Chuen-Fu Lin

Central Taiwan University of Science and Technology, 臺中市, Taiwan, Taiwan

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Publications (9)17.17 Total impact

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    ABSTRACT: The complete nucleotide sequence of a cryptic plasmid, pK50-2, from Lactobacillus reuteri K50 had been determined. It consisted of an 1866bp circular molecule with a G+C content of 35%, from which two putative open reading frames (orfs) could be predicted. Based on sequence similarity, the orf1 was not homologous to any known protein, while the N-terminus of the orf2 shared 56% and 64% identities with RepB proteins of plasmid pAR141 and an unnamed plasmid in L. reuteri strain PA-16, members of the rolling-circle replication (RCR) pMV158 family, respectively. Downstream of orf2, a sequence containing two conserved regions (i.e., bind and nick), possibly involved in the binding and nicking of Rep protein, similar to the dso (double strand origin) of RCR-pMV158 family was also identified. Furthermore, a sequence capable of forming the characteristic secondary structure of ssoT (single-strand origin type T) was subsequently determined upstream of the orf2 gene. Thus, the three elements essential for a RCR plasmid (i.e., dso, sso, and rep gene) were all deducible in the pK50-2. Noteworthy was that a conserved alpha helix-turn-alpha helix (HTH) motif, thus far only seen in theta-type plasmids, was for the first time identified in Rep protein of RCR plasmid, pK50-2. To estimate the pK50-2 could be an expression vector to deliver exogenous antigens, a shuttle vector pK50-S containing both pK50-2 and pUE80 (-) was used to analyze the segregational stability and copy-number, which were shown that pK50-S in L. reuteri DSM 20016 were estimated to be 98%, 77%, and 75% after 36, 72, and 100 generations and about 50 copies per chromosome equivalent by real-time PCR.
    Plasmid 09/2012; · 1.28 Impact Factor
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    ABSTRACT: Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome. Two major PCV2 genotypes, PCV2a and PCV2b, have been identified. To explore the prevalence of different subgroups of PCV2 in Taiwan, 37 PCV2 isolates collected during 2002-2011 were analyzed. The genotypes of the PCV2 isolates collected before 2007 belonged to either PCV2a or PCV2b. However, all of the isolates collected after 2008 were PCV2b. Most of isolates obtained since 2008 have been classified into a novel genotype within subgroup of PCV2b based on complete ORF2 sequence analysis. Moreover, analysis of the PCV2 isolates from the same pig farm but from different years revealed that the viruses shifted from a PCV2b genotype to a novel subgroup of the PCV2b genotype. Collectively, PCV2b was the dominant PCV2 genotype in Taiwan currently and that the viruses have shifted into a new emerging subgroup within the PCV2b genotype.
    Journal of Veterinary Medical Science 06/2012; · 0.88 Impact Factor
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    ABSTRACT: The spike (S) protein, containing two subunits, S1 and S2, is the major immunity-eliciting antigen of avian infectious bronchitis virus (IBV), a highly contagious disease of chickens. Several immunogenic regions, mainly located within the S1 subunit, have been identified. Nonetheless, these immune-dominant regions were defined using selected monoclonal antibodies or using a short peptide approach that involves only certain limited regions of the S protein. In addition, some immune-dominant regions are located in hypervariable regions (HVRs) which are not present in all serotypes. Hence, the aim of this study was to determine a broader range of antigenic regions that have strong antibody eliciting ability; these could then be applied for development of an IBV-diagnostic tool. Initially, the S1 and part of the S2 subunit protein (24-567 amino acids) were expressed as five fragments in prokaryotic system. The antigenicity was confirmed using IBV immunized sera. Performance of the S subfragments was evaluated by ELISA using a panel of field chicken sera with known IBV titres determined by a commercial kit. This indicated that, among the five antigenic recombinant proteins, the region S-E showed the highest specificity and sensitivity, namely 95.38 % and 96.29 %, respectively. The κ value for the in-house ELISA using the S-E fragment compared to a commercial kit was 0.9172, indicating a high agreement between these two methods. As region S-E harbors strong immunogenicity within the spike protein, it has the potential to be exploited as an antigen when developing a cost-effective ELISA-based diagnosis tool.
