107 年度春季研討會 口頭論文競賽組 (OA)

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OA-01

Isolation and Characterization of Mycobacterium from Diseased Clownfish

Miguel Flemming1, Shih-Chu Chen1,2

1International Degree Program of Ornamental Fish Science and Technology, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan

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

The three species Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium chelonae have dominated the literature on reports of Mycobacterium spp. isolated from fish. Mycobacterium is a gram-negative acid fast bacilli which seems almost impossible to eradicate and can contribute to severe economic loss. Mycobacterium is one of the main bacterial species isolated from ornamental fish. Amphiprion spp. is a favorite in the ornamental fish industry due to their beauty, easy management and reproduction in captivity. There have been no reports on Mycobacterium isolated from clownfish but this study revealed that Mycobacterium is a disease-causing agent in clownfish. In this study, two Mycobacterium species (Mycobacterium marinum and Mycobacterium fortuitum) were isolated from 37 cases of diseased clownfish. The isolated species were characterized morphologically (photochromogenecy), phenotypically (growth on the media) and biochemically (nitrate, urease, catalase). Löwenstein–Jensen medium was used for isolation and genus and species were identified using acid fast staining and sequencing of the 16S rRNA, rpoB and Heat shock protein genes. DNA sequencing and analysis followed by an NCBI-BLAST search comparison revealed that 77% of the isolates were Mycobacterium marinum and 23% were Mycobacterium fortuitum. Morphological results indicated that the Mycobacterium marinum isolates are photochromogenic while Mycobacterium fortuitum isolates were nonphotochromogenic. Physiological analysis shows that most isolates were able to grow on other medium and all isolates were catalase positive. Mycobacterium fortuitum is a fast-growing species and this was observed during the culture process and Mycobacterium marinum grew slower. These results are similar to previous reported studies and are all important practices for bacteria characterization. This study proves that Mycobacterium infection is a threat to clownfish and fish hobbyists must take precautions during handling to prevent infection.

Keywords: Mycobacterium, characterization, clownfish, photochromogenecy

 

OA-02

Development of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Vaccine

Chia-Hsin Liu1, Kuo-Pin Chuang1

1Graduate Institute of Animal Vaccine Technology, College of Veterinary Medicine, Nation Pingtung University of Science & Technology, Pingtung, Taiwan

The porcine reproductive and respiratory syndrome virus (PRRSV) causes acute respiratory disease and persistent infection in piglets and reproductive failure in pregnant sows, which can lead to serious economic losses to the swine industry worldwide. PRRSV-infected pigs develop a delayed appearance of neutralizing antibodies and a weak cell-mediated immune response. The heterodimer of glycoprotein (GP5) and non-glycosylated matrix protein (M) are the leading targets for the development of new generation of vaccines against PRRSV infection. It has been hypothesized that the formation of heterodimers of GP5 and M proteins may play a critical role in assembly of infectious PRRSV. Many studies have been corroborated that co-expressing GP5 and M proteins as a fusion protein trigger better immunogenicity than that expressing GP5 or M protein alone. The GP5/M protein fused with porcine granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrated that GM-CSF fused with GP5 and M protein of PRRSV could significantly enhance the humoral and cellular immune responses and provide protection against PRRSV challenge in pigs. Adenoviruses are often used as vectors for gene therapy and foreign gene expression due to the reason that the adenovirus vector has the advantages of easy culture to high titer, high transduction efficiency in splitting and non-dividing cells and induce great T cell immunity response. The recombinant adenovirus and GP5/M-GM-CSF recombinant protein were produced by HEK293pTP mammalian cells using the AdEasy system as vaccine. We successfully recombined the target fragment GP5/M-GM-CSF gene into BJ5183 bacteria. The recombinant vector was also transfected into HEK293pTP cells, and significant fluorescence was observed under fluorescence microscopy. In addition, Confirmation of protein results by western blotting. The vaccine efficacy will be revealed by animal testing via subcutaneous injection and intranasal immunization. Moreover, the immunoassay was tested by ELISA and neutralizing antibodies test.

