OA:  口頭報告競賽組

Oral Presentation for Competition

OA-01

Effects of Trivalent Chromium Alone and Combined with Estradiol on Bone Metabolism in Ovariohysterectomized Rats

Lin-Jing Kong¹, Dan-Wei Shao¹, Wen-Yuan Xue¹, Hsiang-Cheng Ho¹, Hui Huang¹, Hong-Yu Chen¹, Jia-Li Liu¹, Wei-Ming Lee^1,2

¹Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University

²Veterinary Medical Teaching Hospital, National Chung Hsing University

In postmenopausal women, the dramatic decrease of estrogen results in a high morbidity of osteoporosis as well as other metabolic disorders like diabetes and obesity. Apart from the direct effect of estrogen on bone metabolism, diabetes and obesity also affect it indirectly. Trivalent chromium (Cr³⁺), for its benefit to carbohydrate, protein and lipid metabolism, is widely used in the treatment of type 2 diabetes mellitus. The traditional treatment of women postmenopausal osteoporosis is hormone replacement therapy (HRT), a supplement of maintaining dose estradiol (E2). However, whether a supplement of Cr³⁺ alone or an addition of Cr³⁺ in HRT will make some differences on bone mineral density remains uninvestigated. Therefore, the aim of this study is to figure out the effects of Cr³⁺ alone and with E2 on bone metabolism in the model of ovariohysterectomized rats. Thirty-six 10-week-old female Sprague-Dawley rats were randomly divided into 6 groups after ovariohysterectomy: Control, Cr (Cr³⁺ supplement only), LE2, LE2+Cr (a supplement of maintaining-dose E2 with or without Cr³⁺), HE2 and HE2+Cr (a supplement of 20X maintaining-dose E2 with or withoutCr³⁺). Body weight was measured weekly during 15 weeks’ experiment. Plasma Calcium (Ca), Phosphorus (P), parathyroid hormone (PTH) and osteocalcin (OC) levels were measured to evaluate bone metabolism. Bilateral femurs and tibiae were dissected to measure bone volume(BV/TV). The results showed an E2-dose-related relationship in variant body weight ratio, Ca, P, PTH and BV/TV within None Cr groups (Control, LE2 and HE2 groups) and only high-dose E2 had a significant (P<0.05) effect on preventing osteoporosis according to BV/TV. The Cr group shared similar data (P>0.05) with LE2 group on all the parameters. Moreover, an addition of Cr³⁺tended to narrow the E2-dose-related difference between None Cr groups. It is indicated that Cr³⁺ has a similar effect to maintaining-dose E2 on bone metabolism, the same as the combination of Cr³⁺ and maintaining-dose E2. However, an addition of Cr³⁺ can weaken the bone protection effect of high-dose E2. Keywords: bone metabolism, estradiol, trivalent chromium

OA-02

Avian Bornavirus Infection among Psittacines in Taiwan

Shang-Han Wu¹, Sheng-Hsun Yang¹, Hui-Wen Chen¹

¹Department of Veterinary Medicine, National Taiwan University

Parrot bornavirus (PaBV) infection is an emerging, life threatening neuropathic disease in psittacine birds, also known as the cause of proventricular dilation disease (PDD), and can be easily confused with other findings, including coma, arrhythmias, vomiting and sudden death. PaBVs belong to the genus Bornavirus, and grouped as 2 different species: Psittaciform 1 bornavirus (PaBV-1, 2, 3, 4 and 7) and Psittaciform 2 bornavirus (PaBV 5). The epidemiology information of PaBV infection in Taiwan is largely unknown. To investigate the presence of virus in the field, we have initiated a surveillance program of avian bornavirus in psittacine samples collected from multiple regional and local veterinary hospitals island wide. To this date, the virological prevalence rate based on our study is approximately 16%. Among the 30 bird samples identified positive by nested RT-PCR, the phylogenetic analysis of the M and N genes revealed that PaBV-2 (13.3%, 4/30), PaBV-4 (83.3%, 25/30) and a rare PaBV-5 (3.3%, 1/30) co-circulate in the field of Taiwan. Viral antigens in infected tissues were evidenced with immunohistochemistry staining using diseased bird serum. With above findings, a quail muscle cell line QM7 was adopted for virus isolation. We have successfully isolated all three types of PaBVs from infected tissue homogenates, and the intracellular localized viruses have been validated by western blot and RT-PCR. Moreover, recombinant nucleoprotein (p40) of avian bornavirus has been expressed and purified for antiserum production and future development of diagnostic assays. Collectively, a survey of avian bornavirus infection among psittacines is conducted for the first time in Taiwan, and multiple types of PaBV have been isolated and characterized. Further effort will be made on developing essential strategies towards disease management and control.

Keywords: avian bornavirus, PaBV, virus isolation, p40 protein

OA-03

Streptococcus dysgalactiae Surface Immunogenic Proteins, Phosphoenolpyruvate Protein Phosphotransferase and Glyceraldehyde-3-phosphate Dehydrogenase, Serve as Highly Effective Vaccine Candidates

Thuy Thi Thu Nguyen¹, Hai Trong Nguyen¹, Tan Duc Nguyen², Pei-Chi Wang¹, Shih-Chu Chen¹

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

²Institute of Veterinary Research and Development of Central Vietnam, Vietnam

An immunoproteomic strategy was undertaken for screening and identifying surface immunogenic proteins of Streptococcus dysgalactiae, a causative agent of massive mortality in a wide range of farmed fish species. Outer surface proteins of this pathogen were extracted and immunogenic targets detected by western blot were subjected to peptide sequencing using NanoLC-MS/MS. Two surface proteins, namely phosphoenolpyruvate protein phosphotransferase (PtsA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showed strong reactions with cobia (Rachycentron canadum) antisera against S. dysgalactiae. Recombinant proteins were produced in Escherichia coli cells and their protective efficacies were investigated in cobia. Fish immunised with recombinant proteins, rPtsA + ISA and rGAPDH + ISA, showed remarkably higher specific antibody responses and lysozyme activity. In addition, the expression levels of IL-1β, TNF-α, IL-8, and Mx in vaccinated fish were higher than those in control fish at 24 h post S. dysgalactiae challenge. Notably, vaccinated fish were protected from lethal challenge with relative percentage of survival (RPS) values for rPtsA + ISA and rGAPDH + ISA groups being 91.67% and 83.33%, respectively. The obtained results demonstrated that two novel protective antigens from S. dysgalactiae were successfully determined using the surface immunoproteomic approach.

Keywords: phosphoenolpyruvate protein phosphotransferase (PtsA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Streptococcus dysgalactiae, outer surface proteins, immunoproteomic

OA-04

Immune Responses and Effectiveness of Outer Membrane Protein Vaccines against Vibrio harveyi in Orange-spotted Grouper

Hai Trong Nguyen¹, Thuy Thi Thu Nguyen¹, Yan-Chun Chen¹ , Tan Duc Nguyen², Pei-Chi Wang¹, Shih-Chu Chen¹

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

²Institute of Veterinary Research and Development of Central Vietnam, Vietnam

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing recombinant V. harveyi outer membrane protein K (rOmpK), outer membrane protein U (rOmpU), and rOmpK-OmpU fusion protein in addition to the metabolisable montanide^TM ISA 763 A VG adjuvant were evaluated in the orange-spotted grouper. The results indicate that immunization of the recombinant V. harveyi protein-based vaccines resulted in a remarkably higher expression of IL-1β and IL-8 at 24 h, and greater antibody production, as early as 2 weeks post-immunisation. Notably, an enhanced immune response and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine-injected fishes with relative percent survival value of 81.8%. Additionally, the rOmpK-OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also V. parahaermolyticus and V. alginolyticus. Our results demonstrated that the fusion protein rOmpK-OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.

Keywords: V. harveyi, fusion protein, ISA 763 A VG, vaccine, vibriosis

OA-05

Cranial Cruciate Ligament Reconstruction in a Dog

Shao-Wen Hung¹, Wan-Yun Tsai², Shih-Ting Chen², Jhin-Wei Chen¹, Wei-Ming Lee^1,2

¹Department of Veterinary medicine, College of Veterinary Medicine, National Chung Hsing University

²Veterinary Medical Teaching Hospital, College of Veterinary Medicine, National Chung Hsing University

The patient was a three-year-old intact male mixed dog, weighing 16.85 kg, with complete vaccination record and regular ecto/endo-parasitic prophylaxis. The left hind limb lameness caused by unknown reason was noticed after dog walking outdoor on June 24^th, 2017, and then the dog was diagnosed as left cranial cruciate ligament rupture after physical examination at local animal hospital on next day. The owner came to VMTH-NCHU for further consultation on June 26^th 2017. At clinic, the patient was in good spirit and normal motility, and revealed a lame but weight-bearing left hind limb by physical examination. Cranial cruciate ligament rupture was diagnosed by positive drawer test and tibial compression test at stifle joint. Ruptured cranial cruciate ligament reconstruction of left stifle joint was recommended and performed on June 28^th, 2017. We used fascia to replace the ruptured cranial cruciate ligament in this surgery. Due to normal physical status with improved left hind limb weight-bearing activity, the patient was discharged on June 30^th, 2017, and activity restriction was recommended. Skin sutures were removed on the 19^th day post-operation, the patient could weigh on the affected limb, and drawer test and tibial compression test was negative during palpation.

Keywords: Cranial cruciate ligament, dog, reconstruction

OA-06

Molecular Cloning, Expression and Purification of the Autoclavable TTSS Gene spaS from Salmonella Choleraesuis

Xin-Xin Liu¹, Chun-An Chen¹, Cheng-Hsun Chiu², Tien-Huan Hsu³, Yu-Chung Chang ¹

¹Department of Biotechnology, Ming Chuan University

² Department of Pediatrics, Chang Gung Children's Hospita

³Department of Veterinary Medicine, School of Veterinary Medicine, National Chung Hsin University

Salmonella Choleraesuis is a major pathogen that causes gastroenteritis in human and animals. It is well-known that salmonellae can use the type three secretion system (TTSS) to invade host cells. One of them, the spaS, is located on Salmonella pathogenicity island I (SPI-I). Previous studies have showed that the SpaS of Salmonella is highly conserved to the EscU of Escherichia coli, and both of them might undergo autocleavage, a process involves the cyclization of asparagine (Asn). In addition, the SpaS plays vital roles as switch protein of TTSS translocon and its needle. It also plays an important role in the secretion of effector proteins. In this study, the spaS of Salmonella Choleraesuis was cloned and expressed to clarify its expression within E. coli. Initially, the spaS was cloned into the protein expression vector pQE-30, but no protein band consistent with the full-length SpaS can be seen on the acrylamide gel after running SDS-PAGE. The reason why SpaS protein was not expressed properly might be due to the fact that it undergoes autocleavage. Therefore we also introduced a point mutation into the supposely cleavage site of SpaS to obtain △spaS in pMAL-c2X. Mutation of spaS caused its 258^th amino acid residue to be changed from Asn to Lys. This mutation might abolish its ability to undergo autocleavage, but it still cannot find any expression of protein △SpaS in E. coli. To avoid the transmembrane region of SpaS will interfere the expression of △SpaS, the △SpaS-CTD gene was subcloned into the expression vector pMAL-c2X. This time the △SpaS-CTD was successfully expressed in E. coli. Our results indicate that the full-length SpaS seems hard to be expressed in E. coli, and also demonstartes that mutation of spaS in the 258^th amino acid residue will abolish autocleavage of SpaS. Moreover, antibodies against purified △SpaS-CTD were prepared in immunized mice and will be used to unravel the potential roles played by SpaS of the TTSS apparatus.

