PB-01

Purification of Porcine Circovirus Type 2 Virus-Like Particle of Evaluation Immune Efficacy

An-Ting Tong, Chi-Chi Wen and Guan-Ming Ke

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

Porcine circovirus type II (PCV2) is a non-enveloped single-stranded circular DNA virus. Among them, PCV2b and mPCV2b are highly virulent and usually infect pigs of 4 to 12 weeks old. This disease is known as porcine circovirus-associated disease (PCVAD). Clinical symptoms include marasmus and lymphadenopathy, etc. In this study, we used the baculovirus expression system to express the mPCV2b open reading frame 2 (ORF2) protein as an antigen and to improve the purity of the antigen by purification. Purified proteins, unpurified proteins and commercial vaccines were used to immunize 3-week-old piglets, and the other group was used as a control group. Boost was done on day 14 and challenged on day 28. Blood, feces, body temperature and body weight were collected weekly to observe piglets. Sacrifice was done on day 49, and collected the organ. As can be seen from the results, the antibody titer of the purified protein group and the unpurified protein group was higher than that of the commercial vaccine group, and the titer of the purified protein group increased by one week earlier than the other immunized groups. The purified protein increased more than the other immunized groups in terms of changes in body weight after challenge. No viral DNA was detected in the immunized group, as opposed to the positive reaction in piglets in the control group. Antibody titers of mPCV2b were further investigated using immuno-fluorescent stain.

Keywords: mPCV2b, ORF2, PCVAD, purification, antibody titer

 

PB-02

Development of Avian Infectious Bronchitis Vaccine Using Cell Culture Production

Yu-Fen Lin, Meng-Jung Chiang and Guan-Ming Ke

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

Avian infectious bronchitis virus (IBV) belongs to Coronavirus. It is an enveloped, single-stranded, positive-sense RNA virus with a diameter of about 70-200 nm. Infection by Avian infectious bronchitis virus would causes poultry respiratory distress, renal failure, tubal atrophy hypoplasia, a drastic drop in egg production, and about 15% to 90% mortality. Because different serotypes of the virus doesn’t give cross-protection, it is necessary to develop a vaccine against the new serotype IBV. IBV can proliferate in chicken embryos. At present, however, it is not easy to obtain the embryo of the SPF chicken since the cost is too high. Waste treatment of chicken embryo is also problemstic. Therefore, this study will establish a suitable cell line to produce IBV antigens, and use real-time quantitative polymerase chain reaction (qPCR) and Tissue culture 50% infectious dose (TCID50) for virus quantification and titer detection. For now, we have successfully cultured and passaged IBV using African green monkey kidney cells and the current titer of IBV up to 106 TCID50/ml after 30 generations of passage. The cells can observe the CPE phenomenon of obvious fusion, vacuolization, shedding and death. Finally, we will prepare live attenuated vaccine to immunize in SPF chickens and will conduct safety and efficacy trials to assess the effectiveness of the vaccine.

Keywords: Avian infectious bronchitis virus, cross protection, African green monkey kidney cells, live attenuated vaccine

 

PB-03

A Nested PCR Assay for the Simultaneous Detection and Differentiation of Babesia gibsoni and Babesia vogeli Infecting Dogs

Ping-Jun Chung and Pan-Chen Liu

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

Canine babesiosis is a widespread tick-borne disease caused by haematozoan parasites of the genus Babesia, including the so-called smallform (B. gibsoni) and the large forms (B. vogeli, B. canis and B. rossi). The severity of the disease depends on various factors such as the Babesia species involved, the age and the immune status of the host. Diagnosis of babesiosis is often impeded by the transient presence of the parasites in peripheral blood, as well as by their pleomorphic nature. Given the reports of an expanding geographical distribution of B. gibsoni and B. vogeli in dogs in Asia, the development of time-efficient and cost-effective diagnostic tools to detect and differentiate these two species is warranted. In this study, we designed and developed a novel polymerase chain reaction (PCR) assay targeting the 18S rDNA-ITS1-5.8S rDNA region of the parasite, for the simultaneous detection and differentiation of B. gibsoni and B. vogeli. The analytical specificity of the assay was assessed using blood samples positive for Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Hepatozoon canis, Toxoplasma gondii, and Leptospira interrogans. No amplification occurred for any of these samples. The analytical sensitivity of the PCR was evaluated using serial dilutions of genomic DNA extracted from individual canine blood samples infected by B. gibsoni (141 infected erythrocytes/ml) and B. vogeli (113 infected erythrocytes/ml). The PCR assay developed here in detected a low number of infected erythrocytes (i.e., 1.4 × 10−2 B. gibsoni, 1.1 × 10−2 B. vogeli ie/ml). The clinical specificity of the PCR assay was evaluated on more than 560 blood samples from dogs. Species-specific bands of the expected sizes (i.e., 401 bp for B. gibsoni and 298 bp for B.vogeli), and two bands in the co-infected blood samples were obtained. Of the 560 blood samples, 52 (9.3%) were positive for B. gibsoni only and 43 (7.7%) for B. vogeli only, whereas 3 (0.5%) was positive for both. This PCR assay represents a sensitive and reliable tool for the diagnosis of B. gibsoni and B. vogeli in areas where these species occur in sympatry, and will assist future epidemiological investigations aimed to track the expanding range of these parasites.

Keywords: Nested PCR assay, Babesia gibsoni, Babesia vogeli, dog

 

PB-05

Compare the Cytotoxic Effects of Single and Divided Treatment of Cyclophosphamide at the Same Total Dosage in Canine Lymphoma Cell Lines.

Chi Liang 1 and Shang-Lin Wang 2,3

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

2National Taiwan University Veterinary Hospital, College of Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan

3Graduate Institute of Veterinary Clinical Science, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

Cyclophosphamide is a commonly used chemotherapy drug for lymphoma treatment, while sterile hemorrhagic cystitis (SHC) is one of its major side effects. Previous study found that divided maximum tolerated dose of orally cyclophosphamide to three days can reduce the incidence of SHC. However, the exact therapeutic effect of divided cyclophosphamide dosage was not test since the patient received multidrug chemotherapy.

The half maximal effective concentration (EC50) of cyclophosphamide in three canine lymphoma cell lines (CLBL-1, CLC and UL-1) were evaluated and used for further examinations. Three cell lines were cultured with EC50 of Cyclophosphamide for two days. The other study group was cultured with half EC50 of Cyclophosphamide for two times on consecutive two days.

The results revealed cyclophosphamide was effective to B and non-B, non-T lymphoma cell lines than T lymphoma cell line. Full dose of cyclophosphamide was significantly killed more lymphoma cells than divided treatment (P = 0.018 and 0.031). The similar result was not found in T lymphoma cell line (P = 0.328). Our study suggest that use divided dosage of cyclophosphamide to reduce the SHC will influence the effect of chemotherapeutic drug. To use maximum tolerated dose of cytotoxic drug is still recommended.

Keywords: Canine, cyclophosphamide, lymphoma

 

 

 

PB-06

Cloud-Based Temperature Monitor System of Companion Animals

Po-Kai Chen1, Pei-Yi Liu1, Cheng-Shu Chung1,2 and Lee-Shuan Lin1,2
1Department of Veterinary Medicine, National Pingtung University of Science and Technology
2Veterinary Medical Teaching Hospital, National Pingtung University of Science and Technology

Body temperature measurement is an important part of physical examination in companion animals. Although there are various methods to measure core temperature, rectal temperature measurement (RTM) is the most common way. There are still many disadvantages in RTM, therefore, the aim of this study was to assess if cloud-based temperature monitor measurement could be a reliable and more comfortable method for hospitalized animals and to reduce measuring time to increase medical quality. In this study, paired rectal and skin temperature (STM) were measured in 30 dogs and 18 cats. A higher correlation was found between RTM and STM (r=0.8617; p<0.0001) in dogs; however, a moderate correlation was found (r=0.6689; p<0.0001) in cats. When using the device, it should be in an appropriate ambient temperature because we found STM was be affected by the ambient temperature easily. We guessed the reason why the bias in the cats was bigger than the dogs was that cats were more stressful during RTM and this measurement may rise more rapidly than STM. The number of samples in this study is insufficient, so it is hoped that more samples can be collected to evaluate the possibility of the temperature monitor for future use in clinical medicine.

