OA-01

Molecular Serotyping of Glaesserella parasuis Isolated from Diseased Pigs and the Relationship between Serovars and Pathological Patterns in Taiwan

Wei-Hao Lin1,2, Hsing-Chun Shih1, Chao-Nan Lin1,2 and Ming-Tang Chiou1,2,3

1Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

2Animal Disease Diagnostic Center, National Pingtung University of Science and Technology, Pingtung, Taiwan.

3Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Glaesserella (Haemophilus) parasuis (G. parasuis) is the etiological agent of Glässer’s disease, and causes severe economic losses in the swine industry. Serovar classification is an indicator of virulence and pathotype and is also crucial for vaccination programs and vaccine development. The aim of this study was to serotype G. parasuis isolates from diseased pigs in Taiwan using a molecular serotyping assay and to analyze the relationship between serovars and pathological patterns. From August 2013 to February 2017, a total of 155 diseased animals from 124 infected herds were examined. One hundred thirty three isolates of G. parasuis were recovered, serotyped by multiplex PCR and correlated with pathological lesions. The dominant G. parasuis were serovars 5/12 (37.6%), 4 (27.8%) and 13 (15%) followed by molecular serotyping non-typable (MSNT) isolates (13.5%). Those prevalent serovar isolates prefer to cause both serosal and pulmonary lesions rather and only pulmonary lesions. The percentage of lung lesions (30.4%) showing G. parasuis infection was significantly higher than that of each serosal lesions, including meninges, meninges, pleura, pericardia, peritonea and synovial cavities of joints.

Keywords: Glaesserella parasuis, polyserositis, serovar, molecular serotyping

 

OA-02

Genotypic Characterization of Glaesserella parasuis Isolates from Taiwan Using Enterobacterial Repetitive Intergenic Consensus PCR and the Presence of the Virulence Associated Trimeric Autotransporters Marker

Wei-Hao Lin1,2, Hsing-Chun Shih1, Chao-Nan Lin1,2* and Ming-Tang Chiou1,2,3

1Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

2Animal Disease Diagnostic Center, National Pingtung University of Science and Technology, Pingtung, Taiwan.

3Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Glaesserella (Haemophilus) parasuis (G. parasuis) causes severe economic losses in the swine industry. Multiple G. parasuis isolates can exist in single animals. Typing techniques are required in order to identify G. parasuis isolates. Different isolates within a serovar display varying virulence. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) can assess the heterogeneity. The group 1 virulence associated trimeric autotransporters (vtaA) gene is an indicator of virulence. The aim of this study was to characterize Taiwanese G. parasuis isolates via molecular serotyping, vtaA PCR, and ERIC-PCR. One hundred forty five isolates were collected between November 2013 and March 2017 in Taiwan and examined. The dendrogram revealed heterogeneous genetic diversity with many clusters. Partial correlation between the ERIC-PCR clusters of different isolates, serovars and lesion patterns was observed. Twelve herds (8.3%) infected with more than one isolate. Group 1 vtaA positive rate was 98.6%. This study showed the high genetic diversity of G. parasuis in Taiwan by a high discriminatory capability of ERIC PCR. Group 1 vtaA commonly exists in G. parasuis isolates and may be important in the pathogenesis of Taiwanese G. parasuis isolates.

Keywords: Glaesserella parasuis, polyserositis, genotype, virulence factor

 

OA-03

Sequence Analysis of Polysaccharide Biosynthesis Loci of Glaesserella parasuis Isolates from Diseased Pigs in Taiwan

Wei-Hao Lin1,2, Hsing-Chun Shih1, Chao-Nan Lin1,2* and Ming-Tang Chiou1,2,3

1Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.

2Animal Disease Diagnostic Center, National Pingtung University of Science and Technology, Pingtung, Taiwan.

3Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan.

