107 年度春季研討會 壁報論文競賽組 (PB)

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PA-01

Identification and Characterization of the Serotype-determining Sequences of the Hemagglutinin Protein from Avibacterium paragallinarum

Shu-Wan Huang¹, Jui-Hung Shien², and Poa-Chun Chung¹

¹Graduate Institute of Microbiology and Public Health, National Chung Hsing University,

²Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan

Avibacterium paragallinarum is the causative agent of infectious coryza, an important acute respiratory disease associated with growth retardation and reduced egg production in egg-laying hens. The hemagglutinin (HA) protein plays a key role in virulence and immunogenicity of Av. paragallinarum. HA has been subtyped into three serovars: A, B, and C. Though hemagglutination-inhibition (HI) assay is the golden standard for serotyping of Av. paragallinarum, this method is complicated and technically demanding. The purpose of this study is to identify the serotype-determining sequences of the HA protein and use phylogenetic analyses to identify the serotypes of Av. paragallinarum. Towards this goal, we constructed strains of Av. paragallinarum that express a series of hybrid HA proteins containing different portion of HA sequences from strain 221(serotype A) and strain TW07 (serotype C). HI tests using HA antigens prepared from these strains revealed that the amino acid sequences that determine the serotypes of A and C are located at residues 240-614 of the HA protein. We then used prokaryotic system to express a recombinant protein containing HA240-614 sequences. Western blot analyses showed that anti-serum against strain 221 can distinguish between HA240-614 from strain 221 and that from strain TW07. Serotyping of different Taiwanese field strains of Av. paragallinarum using phylogenetic analysis of HA240-614 sequences is consistent with that of HI testes. This study is the first report showing that HA240-614 contains the serotype-determining sequences of Av. paragallinarum. This finding can be used to develop a method for rapid serotyping of strains of Av. paragallinarum.

Keywords: Avibacterium paragallinarum, serotype-determining sequences, hemagglutinin

PA-02

Safety Assessment of Edible Oil Before and After High Temperature Processing by Medium-Term Hepatocarcinogenesis in Rats

Yun-Chieh Tuan¹, Yi-Hui Su¹, Yan-Xiu Lin¹, Tony Ji Fang², Jiunn-Wang Liao^{1, 3}

¹Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan R.O.C

² Food Industry Research and Development Institute, Hsinchu, Taiwan R.O.C.

³Animal Disease Diagnostic Center, National Chung Hsing University, Taichung, Taiwan R.O.C.

Nowadays, news that focus on health effects, including carcinogenesis, caused by eating the fried foods has been reported more frequently, which raised the question that whether long-term intakes the fried foods increase the risk of cancer? Therefore, the aim of this study is to evaluate the potential carcinogenicity of oils via medium-term bioassay model in rats. 8 week-old, SD rats, 6 males per group were used. This experiment was divided into five groups: Blank Control (BC), Initiator DEN Control (DC), Positive Control (PC), UHOil and HOil groups. Firstly, Diethylnitrosamine (DEN) 200 mg/kg was intraperitoneally injected on day 1 as an initiator. At the second week, administered the samples by gavage in UHOil and HOil groups, and added 2-AAF 200 ppm in diet in the PC only, and oil samples were gavaged for the consecutive 7 weeks. At the third week, 1/3 partial hepatectomy was performed to be a promoter. The body weight and food consumption, urinary, complete blood counts, biochemistry, gross and histopathology were recorded at the end of experiment. In addition, the tumor marker of GST-P was detected in liver by immunohistochemistry (IHC) and Western blot analysis for the carcinogenicity. Except rats in the PC was successfully induced hepatoma, neither the UHOil nor HOil-treated groups posed hepatocarcinoginicity in male rats. Based on the above results, both the unheated and heated edible oils are classified as “not carcinogenic” in the medium-term bioassay model in rats.

Keyword: GST-P, heated, carcinogenicity, medium-term bioassay, soybean oil, rats

PA-03

Retrospective Pathological Reports of Splenic Tumors in Domestic Hamsters

Yun-Chieh Tuan¹, Ruo-Chan Wang², Ju-Po Kao^{2, 3}, Kimimasa Takahashi⁴, Jiunn-Wang Liao^{1, 5}

¹Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan R.O.C

²Veterinary Medical Teaching Hospital, National Chung Hsing University, Taichung, Taiwan R.O.C.

³Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan R.O.C.

⁴ Department of Veterinary Pathology, Nippon Veterinary and Animal Science University, Musashinoshi, Tokyo, Japan.

⁵Animal Disease Diagnostic Center, National Chung Hsing University, Taichung, Taiwan R.O.C.

A total of 15 splenic biopsy specimens in 212 cases of hamsters from the Division of Wild (Exotic) Animal Medicine, Veterinary Medical Teaching Hospital, National Chung Hsing University, between 2010 and 2017 were studied for the retrospective pathological study. The incidence in the spleen was 7.1% (15/212), including 15 Phodopus hamsters. Ten neoplasms and 5 non-neoplastic lesions in the Phodopus hamsters occurred. For neoplastic lesions, the prevalence in Phodopus hamsters was higher than in Syrian hamsters (found 3 cases in VMTH, NCHU), which are similar to laboratory hamsters. In Phodopus hamsters, the most common tumors are histiocytic sarcoma (HS), lymphoma, malignant fibrous histiocytoma (MFH) and hemangiosarcoma. In addition, hemangiosarcoma and histiocytic sarcoma were found in Syrian hamsters. The non-neoplastic lesion was a fibrotic nodule. The mean age of affected hamsters was 16.6 months, and females were affected more than males. In immunohistochemical staining, histiocytic sarcoma and malignant fibrous histiocytoma were positive for lysozyme, lymphoma was negative for CD79a and CD20, one case of lymphoma was positive for CD3, and hemangiosarcoma was positive for Von Willebrand factor (VWF). In terms of non-neoplastic lesions, the most common are fibrotic nodules and all occur in aging female hamsters. The nodules consist of collagen fiber that can be identified with Masson's trichrome stain, and are related to repairing of trauma in the spleen. To our knowledge, there have been no retrospective pathological reports of splenic tumors in domestic hamsters. Therefore this study can provide some valuable analytical data about spleens in hamsters.

Keyword: tumors, non-neoplastic lesion, retrospective pathological reports, spleen, hamsters

PA-04

Development of Porcine Epidemic Diarrhea Virus Particle-Type Oral Vaccines by Using Sodium Alginate and Chitosan as Biological Materials

Lian-Xiu Liu¹, Yao chi Chung¹

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

The porcine epidemic diarrhea virus (PEDV) particle-type oral vaccines can be more convenient and safe than traditional vaccine, and can reduce the stress caused by administration by injection, also can effectively induce the better mucosal immune response than that induced by conventional injection. PED mainly caused by fecal infection. The infected pigs will cause diarrhea, vomiting, and even death by dehydration. Therefore, this study used chitosan and sodium alginate to develop particles-type oral vaccine that could induce intestinal mucosal immunization of pigs to against PEDV infection. The chitosan and sodium alginate are both biodegradable and not toxic to the immunized animals. In this study, we cloned the PEDV S protein which is able to induce neutralizing antibody response as target antigen. Preliminary results have confirmed the size and antigenicity of protein products using SDS-PAGE and Western blotting. The particle size and shape integrity of particle-type oral vaccines were analyzed by using scanning electron microscopy. After the protein was coated in oral vaccines, the amount of coated protein, coating efficiency, and release efficiency were analyzed. Animal test were performed by oral immunization with mice, the protective efficacy of the vaccine was evaluated by ELISA antibodies, cytokine expression.