Keywords: Companion animals, rectal temperature, skin temperature, cloud-based temperature monitor

 

PB-07

The Etiologic and Pathologic Analysis of Swine Arthritis

Cheng-Yu Tsai1, Chao-Nan Lin1 and Ming-Tang Chiou1,2

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

2Research Center for Animal Biologics, National Pingtung University Science and Technology, Taiwan

The symptoms of arthritis include joint pain and stiffness. It not only affects the welfare of the animals and the appearance of carcasses but also causes severe economic losses in the swine industry. The purpose of this study was to analyze the gross lesions and histopathologic lesions of arthritis caused by different pathogens to provide initial direction of diagnosis for veterinarians. The suckling and nursery pigs with swollen joints were collected from Animal Disease Diagnosis Center of National Pingtung University of Science and Technology during 1/21/2017 to 2/1/2018 for molecular detection of Haemophilus parasuis (Hps), Streptococcus suis (Ss) and Mycoplasma hyorhinis (Mhr), and pathologic lesions examination. In a total of 152 samples, 63 samples were detected. The detection rate of Mhr, Hps and Ss were 25%(38/152), 9.9%(15/152), 1.3%(2/152), respectively, and at least two pathogens co-infection detection rate was 5.3%(8/152). The results of gross lesions showed that increased yellowish synovial fluid with clean to slight turbidity was most likely caused by Mhr or Hps. Yellowish with severe turbidity was most likely caused by Ss. Histopathologic lesions of Mhr and Hps revealed that non-suppurative and fibrinous synovitis, and moderate to severe hyperplasia of synovial membrane. Suppurative and fibrinous synovitis were seen in Ss. In summary, the predominant pathogen causing arthritis in suckling and nursery pigs is Mhr followed by Hps, and both of them present clean to slight turbidity of synovial fluid.

Keywords: Haemophilus parasuis, Streptococcus suis, Mycoplasma hyorhinis, arthritis

 

PB-08

Application of Closed Reduction and Physical Therapies to Hip Dysplasia in a Dog

Kai-Xuan Wang1, Jui-Te Wu1,2, Kuan-Lun Wu2, Ming-Han Huang1, Yao-Chi Su1 and Han Hsiang Huang1,2

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

2 Veterinary Teaching Hospital, National Chiayi University, Chiayi, Taiwan

Canine hip dysplasia (CHD) is one of the most prevalent developmental orthopedic diseases, particularly in large breed dogs whereas it can also occur in smaller breeds. CHD is characterized by a complex and abnormal development of the hip joint with different degrees of laxity and subluxation and eventually results in secondary osteoarthritis (OA). An eight-month-old, intact female Golden retriever presented with acute weight-bearing left hindlimb lameness (score 2) and abnormal gait. Positive Ortolani sign was found while craniocaudal views of radiographic examinations showed subluxation at left coxofemoral joint. Norberg angle (NA) was less than 105° as femoral head coverage (FHC) was lower than 50% in radiographic evaluation. Hip dysplasia and subluxation at the left hindlimb was diagnosed in this dog. The external fixation Ehmer sling and physical therapies were selected as remedies to manage the condition. Two-week Ehmer sling application was selected to accomplish the closed reduction. During Ehmer sling application, transcutaneous electrical nerve stimulation (TENS) and manual therapy were performed for the dog. Physical therapies included therapeutic ultrasound, TENS, passive/assisted exercise, manual therapy, cryotherapy and gait evaluation were conducted for 6 weeks after the sling was removed. Re-luxation of the hip did not occur available for hospital follow-up from Jan 2017 to Oct 2018.

Keywords: canine hip dysplasia, subluxation, closed reduction, physical therapies

 

 

PB-09

Real-Time Surveillance of Swine Bacterial Pathogens by Using Surface-Enhanced Raman Scattering System

Ruei-Chen Hung1, Chia-Chi Chen2, Chien-Chao Chiu1, Jen-Wei Lin1, Ping-Min Huang1, Ying-Ching Hung1, Pin-Tasn Tsai1, Cheng-Yao Yang3, Cho-Lin Li2, Chia-Yu Lin2 and Shao-Wen Hung2

1Division of Animal Resources, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan

2Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan

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

This shift away from traditional pig farming to large-scale intensive methods has resulted in the increasing cases of complex, mixed infections, or secondary infections. The early diagnostic and disease prevention become more important for pig farm management. However, antibiotic treatment and atypical diseases symptoms might be interfered clinical diagnosis. In order to perform accurate pathology diagnosis, we need to combine the strengthening laboratory diagnosis and traditional diagnostics. Surface-enhanced Raman Scattering (SERS) spectroscopy has recently been shown to be a potentially powerful whole-organism fingerprinting technique and is used for the rapid identification of bacteria. Biosensors based on SERS hold great promise as a platform for sensitive and rapid detection of bacterial pathogens and decrease time of diagnosis. In this study, we tried to use SERS to rapidly characterize and evaluate for the detection and identification of pathogens in the samples of SPF mice infected with two zoonotic bacterial pathogen infections and four kinds of swine bacteria mixed in the sera and feces. Spectra of bacteria recovered from sera and feces were compared to pure culture bacteria. The results indicated that successful detection, identification, and classification of these bacteria from mice specimens (sera and feces) and swine specimens (sera and feces) by using SERS. The approach may be a new and powerful tool for real-time surveillance of animal bacterial disease pathogens in clinics.

Keywords: Real-time surveillance, Surface-enhanced Raman scattering system, Swine bacterial pathogens, Zoonotic bacterial pathogens

 

PB-10

Establishment of the Mouse Model with Parkinson’s Disease and Drug Screening System

Chia-Chi Chen1, Jen-Wei Lin2, Chien-Chao Chiu2, Ruei-Chen Hung2, Ping-Min Huang1, Ying-Ching Hung1, Pin-Tasn Tsai1, Cheng-Yao Yang3, Cho-Lin Li1, Chia-Yu Lin1 and Shao-Wen Hung1

1Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan

2Division of Animal Resources, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan

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

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases that affects about 1.5% of the global population over 65 years of age. PD animal models have contributed a large part to our understanding and therapeutics developed for treatment of PD. Hence, the establishment of a PD model in murine for R&D of anti-PD drugs is much needed. Here, we have successfully established a mouse model with PD and drug screening system. Via using the PD model and drug screening system, we can rapidly screen the functional materials in the agriculture. We hope to apply this PD mouse model and drug screening system for R&D of the novel anti-PD drugs and therapeutic strategies in the future.

Keywords: Drug screening system, In vivo, Mouse models, Neurodegeneration, Parkinsons disease

 

PB-11

Establishment of the Ectopic Xenograft Liver Cancer-Bearing Mouse Model and Anti-Liver Cancer Drug Screening System

Chia-Chi Chen1, Chien-Chao Chiu2, Jen-Wei Lin2, Ruei-Chen Hung2, Ping-Min Huang1, Ying-Ching Hung1, Pin-Tasn Tsai1, Cheng-Yao Yang3, Cho-Lin Li1, Chia-Yu Lin1 and Shao-Wen Hung1

1Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan

2Division of Animal Resources, Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan

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

Liver cancer is the leading cause of cancer death worldwide. At present, the preclinical trials of liver cancer mouse models have their own advantages and disadvantages, failing to reflect the clinical conditions of most patients. The developed a set of tumor-bearing mouse model and drug treatment strategy for R&D of anti-liver cancer drugs. In this study, tumor cells (1 × 107/100 μL of HUH-7 cells) were implanted ectopically in NOD SCID mice. 5-FU as a reference drug was administrated at 60 mg/kg body weight (thrice/wk for 4 wks) through intravenous injection. Blood samples were collected to analyzed drug safety per week. At the end of experiment, the tumor mass, lung, liver, and spleen were collected. The results showed that after 5-FU was administered three times per week for a total of 12 times, tumor weight and volume as well as animal mortality were reduced. Moreover, the expression of the HUH-7 cell proliferative markers were also ameliorate. To compare with the control groups, 5-FU caused a significant increase in serum levels of GOT and GPT. According to above results, the anti-cancer drug screening system include tumor-bearing mouse model, 5-FU therapy strategy, tumor histopathology and immunohistochemistry analysis, and assessment of serum liver and kidney function. We hope to apply this anti-cancer drug screening system for R&D of the novel anti-cancer drug and therapeutic strategies in the future.