Keywords: Type III secretion system, TTSS, Salmonella, autocleavage, SpaS

OA-07

Development of Newcastle Disease Live Vaccine of Genotype VII and the Virulence Determinantion

Ai-Ping Hsu ^1,2, Yi-Ta Lu², Yu-Hua Shih^1,2, Hsuan Chen², Wen-Jane Tu², Chun-Hsien Tseng² and Hsiang-Jung Tsai¹

^1.Graduate Institute of Veterinary Medicine, National Taiwan University
^2.Animal Health Research Institute, Council of Agriculture

Newcastle disease (ND) is a highly transmissible disease of poultry caused by Newcastle disease virus (NDV). Animal Health Research Institute (AHRI) has invented a new ND live vaccine strain of genotype VII based on artificially serial attenuations and genetically recombinant techniques, and further investigated the virulence reduction mechanism of this virus strain. One specific amino acid on L protein was found to be involved with the virus virulence. This specific amino acid was moreover modified and recombinant virus (477A-Plusafe) was recovered as well. Multiple passage manipulations (more than 40 passages) in the embryonated eggs didn’t bring to the increase on the intracerebral pathogenicity index (ICPI) of 477A-Plusafe; always being below 0.25. In the study to assess potential of regaining virulence, the organ emulsion collected from the chicks inoculated with 477A-Plusafe (10⁸ EID₅₀ per chick) could be only detected the infinitesimal viral RNA, but failed to recover the virus. 477A-Plusafe has also to be demonstrated highly effective as 11 or 14 days post one shot immunization of 10⁵ EID₅₀ could lead to 100% protection against challenge of wildtype NDV. All above concludes 477A-Plusafe is a safe and ideal vaccine candidate for ND market of genotype VII.

Keywords: Newcastle disease, vaccine, reverse genetics, genotype VII

OA-08

Development of Chicken Coccidiosis Subunit Vaccine by Using Outer Membrane Vesicle Expression System of Escherichia coli

Yi-Hsuan Kuo, Kuo-Pin Chuang

Graduate Institute of Animal Vaccine Technology, National Pingtung University of Science and Technology

Outer membrane vesicles (OMVs) are spherical vesicles secreted from the outer membrane of Gram-negative bacteria. These vesicles which play a role in pathogen promotion, bacterial survival during noxious conditions, and regulation of microbial interactions within bacterial community, have been recently explored as a platform for vaccine development. Chicken coccidiosis, caused by various parasite mostly Eimeria species, infecting various sites in the intestine, is a serious disease in the poultry industry, and lead to the tremendous economic loss annually. In the present study, four genes of Eimeria were cloned and expressed, and the antigenicity and immunogenicity, as the antigens of this research, were investigated. The x gene of E. coli strain BL21 was deleted by homologous recombination and compared with wild-type BL21, the gene modified bacteria was shown to secrete more OMVs by the test of protein concentration. Furthermore, Transmission Electron Microscope was used to check the bacterial appearance and the OMVs of modified-BL21 (m-BL21). The gene of Eimeria antigen was expressed by the m-BL21, and the result of Western-Blot demonstrated that OMV can cover the heterologous protein. The secreted OMVs containing the protein products were collected, and were injected to the chickens. After the animal test, the results of weight gain shown that OMV vaccine has no influence on weight of market. This study suggests that OMV can be a vaccine candidate of chicken in the future.

Keywords: chicken, Coccidiosis, Eimeria, subunit vaccine, OMVs

OA-09

Identification of the Binding Site of FimW in Salmonella enterica Serovar Typhimurium

Zheng-Fang Liu¹, Kuang-Sheng Yeh^1,2

¹Department of Veterinary Medicine, National Taiwan University

²National Taiwan University Veterinary Hospital

Salmonella enterica serovar Typhimurium is one of the most important causative agents responsible for salmonellosis in both human and animals. Transmission of S. Typhimurium is by oral-fecal route and this microorganism adheres epithelial cells of intestinal tract by fimbrial structures. There are 13 different fimbrial gene clusters that have the potential to encode these filamentous structures of S. Typhimurium. The fim gene cluster that encodes type 1 fimbriae is composed of six structural genes and five regulatory genes. FimW plays a repressive role in type 1 fimbrial production. Amino acid sequence of FimW suggests it a transcriptional regulator and C-terminal part of FimW possesses a helix-turn-helix motif. No specific binding site of FimW has yet been identified. Complementation test indicated that this fimbrial phenotype was conferred by fimW. A FimW fusion protein was constructed using a pET101 system. However, western blot analysis indicated that FimW was located in the bacterial pellet portion. The DNA fragments ranging from 200-300 bp upstream of bcfA, stbA, stdA, stiA, stfA, lpfA, and fimA had been PCR amplified and purified. An electrophoretic mobility shift assay will be performed using FimW and the abovementioned DNA pieces. If no binding between FimW/fimbrial promoter DNA is observed, chromatin immunoprecipitation assay (ChIP assay) will serve as an alternative to locate the FimW-binding site within S. Typhimurium genome. Different fimbrial expression with one bacterium requires a delicate regulatory network; our investigation in FimW may shed light on the cross talk mechanism between type 1 fimbriae and other fimbrial systems.

Keywords: Salmonella Typhimurium, type 1 fimbriae, FimW, DNA binding site

OA-10

Investigation of Silver (Ag) Deposition and Concentration in Tissues from Stranded Cetaceans by Autometallography (AMG)

Bang-Yeh Liou¹, Wei-Cheng Yang^2,3, Meng-Hsien Chen⁴, Wen-Ta Li^1,2, Hui-Wen Chang¹, Victor Fei Pang¹, and Chian-Ren Jeng¹

¹Graduate Institute of Molecular and Comparative Pathobiology, National Taiwan University,

²Taiwan Cetacean Society

³College of Veterinary Medicine, National Chiayi University

⁴Department of Oceanography and Asia-Pacific Ocean Research Center, National Sun Yat Sen University

Silver (Ag), including silver nanoparticles (AgNPs), has been used in various commercial products and may be released into environment. Whether Ag deposition cause adverse effect on biological systems or animal health has been a serious concern. Cetaceans acting as apex predator in ocean may accumulate a high level of Ag in their bodies due to bioaccumulation, and could acquire negative impact on their health. To investigate the deposition of Ag and Ag compounds in cetacean, we utilized autometallography (AMG) to localize the Ag in the formalin-fixed paraffin-embedded (FFPE) tissue of stranded cetacean. The present study established a model to estimate Ag concentration in the liver and kidney of stranded cetacean by correlation the result of inductively coupled plasma mass spectrometry (ICP-MS). The results revealed that silver is mainly deposited in hepatocyte, Kupffer cells, and epithelial cells of some proximal renal tubules. In high concentration level of silver deposition individuals of all cetacean species investigated, AMG signal could be found in glial cells, pneumocytes, vascular endothelium of heart and brain, adrenal cortex and medulla. Silver deposition also could be found in myocardium and neuron in some cases. Comparing with previous studies, the patterns of silver deposition in cetacean is different from the pattern conducted in laboratory rodents. It suggests that cetaceans may have different silver metabolic pathway. The present result indicates that Ag contamination in cetacean in North-western Pacific Ocean may be more severe than previous evaluation. Based on the present result, the negative effects caused by Ag in cetacean should be further investigated.

Keywords: Autometallography, cetacean, inductively coupled plasma mass spectrometry (ICP-MS), silver (Ag)

OA-11

Mosquito-Borne Diseases Surveillance of Mosquito Captured in Farms and Boundaries in Taiwan

Yi-Chi Luo, Hui-Wen Chang, Victor Fei Pang, Chian-Ren Jeng

Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University

In the tropical and subtropical countries, mosquito-borne diseases (MBDs) represent an important problem which may cause diseases and economic losses, both in human and animals. According to geographic location of Taiwan, it has high risks epidemics for MBDs. Further, attributed to the rapid growth of human traveling and international commerce activity, the possibility of new MBDs introduction is increasing by transmissible mosquitoes. Therefore, the aim of this study was to investigate the prevalence of important MBDs and transboundary MBDs in mosquitoes in the area including cattle and goat farms, airport, and harbors. The CDC light net traps were placed in the study areas, and samples were collected during May to October, 2017. A total of 1722 mosquitoes was captured, subclassified to species level, pooled and tested by PCR and RT-RCR for targeting pathogens including malaria, filarial worms,  Flaviviridae (Dengue, West Nile, yellow fever, Japanese encephalitis, zika), Togaviridae (Chikungunya) and Bunyaviridae (Rift valley fever) virus. Each mosquito pools were formerly tested for housekeeping gene Actin-1 for ensuring the DNA and RNA quality. A total of 46 DNA mosquito pools and a total of 22 RNA mosquito pools were tested, respectively. The results showed all pathogens were confirmed to be negative. The study indicates the importance of monitoring the high risk of transboundary MBDs in mosquitoes in Taiwan.

Keywords: mosquito, mosquito-borne disease, PCR

OA-12

Construction of Swine Norovirus P Particle as a Vaccine Platform for the Expression of Salmonella Antigens

Zheng-Jie Gong¹, Ting-Chih Chen¹, Xin-Xin Liu¹, Shin-Bei Weng¹, Wan-Zhen Zhu¹, Chia-Jung Hsieh¹, Tien-Huan Hsu², Yu-Chung Chang*¹  

¹Department of Biotechnology, Ming Chuan University

²Department of Veterinary Medicine, School of Veterinary Medicine, National Chung-Hsin University

Noroviruses (NoVs) are potential pathogens of zoonosis and also the major gastrointestinal pathogens accounting for more than 85% of the incidence of human viral enteritis. Till now, there are 33 serotypes of noroviruses and can be divided into six genogroups (GI-GVI). Former reports have demonstrated that members of GII noroviruses can infect human, swine and dogs. The ORF2 of the norovirus is translated into the major capsid protein (VP1). VP1 can also be subdivided into two domains, i.e., S domain (shell domain) and P domain (protruding domain). The P domain may form a 24 base-pair-lone P particle which can be utilized to develop a multivalent vaccine vector platform. The P particle with octahedral symmetry is extremely stable and highly immunogenic. The P domain of human norovirus contains three loops which can be replaced by restriction enzyme sites. Previous studies have proved that the recombinant human norovirus P particle is an excellent multivalent vaccine platform and have successfully expressed several foreign antigens. In this study, we have also successfully constructed a swine norovirus P particle vector and introduced Salmonella TTSS genes on it. After that animal experiment will be performed to test whether antibodies can be elicited against both swine norovirus and Salmonella antigens.

Keywords: Swine norovirus, P particle, Salmonella, vaccine

*Corresponding author

OA-13

OA-14

OB: 口頭報告非競賽組

Oral Presentation for Non-Competition

OB-01

Investigation of The Correlation of Meridians Length between Canine and Human Based on The “Shu-shu” Principle: Using Toy-Breed Poodles as an Example

Chen-Yin Liao ¹ , Cheng-Hung Lai ¹ , Han-Wen Cheng ^3,4, Tung-Ti Chang ^2,3

¹Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University.

²School of Post-baccalaureate Chinese Medicine of China Medical University

³Master’s Program for Traditional Chinese Veterinary Medicine of China Medical University

⁴The Chinese Society of Traditional Veterinary Science  

Using acupuncture to carry out animal health in the East and West world has become a wave. “Human acupuncture point transformation” method is widely used and accepted. However, since there exists a paucity of investigations on the reality of meridians in either human or animal studies, no conclusion can be found on this issue. It is therefore impossible to understand the meridians correlation between canine and human based on the reported literature. Jye-Chou Yang, the author of “Accomplishments of Acupuncture”, said, “Track back to the origin, study the flow direction, and we understand the truth”. Hence, this thesis has started with the fundamental theory in Chinese medicine and designed the experiments based on the “Shu-shu” Principle. Canine ratio of Ying/Yang meridians between front and hind limps were measured in toy-breed poodles, whereas the corresponding human ratios were also measured for comparison. The results indicate that these ratios can be well explained by the “Shu-Shu” model. This conclusion confirms the solid correlation between canine and human meridians, and lends scientific supports to the “human acupuncture point transformation” method adopted by clinical Chinese medicine veterinary surgeons.

