Genetic diversity of porcine reproductive and respiratory syndrome virus in Korea

The high genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been an obstacle to developing an effective vaccine for porcine reproductive and respiratory syndrome (PRRS). This study was performed to assess the degree of genetic diversity among PRRSVs from Korean pig farms where wasting and respiratory syndrome was observed from 2005 to 2009. Samples from 786 farms were tested for the presence of PRRSV using reverse transcription PCR protocol. A total of 117 farms were positive for type 1 PRRSV while 198 farms were positive for type 2. Nucleotide sequences encoding the open reading frame (ORF) 5 were analyzed and compared to those of various published PRRSV isolates obtained worldwide. Sequence identity of the ORF 5 in the isolates was 81.6~100% for type 1 viruses and 81.4~100% for type 2 viruses. Phylogenetic analysis of the ORF 5 sequences showed that types 1 and 2 PRRSVs from Korea were mainly classified into three and four clusters, respectively. The analyzed isolates were distributed throughout the clusters independent of the isolation year or geographical origin. In conclusion, our results indicated that the genetic diversity of PRRSVs from Korean pig farms is high and has been increasing over time.

Epidemiological Prevalence of Avian Pathogenic Escherichia coli Differentiated by Multiplex PCR from Commercial Chickens and Hatchery in Korea

We examined 216 Escherichia coli (E. coli) isolated from chickens and environmental specimens from hatcheries between 2005 and 2006 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Korea tentatively by multiplex PCR. The multiplex PCR which was used as tentative criteria of APEC targets 8 virulence-associated genes; enteroaggregative toxin (astA), increased serum survival protein (iss), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. It was demonstrated that E. coli strains already typed as APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of APEC is a possession of more than 5 virulenceassociated genes, we discriminated 24 APEC strains among the 216 E. coli strains. Contamination rates of APEC in the field were 31.3% in layers, 14.0% in broilers, 2.7% in broiler breeders, and 0.0% in environmental specimens from hatcheries. The combinational tendency of APEC examined is a fundamental possession of astA, iss and iucD genes and addition of cva/cvi, tsh, vat, and irp2 genes which have a critical importance for virulent traits of APEC. Compared with intravenous chicken challenge or embryo lethality assay, multiplex PCR method could be useful to discriminate APEC rapidly for convenient diagnosis.

Development of a Lightcycler-based reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus

One step TaqMan real-time reverse transcription polymerase chain reaction (R/T RT-PCR)using a set of primers/probes was developed for the detection of foot-and-mouth disease (FMD)virus. The gene-specific probes labeled fluorogen for the internal ribosomal entry site, Leader sequence and 2B regions were used to detect FMD virus (FMDV). This assay specifically detected FMDV both in cell culture preparations and clinical samples, and was capable of distinguishing FMD from other viral diseases similar to clinical signs (swine vesicular disease, vesicular stomatitis and bovine viral diarrhea). This assay was shown to be 1000-fold more sensitive than the conventional RT-PCR method. The detection limits of this assay was 1 TCID 50 /ml of the FMDV RNA concentration. Quantification was obtained by a standard curves plotting threshold cycle values versus known infectivity titer. The assay was sensitive, specific and rapid enough to detect FMDV RNA genome in probang samples. As such, the described method is reliable and provides faster disease diagnostics than the conventional RT-PCR procedure to detect FMDV.

Identification and antigenic site analysis of foot-and-mouth disease virus from pigs and cattle in Korea

From May to June 2002, a total of 16 foot-and mouth disease (FMD) outbreaks due to the serotype O virus, Pan Asia strain, were recorded in Korea. The viruses were identified by antigen ELISA, RT-PCR and sequence analysis. The overall nucleotide sequence divergence of the VP1 region among the 4 isolates in 2002 was 0 to 1.4%, but between O/SKR/2002 and O/SKR/2000 isolates was 1.9-4.9%. Phylogenetic analysis with the some known strains from East Asian countries showed that the 4 Korean isolates in 2002 formed one distinct cluster, which different from clusters of Korean isolates in 2000, with in the same lineage of the ME-SA topotype strains. Deduced amino acid sequences around neutralizable antigenic site on VP1 site of O/SKR/2002 isolates were aligned and compared with other strains. At the antigenic site 1, the replacements of the critical amino acid residues at position 144 from V to L and at position 152 from A to T were observed in O/SKR/2002 viruses. For antigenic site 2 and 4, there were not significant variations in general. At the antigenic site 3, the substitutions of amino acid residues were present at positions 54 and 56 in O/SKR/2002 isolates and an alternative residue I at position 54 are observed only at the sequence of O/SKR/AS/2002 (cow) virus. And the substitution (L-->P) of significant residue at position 144 was detected at the amino acid sequence of the O/SKR/2002 (cow) virus.

