Pharmacokinetics of amoxicillin trihydrate in cultured olive flounder (Paralichthys olivaceus)

The study was aimed at investigating the pharmacokinetics of amoxicillin trihydrate (AMOX) in olive flounder (Paralichthys olivaceus) following oral, intramuscular, and intravenous administration, using high‐performance liquid chromatography following. The maximum plasma concentration (C_max), following oral administration of 40 and 80 mg/kg body weight (b.w.), AMOX was 1.14 (T_max, 1.7 h) and 0.76 μg/mL (T_max, 1.6 h), respectively. Intramuscular administration of 30 and 60 mg/kg of AMOX resulted in C_max values of 4 and 4.3 μg/mL, respectively, with the corresponding T_max values of 29 and 38 h. Intravenous administration of 6 mg/kg AMOX resulted in a C_max of 9 μg/mL 2 h after administration. Following oral administration of 40 and 80 mg/kg AMOX, area under the curve (AUC) values were 52.257 and 41.219 μg/mL·h, respectively. Intramuscular 30 and 60 mg/kg doses resulted in AUC values of 370.274 and 453.655 μg/mL·h, respectively, while the AUC following intravenous administration was 86.274 μg/mL·h. AMOX bioavailability was calculated to be 9% and 3.6% following oral administration of 40 and 80 mg/kg, respectively, and the corresponding values following intramuscular administration were 86% and 53%. In conclusion, this study demonstrated high bioavailability of AMOX following oral administration in olive flounder.     

A peptide derived from human bactericidal/permeability-increasing protein (BPI) exerts bactericidal activity against Gram-negative bacterial isolates obtained from clinical cases of bovine mastitis

Gram-negative bacteria are responsible for approximately one-third of the clinical cases of bovine mastitis and can elicit a life-threatening, systemic inflammatory response. Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and is largely responsible for evoking the inflammatory response. Antibiotic and anti-inflammatory therapy for treating Gram-negative infections remains suboptimal. Bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived protein with antimicrobial and LPS-neutralizing properties. Select peptide derivatives of BPI are reported to retain these properties. The objective of this study was to evaluate the antimicrobial activity of a human BPI-derived synthetic peptide against clinical bovine mastitis isolates of Gram-negative bacteria. A hybrid peptide was synthesized corresponding to two regions of human BPI (amino acids 90-99 and 148-161), the former of which has bactericidal activity and the latter of which has LPS-neutralizing activity. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of this peptide against various genera of bacteria were determined using a broth microdilution assay. The MIC's were determined to be: 16-64 microg/ml against Escherichia coli; 32-128 microg/ml against Klebsiella pneumoniae and Enterobacter spp.; and 64-256 microg/ml against Pseudomonas aeruginosa. The MBC's were equivalent to or 1-fold greater than corresponding MIC's. The peptide had no growth inhibitory effect on Serratia marcescens. The antimicrobial activity of the peptide was retained in the presence of serum, but severely impaired in milk. Further functional evaluation of the peptide demonstrated its ability to completely neutralize LPS. Together, these data support additional investigations into the therapeutic application of BPI to the treatment of Gram-negative infections in cattle.     

Isolation and characterization of Streptococcus sp. from diseased flounder (Paralichthys olivaceus) in Jeju Island

Streptococcus sp. is gram-positive coccus that causes streptococcal infections in fish due to intensification of aquaculture and caused significant economic losses in fish farm industry. A streptococcal infection occurred from cultured diseased olive flounder (Paralichthys olivaceus) in May, 2005 at a fish farm in Jeju Island, Korea. The diseased flounder exhibited bilateral exophthalmic eyes and rotten gills; water temperature was 16~18℃ when samples were collected. Of the 22 fish samples collected, 3 samples were identified as Lactococcus garvieae and 18 samples were identified as Streptococcus parauberis by culture-based, biochemical test. Serological methods such as slide agglutination, hemolysis and antimicrobial susceptibility test were also used as well as multiplex PCR-based method to simultaneously detect and confirm the pathogens involved in the infection. S. parauberis and L. garvieae have a target region of 700 and 1100 bp., respectively. One fish sample was not identified because of the difference in the different biochemical and serological tests and was negative in PCR assay. In the present study, it showed that S. parauberis was the dominant species that caused streptococcosis in the cultured diseased flounder.     

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.     

Application of a solid-phase fluorescence immunoassay to determine oxytetracycline and tetracycline residues in tissue of olive flounder (Paralichthys olivaceus)

Parallux, a solid-phase fluorescence immunoassay (SPFIA) developed for antibotics residue detection in milk, was applied for analysis of fish tissue. The recommended therapeutic doses of oxytetracycline (OTC, 100 g/ton water, withdrawal period 30 days) and tetracycline (TC, 150 g/ton water, withdrawal period 30 days) were treated to a group of 35 olive flounders (Paralichthys olivaceus) using dipping administration. Muscle was sampled before and after drug treatment 1st, 2nd, 3rd, 5th, 7th, and 14th day. The concentration of oxytetracycline in muscle, determined by SPFIA, was compared with that of internal standard (100 ppb as oxytetracycline). The S/C ratio of sample inhibition value to cutoff inhibition value was employed as an index to determine the muscle residue in olive flounder. To investigate the recovery rate, and standard solutions were added to muscle samples to give final concentrations in muscle of 0.1 and 0.5 microg/ml. The recovery rates of all spiked samples were >89% of the spiked value. OTC and TC were detected in muscle of fishes treated until the 3rd day of withdrawal period. The present study showed that the SPFIA can be easily adopted in predicting tissue residues for OTC and TC in farmed fishes.     

The efficacy of amoxicillin sodium against streptococcosis in cultured olive flounder Paralichthys olivaceus and its pharmacokinetics

The efficacy of amoxicillin sodium for controlling field and experimental Streptococcus iniae and S. parauberis infections in olive flounder (Paralichthys olivaceus) was evaluated after a single intramuscular administration. Furthermore, the minimal inhibitory concentrations (MIC) against 21 Streptococcus strains were determined. In addition, the pharmacokinetics and residue depletion in olive flounder were investigated. Single intramuscular doses of amoxicillin sodium at 20, 40, 80, and 160 mg/kg b.w. fish significantly reduced cumulative mortality rates to 18.8–31.3% (P < 0.05) for S. iniae and to 5.0–15.0% (P < 0.01) for S. parauberis, whereas the S. iniae‐ and S. parauberis‐infected positive control groups showed cumulative mortality rates of 68.8% and 60.0%, respectively. In a S. parauberis outbreak, amoxicillin sodium reduced the cumulative mortality rate to 7.5% and 4.8% at 20 and 40 mg/kg b.w. fish, respectively, whereas that of the untreated control group was 35.2%. Peak plasma concentrations (C_max) following a single intramuscular dose of 40 and 80 mg/kg b.w. fish were 62.64 (T_max, 1.59 h) and 87.61 (T_max, 3.02 h) μg/mL, respectively, with large AUC_0?t/MIC and C_max/MIC ratios, and sufficient T > MIC (time for maintaining plasma drug concentration greater than MICs) for S. iniae and S. parauberis. The estimated withdrawal period of amoxicillin sodium from muscle of olive flounder was about 8 days at 40 mg/kg b.w. fish (at 22 ± 1 °C). These results demonstrated a single intramuscular administration of amoxicillin sodium to be effective against streptococcosis in olive flounder.     

Identification and purification of a novel adhesion-associated protein in a new strain of Lactobacillus, L15, from flounder (Paralichthys olivaceus)

Adhesion is a crucial and prerequisite step for Lactobacillus colonization in the digestive tract which subsequently confers its probiotic effects on the host. The aim of this report was to identify and purify a novel adhesion-associated protein which mediates the adherence of a new strain of Lactobacillus, L15, to intestinal mucus from flounder. It was shown that surface proteins were involved in the adhesion of L15 to flounder mucus. The adhesion efficiencies of this strain were significantly decreased from 16.0% to 5.8% after extraction of L15 with 5 M LiCl and were further inhibited to 3.6% by blocking with an L15 cell extract containing surface proteins. The adhesion-associated protein in the cell extract was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and was identified by Western blotting with sulfo-N-hydroxysuccinimide-biotin (B-NHS)-labeled crude mucus as a 61.8 kDa protein. The identical protein could be purified from L15 whole cell proteins by affinity chromatography using Sepharose, covalently coupled with crude mucus. It was demonstrated that the adhesion-associated protein was a new adhesive protein. Its characteristics and similarities with other known adhesive proteins need further investigation.     

Potential of auxotrophic Edwardsiella tarda double-knockout mutant as a delivery vector for DNA vaccine in olive flounder (Paralichthys olivaceus)

To evaluate potential of an auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine in fish, olive flounder (Paralichthys olivaceus) were immunized with the E. tarda mutant harboring plasmids (pG02-ASD-CMV-eGFP) for eukaryotic expression of the enhanced green fluorescent protein (eGFP) gene through either intraperitoneal (i.p.) or oral route, and the expression of eGFP in the internal organs and generation of antibody against eGFP in fish were analyzed. In fish i.p. injected with 2×10(7)CFU/fish of Δalr Δasd E. tarda harboring pG02-ASD-CMV-eGFP, expression of eGFP was detected in liver, kidney, and spleen from 1 day to 28 days post-injection. In fish orally administered with 1×10(9)CFU/fish of the bacteria, the eGFP band was detected in liver, kidney, and spleen from 1 day to 14 days post-administration, whereas, in intestine, the band was detected only at 1 day post-administration. Either oral or i.p. immunization of olive flounder with recombinant E. tarda that carried eGFP-expressing eukaryotic plasmids was successful to induce humoral adaptive immunity against not only E. tarda that was used as a delivery vehicle but also eGFP that was used as the reporter protein of DNA vaccine, suggesting attenuated E. tarda-vectored DNA vaccine has a potential to be used as a combined vaccine against infectious diseases in fish.     

Recombinant bactericidal/permeability-increasing protein (rBPI21) for treatment of parvovirus enteritis: a randomized, double-blinded, placebo-controlled trial

We evaluated the ability of an antimicrobial and endotoxin-neutralizing agent, the recombinant amino terminal fragment of bactericidal permeability-increasing protein (rBPI21), to decrease plasma endotoxin concentration and severity of clinical signs of canine parvovirus and to improve survival. This randomized, double-blinded, placebo-controlled clinical trial included 40 client-owned dogs and 9 normal puppies from a closed research colony. Dogs weighing >5 kg (11 lb) with fecal antigen-confirmed parvovirus and clinical signs of vomiting and diarrhea were randomly assigned to receive placebo or rBPI21 infusion over 6 hours. Plasma endotoxin concentration was measured at 0, 3, and 6 hours of infusion. Owners chose continued medical care with either the Veterinary Hospital of the University of Pennsylvania Internal Medicine Service or a local veterinarian. Telephone follow-up was conducted at 14 days. Surviving dogs were reevaluated at >30 days (recovered group), at which time plasma samples for measurement of endotoxin concentration were obtained. Plasma endotoxin concentrations were significantly higher in dogs with parvovirus than in normal or recovered dogs. Despite 90% survival, the rBPI21 treatment did not have a significant effect on outcome, duration of hospitalization, or plasma endotoxin concentrations. Treatment in a tertiary care hospital, however, significantly improved survival but resulted in a significantly increased duration of hospitalization. Endotoxemia occurs in dogs with parvovirus enteritis, but rBPI21 is not associated with improved survival.     

绵羊感染支原体后杀菌通透性增加蛋白(BPI)mRNA表达水平的变化

为了检测巴什拜羊和盘羊杂交羊在感染绵羊肺炎支原体(MO)前后杀菌通透性增加蛋白(BPI)表达水平的变化,对试验羊攻毒MO,在感染前(第0天)及感染后的第2、5、7、14及21天,颈静脉采血分离中性粒细胞,采用Real-time q PCR方法检测BPI的表达水平。结果显示,感染后第5天,2组BPI相对表达量升高。巴什拜羊BPI持续升高,杂交羊在第7天表达水平最高,在14-21天则逐渐降低。在第7天,巴什拜羊高于杂交羊(P0.05),在第14-21天,巴什拜羊极显著高于杂交羊(P0.01)。说明绵羊感染MO后初期BPI有明显升高趋势,在后期巴什拜羊和杂交羊的BPI变化有差异,这对研究支原体肺炎发病机理提供参考。     

Molecular cloning, expression and characterization of an interleukin 27 p28 homolog in pig (Sus scrofa)

IL-27 is the newest member of the IL-12 cytokine family and plays an important role in the immune regulation. It is composed of two subunits, p28 and EBV-induced gene 3(EBI3). Although human and mouse IL-27 p28 genes have been cloned, pig IL-27 p28 gene has not ever been reported. In the present study, we have cloned and characterized the full-length cDNA of IL-27 p28 from pig. The open reading frame of pig IL-27 p28 gene is 720 bp, which encodes a protein of 239 amino acids with a predicted molecular mass of 26.6 kDa. The deduced amino acid sequence of pig IL-27 p28 showed a high degree of homology to human (63%) and mouse (58%). It was a 4-helix cytokine and belonged to 4-helix cytokine superfamily. Pig IL-27 p28 has one transmembrane region, one signal peptide, and one N-glycosylation site, two Protein kinase C phosphorylation sites, three Casein kinase II phosphorylation sites and one N-myristoylation site. For the expression of pig IL-27 p28 protein in a eukaryotic expression system the recombinant plasmid was constructed. The expression of pig IL-27 p28 in mammalian cells were confirmed by flow cytometry analysis, immunofluorescence and Western blot. The analysis also confirmed a cross reactivity with anti-mouse IL-27 p28 antibody. This is the first report of cloning and characterization of IL-27 p28 in pig.     

多年生黑麦草LpGCS基因克隆及其在烟草中的初步功能验证

本研究以多年生黑麦草‘高帽2号’叶片为试材,根据同源基因的CDS序列设计简并引物,采用RT-PCR和RACE技术,克隆了多年生黑麦草γ-谷氨酰基半胱氨酸合成酶基因LpGCS。该基因全长为1674 bp(GenBank登录号:KJ551844),具有完整的开放阅读框(ORF),共1125 bp;对其氨基酸序列特性、结构以及与其他8种同源基因氨基酸序列间的同源性进行了研究,预测该蛋白分子量为42.86 kDa,属于不稳定类蛋白质,其蛋白质二级结构以α-螺旋为主;蛋白质氨基酸序列与其他8种同源基因氨基酸序列间同源性都比较高;此外,成功构建了该基因的正、反义植物表达载体pCAMBIA-Ubi-LpGCS+和pCAMBIA-Ubi-LpGCS-,并通过农杆菌介导法获得转正、反义基因烟草。对转基因植株和野生型烟草进行镉离子胁迫试验,生理生化指标测试结果表明镉离子胁迫处理10 d后,转LpGCS+植株中MDA含量低于野生型,光合色素含量与POD、SOD、CAT活性均高于野生型;而转LpGCS-植株中MDA含量高于野生型,光合色素含量与POD、SOD、CAT活性均低于野生型。综上所述,LpGCS基因在烟草中的过量表达可以提高植株的耐镉胁迫能力,为进一步利用该基因转化多年生黑麦草培育抗重金属植株奠定基础。     

Molecular cloning and functional characterization of porcine IFN-beta promoter stimulator 1 (IPS-1)

The IFN-beta promoter stimulator 1 (IPS-1), also known as MAVS/VISA/Cardif, is an adaptor molecule for the retinoic-acid-inducible protein I (RIG-I) or melanoma-differentiation-associated gene 5 (MDA5) that recognizes intracellular double-stranded RNA (dsRNA) and triggers a signal for producing type I IFN. In the present study, porcine IPS-1 cDNA was cloned, using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR, from porcine peripheral blood mononuclear cells. The open reading frame of porcine IPS-1 consists of 1575bp encoding 524 amino acids. The putative porcine IPS-1 protein contains a N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to mouse, rat, monkey, human and cattle counterparts, ranging from 59% to 79%. Semi-quantitative RT-PCR showed that porcine IPS-1 mRNA was widely expressed in different tissues. Porcine kidney (PK-15) cells transfected with a DNA construct encoding porcine IPS-1 produced type I IFN, and activated IRF3 and NF-kappaB. Deletion mutant analyses further revealed that both the CARD-like domain and transmembrane domain are essential for these functions. In addition, poly(I:C)-induced porcine IFN-beta promoter activation in PK-15 cells was significantly reduced by siRNA targeting IPS-1, indicating that IPS-1 is an important immunoregulator in the porcine innate immune system. The availability of porcine IPS-1 and establishment of its function in the type I IFN signaling pathway provides a useful molecule for defining its role during the course of pig infectious diseases.     

Identification and characterization of Japanese flounder, Paralichthys olivaceus interferon-stimulated gene 15 (Jf-ISG15)

Previously, using cDNA microarray analysis, we demonstrated that an EST clone of Japanese flounder (Paralichthys olivaceus) with homology to mammalian interferon-stimulated gene 15 (ISG15) was strongly induced by treatment with DNA vaccine encoding the glycoprotein gene of Hirame rhabdovirus (HIRRV). In this study, we conducted molecular cloning and expression analysis of the Japanese flounder ISG15 (Jf-ISG15). Jf-ISG15 encoded two exons. The first exon was non-coding, while the second exon encoded a protein of 158 amino acids. The coded protein has two tandem ubiquitin-like domains with a carboxyl-terminus conjugation motif “LRLRGG”. Phylogenetic analysis revealed an evolutionary relationship among Jf-ISG15, mammalian and fish ISG15 orthologues. The interferon-stimulated response element (ISRE) sites were conserved among DNA sequences of Jf-ISG15 and mammalian ISG15 promoter regions. An RT-PCR analysis of healthy tissues showed that Jf-ISG15 mRNA was notably strongly expressed in gills, PBLs and spleen. Expression of Jf-ISG15 was strongly induced by poly-I:C treatment in head-kidney cells, peripheral blood leukocytes (PBLs) and spleen cells, and by HIRRV infection in kidney of juvenile fish suggesting that Jf-ISG15 plays a role in fish antiviral response.     

Molecular cloning and characterization of the alphaX subunit from CD11c/CD18 horse integrin

This work reports the cloning and sequence determination of the horse alpha subunit of the integrin CD11c/CD18, a marker of dendritic cells. A cDNA clone of 4582 base pairs was obtained. It encodes a protein segment of 1086 amino acid residues of the extracellular domain with 10 potential sites of glycosylation, a transmembrane domain of 32 residues and a C-terminal cytoplasmic tail of 24 residues. A phylogenetic analysis of this integrin shows close similarity (83%) with that of Canis familiaris.