Gene mapping, gene-set analysis, and genomic prediction of postpartum blood calcium in Holstein cows.

The onset of lactation results in a sudden irreversible loss of Ca for colostrum and milk synthesis. Some cows are unable to quickly adapt to this demand and succumb to clinical hypocalcemia, whereas a larger proportion of cows develop subclinical hypocalcemia that predisposes them to other peripartum diseases. The objective of this study was to perform a comprehensive genomic analysis of blood total Ca concentration in periparturient Holstein cows. We first performed a genomic scan and a subsequent gene-set analysis to identify candidate genes, biological pathways, and molecular mechanisms affecting postpartum Ca concentration. Then, we assessed the prediction of postpartum Ca concentration using genomic information. Data consisted of 7,691 records of plasma or serum concentrations of Ca measured in the first, second, and third day after parturition of 959 primiparous and 1,615 multiparous cows that calved between December 2015 and June 2020 in 2 dairy herds. All cows were genotyped with 80k SNPs. The statistical model included lactation (1 to 5+), calf category (male, females, twins), and day as fixed effects, and season-treatment-experiment, animal, and permanent environmental as random effects. Model predictive ability was evaluated using 10-fold cross-validation. Heritability and repeatability estimates were 0.083 (standard error = 0.017) and 0.444 (standard error = 0.028). The association mapping identified 2 major regions located on Bos taurus autosome (BTA)6 and BTA16 that explained 1.2% and 0.7% of additive genetic variance of Ca concentration, respectively. Interestingly, the region on BTA6 harbors the GC gene, which encodes the vitamin D binding protein, and the region on BTA16 harbors LRRC38, which is actively involved in K transport. Other sizable peaks were identified on BTA5, BTA2, BTA7, BTA14, and BTA9. These regions harbor genes associated with Ca channels (CACNA1S, CRACR2A), K channels (KCNK9), bone remodeling (LRP6), and milk production (SOCS2). The gene-set analysis revealed terms related to vitamin transport, calcium ion transport, calcium ion binding, and calcium signaling. Genomic predictions of phenotypic and genomic estimated breeding values of Ca concentration yielded predictive correlations up to 0.50 and 0.15, respectively. Overall, the present study contributes to a better understanding of the genetic basis of postpartum blood Ca concentration in Holstein cows. In addition, the findings may contribute to the development of novel selection and management strategies for reducing periparturient hypocalcemia in dairy cattle.

Diverse ß-lactam antibiotic-resistant bacteria and microbial community in milk from mastitic cows.

Intramammary bacterial infection, the most common cause of mastitis, is the most costly disease in dairy cattle in the US and reason for antibiotic usage. Ceftiofur, a third-generation cephalosporin, is generally used to treat such disease, but it has a high treatment failure rate. Though the reason is not known clearly, it is hypothesized that multiple factors are associated with the treatment failure. In this study, we analyzed 169 milk samples from cows with mastitis in two independent dairy farms (Farm A and B) in which 19.4% (Farm A) and 14.3% (Farm B) of the antibiotic treated cows were not cured. The prevalence of cephalosporin-resistant bacteria (CRB) in milk was 72.0% and 42.1% in Farm A and B, respectively. Nineteen and nine bacterial genera were identified in Farm A and B respectively, with the most abundant genus being Staphylococcus (27.1%; Farm A) and Bacillus (63.5%; Farm B). However, no strong relationship between the treatment failure rate and the CRB prevalence was observed. Furthermore, the metagenomic analysis showed no significant differences in the α- and ß-diversities of microbiota in milk samples from cured and uncured cows, suggesting that antibiotic-resistant bacteria were not the sole reason for the antibiotic treatment failure. KEY POINTS: • The mastitic milk samples had high prevalence of cephalosporin-resistant bacteria (CRB). • The CRB identified belong to diversified species. • Antibiotic treatment failure was not solely caused by the abundance of CRB.

Symposium review: Transition cow calcium homeostasis-Health effects of hypocalcemia and strategies for prevention.

