The prevalence and genetic characteristics of porcine circovirus type 2 and 3 in Korea.

BACKGROUND: Porcine circovirus-associated diseases (PCVAD), caused by porcine circovirus type 2 (PCV2), threaten the pig industry worldwide. Five genotypes of PCV2 were recently identified: PCV2a, PCV2b, PCV2c, PCV2d and PCV2e. In addition, a novel porcine circovirus from a case of a sow with dermatitis, nephropathy syndrome and reproductive failure has been identified based on metagenomic analysis and classified as porcine circovirus type 3 (PCV3). Therefore, the current study was conducted to determine the prevalence and genetic characteristics of PCV2 and PCV3 in clinical samples. RESULTS: A total of 471 samples (161 tissue samples of lungs and lymph nodes from 34 farms and 310 serum samples from 47 farms) were tested for PCV2. Among them, 171 samples from 59 farms that had been positive for PCV2 were genotyped. Another 690 samples (296 tissue samples of lungs and lymph nodes from 91 farms, 108 samples of aborted foetuses from 26 farms, and 286 serum samples from 47 farms) were tested for PCV3. Based on PCV2 genotyping results, PCV2d was the most prevalent genotype (107 of 171 samples), and co-infections with combinations of PCV2a, 2b and 2d were identified in 48 samples from 17 farms. A total of 14 samples from 11 farms were also positive for both PCV2 and PCV3. For PCV3, 57 samples (9.8%) from 32 farms (23.2%) were positive. Among the 108 aborted foetuses from 26 farms, only 2 samples were positive for PCV3. Based on sequence comparisons, PCV2d shares 89.6-91.0% and 93.2-94.3% homology with PCV2a and PCV2b, respectively; 98.6-100% homology is shared among PCV2d strains. The PCV3 strains identified in this study share 98.0-99.5% homology. CONCLUSIONS: Our study concludes that PCV2d has become the most predominant genotype in Korea. PCV3 was also identified in clinical samples, though no significant association with clinical symptoms was observed in PCV3-positive cases.

Primary B-Cell Lymphoma of Submandibular Lymph Node in a Water Deer ( Hydropotes inermis ).

We describe a B-cell lymphoma of a submandibular lymph node with metastasis to the lung and facial subcutaneous tissues in a water deer ( Hydropotes inermis ). Neoplastic cells contained pleomorphic lymphocytes that were positive for CD79a, consistent with B-cell lymphoma. PCR for bovine leukemia virus was negative.

Proteomic Analysis to Elucidate the Antibacterial Action of Silver Ions Against Bovine Mastitis Pathogens.

Silver ions act as a powerful, broad-spectrum antimicrobial agent and are known to kill over 650 different kinds of pathogens. We investigated the protein expression pattern and identity after silver ion treatment in Escherichia coli and Staphylococcus aureus, which are primarily responsible for the majority of bovine mastitis cases using proteomics. Two-dimensional electrophoresis showed that silver ion treatment significantly reduced 5 spot's density in E. coli and S. aureus, respectively. We identified 10 proteins (alkyl hydroperoxide reductase C22 subunit, phosphoglucomutase, fructose-1-phosphate kinase, putative carbamoyl transferase, alpha-galactosidase, carbamate kinase, ornithine transcarbamoylase, fumarate hydratase class II, alcohol dehydrogenase, and conserved hypothetical protein) by matrix-assisted laser desorption ionization time of flight (MALDI-TOF). These results demonstrated that silver ions have bactericidal effects through energy deprivation, inhibition of DNA replication, and accumulation of oxidants in bovine mastitis pathogens and suggested that silver ions can be applied for the treatment of bovine mastitis.

Caffeine prevents human prion protein-mediated neurotoxicity through the induction of autophagy.

The human prion protein (PrP) fragment PrP(106­126) possesses the majority of the pathogenic properties associated with the infectious scrapie isoform of PrP, known as PrPSc. The accumulation of PrPSc in the brain of humans and animals affects the central nervous system. Recent epidemiological studies have suggested that caffeine, one of the major components of coffee, exerts protective effects against the development of neurodegeneration. However, the protective effects of caffeine against prion disease have not been reported to date. In this study, we therefore investigated the effects of caffeine on PrP-mediated neurotoxicity. The protein expression of the autophagosomal marker, LC3-II, was increased by caffeine in a dose-dependent manner, and the autophagy induced by caffeine protected the neuronal cells against PrP(106­126)­induced cell death. On the contrary, the downregulation of LC3-II using the autophagy inhibitors, 3-methyladenine (3-ΜΑ) and wortmannin, prevented the caffeine-mediated neuroprotective effects. To the best of our knowledge, the present study provides the first evidence that treatment with caffeine protects human neuronal cells against prion­mediated neurotoxicity and these neuroprotective effects are mediated by caffeine-induced autophagy signals. Our data suggest that treatment with caffeine may be a novel therapeutic strategy for prion peptide­induced apoptosis.

Lactoferrin protects against prion protein-induced cell death in neuronal cells by preventing mitochondrial dysfunction.

Prion disorder-related neurodegenerative diseases are characterized by the accumulation of prion protein (PrP) scrapie isoform (PrPsc) within the central nervous system. PrPsc induces neuronal cell death by increasing intracellular generation of reactive oxygen species (ROS). Lactoferrin (LF) is an 80 kDa protein, which has antioxidant abilities due to the scavenging of ROS. The effects of LF treatment on PrP (106-126)-mediated neurotoxicity and ROS generation were the focus of this study. LF treatment protected against PrP (106-126)-induced neuronal cell death and decreased ROS generation. The reduced ROS generation prevented PrP (106-126)-induced mitochondrial dysfunction. Moreover, PrP (106-126)-induced protein activation including c-Jun N-terminal kinase and caspase-3 were blocked by LF treatment. These results demonstrated that LF protects neuronal cells against PrP (106-126)-mediated neurotoxicity through the scavenging of ROS and provide evidence that LF treatment prevents neuronal cell death caused by PrP (106-126).

Insulin-like growth factor-1 protects against prion peptide-induced cell death in neuronal cells via inhibition of Bax translocation.

Insulin-like growth factor-1 (IGF-1) is one of the most important components of bovine colostrum. It exhibits antiapoptotic and antioxidative activities. Prion diseases are neurodegenerative disorders caused by cell death through mitochondrial dysfunction and increasing generation of reactive oxygen species (ROS). This study examined the protective effect of IGF-1 on residues 106-126 of the cellular prion protein [PrP (106-126)]-mediated mitochondrial neurotoxicity and oxidative stress. In SH-SY5Y human neuronal cells, treatment with PrP (106-126) decreased the cell viability and IGF-1 pretreatment markedly blocked the PrP (106-126)-induced neuronal cell death. IGF-1 inhibited PrP (106-126)-induced intracellular ROS generation and mitochondrial oxidative stress. In addition, IGF-1 blocked the translocation of the Bax protein to the mitochondria induced by PrP (106-126). These results demonstrate that IGF-1 protects neuronal cells against PrP (106-126)-mediated neurotoxicity through an antioxidative effect and blockage of mitochondrial Bax translocation. The results also suggest that regulation of IGF-1 secretion may have a therapeutic potential in the management of mitochondrial dysfunction and oxidative stress-induced neurodegeneration.

Autophagy induced by resveratrol prevents human prion protein-mediated neurotoxicity.

Our previous study revealed that resveratrol blocks prion protein peptide PrP(106-126)-induced neurotoxicity. However, the mechanism of resveratrol-mediated neuroprotection in prion diseases is not clear. Resveratrol initiates neuroprotective effects via the activation of autophagy, which protects organelles, cells, and organisms against misfolded protein-disorders, including Alzheimer's disease and Parkinson's disease via regulation of mitochondrial homeostasis. Thus, we focused on elucidating the mechanisms responsible for resveratrol-mediated neuroprotection related to mitochondrial homeostasis as a result of autophagy activation. Resveratrol prevented PrP(106-126)-induced neuronal cell death by activating autophagy. Moreover, resveratrol-induced autophagy prevented the PrP(106-126)-induced reduction in mitochondrial potential and translocation of Bax to the mitochondria and cytochrome c release. Our results indicate that treatment with resveratrol appears to protect against neurotoxicity caused by prion protein peptides and the neuroprotection is induced by resveratrol-mediated autophagy signals.

Silver ion treatment of primary cultured bovine mammary gland epithelial cell (BMEC) damage from Staphylococcus aureus-derived alpha-toxin.

Bovine mastitis remains the largest hazard in the global dairy industry and has facilitated the development of various therapeutic strategies. Silver is a well-known disinfectant that is widely used in the treatment of clinical disease. In this study, we separated bovine mammary gland epithelial cells (BMEC) using an enzyme probe. We also examined safe concentrations for the application of silver ions in bovine mastitis, particularly in cases induced by Staphylococcus aureus. S. aureus-derived alpha-toxins induced cell damage through DNA fragmentation, reactive oxygen species (ROS) generation, and the dissipation of mitochondrial transmembrane potential (MTP) in BMEC. Silver ion treatment doses of lower than 2 ppm did not induce BMEC damage, but silver ion concentrations greater than 4 ppm was accompanied by DNA fragmentation. Furthermore, silver ions doses below 2 ppm inhibited alpha-toxin-induced cell damage through the reduction of ROS generation. Recognizing this, it demonstrate that low doses of silver ions inhibit alpha-toxin-induced BMEC damage and suggest that silver ions may be a potentially beneficial treatment against bovine mastitis, particularly in cases induced by S. aureus.

Mitochondrial transmembrane potential and reactive oxygen species generation regulate the enhanced effect of CCCP on TRAIL-induced SNU-638 cell apoptosis.

TRAIL is a member of the tumor necrosis factor family and engages apoptosis via recruitment and rapid activation of caspase-8. This study investigated the effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a classic uncoupler of oxidative phosphorylation, on TRAIL-induced apoptosis in SNU-638 cells derived from human gastric cancer cells. It was found that treatment with CCCP followed by incubation with TRAIL markedly enhanced apoptosis by 2 fold compared with treatment with TRAIL alone. This effect was accompanied by reduction in mitochondrial transmembrane potential and generation of reactive oxygen species. This sensitization was inhibited by N-acetyl-l-cysteine, which restored the mitochondrial transmembrane potential and reduced reactive oxygen species generation. Treatment with N-acetyl-L-cysteine also inhibited expression of apoptotic proteins such as Bax and Smac and abrogated caspase-8 activation. Moreover, treatment with N-acetyl-L-cysteine prior to induction with TRAIL increased expression of the anti-apoptotic Bcl-2 protein. These data indicate that CCCP enhanced TRAIL-induced apoptosis by dissipation of mitochondrial transmembrane potential and reactive oxygen species, suggesting that treatment with CCCP combined with that with TRAIL can be an efficient method to induce death of tumor cells, particularly cells that are resistant to TRAIL-induced apoptosis.

Batch and continuous culture kinetics for production of carotenoids by beta-ionone-resistant mutant of Xanthophyllomyces dendrorhous.

A beta-ionone-resistant mutant strain isolated from the red yeast Xanthophyllomyces dendrorhous KCTC 7704 was used for batch and continuous fermentation kinetic studies with glucose media in a 2.5-1 jar fermentor at 22 degrees C and pH 4.5. The kinetic pattern of growth and carotenoid concentration in the batch fermentations exhibited a so-called mixed-growth-associated product formation, possibly due to the fact that the content of intracellular carotenoids depends on the degree of physical maturation toward adulthood. To determine the maximum specific growth rate constant (microm) and Monod constant (k(s)) for the mutant, glucose-limited continuous culture studies were performed at different dilution rates within a range of 0.02-0.10 h(-1). A reciprocal plot of the steady-state data (viz., reciprocal of glucose concentration versus residence time) obtained from continuous culture experiments was used to estimate a microm of 0.15 h(-1) and k(s) of 1.19 g/l. The carotenoid content related to the residence time appeared to assume a typical form of saturation kinetics. The maximum carotenoid content (Xm) for the mutant was estimated to be 1.04 microg/mg dry cell weight, and the Lee constant (k(m)), which was tentatively defined in this work, was found to be 3.0 h.

Comparison of two types of CIDR-based timed artificial insemination protocols for repeat breeder dairy cows.

This study compared two types of controlled internal drug release (CIDR)-based timed artificial insemination (TAI) protocol for treatment of repeat breeder dairy cows. In the first trial of the experiment, 55 repeat breeder cows were randomly assigned to the following two treatments. (1) In the EB group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 1 mg estradiol benzoate (EB) plus 50 mg progesterone (P4; Day 0). On Day 7, they were given an injection of PGF(2alpha) and the CIDR device was removed. The cows were given an injection of 1 mg EB on Day 8 and were subjected to TAI 30 h later (n=27). (2) In the gonadotrophin releasing hormone (GnRH) group, a CIDR device was inserted into the cows, and then the cows were administered an injection of 250 microg gonadorelin (GnRH; Day 0). On Day 7, they were given an injection of PGF(2alpha) and the CIDR device was removed. The cows were given an injection of 250 microg GnRH on Day 9 and were subjected to TAI 17 h later (n=28). In the second trial, 41 repeat breeder cows that were confirmed as not pregnant in the first trial were randomly assigned to the same two treatments used in the first trial (an EB group of 20 cows and a GnRH group of 21 cows). The ovaries of 15 cows from each group were examined by transrectal ultrasonography in order to observe the changes in ovarian structures, and blood samples were collected for analysis of serum P4 concentrations. The pregnancy rates following TAI in the first (18.5 vs. 32.1%) and second (40.0 vs. 38.1%) trials and the combined rates (27.7 vs. 34.7%) did not differ between the EB and GnRH groups. The proportions of cows with follicular wave emergence within 7 days did not differ between the EB (12/15) and GnRH groups (13/15). The interval to wave emergence was shorter (P<0.01) in the GnRH group than in the EB group, but there was no difference in the mean diameters of dominant follicles on Day 7 between the groups. Moreover, the proportions of cows with synchronized ovulation following a second EB or GnRH treatment did not differ between the groups. In conclusion, treatment with either EB or GnRH in a CIDR-based TAI protocol results in synchronous follicular wave emergence, follicular development, synchronous ovulation, and similar pregnancy rates for TAI in repeat breeder cows.

The effects of administering estradiol benzoate plus progesterone during the growth or static phases of the dominant follicle in CIDR-treated lactating dairy cows.

We studied the effects of administering estradiol benzoate (EB) plus progesterone (P4) as part of a CIDR-based protocol during the growth or static phases of dominant follicle development on follicular wave emergence, follicular growth, synchrony of ovulation and pregnancy rate following CIDR withdrawal, treatment with PGF(2alpha) and GnRH, and fixed-time artificial insemination (TAI). Forty-one previously synchronized lactating Holstein dairy cows were randomly allocated to three treatment groups. The control group (n=14) received a CIDR on the third day after ovulation only (Day 0). The two treatment groups were administered CIDRs comprising 2 mg EB and 50 mg P4 either on the third (T1, n=14) or eighth day (T2, n=13) after ovulation (Day 0). All cows received PGF(2alpha) after CIDR removal on Day 7, GnRH on Day 9, and TAI 16 h after GnRH treatment. The proportion of cows with follicular wave emergence within 8 days of treatment differed (P<0.01) among the control (14.3%), T1 (85.7%), and T2 groups (92.9%). However, the mean intervals between treatment and wave emergence were not significantly different. There were significant differences in the diameters of the dominant follicles on Day 7 (P<0.01) and in preovulatory follicles on Day 9 (P<0.01), with the largest follicles observed in the control group and the smallest follicles observed in the T2 group. In contrast, the numbers of cows showing synchronous ovulation after GnRH treatment (92.9 to 100.0%) and pregnancy following TAI (46.2 to 50.0%) were similar between the treatment groups. The results showed that, irrespective of the phase (growth or static) of the dominant follicle, administration of 2 mg EB plus 50 mg P4 to CIDR-treated lactating dairy cows induced consistent follicular wave emergence and development, synchronous ovulation after GnRH administration, and similar pregnancy rates following TAI.