Glaesserella (Haemophilus) parasuis (G. parasuis) causes severe economic losses in the swine industry. Serovar classification is an indicator of virulence and pathotype and is also crucial for vaccination programs. Serovars of G. parasuis is determined by capsule based on the polysaccharide biosynthesis locus. One serovar 5 isolate and two molecular serotyping non-typable (MSNT) isolates subtyped into different genotypes by enterobacterial repetitive intergenic consensus polymerase chain reaction were sequenced for analysis of gene compositions in the polysaccharide biosynthesis loci. In the polysaccharide biosynthesis loci of three G. parasuis isolates, the export regions remained conserve but the putative serovar specific regions varied compared to serovar reference strains. Serovar 5 isolate existed two function unknown genes contained in serovar 6 and 8 reference strains. One MSNT isolate existed not only glycosyltransferase genes gltM and gltL as serovar specific markers of serovar 9 but also glycosyltransferase gene wbgY and function unknown genes as specific markers of serovar 6 and 8. The other MSNT isolate existed lstB and lstA as serovar specific markers of serovar 4 but lacked glycosyltransferase and polysaccharide biosynthesis protein genes. Our results suggest the molecular serotyping assay is worthy of improving to serotype Taiwanese field isolates and the cross-protection among different serovars and MSNT isolates should be further investigated for vaccine strategies.

Keywords: Glaesserella parasuis, polyserositis, polysaccharide biosynthesis locus, capsule, sequence

 

OA-04

Effectiveness of Formalin-Killed Francisella noatunensis subsp. orientalis Vaccine against Francisellosis in Tilapia (Oreochromis spp.)

Theeraporn Pulpipat1, Pei-Chi Wang1,2 and Shih-Chu Chen1,2,3,4

1Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

2Southern Taiwan Fish Disease Research Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

3International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan

4Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan

Francisella noatunensis subsp. orientalis (Fno) is an intracellular pathogen affected to freshwater and seawater fish. Tilapia (Oreochromis spp.) is a major susceptible species. Fno causes systemic granulomatous disease resulting in high mortality and huge economic losses.  Few studies have been carried out of vaccine research. Live attenuated vaccine strain was implied in general. Thus, it may possibly cause reversion to become virulent. Regarding the adverse effects of vaccine is concerned about vaccine safety and efficacy. This study was aimed to set a pilot study of francisellosis vaccine. We conducted an experiment of a formalin-killed Fno vaccine test in cultured tilapia. Fno was isolated from diseased tilapia, inactivated with formalin and mixed with a mineral oil base adjuvant (MontanideTM ISA76AVG). Total of 120 tilapias with bodyweight of 35 ± g were divided into 3 groups.  Each group had 40 fish were intraperitoneal injected with 0.1 ml 1) tested of vaccine in adjuvant, 2) control of PBS and 3) control adjuvant in PBS, respectively. Booster were done at weeks 2 post-vaccination (PV).  Serums were collected and evaluated agglutination antibody titer at weeks 2, 4, and 6 PV.  Tilapias were challenged at weeks 8 PV, recorded mortality for 21 days and calculated relative percentage of survival (RPS). Our vaccine induced greater agglutination titer and the RPS values for the vaccine and adjuvant group were 60.78 % and 9.08 %, respectively, compared to control group. The results of this study provide essential resources for further studies on inactivated Fno vaccine.

Keywords: Francisella noatunensis subsp. orientalis, formalin-killed vaccine, agglutination antibody titer, relative percentage of survival, and Oreochromis spp.

 

OA-05

Characteristics of the Extended-Spectrum β-Lactamases-Producing Escherichia coli Isolated from the Dogs and Cats in National Taiwan University Veterinary Hospital from 2014 to 2017

Yi-Hsuan Huang 1 and Kuang-Sheng Yeh 1,2

1Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

2National Taiwan University Veterinary Hospital, Taipei, Taiwan

Extended-spectrum β-lactamases (ESBLs) are the enzymes that mediate resistance to the newer β-lactam antibiotics, including the extended-spectrum cephalosporins and monobactams. Production of ESBL is primarily plasmid mediated and such plasmids often comprise the genes that encode resistance to other classes of antimicrobials like aminoglycosides and fluoroquinolones. Therefore, ESBL-producing microorganisms leave clinicians only limited therapeutic options in both human and veterinary medicine. Compared to human medicine, information regarding the ESBL-producing microorganisms is limited in veterinary medicine. Our research goal is to screen for ESBL-producing Escherichia coli from dogs and cats admitted to National Taiwan University Veterinary Hospital during 2014 to 2017 and further analyze the genotypes and phylogenetic traits of these ESBL-producers. The double disc tests specified by Clinical and Laboratory Standards Institute (CLSI) were performed on 304 E. coli isolates and revealed 69 E. coli with the ESBL phenotype (26.7%). The CTX-M-1 was the most commonly found ESBL gene group. The ESBL-producing E. coli all demonstrated multi-drug resistance phenotype. Multilocus sequence typing (MLST) had identified 14 sequence types, namely ST38, ST131, ST345, ST359, ST405, ST428, ST457, ST648, ST5640, ST5685, ST5703, ST5865, ST6470, and ST8171. Among the abovementioned STs, the ST38, ST131, ST345, ST359, ST405, ST428, ST457, and ST648 were also documented to infect humans; especially the E. coli ST131 is a zoonotic global clone of public health concern. Since companion animals like dogs and cats are in close contact to humans, characterization of the ESBL-producers from them is important to provide effective therapy and further reduce human exposure risk. 