The effects of subclinical hypocalcemia have been explored in numerous observational and mechanistic studies in recent years. Besides obvious, well-known effects on muscle contractility, the role of Ca with respect to immune function and intermediary metabolism explains the contribution of subclinical hypocalcemia to the development of several diseases observed in early lactation and underlines its importance in high-performing dairy cows. The present review aims at integrating recent scientific progress, such as discoveries about the role of the mammary gland in regulating bone mobilization, into generally accepted aspects of the endocrine control of Ca homeostasis. We will discuss Ca transport mechanisms through absorption, resorption, secretion, and mobilization, as well as the physiological regulation of Ca through parathyroid hormone, 1,25-dihydroxyvitamin D, fibroblast growth factor 23, and serotonin, in addition to dietary mineral requirements. To improve hypocalcemia prevention strategies, our knowledge of the physiological mechanisms necessary to maintain normocalcemia and their endogenous regulation should be combined with data derived from herd-level studies. Using such studies, we will discuss prepartum nutritional strategies aimed at reducing the incidence of subclinical hypocalcemia, as well as options for postpartum Ca supplementation and their effects on early-lactation health and production. Especially in respect to approaches that might interfere with endogenous adaptation processes, such as supplementation with vitamin D metabolites or large doses of Ca, a thorough understanding of the underlying mechanisms that might induce unwanted hypocalcemia rebound effects will be crucial to ameliorate our future management of transition cows. Continued efforts by researchers to understand the interaction of Ca homeostasis with prevention strategies is necessary to optimize cow health and support copious milk production.

MILK Symposium review: Identifying constraints, opportunities, and best practices for improving milk production in market-oriented dairy farms in Sri Lanka.

Dairy is the most important subsector in the Sri Lankan livestock industry, due to the need to address the growing demand for fresh milk and milk products, and because of its potential influence on the rural economy. The USDA Food for Progress program awarded a 4.5-year Market-Oriented Dairy project to International Executive Service Corps, a not-for-profit organization based in Washington, DC. The objective of the Market-Oriented Dairy project is to support Sri Lanka's dairy sector and catalyze sustainable growth by strengthening the dairy sector through better technological, financial, and management practices benefiting all stakeholders and consumers along the dairy value chain. The University of Florida is working with International Executive Service Corps as technical experts in conducting dairy value chain assessments, identifying gaps and challenges in dairy management practices, extension services, milk quality management standards, and artificial insemination services. Assessment of the dairy value chain in 2018 identified a lack of good quality and quantity of feed, along with poor dairy management practices and ineffective extension services as major constraints to improving dairy productivity in Sri Lanka. In addition, lack of national milk quality standards that are consistent with international benchmarks and inadequate cooling facilities are significant challenges to improving milk quality. The nutritional status of cows is not suitable for optimal reproductive performance, compromising the success of artificial insemination in Sri Lanka. Based on these findings, we developed a dairy assessment tool and provided comprehensive training sessions targeting extension agents, veterinarians, and farmers to promote best practices in dairy management. Beyond training, however, industry support for standardization and monitoring of milk and feed quality are needed, providing opportunities for private investment to support the dairy industry. Similar opportunities are available for forage production and delivery to producers. The broader aim of the Market-Oriented Dairy project intervention is to reduce Sri Lanka's dependency on imported milk and contribute toward the goal of a safe, self-sufficient fresh milk supply.

Feeding supplemental 25-hydroxyvitamin D3 increases serum mineral concentrations and alters mammary immunity of lactating dairy cows.

