The protective effect of chlorogenic acid on bovine mammary epithelial cells and neutrophil function

Chlorogenic acid (CGA) is the ester of caffeic acid and quinic acid and plays an important role in antibacterial activity and anti-inflammatory properties. The objective of this study was to examine the effects of CGA on the growth of Staphylococcus aureus and the mRNA levels of the genes encoding the inflammatory response cytokines, κ-casein, and neutrophil function in bovine mammary epithelial cells (BMEC) exposed to S. aureus. Chlorogenic acid has important antibacterial, antioxidant, and anti-inflammatory functions; however, the effect of CGA on BMEC and neutrophils exposed to S. aureus has not been investigated previously. Our results demonstrated that 10, 20, and 30 μg/mL CGA had no cytotoxic effects on BMEC in culture, and that 20 μg/mL CGA enhanced the viability of BMEC exposed to S. aureus, whereas 30 μg/mL CGA reduced S. aureus growth after 9 h compared with controls. The rate of S. aureus invasion into BMEC was also attenuated by 30 μg/mL CGA compared with controls, whereas this treatment led to reduced abundance of IL6, IL8, and TLR2 mRNA in S. aureus-exposed BMEC. Migration of bovine polymorphonuclear leukocytes was significantly decreased in S. aureus-exposed BMEC with 10 and 20 μg/mL CGA treatment when compared with S. aureus treatment alone. In addition, incubation with 20 or 30 μg/mL CGA enhanced the phagocytic ability of polymorphonuclear leukocytes compared with the control group. Importantly, levels of κ-casein were enhanced by treatment of S. aureus-exposed BMEC with CGA. Our results suggest that the use of CGA may be a potent therapeutic tool against bovine mastitis caused by S. aureus.     

Sirtuin 3 inhibits nuclear factor-κB signaling activated by a fatty acid challenge in bovine mammary epithelial cells

Susceptibility to mastitis is highest during the peripartal (transition) period and is often concomitant with other comorbidities such as ketosis. Although infection with pathogenic microorganisms and immune-dysfunction around calving clearly play key roles in mastitis development, other metabolic factors also contribute. Sirtuin 3 (SIRT3), a mitochondrial deacetylase regulating energy and redox homeostasis, antagonizes the lipotoxic effects of nonesterified fatty acids (NEFA). Thus, we hypothesized that increases in circulating NEFA concentrations, as observed in the transition period, provokes inflammatory responses that can be reversed via activation of SIRT3. Here we aimed to study (1) proinflammatory NF-κB signaling and SIRT3 abundance in mammary tissue of ketotic cows and healthy controls, and (2) the effect of SIRT3 on NF-κB activation in bovine mammary epithelial cells (BMEC) treated with high levels of NEFA. The mammary gland biopsy samples were from a previous study, which included 15 healthy cows and 15 ketotic cows. Primary BMEC were isolated from 3 healthy Holstein cows with collagenase III digestion. Purified BMEC were incubated with or without SIRT3 overexpression adenovirus for 48 h, then treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 24 h. Mammary tissue of ketotic cows was associated with lower protein abundance of SIRT3 along with greater NF-κB P65 phosphorylation levels (p-NF-κB P65), p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. In BMEC, exogenous NEFA dose-dependently reduced protein abundance of SIRT3, but increased p-NF-κB P65, p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. Compared with green fluorescent protein adenovirus vector + NEFA, overexpression of SIRT3 in NEFA-treated BMEC downregulated p-NF-κB P65 and mRNA abundance of IL1B and IL6. Immunofluorescence indicated that overexpression of SIRT3 inhibited nuclear translocation of NF-κB P65. Overall, our data demonstrated that ketosis is associated with a reduction in SIRT3 abundance and activation of NF-κB signaling in the mammary gland. In vitro data provided evidence that high NEFA concentrations inhibit SIRT3, which contributes to enhanced NF-κB signaling including nuclear translocation and a pro-inflammatory response. The data suggest a promising role of SIRT3 as a target for helping alleviate localized inflammation of the mammary gland resulting from exposure to high concentrations of NEFA.     

