107 年度春季研討會 壁報論文非競賽組 (PB)



Hyperadrenocorticism and Diabetes Mellitus in a Dog

Wan-Hsuan Lai1, Ming-Huei Fan2, Kun-Wei Chan*1,2

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

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

Diabetes mellitus (DM) is one of the most common complications of hyperadrenocorticism. In this case, a six-year-old, female, neutered Chihuahua dog presented with anemia and requested for transfusion. It was diagnosed with diabetes mellitus by local animal hospital six months ago. Since then, a dose of 1.9 U/kg Caninsulin® was injected subcutaneously after every 12 hours. Based on the history of high insulin dose and physical examinations such as pendulous abdomen and bilateral symmetrical alopecia without pruritus, hyperadrenocorticism was suspected. A diagnosis of hyperadrenocorticism was made at the veterinary teaching hospital via complete blood count, serum biochemisty examination, low-dose dexamethasone suppression test, ACTH stimulation test, ultrasonography and computed tomography (CT). Administration of a small dose of dexamethasone to a dog with pituitary-dependent hyperadrenocorticism (PDH) causes the serum cortisol concentration to be variably suppressed. However, it is no longer suppressed 8 hours after dexamethasone administration, compared with the response seen in normal dogs. Abdominal ultrasonography showed the finding of bilateral symmetrical adrenomegaly served as a evidence of adrenal hyperplasia caused by PDH. However, the pituitary gland did not show obvious enlargement in the CT image. Trilostane was used as the primary treatment modality for this dog. After four months, the dog’s cortisol and fructosamine levels were well controlled.

Keywords: Hyperadrenocorticism, diabetes mellitus, Caninsulin®

*is the corresponding author. Email: Kwchan@mail.ncyu.edu.tw



Antimicrobial Susceptibility of Streptococcus-like Bacteria Isolates from Cultured Taiwan Tilapia (Oreochromis sp.) in Chiayi County

Hsien-Yi Tu1,2, Yi-Jing Xue1, Shu-Yung Chiu3, Jian-Lin Lee3, Jiann-Hsiung Wang1



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

2Animal and Plant Disease Control Center of Yunlin County, Yunlin, Taiwan2,

3Livestock Disease Control Center of Chiayi County, Chiayi, Taiwan

Streptococcosis is an important bacterial disease in cultured Taiwan tilapia (Oreochromis sp.) and caused serious economic losses. In this study, we obtained and identified 67 isolates of Streptococcus-like bacteria of Taiwan tilapia from Chiayi county by multiplex polymerase chain reaction, and conduct the minimum inhibitory concentration test of 7 kinds antibiotics (amoxicillin, ampicillin, doxycycline, erythromycin, florfenicol, lincomycin and oxytetracycline). According to the identify result of 67 Streptococcus-like isolates, 1 strain is Lactococcus garvieae, 57 for Streptococcus agalactiae, and 9 for Streptococcus iniae. In conclusion, S. agalactiae is the main pathogen of Streptococcosis in Taiwan tilapia (Oreochromis sp.). Antimicrobial susceptibilities of 67 isolates were determined by micro broth dilution method. It showed well effective of amoxicillin, ampicillin, doxycycline, erythromycin, lincomycin and oxytetracycline to L. garvieae, the MIC90 results between 0.25-0.125 mg/mL. Same results occur in amoxicillin, doxycycline, erythromycin, lincomycin and oxytetracycline to S. agalactiae. In addition, MIC90 results are 0.125 mg/mL in amoxicillin, doxycycline, erythromycin, lincomycin and oxytetracycline to S. iniae. All isolates shows no resistance to 7 kinds antibiotics.

Keywords: Streptococcus agalactiae, Taiwan tilapia, Ch iayi, Antimicrobial susceptibility


Immune-Mediated Hemolytic Anemia in a Dog

Chung-Li Liao1, Ming-Huei Fan2, Kun-Wei Chan*1,2

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

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

*is the corresponding author. Email: kwchan@mail.ncyu.edu.tw

Autoimmune hemolytic anemia, or immune-mediated hemolytic anemia (IMHA), is a complex disease in which hemolysis occurs because of antierythrocyte antibody production, and it can be classified as Type II hypersensitive reaction. In this case, a four-year-old male intact poodle was referred to National Chiayi University Animal Hospital on May 17, 2017. The dog presented with symptoms of poor appetite, depressed, brown-colord urine, pale mucous membrane, bloody stool and hematuria. The severe anemia (PCV: 13.5%) and leukocytosis was shown in complete blood count, and blood parasites could be ruled out by IDEXX®SNAP 4Dx test and nested polymerase chain reaction (Babesia canis and Babesia gibsoni). The blood smear showed spherocytosis. Additionally, a direct antiglobulin test showed a strong positive result, which manifested the presence of antierythrocyte antibody. Therefore, the dog was diagnosed with Primary IMHA. After the one month immunosuppressive therapy (prednisolone 2.0 mg/kg/day), the PCV and WBC count returned to normal range. In conclusion, the condition was controlled because the dog showed good response to immunosuppressive therapy, and the dog did not suffer a relapse since discharged.



Non-effusive Feline Infectious Peritonitis

Pei-Yu Su1, Ming-Wei Lee2, Kun-Wei Chan*1, 2

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

2Veterinary Teaching Hospital, College of Veterinay Medicine, National Chiayi University, Chiayi, Taiwan.

*is the corresponding author. Email: kwchan@mail.ncyu.edu.tw.

Feline infectious peritonitis (FIP) is a highly transmissible and fatal disease, and the virus mutates from feline coronavirus (FcoV). Unfortunately, not only is the diagnosis difficult, there is also no effective treatment. Nowadays, the gross lesions, histopathological examinations and immunohistochemistry are generally regarded as the “gold standard” for FIP diagnosis. In this case, a five-month-old intact male mixed breed cat presented with symptoms of vomiting, diarrhea, fever and depression. Two masses were palpated on the left and right abdominal cavity which gradually expanded. From abdominal ultrasonographic examination performed at a local veterinary clinic, thickening of the small and large intestinal walls, indistinct intestinal delamination, and irregular serosal surface were noted. The complete blood count results indicated mild anemia. Serum biochemistry examination indicated hypoalbuminemia (2.1 g/dL) and reduction of A/G ratio (0.5) as well as blood urea nitrogen (11 mg/dL). A one-week oral medication was prescribed, but the patient’s condition did not improve. Thus, the patient was referred to the veterinary teaching hospital of National Chiayi University. Non-effusive FIP was diagnosed, and this patient was euthanized. Hence, it is important for us to further explore the diagnostic methods and treatment of FIP.

Keywords: Feline coronaviruses, feline infectious peritonitis virus, gold standard.



Development of a New Type Vaccine Against Porcine Rotavirus

Ya-Wen Tsai1, Guan-Ming Ke1

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

Porcine Rotavirus (PRV) belongs to the Reoviridae family and it is a double-stranded RNA virus without envelope. The Porcine Rotavirus Group A (RVA) is one of the most important pathogens for the viral diarrhea of young animals in the world. In pig farms, they often trigger large-scale swine diarrhea, poor growth performance, and even dehydration death. However, because clinical symptoms are not easily distinguishable from other pathogens, it will increase the cost of diagnosis and treatment, resulting in serious economic losses. The objective of this study was to develop a new type vaccine for porcine rotavirus, exploring the optimal antigen production by evaluating the immune efficiency. In addition, TCID50 and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay were used to detected and quantified the virus. On the other hand, we have selected PRV VP7 protein genes that have the capabilities of inducing neutralizing antibody, using the baculovirus expression system to express VP7 proteins. Finally, we will perform animal experiments to evaluate the protective efficacy of the PRV vaccines. The isolate of PRV was successfully passaged in MA-104 cells. Titer of the virus gradually increased, reaching 108 TCID50/ml after sixteen generations domestication, and the production of PRV VP7 protein can reach up to 92 μg/mL with the baculovirus expression system. When 50 μg of VP7 protein and 106 TCID50 of PRV mix with adjuvant and used to immunize pigs, the ELISA test result showed an increased efficacy one week after immunization, and peaked at week 6. Therefore, the present study developed a porcine rotavirus vaccine, when the VP7 proteins mixing with inactivated virus, that still retain its antigenicity for enhanced protection, thus, could be further developed of the vaccine.