Keywords: Shu-shu, Poodle, length of meridians, Meridian ratios, Traditional Chinese Veterinary Medicine

OB-02

Diagnosis and Treatment of Bilateral Esophageal Diverticula in a Dog: Case Report

Yu-Ting Chang¹, Cheng-Shu Chung^1,2, Lee-Shuan, Lin^1,2

¹Veterinary Medical Teaching Hospital, National Pingtung University of Science and Technology

²Department of Veterinary Medicine, National Pingtung University of Science and Technology

A nine year-old intact male Yorkshire Terrier had a history of vomiting and regurgitation immediately after meal for one month. Dehydration, anorexia, lethargy, and weight loss was claimed at first attendance to NPUST VMTH. Barium contrast radiography had been performed by referral veterinarian. This study showed accumulation of the barium contrast in the esophagus, an overlapping cystic-like radiopaque sign adjacent to esophagus between heart base and diaphragm section was noted. Through endoscopic examination, bilateral esophageal diverticula were confirmed and the chicken bone stuck in diverticula was also retrieved. Furthermore, antiemetic medication was given and hospitalized, however, the clinical sign was not improved. Finally, the bilateral esophageal diverticula were surgical excised and the patient recovered well. Bilateral esophageal diverticula is rare in clinical cases. The cause of bilateral esophageal diverticula in this case may be congenital or secondary to foreign body. Accurate diagnosing and surgical intervention may provide good prognosis.

Keywords: dog, esophageal diverticula, regurgitation

OB-03

Evaluation of Activation Effect of Low-Level Laser on Feline Platelet-Rich Plasma

Shu-An Pai¹, Cheng-Shu Chung^1,2, Lee-Shuan Lin^1,2

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

² Veterinary Medical Teaching Hospital, National Pingtung University of Science and Technology

OB-04

Cytokine mRNA Expressions in Bovine Peripheral Blood Mononuclear Cells Suggest Strain Differences of Bluetongue Virus in Inducing Host Responses

Jia-Ling Yang, Fun-In Wang

Graduate Institute of Veterinary Medicine, National Taiwan University

Bluetongue (BT) is an insect-transmitted disease of domestic and wild ruminants caused by bluetongue virus (BTV), and it is one of the economic threat to animal husbandry worldwide. Although BTV infection in Taiwan is often subclinical, it is still a potential risk factor for livestock on Taiwan. To develop an efficient vaccine to induce effective immune responses, it is crucial to know the responses of different lymphocyte populations. Most studies on BTV-induced immune responses focused on the reactions induced by virulent strains, rather than by low virulent strains. In this study, an exploratory investigation of the Th1 and Th2 pathways in the course of the immune response to two Taiwan strains (BTV-2 and BTV-12, both of low virulent) was performed. Relative quantification of cytokine mRNA results showed that high and persisted IL-4 expression, an indication of Th2 pathway, in early stages of BTV2 infection may be detrimental for virus to further replicate in the host. Although IL-4 mRNA is also increased in 4 hours post infection by BTV12, the immune response eventually converted to Th1 virus clearance pathway, indicated by IL-12 and IFN-γ expressions.

Keywords: bluetongue, pathogenesis, cytokine, bovine peripheral blood mononuclear cells (bPBMCs), qRT-PCR

OB-05

Determination of Phthalates in Pork and Chickens by Liquid/

Gas Chromatography-Tandem Mass Spectrometric in Taiwan

Geng-Ruei Chang^1,2

¹Department of Veterinary Medicine, National Chiayi University

²Division of Residual Control, Agricultural Chemicals and Toxic Substance Research Institute, Council of Agriculture

Phthalate esters (PAEs) were summarized as the suspected interference factor of environmental hormone and endocrine interference factor, that have been reported especially for the developmental and reproductive toxicants in the fetus and pregnant females. Firstly, we set up the analyzing phthalates methods of Butyl benzyl phthalate (BBP), Di-n-butyl phthalate (DBP), Di-2-ethylhexyl phthalate (DEHP), Di-isodecyl phthalate (DIDP), Di-isononyl phthalate (DINP), Di-n-octyl phthalate (DNOP) through n-hexane saturated methanol for extraction in this study, which was to investigate the pollution sources from the residues of PAEs in fresh pork and chickens in Taiwan. The detection limits of these phthalates were 0.05 mg/kg for BBP, DNOP, DIDP, and 0.1 mg/kg for DBP, and 0.45 mg/kg for DEHP, respectively. Here, a total of 60 fresh pork and chickens’ samples obtained from the stallholder and slaughterhouse were comprised. The results showed that DEHP was determined in 2 fresh pork samples (0.61-0.80 mg/kg) and in 4 fresh chicken samples (0.11-0.61 mg/kg). These residues of PAEs from our collection samples were also lower than 1 mg/kg. Finally, these findings can be preliminarily revealed that fresh vegetables and fruits were not obviously polluted by PAEs. Furthermore, fresh vegetables and fruits in Taiwan both exhibit good qualities for food safety.

Keywords: Phthalates, Pork, Chicken, Liquid chromatography tandem mass spectrometry, gas chromatography tandem mass spectrometry, Residues

OB-06

A Survey of Tetracyclines, Chloramphenicols, Nitrofurans Metabolites, Malachite Green, and Leucomalachite Green Residues by Liquid Chromatography Tandem Mass Spectrometry in Fishes of Taiwan

Geng-Ruei Chang^1,2

¹Department of Veterinary Medicine, National Chiayi University

²Division of Residual Control, Agricultural Chemicals and Toxic Substance Research Institute, Council of Agriculture

The food safety of seafood is a prominent public health issue for consumers worldwide. In this study, we detected the residues of veterinary drugs in aquatic products by using liquid chromatography tandem mass spectrometry. The veterinary drugs tested included three chloramphenicols, four tetracyclines, four nitrofurans metabolites, malachite green, and leucomalachite green. The method detection limit (MDL) of tetracycline, chlorotetracycline, doxycycline, and oxytetracycline was 2.50 ng/g. In addition, the MDL of chloramphenicol, florfenicol, thiamphenicol, and nitrofurans metabolites was 0.25 ng/g, whereas that of malachite green and leucomalachite green was 0.50 ng/g; these values correspond to the maximum residue limits (MRLs) specified in Taiwan, Japan, and the European Union, and the minimum required performance limit (MRPL) in Taiwan and the European Union. In this study, 80 samples comprising fish were collected from various types of markets and aquatic farms in Taiwan between January 2012 and December 2012. Malachite green and leucomalachite green were detected in one sample of golden thread and exceeded the MRL of Taiwan by 1.25%. The findings of this study can be used for monitoring veterinary-drug residues in aquatic products and as a reference for food safety. In addition, the results also revealed that the residues of veterinary drugs in aquatic products could be detected even when the violation rate was low. Therefore, the continual survey of aquatic samples is required to ensure food safety for consumers.

Keywords: Fishes, Veterinary drugs, Liquid chromatography tandem mass spectrometry, Residues, Taiwan

OB-07

Analysis of Banned Veterinary Drugs Residues in Shellfish by Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS)

Geng-Ruei Chang^1,2

¹Department of Veterinary Medicine, National Chiayi University

²Division of Residual Control, Agricultural Chemicals and Toxic Substance Research Institute, Council of Agriculture

Seafood safety is a crucial public health concern for consumers. In this study, we applied a validated method to analyze the residue of banned veterinary drugs in shellfish, namely chloramphenicol, malachite green, leucomalachite green, and nitrofuran metabolites by LC/MS/MS. The method detection limit (MDL) of tetracycline, chlorotetracycline, doxycycline, and oxytetracycline was 2.50 ng/g. In addition, the MDL of chloramphenicol, florfenicol, thiamphenicol, and nitrofurans metabolites was 0.25 ng/g, whereas that of malachite green and leucomalachite green was 0.50 ng/g; these values correspond to the maximum residue limits (MRLs) specified in Taiwan, Japan, and the European Union, and the minimum required performance limit (MRPL) in Taiwan and the European Union. In total, 42 shellfish samples, which included hard clams, freshwater clams, and oysters, were collected from aquafarms and production areas in Taiwan during 2012. Our results revealed 3.8 ng/g of chloramphenicol in one hard clam, indicating that 2.4% of the samples contained residues from banned veterinary drugs. These data can be used to monitor the residue of veterinary drugs in aquatic organisms and as a reference for food safety.

Keywords: Banned veterinary drugs, Shellfish, Liquid chromatography tandem mass spectrometry, Residues, Taiwan

OB-08

Analysis of Pesticides Residues in Shellfish by Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS) and Gas Chromatography-Tandem Mass Spectrometry (GC/MS/MS)

Geng-Ruei Chang^1,2

¹Department of Veterinary Medicine, National Chiayi University

²Division of Residual Control, Agricultural Chemicals and Toxic Substance Research Institute, Council of Agriculture

Environmental pollution with pesticide residues is evident throughout the world, and a great number of data have been reported. In this study, an adapted QuEChERS method, coupled to a liquid and gas chromatography-mass spectrometry assay, was developed to quantify 418 pesticides, including carbamates, organochlorides, organophosphates, and synthetic pyrethroids in adductor muscles of oysters (Crassostrea gigas) and hard clams (Meretrix lusoria). The detection limits of these pesticides were lower than 10 ng/g for 89.5% analytes in oysters and 89.2% analytes in hard clams, respectively. At lower spiked levels of 50 ng/g for GC-MS/MS and 20 ng/g for LC-MS/MS assay, the average recoveries were in the range of 70% to 120% for 80.9% analytes in oysters and 83.7% analytes in hard clams, respectively. On the other hand, recoveries were between 76.3% in oyster sand 87.8% in hard clams at higher spiked levels of 200 ng/g for GC-MS/MS and 100 ng/g for LC-MS/MS assay, respectively. The relative standard deviations (RSDs) were lower than 20%. The quantification of analytes was carried out using the most sensitive transition for each compound and by matrix-matched calibration standards. This method has been successfully applied to the two shellfish species. In total, 42 shellfish samples, which included hard clams, freshwater clams, and oysters, were collected from aquafarms and production areas in Taiwan during 2012. Our results revealed 19.9–32.1 ng/g of ametryn in two hard clams, 16.1–60.1 ng/g of pendimethalin in four hard clams, and 17.0 ng/g of mefenacet in one oyster, indicating that 16.7% of the samples contained residues from pesticides. Thus, this proposed method is satisfactorily considered for routine monitoring of pesticide residues in oysters and hard clams.

Keywords: Pesticides, Shellfish, Liquid chromatography tandem mass spectrometry, Gas chromatography-tandem mass spectrometry, Residues, Taiwan

OB-09

Synthetic Peptides Mimic Porcine Circovirus Type 2 Capsid and Non-Capsid Proteins Inducing Antibody Response

Ling-Chu Hung^{1, 2}, Ivan-Chen Cheng²

¹Animal Health Research Institute, Council of Agriculture, Executive Yuan

²School of Veterinary Medicine, National Taiwan University

The purpose of this study was to use a reverse vaccinology approach to demonstrate the ideal immunogens relevant for PCV2 peptide vaccine development, initiating from identification of peptides reacting well with pig sera from PCV2-infected herds. Subsequently, those peptides were used as immunogens to inoculate mice. This study showed that mouse anti-PCV2 antisera could be generated by specific synthetic peptides and recognizing PCV2 viral protein. Also, the peptide C3 indeed mimic PCV2 capsid protein (CP) and that is capable of inducing antibody response. It showed that the tertiary or linear form C-terminal sequence (C3) of PCV2 capsid peptide only appeared a local distribution in the nucleus of PCV2-infected PK cells, virus-like particles of PCV2 major appeared a local distribution in the cytoplasm, and ORF 6 protein of PCV2 were shown unusually in cytoplasm. Furthermore, most residues of the peptide C1 and the peptide C3 were presented on the surface of PCV2 CP, in the view of 3-D structure of the CP. Those results showed some specific synthetic peptides (C3 and N2) could mimic viral proteins of PCV2 in nature. This study also focused on the nature of the binding between the antigen (peptides or viral protein) and antibodies which come from either pig sera or anti-peptide mouse sera. Moreover, those results demonstrated that suckling newborn piglets absorb high concentration of maternal transferring antibodies (C3-specific IgA and IgG) from colostrum and milk in the first 24 h, and it might protect themselves from PCV2 infection during neonatal age.