Objectives were to determine the effects of feeding supplemental 25-hydroxyvitamin D3 [25(OH)D3] on concentrations of vitamin D metabolites and minerals in serum, mammary immune status, and responses to intramammary bacterial infection in dairy cows. Sixty multiparous, pregnant lactating Holstein cows with somatic cell count <200,000/mL were blocked by days in milk and milk yield and randomly assigned to receive a daily top-dressed dietary supplement containing 1 or 3 mg of vitamin D3 (1mgD or 3mgD), or 1 or 3 mg 25(OH)D3 (1mg25D or 3mg25D) for 28 d (n = 15/treatment). Cows were kept in a freestall barn and fed a total mixed ration in individual feeding gates. Individual dry matter intake (DMI) and milk yield were recorded daily, and milk and blood samples were collected at 0, 7, 14, and 21 d relative to the start of treatment. At 21 d, cows fed 1mgD and 3mg25D received an intramammary challenge with Streptococcus uberis. Cows were observed for severity of mastitis, and blood and milk samples were collected every 12 h to measure inflammation. The 1mg25D and 3mg25D cows had greater serum 25(OH)D3 concentrations at 21 d compared with 1mgD and 3mgD cows (62 ± 7, 66 ± 8, 135 ± 15, and 232 ± 26 ng/mL for 1mgD, 3mgD, 1mg25D, and 3mg25D, respectively). The 3mg25D cows had greater concentrations of Ca and P in serum at 21 d compared with other treatments (Ca = 2.38, 2.4, 2.37, and 2.48 ± 0.02 mM, 1.87, 1.88, and 2.10 ± 0.08 mM for 1mgD, 3mgD, 1mg25D, and 3mg25D, respectively). Yields of milk and milk components, DMI, body weight, and concentrations of 1,25-dihydroxyvitamin D and Mg in serum did not differ among treatments. Abundance of mRNA transcripts for interleukin-1ß (IL1B) and inducible nitric oxide synthase (iNOS) in milk somatic cells before S. uberis challenge were increased in cows fed 25(OH)D3 compared with cows fed vitamin D3. Furthermore, IL1B, iNOS, ß-defensin 7, and ß-defensin 10 in milk somatic cells increased as concentrations of 25(OH)D3 increased in serum. Cows fed 3mg25D had less severe mastitis at 60 and 72 h after challenge with S. uberis compared with cows fed 1mgD. Concentrations of bacteria, somatic cells, and serum albumin in milk after challenge did not differ between treatments; however, an interaction between treatment and day was detected for lactate dehydrogenase in milk. Expression of adhesion protein CD11b on milk neutrophils after the S. uberis challenge was greater among 3mg25D cows compared with 1mgD cows. Transcripts of CYP24A1 and iNOS in milk somatic cells during mastitis also were greater in 3mg25D cows compared with 1mgD cows. Feeding 25(OH)D3 increased serum 25(OH)D3 more effectively than supplemental vitamin D3, resulting in increased serum mineral concentrations, increased expression of vitamin D-responsive genes, and altered immune responses to intramammary bacterial challenge.

Use of a Fluorescent Analog of Glucose (2-NBDG) To Identify Uncultured Rumen Bacteria That Take Up Glucose.

Few characteristics are more important to a bacterium than the substrates it consumes. It is hard to identify what substrates are consumed by bacteria in natural communities, however, because most bacteria have not been cultured. In this study, we developed a method that uses fluorescent substrate analogs, cell sorting, and DNA sequencing to identify substrates taken up by bacteria. We deployed this method using 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose analog, and bacteria of the bovine rumen. This method revealed over 40 different bacteria (amplicon sequence variants [ASVs]) from the rumen that take up glucose. Nearly half of these ASVs represent previously uncultured bacteria. We attempted to grow these ASVs on agar media, and we confirmed that nearly two-thirds resisted culture. In coculture experiments, the fluorescent label of 2-NBDG was not transferred to nontarget bacteria by cross-feeding. Because it is not affected by cross-feeding, our method has an advantage over stable isotope probing. Though we focus on glucose, many substrates can be labeled with the fluorophore NBD. Our method represents a new paradigm for identifying substrates used by uncultured bacteria. It will help delineate the niche of bacteria in their environment.IMPORTANCE We introduce a method for identifying what substrates are consumed by bacteria in natural communities. Our method offers significant improvement over existing methods for studying this characteristic. Our method uses a fluorescently labeled substrate which clearly labels target bacteria (glucose consumers in our case). Previous methods use isotope-labeled substrates, which are notorious for off-target labeling (due to cross-feeding of labeled metabolites). Our method can be deployed with a variety of substrates and microbial communities. It represents a major advance in connecting bacteria to the substrates they take up.

Short communication: Inhibition of DNA methyltransferase and histone deacetylase increases ß-defensin expression but not the effects of lipopolysaccharide or 1,25-dihydroxyvitamin D3 in bovine mammary epithelial cells.

Antimicrobial peptides are a common defense against bacterial infections in many species and a significant part of the innate immune response of the bovine mammary gland. The objective of this study was to investigate the influence of epigenetic factors on vitamin D and toll-like receptor-mediated induction of ß-defensins in mammary epithelial cells. Primary bovine mammary epithelial cells were treated with lipopolysaccharide (LPS, 0 or 100 ng/mL), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 0 or 10 nM], and 5-aza-2'-deoxycytidine (5-Aza, inhibitor of DNA methyltransferase, 0 or 5 µM) or trichostatin A (TSA, inhibitor of histone deacetylase, 0 or 80 nM) in a factorial arrangement. Effects of treatments on ß-defensin gene expression along with genes for cytokines and enzymes known to be induced by LPS or 1,25(OH)2D3 were evaluated by quantitative PCR. The LPS treatment induced expression of ß-defensin (DEFB)3, DEFB5, DEFB7, DEFB10, enteric ß-defensin (EBD), lingual antimicrobial peptide (LAP), and tracheal antimicrobial peptide (TAP); whereas, the 1,25(OH)2D3 treatment increased DEFB5 and DEFB7 expression and decreased LAP. The 5-Aza treatment increased expression of DEFB3, DEFB5, DEFB10, EBD, LAP, and TAP in the presence and absence of LPS. The TSA treatment increased expression of DEFB3, DEFB4, DEFB5, DEFB7, and DEFB10 in the absence of LPS but decreased LPS-induced expression of and LAP and TAP. Together these results indicate that ß-defensin expression in bovine mammary epithelial cells is likely influenced by DNA methylation and histone acetylation. Investigation of environmental and nutritional factors that influence epigenetic control of ß-defensins in the mammary gland may be beneficial for improving resistance to intramammary infections.

Neutrophil ß-defensin gene expression of postpartum dairy cows is altered by prepartum dietary cation-anion difference.

The objective of this study was to evaluate expression of a cluster of genes encoding ß-defensin antimicrobial peptides in neutrophils of postpartum cows in relation to prepartum dietary cation-anion difference (DCAD), vitamin D, and postpartum disease. Pregnant dry Holstein cows (28 nulliparous and 51 parous) at 255 d gestation were blocked by parity and randomly assigned to 4 prepartum diets of positive (+130 mEq/kg) or negative (-130 mEq/kg) DCAD and either 3 mg vitamin D3 or 3 mg of 25-hydroxyvitamin D3 per 11 kg of dry matter/d. Treatment diets were fed from 255 d of gestation until calving. Peripheral blood neutrophils of 35 parous cows were collected at 0 and 3 d after calving and stimulated with 0 or 100 ng/mL of lipopolysaccharide (LPS). Furthermore, serum Ca and incidences of postpartum diseases were recorded for all cows. The mRNA transcripts of ß-defensin genes were quantified by real-time PCR, and data were analyzed with a general linear mixed model to test for fixed effects and interactions of day, level of DCAD, source of vitamin D, and incidence of disease. Effects of DCAD and vitamin D on neutrophil oxidative burst and phagocytosis were previously reported but were analyzed for effects of disease in the present study. Transcripts for DEFB1, DEFB3, DEFB4, DEFB5, DEFB7, DEFB10, and lingual antimicrobial peptide (LAP) in neutrophils were upregulated by LPS at 0 d but not at 3 d. Transcripts for DEFB4 and DEFB7 in LPS-stimulated neutrophils were greater in cows fed negative DCAD diets compared with positive DCAD. Source of vitamin D (vitamin D3 vs. 25-hydroxyvitamin D3) did not affect expression of ß-defensins in neutrophils. Cows with postpartum subclinical hypocalcemia (serum Ca <2.0 mM) had decreased DEFB3, DEFB4, DEFB6, DEFB7, DEFB10, and LAP expression in LPS-stimulated neutrophils compared with cows that did not experience subclinical hypocalcemia. Likewise, DEFB4, DEFB6, DEFB7, DEFB10, and LAP in LPS-stimulated neutrophils at 3 d postpartum were positively associated with serum Ca at 0 d postpartum. Transcripts for DEFB7, DEFB10 and LAP also were less abundant in neutrophils from cows with metritis compared with healthy cows. In conclusion, feeding a prepartum negative DCAD to improve postpartum serum Ca resulted in greater neutrophil ß-defensin expression, and greater neutrophil ß-defensin expression was positively associated with postpartum health.

Intramammary 25-hydroxyvitamin D3 treatment modulates innate immune responses to endotoxin-induced mastitis.