Invited review: Microbiota of the bovine udder: Contributing factors and potential implications for udder health and mastitis susceptibility

Various body sites of vertebrates provide stable and nutrient-rich ecosystems for a diverse range of commensal, opportunistic, and pathogenic microorganisms to thrive. The collective genomes of these microbial symbionts (the microbiome) provide host animals with several advantages, including metabolism of indigestible carbohydrates, biosynthesis of vitamins, and modulation of innate and adaptive immune systems. In the context of the bovine udder, however, the relationship between cow and microbes has been traditionally viewed strictly from the perspective of host-pathogen interactions, with intramammary infections by mastitis pathogens triggering inflammatory responses (i.e., mastitis) that are often detrimental to mammary tissues and cow physiology. This traditional view has been challenged by recent metagenomic studies indicating that mammary secretions of clinically healthy quarters can harbor genomic markers of diverse bacterial groups, the vast majority of which have not been associated with mastitis. These observations have given rise to the concept of “commensal mammary microbiota,” the ecological properties of which can have important implications for understanding the pathogenesis of mastitis and offer opportunities for development of novel prophylactic or therapeutic products (or both) as alternatives to antimicrobials. Studies conducted to date have suggested that an optimum diversity of mammary microbiota is associated with immune homeostasis, whereas the microbiota of mastitic quarters, or those with a history of mastitis, are considerably less diverse. Whether disruption of the diversity of udder microbiota (dysbiosis) has a role in determining mastitis susceptibility remains unknown. Moreover, little is known about contributions of various biotic and abiotic factors in shaping overall diversity of udder microbiota. This review summarizes current understanding of the microbiota within various niches of the udder and highlights the need to view the microbiota of the teat apex, teat canal, and mammary secretions as interconnected niches of a highly dynamic microbial ecosystem. In addition, host-associated factors, including physiological and anatomical parameters, as well as genetic traits that may affect the udder microbiota are briefly discussed. Finally, current understanding of the effect of antimicrobials on the composition of intramammary microbiota is discussed, highlighting the resilience of udder microbiota to exogenous perturbants.     

Estimation of prevalence and incidence of subclinical mastitis in a large population of Brazilian dairy herds

The objectives of this study were to estimate the prevalence and incidence of subclinical mastitis (SM) in a large population of Brazilian dairy herds and to describe how these indices changed over time. A data set comprising individual cow somatic cell counts (SCC) from 18,316 test days (TD) of 1,809 herds that participated in a Dairy Herd Improvement Association (DHIA) program between January 2011 and May 2015 was available for analysis. Only tests that had ≥10 lactating cows and that were performed at 30 ± 10-d intervals were used for analysis. The final data set included 8,285 TD from 517 herds located in 5 regions of the country. Prevalence (%) of SM was defined as the number of cows with SCC ≥200,000 cells/mL divided by the total number of tested cows on a given TD. The incidence of SM was defined as the number of cows whose SCC increased from <200,000 to ≥200,000 cells/mL over 2 consecutive TD divided by the sum of each cow's days at risk during this interval, expressed as new cases per cow month at risk. Prevalence and incidence of SM were compared among years, regions, herd size categories, and frequency of DHIA testing during the study period. The overall mean prevalence and incidence of SM including all tests performed during the study period was 46.4% and 0.17 new cases per cow month at risk, respectively. The prevalence of SM varied little from 2011 to 2015, and an increasing trend was observed over the years. Prevalence was lower in herds that performed ≥60 DHIA tests during the study period than in those that performed fewer tests and was not different among regions or herd size categories. Incidence of SM varied little over the years and was not different among the regions studied. Prevalence and incidence of SM in the 517 herds studied were high and did not improve over the years. These trends were observed across all herd size categories and regions studied. Producers who had more DHIA tests performed per herd during the study period had lower prevalence of SM. Results of this study highlight the need to establish large-scale milk quality programs in Brazil.     

Association of bovine leukocyte antigen (BoLA) DRB3.2 with immune response, mastitis, and production and type traits in Canadian Holsteins