Keywords: porcine circovirus type 2, peptide, immunogen, capsid protein, open reading frame (ORF) proteins, antibody

OB-10

A Constructed Porcine Teschovirus with Deleted Highly Conserved “RNNQIPQDF” Epitope of VP1 has Potential to Serve as DIVA Vector

Arthur Tung-Hsuan Tsai ¹, Chia-Yi Chang² and Fun-In Wang ¹*

¹ School of Veterinary Medicine, National Taiwan University

² Animal Health Research Institute

Differentiation of infected from vaccinated animals (DIVA) is an important strategy for disease control. Development of a specific epitope-erased marker vector and accompanying diagnostic serum may improve the efficiency of DIVA. Porcine teschovirus (PTV) is well suited for serving as an immunization vector due to its: 1. Fecal-oral or intranasal route of infection. 2. Pantropism in swine host. 3. Generally low virulence and minor epidemic impact on swine herds worldwide. 4. DIVA purpose as proposed above. Here, a monospecific antibody was found to recognize a conserved “RNNQIPQDF” epitope spanning amino acids 188–196 of capsid protein VP1 of PTV1/PS 34 strain. The epitope was deleted and replaced by a reverse genetics procedure. The ability of this monospecific antiserum to differentiate the epitope-erased marker PTV from parental virus shows its potential to serve as an immunization vector for swine diseases. This is the first report of a DIVA possibility based on an anti-structural protein antibody of PTV. In general, recombined picornaviruses have DIVA potential on using as vector-based vaccines.

Keywords: Porcine teschovirus, DIVA, vector

*Corresponding author

OB-11

Phylogenetic Analysis of Newcastle Disease Viruses in Taiwan

Yu-Pin Liu ^1,2, Yu-Ju Lin ¹, Li-Hsuan Chen ¹, Wan-Chen Li ¹, Yen-Ping Chen ¹, Fan Lee ¹, Wen-Jane Tu ¹, Hsiang-Jung Tsai ²

¹Animal Health Research Institute

²School of Veterinary Medicine, National Taiwan University

Thirty Newcastle disease virus (NDV) isolates from domestic poultry and wild birds during 2012 to 2017 in Taiwan were genetically characterized. The phylogenetic analysis of the variable region of the F gene indicated that there are at least five genotypes of NDV circulating in domestic poultry and wild birds. Five isolates belonged to class II genotype I, four to genotype II, nine (isolated from pigeons) to genotype VI, four to genotype VII, and the remaining seven isolates belong to class I NDV. At the F0 cleavage site, the class I and class II genotypes I, II, VI, VII NDV isolates had the sequence motifs of E-Q/R-Q-E-R↓L, G-K/E-Q-G-R↓L, G-R-Q-G-R↓L, R-R-Q-K-R↓F, and R-R-K-K-R↓F respectively. The isolates from poultry farms were classified into genotype I, II, and VII. Base on the phylogenetic analysis and the motif of the fusion glycoprotein cleavage site, the genotypes I and II isolates were lentogenic NDV vaccine strains. All of the genotype VII velogenic isolates belong to the sub-genotype VIIe, and these results suggest that sub-genotype VIIe NDV is the prevalent virulent strains circulating among chicken farms in Taiwan. In this study, half of the NDV isolates were collected from wild birds, and our results indicate that wild bird population carries avirulent NDV with genetic divergence regularly and may act as one of the important reservoirs.

Keywords: Newcastle disease, paramyxovirus, phylogenetic analysis

PA:  壁報展示競賽組

Poster Presentation for Competition

PA-01

The Evaluation of Antitumor Effect of STAT3 Decoy- Oligodeoxynucleotide (deODN) on Canine Mammary Gland Tumors

Sinling Wang^{1, 3}, Albert Taiching Liao^{2, 3}

1 Department Institute of Molecule and Comparative Pathobiology Schoool of Veterinary Medicine National Taiwan University

2 Department and Graduate Institute of Veterinary Medicine, National Taiwan University

3 Animal Cancer Center, National Taiwan University

Canine mammary gland tumors (MGTs) are the most common neoplasms in female dogs and half of them are malignant. Various factors have influence on the development of MGTs, such as age, breed, hormone, growth factor, and diet. Surgery, chemotherapy and radiotherapy are the traditional therapies for MGTs. However, recurrence and metastasis are common in most malignant MGT patients after traditional therapies. Several novel therapies are under developing and targeted therapy is one of them.

Signal transducer and activator 3 (STAT3), one of transcription factor, has been described as a key regulator of cell survival and proliferation. The functions of activated STAT3 are involved in cell growth, differentiation, development, apoptosis and angiogenesis. Constitutive activation STAT3 has been found in a variety of human tumors including prostate, breast, head and neck cancer, multiple myeloma and glioma etc. Interleukin-6 (IL-6), through the Janus kinase (JAK)–dependent signaling cascade, is the major cytokine to stimulation the activation of STAT3 in the tumor microenvironment. Activation of STAT3 causes tumor cell proliferation, survival, angiogenesis, invasion and metastasis.

Decoy oligodeoxynucleotide (deODN) is a short DNA fragment which mimic and to compete with the binding site of targeted transcription factors. As soon as it binds to targeted transcription factor, the function of targeted transcription factor is fail. deODNs targeting various transcription factors have been applied to a variety of human cancers and induced tumor cell death successfully. In this study, we are going to design deODN targeting STAT3 and evaluate its antitumor effect on canine mammary tumors.The aim of the study is to evaluate the antitumor effect of STAT3 decoy ODN on canine mammary gland tumors

IL-6, pSTAT3 and STAT3 are expressed on MGT tissue by IHC stain. And in cell biology, the best condition of transfection was confirmed. Migration of MGT cells is inhibited by STAT3 deODN. However, STAT3 deODN fails to inhibit the proliferation of MGT cells. Further investigation on the evaluation of the inhibition of biological function on MGT by deODN is needed.

Keywords: MGT; STAT3; transcription factor; Decoy oligodeoxynucleotide; Migration

PA-02

Development of Mycoplasma synoviae Bacterin – Vaccine Safety and Efficacy Studies in Field

Guo-wei Lee, Sheng-Xiang Huang, Hsiao-Chun Lin, Zeng-Weng Chen, Jyh-Perng Wang, Jiunn-Horng Lin, Ho-Yuan Chou

Animal Technology Laboratories, Agricultural Technology Research Institute, Taiwan

Mycoplasma synoviae (M. synoviae) infection is one of the most common disease found in turkey and chicken farm around the world, and can causes respiratory diseases, arthritis, and growth retardation. Based on our previous studies, M. synoviae bacterin vaccine can significantly protect SPF chickens from M. synoviae challenge. In the present studies, we applied our vaccine on chickens at field farm to further confirm its safety and effectiveness. In this study, the M. synoviae bacterin vaccine developed in our lab were applied to the field chicken farm. Our results showed that this novel vaccine not only very safe, but also improve growth performance and survival rate in commercial chickens.

Keywords:Mycoplasma synoviae, vaccine, commercial chickens

PA-03

Comparison of the Protective Efficacy of Mycoplasma gallisepticum Subunit Vaccines with Commercial Inactive Vaccine in Chicken

Sheng-Xiang Huang, Guo-wei Lee, Hsiao-Chun Lin, Zeng-Weng Chen, Jyh-Perng Wang, Jiunn-Horng Lin, Ho-Yuan Chou

Animal Technology Laboratories, Agricultural Technology Research Institute, Taiwan

Mycoplasma gallisepticum (M. gallisepticum) commonly causes chronic respiratory disease (CRD) on turkey and chickens of all ages. The typical symptoms, such as sneeze, rapid breathing, airsacculitis and growth retardation, cause serious economic losses in poultry industry. The purpose of this study is to compare the subunit vaccine developed in our lab with commercial inactive vaccine of M. gallisepticum. In this study, we confirm again that our novel M. gallisepticum subunit vaccine not only can induce high ELISA and HI antibody titer in chicken, but also prevent average daily gain reduction and decrease air sac and trachea lesion after M. gallisepticum challenge. In addition, our M. gallisepticum subunit vaccine can provide better protection when compared to the commercial M. gallisepticum inactivated vaccine (C1 and C2).

Keywords: M. gallisepticum, subunit vaccine, commercial inactive vaccine

PA-04

Avian Reovirus σA Protein Increases ATP Formation by Enhancing Glycolysis and TCA Cycle Through Activation of IDH3B/mTOC1/eIF4E/HIF-1α pathway and Inhibition of LDHA Benefiting Virus Replication

Pei-I Chi and Hung-Jen Liu

Institute of Molecular Biology, National Chung Hsing University

This study provides a novel insight on the avian reovirus σA protein-modulates glycolysis and TCA cycle enhancing virus replication. Adenosine triphosphate (ATP) has been shown as an energy source for many types of viruses for facilitating virus replication. This is the first report to demonstrate that the structural protein σA of avian reovirus (ARV) functions as an activator of cellular energy. In this work, three cellular factors, including isocitrate dehydrogenase 3 subunit beta (IDH3B), lactate dehydrogenase A (LDHA), and vacuolar-type H+-ATPase (v-ATPase) co-immunoprecipitated with the σA antibody were identified by 2D-LC/MS/MS. We found that ARV σA protein activates several key enzymes in glycolysis pathway to promote the cellular ATP levels in IDH3B/mTORC1/HIF-1α-dependent manner. ARV σA enhances both IDH3B and v-ATPase to trigger the mTOC1/eIF4E pathway to assist HIF-1α translation. Besides, σA upregulates the level of pyruvate kinase while LDHA is decreased, which suggest that ARV enhances the glycolysis/TCA cycle flux to increase the ATP formation. The σAR155/273A mutant loses its ability to enter nucleolus, impairing its ability in glycolysis enzymes regulation, thereby decreasing the ATP generation. Furthermore, we discovered that both 5’ and 3’ of untranslated regions (UTR) in ten dsRNA genome segments play a critical role in viral protein synthesis in an ATP-dependent manner. Deletion of either 5’ or 3’ UTR impaired virus replication. Knockdown of σA with an shRNA reduced the ATP level and significantly decreased virus titer. Taken together, our results suggest that ARV σA protein modulates both LDHA and IDH3B/mTORC1/eIF4E/ HIF-1α pathways to upregulate glycolysis and TCA cycle for enhancing the level of cellular energy and benefiting virus replication.

