Comparison of prepartum blood parameters in dairy cows with postpartum ketosis and new risk prediction candidates.

Introduction: Ketosis is a predominant metabolic problem and a risk factor for several postpartum diseases. This retrospective study aimed to evaluate the complete blood count (CBC), plasma biochemistry, and osteocalcin and identify significant prepartum and early postpartum values expressed in ketotic cows. Methods: In 135 Holstein Friesian cows, 210 parturitions of 114 primiparous and 96 multiparous cows were examined. According to the plasma concentrations of ß-hydroxybutyrate (BHB; ≥ 1.4 mmol/L) or non-esterified fatty acids (NEFA; ≥ 0.7 mmol/L) in the postpartum period, cows were divided into healthy cows (CON) and ketotic cows (KET). Analyses of CBC and biochemistry profiles were performed from -6 to 4 weeks of parturition every 2 weeks (prepartum; BW-5, BW-3, and BW-1, postpartum; BW1 and BW3), and osteocalcin ELISA tests were performed using blood samples from -2 to 2 weeks of parturition (BW-1 and BW1). Results: In primiparous KET (n = 114) before parturition, lower lymphocyte (Lym) in BW-5 and BW-3, lower red blood cell (RBC) in BW-5, higher mean corpuscular volume (MCV) in BW-1, and higher NEFA in BW-3 were significant compared with CON. Primiparous KET showed lower carboxylated osteocalcin (cOC) levels and a significant decrease after parturition. In multiparous KET (n = 96) before parturition, lower neutrophil (Neu) in BW-5, higher hemoglobin (HGB) in BW-5, higher MCV in BW-5 and BW-1, higher MCH in BW-5, lower total cholesterol (TC) in BW-5, higher triglyceride (TG) in BW-3, higher NEFA in BW-1, higher glucose (Glu) in BW-3, lower γ-glutamyl transferase (GGT) in BW-5, lower inorganic phosphate (iP) in BW-3, and higher body condition score (BCS) in BW-5 and BW-3 were significant compared with CON. Multiparous KET showed decreased cOC and uncarboxylated osteocalcin (ucOC) after parturition, which was lower than that in the CON group. Discussion: The blood parameters expressing different values between CON and KET in prepartum or early postpartum periods are presumed to show individual nutrition and health states, liver function, and overweight status. These parameters could be valuable indicators that can be used to prevent the occurrence of ketosis and improve management practices by recognizing these differences in ketotic cows before calving.

One-year Aerobic Interval Training Improves Endothelial Dysfunction in Patients with Atrial Fibrillation: A Randomized Trial.

Objective To evaluate the effects of one-year aerobic interval training on endothelial dysfunction in patients with atrial fibrillation. Methods Seventy-four patients with atrial fibrillation (53 men, 21 women; mean age 63±6 years old) were randomized into a 1-year continuous aerobic interval training (CT), 6-month detraining after 6 months of aerobic interval training (DT), or medical treatment only (MT) group. Aerobic interval training was performed 3 times a week for 1 year or 6 months, with an exercise intensity of 85-95% of the peak heart rate. The primary outcome was a change in biomarkers of endothelial dysfunction from baseline at six months or at the one-year follow-up. Results Six-month aerobic interval training reduced von Willebrand factor (CT: 103.7±30.7 IU/dL and DT: 106±31.2 IU/dL vs. MT: 145±47.7 IU/dL, p=0.044). Improvements were maintained with continuous aerobic interval training; however, the values increased again to the baseline levels upon detraining (CT: 84.3±39.1 IU/dL vs. DT: 122.2±27.5 IU/dL and MT: 135.9±50.4 IU/dL, p=0.002). Interleukin 1 beta levels decreased after 6 months of aerobic interval training (CT: 0.59±0.1 pg/mL and DT: 0.63±0.09 pg/mL vs. MT: 0.82±0.28 pg/mL, p=0.031), and the improvement was maintained with continuous aerobic interval training and even after detraining (CT: 0.58±0.08 pg/mL and DT: 0.62±0.09 pg/mL vs. MT: 0.86±0.28 pg/mL, p=0.015). Conclusion One-year aerobic interval training improves endothelial dysfunction in patients with atrial fibrillation and is primarily associated with the reduction in circulating thrombogenic and pro-inflammatory factors. A definitive way to sustain these improvements is the long-term continuation of aerobic training.

Protective Immunity against Listeria monocytogenes in Rats, Provided by HCl- and NaOH-Induced Listeria monocytogenes Bacterial Ghosts (LMGs) as Vaccine Candidates.

Listeria monocytogenes (Lm) bacterial ghosts (LMGs) were produced by the minimum inhibitory concentration (MIC) of HCl, H2SO4, and NaOH. Acid and alkali effects on the LMGs were compared by in vitro and in vivo analyses. Scanning electron microscope showed that all chemicals form lysis pores on the Lm cell envelopes. Real-time qPCR revealed a complete absence of genomic DNA in HCl- and H2SO4-induced LMGs but not in NaOH-induced LMGs. HCl-, H2SO4- and NaOH-induced LMGs showed weaker or missing protein bands on SDS-PAGE gel when compared to wild-type Lm. Murine macrophages exposed to the HCl-induced LMGs showed higher cell viability than those exposed to NaOH-induced LMGs or wild-type Lm. The maximum level of cytokine expression (TNF-α, iNOS, IFN-γ, and IL-10 mRNA) was observed in the macrophages exposed to NaOH-induced LMGs, while that of IL-1ß mRNA was observed in the macrophages exposed to HCl-induced LMGs. To investigate LMGs as a vaccine candidate, mice were divided into PBS buffer-injected, HCl- and NaOH-induced LMGs immunized groups. Mice vaccinated with HCl- and NOH-induced LMGs, respectively, significantly increased in specific IgG antibodies, bactericidal activities of serum, and CD4+ and CD8+ T-cell population. Antigenic Lm proteins reacted with antisera against HCl- and NOH-induced LMGs, respectively. Bacterial loads in HCl- and NaOH-induced LMGs immunized mice were significantly lower than PBS-injected mice after virulent Lm challenges. It suggested that vaccination with LMGs induces both humoral and cell-mediated immune responses and protects against virulent challenges.

Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli.

A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 103 copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 104 copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

In Vitro Antioxidant and Anti-Propionibacterium acnes Activities of Cold Water, Hot Water, and Methanol Extracts, and Their Respective Ethyl Acetate Fractions, from Sanguisorba officinalis L. Roots.

Identification of medicinal plants and naturally derived compounds as new natural antioxidant and antibacterial sources for topical acne treatment has long been important. To determine anti-Propionibacterium acnes activity and in vitro antioxidant activities, Sanguisorba officinalis L. root (SOR) was extracted with cold water (CWE), hot water (HWE), and methanol (ME), and each extract was fractionated successively with hexane, ethyl acetate (EA), and butanol to determine whether the activities could be attributed to the total phenolic, flavonoid, terpenoid, and condensed tannin contents. Pearson's correlation coefficients were analyzed between the respective variables. The SOR CWE, HWE, ME, and their respective EA fractions showed anti-P. acnes activity based on the paper disc diffusion method on agar plates, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC). The MIC against P. acnes had a moderate (+) correlation with the total phenolic content, but not with the other measures. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity (SC) had a strong (⁻) correlation with the total phenolic content and a moderate (⁻) correlation with the total flavonoid content. The total antioxidant capacity had a strong (+) correlation with the condensed tannin content. Linoleic acid peroxidation inhibition had a strong (⁻) correlation with the total phenolic content. To elucidate the major active phytochemicals in the CWE-EA, HWE-EA, and ME-EA fractions, high performance liquid chromatography-ultraviolet (HPLC-UV) and ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) were performed. The HPLC-UV analysis showed the presence of nine compounds in common (arjunic acid and/or euscaphic acid, gallic acid, kaempferol, caffeic acid, ferulic acid, tannic acid, and coumarin, quercetin). The UHPLC-QTOF-MS analysis showed the presence of nine compounds in common (gallic acid; caffeic acid; umbelliferone; arjunic acid, euscaphic acid, and/or tormentic acid; pomolic acid; rosamultic acid; and benzoic acid). When standards of the identified phytochemicals were tested against the same bacterium, quercetin, coumarin, and euscaphic acid showed antibacterial activity against P. acnes.

Expression of Aspergillus nidulans phy gene in Nicotiana benthamiana produces active phytase with broad specificities.

A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.

Chemically induced Salmonella enteritidis ghosts as a novel vaccine candidate against virulent challenge in a rat model.

Salmonella enteritidis ghosts (SEGs), non-living empty bacterial cell envelopes were generated by using the minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH) and investigated as a vaccine candidate in rats. To determine the immunogenicity and protective efficacy of SEG vaccine, rats were divided into four groups: group A (non-vaccinated control), group B (orally vaccinated), group C (intramuscularly vaccinated) and group D (intramuscularly vaccinated with complete Freund's adjuvant). Vaccination of rats with SEGs induced significant immune responses before and after virulent challenge. Rats vaccinated with SEGs showed significant increases in serum IgG antibodies after challenging with virulent S. enteritidis on week 8 and week 10 (P<0.01). During the vaccination period, groups B, C and D showed significantly higher serum bactericidal activity (SBA) compared to group A (P<0.01). Most importantly, bacterial loads in vaccinated groups were significantly lower than in the non-vaccinated group (P<0.01). In conclusion, these results show that the chemically induced SEGs as a vaccine candidate against virulent challenge.

PCR-RFLP-based typing for differentiation of Tomato yellow leaf curl virus (TYLCV) genotypes from infected host plants in Korea.

A polymerase chain reaction (PCR) using two sets of primers designed from published Tomato yellow leaf curl virus (TYLCV) genomes was developed to distinguish from the TYLCV-IL groups. The specificity of the two sets of primers was proven by testing against control TYLCV genomes and the symptomatic leaves of 34 different tomato cultivars naturally infected with TYLCV in greenhouses. One set for TYLCV-IL strain-specific primers (TYLCV-UNI-F and TYLCV-UNI-R) amplified full-length genome fragments from all the 34 tomato cultivars. Another set for TYLCV-IL group-II strain-specific primers (TYLCV-GPII-F and TYLCV-GPII-R) amplified target DNA fragments from only 9 tomato cultivars. Digestion by BglII and EcoRV of the PCR amplicons produced restriction fragment length polymorphism pattern that distinguished the TYLCV-IL group-I with two fragments from the TYLCV-IL group-II with no digested fragment. PCR coupled with BglII and EcoRV digestion confirmed that the 9 tomato cultivars were infected with the TYLCV-IL group-II and the remained 25 tomato cultivars were infected with the TYLCV-IL group-I.