Bali, February 2020

Arranged by Udayana University: I Nengah Kerta Besung Kadek Karang Agustina

Edited by: Koichi SATO (Yamaguchi University) Hiroshi SATO (Yamaguchi University) Srihadi Agungpriyono (IPB University)

Compiled by: Leni Maylina (Yamaguchi University and IPB University)

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CONTENTS List of Presentation

ORAL PRESENTATION

KEYNOTE PRESENTATION NO PAGES AUTHORS TITTLE KEYNOTE 2 Prof. I Nyoman Mantik Control And Eradication Of Rabies Astawa In Animals In Bali

DEAN PRESENTATION NO PAGES AUTHORS TITTLE DO-1 3 Prof. Koichi SATO, DVM, Research And Study In Yamaguchi PhD University DO-2 5 Prof. Yoshiaki HIKASA, Research And Development Of DVM, MSc, PhD Joint Graduate School Of Veterinary Sciences In Tottori University DO-3 7 T. Unno About Gifu University/Tottori University Joint Graduate School Of Veterinary Sciences DO-4 9 I Nengah Kerta Besung, I Faculty Of Veterinary Medicine, Wayan Suardana, I Udayana University (Future Nyoman Suartha, Ida Collaboration In Academic And Bagus Ngurah Swacita Research) DO-5 11 Siti Isrina Oktavia Salasia, Research And Academic Agung Budiyanto, Teguh Collaboration Faculty Of Budipitojo, Rini Veterinary Medicine Universitas Widayanti, Widagdo SN Gadjah Mada DO-6 13 Pudji Srianto, Fedik A. Improving And Collaboration On Rantam, Mufasirin, Academic And Research Activities, Suwarno Faculty Of Veterinary Medicine, Universitas Airlangga Surabaya, Indonesia DO-7 15 Srihadi Agungpriyono, Establishment Of Three Centers Deni Noviana Services Initiative In Ipb University Veterinary Teaching Hospital To Support Hospital Based Education

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NO PAGES AUTHORS TITTLE DO-8 17 Maxs U.E. Sanam, Diana Faculty Of Veterinary Medicine, A. Wuri and Johanes Universitas Nusa Cendana: T.R.M.R. Simarmata Partnership Towards Improving The Quality Of Learning, Graduates And Research Capacity DO-9 18 Dwi Kesuma Sari The Potential Of The South Sulawesi Region As A One Health- Based Research Development DO-10 19 Kholik, Kunti Tirtasari, Faculty Of Veterinary Medicine Candra Dwi Atma Pendidikan Mandalika University: Academic Collaboration And Future Research DO-11 20 Aulanni’am, Dyah Ayu Faculty Of Veterinary Medicine Oktavianie, Herawati, Universitas Brawijaya: Towards Wawid Purwatiningsih, Global Competitiveness In Fajar Shodiq Permata Academic And Research DO-12 22 Rohiman Sasmita, Lusia Canine Parvovirus Infections With Adityaningtyas, Rondius Colibacillosis Infection And Solfaine Toxocara Canis Infestation In Dogs DO-13 29 Dean The Veterinary Medicine Study Program Of Faculty Of Medicine, Universitas Padjadjaran, Indonesia

SCIENTIFIC PRESENTATION-1 NO PAGES AUTHORS TITTLE SO1-1 30 Takuya MIZUNO Development Of New Treatment Strategy For Canine Cancers SO1-2 31 Aiko IGUCHI, Takashi Development Of In Vitro TAKEUCHI Atovaquone-Resistant Babesia Gibsoni And The Epidemiological Survey For Atovaquone Resistant Related Gene SO1-3 32 Teguh B, Yosua KA, Irma Correlation Between Forefoot And P Back Foot Length Against Body Length Of Sunda Porcupine From Central Java

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NO PAGES AUTHORS TITTLE SO1-4 36 Deni Noviana, Frizky Continuing Professional Amelia, Leni Maylina, Development: The Crucial Roles Of Srihadi Agungpriyono Veterinary Teaching Hospital In Facilitating A Long Life Learning Of Veterinarians SO1-5 38 Hiroya KADOKAWA Reproductive Physiology And Management For Domestic Animal SO1-6 40 Kenji TAKAHASHI, Toshio Cytotoxic Effects Of Menthol OHTA SO1-7 41 Albiruni Haryo, Miranti Change Observation Condistion Verdania, Mimin Of Rabbit Organs In Intervals Wulandari. Lutfiana Different Time Pratiwi, Vilinda Maya (Post Interval Mortem)

SO1-8 42 Agung Budiyanto, Erif The Study Of Calf Weight On The Maha Nugraha Estrous Postpartum (Eep) Period Setyawan, Soedarmanto On Peranakan Ongole (Po) Cattle Indarjulianto Jumaryoto Topas Wicaksono Priyo Jr SO1-9 45 Safitri Ummahatul Extract Of Artocarpus Altilis Leaves Mukminah, Djoko Improved Kidney Function Of Winarso, Fajar Shodiq Streptokinase Induced Acute Glomerulonephritis Mice Based Permata On Bun And Creatinine Level SO1-10 49 Nururrozi, S. Prevalence And Associated Risk Indarjulianto, Factor Of Feline Lower Urinary Yanuartono, S. Hartati, Tract Disease In Cats Attending H. Wuryastuty, Yuriadi, I. Primary Care Veterinary Practices Widiyono, I. Tjahajati, H. In Yogyakarta Purnamaningsih, G.T. Mulyati, and S. Raharjo SO1-11 55 Ida Fitriana, Agustina Pharmacokinetic Of Tylosin And Dwi Wijayanti, Dwi Enrofloxacin Combination Cahyo Budi Setiawan, Following Oral Administration In Anggi Muhtar Pratama, Broiler and Sugiyono SO1-12 56 I K. Berata, I W. Sudira, I Lead And Cadmium M. Kardena, I.B.O. Contamination In Blood Cattle Winaya Which Is Slaughtered At The Traditional Slaughterhouse

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SCIENTIFIC PRESENTATION-2 NO PAGES AUTHORS TITTLE SO2-1 59 HAYASAKA D, SHIMODA Epidemiological Survey And H, TAKANO A Elucidation Of The Pathogenic Mechanism Of Zoonotic Viruses SO2-2 60 M BABA, T BATANOVA, K Adhesion Of Toxoplasma Gondii KITOH, Y TAKASHIMA Tachyzoite-Infected Vehcle Leukocytes To Capillary Endotherial Cells Triggeres Timely Parasite Egression SO2-3 64 A. Winarso, M.G.M. Jo, Prevalence Of Trematodosis In Y.R. Nadja, D.T. Dhiu, M. Bali Cattle In West Timor Cantona, L.D. Amleni, S.J. Koanak, Y.V. Nawa, A.N. Daki, J.F.T.L. Pora SO2-4 67 Okti H, Marla A, Sidna A, Screening Of Sparrows (Passer Tri U Domesticus) For Salmonella Sp Infection In Yogyakarta With Molecular And Cultivication Metho SO2-5 71 Tsuyoshi YAMAGUCHI, What Transmits Highly Tatsufumi USUI, Yukiko Pathogenic Avian Influenza Virus TOMIOKA and Toshihiro To Poultry House Chickens? ITOH SO2-6 73 Supriyono, Hiroshi Isolation And Characterization Of Shimoda, Ryusei Kuwata, Heramatsu Virus (Reoviridae: Ai Takano, Keita Noguchi, Orbivirus) From A Bat (Miniopterus Fuliginosus) In Japan Kenzo Yonemitsu, Shohei Minami, Ngo Thuy Bao Tran, Yudai Kuroda, Kango Tatemoto, Milagros Virhuez Mendoza, Daisuke Hayasaka, Ken Maeda SO2-7 75 Siti Gusti Ningrum, Detection Of Arcanobacterium Osama Sammra, Pluranimalium In Muskox Using Christoph Lämmler, Amir Loop-Mediated Isothermal Abdulmawjood Amplification (Lamp) Assay Targeting The Pla Gene

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NO PAGES AUTHORS TITTLE SO2-8 77 I Gede Hendra Prasetya Pathological Changes Of Liver And Wicaksana, Anak Agung Small Intestine In Chicken Embryo Ayu Mirah Adi2, I Made After Inoculated With Virulent Kardena Isolate Of Newcastle Disease Virus SO2-9 81 I Made Kardena, Anak Socio-Cultural Practices In Bali Agung Ayu Mirah Adi, Are Potentially Associated With Michael Laurence, Mark Clinical Cases Of Acute O’ Dea, Mieghan Bruce Encephalitis Syndrome Related To Japanese Encephalitis Infection SO2-10 82 Yosua KA, Irma P, Teguh Correlation Between Body Length B And Body Weight Against Uterine Length And Testicular Weight Of Sunda Porcupine From Central Java SO2-11 87 Irma P, Yosua KA, Teguh The Correlation Of Body Length B With Total Intestine Length Of Sunda Porcupine SO2-12 91 Tri Komala Sari, Satriana A Novel Specific Role Of A. Gianopulos, Seth. M. Glycoprotein C In Herpes Simplex Schneider, Darin J. Weed, Virus Infectivity Suzanne M. Pritchard, Anthony V. Nicola

POSTER PRESENTATION NO PAGES AUTHORS TITTLE P-1 97 Sonoko Matsumoto, Identification Of Host Factors In Takashi Shimizu, Kenta Francisella Tularensis Infection Watanabe, Masahisa Watarai P-2 99 Marla Anggita, Okti Molecular Detection Of Momp Herawati, Widya Asmara, Gene Of Chlamydia Spp. From A.E.T.H. Wahyuni, Tri Wild Bird’s Fecal Sample In Untari, Michael Haryadi Yogyakarta Wibowo, Sidna Artanto P-3 102 Inoru Yokoe, Kazuo Combination Of Lipid Bubble And Maruyama, Tomohiro Ultrasound Irradiation Enhances Osaki Anti-Tumor Effect Of Liposomal Doxorubicin In Vivo

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NO PAGES AUTHORS TITTLE P-4 103 Arief P Mihardi, Anita The Diarrhea Symptom In Calves Esfandiari, Sus D Infected By Escherichia Coli K99 Widhyari, Sri Murtini, And Received Suspension Of Igg Retno Wulansari, Anti Escherichia Coli K99 Leni Maylina P-5 104 M Kawasaki, N Ito, Y Comparison Of Three Culture Okamoto Media In Maintaining Corneal Epithelial Cells Derived From Four Animal Species In-Vitro P-6 105 Agus Widodo, Lita Morphometric Analysis Of The Rakhma Yustinasari, Eka Intestine Of Domestic Quails Pramyrtha Hestianah (Coturnix Coturnix Japonica) Treated With Different Levels Of Clam Shell Flour Of Placuna Placenta

P-7 108 Y. Matsushita, M. Activation Of Α2 Adrenergic Manabe, N. Kitamura, I. Receptors Inhibits Trpv1 Activities Shibuya Resulting In Analgesia P-8 109 Leni Maylina, Ratyan Tri Evaluation Of Hematological Widowati, Febri Fadhli Profiles In The Police Dogs Zumaro, Alyssa, Agus Infected By Ehrlichia Canis That Wijaya, Sus Derthi Given Treatment Combination Widhyari, Rini Madyastuti, Anita Esfandiari, Retno Wulansari, Arief Purwo Mihardi, Jeanni Dumayanti P-9 111 Lalu Unsunnidhal Design Of Feed Development Technology In Pumice Soil In North Lombok To Realize Ntb Bss P-10 112 A. Ayu Nur Ramadhani, Detection Of Heavy Metal Lead Sitti Arifah, Erdi (Pb) In Sepat Siam Fish Purwanto, Sharifuddin (Trichopodus Pectoralis) Meat In Bin Andy Omar, Zainal Lake Tempe, South Sulawesi Abidin Kholilullah P-11 113 Imron Rosyadi, Diverse Morphotypes Of Muchammad Yunus, Trypanosomes In Frogs And Toads Kholik, Hiroshi Sato From Surabaya And Lombok Island, Indonesia

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NO PAGES AUTHORS TITTLE P-12 115 M. Yunus, Kholik, K. Phylogenetic Analyses Of Some Kamimura, L. A. Jimenez, Intestinal Flukes Of Frogs And H. Sato Collected In Surabaya And Lombok Is., Indonesia, And Mati, Mindanao Is., Phillipines P-13 116 Yumi Sartika, Zulkarnaen, Antimicrobial Resistance Of Rahmawati, Kholik Pasteurella Multocida Isolated From Stray Dog In Mandalika Dog Shelter To Various Antibiotics P-14 117 Anggi Muhtar Pratama, Blood Urea Nitrogen (BUN) and Agustina Dwi Wijayanti, Creatinin values of Mus musculus Dwi Cahyo Budi given by compound Four Species Setiawan, Ida Fitriana, of Zingiberaceae Aria Ika Septana

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020

ORAL PRESENTATION

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 KEYNOTE

CONTROL AND ERADICATION OF RABIES IN ANIMALS IN BALI

I Nyoman Mantik Astawa Faculty of Veterinary Medicine, Udayana University, Bali, Indonesia

Abstract Rabies is a zoonotic viral disease causing deaths of animals and human worldwide. The virus infects warm-blooded animals causing severe encephalitis with case fatality rate reaching 100% when clinical symptoms have occurred. At present, only 9 out of 34 provinces in Indonesia are declared free from rabies which include East Java, Yogyakarta, Jakarta, Central Java, Riau Island, Bangka Belitung, West NusaTenggara, Papua and West Papua. In Bali, first rabies outbreak was reported in late 2008 in South Bali, and it then spread quickly to all 9 districts (regencies) in Bali causing many deaths of both animals and human. The massive efforts have been taken to control and eradicate rabies from Bali. These include massive vaccination program, surveillance, animal population control and strict control of animal transports. Such control and eradication programs seemed to be able to reduce the rabies infected areas as at present only 105 (around 15%) out of 716 villages still infected by rabies. However, rabies cases in Bali are still very high in which in 2018 and 2019 as many as 149 and 169 cases respectively were reported in dogs. It is suggested that such cases occur mainly in unvaccinated dogs. Three districts, Karangasem, Buleleng and Bangli which still have high rabies cases, are considered as rabies hot spot in Bali. Vaccination programs in Bali is mainly conducted by intramuscular injection. In the future, an alternative vaccine such as oral rabies vaccine need to be taken into consideration as studies have shown that such oral vaccine can be effective for vaccination of stray dogs.

Keywords: Rabies, control, eradication, Bali

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-1

RESEARCH AND STUDY IN YAMAGUCHI UNIVERSITY

Prof. Koichi SATO, DVM, PhD* Dean of Yamaguchi University Laboratory of Veterinary Pharmacology, Joint Graduate School of Veterinary Medicine, Yamaguchi University, Yamaguchi city, Japan *Corresponding author: [email protected]

Abstract Establishment of veterinary education in Yamaguchi prefecture was first founded in June 1883 as the Department of Veterinary Science at Yamaguchi Cultivation Test Center. Later in 1944, it developed as Yamaguchi Veterinary Medical School, and joined as the Department of Veterinary Medicine, Faculty of Agriculture at Yamaguchi University (YU). In 1984, a four-year program of veterinary education in Japan became a six-year program. In 2008, Ministry of Education, Culture, Sports, Science and Technology (MEXT) allowed the modifications of the various operating structures of the universities and created new forms of academic positions. Therefore, YU and Kagoshima University (KU) established a Joint Faculty of Veterinary Medicine (JFVM; which is named VetJapan South in Europe) in April 2012 to enhance veterinary training and for providing veterinary education at an international standard. Finally, on December 11th 2019, VetJapan South acquired certification “first in Asia” from European Association of Establishments for Veterinary Education (EAEVE), which evaluates quality of veterinary education. The United Graduate School of Veterinary Science (UGSVS) was established in 1990 as a four-year PhD program with Kagoshima and Tottori University (originally with Miyazaki University). To enforce the research and problem-based learning and to offer a strong research basis to our undergraduate education on the undergraduate course of JFVM-YU and -KU, UGSVS positively disappeared to become a Joint Graduate School of Veterinary Medicine (JGSVM) of YU and KU has been established in April 2018. Graduate students of JFVM who wish to continue their research activities can be included in JGSVM. JGSVM consists of three Units including 19 Laboratories and two centers. Unit of Basic Veterinary Medicine [Laboratories of Veterinary Anatomy, Veterinary System Physiology, Veterinary Biochemistry, Veterinary Pharmacology, Laboratory Animal Science, Veterinary Developmental Biology, Veterinary Microbiology], Unit of Applied Veterinary Medicine [Laboratories of Veterinary Epidemiology, Veterinary Pathology, Veterinary Hygiene, Veterinary Public Health, Veterinary Parasitology], Unit of Clinical Veterinary Medicine [Laboratories of Veterinary Surgery, Veterinary Radiology, Veterinary Internal Medicine, Veterinary Clinical Pathology, Veterinary Theriogenology, Preventive Physiology and Management, Large Animal Clinic]. Centers are consisted by Veterinary Diagnosis and Development Center, and Animal Medical Center. By fostering specialists with highly specialized knowledge and enriched common sense, flexible ways of thinking, wide perspectives, and high motivation to achieve its goal, the JGSVM contributes to the domestic and international societies, especially

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 developments, peace-keeping, and the environmental protection against pandemic threat on animal and human health and economic and social stability by emerging infectious disease in Asia. Education programs in JGSVM correspond to these demands from these special communities. In 2018, JFVM/JGSVM-YU established Association of Japan-Indonesia Veterinary Education (AJIVE) with Airlangga University, Gadjah Mada University and Bogor Agricultural University. The purpose of AJIVE is the development of cultivating program for global veterinary educational researcher in Indonesia by Japanese Veterinary education and research system. The goal of AJIVE is (1) Establish AJIVE and a program to cultivate the young international educational researchers (teachers) of veterinary medicine. (2) Develop the program to the standard of Asia veterinary medical cl education based on EU educational system. (3) Enhancement of the international veterinary medical educational research enforcement system. In this symposium, I will explain the organization, the aim, the research targets of JGSVM, and also explain about the aim, future effort of AJIVE and also about the EAEVE accreditation.

Keywords: VetJapan South, JFVM, JGSVM, EAEVE accreditation

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-2

RESEARCH AND DEVELOPMENT OF JOINT GRADUATE SCHOOL OF VETERINARY SCIENCES IN TOTTORI UNIVERSITY

Prof. Yoshiaki HIKASA*, DVM, MSc, PhD* Laboratory of Veterinary Internal Medicine, Joint Graduate School of Veterinary Sciences, Tottori University, Tottori city 680-8553, Japan *Corresponding AUthor: [email protected]

Abstract Veterinary education at Tottori University (TU) was originated from the Tottori High Agricultural School in 1920, followed by establishment of the Department of Veterinary and Livestock Science in 1939, and was established as the Department of Veterinary Medicine, Faculty of Agriculture, TU in 1949. In 1984, the bachelor course in veterinary medicine in Japan was changed from 4- to 6-year. In 2013, TU established the Joint Department of Veterinary Medicine (JDVM) with Gifu University (GU) to improve education quality in veterinary bachelor course. On the other hand, TU has constituted the United Graduate School of Veterinary Sciences with Yamaguchi and Kagoshima universities in 4-year PhD course from 1990. In April 2019, Joint Graduate School of Veterinary Sciences (JGSVS) in TU was newly established as a standard 4- year PhD course in collaboration with GU. TU JGSVS promotes advanced research in a wide range of fields on animal and human health including the health of the ecosystem, as well as training veterinary science educators and researchers who can contribute to the advanced veterinary science. This JGSVS trains veterinary science specialists with advanced knowledge and technology, as well as expertise and ethics, who can take a leading role in international and regional societies. Under this philosophy, joint education with TU and GU was started. In addition, JGSVS has specific education programs for training “Animal hygiene and public health specialists”, “One Health specialists”, or “Treatment of intractable diseases and drug discovery specialists”, to meet the needs of society. Four affiliated facilities in TU faculty of agriculture, “Avian Zoonosis Research Center”, “Veterinary Medical Center”, “Field Science Center”, and “Fungus/Mushroom Resource and Research Center”, are connected with education and/or research in the bachelor course and JGSVS PhD course. In addition, JGSVS has the “Research and Development Promotion Center for Veterinary Science Education” for the realization of a smooth collaboration between TU and GU, and for promoting organic collaboration with educational and research institutes in Japan and overseas. In TU JDVM, 18 laboratories are arranged in four divisions, i.e., “Basic veterinary science”, “Veterinary pathobiology”, “Applied veterinary science”, and “Clinical Veterinary science”. Each division consists of 3 to 7 laboratories. Thirty-six faculty members are attached to the department. Many members belong to Applied- and Clinical-divisions, because they are also managing the Avian zoonosis research center and Veterinary medical center. TU JGSVS has 3 departments, “Basic Veterinary

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Science”, “Veterinary Pathobiology and Applied Veterinary Science”, and “Clinical Veterinary Science”, and have twenty major supervisors. TU JGSVS will promote new international academic exchanges, acquiring international students, presentations at international conferences, participation in international symposiums, joint research both in Japan and overseas, and developing an expansive global project as a new graduate school. TU JGSVS contributes to the development of veterinary science with a global perspective.

Keywords: TU, JGSVS, Research, Development

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-3

ABOUT GIFU UNIVERSITY/TOTTORI UNIVERSITY JOINT GRADUATE SCHOOL OF VETERINARY SCIENCES

T. UNNO* Dep. Vet. Pharmacol., Gifu University, Japan. *Corresponding author: [email protected]

On April 1, 2019, the Gifu University/Tottori University Joint Graduate School of Veterinary Sciences was newly established. Gifu University had been organizing a Joint Graduate School of Veterinary Sciences with Obihiro University of Agriculture and Veterinary Medicine, Iwate University, and Tokyo University of Agriculture and Technology, while Tottori University had been organizing a Joint Graduate School of Veterinary Sciences with Yamaguchi University and Kagoshima University to conduct graduate school education, and they have been responsible for the training of researchers in various fields of veterinary science. On the other hand, each of these universities that make up the Joint Graduate School of Veterinary Sciences have been working to enhance undergraduate education by combining the two universities to form a joint education curriculum which complies with the veterinary science education model core curriculum. Gifu University opened the Joint Department of Veterinary Sciences in April, 2013 with Tottori University, and the first year students graduated in March, 2019. Under these circumstances, the universities that are a part of the joint education curriculum have decided to open a new joint graduate school. This enables human resource training based on a consistent educational philosophy from undergraduate to graduate school. The Joint Major in Veterinary Sciences by Gifu University and Tottori University Graduate Schools will take advantage of the characteristics of existing research activities from both universities, and aims for the training of “Animal hygiene and public health specialists”, “One Health specialists”, and “Treatment of intractable diseases and drug discovery specialists” (for details, see below). The objectives are to train “Animal hygiene and public health specialists” who can take the lead in the fight against infectious diseases such as classical swine fever, foot-and-mouth disease, and so on; “One Health specialists” with the ability to think about maintaining worldwide health by taking internationally leading roles with the ability to see things from the perspectives of humans, animals, and the environment as a whole; and “Treatment of intractable diseases and drug discovery specialists” for taking the lead among clinical veterinarians and having the ability to promote translational research to clinically apply results from basic research to technologies and drug discovery. We, the faculty and staff, will continue to make tireless efforts to develop and enhance education and research in the Joint Graduate School of Veterinary Sciences. Thus, we expect you to enter our university and to study together about veterinary sciences. In this presentation, I will introduce the characteristics of curriculum, flow of degree acquisition, entrance examination, scholarships from our university and Japanese ministry of education, and life in Gifu.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-4

FACULTY OF VETERINARY MEDICINE, UDAYANA UNIVERSITY (FUTURE COLLABORATION IN ACADEMIC AND RESEARCH)

I Nengah Kerta Besung, I Wayan Suardana, I Nyoman Suartha, Ida Bagus Ngurah Swacita Faculty of Veterinary Medicine, Udayana University Corresponding Author: [email protected]; [email protected]

Abstract The Faculty of Veterinary Medicine at Udayana University (FVM-Unud) is located in Denpasar City, Bali. The faculty belongs to one of the top five of the 12 Faculties of Veterinary Medicine in Indonesia including the Faculty of Veterinary Medicine at Bogor Agriculture University, Gadjah Mada University, Airlangga University and Syah Kuala University. The FVM Unud has a vision of producing professional graduates in the field of veterinary medicine based on ethics and culture. FVM Unud has a motto of “Manusya Mriga Satwa Sewaka” which means “Serving for human welfare through the animal world”. Initially, FVM-Unud has carried out two educational programs which are the Undergraduate Program of Veterinary Medicine and the Veterinary Profession Program (VPP). Since 2010, FVM Unud also conducted Veterinary Magister program. Furthermore, In the implementation of teaching and learning, FVM-Unud supported by 71 lacademic staffs and 41 support staffs. The academic staffs (lecturer) consist of 15 professors (21,13%), 28 doctor (39,44%) and 28 Magister (39,44%). The implementation of education in FVM-Udayana is supported by facilities such as lecture hall, laboratory and animal hospital. Recently, the undergraduate and profession programs have been acrredited on excellent grade by Higher Education Independent Accreditation Board of School of Medicine (LamPTKes), as well as for magister program which is still categorized as an excellent by National Accreditation Board for Higher Education (BAN PT). In 2020, FVM-UN plan to register on Quality Assurance System by asean University Network (AUN-QA). As it has been recorded until the end of October 2019, there has been as many as 2677 alumni which consist of 1407 under graduates, and 1176 veterinary professions. Aditionally, research activities at FVM-Unud are run dynamically by involving various stakeholders as well as partners of cooperation both from within the country and overseas. Lecturer researches are conducted in cooperation with several related Faculties at the university and overseas. FVM Unud alumni have also worked in various sectors both in government and private sectors such as the Directorate General of Animal Husbandry and Animal Health, Animal Quarantine, Provincial and District Livestock Service Office, Animal Disease Investigation Center, lecturers, researcher, the west national bali park, and etc. While in private sectors including independent practices, animal food companies, as well as practices in national and international animal hospitals such as in the Netherland, Iceland, Abu Dhabi, and Malaysia. Various research and colaboration grants have been obtained by FVM-Unud include: Grants from Ministry of Research, Technology and Higher Education, Grants from the Ministry of Agriculture, Grants from the Ministry of Health, and cooperation funds from abroad such as Collaborative research with Murdoch University, The University of Tokyo, etc. Opportunities for recent collaborations between FVM

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Udayana with other universities include: 1) Research field on Liberation program of Bali as a rabies free area, development of Bali cattle as prime quality meat producer like wagyu, development of diagnostic kit for detection of zoonotic agent, research for Kintamani dog, research for bali pig, etc; 2) Field of education: opportunities for exchange of lecturers and students, International seminars, etc.

Keywords: Collaboration, Veterinary medicine, Udayana University

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-5

RESEARCH AND ACADEMIC COLLABORATION FACULTY OF VETERINARY MEDICINE UNIVERSITAS GADJAH MADA

Siti Isrina Oktavia Salasia*, Agung Budiyanto, Teguh Budipitojo, Rini Widayanti, Widagdo SN Faculty of Veterinary Medicine, Universitas Gadjah Mada, Jl. Fauna 2, Karangmalang, Yogyakarta 55281, Indonesia. *Corresponding author: [email protected] (Presented in the 4th International Symposium in Veterinary Science, Denpasar, February 13th, 2020)

Abstract Faculty of Veterinary Medicine, Universitas Gadjah Mada (FVM UGM), was established on September 20th, 1946. It is dedicated to educating veterinarians, to provide and to support research, also to give community services in the field of animal health and food hygiene. Veterinary Study programs of UGM have been accredited by National Accreditation Board for Higher Education (BAN-PT) and also by International Accreditation Agency (ASIIN Germany), hold certification for Quality Assurance System by ASEAN University Network (AUN-QA), and ISO 9001:2008. In 2019 there were 138 research titles, 35 research titles funded by the Ministry of Technology Research and Higher Education, 36 by Universitas Gadjah Mada, and 67 supported by Faculty. The research themes are including: A. Wild Animals (Preservation of species and genetic resources; Genomic, proteomic, and metabolomic; Exploration of the structure and functionalities of endemic animals in Indonesia). B. Protection of animal products and biosafety (zoonosis; drug residue and antimicrobial- resistant; forgery of animal products). C. Health and medicine (mastery of vaccines; stem cell; development of medical devices diagnostic; development of phytopharmaca based on local resources). From this research have been published 60 international journals and 32 national journals. To give international experiences to the students, we have sent students to various universities overseas, including to Chiang Mai University, Kasetsart University, Mahidol University, Khonkaen University, Chulalongkorn University (Thailand), Putra Malaysia University, Murdoch and Sydney University (Australia). On the other side, some universities have also sent their students to FVM UGM as an implementation of cooperation between FVM and several veterinary faculties in different countries and also to support the UGM program towards World Class University. FVM UGM has also established several international collaboration with overseas universities such as as Prince Songkhla University, Khon Kaen University, Chiang Mai University (Thailand), Universiti Malaysia Kelantan, Hiroshima University, Gifu University, Yamaguchi University, The Primate Research Institute Kyoto University, Antimicrobial Resistance Research Center National Institute of Infectious Diseases (NIID) Japan, Australia Centre for International Agricultural Research (ACIAR), International Livestock Research Institute (ILRI), French Agricultural Research Centre for International Development (CIRAD) and

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Orangutan Veterinary Advisory Group (OVAG). The list of the student's mobility of FVM and International collaborations that have been signed an MoU with FVM are available on the web page http://fkh.ugm.ac.id/international-colaboration/. We want to expand our cooperation through Association of Japan and Indonesia Veterinary Education (AJIVE) forum for inviting experts from Yamaguchi, Kagoshima, Tottori, Kagoshima, Miyazaki, Gifu Universities Japan, human resources development in the field of animal health and veterinary science (short term training, Master and Doctor program), joint research and partnership program.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-6

IMPROVING AND COLLABORATION ON ACADEMIC AND RESEARCH ACTIVITIES, FACULTY OF VETERINARY MEDICINE, UNIVERSITAS AIRLANGGA SURABAYA, INDONESIA

1Pudji Srianto, 1Fedik A. Rantam, 1Mufasirin, 1Suwarno 1Faculty of Veterinary Medicine, Universitas Airlangga Surabaya, Indonesia Corresponding Author: Pudji Srianto

Abstract The history of the Faculty of Veterinary Medicine Universitas Airlangga officially was established on 1969 in Malang, Finally, with the Decree of the Minister of P & K No. 055/01/1972 commencing January 1, 1972, it was inaugurated by the Minister of P & K that at that day was become a first established Faculty of Veterinary Medicine, Universitas Airlangga. Faculty of Veterinary Medicine is one of the best in Indonesia and Southeast Asia, and it was the sixth faculty that was established at the Universitas Airlangga, and now they have nine departments; Department of Basic Veterinary Medical Science, Department of Veterinary Anatomy, Department of Veterinary Pathology, Department of Veterinary Parasitology, Department of Veterinary Microbiology, Department of Veterinary Public Health, Department of Veterinary Reproduction, Department of Veterinary Clinic, and Department of Animal Husbandry, and has four master program, and one Doctoral Program of Science Veterinary. The education system in Faculty of Veterinary Medicine assured under Airlangga Integrated Management System, Malcom Baldrige – MBN QA 2011-2012, Quality Management System ISO 9001 : 2008, and certified by Asean University Network (AUN), certified by IKTIRAF Malaysia, accredited by BAN-PT Ministry research, Technology and Higher Education, and since 2019 accredited by ASIIN, Germany. Currently Education system in Faculty of Veterinary Medicine Universitas Airlangga is based on modified student center learning and there are two type of class; regular class and international class. We have adequate facilities of equipment’s and some center for supported in teaching learning process like Animal Teaching Hospital, feed formulation center, BSL3 for animal and GLP for animal experiment model, informatics technology, and Institute of Tropical Disease (ITD), and Airlangga Disease and Prevention Center (ADPRC- OHCC). Based on human resources this faculty has 110 faculty members 76 doctors and 29 professors with various specificity of experts in research such as; frozen semen unit, diagnostic kit development, avian influenza research center, stem cell research unit, zoonotic disease, probiotic development, and nutrition. Recently our faculty start focusing on five fields of research; 1) Biomedical Science (seed vaccine development, diagnostic kit development, natural product based pharmacology), 2) reproductive science (sperm extender development, genetic, embryo transfer, hormones), 3) Nutrition and Animal Science (probiotic development, feed formulation for specific

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 purposes), 4) One Health (Interdisciplinary on environment, human and animal health, include development of science communication, economic and social science, , bio– eco-socio culture approach, biosecurity and biosafety, Zoonosis, wild infection, antibiotics microbial resistant (AMR), Emerging Infectious Disease (EID)) and 5). Clinical Science. Finally, our concerned will formulize the new approach called Bio- X). In The other hand, we held the service on community by established the community research center such as, Taman Safari II Prigen Pasuruan (harmonization between the society nearby of Safari park by one health approach), Banyuwangi (goat and sheep development) and Trenggalek (Livestock health service to the community). Based on these various activities on education, research and community service we are also established an agreement to be collaborate with various country around the world such as universities in Japan coordinated by AJIVE namely Universities Yamaguchi University, Kagoshima University, Totori University, Miazaki University, Malaysia (UPM, UMK); Thailand (Chulalongkorn University, Khon Kaen University, Kasetsart University, Chiang may University); Philippines (Tarlac Agriculture University); South Korea (Seoul National University), Australia (Murdoch University, Melbourne University), and then Tuft University, USA, Minnesota University, USA under coordinated by INDOHUN, and also Pennsylvania University USA; Freie Universitaet Berlin, Berlin Germany; ONIRIS Ecole Nationale Veterinaer Nantes, France; NTU, Tsung Shing University, Taiwan In the future we still need to strengthen the collaboration with another party to support the development of faculty members, research and community service, staff mobility, student mobility, junk professor from anothers countries such as Japan Universities, USA Universities, Malaysia Universities, Australian Universities, Germany Universities, France Universities, Thailand Universities, Korean Universities, Gryphone Institute, USA, OIE, also Taiwan Universities.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-7

ESTABLISHMENT OF THREE CENTERS SERVICES INITIATIVE IN IPB UNIVERSITY VETERINARY TEACHING HOSPITAL TO SUPPORT HOSPITAL BASED EDUCATION

Srihadi Agungpriyono1*, Deni Noviana2 1 Dean Faculty of Veterinary Medicine, Faculty of Veterinary Medicine, IPB University 2 Director of Veterinary Teaching Hospital, Faculty of Veterinary Medicine, IPB University Corresponding Author: [email protected]

Abstract The industrial era 4.0, provides challenges as well as new opportunities for veterinary education. This underlies the importance of applying three literacies namely big data, biotechnology, medical information and communication technology (medical ICT) in the process of education, research and community service. The Role of Veterinary Teaching Hospital of the Faculty of Veterinary Medicine (VTH FVM) of IPB University is very important because it is the core and backbone of hospital-based veterinary education as well as providing services to community. To facilitate this function, three Centers Services Initiative of VTH FVM IPB University have been recently developed in 2019, namely Cardiology Center Services, Clinical Skills Center and Diagnostic Imaging Center. The Cardiology Center Services offers the most advanced diagnostic tests for diagnosing and treating heart disease in animals. This center is a one stop service and handles a variety of referral patients from veterinarians or other veterinary clinics that require further diagnosis and treatment. The existence of center is also very important in supporting hands-on clinical learning process for veterinary professional education programs (clinical rotation co-assistant), recidency student, post graduate students as well as participating veterinarians in continuing education (continuing professional development) carried out by VTH FVM IPB University. Various heart cases can be seen in realtime or in the form of e-files that can be accessed by students and course participants through digital applications. The Clinical Skills Center is an area and rooms designed for students to learn a variety of practical and clinical skills and provide a means to overcome the problem of animal usage and welfare. The Clinical Skills Center is centered for students and provides several special benefits, including continuous access and availability to practice repeatedly in a safe and relatively stress-free environment, where there is no risk of injuring animals. This is in line with the principle of 3R on the reduce, refinement and replacement the use of animals and, 5F, especially aspects must be free from pain and free from discomfort. This center use manikins, models, and other simulators to reduce animal use along with preparing the students to perform real procedures

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Diagnostic Imaging Center (Teleradiology) offers consulting service for radiological patient images, such as x-rays (radiography), ultrasonography, CTs, and MRIs, who provides high quality diagnostic imaging interpretation. Indonesia is very big country, therefore teleradiology practices do provide benefits in terms of overcoming geographic and coverage challenges. Teleradiology improves patient care by allowing radiologists to provide services without actually having to be at the location of the patient. In this center, active involvement of students in clinical rotation, post graduate and residency programs become a very important thing. To support this learning process, SmartTV, computers, internet access and various reference books, electronic journals and application-based 3D diagnostic imaging are provided at this center.

Keywords: Cardiology Center Services, Clinical Skills Center, Diagnostic Imaging Center

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-8

FACULTY OF VETERINARY MEDICINE, UNIVERSITAS NUSA CENDANA: PARTNERSHIP TOWARDS IMPROVING THE QUALITY OF LEARNING, GRADUATES AND RESEARCH CAPACITY

Maxs U.E. Sanam, Diana A. Wuri and Johanes T.R.M.R. Simarmata The Faculty of Veterinary Medicine of Universitas Nusa Cendana

Abstract The Faculty of Veterinary Medicine of Universitas Nusa Cendana or FKH Undana is one of the veterinary education institutions in the eastern region of Indonesia. Located in Kupang City, East Nusa Tenggara Province, an archipelago province that also has the potential of developing extensive livestock farming, especially cattle and horses farms because it has a vast expanse of grassland throughout the region. The NTT province also has exotic animals such as Komodo dragons, snake-head turtles, Timor deer, birds and others. The Faculty of Veterinary Medicine at Undana was originally established as a veterinary study program in 2010 and was developed into a faculty in the year 2013. Its vision is to be a developed and leading veterinary education institution producing human resources, and solutive and innovative researchs against animal diseases in the archipelagic dry land. FKH Undana has 27 lecturers and a total of 300 students distributed in two study programs, academic and profession. The curriculum used is developed to form the competency of graduates in working independently to support the specific needs and demands of employment. Learning by utilizing information technology is encouraged to broaden insight and improve learning access for both students and lecturers. The involvement of lecturers and guest experts as well as industrial internship programs and on-campus educational rides are taken to achieve the intended purpose. To date, FKH Undana has produced more than 100 veterinarians working in various fields, as private practitioners, civil servants, teachers and lecturers, technical consultants at livestock and medicinal companies, and zoos. Current FKH Undana research topics includes Genetic diversity of B. Anthracis; Strategy of vaccination agains anthrax and Haemorhagic Septicaemia for livestock in semiarid region; Antibiomicrobial resistances, Dynamics of endo and ectoparasites on livestock in semiarid region; The use of herbal as anthelmintic and antibacterials; Food safety and public health; and Zoonotic parasites in fishes. The research topics to be conducted in the future are Community empowerment for rabies eradication in Flores, and Diseases and conservative issues of Komodo (Varanus komodoensis) in its natural site. Research cooperation with experts both international and national is necessary to improve the competency and research capacity of young researchers at FKH Undana. Its main cooperation is in the form of increased academic qualifications of lecturers for doctoral studies, and research cooperation as well as publication of international journals.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-9

THE POTENTIAL OF THE SOUTH SULAWESI REGION AS A ONE HEALTH-BASED RESEARCH DEVELOPMENT

Dwi Kesuma Sari Study Program of Veterinary Medicine, Faculty of Medicine, Hasanuddin University, Jl. Perintis Kemerdekaan Km. 10, Makassar, 90245 Email: [email protected]

Abstract

South Sulawesi as one of the regions in Indonesia is an area with certain peculiarities because it is located in the Wallacea line region so that both the flora and fauna contained in the area are very specific and are endemic animals that are not found in other regions. Hasanuddin University as one of the universities in the Sulawesi region, especially South Sulawesi, is trying to develop a number of endemic animal- based studies, for example the Sulawesi Medaka fish (Oryzias celebensis) that was developed into a model animal candidate that can replace Zebrafish fish. In addition, several other animals that have the potential to become a source of zoonotic and food borne disease transmission are also being carried out further research such as microbial contamination in the bird's nest wallet which is commonly found in South Sulawesi, mapping of rabies and rabies carrying animals or HPR which are endemic diseases in the South Sulawesi region. Other research developed is the interaction between humans, monkeys and vector disease carriers in the incidence of Malaria and Filariasis, which also includes one of the tropical diseases that are often found. In addition, the potential of bat birds as disease-carrying animals can also be developed due to a special area in South Sulawesi, Soppeng Regency, where bats are easily found in human settlement areas. The potential for disease transmission from animals to humans can occur so that the participation of all parties in the One Health collaboration is expected so that the incidence of zoonotic diseases can be avoided.

Keywords: One Health, zoonotic disease, animals, endemic area

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-10

FACULTY OF VETERINARY MEDICINE PENDIDIKAN MANDALIKA UNIVERSITY: ACADEMIC COLLABORATION AND FUTURE RESEARCH

Kholik1*, Kunti Tirtasari1, Candra Dwi Atma1 1 Faculty of Veterinary Medicine, Pendidikan Mandalika University, Mataram 83125, West Nusa Tenggara Province, Indonesia Corresponding author: [email protected]*

Abstract Faculty of Veterinary Medicine Nusa Tenggara Barat University (FVM UNTB) has been changed to Pendidikan Mandalika University (FVM PMU) Since October 10, 2019. FVM Pendidikan Mandalika University is the only veterinary school in West Nusa Tenggara (NTB) Province. FVM UNTB is equipped with sufficient facilities for learning activities, such as Animal Hospital, Laboratory, the Equine Clinical Skills Centre (CSC) and Teaching Farm. Every learning activity in FVM PMU is under the guidance of the Association of Indonesian Veterinary Faculties (AFKHI). Faculty of Veterinary Medicine Pendidikan Mandalika University (FVM PMU) consists of 6 divisions. The six divisions are Anatomy Physiology and Pathology, Veterinary Public Health, Microbiology and Parasitology, Animal Reproduction, Clinic, and Diagnostic Laboratories. FVM PMU has collaborated both nationally and internationally in academic and research with many other universities. A veterinary teaching hospital of FVM PMU has collaborated with FVM Bogor Agricultural University to enhance the hospital-based learning education system. The international collaborations have been created with Japan Animal Trust (Osaka, Japan) and SPANA (United Kingdom) for the Equine Clinical Skills Centre (CSC). FVM PMU has main research in zoonotic diseases in farm and wildlife, also antimicrobial and anthelmintic resistant, especially in livestock and poultry farm to support Bumi Sejuta Sapi (BSS) Program. The Bumi Sejuta Sapi, which is literally translated as land with one million cattle, This is the main program of NTB province to develop economic growth. For future research, We try to make research on possible routes of pathogen transmission pathways between livestock, vector and reservoir animal such as frogs. Frogs can be a reservoir of waterborne pathogens from feces and manure of livestock. We are so happy and we need to make new collaboration with Japan-Indonesia Veterinary Education (AJIVE) members in academic and research activities to develop the academic atmosphere and research of our new university.

Keywords: FVM, PMU, Academic, Research, Collaboration

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-11

FACULTY OF VETERINARY MEDICINE UNIVERSITAS BRAWIJAYA: TOWARDS GLOBAL COMPETITIVENESS IN ACADEMIC AND RESEARCH

Aulanni’am*, Dyah Ayu Oktavianie, Herawati, Wawid Purwatiningsih, Fajar Shodiq Permata Faculty of Veterinary Medicine, Universitas Brawijaya, Malang *Corresponding Author: [email protected], [email protected]

Abstract Faculty of Veterinary Medicine Universitas Brawijaya (FVM UB) is the thirteenth Faculty of fifteen Faculty at Brawijaya University. Veterinary Medicine School of Brawijaya University was established in 2008 with recognition of Directorate General of Higher Education, Ministry of National Education in 2008 (SK DIKTI No. 2953/D/T/2008, 4 Sept 2008), then the extension was approved in 2010 (SK DIKTI No: 4860/D/T/K-N/2010). Veterinary Medicine education at FVM BU consists of two Programs: Bachelor of Veterinary Medicine (B.VSc) and Doctor of Veterinary Medicine (DVM). The curriculum of the undergraduate Program of FVM BU consists of 149 credits, including lectures and practical work, on-campus and off-campus that are taken within eight semesters. Followed by Profession Study Program (Internship School) to get the authority of Doctor of Veterinary Medicine, it consists of 38 credits. Both of the two Study Program at FVM BU have accredited as “Good” from National Accreditation Board for Higher Education (BAN-PT), valid until 2021. FVM BU plays an essential role in produce competent veterinarian graduates to meet the necessity of veterinarians in Indonesia. To running its purpose, the education in the Faculty of Veterinary Medicine is directed towards producing veterinarian graduates output with veterinary public health and biomolecular insight as a competent, tough and competitive clinician and able to be developed into academics, researchers and technopreneur. The learning process is conducting in Competence-Based Curriculum through Problem Based Learning (PBL) strategy. This strategy is selected in regards to achieved graduate competencies based on National Competency Standard also to deliver Day 1 competencies of DVM based on OIE regulations. Veterinary Teaching Hospital and Animal Clinic strengthened the learning process in FVM UB and 13 Laboratory including Animal Disease Diagnostic (ADD) laboratory as the prime laboratory, sharing with the related laboratories in Brawijaya University and also supporting by institution both governments and private in Malang and East Java. FVM UB has 40 lectures, including a Master and Ph.D. degrees, and 30 staff. FVM UB has established international collaboration in academics and research, such as with the National Pingtung University of Science and Technology (NPUST) Taiwan, Tarlac Agricultural University Philippines, as well as with international organizations such as SEAVSA, AAVS, FAO, and SEOHUN.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 In research, FVM BU has a Research Roadmap that directed to the development of herbal medicine and alternative therapy for various diseases in humans and animals, development of animal models, and development of vaccine and detection kit for animal disease. It also supports a Research Group in FVM BU which is Biopeptide, Immunodetection and Vaccines (BIODET-Vaccines) which develops biosimilar based material, medical devices for detection kit and vaccines. Some works of this researches are the development of detection kits for Brucellosis, Pregnancy detection for a pet animal and large animal as well, Autoimmune Thyroiditis (AITD), also the development of a vaccine for male immunocontraception.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-12

CANINE PARVOVIRUS INFECTIONS WITH COLIBACILLOSIS INFECTION AND TOXOCARA CANIS INFESTATION IN DOGS

Rohiman Sasmita1*, Lusia Adityaningtyas2, Rondius Solfaine3 1Laboratorium Of Parasitology, Faculty of Veterinary medicine, University Wijaya Kusuma Surabaya 2Laboratorium Of Animal Health and Fishery, Blitar District Veterinary Office, East Java 3Laboratorium Of Pathology, Faculty of Veterinary medicine, University Wijaya Kusuma Surabaya Corresponding: [email protected]

Abstract A dog from a local owner in Surabaya, East Java was examined at the diagnostic laboratory of the UWKS Veterinary Faculty at 2.5-months old, male mixbreed, with of the history of no appetite, diarrhea and weakness for three days. The dog never been vaccinated and had not been given worm medicine. The clinical symptoms was decreased skin turgor, dehydration, anorexia, diarrhea, melena, weakness, pale in both conjunctiva. Based on the prognosis that shows infausta, dogs are sacrificed for autopsy to determine the cause of the disease. From on pathology examination, macroscopic organs of the lungs appear blackened and hardened lungs at the edges, microscopically serous fibrinous fluid, pulmonary alveoli filled with inflammatory cells and infiltration of inflammatory cells occurs. In the small intestine macroscopically occur segmentally in the form of color changes in the intestine due to congestion and bleeding of the outer lining of the intestinal mucosa, the small intestine appears hyperemic and there is hemorrhage in the intestinal mucosa. The results of the rapid test kit for CPV detection of dog faeces with protocol number AJ-03 obtained two strips on the CPV test kit (positive sample). Samples from small intestine and liver swabs from dogs were specific to the pathogenic bacteria E. coli. Based on blood serum examination, it is known that dogs suffer from hypochromic anemia, leukopenia, neutropenia and lymphopenia. Whereas on parasitological examination, showing the morphology of the Toxocara canis worm. Based on pathology and microbiological examination, it was concluded that the dog was suspected to be infected with Canine Parvo Virus with secondary infection of Colibasiolosis and Toxocara canis infestation.

Keywords: Canine parvovirus, Colibacillosis, Toxocara canis,

Introduction Dog is one of the pets that have a high level of intelligence so that it becomes part of people's lives. Viral and bacterial diseases are one of the main causes of death in puppies, because puppies have very low endurance. Diseases in puppies are usually accompanied by symptoms of anorexia, diarrhea, dehydration or gastroenteritis, this can be caused by a viral infection, bacterial infection or worm infestation in the digestive tractus.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Diarrhea can be caused due to a viral infection. Parvovirus, coronavirus, and Rotarivirus are known to cause viral enteritis and diarrhea in dogs and cats. Parvovirus in dogs often attacks all ages and all types of dogs. In general, the virus attacks puppies aged 2-6 months (Chappuis, 1982; Ettinger and Feldman, 1995). Canine Parvovirus infection occurs mainly in puppies that are not protected by adequate maternal antibodies and vaccinations (Panda et al., 2008). In addition, there are trends in several breeds of dogs such as Rottweiler, Doberman Pinscher, Labrador Retriever, American Staffordshire Terriers, and German Sherpherds who have susceptibility to Canine Parvovirus infections (Nelson and Couto., 2009). As a result of this viral disease is a very rapid transmission between dogs and dogs that are sensitive to this disease, causing financial and emotional damage to dog breeders and dog owners. Canine Parvovirus is transmitted from one dog to another through direct or indirect contact with the feces of patients containing the virus (Meunier et al., 1985). In maternal conditions, antibodies that decrease with symptoms of diarrhea, it is possible to have a secondary infection from another disease, Colibacillosis. This disease is a the digestive tractus caused by E. coli bacteria which can damage the intestinal wall, causing diarrhea and can cause death if no treatment is taken. According to Jawetz et al., (1995) E. coli becomes a pathogen if the number of these bacteria in the digestive tract increases or is outside the intestine. E. coli produces enterotoxins which cause several cases of diarrhea. E. coli is associated with enteropathogenic production of enterotoxins in epithelial cells. E. coli is a member of the normal intestinal flora. E. coli plays an important role in the synthesis of vitamin K, the conversion of bile pigments, bile acids and absorption of food substances. E. coli is included in heterotrophic bacteria that obtain food in the form of organic substances from their environment because they cannot arrange the organic substances themselves. Organic substances are obtained from the rest of other organisms. These bacteria break down organic substances in food into inorganic substances, namely CO2, H2O, energy, and minerals. In the environment, these spoilage bacteria function as decomposers and provide nutrition for plants (Ganiswarna, 1995). Objective The purpose of this examination is to determine the cause of disease in dogs by conducting general pathology examinations including macroscopic and microscopic changes, parasitology, microbiology, and clinical pathology to make definitive diagnoses. Methods The sample in this case used a 2.5 month old dog weighing 1 kg with protocol number AJ-03. The materials used are EDTA, methanol, giemsa coloring, physiological NaCl, Turk reagents, emersion oil, sterile PBS, blood agar plate, 70% alcohol, saturated NaCl solution and saturated use solution, gram staining, EMBA Media, Blood Media agar and formalin 10% for the preparation of histopathological preparations. This study use plastic trays, blood tubes eppendoof, a set of necropsy tools, scales for weighing feces, measuring cups, glass beakers, single and double object glass cups, deck glass, microscopes, petri dishes, pipettes , mortars, test tubes, ose, stirrers, syringe counters with needles, spiritus lamps, leukocyte pipettes, erythrocyte pipettes, waterbaths, microhematocrit counting rooms and spectrophotometers. Blood examination blood smear preparations Making blood smear preparations with Wright Giemsa staining. Fresh blood samples without anticoagulants are then dropped on the tip of the glass object (2 µl), then take a slide spreader and position it in front of the blood drop at an angle of 45o.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 pull the slide spreader backward until it touches the drop of blood and wait for blood to flow to both sides of the slide, then push the slide spreader forward to smear. Parasitology Examination Parasitology examination was carried out using faecal, intestinal, blood smear samples, intestinal scrapings, and proventriculus. Stool examination is carried out by the natif and centrifuge method. The native method is done by grinding a small amount of feces and adding enough water. Drop the solution over a glass object and cover with a glass deck, then observe under a microscope (10x10). The centrifuge method is carried out by mixing 2-3 grams of stool with water, pouring into a centrifuge tabung section and centrifuge for 5 minutes. Discard the supernatant and add saturated NaCl ¾ part of the centrifuge tube and stir with a stirrer. Return the centrifuge for 5 minutes, place it on the tube rack and add saturated NaCl over the liquid until the surface is convex and leave it for 3 minutes. Attach the glass object to the convex surface, turn quickly and immediately observe it under a microscope (10x10). Results In the macroscopic organ, the lungs appear blackened and hardened at the edges, microscopically (in figure 1) with 40x magnification there is serous fibrinous fluid, pulmonary alveoli septa filled with inflammatory cells and inflammatory cell infiltration occurs. Thickened septa alveoli due to erythrocytes found in the lungs indicate a secondary infection caused by coliform bacteria ((Turk et al., 1990). In the acute condition pulmonary edema is found. Histopathologically, according to Saif (2008) the presence of a viral infection causes pulmo to experience hemorrhage, congestion and edema as a result of circulatory disorders that occur due to viremia and secondary bacterial infections. Changes in the small intestine can be found in all dogs sufferers. The main target of CPV is small intestinal epithelium, lysis infection resulting in desquamation, bleeding and shortening of the deudenous villi, jejenum and ileum. Damage to the gastrointestinal epithelium can stimulate secondary infection by E. coli bacteria. Circulation of bacteria and endotoxins in the bloodstream causes a condition called coliform septicemia. If this condition cannot be resolved, it can continue to be a septic shock that ends in death (Turk et al, 1990) as well as clinical symptoms of blood defecation in dogs sufferers are a consequence of endotoxemia and cytokine proliferation (Isogai et al,1999). In dogs infected with CPV the older age, the division of cells in the heart begins to decrease while the division in the intestine begins to increase. This causes CPV-infected dogs at> 2 months of age to suffer more forms of enteritis with symptoms such as diarrhea and vomiting (Klinkam, 2006) This form of enteritis occurs in dogs aged> 2 months characterized by vomiting and diarrhea. This is caused by CPV damaging intestinal villi and intestinal kripta. In addition, there are other factors such as complications, malnutrition, secondary infections and others that can affect the condition dog's body. If the body's condition is weak then the virus will easily infect the dog (Honkins, 1995). The existence of secondary infection of colibacilosis is characterized by intestinal inflammation and small bowel distention (Pfizer, 1990). Swelling occurs as a result of plasma filtrate that accumulates in the intestine region of the intestinal organ that is inflamed. Intestinal distension occurs due to accumulation of fluid and gas increases in the intestine. E. Coli bacteria that have villi will stick to the small intestine and then release enterotoxins which result in decreased absorption of sodium and intestinal lumen stretch and an increase in intestinal peristalsis which will cause diarrhea (Buxton and Fraser, 1977).

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 A B C D

Figure 1. Macroscopic and Figure 2. Macroscopic and Microscopic microscopic changes in the lungs. A. Examination of Heart Organs. A. The tip of The lung is blackened (white arrow) the heart is blunt and the heart is enlarged and the tip of the lung is hardened (white arrow), the heart muscle is dilated (black arrow). B. (a) Serous fibrinous (black arrow) fluid (b) hemorrhage (c) infiltration of B. (a) Normal heart cells (b) myocardial inflammatory cells in the lungs infarction (c) necrosis (d) necrosis E F G H

Figure 3. Macroscopic and Figure 4. Macroscopic and Microscopic microscopic changes of the small Fine Intestines (Jejunum). A. Hemorrhage intestine (Duodenum). A. Congestion occurs in the intestinal mucosa. B. (a) and hemorrhage (black arrow) Hemorrhage (b) necrosis (c) infiltration of occurs.B. (a) Rupture of the intestinal inflammatory cells villi

Microbiological examination is done by taking samples of small intestine swabs and liver organs to isolate the presence of Escherichia coli which is suspected to be a cause of secondary infections in dogs that cause diarrhea. Samples of small intestine and liver swabs from Aj-03 dogs were planted on a blood agar plate and EMBA were then incubated at 37 C for 24 hours. In the isolation plate so that blood is obtained by a colony with a shiny colony, whitish, opaque, size 2-4 mm and looks to demolish blood. This means that E. coli is a pathogenic bacterium. On eosin-methylen blue agar (EMBA) bacteria, a shiny green colony of bacteria, this is in accordance with Saif et al (2008) Escherichia coli has a dark green black metal glitter on eoasin methylen blue agar (EMBA). Toxocara canis worm eggs have a thick (outer layer) second egg shell. The egg can hold for years in the feces of infected dogs and wolves (Levine, 1994). The size of the 85 x 75 μm Toxocara canis worm egg is oval in shape with jagged surface, light brown color, and thick wall (Soedarto, 2003). The Toxocara canis worm lives in the dog's small intestine and is the largest nematode. An adult Toxocara canis female worm can produce approximately 200,000 eggs per day (Subronto, 2006). Infective eggs are removed with feces. Stool containing Toxocara sp falls on the ground with a temperature of 10 - 35ºC and humidity of 85% and optimal conditions then in at least 5 days will develop into infective eggs containing embryos (Levine, 1994). The body of

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Toxocara canis is white, with long and narrow cervical alae (Levine, 1994). The shape of the tail of Toxocara canis for the male sex is shaped like a hand with a finger pointing (digitiform), while for the female sex the shape of the tail is pointed and round (Manurung and Siahaan, 2012). The Toxocara canis worm has a slightly different shape from the type of the Toxocara sp. the other. Where male worms have not the same size, bent, winged wings and a length of 750-1300 microns (Levine, 1994).

A B

Figure 5. Isolate E. coli in blood agar media. Visible hemolysis, shiny whitish, opaque. B. Isolation of E. Coli bacteria on EMBA media. Growing shiny green bacterial colonies C D

Figure 6. Toxocara Canis worm eggs which are colored in Lactophenol. Morphology of Toxocara canisA. Anterior section of Cephalic alae and Posterior section

In Toxocara canis there are cervical wings that are shaped like lancet, whereas in Toxocara cati the wings are wider, so that the head resembles the head of a cobra. The shape of the tail of Toxocara canis and Toxocara cati are almost the same, for the male sex the tail is shaped like a hand and with the finger being pointed (digitiform), while for the female sex the shape of the tail is tapered round. The results of routine leukocytes in this study show leukopenia, lymphopenia, and neutropenia. In puppies infected with Canine parvovirus the virus replicates in the lymph nodes then goes to lymphocytes during the first day or two. Many of these CPV- infected lymphocytes eventually die, leading to a reduction in the number of

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 lymphocytes in the circulation of a condition called lymphopenia. In the bone marrow, the virus weakens the body's ability to protect itself by destroying young immune cells and causing a decrease in the number of protective white blood cells. So with the condition that the antibodies are getting weaker the virus easily enters the digestive tractus. Conclusions Based on the results of examinations in the Pathology, Microbiology, Parasitology and Clinical Pathology Laboratory, dogs with protocol number AJ-03 were diagnosed with Canine Parvovirus with secondary infections of Colibacillosis and Toxocara canis worm infestations

References Agna. 2009. Toxocariasis pada Kucing, (on line). (http://dragna.livejournal.com/3275.html. Diakses pada tanggal 27 Januari 2014). Anonymus. 2007. Toxocara embryonated eggs.jpg. (on line). (http://commons. wikimedia.org/ wiki/File:Toxocara_embryonated_eggs.jpg. Diakses pada tanggal 10 februari 2014). CDC. Control Desease and Prevention. 2013. Parasites - Toxocariasis (Also Known As Roundworm Infection). USA.(http://www.cdc.gov/parasites/ toxocariasis/. Diakses pada tanggal 26 Januari 2014). Chappuis G. 1982. Etiopathogenie des affections digestives d’origine virale chez le chien. Rec Med Vet 158(1): 107-13. Davey P., 2006. At a Glace Medicine. Alih bahasa oleh, Rahmalia A., Novianti C. Jakarta: Erlangga. 374-5 Ettinger SJ, and Feldman EC. 1995. Text Book of Veterinary Medicine. Diseases of Dog and Cat. Sixth Ed. Vol. 2. California. Elseiver Sauders. Pp. 405-407.

Harrison, G.J., dan Lightfoot, T. 2006. Clinical Avian Medicine, 1st Edition. Spix Publications. Palm Beach, Florida. Harvey, J.W. 2001.Atlas of Veterinary Hematology. WB Saunders company. Philadelphia Hoskin, J.D. 1997. Update on canine parvovirus enteritis. Vet. Med. 92(8):694-709. Isogai, E., H. Isogai, and M. Onuma. 1999. Escherichia coli associated endotoxemia in dog with parvovirus infection. Jpn. J. Vet. Sci.51(3):597-606. Jawetz, E. et al. (1995). Review of Medical Microbiology. Los Altos, California: Lange Medical Publication. Pages 227-230. Kerr, M.G. 2002.Veterinary Laboratory Medicine: Clinical Biochemistry and Haematology, 2nd ed. Blackwell Science. Oxford Latimer, K.S., 2011. Ducan and Prasse's Veterinary Laboratory Medicine : Clinical Pathology Fift Edition. Lowa; Wiley-Blackwell Leboffe, M.J dan Pierce, B. E.2011. A Photographic Atlas for the Microbiology Laboratory. Edisi ke-4. Marton Publishing Company. United States of America. Pp 63 – 96. Levine N. 1994. Buku Pelajaran Parasitologi Veteriner. Yogyakarta : UGM Press. Maza, L. M., Pezzlo, M. T., & Baron, E. J. 1997. Color Atlas Of Diagnostic Microbiology. Missouri: Mosby-Year Book. Meunier, PC., Cooper, BJ., Appel, MJ., Lanieu, ME., Slauson, DO. 1985. Pathogenesis of Canine Parvovirus Enteritis : Sequential Virus Distribution and Passive Immunization Studies. Veterinary Pathology 22 : 617-624.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Mohyedini S, Jamshidi S, Rafati S, Nikbakht GHR, MalmasiA, Taslimi Y, Akbarein H (2013). Comparison of immunechromatographic rapid test with molecular method in diagnosis of canine parvovirus. Iran. J. Vet. Med. 7(1): 57-61. Nelson, R.W. dan Couto, C.G., 2009. Small Animal Internal Medicine 4th Edition. Philadelphia : Mosby Elsevier. 443-445. Overgaauw PAM. 1997. Prevalence of intestinal nematodes of dogs and cats in the Netherlands . Vet . Quart . 19 : 14-17 . Panda, A. K.; Kumar, A. A.; Singh, S. D.; Sastry, V. R. B., 2008. Growth performance and pathological lesions in broiler chickens fed raw or processed karanj (Pongamia glabra) cake as protein supplement. Indian J. Anim. Sci., 78: 997–1001 Pennisi E. 2002. Canine evolution: A shaggy dog history. [terhubung berkala] http: //www. Dogexpert. Com/popular % 20 press/caning 20 Evulution. Html. [30 mei 2010]. Saif, YM. Fadly AM. Glisson JR. Mc. Dougald LR. Nolan LK. Swayne DE. 2008. Disease of Poultry 12th Edition. Blackwell-Publishing. Oxford. Pp 156-158.

Acknowledgment This work was supported by Laboratory of Diagnostic Faculty of Veterinary Medicine, University Wijaya Kusuma Surabaya Indonesia, 2019.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 DO-13

THE VETERINARY MEDICINE STUDY PROGRAM OF FACULTY OF MEDICINE, UNIVERSITAS PADJADJARAN, INDONESIA

Dean

Abstract The Veterinary Medicine Study Program of Universitas Padjadjaran was established in January 2016 and has been accredited “B” from Indonesia Accreditation Agency for Higher Education in Health in November 2020. The study program vission is being an excellent study program in Veterinary Public Health based on One Health principal. The study program has supporting facilities, that are Central Laboratory and Animal Hospital. The laboratory was equipped with advanced medical laboratory PCR, IHC and Western Blot, animal imaging extraction and purification. The animal hospital has several facilities, which are x-ray, ultrasonography, laboratory analysis and surgery room. The number of student study in this program are 211 persons of 4 academic years. The 4th year students are now conducting research for their last project. The scope of research that has been carried out covers veterinary public health, zoonosis, biomedical, animal health, microbiology, pathology, animal welfare, human-animal interaction, and phytopharmacology. The research was supervised not only by the vet lecturer but also from other study program such as from medicine, biology and dentistry.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-1

DEVELOPMENT OF NEW TREATMENT STRATEGY FOR CANINE CANCERS

Takuya Mizuno* Department of Veterinary Clinical Pathology, Yamaguchi University *Corresponding author: [email protected]

Canine cancer is one of the leading causes of death in adult dogs. Surgery, radiation therapy, and chemotherapy are three most common ways to treat cancer, however we still need a novel therapy to support these therapies. Our laboratory has been trying to develop a novel therapy for canine cancers, such as oncolytic virotherapy, small molecule inhibitors, antibody therapy, and immune cell therapy. In our laboratory, we usually start in vitro study using cancer cell lines to obtain proof of concept (POC) of a novel therapy, followed by mouse study using xenotrasplanted mouse model. We also have an approach to elucidate the molecular mechanism of a novel therapy using the molecular biology, cell biology, molecular immunology, and protein biology techniques. After safety study using healthy beagle dogs, the inhouse small clinical trial is conducted to obtain the POC data. Through these approaches, clinical trials using oncolytic virotherapy and antibody therapy already had been started for cancer-bearing patient dogs in our veterinary teaching hospital.

Examples of the novel therapies developed in our laboratory ・Small molecule inhibitors: Tenovin-6, ATM inhibitors ・Oncolytic virotherapy: reovirus (Reolysin®) ・Antibody therapy: anti-dog PD-1/PD-L1 antibodies, anti-dog CD20 antibody, antibodies targeted canine lymphoma or melanoma ・Immune cell therapy: gene-modified T cell therapy (chimeric antigen receptor-T cells)

Keywords: canine, cancer, novel therapy, antibody therapy, molecular targeted therapy

Selected References since 2016 related to above projects Igase M. et al., Exp Cell Res. 2020; Hwang CC. et al., Biochem BIophys Res Commun. 2019; Igase M. et al., Mol Ther Oncolytics 2019; Igase M. et al., Vet Comp Oncol. 2019; Hwang CC. et al., Vet Comp Oncol. 2018; Nemoto Y. et al., Vet Immunol Immunopathol. 2018; Sakai O. et al., J Vet Med Sci. 2017; Ema Y. et al., Vet Comp Oncol. 2016; Hwang CC. et al., Vet Comp Oncol. 2016; Shosu K. et al., In Vivo 2016; Igase M. et al., Can J Vet Res. 2016

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-2

DEVELOPMENT OF IN VITRO ATOVAQUONE-RESISTANT BABESIA GIBSONI AND THE EPIDEMIOLOGICAL SURVEY FOR ATOVAQUONE RESISTANT RELATED GENE

Aiko Iguchi, Takashi Takeuchi Veterinary Laboratory Medicine, Tottori university *Corresponding author: [email protected]

Abstract Introduction Atovaquone (ATV) is a well-tolerated, metabolically stable and effective agent with broad-spectrum anti-parasitic activity. ATV monotherapy was effective for acute canine B. gibsoni infection; however, it resulted in relapse of ATV-resistant parasites. The characteristics of ATV-resistant B. gibsoni was unknown, and it was also unknown whether they were prevalent in Japan. Objective The study was conducted to develop an in vitro ATV-resistant B. gibsoni by exposing the parasite culture to ATV, and to perform mitochondrial gene analysis. And then, the second study was conducted to detect the M121I variant population in Japan. Methods & Results 1. For development of ATV-resistant B. gibsoni, different concentrations of ATV were exposed to Wild type B. gibsoni in vitro. After 800 nM ATV exposure, parasites showed re-growth. The parasites were less sensitive to ATV than WT B. gibsoni, so were isolated as ATV-resistant B. gibsoni. 2. The nucleotide and deduced amino acid sequence of mitochondrial cytb, COX1, and COX3. Cytb of the ATV-resistant B. gibsoni had a single nucleotide substitution at nt363 (G to T), which resulted in the substitution of methionine with isoleucine (M121I), and there was no substitution at COX1 and COX3. 3. In 73 gDNA samples from B. gibsoni infected dogs, the M121I variant population was measured by using allele-specific real-time PCR. 3/73 samples showed over 5% M121I variant population. Conclusion An ATV-resistant B. gibsoni was developed by exposing WT B. gibsoni to 800 nM ATV for 6 days in vitro. M121I substitution in cytb might be associated with the less sensitive to ATV. Although the M121I variant population is a low percentage, it runs the risk of spreading drug-resistant parasites. References A. Iguchi et al., Development of in vitro atovaquone-resistant Babesia gibsoni with a single-nucleotide polymorphism in cytb. Vet Parasitol 185 (2012). A. Iguchi et al., The epidemiological survey for atovaquone resistant related gene of Babesia gibsoni in Japan

Keywords: Babesia gibsoni, atovaquone, ATV-resistant, the epidemiological survey

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-3

CORRELATION BETWEEN FOREFOOT AND BACK FOOT LENGTH AGAINST BODY LENGTH OF SUNDA PORCUPINE FROM CENTRAL JAVA

Teguh B1*, Yosua Ka2, Irma P1 1Department of Anatomy, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia; 2Department of Reproduction and Obstetrics, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia *Corresponding author: Teguh B, [email protected]

Abstract

Sunda porcupine or Hystrix javanica is wild animal that can be found in shrubland, forest, and terrestrial area. This species is endemic in Indonesia and found only in Java Bali, Sumbawa, Flores, Lombok, Madura, and Tonahdjampea island. Since 2018, Indonesia Government classified this species into protected animal due to decrease of their population in nature. We begin study about anatomical aspect of this animal since 2013. In this paper, we assessed the correlation between forefoot and back foot length against body length of Sunda porcupine. Data from thirty-five Sunda porcupines consist of male and female animals from Tawangmangu and Ngawi, Central of Java Province were used in this analysis. Correlation and regression analysis were performed using Ms.Excel program. Based on the analysis, correlation coefficient (r) of forefoot length and body length was r=0,282426. This showed that there was low correlation between forefoot length and body length. The linear graph showed the upward sloping line that indicated a positive correlation. It means if the forefoot length increased, the body length will increase too. Regression coefficient showed R2=0,0798. It indicated that the forefoot length will affect body length about 7,98%. Regression equation was y=3,1881x+62,306. The correlation coefficient (r) of back foot length and body length was r=0,401003. This showed that there was average correlation between back foot length and body length. The linear graph showed the upward sloping line that indicated a positive correlation. It means if the back-foot length increased, the body length will increase too. Regression coefficient showed R2=0,1608. It indicated that the forefoot length will affect body length about 16,08%. Regression equation was y=3,2965x+51,561. Based on the analysis result, we found that back foot length has higher correlation against body length than forefoot length. This data will help to estimate the body size of Sunda porcupine if we found their footprint in nature. Estimation of body size is important to determine the mature and immature status. However, correlation study between body size and mature and immature status needs to be done furthermore.

Keywords: Sunda porcupine, forefoot length, back foot length, body length, correlation

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Introduction The significant relationship between body height and the length of other body parts, such as bone, has been used for many years to make some physical formula of body height estimation (Grivas et al., 2008; Preece, 1988). Memarian et al. (2016) and Masanovic et al. (2017) reported that in human, foot length also can be used to predict body height. This discovery was very important for some condition, such as forensic medical issue (Giles and Vallandigham, 1991) and neonate prediction (James et al., 1979). In the veterinary field, relationship between body length and the length of other body part also useful, such as in animal conservation and forensic veterinary medical issue. Indonesia has many endemic animals that its population start to decrease in nature. Sunda porcupine or Hystrix janavanica is one of the examples. Sunda porcupine or Hystrix javanica can be found only in Java, Bali, Sumbawa, Flores, Lombok, Madura, and Tonahdjampea island, Indonesia (Van Weers 1979, 1983; Woods and Kilpatrick 2005). Sunda porcupine has habitats and ecology in forests of subtropical/tropical moist lowland and shrubland of subtropical/tropical moist. Study about anatomical morphometry of this species are important to support the conservation effort, thus we can predict the body size based on footprint found in nature. However, the correlation between foot length and body length of Sunda porcupine has never been reported. Objective This study was conducted to analyze the correlation between forefoot and back foot length against body length of Sunda porcupine from Tawangmangu and Ngawi, Central Java Province. Method We got the foot length and body length data of Sunda porcupine from slaughterhouses in Tawangmangu and Ngawi District from 2013 until 2018. Thirty- five data consisted of both, male and female, were obtained by measuring the forefoot length, back foot length, and body length using ruler. The data were analyzed statistically with Ms. Excel program software. Results The correlation coefficient of forefoot length and body length of Sunda porcupine was r=0,282426. The linear graph showed the upward sloping line (Figure 1). The determination coefficient was R2=0,0798. These results showed that there was a positive correlation between forefoot length and body length of Sunda porcupine. Based on the coefficient interval table, the level of relation was categorized as low (Table 1). This means if the forefoot length was increased then the body length will increase too and vice versa. The forefoot length affects the body length of about 7,98%.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020

Correlation: forefoot length and body length 100 y = 3.1881x + 62.306 90 R² = 0.0798 80 Forefoot Length X Body 70 Length 60 Linear (Forefoot Length Body Length (cm) Length Body X Body Length) 50 3 4 5 6 7 8 Forefoot Length (cm)

Figure 1. Linear Graph of Correlation: forefoot length and body length.

The correlation coefficient of back foot length and body length of Sunda porcupine was r=0,401003. The linear graph showed the upward sloping line (Figure 2). The determination coefficient was R2=0,1608. These results showed that there was a positive correlation between back foot length and body length of Sunda porcupine. Based on the coefficient interval table, the level of relation was categorized as average (Table 1). This means if the back-foot length was increased then the body length will increase too and vice versa. The back-foot length affects the body length of about 16,08%. Correlation: back foot length and body length 100 90 y = 3.2965x + 51.561 R² = 0.1608 80 Back foot Length X 70 Body Length

Body Length Body 60 Linear (Back foot Length X Body Length) 50 6 8 10 12 Back foot Length (cm)

Figure 2. Linear Graph of Correlation: back foot length and body length.

This study showed that there was positive correlation between forefoot and back- foot length against body length of Sunda porcupine. However, correlation between back-foot against body length (r=0,401003) was higher than correlation between forefoot length against body length (r=0,282426). Back-foot length has affected the body length more than forefoot length. In Asian elephants (Elephas maximus), body weight can be estimated from heart girth, height at the withers, body length, and foot- pad circumference, while the best predictor is heart girth (Hile et al., 1997). This means that there are many correlations between body part with body size. In Sunda porcupine,

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 we assessed correlation between forefoot, back-foot, and body length and found that back-foot length was better predictor than forefoot length of body length.

Table 1. Coefficient interval table and level of relation. Coefficient Interval Level of Relation 0,000-0,199 Very Low 0,200-0,399 Low 0,400-0,599 Average 0,600-0,799 Strong 0,800-1,000 Very Strong

Conclusion There was correlation between forefoot and back-foot length against body length of Sunda porcupine. Back-foot length has higher correlation against body length than forefoot length.

References Giles, E., Vallandigham, P.H. 1991. Height estimation from foot and shoeprint length. J Forensic Sci. 36:1134-1151. Grivas, T.B., Mihas, C., Arapaki, A., Vasiliadis, E. 2008. Correlation of foot length with height and weight in school age children. J Forensic Leg Med. 15:89-95. Hile, M.E., Hintz, H.F., Erb, H.N. 1997. Predicting body weight from body measurements in Asian elephants (Elephas maximus). J Zoo Wildl Med. 28(4):424- 7. James, D.K., Dryburgh E.H., Chiswick, M.L. 1979. Foot length--a new and potentially useful measurement in the neonate. Arch Dis Child. 54(3):226-230. Masanovic, B., Gardasevic, J., Arifi, F. 2017. Relationship between Foot Length Measurements and Body Height: A Prospective Regional Study Among Adolescents in Central Region of Kosovo. Memarian, A., Soltani, S., Aghakani, K., Mehrpisheh, S., Taftachi, F., Abdolkarimi, L., Zadmehr, A. 2016. Estimation of Body Height by Measuring Foot Dimensions: A Survey on Iranian Adult People. J Med Toxicol Clin Forens Med. 3:1. Preece, M.A. 1988. Prediction of adult height: Methods and problems. Acta Paediatr Scand Suppl. 347:4-11. SSRN: https://ssrn.com/abstract=3077530 or http://dx.doi.org/10.2139/ssrn.3077530 van Weers, D.J. 1979. Notes on southeast Asian porcupines (Hystricidae, Rodentia) IV. On the taxonomy of the subgenus Acanthion F. Cuvier. Beaufortia. 29(356):215- 272. Van Weers, D.J. 1983. Specific distinction in Old World porcupines. Zoologische Garten. 53:226-232. Woods, C.A.,Kilpatrick, C.W. 2005. Infraorder Hystricognathi. In: Wilson, D.E. and Reeder, D.M. (eds), Mammal Species of the World, pp. 1538-1599. The Johns Hopkins University Press, Baltimore, MD, USA.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-4

CONTINUING PROFESSIONAL DEVELOPMENT: THE CRUCIAL ROLES OF VETERINARY TEACHING HOSPITAL IN FACILITATING A LONG LIFE LEARNING OF VETERINARIANS

Deni Noviana1*, Frizky Amelia2, Leni Maylina3,4, Srihadi Agungpriyono5 1Director of Veterinary Teaching Hospital, Faculty of Veterinary Medicine, IPB University 2PhD student, Animal Biomedical Science, Graduate School of IPB University 3Division of Internal Medicine, Faculty of Veterinary Medicine, IPB University 4PhD student, Laboratory of Internal Medicine, Joint Graduate School of Veterinary Science, Yamaguchi University 5Dean of Faculty of Veterinary Medicine, IPB University Corresponding Author: [email protected]

Abstract Students who completed their degree in veterinary medicine and finally become a veterinarian is starting their journey of life-long learning and continuing professional development until retirement. As the veterinary profession is continually evolving, veterinarians need to keep updating the information on new treatment protocols, as well as scientific research and technology associated with the veterinary industry to provide the best care for their patients. Moreover, veterinarians in Indonesia must actively participate in continuing education activities, especially if they wish to apply for an extension of their practice license from the Indonesian Veterinary Medical Association (IVMA). One of the Tridharma functions of the university is to carry out and facilitating educational activities, not only for its undergraduate and graduate students but also for the alumni and veterinarians nation-wide as well. In carrying out its educational activities, the Faculty of Veterinary Medicine IPB University is equipped with special supporting unit, namely IPB University Veterinary Teaching Hospital (IUVTH). The IUVTH was established in 2000 and plays a big role in assisting and providing a platform for continued education for veterinarians. Our hospital holds several continuing professional development programs for veterinarians. We had at least 53 CPD programs that held in the past 4 years (2016-2019) involving more than 1100 participants. During that period, at least as many 23 speakers participated, both prominent national speakers and professional speakers from abroad such as United States of America (USA), Thailand, