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.

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.

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.

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.

Vitamin D status of dairy cattle: Outcomes of current practices in the dairy industry.

The need for vitamin D supplementation of dairy cattle has been known for the better part of the last century and is well appreciated by dairy producers and nutritionists. Whether current recommendations and practices for supplemental vitamin D are meeting the needs of dairy cattle, however, is not well known. The vitamin D status of animals is reliably indicated by the concentration of the 25-hydroxyvitamin D [25(OH)D] metabolite in serum or plasma, with a concentration of 30ng/mL proposed as a lower threshold for sufficiency. The objective of this study was to determine the typical serum 25(OH)D concentrations of dairy cattle across various dairy operations. The serum 25(OH)D concentration of 702 samples collected from cows across various stages of lactation, housing systems, and locations in the United States was 68±22ng/mL (mean ± standard deviation), with the majority of samples between 40 and 100ng/mL. Most of the 12 herds surveyed supplemented cows with 30,000 to 50,000 IU of vitamin D3/d, and average serum 25(OH)D of cows at 100 to 300 DIM in each of those herds was near or above 70ng/mL regardless of season or housing. In contrast, average serum 25(OH)D of a herd supplementing with 20,000 IU/d was 42±15ng/mL, with 22% below 30ng/mL. Cows in early lactation (0 to 30d in milk) also had lower serum 25(OH)D than did mid- to late-lactation cows (57±17 vs. 71±20ng/mL, respectively). Serum 25(OH)D of yearling heifers receiving 11,000 to 12,000 IU of vitamin D3/d was near that of cows at 76±15ng/mL. Serum 25(OH)D concentrations of calves, on the other hand, was 15±11ng/mL at birth and remained near or below 15ng/mL through 1mo of age if they were fed pasteurized waste milk with little to no summer sun exposure. In contrast, serum 25(OH)D of calves fed milk replacer containing 6,600 and 11,000 IU of vitamin D2/kg of dry matter were 59±8 and 98±33ng/mL, respectively, at 1mo of age. Experimental data from calves similarly indicated that serum 25(OH)D achieved at approximately 1mo of age would increase 6 to 7ng/mL for every 1,000 IU of vitamin D3/kg of dry matter of milk replacer. In conclusion, vitamin D status of dairy cattle supplemented with vitamin D3 according to typical practices, about 1.5 to 2.5 times the National Research Council recommendation, is sufficient as defined by serum 25(OH)D concentrations. Newborn calves and calves fed milk without supplemental vitamin D3, however, are prone to deficiency.

Vitamin D and estrogen synergy in Vdr-expressing CD4(+) T cells is essential to induce Helios(+)FoxP3(+) T cells and prevent autoimmune demyelinating disease.

Multiple sclerosis (MS) is a neurodegenerative disease resulting from an autoimmune attack on the axon-myelin unit. A female MS bias becomes evident after puberty and female incidence has tripled in the last half-century, implicating a female sex hormone interacting with a modifiable environmental factor. These aspects of MS suggest that many female MS cases may be preventable. Mechanistic knowledge of this hormone-environment interaction is needed to devise strategies to reduce female MS risk. We previously demonstrated that vitamin D3 (D3) deficiency increases and D3 supplementation decreases experimental autoimmune encephalomyelitis (EAE) risk in a female-biased manner. We also showed that D3 acts in an estrogen (E2)-dependent manner, since ovariectomy eliminated and E2 restored D3-mediated EAE protection. Here we probed the hypothesis that E2 and D3 interact synergistically within CD4(+) T cells to control T cell fate and prevent demyelinating disease. The E2 increased EAE resistance in wild-type (WT) but not T-Vdr(0) mice lacking Vdr gene function in CD4(+) T cells, so E2 action depended entirely on Vdr(+)CD4(+) T cells. The E2 levels were higher in WT than T-Vdr(0) mice, suggesting the Vdr(+)CD4(+) T cells produced E2 or stimulated its production. The E2 decreased Cyp24a1 and increased Vdr transcripts in T cells, prolonging the calcitriol half-life and increasing calcitriol responsiveness. The E2 also increased CD4(+)Helios(+)FoxP3(+) T regulatory (Treg) cells in a Vdr-dependent manner. Thus, CD4(+) T cells have a cooperative amplification loop involving E2 and calcitriol that promotes CD4(+)Helios(+)FoxP3(+) Treg cell development and is disrupted when the D3 pathway is impaired. The global decline in population D3 status may be undermining a similar cooperative E2-D3 interaction controlling Treg cell differentiation in women, causing a breakdown in T cell self tolerance and a rise in MS incidence.