Data collected from 328 Canadian Holsteins in a research herd at the University of Guelph were used to study associations among expression of bovine leukocyte antigen (BoLA) DRB3.2 alleles, immune response, mastitis resistance via somatic cell counts (SCC), and clinical mastitis, as well as to extend these results to production and type traits. Accordingly, groups of cows were evaluated in vivo for both the antibody-mediated immune response (AMIR) and the cell-mediated immune response (CMIR), which generally predominate in responses to extracellular and intracellular pathogens, respectively. Of note was that associations between BoLA DRB3.2 alleles and immune responses tended to be in the opposite sign for the 2 AMIR and CMIR traits examined. For example, alleles DRB3.2*3 and *24 were associated with higher AMIR but lower CMIR, whereas allele *22 was associated with lower AMIR but higher CMIR. This finding is in agreement with the hypothesis that both traits are genetically independent and represent opposing type 1 and type 2 immune responses. Additionally, BoLA DRB3.2*3 and *11 were associated with lower SCC, whereas alleles *22 and *23 were associated with higher SCC. Finally, allele DRB3.2*3 was also associated with less clinical mastitis, whereas allele *8 was associated with higher mastitis risk. Allele *3 was of particular relevance because it was associated with increased antibodies, as well as reduced mastitis and SCC. This could be due to an indirect relationship between the ability to produce a high antibody response and enhanced defense against intrammamary infections caused by extracellular pathogens. Consequently, the BoLA DRB3.2*3 allele should be investigated further as a candidate for resistance to some types of intramammary infections, the important caveat being its association with lower CMIR, particularly with one of the test antigens used to evaluate delayed-type hypersensitivity. The results of associations between BoLA DRB3.2 and production traits were, in some cases, antagonistic in that BoLA DRB3.2 alleles *11 and *23, which are associated with increased production traits, were associated with lower and higher SCC, respectively. Collectively, these findings advocate the use of alleles *3, *23, and *22 as reference points for more detailed mechanistic studies. This does not imply that genetic selection for mastitis resistance should be based on BoLA alleles, but that information on a variety of genes may aid in identification and selection for improved health.     

Staphylococcus aureus induces autophagy in bovine mammary epithelial cells and the formation of autophagosomes facilitates intracellular replication of Staph. aureus

Staphylococcus aureus is an important pathogen causing chronic and subclinical mastitis of cows. Autophagy is an important regulatory mechanism that participates in the elimination of invading pathogenic organisms. Here, we hypothesize that autophagy is involved in the process of Staph. aureus survival in bovine mammary epithelial cells (BMEC). In this study, we detected the expression of autophagy-related proteins during infection and assessed the effect of autophagosome formation and degradation on the proliferation of intracellular Staph. aureus. Infection with Staph. aureus increased the protein expression of microtubule-associated protein 1 light chain 3-II (MAP1LC3, also called LC3-II) and sequestosome-1 (SQSTM1, also called p62) in BMEC. After infection, the formation of the autophagosomes increased but the autophagosomes and lysosomes could not fuse normally to form autolysosomes. When the formation of the autophagosomes was enhanced or the degradation of the autolysosomes was inhibited, the number of Staph. aureus in the BMEC increased. However, the intracellular proliferation of Staph. aureus was slowed when formation of autophagosomes was inhibited. Therefore, autophagy was induced in BMEC challenged by Staph. aureus but the autophagic flux was obstructed. Inhibiting the formation of autophagosomes in BMEC facilitated the clearance of intracellular Staph. aureus, which may offer a new strategy for the treatment of mastitis in cows.     

The National Cohort of Dairy Farms--a data collection platform for mastitis research in Canada

Costs and feasibility of extensive sample collection and processing are major obstacles to mastitis epidemiology research. Studies are often consequentially limited, and fundamental mastitis researchers rarely have the opportunity to conduct their work in epidemiologically valid populations. To mitigate these limitations, the Canadian Bovine Mastitis Research Network has optimized research funds by creating a data collection platform to provide epidemiologically meaningful data for several simultaneous research endeavors. This platform consists of a National Cohort of Dairy Farms (NCDF), Mastitis Laboratory Network, and Mastitis Pathogen Culture Collection. This paper describes the implementation and operation of the NCDF, explains its sampling protocols and data collection, and documents characteristics, strengths and limitations of these data for current and potential users. The NCDF comprises 91 commercial dairy farms in 6 provinces sampled over a 2-yr period. Primarily Holstein-Friesian herds participating in Dairy Herd Improvement milk recording were selected in order to achieve a uniform distribution among 3 strata of bulk tank somatic cell counts and to reflect regional proportions of freestall housing systems. Standardized protocols were implemented for repeated milk samplings on clinical mastitis cases, fresh and randomly selected lactating cows, and cows at dry-off and after calving. Just fewer than 133,000 milk samples were collected. Demographic and production data were recorded at individual cow and farm levels. Health management data are documented and extensive questionnaire data detailing farm management and cleanliness information are also captured. The Laboratory Network represents coordinated regional mastitis bacteriology laboratories using standardized procedures. The Culture Collection archives isolates recovered from intramammary infections of cows in the NCDF and contains over 16,500 isolates, all epidemiologically cross-referenced between linked databases. The NCDF is similar to Canadian dairies in relation to mean herd size, average production, and freestall percentages. Pathogen recovery was greater than anticipated, particularly for coagulase-negative staphylococci and Corynebacterium spp. International scientists are encouraged to use this extensive archive of data and material to enhance their own mastitis research.     

