody-mediated (AMIR) and cell-mediated immune responses (CMIR) to two bovine SCV forms and their parent strains isolated from cows with mastitis. Four groups of healthy cows, five cows/treatment group, were challenged by the intramammary route with naturally occurring bovine SCV Heba3231, its parent strain 3231, a hemB mutant displaying the SCV phenotype or its parent strain, Newbould 305. Blood and milk samples were collected at day 0 before challenge and at days 1, 14, 21 and 36 post-challenge to determine antigen-specific immunoglobulin (Ig) IgG(1) and IgG(2) antibody responses as indicators of type 2 and type 1 responses, respectively. At day 24 post-challenge cows in each group were inoculated with the UV-killed homologous strain intradermally in the neck to induce delayed-type hypersensitivity (DTH) as an indicator of CMIR. The SCV Heba3231 and 3231 strains induced significant IgG(1) and IgG(2) antibody responses in sera and in sera and milk whey, respectively. The hemB SCV mutant and Newbould 305 strains induced significant IgG(1) antibody in milk whey, and in sera and milk whey, respectively. The SCV Heba3231 and 3231 strains induced DTH, the hemB mutant induced intermediate hypersensitivity, and Newbould 305 failed to induce DTH. These results indicate marked differences in immune responses induced by parent and SCV forms of the same strain of S. aureus and by the two wild-type strains. This is the first study to evaluate both AMIR and CMIR in the context of persistent bovine mastitis to different and genetically characterized strains of S. aureus including two SCVs. The findings expand our understanding of immune responses to persistent S. aureus mastitis.

Somatic cell scores and clinical signs following experimental intramammary infection of dairy cows with a Staphylococcus aureus small colony variant (S. aureus SCV) in comparison to other bovine strains.

A recently isolated bovine Staphylococcus aureus small colony variant (SCV) was shown to persist within cultured endothelial cells. However, the clinical importance of S. aureus SCV in persistent infection of the mammary gland is unknown. The hypothesis tested here was a naturally occurring bovine S. aureus SCV, Heba3231, and a Newbould hemB mutant that was an artificially created SCV would establish mild intramammary infection and induce a different host response compared to their isogenic parental strains, 3231 and Newbould 305. Four groups of clinically healthy cows, 5 cows/treatment group, were infected by the intramammary route with SCV Heba3231, strain 3231, the hemB mutant or strain Newbould 305. Three quarters of the mammary gland of each cow were challenged with approximately 5000 colony-forming units of bacteria and the fourth quarter was infused with PBS. Cows were monitored and assigned clinical scores for the first 5 days post-challenge based on rectal temperature, appetite, milk yield, udder uniformity, milk appearance, bacterial culture of foremilk samples and somatic cell score (SCS). All cows were examined for chronic infection up to day 36 post-challenge. Cows challenged with SCV strains developed mastitis that was mild compared with that induced by the parent strains. Cows challenged with strain 3231 developed the most severe clinical mastitis. SCS from all treatment groups were significantly (P<0.05) higher at days 2-5 and 14-36 post-infection compared to day 0 before challenge. This milder host response to SCVs may represent an adaptation of S. aureus to the mammary gland environment.