Keywords: Avian reovirus, ARV, σA, Adenosine triphosphate, ATP, glycolysis, mTOR complex I, eIF4E, HIF-1α, TCA cycle, lactate dehydrogenase A, LDHA, v-ATPase, isocitrate dehydrogenase 3 subunit beta, IDH3B, untranslated region, hexose kinase, phosphfructose kinase, TPI and pyruvate kinase

PA-05

Engineering of Canine YKL40 scFv Antibodies for Canine Cancer Research

Kai-Jie Chang¹, Albert Taiching Liao^{1, 2}

¹Department and Graduate Institute of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University

²Animal Cancer Center, College of Bioresources and Agriculture, National Taiwan University

According to the statistics from Taipei City Animal Protection Office, cancer is now the number one killer of canine in Taiwan. Early detection of cancer is the crucial determiner to successful recoveries. YKL-40, also known as CHI3L1 (chitinase-3-like-1), is supposed to be a cancer biomarker in human cancers, and elevated serum level of YKL-40 is correlated with metastasis, poor survival and bad prognosis in a variety of human cancers. Our study in canine YKL-40 in dog cancer cases has also revealed this similar finding. Inspired by this, we have developed three mouse mAbs (monoclonal antibodies) through hybridoma technology to fight against canine YKL-40. The developed mouse mAbs have presented the ability to a certain degree of inhibition on YKL-40 functions in cellular assays. To preserve these mAbs and expand their applications, the aims of this study are to develop and characterize their scFv (single-chain variable fragment), which can be expressed through the prokaryotic expression system, to facilitate mass production in vitro and possess better penetration, lower immunogenicity, and therapeutic antigen targeting in vivo. To these ends, the scFvs were cloned from the hybridoma cells using SOE RT-PCR (Splicing by overlap extension reverse transcription polymerase chain reaction). Two of scFv, scFv/M2 and scFv/M3, may have potential to block cell migration and invasion elicited by rcYKL-40. Next, we evaluated the protective effect of YKL-40 on canine cancer cells from chemotherapy. Finally, an indirect ELISA was established using these scFvs to detect serum YKL-40 levels of cancer dogs and healthy dogs. In conclusions, the scFvs of mAbs against canine YKL-40 are generated. They can be applied to ELISA, WB and IFA to detect canine YKL-40, and parts of them can be used to reverse biological functions elicited by rcYKL-40. Various assays can be further developed to evaluate scFvs for other applications.

Keywords: YKL-40, single-chain variable fragment (scFv), canine, cancer, ELISA

PA-06

Antiviral Efficacy of Diphyllin Nanoparticles against Influenza Viruses

You-Ting Chen¹, Zih-Syun Fang^1,2, Wei-Shan Chang¹, Che-Ming Jack Hu², Hui-Wen Chen¹

¹Department of Veterinary Medicine, National Taiwan University

²Institute of Biomedical Sciences, Academia Sinica

Influenza virus infections are a major public health concern and cause significant morbidity and mortality worldwide. Conventional treatments against the disease are designed to target viral proteins. However, the emergence of new influenza viral strains carrying drug-resistant mutations that can outpace the development of pathogen-targeting antivirals presents a major clinical challenge. Diphyllin, a novel vacuolar ATPase inhibitor, was previously identified with a broad-spectrum antiviral activity. However, diphyllin’s poor water solubility and its potential adverse effect may limit its clinical application. The central objective of this project is to generate a novel anti-influenza therapeutic strategy, integrating nanoparticle technology to enhance host-targeting antiviral delivery towards improved drug safety and efficacy. In this study, we have prepared a nanoformulation of diphyllin, which the size was measured approximately 200 nm by the transmission electron microscopy (TEM) and the drug encapsulation was analyzed nearly 35% by the high-performance liquid chromatography (HPLC). The nanoparticles showed to have efficient intracellular delivery in multiple cell lines including Fcwf-4, ARPE-19, and MH-S cells. The nanoformulation of diphyllin exhibited lower cytotoxicity as compared to the free diphyllin molecules. Furthermore, diphyllin nanoparticles demonstrated prominent anti-influenza activity in MDCK and MH-S cells. Upon the investigation of in vivo safety study, diphyllin nanoparticles were found well-tolerated in mice, from which the body weight was monitored and blood chemistry parameters were evaluated following intravenous or intranasal administration. Collectively, this work highlights the nanoformulation of diphyllin as a potential effective host-targeted treatment against influenza.

Keywords: influenza virus, diphyllin, vacuolar ATPase, nanoparticles, antiviral

PA-07

Evaluation of the Influences of Different Intravenous Fluid for Delivering Canine Mesenchymal Stem Cells

Cheng-Li Chen¹, Lee-Shuan Lin^1,2, Cheng-Shu Chung^1,2

¹Department of Veterinary Medicine, National Pingtung University of Science and Technology
²Veterinary Medical Teaching Hospital, National Pingtung University of Science and Technology

Mesenchymal stem cells have been used in treating various disease in companion animals. Currently, intravenous delivery of mesenchymal stem cells with advantages of convenience, noninvasive and multi-usable, is the major route for clinical use. However, the appropriate intravenous fluid for delivering canine mesenchymal stem cells (cMSCs) is still unknown, and we would like to evaluate the influence of intravenous fluid when applied for delivering cMSCs. Adipose tissue harvested from the greater omentum of 4 dogs were taken for isolating cMSCs. The cMSCs were incubated at room temperature with 3 different intravenous fluids: Sodium Chloride 0.9%, Lactated Ringer’s and Dextrose-Lactated Ringer’s Injection. After incubation for 1 and 2 hours, the cell suspension was collected to compare the viability by trypan blue stain and the number of adherent cells was also calculated. There is no statistically significant difference between any intravenous fluid at different time point in this study. As a result, the 3 intravenous fluids may be equally suitable for delivering cMSCs.

Keywords: Adipose tissue, Canine, Intravenous fluid, Mesenchymal Stem Cells

PA-08

Heat Sock Protein 90 Regulates The Avian Reovirus P17 Protein Stability Enhancing P17-Induced Autophagy by Activation of The CK2/Cdc37/Hsp90/ NF-κB Pathway

Wei-Ru Huang¹, Ching-Dong Chang², and Hung-Jen Liu¹

¹Institute of Molecular Biology, National Chung Hsing University,

²Department of Veterinary Medicine, National Pingtung University of Science and Technology

This study provides novel insights on chaperone Hsp90 regulated stability of avian reovirus (ARV) p17 enhancing p17-induced autophagy via activation of the CK2/Cdc37/Hsp90/IKK/NF-κB pathway. In the present study, we demonstrate for the first time that the ARV p17 protein functions as a nucleoporin Tpr suppressor leading to p53 and p21 activation in Hela, B16-F10, and A549 cancer cells. In this work, Hsp90 protein co-immunoprecipitated with the p17 antibody was identified by 2D-LC/MS/MS. Co-immunoprecipitation assays revealed that the ARV p17 protein interacts with Hsp90 protein of Vero and B16F10 cells to stabilize IKK from polyubiquitylation and protein degradation, thereby activating NF-κB and inducing autophagy. Knockdown of Hsp90 and CK2 with 17-AAG and shRNAs inhibits NF-κB and downregulates Beclin1 and LC3-II in p17-transfected cells, indicating that the ARV p17 protein induces autophagy via activation of the CK2/Cdc37/Hsp90/IKK/NF-κB pathway. We also discovered that ARV p17 protein enhances Cdc37 phosphorylation at Ser13 by CK2 to promote Hsp90 activity. This regulation was reversed while CK2 knockdown with an shRNA. Furthermore, we found that Hsp90 interact with p17 and protects it from polyubiquitination and proteasome degradation. Inhibition of Hsp90 with 17-AAG reduced p17 and σC proteins of ARV.

Keywords: Avian reovirus p17 protein, nucleoporin Tpr, p53, p21, PTEN, Hsp90, NF-κB, Beclin1, autophagy

PA-09

16S rRNA gene-based Profiling of the Captive Dolphin Gut Microbiota

Chia-Chi Tung¹, Kun-Wei Chan ^1,2, Wei-Cheng Yang*^1,2

¹Department of Veterinary Medicine, National Chiayi University

²Veterinary Teaching Hospital - College of Veterinary Medicine, National Chiayi University

The gut microbiota of different animal species is directly affected by the source of ingestion, and plays an important role towards host health and nutrition. In the past, people have had successfully understand the anatomy of cetacean, however, the composition of the alimentary bacterial flora is less discussed; and yet, to us acknowledge, there’s no discussion between the relationship of gut microbiota and the individual health status till present. Fecal microbiota of 8 captured dolphins (1 female Grampus griseus and 7 Tursiops truncatus) in Taiwan by high throughput sequencing of the V1 - V3 region of the 16S rRNA gene. This study aimed to reveal the microbial diversity in the fecal samples of captive dolphins using the 16S rRNA sequencing analysis on the Illumina MiSeq platform. Thirty-five samples collected from 8 captured dolphins of 2 species including 1 female Risso's dolphin (Grampus griseus), and 7 bottlenose dolphins (Tursiops truncatus) living in a controlled environment from Oct 2014 to April 2015. Amount the bottlenose dolphins, 1 male and 6 females, two of the female individual were pregnant during sampling. Operational taxonomic unit (OTU) clustering was performed by identifying an OTU at 97% sequence identity. The alpha and beta diversities were applied to estimate the differences in microbial diversity among the 35 fecal samples. Totally, 111,850 and 3139 OTUs in the fecal samples were identified. The majority of bactreria were generally dominated by phylum Proteobacteria, Fusobacteria and Firmicutes in the 35 samples. By contrast, the degree of change in microbiotas showed no significant relationship with species, gender, pregnancy, and month. Then we compared the health log and the intestinal bacterial flora, hope to understand the “healthy” gut microbiota of capture dolphins, that the observation could be used in dolphin health care, monitoring of physiology status, or contribute to disease prognosis of veterinarian.

keywords : Next Generation Sequencing, 16S rRNA gene, cetacean, fecal microbiota,

PB:壁報展示非競賽組

Poster Presentation for Non-Competition

PB: 壁報展示非競賽組(續)

Poster Presentation for Non-Competition (continued)

PB-01

Evaluate the Potency of Ganoderma lucidum Polysaccharide Extract as an Animal Vaccine Adjuvant

Wei-Hao Xia, Li-Ting Cheng

Graduate Institute of Animal vaccine technology, National Pingtung University of science and technology

Ganoderma lucidum has since been considered to enhance resistance, and modern science has also confirmed that Ganoderma lucidum has anti-tumor and immune regulation of activity. Ganoderma lucidum polysaccharide extract (G. lucidum) has been extensively studied as an immunomodulator and it can cause a strong immune response. In this study, the G. lucidum as an animal vaccine adjuvant to mice as a model for In vitro and In vivo experiments will be investigated. In vitro, the mouse macrophages (RAW 264.7) were stimulated with G. lucidum to evaluate the effects of nitric oxide and cytokines (TFN-𝛂, IL-12, IL-6) produced by mouse macrophages. In the In vitro results, the G. lucidum induced macrophages to produce nitric oxide and stimulate the production of TNF-α, IFN-𝛄. In the future, mice will be immunized with BSA as antigen and G. lucidum as adjuvant. The effects of antibody, cytokine and T cell proliferation were analyzed by ELISA, Real-time PCR and T cell proliferation assay. The results of this study can be used as a basis for the development of G. lucidum adjuvant, and should be applied to other animal vaccine adjuvant.

Keywords: Ganoderma lucidum, RAW 264.7, Polysaccharide, Nitrite Oxide, Cytokines

PB-02

Challenge Model for Porcine Reproductive and Respiratory Syndrome Virus

Yu-Han Jheng^{1, 2}, Chun-Yu Lin¹, Yi-Hsin Lin¹, Yu-Lang Liu¹, Wen-Bin Chung^{1, 2}

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

²Animal Disease Diagnostic Center, College of Veterinary Medicine, National Pingtung University of Science and Technology

Porcine reproductive and respiratory syndrome (PRRS) is categorized as one of the highly contagious and economically damaging swine diseases. In year 1991, the first reported outbreak emerged and caused heavy economic losses among the swine farms in Taiwan. Pigs naturally infected or experimentally challenged with PRRS virus (PRRSV) have been shown the characteristics of persistent infection. This disease became the major cause of death and culling of pigs in swine industry. The purpose of this experiment is to establish a PRRSV inoculation mode in pigs. Immunological and pathological changes in pigs of PRRSV were studied. A Taiwanese PRRSV strain (HY-01) had successfully isolated from sick pigs and sub-cultured to the 14th generation. Viral quantitative real time polymerase chain reaction (Real-time PCR), serum viral neutralization test and enzyme-linked immunosorbent assay (ELISA) were established and applied to the serial challenge studies. No apparent clinical signs and pathological changes were observed in pigs after challenged with the 14th passaged strain. However, typical clinical signs and histopathological changes were observed in pigs challenged with the inocula prepared from first passage of PRRSV in alveolar macrophages. The PRRSV challenge model was successfully established.

Keywords：PRRSV, inoculation tests, challenge experiment

PB-03

Study of Avian Bornavirus of Parrot in Taiwan

Wan-Peng Lee, Ching-Dong Chang, Shyh-Shyan Liu, Hung-Yi Wu

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

Disease Investigation of parrot rarely mentioned to proventricular dilatation disease (PDD) caused by avian bornaviruses. In this paper, we major diagnose and investigate presenting PDD parrot kept in Taiwan. Our laboratory examination including RT-PCR, necropsy and histopathologic examinations. Currently, presenting positive cases have Guaruba guarouba, Ara ararauna, Cockatoo, Psittacus erithacus, and Eclectus roratus, and these cases all have PDD. Not only Avian bornavirus, but also many factors will cause like PDD symptom, so have to do differential diagnosis. Currently, it already having diagnosing PDD in parrot technique in Taiwan. In future, the technique can be used to diagnose if poultry, waterfowl and endemic species birds in Taiwan also infected Avian bornaviruses.

Keywords: parrot, proventricular dilatation disease, Avian bornaviruses

PB-04

Established a Macrophage-mediated Phagocytosis Assay to Evaluate the Effects of Mycoplasma hyopneumoniae Subunit Vaccine-induced Antibody

Huei-Yu Lee, Ming-Wei Hsieh, Jyh-Perng Wang, Tzu-Ting Peng, Jiunn-Horng Lin, Zeng-Weng Chen*

Division of Animal Medicine, Agricultural Technology Research Institute

Mycoplasma hyopneumoniae (Mhp) is the etiological agent of enzootic pneumonia, a chronic respiratory disease characterized with high morbidity and low mortality. Currently, vaccination is the primary strategy to prevent this disease. Vaccine-induced specific antibodies can block the pathogen, but also as a opsonin to promote macrophage phagocytosis. The objective of the study was to evaluate the effects of Mhp vaccine-induced antibody on macrophage phagocytosis. We demonstrated the engulfment of FITC-labeled inactivated Mhp by porcine alveolar macrophages (PAM), and then revealed that the phagocytosis of PAM was most appropriate when treatment with 200 ng/ml Mhp for 30 minutes. Some recombinant antigen’s antibody, Mhp - 1 and Mhp - 6 were found to significantly increase the phagocytosis of PAM. This in vitro phagocytosis system can be used to select potential antigen as a Mhp subunit vaccine candidate.

Keywords: Mycoplasma hyopneumoniae, antibody, macrophage phagocytosis

PB-05

Safety and Efficacy Test of Bivalent Vaccine of Mycoplasma hyopneumoniae plus PCV2 in Mice

Ming-Wei Hsieh, Chin-Shu Chang, Weng-Zeng Huang, Jyh-Perng Wang, Zeng-Weng Chen and Jiunn-Horng Lin

Division of Animal Medicine, Agricultural Technology Research Institute

Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2) are both important infectious pathogens of pig production worldwide. Many research evidences have shown that vaccination is an effective way to control both diseases. In the present study, safety and efficacy test of bivalent vaccine of M. hyopneumoniae plus PCV2 was evaluated in mice. Thirty 4-week-old healthy female BALB/c mice were randomly assigned into three groups (10 mice/group). Mice were immunized with the bivalent vaccine by the intraperitoneal (IP) and subcutaneous (SC) routes. The sera of mice were collected and the anti-M. hyopneumoniae and anti-PCV2 IgG titers were determined by using ELISA and indirect hemagglutination assay (IHA). The results indicated that mice immunized with this bivalent vaccine showed no adverse side effect and can elicit good immune responses toward PCV2 and M. hyopneumoniae.

Keywords: Bivalent vaccine, Mycoplasma hyopneumoniae, PCV2

PB-06

Establishment of an Ectopic Xenograft Lung Cancer Model with Therapeutic Strategies in Mice

Shao-Wen Hung*, Jen-Wei Lin, Chia-Chi Chen, Hua-Wen Lin, Chia-Ying Lin, and Cheng-Yao Yang*

Division of Animal Resources, Animal Technology Laboratories, Agricultural Technology Research Institute

Lung cancer is the leading cause of cancer death worldwide. The major cause of treatment failure and mortality is cancer metastasis. Although these patients with cancer may be removed tumor via surgery and chemotherapy. Unfortunately, even after chemotherapy using the best drugs, the five-year survival rate for lung cancer is between 1% to 49% depend on the lung cancer stage. In the United States, lung cancer is the secondary commonly diagnosed cancer and the first leading cause of cancer death in male and female. In 2016, approximately 13,720 and 11,010 cases of lung cancer will be newly diagnosed in male and female, respectively and 9,710 and 7,340 lung cancer deaths will occur in male and female, respectively. Therefore, the establishment of the suitable lung cancer-bearing animal models and therapeutic strategies for development of more effective treatments for inhibition, not only of proliferation, but also of cancer metastasis is urgently needed. In this study, we presented the successful establishment of an ectopic xenograft lung cancer model with different therapeutic strategies with chemotherapeutic drugs as 5-FU or cisplatin in mice. We hope that the ectopic xenograft lung cancer model with therapeutic strategies with chemotherapeutic drugs will applied to the new anti-cancer drug research and development in the future.

Keywords: Animal model, Cancer, Establishment

*Corresponding author

PB-07

Establishment of Three Orthotopic Cancer Models in Mice: Prostate Cancer, Cervical Cancer, and Ovarian Cancer

Shao-Wen Hung*, Jen-Wei Lin, Chia-Chi Chen, Hua-Wen Lin, Chia-Ying Lin, and Cheng-Yao Yang*

Division of Animal Resources, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan

Cancer is a major public health problem worldwide. Although cancer death rates have been continuously declining for the past 2 decades, it is currently the second leading cause of death in the United States, and is expected to surpass heart diseases as the leading cause of death in the next few years. Overall, the risk of dying from cancer decreased by 22% between 1991 and 2011. In the United States, prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer death. In 2016, approximately 29,530 cases of prostate cancer will be newly diagnosed and 4,450 prostate cancer deaths will occur. In 2016, an estimated 2,290 new cases of invasive cervical cancer and 750 deaths are expected to occur. In addition, 1,370 deaths of ovarian cancer are expected to occur in 2016. Although these patients with cancer may be removed surgically and chemotherapy has generally been adopted after surgery. Unfortunately, even after chemotherapy using the best drugs currently available such as paclitaxel, cis-platinum analogues and doxorubicin. Therefore, the establishment of the suitable cancer-bearing animal models for development of more effective treatments for inhibition, not only of proliferation, but also of cancer metastasis is urgently needed. In this study, we presented the successful establishment of three orthotopic cancer models (prostate, cervical, and ovarian cancer) in mice and detection platform of cancer growth and metastasis. We hope that theses animal models and detection platform will applied to the new anti-cancer drug research and development in the future.

Keywords: Animal model, Cancer, Establishment

*Corresponding author

PB-08

Efficacy of the Mycoplasma hyorhinis Subunit Vaccine with Different Antigen Combination in SPF Pigs

Chiung-Wen Hsu, Cong-You Wu, Wen-Cheng Huang, Hang-Jr Wang, Hui-Jei Lin, Jiunn-Horng Lin, Zeng-Weng Chen, Jyh-Perng Wang *

Division of Animal Medicine, Agricultural Technology Research Institute

Mycoplasma hyorhinis is one of the causative agent of mycoplasmal pneumonia and also induces arthritis and polyserositis in affected pigs. The pathogen causes substantial economic losses to the pig industry. It is believed that use of vaccine can prevent infection of Mycoplasma hyorhinis. In this study, specific pathogen free (SPF) pigs were randomly assigned into vaccine and control groups. Then, vaccination-challenge trials were undertaken. Gross lesions, daily body weight gain and seroconversion were evaluated. The results indicated that pigs immunized with a subunit vaccine composed of three recombinant Mycoplasma hyorhinis antigens can effective reduce the clinical signs after challenged with Mycoplasma hyorhinis.

Keywords: Mycoplasma hyorhinis, Subunit vaccine, efficacy

PB-09

Efficacy of Porcine Reproductive and Respiratory Syndrome (PRRS) Subunit Vaccine in a Porcine Circovirus Type 2 (PCV2) Infected Farm

Tzu-Ting Peng, Huei-Yu Lee, Weng-Zeng Huang, Jyh-Perng Wang,

Jiunn-Horng Lin, Zeng-Weng Chen

Division of Animal Medicine, Agricultural Technology Research Institute, Taiwan

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered to be the major pathogen that cause porcine respiratory disease complex (PRDC) which increased economic losses and antimicrobial use. Vaccination is a common procedure to control the disease. However, there were some PRRSV vaccine failures in fields. The objectives of this study is to investigate the efficacy of a novel PRRS subunit vaccine in a PCV2 infected farm. The study was conducted in a farrow-finish farm. Sows were vaccinated twice with ATRI PRRS vaccine (N=9/group) before parturition and piglets were vaccinated at 3 and 6 weeks old. The results indicated the novel PRRS subunit vaccine can improve the average daily weight gain and survival rate in pig farm. Moreover, the efficacy of the ATRI PRRS vaccine wasn’t interfered by PCV2 infection and has a potential to control PCV2 infection and PRDC.

Keyword: Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV2) infection, Subunit vaccine

PB-10

Effects of Bidens pilosa on Animal Growth Performance in Mice and Broilers

Cheng-Ying Yang¹, Wen-Chin Yang², Chih-Lung Liang³ & Cicero Lee-Tian Chang^1,*  

¹Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University

²Agricultural Biotechnology Research Center, Academia Sinica

³Institute of Microbiology and Immunology, Chung Shan Medical University

Bidens pilosa (BP) is a common Asteraceae plant. In Chinese tradition, Bidens pilosa is an edible plant. It has been used as a food and medicine without noticeable side effects. We have found that BP can ameliorate body weight and adipogenesis in C57/B6 mice. We observed that BP can decreased the size of white adipose tissue and increased the function of brown adipose tissue. But it was different in broilers. When we used BP as feed additives to feed broiler chickens. We saw the chickens are bigger than control group and could for sales earlier. Even in the situation of coccidiosis, BP could prevent the broiler’s body weight loss. This will bring three advantages by shorten the feeding time, reduce feed consumption and reduce the risk of disease. In summary, BP can suppress adipogenesis and increased body weight in animals. BP may make our livestock industry and body fitness methods set off a revolution.

Keywords: Bidens pilosa, adipose, field trial

*Corresponding author

PB-11

Development of efficacious subunit vaccines of against Toxoplasmosis.

Wei Yun Fang, Chung Da Yang

College of veterinary Medicine Graduate Institute of Animal National Pingtung University of Science and Technology

Toxoplasmosis is one of the more common serious parasitic zoonoses world-wide. Its causative agent, Toxoplasma gondii, is a facultative heteroxenous, polyxenous protozoon that has developed several potential routes of transmission within and between different host species. Toxoplasmosis is likely to spread through the blood, tissue transplantation, contaminated drinking water, eating undercooked meat infected. The chemical anti-toxoplasmosis drugs are commonly used to control infection. Drug residue in meat also becomes a major threat affecting food safety. Although there is commercially available attenuated live vaccine to prevent the miscarriage of sheep, but the vaccine is still toxic to restore or pollute the environment concerns. In the study, we aim to develop an effective subunit vaccine against Toxoplasmosis. Toxoplasma tachyzoites surface antigens are currently the most promising protective antigens. The distribution of SAG1 and SAG2 on the surface of the tachyzoites is the highest and both antigens are related to the tachyzoites invasion of host cells. We have cloned the gene fragments of SAG1 of T. gondii into the pET24a plasmid and then expressed their recombinant proteins. These E. coli-based recombinant proteins will be prepared to evaluated their protective efficacy. We expect these recombinant proteins may be to develop as effective vaccines against T. gondii.

Keywords: Toxoplasma gondii , subunit vaccines, surface antigens

PB-12

The Values of Alpha-1-Acid Glycoprotein (fAGP) in Healthy Domestic Cats and Its Variation after Surgical Intervention

Chih-His Tsai¹, Yee-Von Tan¹, Lee-Shuan Lin^1,2, Cheng-Shu Chung^1,2

¹Department of Veterinary Medicine, National Pingtung University of Science and Technology
²Veterinary Medical Teaching Hospital, National Pingtung University of Science and Technology

Acute phase protein, Alpha 1-acid glycoprotein (AGP) which is a moderate acute phase protein in cats, increases in response to inflammation. However, limited reports describing its clinical values in cats and the values may be altered by environmental factors. Therefore, this study intended to evaluate the basic properties of AGP in cats in Taiwan. First, 40 cats, including 20 males and 20 females, were used for evaluating the AGP values in healthy cats. Further, 6 cats underwent OHE (n=3) and castration (n=3) were used for evaluating the variation of AGP after the surgical intervention. Plasma was collected for examination and the correlation between the concentration of plasma AGP and WBC was also evaluated. Results showed the AGP value in heathy female cats was less than 806.2mg/L, while AGP in male cats was less than 759.1mg/L. AGP values increased maximally after surgery approximately 1.9-2.6 folds on second day post-surgery. The variation of AGP through different spay procedures was similar. Moderate positive correlation between AGP and WBC was also noted. In conclusion, AGP values in healthy cats here was similar to previous studies, but for sterilized spay procedures induced mild injures, AGP may be less valuable to evaluate the recovery. Further studies were needed for other clinical applications.

Keywords: Acute phase protein, Alpha 1-acid glycoprotein, Cats

PB-13

Development an Inactivated Vaccine Against Edwardsiella tarda in Fish

Shih-Hua Liu, Chung-Da Yang,

Graduate Institute of Animal Vaccine Technology, National Pingtung University of Science & Technology

Edwardsiella tarda has been recognized as one of the important bacterial pathogens to the cultured freshwater and marine fish worldwide. Infection with E. tarda often leads to the development of a systematic disease called Edwardsiellosis, characterized by symptoms of anal swelling, hemorrhagic, exophthalmia, liver and kidney severe lesions of internal organs. Since E. tarda is an intracellular pathogen, most antibiotics are not effective for this disease. Although development of immunizing agents is being studied, effective preparations are not currently available. In this study, the E. tarda strain of the Food Industry Research and Development Institute was used as a vaccine candidate strain, and the antigen was prepared by formalin inactivation and treated with sodium alginate as a microparticles vaccine. A virulence test was performed using wild plants as a virus for the attack and a Median Lethal Dose (LD₅₀) was calculated. In the study, Biodegradable and biocompatible alginate gels have been used as safe, potent adjuvants or delivery systems to encapsulate antigens for preparing controlled-release microparticle vaccines. The purpose of this work is to prepare alginate microparticles that can sustain stable release of proteins of inactivated E. tarda. The morphology, size, entrapment efficiency, loading level and release rate of alginate microparticles will be studied. The humoral and cell-mediated responses will be measured to evaluate anti-E. tarda protection in fish. We anticipate that the alginate microparticles capable of sustaining release of E. tarda proteins can be employed to develop effective microparticle vaccine against E. tarda.

Keywords: Edwardseilla tarda, inactivated vaccine, LD₅₀, Alginate microparticles

PB-14

Residue Depletion Study of Oxolinic Acid in Cobia (Rachycentron canadum)

Re-Shang Chen^1,2, Yi-Jing Syue³, Shi-Yuan Sheu^4,5, Jiann-Hsiung Wang^3,*, Chung-Hsi Chou^1,*, Tzong-Fu Kuo^6,*

¹ School of Veterinary Medicine, National Taiwan University

² Animal Health Research Institute, Council of Agriculture, Executive Yuan

³ Graduate Institute of Veterinary Medicine, National Chiayi University

⁴ School of Medicine, Chung Shan Medical University

⁵Department of Integrated Chinese and Western Medicine, Chung Shan Medical University Hospital

⁶ Department of Post-Baccalaureate Veterinary Medicine, Asia University

The experiments were performed to establish an adequate depletion period of oxolinic acid (OA) in cobia by determining the residues of oxolinic acid post feeding with multiple doses. The dosages of OA were applied via oral administration at the dose of 30 and 60 mg/kg body weight for 5 consecutive days. The drug concentration was measured on 1, 3, 5, 7, 10, 14, 21th days post-treatment. Those samples were taken to analyze OA concentrations by using liquid chromatograph/tandem mass spectrometer (LC/MS/MS) in multiple reaction monitoring (MRM) mode. This method showed that the quantitation limit (LOQ) of 0.01 μg/g and that the mean recovery of the method was 95.8% in muscle and 86.55% in liver. The liner regression equation of signals and drug concentrations were Ŷ = 115139.57 x + 455100.54 (R² = 0.9989) in spiked muscle and Ŷ = 25239.75 x – 37040.26 (R² = 0.9992) in spiked liver of cobia. After the 30 mg/kg dose, muscle and liver samples at 24 h following termination of medication had a peaked mean (n = 5) concentrations of 0.06 ± 0.01 and 0.06 ± 0.02 μg/g, respectively; while for 60 mg/kg dose the peak mean concentrations were 0.20 ± 0.05 μg/g and 0.26 ± 0.11 μg/g, respectively. Based on the maximum residue level (MRL) of 0.05 μg/g in edible tissue for fin fish, the withdrawal times of OA in muscle and liver were estimated to fall below the MRL after a withdrawal period of 15 days following the multiple-dose administration. These results may be helpful to regulatory agencies as they determine what tissues to be monitored to ensure that the established residue safety tolerance levels are not exceeded.

Keywords: Oxolinic acid, Residues, Cobia

* These authors contributed equally to this work

PB-15

The Effects of Low-Level-Laser on Canine Platelet-Rich-Plasma

Ping-Tzu Wu¹, Ho-Lin Hsieh¹, Cheng-Shu Chung^1,2, Lee-Shuan Lin^1,2

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

²Veterinary Medical Teaching Hospital, National Pingtung University of Science and Technology

Platelet-rich plasma (PRP) is an activated autologous product that aggregates and concentrates platelets in a small volume of plasma. Healing process can be stimulated by growth factor released by PRP. Low-level laser therapy (LLLT) had showed various biostimulatory effects, however, the molecular mechanism associated with the stimulatory effects of LLLT has not been fully clarified. According to previous study, intracellular ATP and calcium (Ca²⁺) concentration were proven to be elevated under the effect of LLLT and calcium signaling plays a major role in platelet activation and aggregation. Therefore, the purpose of the study is to determine the relation between calcium concentration and platelet activity, and additionally, the effects of LLLT on activation of PRP. PRP was prepared from 4 adult beagles and stimulated by different procedures afterwards: PRP+54J laser+CaCl₂; PRP+CaCl₂+54J laser; PRP+DD water; PRP+CaCl₂. ELISA kit was used to quantify PDGF-BB levels of activated PRP. Furthermore, spectrophotometer was utilized for intracellular calcium level detection. Lastly, Friedman test was applied for statistical analysis. The results indicated that the intracellular Ca²⁺ concentration does not increase significantly with stand-alone application of LLLT only, while combination of LLLT and CaCl₂ activated PRP can enhance the releasing of growth factor significantly. The enhancement is achieved by indicating LLLT followed by CaCl₂, but not vice versa in sequence.

Keywords: CaCl₂, Calcium, LLLT, PDGF-BB, PRP

PB-16

Effects of Low-Level Laser Irradiation on the Proliferation of Canine Adipose-derived Mesenchymal Stem Cells

Ming-Sam Chih¹, Lee-Shuan Lin^1,2, Cheng-Shu Chung^1,2

¹Department of Veterinary Medicine, National Pingtung University of Science and Technology
²Veterinary Medical Teaching Hospital, National Pingtung University of Science and Technology

Due to the limited amount and growth rate of canine mesenchymal stem cells, there are obstacles when it comes to clinical applications. Low-level laser irradiation has been used in improving the proliferation of variable kinds of cells. Therefore, this study was going to evaluate the effects of low-level laser irradiation on the proliferation of canine adipose-derives mesenchymal stem cells (cASCs). Greater omentum fat tissue was harvested from three dogs. After the isolation of cASCs, the cells were used here until passage two. cASCs were divided into three groups, group A was irradiated with a dose of 0.5 J/ cm², B group was irradiated with a dose of 1.0 J/ cm², and C group didn’t receive any laser irradiation. Laser irradiation was given at 0 and 48 hours. At 96 hours, the cell’s morphology was analyzed and the trypan blue assay was used to evaluate the viability and growth rate. Results revealed that there were no morphological alternations observed among groups. After receiving laser irradiation, group A and B have higher cell growth rate compared to group C, and there were no significant differences between group A and B. According to the result, low-level laser irradiation stimulated the proliferation of cASCs and may decrease the waiting time during autogenous stem cell therapy, however higher laser doses didn’t improve the effect. More, the healing effect of low-level laser therapy might be resulted from the proliferation of stem cells dispersed among the surrounding tissue.

Keywords: Adipose-derived mesenchymal stem cells, Canine, Cell proliferation, Low-level laser irradiation

PB-17

The Analysis of Gene Expression of Immature Feline Kidney

Bing-Jhen Jhuang¹, Lee-Shuan Lin^1,2, Cheng-Shu Chung^1,2

¹Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan
²Veterinary Medical Teaching Hospital, National Pingtung University of Science and Technology, Pingtung, Taiwan

Although feline acute and chronic kidney diseases have high prevalence in clinical practice, the ordinary therapies still can’t restore the renal function effectively. Recently, the novel therapies like regenerative medicine, stem cell-based therapies have been proved to have many benefits to kidney, like anti-inflammatory, pro-angiogenic, anti-apoptotic, immune-modulatory, and to differentiate into kidney cells. The embryo-developmental gene expression is commonly used as a roadmap for most differentiation protocols, but only a few related researches on cats. For these reasons, the purpose of this research is to evaluate the gene expression of feline immature kidney. A 30-day feline fetus was used in this study. The kidney of fetus was minced and homogenized. After that, mRNA was extracted and cDNA was synthesized with commercial kit. Further, real-time PCR was used to detect the expression of genes in different stages, immature stage: SOX2, NANOG, OCT4; intermediate stage: T, SIX2, OSR1, EYA1, WT1; and mature stage: AQP1, SLC12A3. Relative quantification, ΔCt method was used to analyze the results. Results showed that all the genes we selected were expressed by real-time PCR. This is a pilot study to identify the gene expression of feline immature renal tissues and it could contribute to establish the roadmap of gene expression during the development of feline kidney. 

Keywords: Cat, Gene expression, Immature, Kidney,

PB-18

Serologic Investigation of Foot-and-Mouth Disease in Cattle and Goats in Kinmen in 2016

Ming-Chang Li^1,2, Shih-Ping Chen¹, Yeou-Liang Lin³, Hsiang-Jung Tsai²

¹ Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute.

² Department of Veterinary Medicine, National Taiwan University

³ Animal Health Research Institute, Council of Agriculture, Executive Yuan

On 27 January 2012, a Foot-and-Mouth disease (FMD) outbreak with serotype O SEA was confirmed in pigs but cattle and goats not included in Kinmen. However, this strain of FMD virus was found from pigs and ruminants in Korea and Japan in 2010. Unfortunately, another Foot-and-Mouth disease (FMD) outbreak with serotype A in cattle in Kinmen was notified to OIE on 8 May 2015. In order to continue evaluate the impact of those FMD outbreak for cattle and goats, total of 1,589 sera collected from 81 farms of cattle and goats were surveyed during 2016. Sera were tested by ELISA for detecting non-structure protein (NSP) specific antibodies. Six of 1,385 sera (0.43%) collected from 61 cattle farms were positive. One of 204 sera (0.49%) collected from 20 goat farms was shown positive. The serological results suggested FMD virus might have infected the cattle and goats in Kinmen. However, contrast to our serologic data, FMD virus has never been detected from the oesophagopharyngeal fluid by virus isolation and polymerase chain reactions. Meanwhile, there are no clinical observations found in cattle and goats. The comparative findings indicated FMD viruses not circulated in cattle and goats even though the very few of positive serological evidences found in cattle and goats.

Keywords: FMD, NSP, Kinmen

PB-19

Comparison of Rodenticides Used to Control the Field Rodents in Southern Taiwan

Li-Syuan Wu¹, Kun-Wei Chan ¹, Ming-Jeng Pan², Jui-Te Wu¹, Shih-Jen Chou¹, and Chuen-Fu Lin*¹

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

² Department of Optometry, Central Taiwan University of Sciences and Technology, Taichung, Taiwan

Field rodent is one of important carrier in zoonosis. Besides, Tainan County is agricultural center in Taiwan, there are many farms and variety of crops can support suitable environments for field rodents. Therefore rodenticides are important to eradicate rodents to reduce crops damage. Anticoagulant rodenticides resistance of field rodents is an emerging problem whose spread is constantly increasing. Brodifacoum^® is the traditional anticoagulant rodenticide to eradicate field rodents, but not ecologically safe for carnivores and human. However, the non-anticoagulant rodenticide, EradiRat^®, is a kind of cellulose-based bait which is harmless to other animals except for field rodents. Consequently, we selected these two different kinds of rodenticides to evaluate the efficiency of control field rodents at the crops in Tainan County. Results could be assumed that the EradiRat^® was extremely unpalatable for field rodents causing only 13% control rate. In contrast, Brodifacoum^® got 91 % control rates for field rodents before plant crops.

Keywords: rodenticides, EradiRat^® , Brodifacoum^®, field rodents

PB-20

Awareness of Veterinary Medical Dispute Risks in Veterinarians and Clients - A Preliminary Study

Zih-Fang Chen, Chung-Hsi JiuJiu Chou¹, Yi-Hsin Elsa Hsu², Jih-Jong Jack Lee¹

¹ School of Veterinary Medicine National Taiwan University

² School of Health Care Administration, Taipei Medical University

In an increasingly litigious society, there are numerous cases of serious medical dispute problems to physicians and veterinarians. This study is expected to explore the awareness of companion animal’s medical dispute factors in veterinarians and clients. The questionnaires are developed by 10 Taiwanese anonymous participants including veterinarians, professors, and clients. The validity and reliability tested questionnaires used Likert-scale scoring from 1 to 5, where higher score presents more risk to induce dispute. A total of 160 participants were included in this study where, 77 were clients, 69 were veterinarians and 14 were veterinary medicine students. Two of the possible causes leading to animal medical related disputes or customer complaints, “ms1: misdiagnosis leading to worsening of patient condition” and “ms2: inappropriate/improper hospital care or treatment procedures”, has a decreasing significance (p<0.05) from students to veterinarians. From students to veterinarians, there are changes in the possible causes leading to disputes or complaints, such as clients’ sticking to subjective perception and lack of trust instead of the risk of misdiagnosis and improper treatment procedures. Besides, the resolution of clients’ suggestions have significantly (p<0.05) higher score than veterinarians’, those are “complaints to be quickly resolved before they become worse”, “educating the veterinarian to have empathy towards their patient and their clients”, and “use of checklist during treatment as precaution to avoid negligence.” Therefore, medical skills and informed consenting may not be considered as the main preventions from disputes or complaints, on the contrary, communication skills such as showing more empathy, clarifying clients’ subjective perception, and dealing with complaints immediately seems to be considered more seriously.

Keywords: veterinary medical disputes, risk factors, questionnaire study

PB-21

Renal Chymase Alteration of the Kidney Injury Induced by Aristolochic Acid in Angiotensin Converting Enzyme II Knockout (ACE2 KO) Mice

Ching-Chang Cho ¹, I-Hua Tseng ¹, Hsi-Tien Wu ², Wan-Hsuan Chuang ³, Wen-Yeh Hsieh ³, Chih-Sheng Lin ¹

¹ Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan

² Department of BioAgricultural Science, National Chia Yi University, Chiayi, Taiwan

³ Department of Internal Medicine, Hsinchu Mackay Memorial Hospital, Hsinchu, Taiwan

Angiotensin converting enzyme (ACE) and angiotensin converting enzyme II (ACE2) are the primary enzyme to convert angiotensin I (Ang I) to angiotensin II (Ang II) and convert Ang II to angiotensin-(1-7) (Ang-(1-7)) in renin angiotensin system (RAS). Chymase also plays an important role to degrade Ang I to Ang II in the progression of kidney disease. The aim of this study was to know whether chymase may play a critical role in aristolochic acid I (AAI)-induced nephropathy under the condition of ACE2 deficiency. Male ACE2 KO mice were used for the study. The nephropathy was induced by AAI and sacrificed 2 weeks and 4 weeks later for nephropathy development, and urine, blood and kidney tissues were sampled for biochemical and pathological assays. Serum ACE and chymase levels were detected using fluorogenic substrates assay. Histological analyses were determined by H&E and PAS staining of renal tissue.

AAI-treated mice decreased body weight, increased serum creatinine and urea nitrogen levels, and increased urine creatinine, protein and albumin levels. The physiological and biochemical determinations indicated that the nephropathy is induced in the mice treated with AAI. In the renal tissue sections, high amount of inflammatory cells and much fibrosis in the interstitial tissue were observed. It was found that a significantly increased chymase activity in the kidney tissues of AAI-treated mice, but the ACE activity did not show significant changes. The results hint that renal chymase might play an important role of ACE independent Ang II generation in the nephropathy progression induced by AAI.

Keywords: Angiotensin converting enzyme, Angiotensin converting enzyme II, Aristolochic acid I, Chymase, Nephropathy, Renin angiotensin system

PB-22

Virus replication, pathological change and tissue tropism related to the attenuation of infectious bronchitis virus TW2575/98 in chicken embryos

Cheng-Ta Tsai¹, Ming-Chang Lee², Hue-Ying Chiou³ and Ching-Ho Wang¹

¹School of Veterinary Medicine, National Taiwan University

²Division of Animal Industry, Agricultural Technology Research Institute

³Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University

An attenuated Infectious bronchitis virus vaccine strain TW2575/98 was successfully developed using 75 serial passages in embryonated eggs. Pathological change, tissue tropism and dwarfing effect on embryos or early death caused by the attenuated vaccine strain are discussed in this study. The attenuated vaccine strain exhibited better proliferation efficiency than the wild strain. The histopathological changes in embryos were not significantly different between the two strains, which included lung edema, epithelium damage of parabronchus with cell infiltration, and focal necrosis without inflammatory cell infiltration but with increased numbers of hematopoietic cells around the portal vein of the liver. The liver lesion in embryos challenged with vaccine strain was almost not found due to early death of embryos. The immunohistochemistry results revealed that the target sites of both viruses were mainly located at the epithelium of the chorionallantoic membrane, lung parabronchus and renal tubules and some in spleen and heart serosa. The positive rate of infection from the vaccine strain in the kidney was significantly lower than that of wild strains due to early death. Thus, the dwarfing effect is related to the virus growth titers but not to the tissue tropism in this study.

Keywords: infectious bronchitis, virus replication, pathological change, tissue tropism, dwarfing effect

PB-23

The Interleukin 6 Receptor of Red-Spotted Grouper (Epinephelus coioides) Expressed in Various Organs Under Different Immunestimultant Treatment

Ting-Yu Chuang¹, Chieh-Hung Su², John Han You Lin^1,2

¹ School of Veterinary Medicine, National Taiwan University

²Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University

Interleukin-6 (IL-6) has been proved various effects on innate immunity and adaptive immunity in previous studies. Activation of IL-6 signaling needs combination between target cell and its receptors such as IL-6R and gp130. It’s known that IL-6, gp130 and IL-6R will aggregate as a complex which turns on the signaling transduction by phosphorylated molecules such as STAT-3. There are two signaling pathways: trans-signaling and classic-signaling pathway which due to combination between soluble or membrane-bound form of IL-6R. IL-6 receptor producing cells that were primarily derived from immune cells and gp130 only cells that almost expressed in all cells are participated in these two IL-6 signaling pathways. It’s still remained unclear between these two IL-6 signaling induced immune responses in mammalian, not to mention in teleost. In this study, the various cell types expressed il6r among different immune organs will be figured out. And the mRNA level of bacterial induction last much longer than viral infection. The ligand boost may modulate IL-6 receptor expression. With lymphocyte cells aggregation, the IL-6 signaling will be reinforced if required. These results will be the cornerstones for studying teleost IL-6 trans-signaling and classic-signaling pathway mechanism in the future.

Keywords: Epinephelus coioides, interleukin-6 receptor, gene expression.

PB-24

The Pathogen Resistance Mechanism of Recombinant Interleukin 6 Treated Grouper (Epinephelus coioides)

Yi-Fan Fang¹, John Han You Lin^1,2

²Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University

² School of Veterinary Medicine, National Taiwan University

Innate immune system is important for the disease resistance of fish. The innate immunity of fish is divided in three parts: physical barriers, humoral and cellular components, which are common to resistance variety water-pathogen infection and maintain the healthy statement of fish. Interleukin-6 (IL-6) is a cytokine of the lymphocytes hormone (lymphokine), mainly induce lymphocytes to reaction. It plays important roles in the innate and acquired immunity. In the research of mammalian, IL-6 can induce acute phase proteins (APPs) and antimicrobial peptide genes expression as well as stimulated the macrophage and neutrophil activation and regulate the inflammatory cytokines expression in the innate immunity, then enhance the resistance to bacterial disease. Regarding to the acquired immunity, IL-6 promotes T cell and B cell differentiation and increases the antibody production. In teleost fish, the functions of IL-6 are not confirmed yet. Previous studies in our laboratory use Epinephelus coioides to be the animal model, confirmed that IL-6 induce the Th2 pathway in the acquired immunity and enhance antibody production. In my study, investigate the mechanism of IL-6 in innate immunity and evaluate its efficacy of disease resistance. In the current study, inject of recombinant IL-6 (rIL-6) regulate some inflammatory cytokine, antimicrobial peptide and other innate immunity related genes. Further, the antibacterial activity in serum by rIL-6 induction was found, that could reduce the bacterial number in the in vitro test, and enhance the phagocytosis of head kidney leukocytes. Finally, in the in vitro challenge test, fish injection of rIL-6 was higher survival in the Vibrio campbellii and Nervous Necrosis Virus challenge trials, and reduced the pathogen. In summary, grouper IL-6 was a molecular that could induce multiple innate immune reactions, which will help to resistant the pathogen invasion.

Keywords: Epinephelus coioides, grouper, interleukin-6, innate immunity, anti-microbial peptides, immune cells.