Keyword: Porcine reproductive and respiratory syndrome virus (PRRSV), Recombination adenoviruses, AdEasy system, Mucosal immunity

 

OA-03

Efficacy Study of Virus-like Particles Vaccine for Pigeon Circovirus

Ching-Yi Tsai1, Yen-Lin Chuang1, Hsin-Yi Tsai1, Kuo-Pin Chuang1

1Graduate Institute of Animal Vaccine Technology, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

Pigeon Circovirus (PiCV) has been reported worldwide ever since it was reported in the 1980s. The virus attacks the pigeons' immune system specifically the bursa, which can cause immunosuppression and subsequently lead to secondary bacterial, viral or protozoan infections. PiCV infects young and old pigeons, but easily found in four to six months old pigeons therefore it is also known as young pigeon disease syndrome (YPDS). Virus-like particles (VLPs) are produced by the self-assembly of structural proteins of viruses. It is similar to natural viruses but without the viral genome. Compared with the traditional subunit vaccine, the VLP vaccine can be more effectively recognized by the antigen-presenting cells and induce a stronger immune response. In this study, the PiCV capsid protein gene was cloned into the pET32a and pGS21a vectors. Escherichia coli expression system was used to produce the recombinant PiCV capsid protein. The capsid protein will assemble into VLPs. Following purification, western blot analysis was used to confirm the antigenicity. Then, vaccine was injected in pigeons via subcutaneous injection.

Keyword: Pigeon Circovirus, capsid protein, virus-like particles, prokaryotic expression system

 

OA-04

Evaluation the Efficacy of Swine Multivalent Subunit Vaccine

CI SHUN Whang1, Chun Yen Chu1

1Graduate Institute of Animal Vaccine Technology, National Pingtung University of Science & Technology, Pingtung, Taiwan

Pasteurella multocida (P. multocida) can infect 8-12-week piglet, and mainly causes atrophic rhinitis (AR). Pasteurella multocida toxin (PMT) is an important virulence factor. Streptococcus suis (S. suis) causes meningitis, arthritis, septicemia in 4-12-week piglet. The Surface antigen one (Sao) is a cell wall surface-associated protein, and is found in all serotypes of S. suis, can induce a good immune response. Erysipelothrix rhusiopathiae (E. rhusiopathiae) can infect all age pigs causing acute septicemia or chronic disease. The Surface protective antigen A (SpaA) not only induce a good protective antibody response, but also provide cross-protecting against other serotypes. We produce PMT, Sao and SpaA recombinant protein with prokaryotic expression system and antigen purification. Then combine with various adjuvant to evaluate the immune responses. The preliminary result indicated PMT, Sao and SpaA recombinant protein have been purified, the antigenicity was confirmed by Western blotting and combined with ISA206 and CpG ODN to make multivalent vaccines to immune piglet. The immunological methods, enzyme-linked immunosorbent assay, cytokine profile and T cell proliferation will be employed to evaluate the efficacy multivalent subunit vaccines.

Keywords: Pasteurella multocida, Streptococcus suis, Erysipelothrix rhusiopathiae, multivalent, subunit

 

OA-05

Identification and Genetic Characterization of Porcine Circovirus Type 3 in Taiwan

Jia-Yun Zeng 1, Chao-Nan Lin 1,2, Ming-Tang Chiou 1,2

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

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

Porcine circovirus (PCV) belongs to the Circoviridae family, and Circovirus genus. It is a single strand circular DNA virus. So far porcine circovirus type 2 (PCV2) has caused great economic losses in the pork industry worldwide. Recently, there had been some cases of porcine dermatitis and nephropathy syndrome (PDNS) and Reproductive failure (RF) that reported to be PCV2 negative. Within these cases, a new type of PCV was detected by histopathological and molecular biological tests, and it was named Porcine Circovirus type 3 (PCV3). There is limited information about PCV3 infection in Taiwan. Thus, the aim of this study was to analyze PCV3 in Taiwan. A total of 1712 specimens from nursery pigs, sow, and were collected from January 2015 to March 2018. The total specimens of PCV3 positive rate were 7.4% (85/1195) of nursery pigs, 3.1% (3/94) of sows and 4.0% (17/423) abortion fetus, respectively. These results confirmed the presence of PCV3 in Taiwan.

Keywords: Porcine circovirus 3, PCR, Porcine circovirus associated disease

 

OA-06

Evaluation of Immunoregulatory Efficacy of Different Immunization Strategies in Riemerella anatipestifer with Different Type Vaccine

Wan-Chen Chang1, Chun-Yen Chu1

1Graduate Institute of Animal Vaccine Technology, National Pingtung University of Science & Technology

Riemerella anatipestifer (Ra) is the most susceptible to the infection and high mortality in goslings and ducklings. Ra is a Gram-negative bacillus, and at least twenty-one serotypes. Ra causes septicemia and infectious serositis. The infection of Ra causes sneezing, cough, greenish diarrhea, and trembling of the head and neck. Nowadays, commercial vaccines that are used inactivated trivalent bacterins, which can’t provide an effective cross-protection for other serotypes. Research indicates that outer membrane protein A (OmpA) of Ra is the most cross-reactive antigen and cross-serotype of vaccine development. In our research, we expressed recombinant OmpA (rOmpA) by the prokaryotic expression system, cloned the OmpA gene into pTCY vector, and both combine with CpG ODN adjuvant effectively elicit the immune response. The objective of this study will produce DNA vaccine, subunit vaccine with CpG ODN, and commercial inactivated vaccines used the Prime-Boost immunization in duck. Detection antibody titer of ELISA, cytokine expression and percentage of CD4+/CD8+ cells to explore the most suitable combination for Prime-Boost immunization.

Keywords: Riemerella anatipestifer, subunit vaccine, DNA vaccine, CpG ODN

 

 

OA-07

Effect of Different Level of Maternal Antibodies on Haemophilus parasuis Infection

Chia-Liang Hung1,2, Chao-Nan Lin1,2, Ming-Tang Chiou1,2

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

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

Haemophilus parasuis (Hps) is a commensal organism of the upper respiratory tract of pigs that causes Glässer’s disease. Disease prevalence may modulate by concomitant stressors and maternal antibody titers. This work studied the impact of maternal antibodies and cortisol concentration in the infection of Hps on piglets. Piglets were randomly selected from high and low ELISA Hps antibody titer sows. Nasal swabs and blood samples were obtained from the piglets at 1, 3, 6, 9 and 13 weeks of age as piglet mortality rate was recorded. Sows were sampled on the 1 week after farrowing, including nasal and blood samples. Hps load in nasal swab and specific Hps antibody titers were evaluated by qPCR and ELISA, respectively. In case of pigs died during the late nursery stages, necropsies were performed. The weak and healthy piglets saliva samples in both group were determined the cortisol level. Hps load in nasal swab of piglets showed increase gradually at 1 to 9 weeks of age in both groups. Piglets from high group had statistically significant higher levels of antibody titers than low ones in all time points. Mortality rate of high levels of antibody titers group was significantly lower than low one. At necropsy, most of pigs were diagnosed with Glässer’s disease. The cortisol concentration from weak piglets were higher than healthy group. These results showed that high level of maternal antibody titers delay infection by Hps and decrease mortality rate, but do not prevent Hps colonization in the nasal cavity of piglets.

Keywords: Haemophilus parasuis, maternal antibody, cortisol, Hps- qPCR

 

OA-08

The Preparation of Activated Canine Platelet-Rich Plasma by Low-Level Laser and Calcium Chloride

Yi-Fang Wei1, Cheng-Shu Chung1,2, Lee-Shuan Lin1,2

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

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

Platelet-rich plasma (PRP) is principally an increased concentration of autologous platelets suspended in anticoagulant blood after centrifugation. Platelets not only play an important role in hemostasis but are rich in growth factors. It was reported that the treatment effect is related to preparation and activation of platelets. The activation of platelets could be triggered by a variety of substances or stimuli such as low-level laser (LLL), thrombin, calcium chloride or collagen. The aim of the study was to evaluate the activated effects of LLL and calcium chloride on canine PRP. Blood samples were collected from 6 dogs and activated by calcium chloride alone or combine with different energy of low-level laser. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect the concentration of growth factor PDGF-BB, VEGF and TGF-β in activated PRP. Results showed these growth factors were significant upregulated after activation. However, there were no significant differences between the calcium chloride and calcium chloride with LLL groups. Our results clearly point that calcium chloride can leading platelet activation but LLL can't enhance the results. Therefore, using calcium chloride is adequate for preparation of activated canine PRP.

Keywords: calcium chloride, canine, low level laser, platelet-rich plasma

OA-09

Application of the socket-based and implanted prosthesis in veterinary clinical cases

Shih-Jung Kuo1, Ting-Yun Wu1, Cheng-Shu Chung1,2, Lee-Shuan Lin1,2

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

2Veteirnary Medicine Teaching Hospital, National Pingtung University of Science and Technology, Pingtung, Taiwan

Rare study of the animal prosthetic in Taiwan is reported. There are two cases utilizing the prosthetics which are applied with the three-dimension printing technology in National Pingtung University of Science and Technology Veterinary Medical Teaching Hospital (NPUSTVMTH). A male chihuahua was amputated due to the wounded left forepaw, and we designed a socket-based prosthetic. Another case is a male mixed feline whose hindlimb was injured by trap. Custom-made implanted prosthetic was designed and implanted following with amputation. Rehabilitation protocols were designed to help the animals using the prosthetics.

Keywords: Customized, Implanted, Socket-Based, Prosthesis, 3D Printing

 

OA-10

Coagulase Negative Staphylococci, Original from Ruminant Mastitis, Carrying Biofilm genes (eno and ica) Raising the Phenol Coefficients of Didecyldimethylammonium Chloride and Chlorhexidine Gluconate

Pei-Tsen Chen1, Jyh-Mirn Lai1*

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

Coagulase negative staphylococci (CNS) having biofilm ability formation have been the most important mastitis pathogens in dairy industry. The study sought to know the disinfectants: povidone iodine (PI), didecyldimethylammonium chloride (DDAC) and chlorhexidine gluconate (CG), are still proper to kill these CNS. In this study, the phenol coefficient (PC) of these three disinfectants against the CNS carrying biofilm genes (ica, bap and/or eno) (n=44) and CNS not carrying the genes (n=15) were analyzed. The results showed the average PC of PI, DDAC, and CG was 0.24±0.02 (CI= 0.20 - 0.28), 32.7±4.85 (CI= 23.0 - 42.4) and 4.70±0.90 (CI = 2.90 - 6.50), respectively. For the CNS carrying the genes, the values of PI, DDAC and CG was 0.23±0.03 (CI= 0.18 - 0.28), 22.1±4.01 (CI= 14.0 - 30.2), 4.11±1.13 (CI = 1.84 - 6.40), separately. For the CNS not carrying the genes, the values of the disinfectants were 0.25±0.02 (CI= 0.20 - 0.29), 63.6±12.1 (CI= 37.7 - 89.6), 6.41±1.17 (CI= 3.90 - 8.93) by the order. In addition, the median PC of PI against CNS carrying icaC, icaR, was statistically significant. The median was 0.10 (Q1=0.06, Q3=0.22), 0.13 (Q1=0.07, Q3=0.19, respectively. The median PC of DDAC against the CNS carrying icaA or icaB and eno was statistically significant. The median was 13.33 (Q1=7.00, Q3=32.00), 10.00 (Q1=6.19, Q3=13.33), and 32.00 (Q1=10.00, Q3=53.33), respectively. The median PC of CG against the CNS carrying icaA and icaB was statistically significant. The median was 1.67 (Q1=1.21, Q3=3.83), 1.25 (Q1=1.00, Q3=1.83), respectively.

Keywords: Coagulase-Negative Staphylococci, biofilm, phenol coefficient


 

OA-11

Mammalian-adapted Evolution of H6N1 Avian Influenza Viruses During 2000 to 2013

Yi-Chi Wen1, Wan-Zhen Zhu1, Jiu-Yi Peng1, Chuan-Fa Chang2, Ching-Ho Wang1, Hui-Wen Chen1,3

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

2Department of Medical Technology, National Cheng-Kung University, Tainan, Taiwan

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

Avian influenza virus (AIV) H6 subtype is wide-distributed pathogen causing various degrees of illness in poultry. To better understand the biological information of H6 AIVs in the field of Taiwan, we conducted the genetic/antigenic relationship, receptor binding profiles, growth kinetics and pathogenicity of the H6 chicken isolates identified between 2000 and 2013. The epidemiological study revealed that H6 seroprevalence rate was 25.9%, and the virus-prevalence rate was 7.3% in the field during 2013-2014. The analyzed H6 AIVs present frequent substitutions in the globular head domain of the HA protein can be classified into different clades. Most of the H6 chicken isolates carry the mammalian-adapted signatures in HA and NA proteins. Of note, the surface proteins of the new clade isolates share high sequence homology with the human and the dog isolates. Although most of the studied H6 AIVs retained the binding specificity to the avian type receptor, some of the recent strains showed moderate binding to the mammalian type receptor. The growth kinetic study in vitro exhibited these H6 AIVs can grow efficiently in MDCK cells. Upon the experimental chicken infection, the new clade of H6N1 AIVs showed enhanced kidney pathogenesis, rapid shedding, and productive virus replication in the trachea, lung, and kidney; Furthermore, the upregulation of Mx1 gene may play an important role in chicken immune response for clearance of viruses and reduce the pathogenicity of viral infections. Collectively, H6N1 AIVs are the potential economic burden on chicken industry and pose a significant health threat to the public.

Keywords: Avian influenza virus, H6 subtype, sequence analysis, receptor binding, pathogenicity.

 

OA-12

Compare the Differences Between CPV-2 Variants in Histopathological Changes and Tissue Distributions by Using In Situ Hybridization

Chieh-An Yang1,2, Chao-Nan Lin1,2, Ming-Tang Chiou1,2

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

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

Canine parvovirus type 2 (CPV-2) is one of the most important alimentary and immune system diseases in dogs. It induces severe hemorrhagic enteritis and necrosis of peripheral lymphoid tissue, and was also been reported showing relation to myocarditis in puppies. CPV-2 was first reported in 1978, and had become a world-wide infectious disease throughout these years. In the 1980s, new antigenic type CPV-2a and 2b had been discovered, and CPV-2c was been reported in the year 2000. Previous research shows that CPV-2 variant has no significant difference in tissue distribution, but may have some differences in pathogenicity. While during CPV-2 diagnosis, In Situ hybridization has been reported showing high sensitivity, and could directly observe viral infection circumstances within the cells. However, limited information to compared the differences of micro lesions and ISH signal distribution caused by all three types of CPV-2 variants. In the present study, twenty-four CPV-2 (eight CPV-2a, two CPV-2b and fourteen CPV-2c) necropsy cases had been collected, all of these cases are shows the variant severity of CPV-2 infectious lesion, and the histopathology also shows necrosis of crypts epithelium and crypts dilation, the molecular biology diagnosis, genetic sequencing and ISH probe synthesis has been done. The ISH positive signal are mainly found in the necrotic crypt epithelium, and lymphocytes and macrophages in lymphoid tissues. But no significant differences were found in histopathology between the variants, shows that there are no obvious differences of intestinal damage between three variants.

Keywords: Canine parvovirus type 2, In Situ hybridization, histopathology