Keywords: extended-spectrum β-lactamases, Escherichia coli, multidrug-resistance, multilocus sequence typing

 

OA-06

fimU Controls Salmonella enterica Serovar Typhimurium Type 1 Fimbrial Expression at the Translational Level by Recognizing Rarely Used Arginine Codon

Hsing-Jung Wu1 and Kuang-Sheng Yeh1, 2

1Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

2National Taiwan University Veterinary Hospital, Taipei, Taiwan

Expression of the type 1 fimbriae of Salmonella enterica serovar Typhimurium is encoded by the fim gene cluster, which is composed of six structural genes fimA, fimI, fimC, fimD, fimH and fimF, and five regulatory genes fimZ, fimY, stm0551, fimW, and fimU. The regulatory gene fimU encodes a tRNA that recognizes the rarely used arginine codon AGA or AGG. Previous studies had revealed that fimU gene controls type 1 fimbriae expression by recognizing the rarely used arginine codon within FimY. Our sequence analysis revealed that FimI, FimD, FimZ, Stm0551, and FimW also possessed such arginine codons that can be recognized by fimU. The objective of this study is to investigate whether fimU also modulates the translation of FimZ and Stm0551, resulting in regulating expression of type 1 fimbriae in S. Typhimurium. Reverse-transcription PCR indicated that the expression of fimU was identical when S. Typhimurium was grown in static broth and on the agar medium. An isogenic fimU deleted strain is being constructed by allelic exchanged. A fusion protein of FimZ and Stm0551 will be generated using a pMAL Protein Fusion and Purification System and such fusion protein will be transformed into the wild type S. Typhimurium and its fimU mutant background to examine the protein expression level by Western analysis. Changing the rarely used AGA or AGG to the commonly used CGG or CGA arginine codon will also be performed on the fusion protein for comparison. Expression of type 1 fimbriae may be regulated at the transcriptional and translational level.

Keywords: rarely used arginine codon, fim gene cluster, fimU, type 1 fimbriae

 

OA-07

Development of Recombinant Subunit Vaccine Against Nocardia seriolae in Largemouth Bass (micropterus salmoides)

Hoa-Huy Hoang1, Pei-Chi Wang1 and Shih-Chu Chen1,2,3,4

1 Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

2 Southern Taiwan Fish Diseases Research Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

3 International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan

4 Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan

Nocardiosis one of the ubiquitous bacterial infection caused by Nocardia seriolae found in all water bodies ranging with different salinity.  Being major concern in Japan and Taiwan, nocardiosis is also responsible for significant economic losses to the world fishery. Largemouth bass, striped bass, sea bass, and grouper are most susceptible fish species of Taiwan to nocardiosis, recording approximately 25-30% of death during outbreaks.  Live attenuated and inactivated vaccine had weak results on intracellular bacteria to stimulate the cell-mediated immunity henceforth targeted subunit vaccine development become the major concern to control the disease.  In the present study, using the whole genome sequences data of the pathogen, we identified the potential vaccine candidates based on their structural homology of proteins between N. seriolae and other bacterial genome sequences. In total 37 candidates were identified and classified into 9 different families namely: PE/PPE protein family; Heat shock proteins; Mce protein family; Antigen 85 complex; ESAT-like protein; EspC-like protein; Resuscitation promoting factor; Lipoprotein; Protein synthesis. Among 37 predicted candidates, 12 were successfully amplified, cloned, expressed in E. coli BL21.  Thus obtained proteins were purified and tested for efficacy in largemouth bass against N. seriolae infection in laboratory condition. The results showed a various degree of protection from the targeted 12 candidates against an artificial challenge of high dose (1.1x105 cfu/fish) of N. seriolae, and thus the data could be used to find potential recombinant subunit vaccine against the disease.

Keywords: Nocardiosis, Nocardia seriolae, Largemouth bass, recombinant proteins.