Keywords: Xenograft, Ectopic implantation, In vivo, Liver cancer

 

PB-12

Application of Computed Tomographic Quantification in Calves with Naturally Occurring Pneumonia

Yun-Jung Wu1, Cheng-Shu Chung1,2 and Lee-Shuan Lin1,2

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

Calf pneumonia is the primary cause of death of weaning calf. Auscultation, necropsy, pathogen analysis, calf respiratory scoring system, and some imaging tools, such as ultrasound, radiology, computed tomography, are currently applied for calf pneumonia diagnosis. There are many researches and applications of computed tomography in human pneumonia, including disease severity assessment, the relationship between computed tomographic quantifications and clinical symptoms, and the prognosis assessment. In addition to human, computed tomographic quantification also apply for other species, such as dogs and mice. However, the current application of computer tomography in calf pneumonia is still rare, and many stay in the research stage. The present study traced back to 12 calves with naturally occurred respiratory disease. Collected their computed tomographic images, and investigate the relationship between computed tomographic quantifications and clinical physiological values. Then compare to other diagnostic methods of calf pneumonia, including calf respiratory scoring system (CRSC), auscultation score, leukocyte esterase score of alveolar fluid, and pathology scoring system. After individual treatment, the body temperature medium, fever period, and survival time were used as prognostic indicators to evaluate the correlation between therapeutic effects and computed tomographic quantifications, in order to know the applicability of computed tomographic quantifications in prognosis. The results showed that compared with other diagnostic tools, computed tomographic quantifications were significantly associated with auscultation scores, bovine respiratory scoring system, and pathological scoring system; however, the associations with prognostic indicators was weak.

Keywords: calf pneumonia, computed tomography, quantification, prognosis

 

PB-13

Development of Mycoplasma synoviae Bacterin – Vaccine Efficacy Studies on Laying Hen

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

Animal Technology Laboratories, Agricultural Technology Research Institute, Zhunan, Taiwan

Avian mycoplasma infections continue to cause economic losses in poultry industries. Mycoplasma synoviae (M. synoviae) infection not only causes respiratory diseases, arthritis, and growth retardation in turkey and chicken, but also causes altered shell surface, thinning, increased translucency, cracks and breaks in the eggshell apex in laying hen. Based on previous studies, M. synoviae bacterin vaccine developed in our lab can significantly protect specific pathogen-free (SPF) chickens from M. synoviae challenge. This novel vaccine also proved its safety and effectiveness in native chicken at field farm. In the present study, we further applied this vaccine on laying hen and investigated its protective efficacy. Our results showed that this novel M. synoviae bacterin vaccine not only decrease M. synoviae induced mortality and body weight loss, but also improve average egg weight and production in vaccine received laying hen. The M. synoviae bacterin vaccine described herein can be applied on the farm and will be of great benefit to poultry industry.

Keyword: Mycoplasma synoviae, Vaccine, Laying Hen

 

PB-14

Sensitivity of Porcine β2 Integrin Transgenic Mice to Actinobacillus pleuropneumoniae Serotype 2

Jung-Chao Hung1, Zeng-Weng Chen1, Jyh-Perng Wang1, Jiunn-Horng Lin1 and Shih-Ling Hsuan2

1Division of Animal Medicine, Agricultural Technology Research Institute, Zhunan, Taiwan

2Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan

To investigate the susceptibility of wild type (WT) and transgenic (TG) mice to Actinobacillus pleuropneumoniae (AP), mice were intraperitoneally inoculated with AP serotype 2, and the mortality assessed and compared. It was found that the death of TG mice occurred at time points earlier than that of WT mice after challenge. We inferred that the higher susceptibility of TG mice to AP may result from the expression of porcine CD18 (pCD18). Further cytotoxicity assay revealed that pCD18-cells were more susceptible toward ApxII/III comparing to pCD18-cells. Apx toxins induced significant levels of cytotoxicity in a concentration-dependent manner in pCD18-cells, which signifies an indispensable role of pCD18 in ApxII/III cytotoxic effect.

Keywords: Actinobacillus pleuropneumoniae, β2 integrin, transgenic mice

 

PB-15

Establish a Swine Dysentery Challenge Model

Tzu-Ting Peng, Huei-Yu Lee, Jian-Wen Chen, Chin-Yu Lo, Chin-Shu Chang, Jiunn-Horng Lin and Zeng-Weng Chen

Division of Animal Medicine, Agricultural Technology Research Institute, Zhunan, Taiwan

Swine dysentery (SD) is characterized as mucohaemorrhagic colitis and diarrhea. The disease caused severe production losses in pigs and associated with infection by strong beta-hemolytic strain of Brachyspira hyodysenteriae (B. hyodysenteriae). SD only develops in pigs and causes a severe inflammation of the large intestine with a bloody mucous diarrhea. The gross lesions restricted to the large intestine that typically include variable mucosal thickening, hemorrhage, fibrinonecrotic exudate and abundant mucus. The morbidity is up to 90-100% and the mortality is 20-30%. To prevent SD, using feed additives was common in the past. However, considering the risk of antimicrobial resistance and the banning of antimicrobials as feed additives, developing a SD vaccine is critical. This study was to establish a SD challenge model in pigs using an ATCC strain of B. hyodysenteriae. Four 9-week-old commercial pigs were used in this study and challenged with B. hyodysenteriae ATCC strain. Clinical signs of the pigs were recorded daily including activity, appetite, body condition and feces. All pigs were sacrificed at 14 days of post challenge and the gross lesions of large intestines were examined and scored. The results demonstrated that all pigs show typical SD clinical signs, gross lesions and microscopic lesion after challenging with an ATCC strain of B. hyodysenteriae. And the scoring system can use to evaluate the clinical signs and gross lesions. The challenge model could be apply for evaluating the efficacy of SD vaccines or antimicrobial alternatives in the future.

Keywords: Swine dysentery, Brachyspira hyodysenteriae, challenge model

 

PB-16

The Validation of Using Whole Genome Sequencing Data as a Subtyping Tool for Salmonella

Jing-Fang Huang, Jyh-Perng Wang, Jiunn-Horng Lin, and Zeng-Weng Chen

Animal Technology Labroatories, Agricultural Technology Research Institute, Zhunan, Taiwan

The nontyphoidal Salmonella (NTS) which associated with animal products is one of the most common foodborne pathogens. Subtyping forms the basis of surveillance for Salmonella. Although serology and pulsed field gel electrophoresis (PFGE) are widely used for the classification of Salmonella, it can be time consuming and laborious, and still leave a part of untypable isolates. Public health microbiology is currently being transformed by whole genome sequencing (WGS), which opens the door to subtype determination using WGS data. This study has used WGS data to dissect the phylogenetic relationships based on single nucleotide variation (SNV) for Salmonella serotypes. The result indicated that the bioinformatics approach may be provide identification and typing in a single method, and to be a replacement tool of serology and PFGE.