Key words: particle type vaccine, Chitosan, Sodium alginate, porcine epidemic diarrhea virus, S protein, mucosal immunity

PA-05

Study on Antibiotic Susceptibility Test to Riemerella anatipestifer and E. coli in Waterfowl

Ding-Bang Huang¹, Chia-Ying Lin¹, Ching-Dong Chang¹, Shyh-Shyan Liu¹, Chin-En Tsai¹, Hung-Yi Wu¹

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

In 2015,the output value ofthe waterfowl industry was approximately NT$10 billion dollars, and it was mainly in Pingtung County, Changhua County, and Yunlin County.Among them,breeding in Pingtung County was the largest, accounting for one-third of the total.Due to the serious abuse of antibiotics by farmers, the bacterial resistance caused serious economic losses, This experiment counts the results of antibiotic susceptibility test of Riemerella anatipestifer and E.coli from 2016-2017 years to provide veterinarian clinical medication reference.The specimens were collected on the liver and air sac of ducks and geese to bacterial culture and antibiotic susceptibility test. Rimererella anatipestifer (33%, 92/282) was the most common and E.coli (23%, 62/282) Mixed infection (6%，16/282) was using by polymerase chain reaction. Riemerella anatipestifer is most sensitive to Cephalexin (48%), Tilmicosin (84%) is the highest resistance, Trimethoprim Sulfamethxazole (83%) is the second. E.coli is most sensitive to colistin (36%)、Cephalexin(35%), Tilmicosin(97%) is the highest resistance. Riemerella anatipestifer mainly occurs in winter, occurs in 3-10 weeks waterfowl. E. coli can occur at all ages and all years in waterfowl. Occurrence of Riemerella anatipestifer in the case of polyserositis was (68%). The incidence of E. coli was (47%). Riemerella anatipestifer is most effective with Cephalexin, and E. coli is most effective with colistin.

Keywords: Waterfowl, Riemerella anatipestifer, E.coli, Antibiotic Susceptibility Test

PA-06

A Study on Probiotic from Crops of Parrots and Pigeons

Yu-Cheng Chang¹, Chia-Ying Lin¹,Ching D. Chang¹, Shyh-Shyan Lin¹, Chin-En Tsai ¹, Hung-Yi Wu

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

According to the supervisor of Kaohsiung County Bird Watching Promotion Association in 2011, in Taiwan, parrots accounted for 70% of the ornamental bird market and the export value reached as high as 7 billion. Among a variety of bird diseases, the lesions of the crop are one of them; the crop is a chamber in the middle part of the esophagus of some birds, and functions include the storage of food bolus and the decomposition of nutrients by the probiotics. In clinical practice, cases associated with crop contain burns and inflammation of it due to the high temperature of the fed powdered milk. In many cases, the bacteria in the crops have become chaos, causing food groups to stagnate and failing to enter the stomach, causing indigestion and secondary infections. In the paper BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLIGY, M. AndreÍna Pacheco indicated that the strains in the parrots that prefer to eat cereals include Enterococcus, Lactobacillus, Streptococcus, Propionibacterium and Enterobacteria Enterobacteriaceae, in which Lactobacillus have the ability to secreted amylases in order to decompose foods, this reserch want to develop Lactobacillus, which belongs to parrots and pigeons, by understanding the main species of normal lactobacilli in the crops of parrots and pigeons. In this study, the API 50 colonization system was used. We found that Lactobacillus, a common bacillus in the parrot crop, contained Lactobacillus (A), Lactobacillus (B), Leuconostoc (C), Lactobacillus (D), and Lactobacillus (E). Lactobacillus (F) and Lactobacillus (G) are found in the crops of pigeons. Two (33%) samples presented Lactobacillus (A) positive individuals, Positive individuals of Lactobacillus (B), Leuconostoc (C), Lactobacillus (D), and Lactobacillus (E) were each (17%); among them, Lactobacillus (B) and Lactobacillus(C) were detected in the crop one individual. In pigeons, we detected Lactobacillus (F) (50%) and Lactobacillus (G) (50%) in 2 individual samples. This study found that the unique Lactobacillus species of several parrots and pigeons are developing toward clinical applications in the future.

Keywords: Parrot, Pigeons, Crop, Lactobacillus

PA-07

Health Monitoring and Analysis of Formosan Sika Deer (Cervus nippon taiouanus) in Sheding Village of Kenting National Park, 2017

Yu-Hsiang Chen¹, Chia-Ying Lin¹, Ching-Dong Chang¹, Shyh-Shyan Liu¹, Chin-En Tsai¹, Hung-Yi Wu¹

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

The Formosan Sika Deer (Cervus nippon taiouanus), a subspecies of Sika Deer (Cervus nippon), is an endemic species of Taiwan. Since 1984, the Formosan Sika Deer restoration program in Kenting National Park has resulted in the current population size, exceeding 1500 individuals, also, the Sheding Rehabilitation Center has perpetuated the purebred traits of Formosan Sika Deer. In the year 2017, Sheding Rehabilitation Center recalled two groups (a total of 70) of Formosan Sika Deer for the evaluation of individual health conditions. A jugular venous sampling section was carried out, after an immobilization was performed with plastic projectile syringes blown from a blow-pipe, and an analysis on hematological and serum biochemical values was done. For the monitoring of parasites, a parasitic oocyte inspection on fecal samples (randomly sampled) was performed. An Intradermal Tuberculin Test (ITT) was performed on the neck area to monitor Tuberculosis. The results were negative for both parasitic oocyte inspection and ITT. The analysis of hematological and serum biochemical values showed that a number of individuals has shown a significant difference in their values; 50% of individuals was diagnosed with physiologic leukocytosis (mild neutrophilia with normal lymphocyte count or mild lymphocytosis) which might be caused by a stressful capture; 5.71% of individuals was analyzed with a higher packed cell volume (PCV), hemoglobin (Hb) and red blood cell count (RBC) which might be caused by stressors (e.g. immobilization by dart-blowing and blood sampling), taken measures (e.g. food and water limitation) which lead to dehydration; there were 17.14% samples analyzed with an increased mean corpuscular volume (MCV); blood glucose level was low in 2.85% of the samples, and a slight decrease in blood total protein in 4.29% of the samples; there were no simultaneous increase in both BUN and CRE, an increase in either BUN or CRE might be caused by temporary dehydration due to captivity; the microfilaria infection of Setaria spp. was detected in 1.43% of the samples. From the findings, the overall status of the Formosan Sika Deer was fair, although a minority was influenced by stress factors, low nutritive condition and microfilaria infection.

Keywords: Kenting National Park, Formosan Sika Deer, Health care, Hematology, Serum Biochemistry

PA-08

Sterilization Effect of Nano-silver in Geese Against Bacteria

Wei-Lin Shih¹, Chia-Ying Lin¹, Ching-Dong Chang¹, Shyh-Shyan Liu¹, Chin-En Tsai¹, Hung-Yi Wu¹

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

Goose is one of the important agricultural industries in Taiwan, the total number of geese slaughter in 106 years has grown 52.95% compared with 105 years, which shows that the industry is recovering. However, the common problem of bacterial infection has always existed, the genus Salmonella spp. also known as Goose paratyphoid, is a common infectious disease of all kinds of poultry, mainly endanger the young geese, and often through contaminated animal products infect human beings, endanger people and animals very heavy. In the face of bacteria most of the use of antibiotics, not only to worry about the withdrawal of antibiotics, but also may cause abuse of antibiotics, resulting in bacteria to produce drug resistance. Therefore, Nano-silver, which has less influence on environment and biology, is chosen as the subject of experiment. Nano-Silver special sterilization mechanism makes the bacterium not easy to produce the resistance, the silver ion can with the bacterium protein the mercaptan base (-SH) the specific binding force to cause the bacterial protein denaturation, achieved the sterilization function. So, we want to discuss the antibacterial effect of nano-silver on salmonella and geese in various concentrations and at different time points. The study found that the minimum effective dose of nano-silver was 20ppm, and then the nano-silver was used in the same concentration of 10ppm and 100ppm in geese to observe the total amount of feces. Simulation of the effect of nano-silver prevention, the owner of the goose to the silver nanoparticles, due to climatic factors, found that the whole field of bacterial infection, after testing determined, began to trace the total amount of feces, to record whether the nano-silver can effectively inhibit bacteria, the experiment proved that nano-silver in the use of the fourth day could be reduced by 43%. Simulation of the effect of nano-silver treatment, the owner of the test to determine the goose farm has a bacterial infection, and then cast nano-silver, we go to the scene to sample, test the total amount of feces changes. Found that in the treatment of no more than the effect of prevention, the highest can only inhibit the total number of bacteria 8%, the understanding of whether the nano-silver residue in the geese, nano-silver to give one months after the owner of the test, the results of our experimental nano-silver will indeed remain in the body.

Keywords: nano-silver, goose, minimum effective dose, prevention, treatment

PA-09

The Investigation of Cryptosporidium Infection in Dairy Cows and Calves in Taiwan

Jui-Yu Lee¹, Hsu-Hsun Lee¹

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

There are four species of Cryptosporidium commonly reported in cattle; C. parvum, C. bovis, C. ryanae and C. andersoni. C. parvum has the highest pathogenicity among them. The most common clinical sign is diarrhea and causing considerable direct and indirect economic losses. Former studies indicated that calves are susceptible hosts to C. parvum. Moreover, recent studies pointed out the high possibility of calves infected Cryptosporidium right after being delivered due to the carrying of this pathogen by their dams. This research will divide Taiwan into four area and conduct the investigation of the prevalence of Cryptosporidium infection and the number of the oocysts in the environment in the expectant dairy cows and calves using nested polymerase chain reaction (nested PCR). The sampling will focus on feces, drinking water and flooring sample. After DNA extraction, nested PCR will be used to detect Cryptosporidium and the product will send to DNA sequencing. The infection rates of southern Taiwan were 32% (63/197), which is significantly higher (P<0.05) than the infection rates of middle Taiwan (21.5%, 26/121) and extremely significantly higher (P<0.01) than the rate of eastern Taiwan (11.7%, 10/86). No significant differences of environment positive rates were found among four areas of Taiwan. The farm positive rates of calves’ flooring sample was 70% (14/20), which is significantly higher (P<0.05) than the farm positive rats of expectant cows’ flooring sample (15%, 3/20). So, we concluded that calves’ environment plays an important role in calves’ cryptosporidiosis.

Keywords: Cryptosporidum, dairy cow, environment, prevalence

PA-10

Epidemiology of Bartonella, Anaplasma and Borrelia burgdorferi Infections among Small Mammals, Carnivores and Non-human Primates in Taiwan.

Yu-Ting Lo¹, Chen-Chih Chen², Chuen-Fu Lin³, Chao-Chin Chang^4,*

^1,4Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan

²Wildlife Conservation Institute, National Pingtung University of Science and Technology, Pingtung, Taiwan

³Department of Veterinary Medicine, National Chiayi University, Chiayi, Taiwan

Bartonella spp. are fastidious, gram-negative, rod-shaped bacteria that are highly adapted to a wide range of mammalian hosts. In this study, a total of 184 blood samples from 4 animal orders were collected at 16 locations among eight cities in Taiwan during December 2014 to September 2017. Based on the PCR results, Bartonellae were detected in seven animal species, including 30.2% (19/63) of Bandicota indica, 100% (1/1) of Mus caroli, 35.7% (5/14) of Rattus losea, 50% (1/2) of Rattus rattus, 50% (1/2) of Suncus murinus, 5.3% (3/57) of Herpestes urva and 18.2% (2/11) of Macaca cyclopis. Through phylogenetic analysis of Bartonella PCR-positive samples using the partial sequence of the gltA gene, eight Bartonella spp. were identified, including seven known Bartonella species and one potentially novel Bartonella species; it showed the large diversity of Bartonella spp. in Taiwan. By direct blood culture results, 3.4% (3/86) of the small mammals were co-infected with different Bartonella spp. To study other emerging vector-borne agents, such as Anaplasmataceae and Borrelia burgdorferi, conventional or nested polymerase chain reaction assays and sequence analysis were further conducted. The results show that 40.7% (35/86) of the small mammals were infected with A. bovis, and 18.4% (16/87) of the carnivores were with Anaplasma spp. (including 15 A. bovis and one A. platys). Furthermore, 8.7% of the carnivores positive for Anaplasma were co-infected with Bartonella. However, none of the animals showed PCR positivity for B. burgdorferi. To the best of our knowledge, this is the first report on isolation of Bartonella from Herpestes urva and Macaca cyclopis worldwide.

Keywords: Bartonella, Anaplasma, Borrelia, small mammals, carnivores, primates, epidemiology, phylogenetic tree

PA-11

Mycoplasma bovis as an Agent Associated High Severity in Pneumonia Feedlot Calves

Natcha Thongrueang¹, Hsu-Hsun Lee¹

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

Bovine respiratory disease (BRD) is one of the cause of morbidity and mortality in calves. Mycoplasma bovis (Mb) is commonly bacteria related with BRD in calves because it is associated with higher odds of clinical signs, lung consolidation and lower average daily gain. The cost of treatment is high and antimicrobial therapy is often ineffective. A total of 9 pre-weaned feedlot calves that have respiratory clinical signs were sent to diagnosis and treatment at Large Animal Hospital, NPUST. Their clinical signs were scored according to the Wisconsin (WI) clinical scoring systems. Almost all of them were evaluated BRD positive. Deep nasopharyngeal swabs were collected and then nested PCR was used for test Mb. All of them were nested PCR positive. Antibiotic sensitivity test (AST) was used to determine which antibiotic will be the most successful for treatment because they were unresponsive to antibiotic therapy. We found that almost calves resistance to antibiotics such as Ampicillin, Ceftiofur, Gentamicin, Enrofloxacin etc. Necropsies were conducted on all of calves after died. Lungs parenchyma showed dark red, rubbery and atelectasis. Numerous foci of caseous necrosis presented in the cranial and middle lobe of the lungs. Our results demonstrate the importance of Mb that associated with high severity pneumonia feedlots calves and also related with unresponsive to antibiotic therapy. Preventive and control measures in Taiwan should be more considered. However, the other bacteria and virus in the respiratory diseases of calves still remains to be determined and should not be default.

Keywords: Bovine respiratory disease (BRD), Mycoplasma bovis (Mb), feedlot calves, Taiwan

PA-12

Experiment of Chinese Herbal Feed Additives on Prevention of Class A Infectious Disease

Meng-Han Tsai¹, Chia-Ying Lin¹, Ching-D. Chang¹, Shyh-Shyan Liu¹, Chin-En Tsai¹, Hung-Yi Wu¹

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

The annual output value of poultry in Taiwan is about 60 billion NT. dollars each year. It is a quiet stable for the supply to the market and has started to move towards large operation due to the successful development and widespread using of the vaccine. However, in the large and intensive breeding Management, many bacteria, viral infectious diseases began to spread in large numbers, resulting in large-scale poultry deaths, in order to curb the incidence of infectious diseases, farmers began to use large amounts of antibiotics against bacterial infections, which made the food safety problem of drug residues occurred by wrong concept of drug using, and also contribute to the development of multiple drug-resistant bacteria ;the other hand ,using of antibiotics against viral diseases also have not effect, above of all, we try to use compound of herbal medicine feed Additives for infectious disease prevention and carry out experiments. The experiment for the use of SPF chickens fed different doses of Chinese herbal medicine every day, after seven weeks, we made those chickens were infected by the Newcastle disease virus, then observe and record their survival rate, gross lesions, pathological Slides for our diagnosis of disease and prevention Research. The experiment from July 2010 to the end of July 2011, is divided into two experiments, the first experiment give 5 grams of Chinese herbal medicine that group had the highest survival rate (50%), the second test will be the dose of Chinese herbal medicine was adjusted downward from 5 grams to 4 grams, and the survival rate was higher in the group fed with 4 grams (60%) than that in the other groups. The gross lesions were mainly observed in brain, lung, mucosa of small intestine and proventriculus. The result appeared mild symptoms was given 4 and 5 grams of herbs group than other groups and the result of pathological section as well. In this study, it was found that the best effect of group in the range of 4 to 5 grams of Chinese herbal medicine for feeding chickens was significant.

Keywords: Chinese herbal medicine, Newcastle disease, Class A infectious disease, drug-resistant bacteria

PA-13

Monoclonal Antibody Preparation and Cellular Distribution Analysis of Koi Herpesvirus ORF72 Protein

Ting-Yun Wu¹, Ching-Wei Wu², Maw-Sheng Chien³, Chienjin Huang¹

¹Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan

²Research Center for Animal Medicine, National Chung Hsing University, Taichung, Taiwan

³Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan

Cyprinid herpesvirus 3 (CyHV-3), commonly known as koi herpesvirus (KHV), is a recently emerging pathogen of koi and common carp (Cyprinus carpio) that causes significant disease and mass mortality in infected fish. CyHV-3 has been classified as a member of Alloherpesviridae which consists of herpesviruses of fish and amphibians. The previous study has demonstrated that a neutralizing monoclonal antibody MAb-B2 specific to KHV ORF72 was obtained by immunizing mice with purified virus particles suggesting that KHV ORF72 protein might be a candidate for diagnostic tool or KHV vaccine. In the present study, the KHV ORF72 gene was amplified by polymerase chain reaction (PCR) from a KHV-infected fish tissue followed by expression of ORF72 protein in E. coli expression system. Expressed recombinant ORF72 protein was purified by HisPurTM Cobalt resin and sodium dodecyl sulfate (SDS) was added to enhance its solubility. Purified ORF72 protein was used as antigen to immunize BALB/c mice and cell fusion was conducted after the final boost. Hybridoma cells were selected by ELISA, Western blot and immunofluorescence assay and a monoclonal antibody 3E1 was obtained. Further epitope mapping of monoclonal antibody 3E1 was analyzed by a series of C-terminal truncated proteins, including N247, N176, N159 and N124 and two subunit proteins, N125-247 and N248-370. The result showed that the monoclonal antibody 3E1 could recognize all C-terminal truncated proteins suggesting that the epitope may located in the N-terminal 124 residues region. According to the result of immunohistochemistry, the MAb 3E1 can specifically recognize the KHV-infected clinical speciemen suggesting its potential as a diagnostic reagent. Furthermore, cellular distribution of KHV ORF72 protein was conducted by Mitochondria Isolation Kit for Cultured Cells® and was detected by the MAb 3E1 in cytosol, nuclear and mitochondria fractions. Sequences analysis of KHV ORF72 using CBS (Center for Biological Sequence analysis) program revealed a potential nuclear export signal (NES) motif near residues 202-212 (KHV/NES). The function of NES motif was confirmed by nuclear export of the KHV/NES tagged PCV2Cap recombinant protein since the PCV2 Cap protein tends to accumulate in the nucleus due to possessing a strong nuclear localization signal (NLS).

Keywords: Koi herpesvirus, monoclonal antibody, nuclear export signal

PA-14

The Influences of Avian Influenza Viruses NS1 protein on Viral Replication, Host Adaptation and the Immune Response

Wen-Chien Wang¹, Yu-Jing Tseng¹, Yu-Hsuan Yang¹, Chia-HSuan Chang¹, Wei-Li Hsu¹, Shan-Chia Ou¹

¹Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan

Avian influenza viruses (AIVs) cause a significant poultry disease worldwide. In the last three decades, H6N1 virus has been enzootic in chickens locally in Taiwan. Since the initial outbreaks of low pathogenic avian influenza virus (LPAIV) H5 virus in 2003, a variety of H5 AIVs emerged these years, including high pathogenic avian influenza virus (HPAIV) H5N2 viruses in 2012, H5 viruses of clade 2.3.4.4 in 2014 and H5N6 virus in 2017. All of them show different characteristics in virulence and host range. In the current study, sequence alignment revealed high frequency of sequence variations among the non-structural 1 (NS1) of AIVs in Taiwan. Influenza NS1 protein involves in limiting innate immune responses and regarded as one of the host range determinants. Therefore, the goal of this study was set to explore the roles of NS1 in host adaptation and in viral virulence of these emerging AIVs. To address this issue, a series of reassorted viruses carrying the NS segments of different Taiwanese H5 viruses were generated in the backbone of a H6N1 virus by reverse genetics. Growth kinetics in DF-1 and MDCK cells showed the diversity of NS1 makes differences in AIV replication in mammalian and avian cells. Then, the expressing levels of cytokines in AIV infected DF-1, Duck embryo (DE) and A549 cells were measured. Some reassorted viruses resulted in significant differences at 6 hours post infection and the results demonstrated that high expression of cytokines in innate immunity may correlate with poor viral replication. Expression levels of cytokines in chicken embryos were similar to the results of DF-1 cells, and most of reassorted viruses induced high expression levels of cytokines in duck embryo. In conclusion, NS1 variants can affect the growth kinetics, innate immune responses and cytokine expressions, and these differences may be responsible for the different characteristics of AIVs in Taiwan.

Keywords: avian influenza virus, NS1 protein, reverse genetics

PA-15

Producing the Recombinant Structural Protein of Chicken Anemia Virus and Chicken Interleukin-12 by Baculovirus Expression System as a Vaccine Candidate

Ta-Yuan Tseng¹, Yee-Chen Liu², Yu-Hsuan Yang¹, Yu-Chen Hsu¹, Chia-Hsuan Chang¹, Shan-Chia Ou¹

¹Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan

²Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan

Chicken anemia virus (CAV) is one of the important pathogens affecting poultry industry globally. The virus cause mortality, production losses, immunosuppression, sensitive to infections and vaccination failures. Currently, only live attenuated chicken infectious anemia (CIA) vaccines are available for administering in breeders older than 8 weeks that induce maternal antibodies to protect their progeny till 3 weeks. However, the vaccine viruses could induce vertical and horizontal infection to other chickens. These disadvantages of the traditional vaccines induce the idea to develop novel CIA vaccines. According to previous studies, VP1 is the only structural protein forming the viral capsid, and the correct folding of VP1 requires expression of VP1 and VP2 in the same cell. Interleukin-12 (IL-12) is a proinflammatory cytokine, which is responsible for enhancing cytotoxicity of NK cells and T cells. Moreover, IL-12 facilitates the generation of related T_H1 cytokines, and promotes the production of immunoglobulins by stimulating helper T cells. Therefore, IL-12 can be serve as a biological adjuvant. In this study, the baculovirus expression system was used to produce the structural protein of CAV and chicken IL-12 (chIL-12) for developing a subunit vaccine.

The VP1 and VP2 of CAV and the chIL-12 gene codons were optimized for expressing in the insect cells, and the recombinant proteins were expressed by the baculovirus expression system. Western blotting and indirect immunofluorescence assays were used to verify the expression of recombinant proteins. The results showed that VP1 and chIL-12 can be expressed in eukaryotic cells. We found that VP1 predominantly localized in the nucleus, and the expression level needs to be further optimized. Biological adjuvant property of chIL-12 has been tested by high level expression of IFN-γ in the recombinant chIL-12 stimulated splenocytes. Finally, different concentrations of VP1 and chIL-12 has been combined together to assess immune responses in chickens, and the results showed that VP1 combined with 5 ng of chIL-12 achieved highest titers of CAV specific antibody than those of other groups. Taken together, the recombinant VP1 could be a candidate of CAV subunit vaccine, and co-immunization of chIL-12 can induce a strong immune response.

Keywords: chicken anemia virus, interleukin-12, baculovirus

PA-16

Survey of Antimicrobial Characterizations and Risk Factors of Staphylococcus Species Isolated from Canine Superficial Pyoderma wi/o Complicated Atopic Dermatitis

Yu-Ling Hsieh¹, Cheng-Hung Lai¹

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

Superficial pyoderma is a common primary or secondary complication of a range of canine chronic diseases, including allergic, endocrine disorders, etc. The predominant pathogen that causes superficial pyoderma is Staphylococcus pseudintermedius. Treatment of superficial pyoderma frequently involves the use of systemic antimicrobial agents. However, recent studies showed that methicillin-resistant staphylococci are present in increasing frequencies in dogs. The aim of this survey was to investigate the bacterial pathogen of canines admitted to the division of dermatology, VMTH-NCHU from Jun. 2017 to Jan. 2018 and diagnosed as superficial pyoderma. Swabs were taken from the lesion site. The staphylococcus isolates were tested by PCR for the presence of mecA and blaZ genes. A total of 70 isolates were obtained from 65 enrolled dogs. Among 70 isolates, 91.4 % were Staphylococcus spp., being 81.2 % S. pseudintermedius, followed by 15.6 % S. schleiferi subsp. coagulans. The prevalence of mecA and blaZ genes among S. pseudintermedius and S. schleiferi subsp. coagulans were 32.7 %, 82.7 % and 70.0 %, 70.0 %, respectively. There was a significant difference in the susceptibility to all antibiotics we selected between methicillin-resistant S. pseudintermedius (MRSP) and methicillin-susceptible S. pseudintermedius (MSSP). mecA gene did not have an impact on the susceptibility of S. schleiferi subsp. coagulans. There was no significant difference in the antimicrobial susceptibility between cases concurrent with atopic dermatitis and without any chronic diseases. In order to identify the risk factors of MRSP infection, clinical isolates of S. pseudintermedius and their corresponding host signalment and medical data covering 3 years prior to staphylococcal isolation were analyzed. More cases with MRSP (23.1 %) were concurrent with pododermatitis than those with MSSP (0.0 %, p=0.03). The results indicated that Staphylococcus pseudintermedius was the primary pathogen of canine superficial pyoderma, and atopic dermatitis increased the risk of antibiotic resistance

Keywords: superficial pyoderma, MRSP, atopic dermatitis, risk factors

PA-17

An Investigation of Staphylococcus psudintermedius Isolated from Dogs with Superficial Pyoderma and from Their Owners

Yu-Chan Ma¹, Cheng-Hung Lai¹

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

The skin microbiota of dogs includes several species of bacteria, which contributes to the skin defense mechanism. Staphylococcus pseudintermedius is an opportunistic pathogen, which is cultured at high frequency from the skin of dogs with superficial pyoderma. Although antibiotic is the standard medication when dealing with bacterial infection, multiple antibiotic-resistant mechanisms gradually developed by bacteria make treatment more challenging nowadays. If S. pseudintermedius was transferred from dogs to humans, it may not only cause direct infection but also exchange drug-resistant gene with other staphylococci on humans. The aims of this study were to investigate the antibiotic-resistant characteristics, the expression of drug-resistant genes of S. pseudintermedius isolated from dogs with superficial pyoderma, to investigate the prevalence of S. pseudintermedius on dog owners and evaluate possible risk factors of transmission, and to clarify the dru types of Methicicclin-resistant S. pseudintermedius (MRSP). Samples were obtained at the division of dermatology, VMTH-NCHU from Jun. 2017 to Jan. 2018, and 60 pairs of dogs and owners were included. Sixty-five bacteria were isolated from the dogs, which included 47 S. pseudintermedius (72.3 %), 12 other staphylococci (18.5 %), 4 other Gram-positive bacteria (6.2 %) and 2 Gram-negative bacteria (3.1 %). The positive rates of possessing mecA and blaZ of S. pseudintermedius were 34.04 % and 80.85 %, respectively. The expression of mecA or blaZ gene increased the risk of multiple-drug resistance. Dogs which received antimicrobial treatment within a recent month were at significantly higher risk of MRSP infections compared with those which didn’t (p <0.05). The prevalence of S. pseudintermedius on dog owners was 8.33 %, and there were no significant risk factors that associated with S. pseudintermedius colonizing owners could be found in this survey. MRSP isolates belonged to one of the following three dru types: dt11y (29.41 %), dt11a (47.06 %) and dt10cp (23.53 %). In conclusion, the major pathogen of canine superficial pyoderma is found to be S. pseudintermedius in Taiwan, and isolates which are mecA- or blaZ-positive are generally more resistant to commonly used antibiotics. Although S. pseudintermedius might be transferred from dogs to owners, definite risk factors should be further examined in the future study.

Keywords: superficial pyoderma, Staphylococcus pseudintermedius, MRSP, owners

PA-18

Characterization of the Role of TNFAIP2 in Influenza Virus Infection

Yu-En Li ¹, Wei-Li Hsu ¹, Chienjin Huang ¹, Hao-Ping Liu ²

¹Graduate Institute of Microbiology and Public Health, National Chung-Hsing University, Taichung, Taiwan

²Department of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan

Tumor necrosis factor alpha (TNFα)-induced protein type 2 (TNFAIP2) was originally identified as an inducible gene in TNFα-treated human endothelial cells, and thus is likely participate in angiogenesis and pro-inflammatory responses. TNFAIP2 expression can be upregulated transcriptionally via activation of the transcription factor NF-κB, which is known to mediate both pro- and antiviral functions. It’s been reported that viral infection can induce TNFAIP2 expression; however, the functional influence remains largely unknown. In this study, the role of TNFAIP2 in the interplay between H1N1 influenza A virus (IAV) infection and cellular response is characterized in IAV-permissive A549 cells. Infection of A549 cells with IAV led to expression of IAV-encoded NS1 protein in conjunction with activation of NF-κB and phosphorylation of AKT at 12 and 24 hours post infection, whereas TNFAIP2 expression was reduced by IAV infection. Moreover, siRNA-mediated depletion of TNFAIP2 was shown to promote viral NS1 expression and IAV-induced activation of NF-κB. Furthermore, knockout of TNFAIP2 by using CRISPR/Cas9 significantly increased the protein expression of viral NS1 and NP at 12 and 24 hours post infection, in conjunction with the enhanced activation of NF-κB and AKT in cells. The data collectively demonstrate that TNFAIP2 may play an inhibitory role, which may not involve NF-κB activation, in early phase of H1N1 IAV infection. On the other hand, expression vectors for various forms of TNFAIP2 have been generated to better elucidate the function of TNFAIP2. This study will shed light on how TNFAIP2 is involved in cellular responses to virus infection.

Keywords: Tumor necrosis factor alpha-induced protein type 2 (TNFAIP2), influenza A virus (IAV), NF-κB, AKT

PA-19

Establishment of ELISA Kit for Detection of Goose Parvovirus Antibody

Chao-Chi Wang¹, Chia-Ying Lin¹, Ching-Dong Chang¹, Shyh-Shyan Liu¹, Chin-En Tsai¹, Hung-Yi Wu¹

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

According to the Agricultural Committee of the Executive Yuan, in the 3rd quarter of 106 years, the livestock and poultry survey in Taiwan, ducks, geese in the breeding of about 11 million, the annual output value of more than 8 billion NT dollars. Ducklings because of the resistance to the environment and poor adaptability, often infected duck viral hepatitis, diarrhea, colds, coccidiosis, viral enteritis and so on. Among them the death caused by the goose parvovirus is very common, often cause ducks, geese depression, diarrhea, vomiting, sleepy, conjunctival congestion, a serious chick will die in 10 hours. Therefore, we would like to use Elisa to understand the height of antibody of the chicks. In the clinical case, we used the polymerase chain reaction to detect goose parvovirus positive case, and the virus was successfully reproduced and purified by duck embryo. And we obtained the infected goose serum, as the antibody serum source. We diluted second antibody IgY 2000 times, 5,000 times, 10,000 times, 15,000 times, 20,000 times, the appropriate concentration of the test was 15,000 times diluted. Antibodies and viruses were twice times diluted in ELISA, we use the OD value to quantify antigen protein. Then we find the best dilution in the positive control group. We test the goose parvovirus positive Serum and other pathogen serum by ELISA, other pathogen serums were not positive. The antigen and antibody of goose parvovirus can be detected successfully by the ELISA set in this experiment.

Keywords: Goose parvovirus, ELISA, Antibody

PA-20

Chinese Herbal Medicine for Pigeon Circovirus Research Results

Wei-Zhou Huang¹, Chia-Ying Lin¹, Ching D. Chang¹, Shyh-Shyan Lin¹, Chin-En Tsai¹, Hung-Yi Wu¹

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

The pigeon racing industry in Taiwan has three seasons in each of the north and south of the country. There are nearly 70 billion in circulation. From the farmer to the boss of the listed company, the virus is the most threat to pigeons in each season. Pigeon Circovirus (PiCV). Pigeon circovirus attacks the immune organs of pigeons such as the bursa of fabricius, liver and spleen. Its characteristic pathological changes mainly occur in primary and secondary lymphoid tissues, accompanied by severe damage to lymphoid tissues, and causes atrophy of the bursa of infected lymphocytes. Grape basophilic inclusion bodies are produced, resulting in loss of immune cells and immunosuppression. The object of this test is to provide a pigeon loft with scientific Chinese medicine that can enhance the body's immune system. The hope is that giving scientific medicine that enhances the body's immune system can enhance the immunity of pigeons and be helpful for prevention and treatment. The owners of the pigeons who were tested positive for PiCV were divided into four groups and were given 0, 0.5, 1 and 1.5 grams of traditional Chinese medicine respectively. After one month of feeding, the host sent the sample to the laboratory for testing. The positive rate of 0 grams of Chinese medicine was 100% and the survival rate was 80%. The positive rate of 0.5 grams of Chinese medicine was 100% and the survival rate was 100%. The dose was 1 gram of Chinese medicine. The positive rate was 80% and the survival rate was 100%. The positive rate of the Chinese medicine administered 1.5 grams was 100% and the survival rate was 100%. One gram of Chinese medicine was administered and one of the PCR-detected circoviruses was negative. During the test, 0 g of the Chinese medicine group showed vomiting, diarrhea, and loss of appetite. A lot of red blood cells, yeast-like cells, and bacilli were found in the stool smear test. After the onset of death, a macroscopic examination revealed that intestinal flushing and enlargement of the spleen, examination of the lower part of the examination revealed intestinal hemorrhage, spleen hyperemia associated with lymphocyte loss, loss of fluidic lymphocytes associated with basophilic inclusion bodies, and combined bacterial identification results were Escherichia coli. The results of this experiment Chinese medicine for only 1 g of infection with the circular virus, 20% of pigeons PiCV negative but the survival rate of 100% higher than the non-administered group.

Keywords: Pigeon, Pigeon Circovirus (PiCV), Chinese herbal medicine

PA-21

Seroprevalence of Neosporosis in Dairy Cows in Taiwan

Po-An Yueh¹, Hsu-Hsun Lee¹

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

Neosporosis cause abortion in cattle and serious economic impact in dairy industry in Taiwan. The aim of this survey was to demonstrate the prevalence of neosporosis in dairy cows in Taiwan. From April 2017 to February 2018, a total of 401 blood samples collected from 34 dairy farms detected by Enzyme-linked immunosorbent assay. We also compared the results with the basic information of farms and cattle. The seroprevalence was 25.9% (104/401), and the positive farm rate was 70.6% (24/34). The seroprevalence varied a lot among different farms. There is also an extreme significant difference of the seroprevalence between the farms occurred severe abortion in the past or not, and 85.7% (6/7) of the farm had severe abortion history are defined as abortion farms in this study. Divide abortion history of cattle and abortion rate of farms into group, one has an extreme significant difference (P<0.01), the other have a significant difference between groups (P<0.05). Although there was no significant difference of seroprevalence between the farms kept dogs or not, but there was an extreme significant difference of seroprevalence between the farms kept dogs in a free-ranging way or not (P<0.01). Neosporosis is still present in dairy cows in Taiwan and still related to abortive issue, but the prevalence decreased significantly compared with previous research. Dairy farms in Taiwan usually kept dogs, they need to set up the range of dogs inside or around the farms, that will be a useful method to control neosporosis.

Keywords: Neosporosis, abortion, ELISA

PA-22

A Study on the Avian Polyoma Virus, Psittacine Beak &amp; Feather Disease of Parrot and Pigeon case Statistics

Cheng-Hao Su¹, Chia-Ying Lin¹, Ching D. Chang¹, Shyh-Shyan Lin¹, Chin-En Tsai¹, Hung-Yi Wu¹

¹Pingtung University of Science and Technology

Watching the pet bird industry has high production value in Taiwan. Nowadays, many people use breeding bird watching as their sideline business. According to statistics, parrots have an output value of only 7 billion yuan. The average monthly turnover of a bird shop is about 2 million yuan. Parrot APV and PBFD are the causative agents of the parrot&#39;s important disease. The pigeons have a production value of up to 70 billion in Taiwan and their bacterial pathogens. (eg E. coli, Salmonella) will cause pigeons to squat, water squeaky, green feces, loss of appetite, indigestion, etc., making the pigeons lose the value of the game. Therefore, this study explores the more common diseases of these two parrots: Parrot APV and PBFD, based on the incidences of different months, bird species, and regions were counted, respectively, and data were used to indicate that this study also counted the effective drugs for the drug sensitivity test of pigeon racing cases. The statistical results of this study provided the owner of the disease

to the disease prevention and future veterinarians for the disease. Treatment analysis and diagnosis basis. The statistics of this study were completed since January 106 in December 106. The incidence of seasonal analysis of A virus infection in the double virus results: 7% in spring (February to April), 13% in summer (May to July), and 31% in autumn ( August to October), 4% in winter (November to January); Seasonal analysis of PBFD infection. Results: Spring 1%, Summer 15%, Autumn 11%, Winter 0%. APV infections in bird’s breed analysis of the incidence of results: 8% of Macaw, 12% of African Grey Parrot, 5% of Sun Parakeet, 5% of Monk Parakeet, 5% of White-bellied Parrot; PBFD infection in birds breed analysis of the incidence of results: 4% of macaws, 5% of African Grey Parrot, 6% of Monk Parakeet, 6% of White-bellied Parrot, 4% of Lory Parrot, and APV of double virus to regional analysis The incidence rate results are: 2% in the north, 13% in the middle, 35% in the south, and 0% in the east. The incidence of PBFD by region analysis results: 1% in the north, 7% in the middle, 24% in the south, and 0% in the east. Effective rate of pigeon susceptibility: Kanamycin 8%, erythromycin 8%, doxycycline 8%, cephalexin 25%, ceftiofur 17%, chloramphenicol 8%, norfloxacin 25 %, Colistin 46%. Based on this analysis, the incidence of parrot APV virus and PBFD virus was significantly higher in summer and autumn. The APV virus had a higher incidence in macaws and gray parrots. PBFD virus was higher in monk parrots and gold headed parrots. The incidence of the two occurs more often in the southern region. The results of pigeon susceptibility tests were most effective with colistin and cephalexin.

Keywords: Parrot,Avian Polyoma Virus,Psittacine Beak &amp; Feather Disease,Pigeon

PA-23

Different Sources of PRP on Cell Growth and Wound Healing

Chung-Jui Chen¹, Chia-Ying Lin¹, Ching-Dong Chang¹, Shyh-Shyan Liu¹, Chin-En Tsai¹, Hung-Yi Wu¹

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

Since the 90, there has been a doctor to use Platelet-Rich Plasma (PRP) in the treatment of ligament injury, due to good results, gradually accepted by mainstream medicine, further become the subject of research subjects. In scientific journals around the world, their research covers orthopedic and diabetic wound management. The greatest advantage of PRP is that the "platelets" used in the treatment are from the patient itself, not the drug, and the platelet is originally the patient itself, after treatment to increase its concentration, and then injected to the desired location or apply to the wound. This study evaluated the effect of wound healing by using PRP produced by goat blood in mice of different species. Using PRP to evaluate the growth effectiveness of BHK21 cell lines, the relationship between cell growth rate and PRP concentration was explored. In this study, the PRP preparation produced by the equipment in the laboratory was divided into seven groups in the cell test. (A) No culture liquid (B) PRP (C) DMEM (D) DMEM+2%PRP (E) DMEM+5%PRP (F) DMEM+10%PRP (G) DMEM+20% PRP. In group E, the effect of cell healing was most significant, while Group F and Group G had an undesirable effect on cell healing due to liquid osmotic pressure, and in the proportion of (E) DMEM+5%PRP, the best growth effect could be achieved. In animal Experiment, the wound healing effect of 5~10% PRP was the best 20%PRP on the wound and the wound healing was not obviously helpful. The effect of cell growth and the effect of wound healing in mice was the most significant in 5~10%PRP concentration.

Keywords: PRP, BHK21, Mice, Wound healing

PA-24

Comparison of hCG and GnRH on Induced Ovulation during Early and Middle Diestrus in Lactating Dairy Cows

Chia-Tang Ho¹, Tsung-Ching Liu¹, Jacky Peng-Wen Chan¹

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

The aim of this study is to evaluate the effect of hCG on induced ovulation during early and middle diestrus in dairy cows. A total of 16 lactating cows were synchronized and randomly assigned to groups of early or middle diestrus that received the first PGF_2α administration (Day 0) on 5.5-6.5 days or 9-10 days after ovulation. In stage 1, cows were treated with 375 and 250 mg PGF_2α on Day 0 and Day 1, respectively, then administered with 250 mg GnRH or 3000 IU hCG in groups of 9-10 day (G and H group) and groups of 5.5-6.5 day (OvG and OvH group) on Day 2. Ovulatory response was consecutively monitored by ultrasonography on Day 3. In stage 2, cows were treated in the same manner converse to that in stage 1. The result demonstrated that 96.6% of the cows showed complete luteolysis, while only one cow failed in the OvH group. Although there was no significant difference in time of ovulation among four groups, the ovulation time after hCG injection (33.4 ± 2.7 h) was later (P < 0.01) than that of GnRH administration (29.2 ± 0.5 h). Ovulation rate was 93.1% (27/29) in total and had no significant difference among groups. To sum up, though hCG group have longer time of ovulation compared to that in GnRH group, both hormones were effective for the fixed time of induced ovulation during early or middle diestrus in dairy cows.

Keywords: hCG, ovulation, dairy cows

PA-25

Coryneform Hyperkeratosis in NOG Mice

Yan-Xiu Lin¹, Yun-Chieh Tuan¹, Fang-Yi Tsai², Yi-Hui Su¹, Ying-Chen Wu², Shu-Jung Chang³, Yun-Jie Hung³, Ji-Hang Yin², Jiunn-Wang Liao^1,2  

¹Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung.

²Animal Disease Diagnostic Center, National Chung Hsing University, Taichung.

³Chang Bing Show-Chwan Memorial Hospital, Changhua.

Male NOG mice, 14-week old, showed multiple pale tan scale-like crusts on the shaved area at the dorsal aspect of the skin. The morbidity was 9.8% (5/51). Grossly, multifocal tan to brown scales were found on the dorsal back skin and were measured approximately 0.1 x 0.2 x 0.1 cm in size. Histological finding in the affected haired skin was multifocal, moderate chronic hyperkeratosis with moderate acanthosis. In addition, basophilic bacterial clumps were found in the keratin layer. Impression smear from the scrapping through the lesional areas stained with Diff-Quik revealed gram positive, clubbed shaped and V shaped arrangement rod bacteria, which was identified through 16S rRNA gene sequencing and had 98% similarity with Corynebacterium mastitidis. Furthermore, same bacteria were cultured from the hair in the razor. Differential diagnoses in this case including scaly skin disease, etiology consistent with Corynebacterium bovis, and particularly frequent observed in the experimental mice, bacterial infection such as Staphylococcus xylosus, Proteus and opportunistic bacteria, and low ambient humidity. However, Corynebacterium mastitidis was cultured predominately in this case and the environment the NOG mice habituated were reported adequately. Therefore, Coryneform hyperkeratosis in NOG mice was made in this case.

Key Word: Corynebacterium mastitidis, Hyperkeratosis, NOG mice

PA-26

The Comparison of the Effects of Nano Silver in the Prevention and Treatment of the Simulated Native Chicken Infected with Salmonella pullorum

Sheng­­-Wen Chen ¹, Chia-Ying Lin¹, Ching-Dong Chang¹, Shyh-Shyan Liu¹, Chin-En Tsai¹, Hung-Yi Wu¹

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

Salmonella spp. is a pathogenic bacterium that infects a wide range of hosts. Avian Salmonellosis has been the focus of the local health authorities. Pullorum Disease (PD), caused by Salmonella pullorum, has a high mortality rate and it is transmitted vertically (transovarian) to the next generation, infected juvenile might experience symptoms such as enteritis and white diarrhea. Human Salmonellosis might be caused by the consumption of contaminated poultry products, symptoms may include abdominal pain, vomiting, nausea and diarrhea and it might be fatal in severe cases, harming both animals and humans. The domestic poultry farm has been lacking efficient Salmonella-elimination strategies, however antibiotics has been widely used to eliminate Salmonella, but there are consequences such as producing antibiotic-resistant bacteria, the abuse of antibiotics and antibiotic withdrawal period. To overcome the difficulties of eliminating Salmonella, field trials were conducted in this study to investigate the effects of different concentration of nano-silver in the prevention and treatment of the simulated native chicken infected with Salmonella pullorum. For the prevention, comparison was made between different concentration of the nano-silver solution (diluted to 1000 times (1 ppm), 2000 times (5 ppm) and 500 times (20 ppm)), a continuous administration of nano-silver solution was given before introducing the bacteria strain, another continuous administration of nano-silver solution was given for seven days after the infection was confirmed, then, an observation of macroscopic changes and an examination of the presence of nano-silver residue in the muscles and liver were carried out. From the results, the most effective nano-silver dilution was 500 times (20 ppm). Through the macroscopic observation, 5 of the 6 dissected simulated native chicken showed fewer intestinal lesions and diarrhea (reduced by approximately 83.3%) compared to those without the injection of nano-silver solution. Nano-silver residue was detected in the muscles and liver with the concentration of 1 ppm and 5 ppm respectively. For the treatment, comparison was made between different concentration of the nano-silver solution (diluted to 1000 times (10 ppm), 500 times (20 ppm) and 250 times (40 ppm)), first, the bacteria strain was introduced, then, a continuous administration of nano-silver solution for seven days was given after a confirmation of the disease was made by Polymerase Chain Reaction (PCR) test. From the results, the most effective nano-silver dilution was 250 times (40 ppm). Through the macroscopic observation, 4 of the 6 dissected simulated native chicken showed fewer intestinal lesions and diarrhea (reduced by approximately 66.7%) compared to those without the injection of nano-silver solution. The nano-silver residues were absent in the muscles for all concentrations, but there was nano silver residue detected in the liver with the concentration of 10 ppm. The results of this study showed that the most effective concentration for prevention is diluted to 500 times (20 ppm), there is a positive correlation between the nano silver residues and application period.

Keywords: nano-silver, Salmonella spp., prevention effects, treatment effects, nano-silver residue

PA-27

Antimicrobial Sensitivity Test of Clinical Cases of Canine and Feline Samples

Chia-Wen Hsieh, Yu-Shin Huang and Chin-En Tsai^*

¹Department of Vaterinary Medicine, National Pintung University of Science and Technology, Pintung, Taiwan

Nowadays, most clinical veterinarians treat animals with antibiotics based on experience rather than ensuring that these antibiotics can really inactivate bacterial pathogens. This type of practice contributes to more antibiotic resistance bacteria. To investigate the relationship of bacteria and treatment with antibiotics, various specimens were collected from animal hospitals throughout Taiwan. Isolates were confirmed using standard biochemical tests and polymerase chain reaction using specific primers. The disk diffusion method was used to investigate antimicrobial susceptibility. The preliminary results showed that the most common isolates were Escherichia coli, and Staphylococcus spp. The most effective antibiotics were amikacin and enrofloxacin. In conclusion, the results demonstrated that amikacin and enrofloxacin can be used to inhibit canine and feline bacterial isolates. These results then, provide clinical veterinarians options with the most effective antibiotics.

Keywords: Antibiotics, Disk diffusion test, antimicrobial sensitivity test

PA-28

Effect Evaluation of Leukoreduction Filters on Canine Whole Blood

Chi-Ya Wang, Hsu-Hsun Lee, Lee-Shuan Lin, Cheng-Shu Chung, Yi-Lun Tsai

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

The most commonly seen transfusion reaction during blood transfusion is febrile non-hemolytic reaction, which is highly associated with the accumulation of cytokines and chemokines released from the donor leukocytes and platelets. However, leukoreduction has not yet been broadly utilized in the field of veterinary transfusion medicine. In this study, 20 canine whole blood samples anticoagulated with CPDA-1 were filtrated through the electrical-charge selection leukoreduction filters. Evaluations on the hematology and erythrocyte morphology from each sample at three time-points (pre-filtration, post-filtration and 35-day post-storage) were conducted. The values acquired at the three time-points were also statistically analyzed with Kruskal-Wallis test followed by post-hoc tests. The hematologic values were measured with IDEXX Procyte Analyzer, and the morphology examination was done manually under light microscopy according to the morphological evaluation scale. According to the analyses on the collected samples, the whole blood samples maintained 92% of the original erythrocyte counts without significant variation in erythrocyte morphology. The WBC manual counts at the three different timepoints were measured as 1.93´10⁹/ 250mL (Pre-filtration), 5.29×10⁷/ 230mL (Post-filtration) and 4.0×10⁷/ 230mL (Post-storage). The WBC and platelet counts were both significantly reduced by the leukoreduction filters. The filtration using the leukoreduction filters showed the success in depleting more than 90% of the WBC and platelet counts from the original whole blood products while maintaining over 85% of the RBCs without causing significant morphological changes.

Keywords: Canine whole blood, transfusion reaction, leukoreduction, hematology, erythrocyte morphology

PA-29

Development of ELISA Coated with PRRS Virus N Protein Expressed by Baculovirus System

Chang-Feng Shen, Pei-Fang Hsieh, Da-Yuan Tseng, Shan-Chia Ou, Ming-Kun Hsieh

¹Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan

Porcine reproductive and respiratory syndrome virus (PRRSV) is the leading cause of porcine reproductive and respiratory syndrome, which has significant negative economic impact on the swine industry. The nucleocapsid (N) protein of PRRSV, which contains rich B cell and T cell epitopes, can induce abundant long-lasting antibody at the early time of infection and be a suitable antigen for ELISA to detect antibody against PRRSV. Baculovirus expression system using many of the protein modification, processing, and transport in higher eukaryotic cells is usually applied for expressing virus proteins. The objective of this study was to express N protein of PRRSV by baculovirus expression system for development of PRRS ELISA. The N gene was amplified from PRRS vaccine strain by reverse transcription PCR and cloned into to the pFastbac Dual vector assigned as a pFastbac Dual/N plasmid. The pFastbac Dual/N plasmid was transformed into DH10Bac competent cell to construct recombinant N bacmid and this recombinant N bacmid was transfected into SF9 cell to generate recombinant N baculovirus. The expression of N protein was confirmed by Immunofluorescence assay (IFA) and western blotting. Green fluorescence in IFA and a band of 15 kDa on western blots were observed in SF9 cells infected with recombinant N baculovirus and stained with anti-His MAb and anti-PRRSV serum. The results indicated that N protein of PRRSV was successfully expressed by baculovirus expression system and maintain its proper antigenicity.

Keywords: PRRS ELISA, Nucleocapsid protein, Baculovirus expression system

PA-30

Development of Triage Criteria for First Aid in Wild Bird Casualties

Chia-Ying Wu¹, Pin-Huan Yu¹, Chau-Hwa Chi¹

¹Institute of Veterinary Clinical Science, National Taiwan University, Taipei, Taiwan

To overcome high mortality of wild bird causalities and achieve early prediction of prognosis, it is necessary to set up a triage system and first aid criteria. A scoring system was established to evaluate the outcomes of different levels of stabilization manner under different patient factors. Signalmen and physical examination parameters including, heart rate, respiratory rate, mentation, body condition score, severity of symptoms and hydration state of 156 rescued birds from several wildlife rescue facilities in Taiwan were collected from 2016 to 2018. Outcome and stabilization manner based on invasiveness including overnight stabilization, oxygen support, fluid therapy (subcutaneous or intravenous and intraosseous) during the first 48-hour-hospitalization were also recorded. To evaluate the correlation between physical parameters, medical managements and prognosis, logistic regression analysis, Kruskal-Wallis Test and sensitivity analyses were performed. The result indicates the heart rate score and mentation score have positive correlation with mortality rate (P<0.05). The system could be applied to or referenced in management of wild bird rescue in the first 48 hours.

Key words: Avian casualty, initial stabilization, triage, management, scoring system, wild bird

PA-31

Construction of Recombinant Infectious Bursal Disease Viruses Expressing Foreign Proteins by Reverse Genetics Technology.

Yu-Hsin Wang¹, Pei-Fung Hsieh¹, Tsung-Ting Chiu¹, Bing-Yi Chen^1,2, Jui-Hung Shien³, Ming-Kun Hsieh¹

¹Institute of Microbiology and Public Health, National Chung Hsing University, Taichung,

²Institute of Animal Health Research, Animal Drugs Inspection Branch, Miaoli,

³Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan

Reverse genetics technology has been successfully developed for numerous RNA viruses as virial vectors for foreign antigen delivery. Recently, infectious bursal disease virus (IBDV), a causative agent of infectious bursal disease (IBD) in chicken, has been explored for its potential to serve as a deliver system. The purpose of this study was to construct a recombinant IBDV as a foreign antigen delivery system by revers genetics technology. The sequences containing 4 virus-neutralizing (VN) epitopes of Newcastle disease virus (NDV) fusion (F) protein was inserted into the N-terminus of the nonstructural VP5 of IBDV. The F gene of NDV or enhanced green fluorescence protein (EGFP) gene was used to substitute partial sequences of IBDV VP3. By using the reverse genetics technology, recombinant IBDVs containing insertions or substitutions of sequences encoding the foreign proteins were engineered base on the backbone of very virulent IBDV strain, P3009. The recombinant IBDVs containing VN epitopes or EGFP were successfully rescued and the expressed foreign proteins were recognized by indirect immunoflurecence assay and western blotting. However, the rescue of the recombinant IBDV containing F protein had failed. This failure might be due to that the F protein is too large to be hold in the viral particle. These results indicated that VP3 and the N-terminus of VP5 of IBDV has shown to be capable of tolerating the substitution and insertion of small proteins, respectively. Thus, IBDV is potentially employed as a viral vector to carry foreign proteins fused with VP5 or VP3 to develop bivalent vaccines.

Keywords: Infectious bursal disease virus, Reverse genetics, viral vector

PA-32

Protein Expression and DNA Transport of Chicken IL-2 Fusion Cytokines

Yin-Chuan Huang¹, Hsiao-Wen Chiang¹, Han-Jung Lee² , Yi-Hsin Fan¹, Pei-Shan Wu¹, Kuan-Chih Chow³, Shiow-Her Chiou¹

¹Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan.

²Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien, Taiwan.

³Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan

The specific aims of this study are to express and transport chicken interleukin-2 (IL-2) fusion cytokines in prokaryotic and avian systems. IL-2, is an important cytokine mainly produced by T helper cells to modulate immune responses, in particular, to stimulate the proliferation of a variety of leukocytes. In veterinary medicine, the use of IL-2 as vaccine adjuvants has been investigated. In this study, a chemokine that can attract leukocytes to target locations was fused to IL-2 with a peptide linker by employing recombinant polymerase chain reaction (PCR) technique. Subsequently, the resultant DNA chemo-IL2 fragment was cloned into a prokaryotic expression vector, pET32a, to express recombinant chemo-IL2 fusion cytokine in Escherichia coli BL21 (DE3). After induction by using isopropyl β- D-1-thiogalactopyranoside (IPTG), chemo-IL2 fusion cytokine was harvested and purified by nickel (Ni2⁺) chelation affinity chromatography. We went on to examine the bioactivities of chemo-IL2 based on chemotactic and proliferating activities of chemokine and IL-2, respectively. On the other hand, cationic or amphipathic cell penetrating peptides (CPPs) that have been used to introduce biomolecules into a number of human cell lines may as well be exploited to carry chemo-IL2 DNA into chicken cells. As a feasibility test, we confirmed that CPP could carry pEGFP-N1 into a human cell line, CNE2. We constructed eukaryotic IL-2 fusion protein expression plasmids (pVAX- and pEGFP-chemo-IL2) and transported these plasmids into chicken cell line, DF-1, to express chemo-IL2 fusion cytokines in avian system. In the future, these fusion cytokines can be applied in veterinary medicine.

Keywords: IL-2, fusion protein, cell penetrating peptide

PA-33

Expression and Transport of Chicken CD40 Ligand Fusion Cytokines in Prokaryotic and Avian Systems

Hsiao-Wen Chiang¹, Yin-Chuan Huang¹, Han-Jung Lee², Yi-Hsin Fan¹, Pei-Shan Wu¹, Kuan-Chih Chow³, Shiow-Her Chiou¹

¹Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan

²Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien, Taiwan

³Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan

The specific aims of this study are to express and transport chicken CD40 ligand (CD40L) fusion cytokines in prokaryotic and avian systems. A chemokine that can attract and guide a variety of leukocytes to target locations was fused to CD40L, which is a cytokine that regulates immune responses such as macrophage activation. By employing recombinant polymerase chain reaction (PCR), both chicken chemokine and CD40L cDNAs are linked by a DNA fragment encoding a peptide linker. The resultant construct was cloned into pET32a to express chemo-CD40L fusion protein in Escherichia coli BL21 (DE3). After isopropyl β-D-thiogalactoside (IPTG) induction, we harvested recombinant chemo-CD40L fusion cytokine and purified it by nickel chelation affinity chromatography. In ongoing studies, we measured the chemotactic and macrophage activating activities of recombinant chemo-CD40L based on a chemotaxis assay and a nitric oxide production assay. To express this chicken fusion cytokine in avian system, we performed a feasibility test by using a cell-penetrating peptide (CPP) to carry pEGFP-N1 into the chicken fibroblast cell line, DF-1. The result showed that CPP was able to transport pEGFP-N1 into DF-1 cells and successfully expressed enhanced green fluorescent protein (EGFP) encoded by pEGFP-N1. We went on to clone chemo-CD40L DNA into pEGFP-N1 or pVAX1 eukaryotic expression vector and express chemo-CD40L cytokines in DF-1 cells. In the future, these fusion cytokines may serve as immunomodulatory molecules for avian vaccines in poultry industry.

Keyword: CD40L, fusion protein, cell penetrating peptide