 

OA-08

Expression, Purification, Characterization, and Protection Efficacy of Nocardia seriolae  Resuscitation-Promoting Factor RpfA Protein

Hoa Huy Hoang1, Pei-Chi Wang1 and Shih-Chu Chen1,2,3,4

1 Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

2 Southern Taiwan Fish Diseases Research Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

3 International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan

4 Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan

Vaccines are needed to control nocardiosis, the bacterial infectious disease that is responsible for significant economic losses of Japanese and Taiwanese fish culture. In actinobacteria, resuscitation promoting factor (Rpf) proteins have been described as having the ability to increase the viable count of dormant cultures and stimulate the growth of vegetative cells through lag phase reduction. Nocardia seriolae proteins for which a possible role in the reactivation process has been hypothesized are the four homologs of Rpf genes of Mycobacterium tuberculosis, namely rpfA, rpfB, rpfC, and rpfE. In this study, the completed rpfA gene was successfully amplified and cloned into pET151D-Topo expression vector, then a recombinant form of RpfA was expressed in E. coli BL21. The vaccine containing recombinant RpfA in addition to the metabolized MontanideTM ISA 763 A VG adjuvant was developed and evaluated in Largemouth bass. The results indicate that RpfA based sub-unit vaccine resulted in a remarkable higher antibody production as early as 2 weeks and still remain at a high level after 4 and 6 weeks post vaccination. Analysis of immune-related genes also show a significant up-regulated of Il-1β, Tnf-α, and Il-12p40 that than of control group at 12h post immunization. Notably, significant protective efficacy against an artificial challenge of N. seriolae was observed in vaccinated fish with relative percent survival value of 62.85%. Our results demonstrated that recombinant protein RpfA is a promising candidate for nocardiosis vaccine development.

Keywords: Nocardiosis, Nocardia seriolae, Resuscitation promoting factor, RpfA, recombinant protein.

 

OA-09

Evaluation of Vaccine Efficacy with Formalin Killed Cells of Lactococcus garvieae against Grey Mullet (Mugil cephalus)

Shreesha Rao1, Pei-Chi Wang1 and Shih-Chu Chen1,2,3,4 *

1Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

2Southern Taiwan Fish Diseases Research Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

3International Degree Program of Ornamental Fish Science and Technology, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan

4Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan

Lactococcosis is a hyper-acute systemic disease that can occur in fishes of various aquatic environments of most countries that sustain an active aquaculture. Lactococcus garvieae are Gram-positive cocci shaped bacteria, the main pathogenic species responsible for warm water Lactococcosis. Lactococcosis is septicemic processes in fishes, caused by L. garvieae, one of the important fish pathogen causing high mortality and economic loses in the fishery industry. Vaccination of susceptible populations is the best measure to control bacterial diseases in fish. The goal of this study is to produce efficient inactivated vaccine of L. garvieae against Grey mullet (Mugil cephalus) - the most susceptible fish species for lactococosis infection in Taiwan. The present study was designed to evaluate the efficacy of the formalin-killed inactivated vaccine in for one month and three months of cultured fish. Formalin-inactivated bacteria were found to induce high antibody titer in gram-positive bacteria, the protection will long last for 2 to 3 months. Commercialized adjuvant-MONTANIDE ISA 763 A VG ® was used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells. Cumulative mortality was recorded for the vaccinated fishes by challenging the vaccine with homologous strains after one month and three months of post vaccination. The Relative percentage of survivelity was recorded 100% both culture periods proving efficacy better for commercialization.

Keywords: Lactococcosis, Mugil cephalus, Lactococcus garvieae, Vaccine

 