Bovine leukemia virus alters growth properties and casein synthesis in mammary epithelial cells

Bovine leukemia virus (BLV) is widespread in US dairy herds, yet only about 1% of infected cattle develop bovine leukosis and are culled from the herd. A major concern is whether BLV infection of dairy cows alters milk yield. Although several studies have examined the effect of BLV on milk production in vivo, the results were inconclusive. No in vitro studies have been done. The discovery of BLV in mammary epithelial cells (MEC) of infected cows raises the possibility that the virus could affect these cells directly. The purpose of this study was to use an in vitro system to determine if BLV could alter milk yield by altering cell number and/or milk production per cell. A short-term cell line established from the MEC of a BLV-negative cow, and a proven casein-producer mouse cell line, Comma D, were stably transfected with a plasmid containing the entire BLV genome. Untransfected parental lines served as negative controls. The BLV-containing bovine MEC line has a reduced population-doubling time, higher saturation density, and increased longevity. The Comma D line is an already-transformed cell line, and growth properties did not change after transfection with BLV. Under appropriate differentiation conditions, both the bovine and mouse MEC transfected with BLV displayed decreased casein production and mRNA synthesis compared with control cell lines without BLV. Our results suggest that effects of BLV infection on milk production may not be related solely to overall animal health but may also be mediated directly at a cellular level.     

Differences in udder health and immune response traits of Holstein-Friesians, Norwegian Reds, and their crosses in second lactation

The objective of this study was to investigate potential differences in udder health and immune response traits among Holstein-Friesian (HF), Norwegian Red (NR), and NR × HF (NRX) cows on 30 commercial Irish dairy farms. A total of 648 second-lactation cows (HF n = 274, NR n = 207, and NRX n = 167) were immunized with hen egg white lysozyme (HEWL) to induce antibody-mediated immune response (AMIR). Candida albicans was used to induce a cell-mediated immune response, with in vivo delayed-type hypersensitivity used as the indicator. Antibody response to HEWL was measured by ELISA. Udder health defined as mean somatic cell score (SCS) over the lactation, mean SCS within 30 d of the beginning of the immunization, peak SCS for each individual cow during lactation, and incidence of mastitis were statistically superior for the NR. The NR had a greater primary AMIR, producing greater concentrations of anti-HEWL immunoglobulin G on d 14 compared with HF and NRX. No difference was observed among the breed groups in the magnitude of secondary AMIR (d 21 post-immunization) or cell-mediated immune response. The proportion of high and low responders was similar across breed groups. Cows with high AMIR and high cell-mediated immune response had significantly lower mean SCS within 30 d of the start of the immunization, but greater occurrence of clinical mastitis, recorded as a binary trait over the course of the lactation. Otherwise, no significant difference in udder health was evident between cows designated as high and low responders. Although differences in mean breed group SCS values were in line with group mean AMIR values, no association was found among the traits when correlated on an individual cow basis. Results highlight the superiority of the NR with regard to udder health and suggest that improvements to udder health may result from crossbreeding with the NR. However, the immune response traits investigated proved to be inconsistent indicators of udder health.     

Selenomethionine stimulates expression of glutathione peroxidase 1 and 3 and growth of bovine mammary epithelial cells in primary culture

This study examined the localization of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in lactating mammary tissue and in primary cultures of bovine mammary epithelial cells (BMEC). The effect of selenium as selenomethionine (SeMet) on the growth and viability of BMEC and GPx protein expression and activity were also studied. Single mammary epithelial cells were recovered by serial collagenase/hyaluronidase digestion from lactating bovine mammary tissue and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20, or 50 nM SeMet. Positive immunostaining with anti-cytokeratin and bovine anti-casein confirmed the epithelial nature and differentiated state of BMEC. Addition of SeMet to media facilitated rapid confluence of BMEC and formation of dome structures. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC and localized in the cytoplasm and nucleus. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. Bovine mammary epithelial cells cultured in SeMet-supplemented medium also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. It is apparent that SeMet degradation to release Se for synthesis of selenoproteins is carried out by BMEC. Results indicate that bovine mammary epithelial cells express GPx1 and GPx3 in vivo and in vitro; SeMet enhances expression of these selenoproteins in vitro and the growth and viability of BMEC.     