    Applied Microbiology and Biotechnology 05/2012; 95(1):233-42. · 3.69 Impact Factor
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    ABSTRACT: The in vitro activity of mastoparan-AF, an amphipathic antimicrobial peptide isolated from the hornet venom of Vespa affinis, alone and in combination with various clinically used antibiotics, was investigated against 21 Escherichia coli isolates/strains. Most E. coli isolates tested were detected containing multiple-antimicrobial resistance genes. Antimicrobial activity of mastoparan-AF was measured by MIC, MBC, time-kill kinetic assay and chequerboard titration method. Mastoparan-AF exhibited potent antimicrobial activity against most multiple-antibiotic-resistant E. coli isolates at the concentrations ranging from 4 to 16 μg/ml. Combination studies showed that mastoparan-AF acts synergistically with certain antibiotics, i.e., cephalothin or gentamicin, against some multiple-antibiotic-resistant E. coli isolates. In conclusion, mastoparan-AF alone or in combination with other antibiotics could be promising as alternatives for combating multiple-antibiotic-resistant E. coli in future clinical applications.
    Peptides 04/2012; 36(1):114-20. · 2.52 Impact Factor
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    ABSTRACT: Mucosal surfaces are common sites of pathogen colonization/entry. Effective mucosal immunity by vaccination should provide protection at this primary infection site. Our aim was to develop a new vaccination strategy that elicits a mucosal immune response. A new strain of Enterococcus faecium, a non pathogenic lactic acid bacteria (LAB) with strong cell adhesion ability, was identified and used as a vaccine vector to deliver two model antigens. Specifically, sigma (σ) C protein of avian reovirus (ARV), a functional homolog of mammalian reovirus σ1 protein and responsible for M-cell targeting, was administered together with a subfragment of the spike protein of infectious bronchitis virus (IBV). Next, the effect of immunization route on the immune response was assessed by delivering the antigens via the LAB strain. Intranasal (IN) immunization induced stronger humoral responses than intragastic (IG) immunization. IN immunization produced antigen specific IgA both systemically and in the lungs. A higher IgA titer was induced by the LAB with ARV σC protein attached. Moreover, the serum of mice immunized with LAB displaying divalent antigens had much stronger immune reactivity against ARV σC protein compared to IBV-S1. Our results indicate that ARV σC protein delivered by LAB via the IN route elicits strong mucosal immunity. A needle-free delivery approach is a convenient and cost effective method of vaccine administration, especially for respiratory infections in economic animals. Furthermore, ARV σC, a strong immunogen of ARV, may be able to serve as an immunoenhancer for other vaccines, especially avian vaccines.
    Vaccine 04/2012; 30(33):5019-29. · 3.77 Impact Factor
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    ABSTRACT: To investigate the frequency of tetracycline resistance (Tet-R) lactobacilli in pig intestines, a total of 256 pig colons were analyzed and found to contain typical colonies of Tet-R lactic acid bacteria in every sample, ranging from 3.2 × 10(3) to 6.6 × 10(5) CFU/cm(2). From these samples, a total of 159 isolates of Tet-R lactobacilli were obtained and identified as belonging to 11 species, including Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus ruminis, Lactobacillus kefiri, Lactobacillus fermentum, Lactobacillus sakei, Lactobacillus coryniformis, Lactobacillus parabuchneri and Lactobacillus letivazi. Based on the EFSA (2008) breakpoints, all isolates, after MIC analysis, were qualified as Tet-R, from which the significant high Tet-R MIC(50) and MIC(90) values indicated an ecological distribution of Tet-R lactobacilli mostly with high resistance potency in pig colons. PCR-detection identified 5 tet genes in these isolates, the most predominant one being tet (W), followed by tet (M), (L), (K), and (Q). Their detection rates were 82.0%, 22.5%, 14.4%, 8.1% and 0.9%, respectively. Noteworthily, isolates of the same species carrying identical tet gene(s) usually had a wide different MIC values. Furthermore, strain-subtyping of these isolates by REP-PCR demonstrated a notable genotypic biodiversity % (average = 62%).
    Anaerobe 08/2011; 17(5):239-45. · 2.02 Impact Factor
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    ABSTRACT: In total, 211 isolates of A. pleuropneumoniae were collected from pigs with hemorrhagic pneumonia at slaughterhouses during 2002-2007. Serotypes, antimicrobial susceptibility and minimum inhibitory concentration (MIC) values were determined for each isolate of A. pleuropneumoniae to 10 antimicrobial agents. Serovar 1 of A. pleuropneumoniae was predominant in Taiwan in 138 of the 211 isolates, followed by serovars 2 and 5. More than 90% of collected isolates were sensitive to ceftiofur, cephalothin, and chloramphenical. However, lincospectin and gentamicin were relatively less susceptible with sensitivities of only 2.4 and 5.7%, respectively. Additionally, ceftiofur had the highest in vitro activity with an MIC(50) of 2.2 µg/ml, followed by cephalothin (2.7 µg/ml) and chloramphenicol (7.9 µg/ml). Lincospectin had the least activity with MIC(50) and MIC(90) values of 73.9 and 114.5 µg/ml, respectively. The data indicate that ceftiofur and cephalothin were extremely active against A. pleuropneumoniae and with minimum MIC values. These drugs are suitable for controlling and treating hemorrhagic pleuropneumonia outbreaks in swine.
    Journal of Veterinary Medical Science 02/2011; 73(2):205-8. · 0.88 Impact Factor
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    ABSTRACT: Six 5-week-old porcine circovirus type 2 (PCV2)-free, cesarean-derived, colostrums-deprived (CDCD) pigs were inoculated intranasally with 10(6) TCID(50) of PCV2. Four CDCD pigs were untreated cohabitants. Forty farm-raised pigs from two PCV2-contaminated herds were randomly selected for PCV2 trace investigations. Blood, nasal, oropharyngeal and fecal samples were collected from all tested pigs weekly. The PCV2 DNA shed at 6-11 and 7-12 weeks of age for PCV2-inoculated pigs and cohabitants, respectively. All the CDCD pigs exhibited seroconversion after PCV2 exposure. In the farm-raised animals, PCV2 shed at 9-15 weeks of age and seroconversion started at 11 weeks of age. Collectively, the pigs had a prolonged PCV2 shedding period following viral exposure, and growing pigs were the source of horizontal PCV2 transmission in PCV2-infected herds.
    Journal of Veterinary Medical Science 12/2010; 73(4):521-5. · 0.88 Impact Factor
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    ABSTRACT: The Nisin-controlled gene expression (NICE) system, which was discovered in Lactococcus lactis, was adapted to Lactobacillus reuteri by ligating nisA promoter (PnisA) and nisRK DNA fragments into the Escherichia coli-Lb. reuteri shuttle vector pSTE32. This chimerical plasmid (pNICE) was capable of expressing the heterologous amylase gene (amyL) under nisin induction. Optimization of induction factors for this Lb. reuteri/pNICE system, including nisin concentration (viz. 50 ng/ml), growth phase of culture at which nisin be added (viz. at the early exponential phase), and the best time for analyzing the gene product after inoculation (viz. at the 3rd h), allowed the amylase product to be expressed in high amounts, constituting up to about 18% of the total intracellular protein. Furthermore, the signal peptide (SP) of amyL gene (SPamyL) from Bacillus licheniformis was ligated to the downstream of PnisA in pNICE, upgrading this vector to a NICE-secretion (NIES) level, which was then designated pNIES (Sec+, secretion positive). Characterization of pNIES using an amyL-SPDelta gene (amyL gene lacking its SP) as a reporter revealed the 3rd h after induction as the secretion peak of this system, at which the secretion efficiency and the amount of alpha-amylase being secreted into the culture supernatant were estimated to reach 77.6% and 27.75 mg/l. Expression and secretion of AmyL products by pNIES in Lb. reuteri was also confirmed by SDS-PAGE and immunoblotting analysis.
    Bioscience Biotechnology and Biochemistry 05/2006; 70(4):757-67. · 1.27 Impact Factor