Development of synthetic peptide ELISA based on nonstructural protein 2C of foot and mouth disease virus

It was reported that the sera of convalescent animals contain antibodies to foot and mouth disease (FMD) virus (FMDV) 2C, highly conserved nonstructural protein (NSP), whereas the sera of vaccinated animals do not. But ELISA methods using this protein were not reported and developed until recently. In this study, NSP 2C peptides were synthesized within the amino acid sequence of the conserved 2C nonstructural region of FMDV according to the sequences from Genbank database and used for identifying antigenic determinants. One of the synthesized thirteen peptides gave strong positive reactivity with most of the sera from 13 FMD infected farms, but not with sera from vaccinated and non-infected animals. Moreover, with the sera collected through serial bleedings from four cattle and five goats infected with FMDV O/SKR/2000 experimentally, positive results were obtained in two species after 10 days post infection (DPI). Therefore, we tried to develop and evaluate this ELISA based on 2C peptides. In comparison with the commercial NSP ELISA, the 2C peptide based ELISA method showed good specificity and sensitivity. These results demonstrate that the synthetic 2C peptide ELISA can be a complementary marker to differentiate FMDV-infected from vaccinated on a herd basis.

Comparision of the Hemodynamic Effects of Dopamine and Milrinone in Patients Undergoing Off-Pump Coronary Artery Bypass Grafting / 대한마취과학회지

BACKGROUND: Off-pump coronary artery bypass grafting (OPCAB) on the beating heart causes hemodynamic compromise during displacement of the heart for graft anastomosis. To overcome hemodynamic unstability, volume loading, Trendelenburg position, inotropic and vasodilator supports etc. are selected as usual. This study was designed to compare the hemodynamic effects of milrinone and dopamine on OPCAB anesthesia. METHODS: Twenty patients (13 men, 7 women; mean age 63.4 +/- 18.2 years old) who underwent OPCAB were enrolled in this study. The patients were randomly placed in a dopamine group (n = 10) and in a milrinone group (n = 10). Basic doses of each drug was administered during left anterior descending artery (LAD), posterior descending arterty (PDA), and left circumflex artery territory (LCX) anastomosis. Hemodynamic variables such as; heart rate (HR), mean arterial pressure (MAP), mean pulmonary arterial pressure (MPAP), cardiac index (CI), systemic vascular resistance (SVR), pulmonary vascular resistance (PVR), central venous pressure (CVP), and pulmonary capillary wedge pressure (PCWP) were collected before mechanical stabilization (T1), during anastomosis (T2), and 5 minutes after anastomosis (T3) of LAD and LCX in each patient. RESULTS: During LAD anastomosis, HR in the D group increased significantly at T2 (12.6%(upword arrow)) and T3 (9.8%(upword arrow)) but didn't show significant changes in the M group. CI in the M group showed significant increase at T3 (21.7%(upword arrow) compared to T3 and 18.0%(upword arrow) compared to the D group). During LCX anastomosis, HR didn't show significant changes in the M group, MAP in the D group showed significant decrease (19.3%) at T2 but it was insignificant in the M group. MPAP and PVR showed significant increase at T2 in the D group compared to T1 and T2 in the M group. PCWP showed similar changes of PVR during LCX anastomosis CONCLUSIONS: Hemodynamic changes during OPCAB are more remarkable during LCX anastomosis than LAD anastomosis. These changes can be successfully relieved by inotropic supports with continuos milrinone infusion during anastomosis.

The Experimental Study Using Beagle Dogs for the Clinical Application of Poloxamer-solutol Propofol / 대한마취과학회지

BACKGROUND: To reduce side effects such as hyperlipidemia, pain on injection, and bacterial growth of the present formation of propofol, many attempts to change its formulation have been tried. We have developed a newly formulated poloxamer-solutol propofol, which is includes soy bean oil and egg phosphatide as sufactants. The aim of this study was to evaluate the poloxamer-solutol propofol regarding its pharmacokinetic and pharmacodynamic characteristics and bacterial growth compared to original propofol. METHODS: Thirty Beagle dogs weighing around 10-15 kg were randomly assigned to one of two groups. Group 1 received Diprivan propofol 1% (AstraZeneca Co. UK), Group 2 received poloxamer-solutol formulated propofol by continuous intravenous infusion at 35 mg/kg/h for 3 hours. Three, 6, 9 and 12 hours after the discontinuation of the propofol infusion, venous samples from the anterior tibial vein were analysed for liver and renal function test. Also, blood lipid levels were checked after 3 hours of infusion and blood propofol concentrations were checked every hour during infusion. Eye opening time and orientation time, represented by walking on four legs, were evaluated. Also, broth cultures (100microliter) of four standard preservative efficacy test organisms (Staphylococcus Aureus, Pseudomonas Aeruginosa, Escherichia Coli, Candida Albicans) were added to 9.9 ml of four test formulations at approximately 200 colony forming units/ml. The subjected formulations were; original propofol (AstraZeneca Co, 1% solution, UK), EDTA added propofol (0.0055% EDTA added propofol), Poloxamer-Solutol formulated propofol (poloxamer 188/407 and solutol mixture), and normal saline. The test formulations were incubated at 25degrees C and 32.5degrees C (Tryptic soy agar medium for bacteria and Sabrouraud dextrose agar medium for fungus) and tested for viable counts after 24 and 48 hours. RESULTS: Poloxamer-solutol propofol showed no increase of triglyceride and the propofol concentrations showed no difference between the two groups. Also the original propofol supported the growth of all microorganisms at both temperatures and times. EDTA added propofol inhibited the growth of microorganisms more than the original propofol, but not as much as the poloxamer-solutol formulated propofol. Saline showed a similar pattern as the propofol with added EDTA. CONCLUSIONS: The poloxamer-solutol formulated propofol has advantages by pharmacokinetic-pharmacodynamic studies in terms of the initial TG level during propofol infusion, and shows more bacteriostatic activity against all four microorganisms than the original propofol and the propofol with added EDTA.