Keywords: Salmonella, subtyping, whole genome sequencing

 

PB-17

Efficacy Test of Bivalent Vaccine of PCV2 and Mycoplasma

hyopneumoniae in Primary SPF Pigs

Ming-Wei Hsieh, Shiang Li Ru, Chin-Shu Chang, Weng-Zeng Huang,

Jyh-Perng Wang, Jiunn-Horng Lin and Zeng-Weng Chen

Division of Animal Medicine, Agricultural Technology Research Institute, Zhunan, Taiwan

Porcine respiratory disease complex (PRDC) is a multifactorial and complex disease caused by combination of infectious viral or bacterial pathogens. Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2) are important pathogens to cause PRDC. Evidences show that vaccination is an effective way to control both diseases. In the present study, eighteen primary SPF pigs were immunized with a bivalent vaccine consisted of Mycoplasma hyopneumoniae bacterin and recombinant PCV2 proteins. Followed by dual challenged by Mycoplasma hyopneumoniae and PCV2. The results indicated that pigs immunized with bivalent vaccine showed no adverse effect and elicited good immune responses toward PCV2 and M. hyopneumoniae. Reduced M. hyopneumoniae lung lesion score were noted in vaccinated pigs and the level of PCV2 viremia were lower in compared to unvaccinated pigs. This study demonstrates that the bivalent vaccine is able to protect pigs against either PCV2 and M. hyopneumoniae dual challenge based on immunological and pathological evaluation in this study.

Keyword: Bivalent vaccine, PCV2, Mycoplasma hyopneumoniae

 

PB-18

Dose Determination of a Mycoplasma hyopneumoniae and Mycoplasma hyorhinis Bivalent Vaccine in Specific Pathogen Free Pigs

Chiung-Wen Hsu, Yu-Hao Lee, Wen-Cheng Huang, Hui-Jei Lin, Chin-Shu Chang, Jiunn-Horng Lin, Jyh-Perng Wang and Zeng-Weng Chen

Division of Animal Medicine, Agricultural Technology Research Institute, Zhunan, Taiwan

Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are the causative agents of Mycoplasmal pneumonia. Of them, Mycoplasma hyorhinis also induces arthritis and polyserositis in piglets. Vaccination is considered to be one of the most effective strategies to control swine Mycoplasma infection. In the present study, different doses of bivalent vaccines were evaluated for safety, serological conversions and protective efficacy against Mycoplasma hyopneumoniae in specific pathogen free (SPF) pigs. The results indicated that all immunized groups showed no adverse effect and antibodies again Mycoplasma hyopneumoniae and Mycoplasma hyorhinis were induced. Also, reduced lung lesion scores were observed in vaccinated pigs as compared to non-vaccinated pigs. This study demonstrated small amount of Mycoplasma hyopneumoniae bacterin in the bivalent vaccine is still effective in protecting piglets from virulent Mycoplasma hyopneumoniae challenge.

Keyword: Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, vaccine

 

PB-19

The Prevalence and Ribotypes of Clostridium difficile in Dogs in Changhua Area

Li-Chiung Hung1, Ying-Chen Wu2, Ter-Hsin Chen2 and Chung-Hsi JiuJiu Chou1

1Food Safety Research Unit, School of Veterinary Medicine National Taiwan University, Taiwan

2Graduate Institute of Veterinary Pathobology, College of Veterinary Medicine, National Chung Hsing University, Taiwan

Clostridium difficile is an anaerobic, spore-forming Gram-positive bacillus that is able to cause self-limited diarrhea in humans. Serious infection may cause pseudomembranous colitis and toxic megacolon, and may lead to sepsis or even death in patients. Although Clostridium difficile infection (CDI) has traditionally been considered as an hospital-associated disease that due to medical intervention, in recent years, the ratio of community-acquired C. difficile infection (CDI) has an upward trend. Several C. difficile ribotypes isolated from animals such as cattle, pigs, horses and dogs were overlap in human patients, therefore, C. difficile was considered had zoonotic potential. Due to dogs are the most intimate companion animals, this study surveyed the prevalence of C. difficile from companion dogs and stray dogs in Changhua area, and further carried out the detection of ribotypes, toxin genes and antimicrobial susceptibility tests. Stool samples were collected between January to May 2014, and the number of samples is 435. C. difficile was isolated from 31 out of 435 (7.13%). The prevalence of C. difficile in companion dogs and stray dogs were 14.14%(28/198) and 1.27%(3/237) individually. And the tcdA+/tcdB+/cdtA-/cdtB- gene prevalence of companion dogs and stray dogs were 58.06%(18/31) and 33.33%(1/3) individually. The results of antimicrobial susceptibility tests showed that there were 4 isolated strains were intermediate and another 4 isolated strains were resistance to moxifloxacin. In addition, all isolated strains were susceptible to metronidazole and vancomycin. This study is the first report of C. difficile prevalence in dogs in Taiwan.

Keywords: Clostridium difficile, ribotype, toxin gene

 

PB-20

Recovery from Paraparesis by Acupuncture and Physical Therapies after Hemilaminectomy for Thoracic Hansen Type I Intervertebral Disk Disease in a Dog

Meng-Hua Tsai1, Chi-Hsien Chien2, Kun-Wei Chan1,3, Ming-Huang Chang1, Wei-Kai Lin1, Yu-Shan Tsai1, Cheng-Feng Tsai3 and Han Hsiang Huang1,3

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

2Department of Post-Baccalaureate Veterinary Medicine, Asia University, Taichung, Taiwan

3Veterinary Teaching Hospital, National Chiayi University, Chiayi, Taiwan

Thoracolumbar intervertebral disk disease (IVDD) is a common and crucial neurologic disease in dogs. A six-year-old, female neutered Dachshund presented with emergent paraparesis on 2017/8/31 and was presented to a neighboring animal hospital. On the next day, paraplegia with loss of deep pain perception was observed and the neurological grading was identified as grade 5. Images of computed tomography showed the disc herniation was at the intervertebral disc space between T12 and T13. Subsequently the operation of hemilaminectomy was conducted and completed at night of 2017/9/1. The dog was admitted to National Chiayi University Animal Hospital on 2017/9/12 with ambulatory paraparesis and continuous non-weight-bearing lameness (lameness score 5). A combination of acupuncture with physical therapies was performed to treat and manage the conditions. Dry needle acupuncture, electrical acupuncture and aquapuncture was applied alternately on this dog. The acupoints HTJJ (Hua-Tao-jiaji), GB-30 (Huan Tiao), KID-1 (Gushing Spring), KID-3 (Taixi), and Bai-hui were stimulated. The dog underwent physical therapies including transcutaneous electrical nerve stimulation (TENS), neuromuscular electrical stimulation (NMES), passive/assisted exercise, manual therapy, cryotherapy and gait evaluation for approximately 3 months. Afterward, hindlimb lameness was not observed (lameness score 0) in this dog.

Keywords: Hansen Type I Intervertebral Disk Disease, hemilaminectomy, paraparesis, acupuncture, physical therapies

 

 

 

PB-21

Establishment and Application of Computed Tomography and Three-Dimension Printing Technology for Surgical Guides for Dental Implant

Siang-Ru Chen1, Lee-Shuan Lin1,2 and Cheng-Shu Chung1,2

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

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

Trauma and periodontal disease are the main causes of missing teeth in dogs and cats. In the absence of functional teeth, it may affect the normal chewing of animals. Dental implants should be a better choice with a high degree of success to replace missing teeth due to the different dentition and occlusion of animals and humans. The angle of implantation will affect the success rate of the operation. Most of them use surgical guidance related technology to assist in the implementation, so as to increase the accuracy of the implantation position and shorten surgical exposure time, and improve patient's prognosis.

In this study, the computed tomography (CT) image and the 3D printer were used to design the dog surgical guides for dental implant. We used the CT image which perform routine health examinations in Pingtung Veterinary Teaching Hospital. The lower first molar was used to simulate dental implant targets. The study aimed to design a dental model and a single-implant surgical guide plate. After placing the implant, take a CT scan for accuracy testing. We found that the best outreach of the guide plate was 0.5 mm and 0.6 mm, and that the implantation angle of the implant was verified by CT to meet our expectations. The Standard Operating Procedure (SOP) for surgical guides for dental implant is to be developed.