Key words: Porcine rotavirus, Diarrhea, Baculovirus, VP7, Subunit vaccine



Characterization of the heparin binding ability of classical swine fever virus (CSFV) glycoprotein Erns

Chih-Yuan Cheng, Chien-jin Huang

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

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is a highly contagious swine disease worldwide. The CSFV envelope glycoprotein Erns possesses RNase activity and has been demonstrated to participate in cell attachment. The CSFV TD96 strain was isolated in 1996 (southern Taiwan) and belongs to the genotype 2.1. In this study, the full-length (N227) and a C-terminal truncated (N190) Erns recombinant protein were expressed by yeast Pichia pastoris. Both N227 and N190 recombinant Erns proteins could bind to heparin column suggesting presence of other heparin binding sites except for the C-terminal site which has been identified in bovine viral diarrhea virus (BVDV). Sequence alignment analysis of Erns gene between CSFV and BVDV revealed an N-terminal potential heparin binding site at the amino acid residues 31-48. Since the basic amino acid residues at 36 and 39 are critical for heparin binding, two mutant recombinant proteins, N227/36,39m and N190/36,39m were constructed by site-direct mutagenesis to substitution of lysine with alanine. No additional fusion tag was present at the recombinant Erns proteins leading to correct conformation. The yeast-expressed Erns proteins were analyzed for their heparin binding ability by gradient NaCl elution with HiTrapTM heparin column. N227 could be eluted at 0.4 M NaCl, whereas N227/36,39m was eluted under 0.2 M NaCl. In addition, N190 could be eluted at 0.3 M NaCl but the N190/36,39m was abundant in the flow-through fraction. Mutation of residues at 36 and 39 resulted in lessening heparin binding ability suggests a potential heparin binding site within this region near the N-terminal of Erns.

Keywords: classical swine fever virus (CSFV), glycoprotein Erns, Pichia pastoris, heparin binding site



Establishment of a Mouse Model with Membranous Nephropathy Induced by Cationic Bovine Serum Albumin

Ruei-Chen Hung, Chia-Chi Chen, Jen-Wei Lin, Chien-Chao Chiu, Pin-Tasn Tsai, Ying-Ching Hung, Shao-Wen Hung, Cheng-Yao Yang

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

With the coming of aging society, the number of patients with end-stage renal disease (ESRD) are also increasing year to year. Data shows that the incidence and prevalence of ESRD in Taiwan were the highest in the world. The expense of treatment for ESRD captures a disproportionate share of health care resources. One of the most common causes leaded to ESRD is membranous nephropathy (MN). An effective method resolving this problem is to establish suitable experimental animal model, it should be useful for studying the mechanism and pathogensis of membranous nephropathy (MN). In this study, we have developed a mouse model of MN induced by cationic bovine serum albumin (CBSA). We treat cyclophosphamide-prednisolone or saline in MN mice by intravenous injection. The result revealed that MN mice treated saline displayed more proteinuria and more severe glomerular injury in renal histopathology than MN mice treated cyclophosphamide-prednisolone. We hope that the mouse model with membranous nephropathy induced by CBSA and therapeutic strategies will applied to the new drug research and development in the future.

Keywords: cationic bovine serum albumin, end-stage renal disease, membranous nephropathy



Establishment of Breast Cancer Drug Screening System

Chia-Chi Chen, Jen-Wei Lin, Chien-Chao Chiu, Ruei-Chen Hung, Pin-Tasn Tsai, Ying-Ching Hung, Shao-Wen Hung, Cheng-Yao Yang

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

Breast cancer is the leading cause of cancer death worldwide. The major cause of treatment failure and mortality is cancer metastasis. Due to the current advances in medicine, cancer patients treat tumors by surgery and chemotherapy etc. The 5-year survival rate of breast cancer is about 98% and the 10-year survival rate is 85%-90%. Therefore, the earlier the breast cancer is found, the less spread or metastasis of cancer cells, and the greater the chance of successful cure. In the United States, breast cancer is the commonly diagnosed cancer and the secondary leading cause of cancer death in female. In 2017, approximately 252,710 cases (30% of new cases of female cancer) of breast cancer will be newly diagnosed in female and 40,610 breast cancer deaths will occur in female (14% of death cases of female cancer). Therefore, the establishment of the suitable breast cancer-bearing animal models and therapeutic strategies for development of more effective treatments for inhibition, not only of proliferation, but also of cancer metastasis is urgently needed. In this study, we presented the successful establishment of the orthotopic and ectopic allograft breast cancer model with different therapeutic strategies with chemotherapeutic drugs as 5-FU or cisplastin in mice. We hope that the orthotopic and ectopic allograft breast cancer model with therapeutic strategies with chemotherapeutic drugs will applied to the new anti-cancer drug research and development in the future.

Keywords: allograft, breast cancer, ectopic, in vivo, orthotopic platform, xenograft



High Protection of A Modified Live Porcine Reproductive And Respiratory Syndrome Vaccine (Fostera® PRRS) In The Experimental SPF Pigs

Shao-Wen Hung1, Chien-Chao Chiu1, Chia-Chi Chen1, Ruei-Chen Hung1, Pin-Tasn Tsai1, Min-Wen Chang2, Ying-Ching Hung1, Chia-Ying Lin1, Hua-Wen Lin1, Jen-Wei Lin1, Cheng-Yao Yang1

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

2Zoetis Taiwan limited, Taipei, Taiwan

The objective of this study was to evaluate the efficacy of commercially available attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine (Fostera® PRRS) against a heterologous type 2 PRRSV challenge. A total of 24, 3 week-old SPF pigs seronegative for PRRSV were randomly divided into 2 groups (control group and vaccine group). Twelve pigs were administrated with 2 mL dose of Fostera® PRRS vaccine intramuscularly and control group were administrated with 2 mL adjuvant. Pigs were kept in separate rooms to serve as negative controls. Four weeks post-treatment (7 weeks old), all pigs were challenged with a local virulent PRRS isolate MD-005 (105 TCID50/mL) via intranasal (1 mL) and intramuscular (1 mL) routes. Pigs were necropsied on 14 dpi and lung lesion scores were visually estimated by observers blinded to groups. Pigs in the vaccine group with PRRSV challenge showed significantly lower levels of mean body temperatures, clinical severity, lung lesion scores, and viremia compared to the pigs in the control group. Vaccine group had significantly higher survival rate and average daily gain. Taken together, this study demonstrates that high protection of Fostera® PRRS in the experimental SPF pigs against the local virulent PRRS isolate MD-005 challenge in growing pigs.

Keywords: allograft, breast cancer, ectopic, in vivo, orthotopic platform, xenograft.



Case Report: Combined Type Osteosarcoma of Humerus in a Dog

Yi-Xin Mao1, Kuan-Lun Wu2, Cheng-Feng Tsai2, Dan-Yuan Lo1, Kun-Wei Chan1,2*

1Department of Veterinary Medicine, National Chiayi University, Taiwan

2Veterinary Teaching Hospital, National Chiayi University, Taiwan


Osteosarcoma (OSA) is the most common primary bone tumor in dogs, generally occurs in middle-aged to older dogs with a median age around 7. About 75 % of canine OSA occur in the appendicular skeleton. Proximal humerus, distal radius, distal femur and proximal tibia are predisposed sites. A 10-year-old neutered male old English sheepdog weighing 24.95 kg was referred to National Chiayi University Veterinary Teaching Hospital due to sudden non-weight-bearing lameness of right forelimb, without any vehicle accident or trauma history. Radiographic examination of the right humerus showed comminuted fracture of the proximal metaphysis and spiral fracture of diaphysis. Osteolytic lesion consistent with a primary bone tumor has also been noticed. High nucleus-to-cytoplasm ratio oval to polygonal cells was seen in cytological examination. After amputation surgery, there was five white nodule-like mass at the cross section of the patient’s proximal humerus, and grade Ⅱ combined type osteosarcoma was diagnosed. OSA is a progressive tumor, some dogs with rapidly growing osteolytic OSAs present acute lameness associated with a pathological fracture. Most dogs either died or were euthanized sine pulmonary metastasis usually occurs early in the course of disease. Nine months after the amputation surgery, nodules had been found in the patient’s thoracic radiographs.

Keywords: Osteosarcoma, primary bone tumor, pathological fracture, osteolysis, pulmonary metastasis



Comparison of Efficacy and Adverse Event of 15-Week CHOP Protocol with 25-Week Protocol in Canine Multicentric Lymphoma of NTUVH: 2010-2018

Chia-Lien Kao1, Jih-Jong Lee1  



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


Lymphoma is the most common hematopoietic tumor in dogs, and multicentric lymphoma serves 84% of dogs diagnosed with lymphoproliferative disease. Although 15-week CHOP protocol has been more frequently used in NTUVH for the recent years, difference between the traditional 25-week protocol in actual efficacy and toxicity profile is still lack of extensive evaluation. In the present study, the medical records of a total of 64 dogs diagnosed with multicentric lymphoma from 2010 January to 2018 February were reviewed retrospectively, with 42 dogs given 25-week protocol and 15 dogs administered with 15-week protocol. No significant difference was found in the distribution of patient characteristics between two groups. The objective response rate was 98.4% for 25-week group and 100% for 15-week group (P=0.466). The median time to progression (TTP) was 242 days for 25-week group and 217 days for 15-week group (P=0.503). The overall survival time (OST) was 354 and 326 days for 25-week group and 15-week group, respectively (P=0.999). For adverse events, neutropenia, anorexia and vomiting were the most frequently presented during the course of treatment. There was no significance in occurrence rate of any adverse events between two groups. For prognostic factors, dogs with body weight higher than median value, Golden retrievers and involvement of lung in radiography were found to be related to lower rate of complete remission (CR). Higher body weight, Golden retrievers and failure to achieve CR were associated with shorter median TTP. However, only CR or not remained significant in multivariate analysis for TTP. For OST, dogs experiencing neutropenia were found more likely to have longer OST (514 days versus 309 days, P=0.029) in univariate analysis. In conclusion, shortening and dose intense regimen of 15-week protocol resulted in a similar efficacy and toxicity profile to those of 25-week protocol in the present study.

Keywords: CHOP protocol, chemotherapy, canine multicentric lymphoma



Cleavage of Coronavirus RNA Is Associated with Cellular Endoribonuclease RNase L

Hsuan-Yung Lin1, Hung-Yi Wu1

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

Cellular RNase L is an endoribonuclease associated with antiviral defense induced by innate immunity. RNase L cleaves RNA predominantly after single-stranded UA and UU dinucleotides. The cleavage of coronaviral RNA and the features for the cleavage have not been previously probed. In an attempt to test whether coronavirus defective interfering (DI) RNA with transcription regulating sequence (TRS) is able to transcribe, a short RNA fragment was unexpectedly found and designed ST RNA. Subsequent study showed that the cleaved site is downstream of TRS, in the loop region and after UU dinucleotides. Additionally, the ST RNA was identified in mock-infected HRT18-cells, indicating that the cellular but not viral factors are responsible for the cleavage. To characterize the features of the cleavage, mutagenesis followed by Northern blot assay was performed and it was found that the sequences upstream and downstream of UU dinucleotides has influence on the efficiency of the cleavage, consisting with the general criteria of cleavage by cellular RNase L. The similar cleavage was also found in A549 cells from which RNase L can be activated. Combined, we for the first time demonstrated that the cleavage of coronavirus genome is correlated to cellular endoribonuclease RNase L. Accordingly, since similar cleavage was found in both active RNase L inducible (A549) and non-inducible (HRT) cells, it is proposed that cells may have basal levels of active RNase L, which may serve as the first line of defense by cleavage of incoming virus RNA in the very early stage of infection.

keywords: Coronavirus, IFN, RNase L



Bacteria Isolation and Antibiotic Selection after Ovariohysterectomy of Canine Pyometra

Ying-Shuo Tang1, 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

Canine pyometra is a common reproductive disease in bitches. The aim of this study is to evaluate the causative bacteria and antibiotic sensitivity test. There were fifty-five pyometra bitches included in this study. Escherichia coli was the most isolated microorganism (63.6%), followed by Streptococcus Spp. (10.9%). Imipenem was the most sensitive antibiotic (95.2%) followed by Gentamycin (85.5%), Amikacin (80.6%), Ceftazidime (72.6%), Nitrofurantoin (72.6%) and Norfloxacin (72.6%) to all 62 isolated pathogens. The microorganisms were most resistant to Lincomycin (96.8%), followed by Penicillin G (85.5%), Trimethoprim + Sulfamethoxazole (58.1%), Amoxicillin (54.8%), Ampicillin (53.2%) and Cefadroxil (50%), respectively. We found the sensitivity of single empirical antibiotic used before sensitivity test was only 51% (25/49). The sensitivity increased to 84.6% (11/13) when we used two antibiotics simultaneously. All pyometra dogs were recovered after ovariohysterectomy, antibiotic administration and supportive therapy. The results of this study provide a reliable information to choose the effective antibiotics before surgery and sensitivity test results. The outcome of canine pyometra was excellent under early diagnosis and appropriate treatment intervention.

Keywords: Antibiotic, bacteria, canine, pyometra



Evaluation of Clinical Effectiveness of a Porcine Reproductive and Respiratory Syndrome Virus Subunit Vaccine in Nursery Pigs

Chen-Huei Yen1, Chih-Cheng Chang1

1Department of Veterinary Medicine, National Chiayi University

The aim of this research was to evaluate the clinical effectiveness of an porcine reproductive and respiratory syndrome virus (PRRSV) subunit vaccine in nursery pigs in the field. Twenty 3-weeks-old pigs were randomly distributed into two groups. Ten pigs in the vaccine group was injected with this subunit vaccine and the other 10 pigs in the control group injected with the same volume of phosphate buffered saline (PBS) . Blood samples of all pigs were collected at 4, 6, 8, 10, 12 and 15 week-old after injection and used to detect the titers of PRRSV and porcine circovirus type2 (PSV2) specific antibodies and the concentrations of Interleukin 10 (IL-10) by Enzyme-linked immunosorbent assay (ELISA); and viral loads of PRRSV and PCV2 by quantitative real-time polymerase chain reaction (qrt-PCR). Average daily grains (ADG), feed conversion rate (FCR) and mortality were counted at age of 12 week-old to evaluate the final clinical effectiveness.  The result showed that pigs in the vaccine group had higher PRRSV antibody titer but lower viral load, and lower PCV2 antibody titter but higher PCV2 positive rate respectively.  IL-10 was only detected in four pigs of vaccine group at age of 12 week-old. The mortality is higher (70 vs 0 %) in the vaccine group when compared with control group. Taken together, the clinical effectiveness of this subunit vaccine was not expected in nursery pigs in the field.

Keywords: rcine reproductive and respiratory syndrome virus (PRRSV), Subunit vaccine, Effectiveness



Characterization of Escherichia coli Isolated from Chicken Fluff in Taiwan Hatcheries.

Shengnan Zhao1, Chung-Hsi Chou1

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

Colibacillosis is an economically important syndromic disease of poultry caused by extra-

intestinal avian pathogenic Escherichia coli (APEC). In order to know whether Escherichia

coli from chicken fluff in Taiwan hatcheries have potential to cause colibacillosis, 47 isolates

from 2953 fluff samples all around hatcheries in Taiwan and 20 poultry clinical extra-intestinal isolates were collected during the period from 2016 to 2017. These isolates were characterized for virulence factors (polymerase chain reaction), antimicrobial susceptibility (broth microdilution method), and genetic relatedness (XbaI pulsed field gel electrophoresis).

The results showed that the most predominant virulence factors were fimC, 89.4% in fluff isolates and 85.0% in clinical isolates. The frequence of several virulence factors in fluff isolates were 8.5%( fyuA), 48.9% (iutA),19.1%(cvaC) and14.9%(astA) compared with 55.0%(fyuA), 75.0% (iutA), 45.0%(cvaC), 30.0%(astA) in clinical isolates. Antimicrobial tests indicated that all of the fluff and clinical isolates were found resistant for ampicillin, amoxicillin, cephalexin, florfenicol and trimethoprim-sulfamethoxazole, however, none of them were resistant to colistin. Similar resistance frequence between fluff and clinical isolates were found in ceftiofur (63.8%,65%), gentamicin (8.5%, 10.0%) and doxycycline (85.1%, 95.0%) while differences were in neomycin (10.6%, 0%), flumequine (42.6%, 80.0%) and nalidixic acid (74.5%, 95%). Based on pulsed field gel electrophoresis, two pairs of fluff isolates shared 100% similarity pattern respectively indicated persistent contamination within these two hatcheries. The most related isolates between fluff and clinical was about 82% similarity. These results suggest that Escherichia coli from chicken fluff in Taiwan hatcheries may have potential to cause colibacillosis and hatchery hygiene should be noticed.

Keywords: fluff, hatcheries, Escherichia coli, virulence factor, drug resistance, pulsed field gel electrophoresis



Uric Acid May Be a Potential Biomarker for Equine Laminitis.

Szu-Ting Lin1, Chia-Chen Chang1, Tsung-Ching Liu2, Hsiao-Chien, Lo2, Yi-Lo Lin1

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

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

Laminitis can cause stress, severe lameness, and irreversible laminae damages to horses. Biomarkers associated with laminitis may provide veterinarians another diagnostic method at the developmental stage of disease progression. Damage-associated molecular patterns (DAMPs) are endogenous danger molecules. High mobility group box 1 (HMGB1), a type of DAMPs, activates pro-inflammation in the development of laminitis. Uric acid, another DAMP, indicates oxidative stress, a feature of laminae damages in laminitis. Apart from DAMPs, lactate acid is an indicator of tissue hypoperfusion, a characteristic of laminae damages in laminitis. Cortisol, a stress hormone, was found significantly higher in horses with acute laminitis than control group. However, researches on the relation of cortisol level and the severity of laminitis are limited. In the present study, we investigated these potential serum biomarkers in non-laminitic and laminitic horses. Serum samples were divided into four groups: control (without laminitis), risk (under risk; without laminitis), mild (Obel grades 1-2), and severe (Obel grades 3-5). Uric acid is significantly higher in laminitic horses, and may be specific to laminitis. HMGB1 might be associated with laminitis with the specificity lower than uric acid. There’s a moderate correlation between HMGB1 and uric acid, indicating that DAMPs may play some part in the pathogenesis of laminitis. Cortisol is significantly higher in severe group, and may be a potential biomarker for stress quantification in laminitis. Finally, besides being specific to equine laminitis, measurement of uric acid is cheaper and easier. All of these advantages make uric acid an ideal biomarker for equine laminitis.

Keywords: laminitis, DAMPs, cortisol, biomarker



Identify Spatial Clusters and Determine Environmental Factors During the Avian Influenza Outbreak in Taiwan, 2015 to 2017

Yu-Chen He1, Wei-Shan Liang1, Hong-Dar Wu2, Day-Yu Chao1

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

2Institute of statistics, National Chung Hsing University, Taichung, Taiwan

Since 2014, new subtypes of high pathogenic avian influenza virus (HPAIV) descendant from H5N1 clade 2.3.4 viruses have emerged from China and spread to worldwide rapidly through the migrating bird flyway.[1] To date, no human case have been reported, but the new clade of HPAIV has caused among poultry farms, even result in huge socio-economic impacts due to the losses of birds killed by the disease or by culling, and from the disruption of trade and market activities imposed by disease control measures such as movement restrictions and a temporary ban of poultry product exports.[2] From 2015 to 2017, there were 1,178 poultry farms confirmed as HPAIV positive in Taiwan. In this study, we identified the spatial clusters through spatial analysis and determine the environmental factors associated with the clustering by logistic regression. The result of spatial autocorrelation analysis, three kilometers radius is the critical distance of spatial clustering for HPAIV outbreaks among the poultry farms. Through Local Moran's I analysis, the main area of hot zone is in the mid-western of Taiwan, especially Yunlin and secondary hot zone is in the north of Pingtung. The stepwise multivariate logistic regression analysis suggested three risk factors strongly associated with the HPAIV hotspot areas throughout 2015-2017, including high poultry farm density, waterfowl-chicken mixed farming density and cropping coverage percentage. Based on the analysis results, we further developed the risk map, which will assist in future surveillance and disease control among the poultry farms in Taiwan.

Keywords: Avian Influenza outbreak, spatial autocorrelation, Local Moran's I, stepwise multivariate logistic regression



Colonization of Bacteroides fragilis Prevents Colonic Neoplasia in Colitis-associated Cancer Model

Yen-peng Lee1, Chien-Chao Chiu2, Shao-Wen Hung2, Tien-Jen Lin3, Yi-Hsun Chen1, Ter-Hsin Chen1, Hsiao-Li Chuang4

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

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

3Department of Neurosurgery, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan.

4National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan

Colorectal cancer (CRC) is a serious health problem in the worldwide. A connection between colorectal carcinogenesis and inflammation is well known in recently studies. The clinical investigation found patients with inflammatory bowel disease (IBD) are at a higher risk for developing colitis-associated cancer than the general population. Chemically induced colitis-associated cancer (CAC) is an outstanding mouse model for studying intestinal inflammation and cancer. This study was aimed to investigate the protective role of Bacteroides fragilis (BF) in a CAC model that by azoxymethane (AOM) and dextran sulfate sodium (DSS) in germ free (GF) mice. The GF mice were colonized with BF for 4 weeks before CRC induction. Our results showed that the BF colonization increased animal survival (100%) and reduced cecum weight. Interestingly, the tumor number and tumor incidence were significantly decreased in BF colonized group. In addition, immunohistochemistry staining found that the numbers of cell proliferation (PCNA) and inflammation (COX2) index were decreased in the colon mucosa of BF group. Taken together, the BF colonization significantly ameliorated AOM/DSS-induced CAC by suppressing the activity of cell proliferation-related molecules and reduced the number of inflammatory cells. These findings suggest that BF might play a pivotal role in maintain gastrointestinal physiologic balance and regulate anti tumorigenesis responses.

Keywords: Colorectal cancer, colitis-associated cancer, symbiotic, Bacteroides fragilis



Activating RAS Accelerates the Pathogenesis of Diabetic Nephropathy in db/db and ACE2 Gene Knockout Mice

Xing-Yang Xie1, Ching-Chang Cho1, Cheng-Yi Chen2, Hui-Fang Chang2, Chih-Sheng Lin1

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

2Department of Internal Medicine, Hsinchu Mackay Memorial Hospital, Hsinchu, Taiwan

Obesity prevalence has been increased and became a main health problem in the world. Previous studies have showed that renin-angiotensin system (RAS) activation is highly related to obesity and hypertension. Therefore, objective of this study is to explore the RAS activation related with the progression of diabetic nephropathy in db/db and angiotensin converting enzyme 2 (ACE2) double gene knockout (db/ACE2-KO) mice. The db/ACE2-KO mice at 8, 12, 16 and 20 weeks old were used in this study.

The results show that fasted blood glucose level in 8-week mice was 256 mg/dl and its level was significantly increased to 368, 409 and 492 mg/dl in mice at 12-week, 16-week and 20-week, respectively. There were significantly increased levels of serum creatinine and urinary albumin, and decreased levels of creatinine clearance in 20-week mice compare with those in 8-week mice. The results indicate that diabetic nephropathy of db/ACE2-KO mice is rigorously developed along with the animal age. The conclusion was supported by the kidney pathological findings that show more expansion of mesangial matrix and increased of tubulointerstitial fibrosis in the kidney of 20-week mice. The renal expression levels of ACE, chymase and Ang II were successively increased from the 8-week to 20-weed db/ACE2-KO mice. RAS activation in the mice with db/ACE2-KO could be confirmed. The renal RAS activation accompanied with the increasing levels of renal p-JNK was detected. According to the results, it might be proposed that RAS activation could accelerate the pathogenesis of diabetic nephropathy via Ang II-AT1R/p-JNK signaling pathway.

Keywords: Diabetic Nephropathy, renin angiotensin system, angiotensin converting enzyme, gene knockout mice



A Study on the Case and Drug Sensitivity of Waterfowl Riemerella anatipestifer

Cheng-Hao Su1, 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

In the case of waterfowl in Taiwan in 2016, ducks, geese, and goslings are the main breeding grounds in the central and southern parts of the country, and Pingtung County, Yunlin County, Changhua County, and Chiayi County are the main cities and counties, and Pingtung County is the county and city. In order to keep the bulk, it accounts for a third of the total number of stations in Taiwan. Pingtung, Taiwan is hot and humid throughout the year, with an average monthly humidity of more than 80%, which is conducive to the growth and spread of pathogenic bacteria. Ducks and geese, in particular, can spread diseases such as excreta, air, wounds, and feeding environments. Infections such as Salmonella, Escherichia coli, Reesei, coccidiosis, etc. The current high bed breeding of ducks can avoid the breeding of pathogens, and the management is easier. However, the intensive feeding and the disease dissemination speed are relatively fast. The current practice of sharing a total drink with geese is only slightly different. This experiment is mainly to investigate the frequency and seasons of waterfowl Riemerella anatipestifer in ducks and geese. Through the polymerase chain reaction (PCR), drug susceptibility testing and pathological diagnosis, it provides research on disease detection and prevention. The experiment was conducted from June of 105 to the end of December of 106. The samples were obtained from the liver, brain, air sac, and pericardium of ducks and geese for bacterial culture, drug susceptibility testing, and case statistics. A total of 178 cases were sent to the necropsy sent by waterfowl. The polymerase chain reaction test results used Riemerella anatipestifer (26%, 46/178) to account for 1/4 of the total. In 46 cases, the drug susceptibility test was Apramycin (24/46, 52%), Flumequine (25/46, 54%), Doxycycline (20/46, 43%), Cephalexin (31/46, 67%), Trimethoprim/sulfamethoxazole (17/46, 37%) and Chloramphenicol (19/46, 41%) are more sensitive (S), but due to the fact that the owner may not comply with the administration and have their own inertia, they may also develop drug resistance. Histopathological diagnosis includes Pericarditis and meningitis. The number of diagnosed cases in winter RA was also the highest, while prophylactic or therapeutic administration of Apramycin, Flumequine, and Cephalexin could control the outbreak of the disease.

Keywords: Waterfowl, Polymerase chain reaction, Drug sensitivity test



Feasibility of Porcine Teschovirus Derived Vector carrying both Foot and Mouth Disease VP1 capsid protein and Classical Swine Fever E2 glycoprotein epitopes as a Vaccine

Chih-Shuan Sheng1, Tung-Hsuan Tsai2, Fun-In Wang1

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

2Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

The family Picornaviridae comprises a large number of animal pathogens, and it is also a model system for the study of positive single-stranded RNA virus biology, pathogenesis, and epidemiology. The development of reverse genetics technology allows the analysis in details of the structure and function of these viruses. Poliovirus has several advantages to serve as vaccine vector, including easy to deliver orally, inherent stability in the intestinal tract, and the ability to induce both humoral and cell immunities. Like poliovirus, porcine teschovirus (PTV) belongs to the family Picornaviridae, which is a positive single-strand RNA virus. The aim of this study was to take the advantages of PTV to develop a vaccine vector. PTV encephalomyelitis is not an OIE listed disease that could be widely detected in swine herds worldwide, and yet shows mild virulence (except for a few recent virgin epidemics), thus lowers the risk of environmental impact and disease prevention by using it as a vaccine vector. Also, PTV has a wide tissue tropism making it feasible to be used as a platform to develop multivalent mucosal vaccine vector by carrying exogenous genes. Its genome length of only 7 kb makes it easier to construct and to present heterogenous proteins. Thus, a recombinant PTV virus was constructed by replacing the B-C loop region on its VP1 protein with both classical swine fever E2 and foot and mouth disease VP1 epitopes, and its G-H loop region by poly-His tag for easier distinction from wildtype virus and easier protein purification and detection if necessary. Immunofluorescent assay showed that the constructed PTV virus was able to express both heterogenous epitopes. These foreign proteins could be detected by both polyclonal and monoclonal antibodies directed to each protein or antigenic sites. Antigenicity and immunogenicity will be tested further in experimental animals.

Keywords: porcine teschovirus, virus-derived vector, immunofluorescent assay



Cytokine Profile of Bovine Peripheral Blood Mononuclear Cells in Challenge Against Low Virulent Bluetongue Virus Taiwan Isolate

Chia-Wei Yen1, Jia-Ling Yang2, Fun-In Wang1

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

2Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

Bluetongue (BT) is an insect-transmitted disease of domestic and wild ruminants caused by bluetongue virus (BTV), and it is one of the economic threats to animal husbandry worldwide. BTV infection in cow and goats has been recorded in Taiwan; although being subclinical, it still poses a potential risk for livestock. Most studies on BTV-induced immune responses focused on the induction by virulent strains of outbreaks in western countries. Least has been studied in low virulent strains as well as the host responses raised in minor susceptible species, like dairy cow and goats in Taiwan. In this study, an exploration of the Th1 and Th2 pathways in the course of the immune response against Taiwan strains (BTV2/KM/2003) of low virulence was performed. Immunofluorescence of anti-NS3 protein demonstrated the replication of BTV in adherent PBMCs 36 hour-post-inoculation. Meanwhile, nuclear immunoreactivity for caspase 3 and membranous signals of annexin V in PBMCs was consistent with morphological changes of apoptosis in the face of viral attack. Relative quantification of cytokine mRNA showed decreased IL-1 and increased IL-4 expression in the early stage of BTV2 infection, suggestive of the initiation of Th2 pathway which is detrimental for viral replication in hosts. The trend of rest of studied cytokines such as TNFα, IL-12 and INF-γ were variable and inconclusive.

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



Analysis of Genome Segment 7 and Amino Acid Sequence of 2018 Bluetongue Virus Isolate in Taiwan

Hao-Che Yen1, Jia-Ling Yang2, Fun-In Wang1

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

2Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

Bluetongue is an arthropod-borne disease, in domestic and wild ruminants, caused by bluetongue virus (BTV), which lead to great economic loss worldwide. Previous studies showed BTV infection in Taiwan is often subclinical, and 2 viruses had been isolated from specimens in Kinmen island and Pingtung district, respectively. Genetic analysis of the 2 isolates showed a positive or diversifying selection of segment 7, suggested that BTV might evolve and adapt when entering Taiwan as a new environment. Genome segment 7 encodes an outermost BTV core protein, VP7, which is the most accessible protein of the BTV core that may participate in cell entry. Taiwan is geographically isolated for a long period of time, and inhabited by several endemic species, including Culicoides, the vector of BTV. The hypothesis is that these endemic Culicoides species in Taiwan provide an evolution pressure to BTV, especially on genome segment 7. Sequence alignment of Taiwan BTV isolates VP7 with isolates from neighboring areas including Japan, China, India and Australia showed amino acid changes in 3 specific sites located close by on three-dimensional model of VP7. The project is to investigate the new viral strains and the distribution of these strains on Taiwan. Blood samples are collected from different districts in Taiwan. RT-PCR and ELISA test were performed. Viral isolation via chicken embryos and BHK21 cell line is in progress.

Keywords: bluetongue, segment 7, VP7, genome alignment



The Status of Mycoplasma suis Infection in Taiwan and the Correlation with Porcine Reproductive and Respiratory Syndrome

Chiun-Te Wu1, Chao-Nan Lin1,2, Ming-Tang Chiou1,2

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

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

Mycoplasma suis (Ms) can cause fever, hemolytic anemia, hypoglycemia and reproductive failure in pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) can lead to reproductive failure and respiratory disease. The main purpose of this study was to investigate on the infection status of Ms in Taiwan and the correlation with the PRRSV. Blood samples were collected from 43 swine farms, in total 1,268 cases, during the April 2016 to March 2018. Sows and nursery pigs at least take 10 samples in each farms. Polymerase chain reaction (PCR) was employed for Ms detection. Ten out of forty-three swine herds were randomly choose for PRRSV determination by reverse transcription quantitative polymerase chain reaction. The Ms positive rate reach 20.9% (265/1268) and 93% (40/43) in total samples and swine farms, respectively. 31.2% (197/632) and 10.7% (68/636) show positive of Ms for sows and nursery pigs, respectively. In the ten out of forty-three swine herds, Ms positive rate reach 19.4% (57/294) and 90% (9/10) of total samples and swine farms, respectively, and 25.5% (38/149) and 13.1% (19/145) of sows and nursery pigs, respectively. PRRSV positive rate was 29.8% (70/235) and 80% (8/10) of total samples and swine farms, respectively, and 1.7% (2/115) and 56.7% (68/120) of sows and nursery pigs, respectively. Collectively, these results showed Ms and PRRS were both high infection rate in Taiwanese swine herds. There was no relationship between these two pathogens in occurrence.

Keywords: Mycoplasma suis, Infectious anemia of pigs, Porcine reproductive and respiratory syndrome virus, PCR



Characteristic and Structural Prediction of Insect-Specific Flaviviruses Envelope Protein

Rong-Xuan Hong1, Shyan-Song Chiou1

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

In last decade, there has been a dramatic increase in the number of insect-specific flaviviruses (ISFs) discovered. ISFs can infect insects and insect cells, but they do not replicate and infect vertebrates or vertebrate cells. Some ISFs have been shown to suppress or inhibit the replication of medically important flaviviruses (MIFs) in mosquito cells. The envelope glycoprotein (E protein) of flavivirus is an important determinant for host range or tissue tropism. But, the characteristics of ISFs E protein are still unclear. Here, comprehensive and total 37 ISF E protein sequences were collected, and analyzed and predicated with public-domain software. The phylogenic tree based on E protein amino acid sequence shown ISFs classified into two groups, classical ISFs (cISFs) and dual-host affiliated ISFs (dISFs), except Paraiso Escondido virus. The length of the E protein amino acid sequence of cISFs (420 to 430 a.a) was shorter than dISFs (490 to 500 a.a) and MIFs (480 to 500 a.a). The MIFs E protein is glycosylated at 1 to 2 amino acids, the potentially N-linked glycosylation sites of dISFs and cISFs were 0~2 and 2 amino acids, respectively. There were six conserved disulfide (SS) bonds in the MIFs E protein, the predicated SS bonds in the dISFs and cISFs E protein were 5 and 6~7 bonds, respectively. The dimer structure of ISFs E protein predicated by using SWISS-MODEL based on the resolved and best template of Japanese encephalitis virus E protein. The MIFs E protein folds into three distinct domains (domain I, II, and III), and forms tight contact dimer (higher buried surface area, BSA). The predicated structure of dISFs E protein was similar to MIFs, but the cISFs shown different characteristics, including domain folding, lower BSA, receptor binding motif, and fusion loop. The analysis of transmembrane region of ISFs is still ongoing. Overall, the dISFs were genetically close-related to MIFs, and the predicated E protein structure also shown similar features. 

Keywords: Flaviviruses, Insect-Specific Flaviviruses, Envelope Protein



A SimpleProbe® Real-time PCR Assay for Differentiating the Canine Parvovirus Type 2 Genotype

Minh Hoang1,2, Hung-Yi Wu3, Ying-Xiu Lien4, Ming-Tang Chiou1,4*, Chao-Nan Lin1,4*

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

2 Department of Anatomy and Histology, Faculty of Veterinary Medicine, Viet Nam National University of Agriculture, Ha Noi, Viet Nam

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

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

*Correspondence: mtchiou@mail.npust.edu.tw; cnlin6@mail.npust.edu.tw; Tel.: +886-8-7740177

Canine parvovirus type 2 (CPV-2) causes an important canine viral disease worldwide. CPV-2

belongs to the Protoparvovirus genus in the family Parvoviridae. An amino acid change at position 426 of the VP2 protein yields different types of CPV-2, designated as CPV-2a (Asn), CPV-2b (Asp) and CPV-2c (Glu). In this study, we compared CPV-2 genotyping results obtained by SimpleProbe® real-time PCR and DNA sequencing analysis. One hundred clinical rectal swab samples were collected from CPV-2 naturally infected dogs and analyzed in parallel using the SimpleProbe® assay and DNA sequencing. CPV-2a (n=23), 2b (n=6) and 2c (n=71) genotyping results obtained by both techniques were identical. In conclusion, the SimpleProbe® assay can be used to detect residue 426 mutations in the CPV-2 VP2 gene from clinical specimens; thus, this assay provides a reliable and sensitive tool for differentiating between CPV-2a, CPV-2b and CPV-2c.

Keywords: protoparvovirus; canine parvovirus type 2; genotype; SimpleProbe®



Study of Immune Mediated Hemolytic Anemia in Domestic Dogs in Taiwan

Ming-Tso Yen1, Jui-Te Wu1,2, Shih-Jen Chou1,2

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

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

Clinical symptoms of anemia may vary due to the severity and the progress rate. Immuno-mediated hemolytic anemia (IMHA) causes severe anemia and has a high mortality rate in dogs. In this study, blood samples were collected from animal hospitals in northern, central and southern Taiwan. Total amount of blood samples was from 220 dogs and their hematocrit (HCT) were below 37%. All samples were examined by hematologic examination, IDEXX SNAP 4Dx® tests, direct antiglobulin test (DAT), and polymerase chain reaction assay for tick-borne haemoparasites, and further evaluate the correlation between test results and IMHA. In results, thirty-seven dogs with anemia, autoagglutination and spherocytosis were diagnosed as positive for IMHA (IMHA+); however, only 14 IMHA+ dogs were positive to DAT (DAT+). All IMHA- dogs were negative to DAT. Contrast to IMHA- dogs, there were significant anemia and leukocytosis in IMHA+ dogs. Babesia gibsoni (57%/51%) was major haemoparasite in IMHA- and IMHA+ dogs, Babesia canis (7%/3%), Dirofilaria immitis (5%/8%) and Mycoplasma Haemocanis (0/3%) were secondary. All IMHA+ dogs were treated with steroids or parasiticides, 82.6% (19/23) and 78.6% (11/14) treatment effects were observed at IMHA+ and DAT+ groups, respectively. The results of this study show that there is a better diagnostic sensitivity for IMHA based on the symptoms of anemia, autoagglutination and spherocytosis.

Keywords: PCV, IMHA, Direct Antiglobulin Test



Liposomes-encapsulated Propranolol Administered by Iontophoresis Enhances Bone Mass at Spine in Ovariectomized Rats

Wei-Hsin Tsai1, Benjamin Teong2, Chung-Hwan Chen2, Shyh-Ming Kuo3, Ming-Yen Hsiao1, Syu-Lun Lin1, Han-Hsiang Huang1*

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

2Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan

3Department of Biomedical Engineering, I-Shou University, Kaohsiung, Taiwan

The authors contributed equally to this study


Propranolol is a nonselective β-adrenoceptor blocker widely used for symptom control such as arrhythmia, hypertension, and angina pectoris. It has been revealed that propranolol is able to enhance bone formation and osteoblastogenesis against age-related bone loss in murine models and epidemiological studies. Nonetheless, propranolol undergoes extensive hepatic first-pass metabolism and thus resulting in poor bioavailability. These drawbacks may largely limit the potential anabolic effects of propranolol on bone in vivo. Therefore, the aim of this study is to investigate and evaluate the anti-osteoporotic effects of liposomal propranolol in combination with iontophoresis at spine in ovariectomized (OVX) rats. We hypothesized that the disadvantages of propranolol can be partially improved by transdermal delivery cooperated with liposomal drug carriers. In the current study, the anti-osteoporotic effects between transdermal delivery and subcutaneous injection were compared as various dosage forms were also evaluated. The results showed that significant improvements in bone volume and total tissue volume (BV/TV) and bone mineral density (BMD) were found in the fourth lumbar spine when low-dose (0.05 mg/kg) liposomal propranolol was iontophoretically administered. Iontophoretic low-dose liposomal propranolol also markedly elevated trabecular thickness in spine. Taken together, liposomes-encapsulated low-dose propranolol delivered by iontophoresis exhibited anabolic effects against bone loss at spine in vivo.

Keywords: propranolol, liposome, bone loss, iontophoresis



Case Report: Comminuted Fracture of the Left Femoral Diaphysis in a Dog

Pao-Hsin Wu1, Cheng-Feng Tsai2, Chia-Ming Chang3, Jui-Te Wu1,2, Kuan-Lun Wu2, Kun-Wei Chan*1,2

1Department of Veterinary Medicine, National Chiayi University

2Veterinary Teaching Hospital, National Chiayi University

3Department of Veterinary Medicine, National Taiwan University

A 12 year-old, 13.6 kg female mixed-breed dog was jumping from a small truck and showed clinical signs of lameness and non-weight-bearing. The same the patient was taken to the Veterinary Medicine Teaching Hospital in National Chiayi University. The patient had a good mentation, and vital signs were normal with no obvious injury. The thoracic and abdomen radiograph examinations showed no specific findings, but the ventral dorsal frog leg view showed fracture of the left femoral diaphysis. It is a comminuted fracture, non-reducible on an old dog and fracture assessment score is 4. The suggested surgery is an open reduction with minimally invasive plate osteosynthesis (MIPO), bridging osteosynthesis, and plate-rod structure and C-arm fluoroscopy as image guide. The immediate postoperative radiographic examinations showed that the alignment, apparatus and apposition were within the acceptable range. Follow up with the patient shows that the gait has gradually gone back to normal and there are no other abnormalities.

Keywords: Minimally invasive plate osteosynthesis, C-arm fluoroscopy, Comminuted fracture

*Corresponding author: kwchan@mail.ncyu.edu.tw



Case Report: Intestinal Intussusception Following Toxicity in a Dog

Cheng-Feng Tsai1, I-Ting Lu2, Ming-Huei Fan1, Jui-Te Wu1,2, Kun-Wei Chan*1,2

1Veterinary Teaching Hospital, National Chiayi University

2Department of Veterinary Medicine, National Chiayi University

A 1-year-old, 13.3 kg male mixed-breed dog was referred here due to a suspicion of having ingested pesticides. Depression, paresis, tremor, salivation nasal discharges, brown-red feces and low temperature were observed during a physical examination. The dorsal-ventral abdomen radiograph of the dog showed a large volume of air in the stomach. The laboratory examinations showed no specific findings. According to this reason, environment, history and clinical signs, the initial suspicion of poisoning leads to gastrointestinal motility and that the metaldehyde is the most likely toxicity agent. After two days, the dog was discovered having suspected intussusception under ultrasound and was suggested laparotomy for treatment of the intussusception. Intussusception was found during surgery and the treatment was with intestinal anastomosis. The next day after surgery, the dog exhibited good attitude and appetite. The dog continued to make an uneventful recovery and was discharged from the hospital four days after surgery. At the time of suture removal 13 days later, the dog was reported to be clinically normal.

Keyword: Intestinal intussusception, Pesticides, Methiocarb

*Corresponding author: kwchan@mail.ncyu.edu.tw



Development of a Neutralizing Mouse-Pig Chimeric Antibody Against PCV2

Jun-Hong Liang, Tsun-Yung Kuo

1Institute of Biotechnology and Animal Science, National Ilan University

PCV2, the causative agent of PCVAD, is a serious economic problem in the swine industry in the world. There are several commercialized PCV2 vaccines are used in the farm currently that can reduce the economic loss. However, PCV2 cannot eradicated since the virus is circulated in the field all the time. In this study, a mouse-pig chimeric antibody was constructed. The variable region sequences of the hybridoma that secret neutralizing antibodies against PCV2 were analyzed, and fused with swine IgG constant region to build up the chimeric antibody. The chimeric antibody was expressed by mammals cells that had been co-transfected with the heavy chain and light chain constructs. The chimeric antibody can be detected by IFA and western blot analysis.

Keywords: chimeric antibody, pig, PCV2



Elimination of Malachite Green in Water by Chlorine Dioxide

Chung-Sheng KUO1, Cheng-Li Wu1, Jun-Ren Ke2, Tony Li3 and Chin-En Tsai*1

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



Aquaculture is still the fastest growing industry in the world. With this growth there are several diseases that hamper production which force farmers to use veterinary drugs. Malachite green (MG) is possible being used as an antiparasitic and antifungal agent in aquaculture. However, there are concerns on residue build up in fish tissues that can be harmful for humans once consumed. Therefore, if the use of such drug is to be continued, elimination from water systems is important to prevent residue build up. Chlorine dioxide is gaining popularity because of its effectiveness of inactivating pathogens and the degradation of harmful chemical residues. Therefore, in this study we investigated the potential of Chlorine dioxide eliminating Malachite Green in water. Five different concentrations 1000, 100, 10, 5 and 2.5 ng/mL of Malachite green were prepared along with the preparation of 50000, 20000, 2000, 200 and 20 ng/mL of Chlorine Dioxide. After mixing together the mixture was allowed to sit for 30 mins then the residual amount was analyzed by using a High Performance Liquid Chromatography (HPLC) machine. The results were then compared with the control group and the minimum effective elimination concentration was concluded. The detection limit of the HPLC was 1 ng/mL. The calibration Curve was,y=0.3475x-2.9502, r2=0.9997. Results show that 2000 ng/mL of Chlorine dioxide can completely eliminate Malachite Green.

Keywords: Malachite green, Chlorine dioxide, HPLC, eliminate



Role of Gut Microbiota in the Modulation of Atherosclerosis in ApoE-deficient Rats

Yi-Hsun Chen1, Ju-Yun Liu2, Jin-Fu Chen2, Chien-Chao Chiu2, Shao-Wen Hung3, Hsiao-Li Chuang2 and Yen-Te Huang2

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

2National Laboratory Animal Center, National Applied Research Laboratories Research Institute, Taipei.

3Division of Animal Resources, Animal Technology Laboratories, Agricultural Technology Research Institute, Miaoli, Taiwan

Atherosclerosis is the main cause of cardiovascular diseases. Previous studies have indicated gut microbiota protects ApoE-deficient mice from atherosclerosis, however, the roles of gut microbiota in the process of atherosclerosis in ApoE-deficient rats still remains unclear. The aim of the study was to clarify the effect of gut microbiota on the development of atherosclerotic lesions by using ApoE-deficient rats reared under SPF and antibiotics treatment. After antibiotic treatment for 26 weeks, there were no significant differences in body weight, serum AST, ALT, HDL, LDL, cholesterol and triglycerides between control, penicillin-treated (P-group) and gentamicin-treated (G-group). However, cecum weight and relative cecum weight in G-group were significantly higher than other groups. Compare to control group, cecum weight and relative cecum weight in P-group were slightly higher but no significant differences. Further investigation of plague in these groups, the plaque index in both G-group and P-group were significantly higher than control group. Compare to P-group, the plaque index in G-group was much higher. In our previous study, the plaque index in germ-free ApoE-deficient rats which associated with enlarged cecum were higher than SPF rats.  Considering of gram-negative bacteria is the target of gentamicin, killing of gram-negative bacteria lead to cecum enlargement. Taken together, the results suggested that gram-negative bacteria may reduce atherosclerosis in ApoE-deficient rats. Moreover, our results provided more information about the effects of gut microbiota on atherosclerosis in ApoE-deficient rats.

Keywords: atherosclerosis, antibiotics, gram-negative bacteria, gut microbiota



Antimicrobial Activity of Curcuma longa Rhizome Extract

Hui-Wen You and Chin-En Tsai*

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

Curcuma longa (turmeric), native to tropical South Asia, belongs to the Zingiberaceae family. Tumeric is very popular in India for its use in culinary and traditional medicine. Several reports published the antifungal, antibacterial, antitumor, anti-inflammatory and antioxidant properties of the plant. In this study, the antibacterial activity of ethanol and water extract of Curcuma longa rhizome was investigated against the standard strains; Staphylococcus aureus, Streptococcus canis, Micrococcus luteus, Bacillus subtilis, Bacillus cereus and Escherichia coli. The Rotary evaporator and steam distiller were used for extraction. The antibacterial activity of the rhizome extracts using the two mentioned methods were evaluated using the disk diffusion assay. Minimum inhibitory concentration and bactericidal concentration of the rhizome extract were determined using the broth microdilution method and major constituents were identified by high performance liquid chromatography (HPLC). Water extract showed antibacterial activity against M. luteus, B. cereus and S. canis and the Ethanol extract showed antibacterial activity against M. luteus, B. cereus, S. canis and S. aureus. The major consitituents found in C. longa rhizome extracts were curcumin, demethoxycurcumin and bisdemethoxycurcumin. In conclusion, the C. longa rhizome extract exhibited antimicrobial activity against some of the tested bacteria. The results reveal the potential use of C. longa rhizome extract in the treatment of infections.

Keywords: Curcuma longa, water extract, ethanol extract, antibacterial activity



Chronic Kidney Disease in a Cat

Jia Xin Yew1, Hsiao-Pei Tsai2, Kun-Wei Chan*1, 2

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

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

*is the corresponding author. Email: kwchan@mail.ncyu.edu.tw

Chronic kidney disease is among one of the most common clinical diagnoses of senior (11-14 years old) and geriatric (≥15-year-old) cats. It is described as structural and/or functional abnormalities of one or both kidneys that have been present for an extended period, usually three months or longer. No specific underlying cause can be identified in most of the cases. In this case, an eleven-year-old intact female mixed breed cat presented with symptoms of lethargy, anorexia, vomiting, polyuria and polydipsia for few months. On physical examination, the patient showed signs of mild dehydration and halitosis. From ultrasonographic examination, both kidneys showed reduced corticomedullary distinction, with the left kidney showing decrease in size and irregular contour. After the correction of dehydration status, mild normochromic normocytic non-regenerative anemia was shown in complete blood count. Serum biochemistry indicated elevation of blood urea nitrogen (74 mg/dL), serum creatinine (8.8 mg/dL), and phosphate level (12.5 mg/dL). Based on the International Renal Interest Society (IRIS) classification, the patient was grouped into stage III/IV of the disease progression. Therefore, the therapy goals were designed to minimize renal azotemia and clinical signs by fluid therapy, diets and prescribed medicines. In addition, regular monitoring of the patient was a necessary part of the course of treatment. After two weeks of treatment, the patient did not show any significant abnormalities during its return visits, and blood urea nitrogen, serum creatinine and phosphate levels were stabilized.

Keywords: chronic kidney disease, creatinine, IRIS



Effect of Feeding Bacillus subtilis to Hematology and Fecal Microbiota of Calves

Yu I Pan, Ya Fang Tseng, Yong-Chang Lyu, Nan-Hui Lin, Hsu-Hsum Lee, Shyh-Shyan Liu*

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

As biocontrol agents, B. subtitis ability to compete with bacteria for nutrients, and release antibiotic substances, makes it widely use to inhibit bacterial growth in plants. Studies have shown that adding B. subtitis to the feed of pigs can effectively inhabit the growth of harmful bacteria in their intestinal tract. When applied on ruminants (Holstein cows), we hope to achieve the same effect, hence, increase production yield. In this experiment, 10 one-to-two weeks old healthy calves were randomly picked, and B. subtitis added to their replacer milk daily for 4 weeks. Test samples collected on the first day was used as control group. During the experiment, stool samples were collected weekly for bacteria count, while hematological examinations carried out on blood samples drawn biweekly were used to monitor the health status of the calves. Results from the stool samples shows that fecal B. subtitis counts were significantly higher and sustained increase at 2nd week, while other intestinal pathogens such as, Ehrlichia coli and Salmonella spp., decreased significantly. Hematological results carried out for TP, ALB, T/G rate, GGT, ALT, CK, BUN, Uric acid, Creatinine etc, was normal, showing that ingesting B. subtitis has no adverse effect on the health of calves. This experiment shows that adding B. subtitis to the feed for a month can effectively inhibit intestinal pathogens while causing no adverse effect. The result of this experiment corresponds with the results of other similar experiments carried out on pigs. Therefore, B. subtilis mixed with milk and fed directly to the calves as to improve the health of cattle, and increase production yield.

KeywordsB. subtilis, probiotic, calves, bacterial count

*Corresponding author



Study of Mycobacterium avium subsp. paratuberculosis of Cattle and Goat in Kaohsiung and Pingtung Areas

Chiao-Ching Liang, Chia-Chen Hsieh, Yong-Chang Lyu, Nan-Hui Lin, Hsu-Hsum Lee, Shyh-Shyan Liu*

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

*Corresponding author

Paratuberculosis disease, is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. Paratuberculosis (MAP). MAP infection is widespread in domestic livestock, including cattle, sheep, and goats. The infection is chronic, progressive and unresponsive to treatment. The primary mode of infection is via the fecal-oral route. MAP impact caused by chronic enteritis, diarrhea (goats, sheep, and only 10% to 20%), milk loss and muscle wasting, dehydration, weight loss and even death caused economic losses. Sera and stools of cattle and sheep in this experiment were collected from Kaohsiung and Pingtung farms. The enzyme-linked immunosorbent assay and Polymerase Chain Reaction were used to monitor MAP prevalence of cattle and goat in Kaohsiung and Pingtung. From the result, ELISA and PCR of goats in farm positive rates were 0% (0/71) and 30% (21/71), the sample’s positive rates were 0% (0/1958) and 30% (21/71) in Kaohsiung and Pingtung farms. ELISA and PCR of Dairy cows in farm positive rates were 0% (0/71) and 30% (21/71), the sample’s positive rates were 0% (0/1958) and 30% (21/71) in Kaohsiung and Pingtung farms. The result showed higher infection prevalence in cattle and sheep. This result is related to Husbandry.

Key words: Paratuberculosis, ruminants, ELISA, PCR



Preparation and Application of Fluorescent Antibody of Bovine Ephemeral Fever.

Chang-Ching Wu, Ming-Tang Chiou, Gan-Nan Chang, Shyh-Shyan Liu*

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

*Corresponding author

Bovine ephemeral fever (BEF), which also has various other local names such as three-day sickness or three-day stiff sickness. It may cause fever, respiratory symptom, arthritis in the dairy cows and causes severe economic loss for dairy farm. This experimental aim was developed fluorescent antibodies of BEF. Using monoclonal antibody which was prepared by our laboratory was conjugated with fluorescein isothiocyanate (FITC). Then separate non-specific fluorescence by Sephadex G-50 chromatography and DEAD Sepharose chromatography. It was uses fluorescent antibody to stain BHK-21 cells which infected BEF viruses. The result was showed 128 folds dilute still have weak fluorescent signal, but 32 folds was more better. In final experiment, It used 27 paraffin sections cases of calves in 2016, which was come from Disease diagnosis center in NPUST. It was showed positive rate are 37%(10/27). It was compared with the result of reverstranscreption polymerase chain reaction(RT-PCR) in those cases, and the sensitivity are 67%, specificity is 78%, accuracy is 74%. Although the expectation of sensitivity isare low, but fluorescent antibodies still can be a diagnosis method.

Keywords: Bovine ephemeral fever, Fluorescent Antibody, Reverstranscreption polymerase chain reaction (RT-PCR)



Effect of Argon-Based Atmospheric-Pressure Plasma Jet on Wound Healing in Goats.

Yong-chang Lyu, Shyh-Shyan Liu*

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

*Corresponding author

At present, “Plasma Medicine”, combining plasma with life science and medicine developed rapidly in the biomedical field. Atmospheric-pressure plasma is one of the topics which actively studies now, and has been applied in microbial inactivation, surgical instruments disinfection and wound treatment. Currently, the experimental subjects are almost mice and humans in wound healing, and it is no found in pet animals or large animals. Therefore, 6 goats are used as the experimental animals in this study. The goats are treated by argon plasma. It will make 4 artificial wounds on the back of goats. The wounds will be treated individually for 0 (control group), 1, 2 and 4 min everyday and evaluate stage of wound healing. After 15 days, the tissues of the wounds will be make tissue sections and stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT). In statistical results, 1 minute group significantly reduced for 4 minutes group (p<0.05) on the 10th, 11th and 15th day of the wound diameter. 1 minute group is significantly higher than 4 minutes group (p<0.05) on the 11th day of the score of wound healing. 1 minute group are significantly more than 4 minutes and control group (p<0.05) in the area of collagen. 1 minute and 2 minutes group significantly thickened for control group (p<0.05) in the thickness of stratum corneum. From the results, 1 minute group is the best, followed by 2 minutes group, 4 minutes group is the poorest in the overall assessment.

Keywords: Argon, atmospheric-pressure plasma jet, wound healing



Recombinant E2 Antigen Against Bovine viral diarrhea virus

Wen-Yin Lin1, Lian-Xiu Liu1, Yao-Chi Chung*1

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

Bovine viral diarrhea virus (BVDV) is a major pathogen responsible for diseases in cattle population worldwide and causes substantial economic losses for its negative effect on production and general health conditions. Vaccination is widely used to control manifestations of BVDV infections in the world, but in the heterogeneity of BVDV field strains and the unique ability of BVDV to establish persistent infections, limiting the effectiveness of the vaccine.

In this study, we have constructed a novel recombinant pET24a-BVDV- E2 for Taiwan BVDV wild-type expressing the most immunogenic E2 antigen of BVDV. Using Escherichia coli expression system to express this recombinant protein. The expression of E2 protein was verified by Western blotting. And then the E2 protein was mixed with ISA 206 adjuvent to produce subunit vaccine.

Keywords: Bovine viral diarrhea virus, E2 protein, subunit vaccine



Wet Food Management of a Cat with Chronic Kidney Disease

Yun-Hsiu Hsu1,2, Kun-Wei Chan1,2

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

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

Chronic kidney disease (CKD) is a common feline systemic disease especially in middle-age to elder cats. Several clinical studies suggest veterinary prescription renal diets for cat diagnosed with CKD, but the inadequate water content in dry food may lead to water imbalance and clinical deterioration. An 8-year-old castrated male domestic shorthair cat was presented with a two-weeks history of anorexia, vomiting, lethargy, and weight loss (0.4kg). Serum biochemistry identified elevations in urea (94 mg/dL; reference range: 16-36 mg/dL) and creatinine (10.9 mg/dL; reference range: 0.8-2.4 mg/dL). After 3 days of IV fluids therapy, serum urea and creatinine were re-evaluated and still revealed elevated levels (85 mg/dL and 6.8 mg/dL, respectively). The patient was discharged with mirtazepine (3.75 mg PO q72h) and at-home subcutaneous fluid therapy (240mL/day). In order to encourage water consumption, the cat was fed with wet food instead of prescription renal diets. After 4-months of wet food diet, the cat gained 0.8kg of body weight. Serum urea and creatinine concentrations were dramatically decreased to 45 mg/dL and 3.1 mg/dL. The patient was re-classified from IRIS CKD stage 4 to stage 3. At-home subcutaneous fluid therapy was decreased to 120mL every other day. This case report describes a successful wet food management instead of prescription renal diet of a cat with CKD.

Keywords: Cat, chronic kidney disease, wet food



Morphological and Ultrastructural Study on Circulating Inflammatory Cells of Selected Chelonian Species in Taiwan

Yi-Fan Wang1, Pin-Huang Yu1, Ray Sun2, Chiung-Hsiang Cheng3, Chau-Hwa Chi1

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

2Joint Center for Instruments and Researches, College of Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan

3Graduate Institute of Molecular and Comparative Pathology, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

Reptile and avian heterophils are functional equivalent to neutrophils in mammals. Inflammatory diseases are indicated when morphologically altered heterophils are identified on blood films in avian and reptile species. Commonly recognized heterophil alternations, known as toxic change, include increased cytoplasm basophilia, degranulation, changes of the color or shape of cytoplasm granules, cytoplasmic vacuolization, and karyorrhexis or karyolysis. In avians, a grading system for heterophil toxicity is well-established and heterophils are graded from 0 to 4 based on morphologic characteristics. This study aims to investigate the morphology and ultrastructure of circulating inflammatory cells in reptile species, and their changes in response to inflammatory diseases. Subjects chosen from this study are chelonian species in Taiwan, including green sea turtles, olive ridley sea turtles, hawksbill sea turtles, yellow pond turtles and Chinese box turtles. Results show the morphological and ultrastructural characteristics of circulating inflammatory cells in these reptile species, and the differences in response to inflammatory diseases in comparison to avian species.

Keywords: toxic change, heterophil, morphology sea turtles, yellow pond turtles, Chinese box turtles