Sustained release system from PLGA particles co-encapsulated with inactivated influenza virus with natural killer T cell agonist α-galactosylceramide.

Vaccines against influenza and many other infectious diseases require multiple boosters in addition to the primary dose to improve efficacy, but this approach is not ideal for compliance. The multiple doses could potentially be replaced by sustained or pulsatile release of antigens encapsulated in degradable microparticles (MPs). The efficacy of a vaccine is improved by adding an adjuvant, which can be co-delivered from the particles to enhance immunogenicity. Here, we developed degradable poly-lactic-co-glycolic acid (PLGA) (7-17 kDa) MPs capable of sustained release of ultraviolet killed influenza virus (A/PR/8/34) (kPR8) vaccine and the natural killer T (NKT) cell agonist alpha-galactosylceramide (α-GalCer) and tested their effectiveness at providing long-term protection against influenza virus infection in mice. Multiple formulations were developed for encapsulating the virus and adjuvant separately, and in combination. The MPs exhibited sustained release of both the virus and the adjuvant lasting more than a month. Co-encapsulation significantly increased the encapsulation efficiency (EE) of the vaccine but reduced the release duration. On the other hand, co-encapsulation led to a reduction in EE for the α-GalCer and a change in release profile to a higher initial burst followed by a linear release compared to a low initial burst and slower linear release. The α-GalCer also had considerably longer release duration compared to the vaccine. Mice injected with particle formulations co-encapsulating kPR8 and α-GalCer were protected from a lethal influenza virus infection 30 weeks after vaccination. This study demonstrates that PLGA MP based vaccines are promising for providing effective vaccination and possibly for replacing multiple doses with a single injection.

Sustained release of inactivated H1N1 virus from degradable microparticles for extended vaccination.

Influenza vaccines administered as intramuscularly injected inactivated viruses or intranasally administered live-attenuated viruses usually provide short-term protection against influenza infections. Biodegradable particles that provide sustained release of the antigen has been studied as an approach to extend vaccine protection. Here, we investigate sustained release of ultraviolet killed influenza virus (A/PR/8/34) (kPR8) loaded into poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles. Particles were prepared using the double emulsion method, and polymer molecular weight (MW), polymer hydrophobicity, polymer concentration in the organic phase, and the amount of killed virus were varied to obtain a range of particles. Formulations included PLGA 50:50 (2-6, 7-17 kDa), PLGA 75:25 (4-15 kDa), and 50/50 PLGA 75:25 (4-15 kDa)/PCL (14 kDa). Additionally, NaOH was co-encapsulated in some cases to enhance particle degradation. The structure of the particles was explored by size measurements and electron microscopy. The kPR8 release profiles were measured using hemagglutinin ELISA. The concentration of the polymer (PLGA) in the organic phase and polymer MW significantly influenced virus loading, while polymer MW and co-encapsulation of NaOH modulated the release profiles. Mice receiving a single intramuscular injection of NaOH microparticle-encapsulated kPR8 were partially protected against a lethal influenza challenge 32 weeks post immunization. Microparticle (MP) vaccination induced a gradual increase in PR8-specific IgGs dominated by IgG1 in contrast to the rapid IgG2a-biased response elicited by soluble kPR8 immunization. Our results indicate that vaccine-NaOH co-loaded PLGA particles show potential as a single dose vaccination strategy for extended protection against influenza virus infection.

Identification of genetic variants and individual genes associated with postpartum hypocalcemia in Holstein cows.

Periparturient hypocalcemia is a complex metabolic disorder that occurs at the onset of lactation because of a sudden irreversible loss of Ca incorporated into colostrum and milk. Some cows are unable to quickly adapt to this demand and succumb to clinical hypocalcemia, commonly known as milk fever, whereas a larger proportion of cows develop subclinical hypocalcemia. The main goal of this study was to identify causative mutations and candidate genes affecting postpartum blood calcium concentration in Holstein cows. Data consisted of blood calcium concentration measured in 2513 Holstein cows on the first three days after parturition. All cows had genotypic information for 79 k SNP markers. Two consecutive rounds of imputation were performed: first, the 2513 Holstein cows were imputed from 79 k to 312 k SNP markers. This imputation was performed using a reference set of 17,131 proven Holstein bulls with 312 k SNP markers. Then, the 2513 Holstein cows were imputed from 312 k markers to whole-genome sequence data. This second round of imputation used 179 Holstein animals from the 1000 Bulls Genome Project as a reference set. Three alternative phenotypes were evaluated: (1) total calcium concentration in the first 24 h postpartum, (2) total calcium concentration in the first 72 h postpartum calculated as the area under the curve; and (3) the recovery of total calcium concentration calculated as the difference in total calcium concentration between 72 and 24 h. The identification of genetic variants associated with these traits was performed using a two-step mixed model-based approach implemented in the R package MixABEL. The most significant variants were located within or near genes involved in calcium homeostasis and vitamin D transport (GC), calcium and potassium channels (JPH3 and KCNK13), energy and lipid metabolism (CA5A, PRORP, and SREBP1), and immune response (IL12RB2 and CXCL8), among other functions. This work provides the foundation for the development of novel breeding and management tools for reducing the incidence of periparturient hypocalcemia in dairy cattle.

Intramammary calcitriol treatment of mastitis alters profile of milk somatic cells and indicators of redox activity in milk.

The objective of this experiment was to determine the effect of intramammary calcitriol treatment on indicators of inflammation during an intramammary bacterial infection. Lactating Holstein cows were challenged with intramammary Streptococcus uberis. At the onset of mild or moderate mastitis, cows were randomly assigned to receive 10 µg of intramammary calcitriol (CAL, n = 7) or placebo control (CON; n = 6) after every milking for 5 days. Data were analyzed by ANOVA with mixed models using the MIXED procedure of SAS with significance declared at P ≤ 0.05. Milk somatic cells, mastitis severity scores, rectal temperatures, and milk bacterial counts did not differ between treatments. Calcitriol decreased the percentage of CD11b+CD14- cells in milk compared with CON (CON = 81 vs. CAL = 61 ± 5%). Antioxidant potential and concentrations of 15-F2t- isoprostanes in milk of infected quarters also were lower in CAL compared with CON. Transcripts for the 25-hydroxyvitamin D 24-hydroxylase and inducible nitric oxide synthase were greater in milk somatic cells of CAL compared with CON, but those for ß-defensin 7, metallothionein 1 A and 2 A, thioredoxin and thioredoxin reductase did not differ between treatments. Although clinical signs of severity did not differ, CAL influenced the composition of milk somatic cells and redox activity in milk of infected quarters.

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.

A novel combination therapy for multidrug resistant pathogens using chitosan nanoparticles loaded with ß-lactam antibiotics and ß-lactamase inhibitors.

Antimicrobial resistance is one of the greatest global threats. Particularly, multidrug resistant extended-spectrum ß-lactamase (ESBL)-producing pathogens confer resistance to many commonly used medically important antibiotics, especially beta-lactam antibiotics. Here, we developed an innovative combination approach to therapy for multidrug resistant pathogens by encapsulating cephalosporin antibiotics and ß-lactamase inhibitors with chitosan nanoparticles (CNAIs). The four combinations of CNAIs including two cephalosporin antibiotics (cefotaxime and ceftiofur) with two ß-lactamase inhibitors (tazobactam and clavulanate) were engineered as water-oil-water emulsions. Four combinations of CNAIs showed efficient antimicrobial activity against multidrug resistant ESBL-producing Enterobacteriaceae. The CNAIs showed enhanced antimicrobial activity compared to naïve chitosan nanoparticles and to the combination of cephalosporin antibiotics and ß-lactamase inhibitors. Furthermore, CNAIs attached on the bacterial surface changed the permeability to the outer membrane, resulting in cell damage that leads to cell death. Taken together, CNAIs have provided promising potential for treatment of diseases caused by critically important ESBL-producing multidrug resistant pathogens.

Impacts of polyclonal antibody preparations from avian origin as a feed additive to beef cattle: immune responses during the step-up transition diets.

This study investigated the effects of feeding an avian-derived polyclonal antibody preparation (PAP; CAMAS, Inc.) against Streptococcus bovis, Fusobacterium necrophorum, and lipopolysaccharides (LPS; 40%, 35%, and 25% of the preparation, respectively) on immune responses (haptoglobin [Hp], serum amyloid A [SAA], rectal temperature [RT], leukocyte counts, and expression of cell adhesion molecules cluster of differentiation [CD] CD11b, CD14, and CD62L) of beef steers during a 21-d step-up adaptation to a high-grain diet. Eight ruminally cannulated Angus crossbred beef steers (658 ± 79 kg of BW) were assigned in a cross-over design and transitioned from a diet containing bermudagrass hay (Cynodon dactylon (L.) Pers.) ad libitum plus 0.45 kg/d of molasses with 0 (CON) or 3 g of PAP to a high-grain diet. Transition consisted of three 7-d steps of increased inclusion of cracked corn (35%, 60%, and 82% of the diet dry matter for STEP1, STEP2, and STEP3, respectively). On each transition day and 7 d after STEP3 (STEP3-7d), RT was obtained every 3 h for a total of 24 h, whereas blood was collected on days 0, 1, and 3, relative to diet transition. There were no effects of PAP inclusion in any of the blood parameters (P > 0.11). However, a tendency for day effect (P = 0.10) was observed for concentrations of Hp, which were greater on days 3 and 7 vs. day 0 relative to the second diet transition (STEP2). Plasma concentrations of SAA were greater on days 1, 3, and 7 compared to day 0 during STEP1 (P = 0.01), while during STEP2 and STEP3, SAA concentrations increased (P < 0.01) from day 0 to 3. During STEP2, PAP steers tended to have lower (P = 0.08) RT than CON steers. Neutrophil and monocyte counts were the least during STEP3 (P < 0.01), whereas expression of CD11b and CD62L was the least through forage feeding (P < 0.01). Concentration of starch in the diet was correlated to all the variables tested (P ≤ 0.01), except for the percentage of B cells (P = 0.22). Yet only ruminal pH, RT, monocyte, and neutrophil counts presented strong correlation coefficients. In conclusion, the step-up transition from forage to high-grain diets triggered systemic inflammation in beef steers as observed by increased plasma concentrations of Hp, SAA, and expression on adhesion molecules in leukocytes. However, feeding polyclonal antibody preparations against S. bovis, F. necrophorum, and LPS did not provide benefits to mitigate inflammation.

Low serum vitamin D concentrations in Spring-born dairy calves are associated with elevated peripheral leukocytes.

A role for vitamin D in the immune system is emerging from human research but data in the bovine is limited. In the current study, 48 Holstein-Friesian calves were randomly assigned to one of 4 groups designed to expose calves to divergent vitamin D levels for a 7 month period and to determine its effects on circulating immunity in young calves. Concentrations of circulating 25-hydroxyvitamin D (25OHD) was measured in serum using a commercial ELISA with validated bovine standards. Results showed that mean circulating concentrations of 25OHD at birth was 7.64 ± 3.21 ng/ml indicating vitamin D deficiency. Neither the injection of Vit D3 at birth nor the elevated levels in milk replacer yield discernible changes to pre-weaning circulating concentration of 25OHD. No calf reached the recommended level of vitamin D immune sufficiencyof 30 ng/ml of 25OHD until at least 3 months of age (T4). Increasing dietary Vit D3 via ration in the post-weaning period significantly elevated 25OHD concentrations in serum in VitD-In calves. Maximal levels of circulating 25OHD were achieved in VitD-Out calves, reaching 60.86 ± 7.32 ng/ml at 5 months of age (T7). Greatest divergence in haematology profile was observed between Ctl-In vs VitD-In groups with Ctl-In calves showing an elevated count of neutrophils, eosinophils, and basophils associated with reduced 25OHD concentrations. Neither IL-8 expression nor ROS production in serum were significantly different between calves with high and low 25OHD, indicating that other vitamin D-dependent mechanisms may contribute to the divergent circulating cellular profiles observed. This novel data on the vitamin D status of neonatal calves identifies a significant window of vitamin D insufficiency which is associated with significant differences in circulating immune cell profiles. Vitamin D insufficiency may therefore exacerbate pre-weaning disease susceptibility, and further work in now warranted.

Effect of IL-8 haplotype on temporal profile in circulating concentrations of interleukin 8 and 25(OH) vitamin D in Holstein-Friesian calves.

Interleukin-8 (IL-8) is an inflammatory chemokine released during the primary innate immune response to recruit neutrophils to the site of infection. Two distinct gene promoter haplotypes have been previously reported to segregate in the Holstein-Friesian breed (IL8-h1 and IL8-h2). Our earlier work showed how these divergent IL8 haplotypes influence IL-8 concentration and other immune response parameters at a systemic level. While a close relationship has been established between vitamin D and IL-8 in other species, the role of genetic haplotype on temporal variation in vitamin D concentrations and its impact on immunity remains unexplored in cattle. Therefore this study had two objectives - 1: to establish the temporal variation in IL-8 concentration profile in healthy calves of each IL-8 haplotype (n = 5/6 per group) and 2: to identify the relationship between systemic 25(OH)D concentration and IL8 haplotype in blood at 10 time points across their first year of life. Elevated IL-8 protein concentration profiles were apparent in IL8-h2 calves at multiple time points throughout the year (P < 0.05). In contrast, circulating concentrations of 25(OH) vitamin D were negatively correlated (0.38) with IL-8, with elevated concentrations in calves of the IL8-h1 haplotype. Increased numbers of innate immune cells - specifically monocytes and basophils, were also detected in blood from IL8-h2 calves (P < 0.05). Importantly, circulating concentrations of vitamin D were substantially below recommended concentrations of 30 ng/mL serum for optimal immunity in the first five months of life, indicating a window of potentially heightened disease susceptibility - particularly in calves of the IL8-h1 haplotype. In conclusion, this study has established that IL8 haplotype confers divergent chemokine concentrations and which contrasts with circulating concentrations of vitamin D. Accounting for both IL8 haplotype and vitamin D concentration may be critical to provide dairy calves with optimal immune protection in early life.

Host genetics exerts lifelong effects upon hindgut microbiota and its association with bovine growth and immunity.

The gut microbiota is a complex ecological community that plays multiple critical roles within a host. Known intrinsic and extrinsic factors affect gut microbiota structure, but the influence of host genetics is understudied. To investigate the role of host genetics upon the gut microbiota structure, we performed a longitudinal study in which we evaluated the hindgut microbiota and its association with animal growth and immunity across life. We evaluated three different growth stages in an Angus-Brahman multibreed population with a graduated spectrum of genetic variation, raised under variable environmental conditions and diets. We found the gut microbiota structure was changed significantly during growth when preweaning, and fattening calves experienced large variations in diet and environmental changes. However, regardless of the growth stage, we found gut microbiota is significantly influenced by breed composition throughout life. Host genetics explained the relative abundances of 52.2%, 40.0%, and 37.3% of core bacterial taxa at the genus level in preweaning, postweaning, and fattening calves, respectively. Sutterella, Oscillospira, and Roseburia were consistently associated with breed composition at these three growth stages. Especially, butyrate-producing bacteria, Roseburia and Oscillospira, were associated with nine single-nucleotide polymorphisms (SNPs) located in genes involved in the regulation of host immunity and metabolism in the hindgut. Furthermore, minor allele frequency analysis found breed-associated SNPs in the short-chain fatty acids (SCFAs) receptor genes that promote anti-inflammation and enhance intestinal epithelial barrier functions. Our findings provide evidence of dynamic and lifelong host genetic effects upon gut microbiota, regardless of growth stages. We propose that diet, environmental changes, and genetic components may explain observed variation in critical hindgut microbiota throughout life.

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.

Vitamin D signaling increases nitric oxide and antioxidant defenses of bovine monocytes.