Keywords: Surgical guide, dental implant, 3D printer, computed tomography

 

PB-22

Serologic Investigation of Foot-and-Mouth Disease in Cattle and Goats in Kinmen in 2017

Ming-Chang Li1,2, Shih-Ping Chen1 and Hsiang-Jung Tsai2

1Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute, Miaoli, Taiwan

2Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

On 27 January 2012, a Foot-and-Mouth disease (FMD) outbreak with serotype O SEA was confirmed in pigs but cattle and goats not included in Kinmen. However, this strain of FMD virus was found from pigs and ruminants in Korea and Japan in 2010. Unfortunately, another Foot-and-Mouth disease (FMD) outbreak with serotype A in cattle in Kinmen was notified to OIE on 8 May 2015. In order to continue evaluate the impact of those FMD outbreak for cattle and goats, total of 498 sera collected from 88 farms of cattle and goats were surveyed during 2017. Sera were tested by ELISA for detecting non-structure protein (NSP) specific antibodies. Four of 363 sera (1.10%) collected from 65 cattle farms were positive. Six of 135 sera (4.44%) collected from 23 goat farms was shown positive. The serological results suggested FMD virus might have infected the cattle and goats in Kinmen. However, contrast to our serologic data, FMD virus has never been detected from the oesophagopharyngeal fluid by virus isolation and polymerase chain reactions. Meanwhile, there are no clinical observations found in cattle and goats. The comparative findings indicated FMD viruses not circulated in cattle and goats even though the few of positive serological evidences found in cattle and goats.

Keyword: FMD, NSP, Kinmen

 

PB-23

Different Ratios of Oleic Acid and Palmitic Acid Affects the Induction

of Intracellular Fat Accumulation in vitro

Yi-Hsun Chen1, Yen-Peng Lee1, Chien-Chao Chiu2, Shao-Wen Hung2,

Wen-Ching Huang3, Ching-Feng Chiu4, Ter-Hsin Chen1 and Hsiao-Li Chuang5

1Graduate Institute of Veterinary Pathobiology, National Chung Hsing University

2Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute

3Department of Exercise and Health Science, National Taipei University of Nursing and Health Sciences

4Graduate Institute of Metabolism and Obesity Sciences, Taipei Medical University

5National Laboratory Animal Center, National Applied Research Laboratories

In recent years, due to changes in dietary habits, the situation of nonalcoholic fatty liver has been increasing year by year. However, the animal model of nonalcoholic fatty liver disease is diet-induced which has the disadvantage that the induction time is too long. On the other hand, Oleic acid (OA) is a common monounsaturated fat and palmitic acid (PA) is the most common saturated fatty acid found in different diet containing. In this study, we used mouse hepatocytic cell line-BNL CL.2 to test the intracellular fat accumulation by incubation with different ratio of OA and PA. Our results found OA alone induced more intracellular fat accumulation than PA alone, and the effects were time- and dose-dependent. The combined treatment with OA and PA in the ratio of 1:1 and 2:1 could induced more lipid droplets accumulated in cytoplasm than other groups by Oil-red staining. These results revealed that OA is more steatogenic than PA and different ratios of OA and PA could affect the intracellular fat accumulation. In MTT assay, the cells incubated with OA alone showed less adverse effect than PA. However, the detail effects of OA and PA induced fatty droplets accumulation and cytotoxicity might require further investigation. The establishment of an in vitro steatosis model can provide a platform for rapid screening of drugs, which can also achieve the purpose of reducing laboratory animal usage.

Keywords: oleic acid, palmitic acid, steatosis

 

PB-24

The Bacteroides fragilis through Toll-Like Receptor 4 to Prevent Colitis-Associated Cancer

Yen-Peng Lee1, Yi-Hsun Chen 1, Chien-Chao Chiu2, Shao-Wen Hung2,

Wen-Ching Huang3, Ching-Feng Chiu4, Ter-Hsin Chen1 and Hsiao-Li Chuang5

1Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan

2Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute, Miaoli, Taiwan

3Department of Exercise and Health Science, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan

4Graduate Institute of Metabolism and Obesity Sciences, College of Nutrition, Taipei Medical University, Taipei, Taiwan

5National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan

Bacteroides fragilis (BF) are Gram-negative anaerobe symbionts present in the colon. Recent studies have reported the beneficial role of BF in stimulating host immunologic development, protecting against intestinal inflammation by pathogenic bacteria. The directly effect of BF on azoxymethane (AOM) and dextran sulfate sodium (DSS) induce colitis-associated cancer (CAC) mouse model in GF mice has studied by our previous experiment. In order to clarify the roles of Toll-like receptor-2 (TLR2) and TLR4, the WT, TLR2 and TLR4 knockout (-/-) GF mice were colonized BF for 28 days, and then administered a single intraperitoneal injection of AOM (10 mg/kg) and received three cycle of 1% DSS. The results showed that the spleen and spleen% of body weight were significantly increased in TLR4-/- mice by BF colonization. Besides the TLR4-/- mice, the immunohistochemistry for cell proliferation-related molecules and number of inflammatory cells were decreased in the WT and TLR2-/- under BF condition. Unlike WT and TLR2-/- mice, the severity of CAC did not improve in TLR4-/- animals after BF colonization. The BF increased survival rate in WT and TLR2-/- group by CAC induction. Moreover, the BF inhibited tumorigenesis in WT and TLR2-/- group compare to GF condition. In contrast, colonized BF could not ameliorate CAC severity in TLR4-/- mice. According to above results, we suggest that the disease-preventing effects of BF in CAC mice model may through the TLR4 signal pathway.

Keywords: Bacteroides fragilis, colitis-associated cancer, Toll-like receptor

 

PB-25

Establish the Protocol of Prosthetic Rehabilitation in Dogs and Cats

Ting-Yun Wu1, Cheng-Shu Chung1,2 and Lee-Shuan Lin1,2

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

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

Text book mentions that dogs and cats can adapt well after they are amputated. But in recent years, some studies showed different severity of problems that may occur in amputated dogs or cats (ex: obesity, neck and back pain etc.). In addition, owners care about their pets wholesome and animal welfare. Above all promote owners to give their pets prosthetics. There are problems when dogs and cats wear prosthetics. Is it suitable for them? How do dogs and cats adapt to it? There isnt a standard protocol and prosthetic rehabilitation assessment. In the present study, we tried to design an appropriate method for dogs and cats in prosthetic rehabilitation. The purpose of this study is to train dogs and cats to learn how to use their prosthetics more smoothly. In this study, two clinical cases were included, a dog who uses a socket-based prosthetic and a cat who uses an implanted prosthetic. The protocol of this study contains three parts, design, rehabilitation and assessment. Because of the difference of species, the design of the prosthetics differs. The method we evaluate rehabilitation effectiveness was calculating the time of the swing phase of the limbs. After more than two months of rehabilitation, the result we found was rehabilitation for amputated dogs and cats who needed to wear prosthetics is beneficial indeed. When working on the prosthetic rehabilitation in dogs and cats, we need observation, patients and accompany. The most important tip is to tailor the most suitable rehabilitation protocol according to their differences. In the future, we expect to use more convenient and accurate methods (ex: Inertial measurement unit, IMU) in rehabilitation assessment.

Keywordsdog, cat, prosthetic, rehabilitation

 

PB-26

Recovery from Lameness by Acupuncture and Physical Therapies after the Surgery of Medial Patellar Luxation in a Dog

Wei-Wu Jian1, Kun-Wei Chan1,2, Kuan-Lun Wu2, Meng-Hua Tasi1, Syu-Lun Lin1 and Han Hsiang Huang1,2

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

2 Veterinary Teaching Hospital, National Chiayi University, Chiayi, Taiwan

Canine patellar luxation is a common orthopaedic stifle disorder in dogs. A 4-year-old, intact male Poodle was admitted to National Chiayi University Animal Hospital on 2018/5/21 with weight-bearing right hind limb lameness. The ventrodorsal view of radiographic examination showed bilateral medial patellar luxation with the femoral neck shaft angles of hindlimbs less than 137°. Bilateral medial patellar luxation (right hind limb grade 3 and left hind limb grade 2) was diagnosed according to physical and radiographic examinations. Reconstructive surgeries including trochlear wedge recession, lateral retinacular reinforcement, medial retinacular release and tibial tuberosity transposition were performed to correct the medial patellar luxation of right hind limb. Acupuncture and physical therapies were selected to manage the post-operative lameness (lameness score 3). Dry needle acupuncture and electrical acupuncture was applied alternately on this dog. The acupoints ST-35a (lateral eye of the knee), ST-35b (medial eye of the knee), GV-4 (Ming-Men), GV-20 (Bai-Hui), GB-30 (Huan-Tiao), KID-3 (Taixi), GB-31 (Feng-Shi), BL-60 (Kun-Lun), BL-40 (Wei-Zhong), GB-34 (Yang-Ling-Quan), ST-36 (Hou-San-Li), LIV-3 (Tai-Chong), Shen-shu and Jian-Jiao were stimulated. Physical therapies including neuromuscular electrical stimulation (NMES), Class IV laser therapy, transcutaneous electrical nerve stimulation (TENS), passive/assisted exercise, manual therapy, cryotherapy and gait evaluation were performed for nearly 2 months after the surgery. Afterward, the dog was not lame when ambling and trotting (lameness score 0). Recurrence of hind limb lameness was not observed available for hospital follow-up by Oct 2018.

Keywords: medial patellar luxation, acupuncture, physical therapies.

 

PB-27

Detection and Differentiation of H5 Avian Influenza Viruses Using Real-time RT-PCR and High Resolution Melting Assays

Yu-Chen Hsu1, Wen-Chien Wang1, Yu-Hsuan Yang1, Chia-Hsuan Chang1, Jui-Hung Shien2 and 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

Avian influenza viruses (AIVs) cause a significant poultry disease worldwide. In the last three decades, H6N1 virus has been confirmed in Taiwan. Since the initial outbreaks of Mexican-like H5N2 low pathogenic avian influenza virus (LPAIV) in 2003, a variety of H5N2 AIVs emerged these years, including Mexican-like H5N2 high pathogenic avian influenza virus (HPAIV) H5N2 viruses in 2008, H5N2, H5N3, H5N8 AIVs of clade 2.3.4.4 in 2015 and H5N6 AIVs in 2017. In the current study, we developed a one-step real-time reverse transcription PCR and high resolution melting (HRM) assays to detect H5 viruses and discriminate between the Mexican-like H5 viruses and the clade 2.3.4.4 H5 viruses. Our results showed that all H5 viruses circulating in the fields can be detected using the new developed real-time RT PCR with the detection limit about 1000 copies. Moreover, the HRM analysis can differentiated the Mexican-like H5 viruses from the clade 2.3.4.4 H5 viruses with the melting temperatures at 77.5˚C and 75.8˚C, respectively. In summary, real-time RT-PCR and HRM assay is developed in this study and it could be valuable for rapid diagnosis and investigation of H5 AIVs in the field.

Keywords: Avian Influenza Viruses, Real-time RT-PCR, HRM.

 

PB-28

Depletion Study Withdrawal Period Calculation of Flumequine in Giant Seaperch (Lates Calcarifer) with Multiple-Dose Oral Administration

Re-Shang Chen1, Yi-Jing Xue2, Shi-Yuan Sheu3,4, Shao-Kuang Chang1Jiann-Hsiung Wang2, Chung-Hsi Chou1 and Tzong-Fu Kuo1,5

1Graduate Institute of Veterinary Medicine, School of Veterinary Medicine,

National Taiwan University, Taipei, Taiwan

2Graduate Institute of Veterinary Medicine, College of Agriculture, National Chiayi

University, Chiayi, Taiwan

3School of Medicine, Chung Shan Medical University, Taichung, Taiwan

4Department of Integrated Chinese and Western Medicine, Chung Shan Medical

University Hospital, Taichung, Taiwan

5Department of Post-Baccalaureate Veterinary Medicine, Asia University,

Taichung, Taiwan

Flumequine contains fluorine in position 6 and is a so-called second-generation quinolone. The fluorination increases the antibacterial potency and activity against gram-positive bacteria. The residue depletion of flumequine (FLU) was evaluated in giant seaperch after 1-21 days of oral administration, at the dose of 20 and 40 mg/kg body weight (bw) every 24 hr for 5 days. The determination of FLU in muscles and liver tissues of giant seaperch was carried out using the solid-liquid extraction for sample preparation, and ultra-high performance liquid chromatography–tandem mass spectrometer (QTRAP®5500 UPLC–MS/MS) operated in scheduled multiple reaction monitoring scan mode for quantitation. The FLU was resolved in < 6 min using a C18 column, with an isocratic mobile phase of 0.05 mol/L sodium dodecyl sulfate – 8% 1-butanol – 0.5% triethylamine buffered at pH 3. There was a good linear correlation between the intensity of signals and FLU concentration in muscle (Ŷ=246x-0.000157, R²=0.9994) and liver (Ŷ=142x–0.000252, R²=0.9997), and the limit of quantification (LOQ) can reach 0.01 μg/mL. The mean recovery of the FLU added to muscle and liver was 103.5% and 99.9%, respectively. After multiple oral administered with a 20 mg/kg bw dose of FLU, muscle and liver levels at 12 hours were 198±41 and 230±38 μg/g (n=5); while for 40 mg/kg bw dose of FLU, the mean muscle and liver concentrations were 257.8±53.3 and 279.9±146.0 μg/g, respectively. The withdrawal period to reach the maximum residue level (MRL) of 600 μg/kg, according to the European Union (EU, 2002) legislative framework to Flumequine, was 2 days.

Keywords: depletion study, flumequine, giant seaperch

 

PB-29

Determination of Phthalates in Pork and Chicken in Taiwan by Using Liquid Chromatography Tandem Mass Spectrometry and a Health Risk Assessment

Hong-You Chang1, Yi-Jing Xue2, Ming-Yang Tsai3, Jyh-Mirn Lai2, Jiann-Hsiung Wang2*, and Geng-Ruei Chang2*

1Ph.D. Program of Agriculture Science, National Chiayi University, Chiayi,

Taiwan

2Department of Veterinary Medicine, National Chiayi University, Chiayi,

Taiwan

3Animal Industry Division, Livestock Research Institute, Tainan, Taiwan

Phthalates are widely used plasticizers that can cause endocrine disruption, mutagenicity, and carcinogenic effects, and can contaminate food from various pathways. Phthalate contamination of organic products can endanger the descendants of those who consume the products. Few studies on dietary exposure of humans to phthalates have investigated livestock and poultry meat. This study examined residual concentrations in unpackaged pork (30 samples) and unpackaged chicken (30 samples) and the association between the health risks of phthalates and meat consumption in the Taiwanese population. The compounds were analyzed, including benzyl butyl phthalate, di-n-butyl phthalate, di-2-ethylhexyl phthalate (DEHP), diisodecyl phthalate, and diisononyl phthalate by using liquid chromatography–tandem mass spectrometr.. The five phthalates had been established tolerable daily intake (TDI) values by the Taiwan Food and Drug Administration (TFDA). DEHP was the major compound detected, which ranged from 0.62 to 0.80 mg/kg in two pork samples, and 0.42 to 0.45 mg/kg in three chicken samples. The concentrations of DEHP were lower than the screening value of 1.0 mg/kg, as defined by the TFDA. The risk was assessed in terms of the estimated daily intake (DI) for potential adverse indices; the DI of DEHP residues was less than 1% of the TDI value. The assessed risk was negligible and considered to be at a safe level, suggesting no association between meat consumption and risk to human health in Taiwan. However, a continuous monitoring program for phthalate residues in meat is necessary to further assess the possible effects on human health.