Expression of F Protein Gene of a Thermostable Isolate of Newcastle Disease Virus Using Baculovirus Expression System

No abstract available.

Flow cytometric evaluation on the age-dependent changes of testicular DNA contents in rats

An age-dependent cellular change of DNA contents in the testis of Sprague-Dawley rats was investigated by flow-cytometric method. Testicular cell suspensions at the age of 4, 5, 6, 7, 8, 10, 12, 16 and 26 weeks were prepared and stained with propidium iodide. The relative proportions in the number of mature and immature haploid (1n), diploid (2n), S-phase and tetraploid (4n) cells were calculated. The proportion in the number of mature haploid cells was sharply increased to the age of 10 weeks (about 38%), thereafter increased slightly to the level of 42% at the age of 26 weeks. The proportion of immature haploid cells was dramatically increased to the age of 6 weeks, then maintained at the level of 20 to 30% thereafter. The proportion of diploid cells was 64% at the age of 4 weeks, then decreased gradually through the age of 26 weeks. The proportion of S-phase cells was increased to the age of 4 weeks, then maintained at a plateau level to the age of 26 weeks. The proportion of tetraploid cells were about 26% at the age of 4 weeks, then decreased gradually to the age of 26 weeks. These results suggest that the proportions of testicular cells may depend on the age of the rat and that the flow cytometric method may be useful in the evaluation of the spermatogenic status with regard to accuracy and sensitivity.

Molecular Cloning and Nucleotide Sequence of the Gene Encoding Fusion(F) Protein of the Thermostable Newcastle Disease Virus Isolated from a Diseased Pheasant

The gene encoding F protein of CBP-1 strain, a heat-stable Newcastle disease virus (NDV) isolated from the diseased pheasants in Korea, was characterized by reverse transcription-polymerase chain reaction (RT-PCR), nucleotide and amino acid sequences. Virus RNA was prepared from the chorioallatoic fluid infected with NDV CBP-1 virus and cDNA was amplified by RT-PCR, cloned and sequenced to analyze. The PCR was sensitive as to detect the virus titer above 25 hemagglutination unit. 1.7kb (1,707bp) size of the cDNA was amplified and cloned into BamHI site of pVL1393 Baculo transfer vector. The nucleotide sequences for F protein were determined by dye terminator cyclic sequencing using four pairs of primers, and 553 amino acid sequences were predicted. In comparison of the nucleotide sequence of F gene of CBP-1 with those of other NDV strains, the homology revealed 88.8%, 98.5% and 98.7% with Kyojungwon (KJW), Texas GB and Beaudette C strains, respectively. As the deduced 553 amino acid sequences of F protein of CBP-1 were compared with those of other NDV strains, the homology appeared 89.9%, 98.7% and 98.9% with KJW, Texas GB and Beaudette C strains, respectively. The putative protease cleavage site (112-116) was R-R-Q-K-R, indicating that CBP-1 strain is velogenic type. The amino acid sequences include 6 sites of N-asparagine-linked glycosylation and 13 cysteine residues. These data indicate that the genotype of CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than KJW strain.

Clinical Application of the Mapleson B System for Controlled Ventilation in Pediatric Patients / 대한마취과학회지

This study was undertaken to observe whether an adult ventilator with a preset volume could be used as a controlled ventrolled ventilator far pediatric anesthesia. 35 Patients ranging in age from 3months to 7 years were divided into two groups based on body weight(Group 1: 5~10kg, 14 cases, Group 2: 11~15kg, 21 cases) and anesthetized with halothane-N2O/O2 - pancuronium using the Mapleson B system. Immediately after induction, the reservoir bay of the Mapleson B system was replaced by the reservoir tube of the adult ventilator (MCM 801). Arterial blood gas studies 30 and 60 minutes after induction were performed, and the data from group 1 was compared with that of group 2. The magnitude of PCO2 increase 30 minutes after induction was not significantly different from that at 60 minutes(p>0.05), and alterations of PCO2 in group 1 were not stati-stically significant with group 2 (P>0.05). In conclusion, it is suggested that the Mapleson B system attached to adult ventilator is an useful and convenient device for controlled ventilation in pediatric patients.