Vitamin D contributes to multiple aspects of bovine immunity and is reported to decrease the effects of mastitis and metritis in dairy cows. We hypothesized that vitamin D signaling in bovine monocytes increases antioxidant responses as part of its immunomodulatory actions. Our objectives were to assess the effects of vitamin D on oxidant and antioxidant responses of bovine monocytes. Monocytes from peripheral blood of nonpregnant, lactating Holstein cows between 90 and 300 d in milk were used for in vitro cell culture experiments. To test the effects of vitamin D on reactive oxygen metabolites (dROM) and antioxidant potential (AOP), monocytes from 14 cows were cultured in replicates for 16 h with 25-hydroxyvitamin D3 [25(OH)D3, 0 or 75 ng/mL] in a factorial arrangement with lipopolysaccharide (LPS, 100 ng/mL) or interferon-γ (IFN-γ, 10 ng/mL) or with no stimulation. Data were analyzed by ANOVA for main effects of 25(OH)D3, stimulant, and interactions between 25(OH)D3 and stimulant. Significant interactions between 25(OH)D3 and stimulant were observed for dROM and AOP of culture supernatants. In unstimulated cultures, 25(OH)D3 tended to increase dROM, but the opposite was observed in stimulated cultures. In contrast, LPS and IFN-γ treatments alone decreased AOP of culture supernatants, but 25(OH)D3 counteracted the decrease in AOP caused by IFN-γ. Abundances of transcripts of genes encoding antioxidant-related proteins were measured by quantitative PCR using RNA from monocytes from 4 cows treated with 25(OH)D3 (0 or 75 ng/mL) in a factorial arrangement with increasing concentrations of LPS (0 to 1,000 ng/mL) or IFN-γ (0 to 10 ng/mL). Treatment with 25(OH)D3 increased transcripts of genes encoding metallothionein 1A and metallothionein 2A in the presence of IFN-γ but not LPS. Furthermore, 25(OH)D3 increased transcripts of genes encoding thioredoxin and thioredoxin reductase, but the effect of 25(OH)D3 did not depend on IFN-γ or LPS stimulation. In conclusion, 25(OH)D3 increased antioxidant capacity of IFN-γ-stimulated bovine monocytes, potentially by increasing metallothionein and thioredoxin activities in monocytes.

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.

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.

Host genetic effects upon the early gut microbiota in a bovine model with graduated spectrum of genetic variation.

Multiple synergistic factors affect the development and composition of mammalian gut microbiota, but effects of host genetics remain unclear. To illuminate the role of host genetics on gut microbiota, we employed animals with a graduated spectrum of genetic variation with minimal environmental influences. We bred 228 calves with linearly varying breed composition from 100% Angus (Bos taurus) to 100% Brahman (Bos indicus), as a proxy for genetic variation, and then raised the offspring in the same environment with identical diets. We hypothesized each breed would harbor distinct gut microbiota due to genetic influence. We found that the gut microbiota of preweaning calves at 3 months old is significantly affected by host genetics, profoundly by paternal genome. We also demonstrate that single nucleotide polymorphisms in host mucin-encoding genes, critical for gut mucosal health, are significantly correlated with both breed composition and mucin-degrading gut bacteria. We further demonstrate host genetics indirectly changes gut microbiota composition via microbe-microbe interactions. These findings indicate a strong contribution by host genetics in shaping the gut microbiota during early life stages, shedding light on impact of animal breeding on gut microbiota, which is associated with animal growth and health.

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.

Vitamin D3-mediated resistance to a multiple sclerosis model disease depends on myeloid cell 1,25-dihydroxyvitamin D3 synthesis and correlates with increased CD4+ T cell CTLA-4 expression.

Microglial cell activation is the earliest biomarker of the inflammatory processes that cause central nervous system (CNS) lesions in multiple sclerosis. We hypothesized that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production by activated microglia and macrophages in the CNS inhibits these inflammatory processes. To test this hypothesis, we targeted the Cyp27b1 gene specifically in myeloid cells, then analyzed the influence of disrupted myeloid cell 1,25-(OH)2D3 synthesis on vitamin D3-mediated resistance to experimental autoimmune encephalomyelitis (EAE). Myeloid cell 1,25-(OH)2D3 synthesis was essential for vitamin D3-mediated EAE resistance. Increased CTLA-4 expression in the CNS-infiltrating CD4+ Tconv and Treg cells and decreased splenic B cell CD86 expression correlated with resistance. These new data provide solid support for the view that vitamin D3 reduces MS risk in part through a mechanism involving myeloid cell 1,25-(OH)2D3 production and CTLA-4 upregulation in CNS-infiltrating CD4+ T cells. We suggest that CTLA-4 serves as a vitamin D3-regulated immunological checkpoint in multiple sclerosis prevention.

A bioactive extract from Olea europaea protects newly weaned beef heifers against experimentally induced chronic inflammation1.

Weaning is one of the most stressful periods in the life of a ruminant. Several factors entrenched within typical management practices pose challenges to the calf gastrointestinal health. Weaning is associated with losses in BW and feed intake. In addition, increasing highly fermentable carbohydrates in the diet at the expense of physically effective fiber after weaning predisposes the development of rumen acidosis and increases the concentration of endotoxin in rumen fluid and the permeability of the lower gut to luminal contents. Endotoxin translocation can elicit immune activation, shifting the metabolic priorities toward the immune system, which if sustained over time can hinder animal health and performance. Strategic supplementation of additives with anti-inflammatory capacity could represent a suitable approach to decrease systemic inflammation, restoring barrier function to luminal contents. Bioactive extracts from Olea europaea have anti-inflammatory activity and have been shown to reduce systemic inflammation in other animal models. A generalized randomized block design was used to evaluate the impact of feeding an olive oil bioactive extract (OBE) to newly weaned heifers injected intravenously with sequentially increasing doses of lipopolysaccharide (LPS). A total of 36 heifers, distributed across 3 experimental periods, were randomly assigned to 1 of 4 treatments that consisted of intravenous injection of either saline (CTL-) or with 6 sequentially increasing doses of LPS (0.10, 0.25, 0.50, 0.75, 1.00, and 1.25 µg/kg of BW) over a 10-d period (CTL+), and CTL+ plus dietary supplementation with a low (OBE-L; 0.04% of diet DM) or a high (OBE-H; 0.16% of diet DM) dose of OBE. Feeding OBE reduced some of the negative effects of prolonged immune activation with LPS, such as improved DMI and decreased intravaginal temperature in some, but not all of the days of LPS challenge (P < 0.05). In addition, feeding OBE reduced circulating concentration of inflammatory markers such as IL-6 and haptoglobin (P < 0.05). Heifers supplemented with OBE had reduced cell surface expression of the cluster of differentiation 14 (CD14) in monocyte cells (P < 0.01), a key receptor for LPS recognition, which was correlated with a faster recovery of immune cell counts in plasma. In conclusion, dietary supplementation with OBE was successful in mitigating the negative effects of sustained immune activation in newly weaned heifers.

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.

Animal Breed Composition Is Associated With the Hindgut Microbiota Structure and ß-Lactam Resistance in the Multibreed Angus-Brahman Herd.

Antibiotics have been widely used in livestock to treat and prevent bacterial diseases. However, use of antibiotics has led to the emergence of antibiotic resistant microorganisms (ARMs) in food animals. Due to the decreased efficacy of antibiotics, alternatives to antibiotics that can reduce infectious diseases in food animals to enhance animal health and growth performance are urgently required. Here, we show that animal genetics is associated with the hindgut microbiome, which is related to fat deposition and beta-lactam resistance in the gastrointestinal tract. We investigated the hindgut microbiota structure in 95 postweaning heifers belonging to the unique multibreed Angus-Brahman herd with breed composition ranging from 100% Angus to 100% Brahman. The hindgut microbial composition of postweaning heifers differed among breed groups. The mucin-degrading bacterium Akkermansia known for promoting energy expenditure was enriched in Brahman calves that contained less intramuscular fat content, while butyrate-producing bacterium Faecalibacterium was linearly positively correlated with Angus proportion. Moreover, the higher relative abundance of beta-lactam resistant genes including ampC gene and arcA gene was associated with the greater Brahman proportion. As the first study aimed at understanding changes in hindgut microbiota among beef cattle with linear gradient of breed composition and its association with marbling in meat, our results suggest that the effects of animal genetics on the gut microbiota structure is associated with fat deposition and potentially a factor affecting the gut antimicrobial resistance.