Keywords: phthalates, pork, chicken, residues, tolerable daily intake

 

 

PB-30

Real-time PCR Assay for Detection Goose Hemorrhagic Polyomavirus

Yu-hua Shih1, 2, Ai-ping Hsu1, Chun-hsein Tseng1 and Chung-hsi Chou2

1Animal Health Research Institute Council of Agriculture, Executive Yuan, New Taipei City, Taiwan,

2School of Veterinary Medicine National Taiwan University, Taipei City, Taiwan

Goose hemorrhagic polyomavirus (GHPV) has identified as the etiologic agent of goose hemorrhagic nephritis and enteritis (HNEG). In March 2015, cases from Chiayi and Tainan had caused nearly half of the geese in three goose farms to die. This disease is not easy to distinguish from waterfowl parvovirus infection. A real-time PCR method developed for rapidly detection GHPV. In this study, the primers and probe selected from the VP1 gene of the GHPV genome conserved sequences. There was no cross-reaction signal found from waterfowl parvovirus, adenovirus, Riemerella anatipestifer, indicating the qPCR assay has good specificity. The standard curve had an error of 0.0101 and efficiency of 102.8%. The detection limit of the assay is 50 genome copies/ mL for GHPV. The results showed the method had good sensitivity and specificity, could be used in GHPV detection.

Keywords: Goose hemorrhagic polyomavirus, hemorrhagic nephritis and enteritis, Real-time PCR

 

PB-31

A One Health Approach for the Surveillance of Antimicrobial Resistance in Animals

Hsiu-Hui Wu, Chin-Shu Chang, Jyh-Perng Wang, Jiunn-Horng Lin

and Zeng-Weng Chen

Animal Technology Laboratories, Agricultural Technology Research Institute, Zhunan, Taiwan

The antimicrobial resistance (AMR) is a worldwide threat, and the Global Health Security Agenda (GHSA) promotes interdisciplinary collaboration under the concept of One Health to develop appropriate strategies for antimicrobial resistance control. To understand the threat in Taiwan, our microbial laboratory has established a platform to monitor the AMR in slaughterhouses and in meat markets in accordance with OIE regulations. Antibiotic resistance profiles are analyzed in accordance with the latest CLSI guidelines. Analytic data are used as a basis for adjustment of antibiotic management guidelines. From 2012 to 2016, a total of 4,019 bacteria isolates were collected and investigated, and 49,573 minimal inhibitory concentration (MIC) assays had been analyzed.

In 2017, the project has established a whole-genome sequencing (WGS) platform to assess AMR in real time which is critical in detection of antimicrobial resistant bacterias. Additionally, the use of WGS can dissect the phylogenetic associations of the bacterium and its antimicrobial resistance genes through the course of an epidemic. Moreover, our laboratory has drafted a white paper on the corresponding strategies for the development of AMR through collaboration with animal/plant/human health in our country, and generates propaganda for AMR awareness.

Keywords: Antimicrobial Resistance, Minimal Inhibitory Concentration, One Health

 

PB-32

Evaluation of Correlation between Purpura and Pathogens Infection of Nursery Pigs in South Taiwan

Joan Wong1, Chao-Nan Lin1 and Ming-Tang Chiou1,2

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

2Research Center for Animal Biologics, National Pingtung University Science and Technology, Taiwan

Purpura results from an extravasation of blood from a vasculature into the skin or mucous membranes. Porcine dermatitis and nephropathy syndrome in growing pigs and thrombocytopenic purpura in weaning pigs may relate to purpura on the hindlimbs and in the perineal area. There are few studies investigating purpura on nursery pigs up to date. Recently, pigs with purpuric lesions in various herds have caused difficulties to local pig farmers. Therefore, the purpose of this study is to evaluate the association among purpura and pathogens causing vasculitis. In this study, 31 nursery pigs (lesions group) and 32 nursery pigs (non-lesions group) were selected from various herds of South Taiwan. Blood samples were collected from those pigs for bacteria and virus detection. The gross and histopathologic lesions of skins were examined. All lesions group of pigs were found their purpura on hindlimbs, perineal area of hindquarter, forelimbs and abdomen. Microscopically, all lesions group pigs had vasculitis of small vessels and dermal hemorrhage. Blood cultures showed no growth of bacteria in lesions group. The detection rate of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and Mycoplasma hyorhinis (Mhr) were 100%, 29% and 54.8% in lesions group, and 69%, 34%, 19% in non-lesions group, respectively. The odd ratios with Haldane-Anscombe 1/2 correction were detected to be 29.4 for PRRSV and 5.2 for Mhr. The conclusion is that PRRSV and Mhr showed a significant correlation with purpura.

Keywords: Purpura, vasculitis, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, Mycoplasma hyorhinis

 

PB-33

Study on Cloning, Expression and Protective Ability of PLP B Protein from Pasteurella multocida

Hsing-Yu Liao and Chung-Da Yang

National Pingtung University of Science and Technology, Pingtung, Taiwan

Pasteurella multocida, a Gram-negative bacterium with a capsule is full of virulence factors, thereby making the mechanism of the disease unclear. After invading from the wound, the rapid propagation of the disease will produce a strong inflammatory reaction. In addition, hemorrhagic septicemia will occur when the host's resistance is low. The main host of the bacteria is economic animals such as chicken, pig, cow, sheep, etc. The clinical symptoms of different hosts are different. The infection caused considerable loss to the global meat market. The bacterial capsular antigen (K antigen) serogroup can be divided into A, B, D, and E, and the bacterial O antigen can be divided into 12 serotypes. We focus on the lipoprotein PLP B on the bacterial outer membrane and would like to clone and express it`s gene (830bp)in the study. So far this sequence is homologous among different sources. Recently, the specific primers were designed to amplify PLP B gene through PCR. The resulting DNA fragment was cloned into pet32a plasmid. The recombinant plasmid was then transformed into BL21(DE3) competent cells. Further effort will be implement to express and purify recombinant protein, PLP B. In addition, the protective ability of recombinant PLP B Protein will be evaluated in mice. We anticipate that the recombinant protein prepared in the study would be an effective candidate to prevent Pasteurella multocida.

Keyworld : Pasteurella multocida, PLP B, E. coli expression system, Protein purification

 

PB-34

The Feasibility of Evaluating Swine Herd Health Status with C-reactive Protein

Hui Sing Soon1, Chao-Nan Lin1, Ming-Tang Chiou1,2

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

2Research Center for Animal Biologics, National Pingtung University Science and Technology, Taiwan

C-reactive protein (CRP) is an acute-phase protein (APP) thought to play an important role in the complement system of mammals. CRP increases significantly before specific antibody titers arise by the stimulation of interleukin-6 (IL-6) during disturbed physiologic homeostasis. Several investigations have indicated that measuring the serum concentrations of the APP aids in monitoring the state of health and welfare in pigs. Therefore, this report is to differentiate healthy, subclinically diseased and clinically diseased pigs by CRP in Taiwan. In total, CRP of 325 serum samples were examinated by AmiShield (ProtectLife International Biomedical Inc., Taiwan) since 2015 to 2016 from 3 main groups: specific pathogen free pigs (SPF), subclinically diseased pigs and clinically diseased pigs. The subclinically diseased pigs were composed of 4 main groups: suckling pigs, nursery pigs, growing pigs and sows. Clinically diseased pigs were composed of 3 main groups which were diagnosed with the following disease: Glässers disease (GD), porcine reproductive and respiratory syndrome, and porcine epidemic diarrhea (PED). The results stated CRP values of clinically diseased pigs were very significantly higher than subclinically diseased pigs (p<0.01). Further analyzing the CRP values of subclinically diseased subgroup pigs overall showed there was an escalating trend with age. The CRP values of pigs with GD were significantly higher than pigs with PED (p<0.05). In conclusion, serum CRP concentrations can be used to evaluate the health status of swine herds.