Vitamin D signaling in response to pathogen-associated molecules contributes to activation of innate immune responses of bovine monocytes. We hypothesized that lipopolysaccharide (LPS) of bacteria associated with mastitis in dairy cows activates the vitamin D pathway in innate immune cells of the udder and that increasing availability of 25-hydroxyvitamin D3 [25(OH)D3] would augment expression of vitamin D-associated genes. The objective of this experiment was to determine the effects of intramammary LPS and 25(OH)D3 treatments on activation of the vitamin D pathway and innate immune responses of mammary immune cells. Individual mammary quarters of 5 lactating cows were treated with placebo control, 100 µg of 25(OH)D3, 5 µg of LPS, or a combination of 100 µg of 25(OH)D3 and 5 µg of LPS. Somatic cells from milk were evaluated for percentage of neutrophil and macrophage populations and expression of genes associated with vitamin D metabolism and innate immunity. Data from samples collected from 4 to 12 h after challenge were analyzed for main effects of LPS and 25(OH)D3 treatments, treatment interactions, and simple effects of 25(OH)D3 treatment. Data from samples collected at the time of challenge were used as covariates. The percentages of neutrophils in milk at 8 h postchallenge were 58 ± 10, 82 ± 11, 89 ± 10, and 63 ± 10% of total cells in milk from control, 25(OH)D3, LPS, and LPS plus 25(OH)D3 glands, respectively, such that the interaction of LPS and 25(OH)D3 was significant. Expression of the vitamin D 1α-hydroxylase (CYP27B1) and vitamin D receptor genes was upregulated by LPS treatment in total cells, macrophages, and neutrophils in milk. In addition, expression of the vitamin D 24-hydroxylase (CYP24A1) gene in milk somatic cells was upregulated by 25(OH)D3 and LPS treatments. The inducible nitric oxide synthase (iNOS), chemokine (C-C-motif) ligand 5 (CCL5), ß-defensin 3 (DEFB3), DEFB7, and DEFB10 genes were upregulated by LPS treatment in total cells and neutrophils from milk. Expression of iNOS in milk somatic cells tended to be affected by the interaction between LPS and 25(OH)D3, such that 25(OH)D3 tended to increase iNOS in the absence of LPS but not in the presence of LPS. Furthermore, expression of CCL5 in macrophages was downregulated by 25(OH)D3. In conclusion, intramammary endotoxin challenge activates the vitamin D pathway in mammary macrophages and neutrophils, and intramammary 25(OH)D3 treatment alters the percentage of neutrophils and expression of immune genes in milk somatic cells.

Symposium review: Targeting antimicrobial defenses of the udder through an intrinsic cellular pathway.

The bovine innate immune system has a strong repertoire of antimicrobial defenses to rapidly attack infectious pathogens that evade physical barriers of the udder. Exploration of the intracrine vitamin D pathway of bovine macrophages has improved understanding of the signals that initiate antimicrobial defenses that protect the udder. In the intracrine vitamin D pathway, pathogen recognition receptors upregulate CYP27B1 mRNA that encodes for the enzyme that converts 25-hydroxyvitamin D [25(OH)D3] to the active vitamin D hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The 1,25(OH)2D3, in turn, is generally known to increase antimicrobial activity and decrease inflammatory responses of immune cells. In cattle specifically, 1,25(OH)2D3 increases nitric oxide and ß-defensin antimicrobial responses of bovine monocytes. Immune activation of the intracrine vitamin D pathway, including induction of inducible nitric oxide synthase and ß-defensin gene expression by 1,25(OH)2D3, has been documented in the mammary glands of lactating dairy cows. Furthermore, intramammary 25(OH)D3 treatment decreased bacteria counts and indicators of mastitis severity in cows experimentally infected with Streptococcus uberis. We propose that vitamin D signaling in the udder contributes to containment of bacterial pathogens and inflammatory responses of the udder. Verification of vitamin D-mediated defenses of the mammary gland potentially provides a path for development of alternative solutions (i.e., nutritional, genetic, therapeutic) to increase mastitis resistance of dairy cows. Continued exploration of the intrinsic cellular pathways that specifically promote antimicrobial defenses of the udder, such as the vitamin D pathway, is needed to support mastitis control efforts for dairy cows.