Evidence for a local effect of leptin in bovine mammary gland

On average, high-energy diets promoting body growth rates above 1 kg/d before puberty impair mammary development by 15 to 20% in cattle. We hypothesized that leptin, a protein produced by adipocytes, mediates the inhibitory effect of high-energy diets on mammary development. Therefore, our objectives were to determine the effect of leptin on mammary epithelial cell proliferation, and the distribution of mRNA for two leptin receptor isoforms in prepubertal bovine mammary glands and other peripheral tissues. Addition of leptin to culture media containing either 5 ng/ml of insulin-like growth factor-I (IGF-I) or 1% fetal bovine serum decreased DNA synthesis of a bovine mammary epithelial cell line (MAC-T) in a dose-dependent manner. The minimal doses of leptin that decreased IGF-I- and fetal bovine serum-stimulated cell proliferation were 64 and 1 ng/ml, respectively. In addition, we determined that MAC-T cells and isolated bovine mammary epithelial cells express the long form of leptin receptor (Ob-Rb) mRNA. Ob-Rb mRNA was detected in all bovine tissues examined. In contrast with reports on other species, mRNA expression of the short form of leptin receptor (Ob-Ra) was detected only in bovine liver, pituitary body, and spleen. These results support the concept that leptin mediates the inhibitory effect of high-energy diets on mammary development.     

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-α, IL-1β, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 β, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.     

Genetic parameters of udder traits, somatic cell score, and milk yield in Latxa sheep

Genetic parameters have been estimated in the Black-Face ecotype of the Latxa breed for udder type traits (udder depth and attachment and teat placement and size) at first or later lactations (considered as different traits), as well as for udder type traits, milk yield, and lactational somatic cell score, including all lactations. Genetic correlations between udder type traits at first or later lactations ranged from 0.85 and 0.95 suggesting that they are nearly identical traits. Udder type traits had moderate heritabilities. Milk yield was estimated to have a genetic correlation of 0.43 with udder depth, 0.10 with udder attachment, −0.25 with teat placement, and −0.10 with teat size, which were unfavorable in general. Genetic correlations of lactational somatic cell score were 0.10 with udder depth, −0.27 with udder attachment, −0.01 with teat placement, and 0.29 with teat size. Genetic correlations between lactational somatic cell score and udder type traits show that udders with good shape are less prone to subclinical mastitis.     

Short communication: Diagnosis and classification of clinical and subclinical mastitis utilizing a dynamometer and a handheld infrared thermometer

In times of ongoing automatization of dairy cow husbandry, objective and reliable tools for mastitis diagnostic are highly in demand. The objective of this study was to investigate the diagnostic value of a handheld dynamometer and an infrared thermometer to diagnose and score clinical and subclinical mastitis and to compare those values with results from palpation of the udder tissue. Overall, 218 cows with clinical mastitis (i.e., 46 mild, 106 moderate, and 66 severe cases), 142 with subclinical mastitis, and 68 healthy cows were enrolled. Our data provide evidence that the dynamometer is an accurate diagnostic tool to differentiate between healthy udder quarters, and those with subclinical and clinical mastitis. Furthermore, the severity score of clinical mastitis can be estimated by dynamometer. The firmness threshold for the detection of clinical mastitis was 1.002 kg. Using a threshold of 1.175 kg in clinical mastitis quarters, it was possible to differentiate between negative and positive bacteriological results. A differentiation between healthy and clinical mastitis quarters with the infrared thermometer was possible, albeit udder surface temperatures were highly influenced by ambient temperature. Udder surface temperature increased by 0.15 to 0.18°C for each degree of ambient temperature. In conclusion, the utility of an infrared thermometer to estimate the udder health status of dairy cows is limited, whereas the handheld dynamometer appeared to be an accurate and objective method.