OA-10

Enhanced Immunomodulatory Activity by Nanoparticulated CpG

Shu-Yi Lin1, Bing-Yu Yao2, Che-Ming Jack Hu2,3, Hui-Wen Chen1,3

1Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

3Research Center for Nanotechnology and Infectious Diseases, Taipei, Taiwan

CpG oligonucleotide (CpG ODN) is a potent immunostimulatory pathogen-associated molecular pattern to be recognized by avian toll-like receptor (TLR) 21 and thereafter triggers antigen presenting cells and B-cell activation, producing Th1 and proinflammatory cytokines. The class B CpG ODN 2007 has been used to enhance vaccine efficacy to against a variety of avian viral pathogens including avian influenza virus, infectious bronchitis virus (IBV) and Newcastle disease virus. In this study, nanotechnology was used to encapsulate CpG ODN 2007 in proprietary hollow PLGA nanoparticles to enhance the stimulatory effects. The Cy5 labeled CpG nanoparticles (CpG-NPs) were firstly treated on the chicken bone marrow-derived dendritic cell (chBMDC) to confirm the intracellular delivery. Then free CpG and CpG-NP were treated on chBMDC to evaluate the cell viability and immune gene stimulation. Finally, free CpG and CpG-NPs were used as an adjuvant to couple with recombinant IBV receptor binding domain (RBD) antigen for chicken immunization. The results showed that CpG-NPs can be efficiently delivered into chBMDC, leading cells to maturation. The CpG-NPs significant enhanced the immune response of chBMDC than free form CpG. Moreover, in vivo vaccination test showed higher virus-specific IgG antibody response induced by CpG-NPs. In conclusion, in contrast with free form CpG, nanoparticulated CpG ODN 2007 exerts an enhanced immunostimulatory effect on chBMDC and on chicken vaccination, demonstrating the potential as a potent chicken vaccine adjuvant.

Keywords: CpG, nanoparticle, vaccine, adjuvant

 

OA-11

Immortalization of Primary Goat Fibroblast by Expression of Simian Virus 40 Large T Antigen for Orf Virus Propagations

Yumiko Yamada, Guan-Ru Liao and Wei-Li Hsu

Graduate Institute of Microbiology and Public Health, National Chung Hsing University , Taiwan

Immortalized cells could grow and divide indefinitely in vitro under optimal culture conditions. Due to the high host range specificity of orf virus (ORFV), primary cells derived from goats and sheep are recommended for isolation and propagation of wild type ORFV that limits the option for study of virus-host interaction. Several methods exist for immortalizing mammalian cells in culture; one such method is induction of immortalization by SV40 T antigen, a viral oncoprotein that could abrogate replicative senescence via interference with cellular survival signaling pathways.

In this study, immortalization of primary goat fibroblast (FB-WT) was achieved by stably expressing SV40 T antigen by means of lentiviral technique.  The presence of SV40 T antigen was confirmed by PCR and Western blot analyses. Growth kinetics and the dependence of serum were determined in both FB-WT and goat fibroblast with T antigen (FB-T) cell lines at different passages. Additionally, cellular signaling pathways that were known to be affected by SV40 T antigen was monitored. Ultimately, susceptibility of ORFV infection was confirmed by viral yield, and plaque formation ability. The successful immortalization of FB-WT will be beneficial to studies on the pathogenesis of ORFV without considering the limited life span of primary goat fibroblast cells.

Keywords: goat fibroblast, SV40 T antigen, orf virus

 

OA-12

Molecular and Preliminary Biological Characterization of Chicken Anemia Virus from Chickens in Taiwan

Suttitas Tongkamsai1, Meng-Shiou Lee2, Ming-Chu Cheng1,Yi-Lun Tsai and Yi-Yang Lien1

1Department of Veterinary Medicine, College of Veterinary Medicine,National Pingtung University of Science and Technology Pingtung, Taiwan

2China Medical University, School of Chinese Medicine Resources, Taichung, Taiwan

During screening of field samples from chickens in southern Taiwan in 2014, two chicken anemia virus (CAV) isolates were confirmed by polymerase chain reaction (PCR) and by isolation of the virus on the MDCC-MSB1 cell. The nucleotide sequence of the full-length viral protein (VP) 1 gene was determined by direct sequencing. Alignment of VP1 amino acid sequence of two Taiwan CAV isolates (04RL05 and 04BF08) with 24 other CAV strains showed 96.4% to 99.5% homology. Phylogenetic analysis of these CAV isolates and strains were also carried out, and were separated into genotype II and genotype III. In motifs associated with virulence, the 04RL05 isolate had virulence-associated motifs composed of 75V, 89T, 125L, 141Q and 144E. The 04BF08 isolate had the motifs composed of 75I, 89T, 125I, 141Q and 144Q, which are commonly found in highly virulent group. The 04RL05 isolate was found closely related to the USA strain. The 04BF08 isolate was closely related to the Cambodia strain. The pathogenicity of two Taiwan CAV isolates were demonstrated in day-old specific pathogen free (SPF) chicks. Experimental inoculation resulted that two Taiwan isolate showed similar pathogenicity. In spite of this, only the 04RL05 isolate was able to produce a mortality case. These data suggest that further study using an extended number of samples is needed to accurately assess the distribution of CAV genotypes and the pathogenicity of these viruses.

Keywords: Characterization, Chicken anemia virus, Taiwan