Keywords: swine, status of swine herds, C-reactive protein, subclinically diseased pigs, clinically diseased pigs

PB-35

The Application of Cat Alpha-1-Acid Glycoprotein on Feline

Lower Urinary Tract Disease

Zheng-Yung Wu1, Yao-Li Lin1, Cheng-Shu Chung1,2, Lee-Shuan Lin1,2

1National Pingtung University of Science and Technology

2National Pingtung University of Science and Technology Veterinary Medical

Teaching Hospital

Feline lower urinary tract disease (FLUTD) is a common disease in cats. The severity of cats varies while they attend. It may not be necessary to stay in hospital for treatment. However, if kidney damage occurs, which is life-threatening, and is treated, we own no effective tool for monitoring without obvious clinical symptoms after the treatment. Acute phase protein (APP) has been widely studied in the veterinary field in recent years. It is elevated in the acute and chronic reaction period of tumor, trauma and inflammation to restore physiological balance. It can be applied to the evaluation of inflammatory response, disease pattern, prognosis, monitoring and so on. Although FLUTD has a wide variety of causes, regardless of the degree of severity, it will produce an inflammatory reaction of the bladder. Meanwhile AGP is one of the effective APPs in cats. Therefore, this study hopes to retrospectively examine whether AGP can reflect the occurrence of inflammation in FLUTD and evaluate the feasibility of clinical application.

We tested AGP on the plasma of six cats with FLUTD at the time of presentation and discharge. The AGP values ​​of all cats were higher than normal at the time of presentation, and there was a rise in white blood cells, azotemia and related clinical symptoms. Patients were discharged after three to eleven days of hospitalization. The clinical symptoms and test data at the time of discharge were significantly improved or returned to normal, but the AGP values were still high. Therefore, based on the results, we found that AGP is sensitive to the inflammatory response induced by FLUTD; general blood test results, clinical symptoms and AGP have no significant correlation; AGP values ​​before treatment cannot be used as prognostic factors; AGP may become a tool to monitor the progression of FLUTD disease, but further research is needed.

Keyword: FLUTD, Acute phase protein, Cystitis

PB-36

Encephalitozoon cuniculiPaper Review

Yu-An Chen1, Cheng-Shu Chung1 and Lee-Shuan Lin1

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

Encehalitozoon cuniculi (E. cuniculi) is an obligate intracellular parasite belonging to the phylum Microsporidia. Through genetic analysis, we now know Microsporidia is the earliest diverging clade of fungi. Although E. cuniculi was first found in rabbits in 1922, it has the capability to infect a wide range of mammals including immunocompromised human. Thus, E. cuniculi is a zoonotic disease worth noting.

E. cuniculi is an important infectious disease to rabbits. The major clinical signs in affected rabbits are neurologicalrenal and ocular manifestation. Histopathological examination is the gold standard for the diagnosis of E. cuniculi infection. However, it is challenging to give an anmortem definite diagnosis. By the detection of specific antibody, rabbits are speculated to have E. cuniculi infection. Nevertheless, high antibody titers represents not only active or subclinical infection, but also exposure to the pathogen. Therefore, researches are dedicated to finding additional serological examinations to optimize the diagnosis of E. cuniculi.

In the treatment of E. cuniculi infection, benzimidazoles are most frequently used in both rabbits and human. Though proved to be effective against E. cuniculi, the influence of benzimidazoles to the host cell causes notable side effects. To avoid undesirable outcome, the dosage and the course of treatment should be within recommendation range. Additionally, the use of chitin synthase inhibitor as a remedy for E. cuniculi should meet limited side effects. 

Apart from treatment, the prevention of this disease is also important under the high seroprevalence(67.8%) in Taiwan. By the use of effective disinfectants, medical preventions and possibly vaccines, we can fight E. cuniculi, and bring about better welfare to rabbits or even other animals.

Key words: Encephalitozoon cuniculi; rabbits; seroprevalence; benzimidazole; chitin synthase inhibitors

 

PB-37

Effects of Everolimus on Papain-Induced Degenerative Osteoarthritis in Sprague Dawley Rats

Yi-Tang Chong and Geng-Ruei Chang

Department of Veterinary Medicine, National Chiayi University

Osteoarthritis is one of the common joint degenerative disorders for which only symptomatic treatment is available. In the present study we have investigated anti arthritic activity of everolimus in papain induced osteoarthritis model which mimics the human arthritis. Osteoarthritis was induced in rats by intraarticular injection of 0.8 mg of papain and 0.03M of cysteine on day 0, 4 and 7. Everolimus was i.p. administered to the animals in the doses of 2 mg/kg for 14 days after the injection of papain and cysteine. Dexamethasone (2 mg/kg) and saline was used as the reference positive control group and negative control group, respectively. The joint activity was assessed by wire hang test and measurement of the knee joint diameter. At the end of 15 days blood samples were collected for the analysis cytokines of IL-1, IL-10 and TNF-α. The animals were sacrificed and the knee of the each animal was collected for the histological studies of the joint. Injection of everolimus produced a significant decrease in joint diameter and significantly increase joint strength as depicted by increase in wire hanging time. Everolimus decrease in swelling of the knee joint along with significant increase in joint strength. There was a significant reduction levels of IL-1, IL-10 and TNF-α in the rats treated with everolimus. Histopathological and immunohistochemistry studies revealed that the reversal of damages in the cartilage induced by papain and cysteine after administration of the everolimus. Our data suggests potential anti-osteoarthritic effect of everolimus on papain induced arthritic rats.

Keyword: Everolimus, Arthritis, Cytokine, Rat

 

PB-38

Detection and Quantitation of the Japanese Encephalitis Virus (JEV) Antigen by ELISA

Qiao-Ru Lan, Tsun-Yung Kuo

Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan

Japanese encephalitis virus (JEV) is an important pathogen that infect the central nervous system. The whole viral inactivated vaccines were developed and commercialized for a long time. In the vaccine manufacturing process, the amount of antigen content is a key parameter for vaccine formulation. Therefore, we conducted a Q-ELISA platform that could detect and quantify JEV antigen. The anti-JEV monoclonal antibodies (mAbs) and rabbit anti-JEV polyclonal antibody (pAb) were generated and characterized. Three hybridoma cell clones were isolated and all three mAbs were IgG1 isotype. An anti-JEV mAb and rabbit anti-JEV pAb were used for development of the Q-ELISA. The results suggest that the serial diluted JEV antigens were detected with excellent linear fits (R2 values 0.99). The best range of antigen detection was from 0.078μg/mL to 2.5μg/mL when anti-JEV mAb as capture and rabbit anti-JEV pAb as detector. However, the range of antigen detection was from 0.31μg/mL to 10μg/mL when the rabbit anti-JEV pAb as capture and anti-JEV mAb as detector.

Keywords: Japanese Encephalitis Virus, monoclonal antibody, polyclonal antibody, Quantitation ELISA

PB-39

Effects of APR Gene Knockout Protecting against Diet-Induced Obesity in Mice.

Zhi-Ying Wong1, Wen-Chin Yang2, Chih-Lung Liang3 and Cicero Lee-Tian Chang1

 

 

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

2Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

3Institute of Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan

Obesity is one of the key risk factors for many chronic diseases, including diabetes, cardiovascular diseases and cancer. It’s recognized as a global epidemic by the World Health Organization (WHO). During chronic caloric excess, adipose tissue expands primarily by enlargement of individual adipocytes, which become stressed with lipid overloading, thereby contributing to obesity-related diseases. Our aim of this study is study the effect of APR gene knockout (APR KO) regulating development of obesity. We observed that APR KO can reduced body weight significantly. When wild-type (WT) mice fed high-fat diet (HFD), white adipose tissue weight was greater than when fed normal diet (ND), whereas this increase was strongly suppressed in APR KO mice. The size of white adipocytes in APR KO mice fed HFD could also decrease significantly. Thus, APR gene knockout could improve the development of obesity.

Keywords: Obesity, adipose, APR gene knockout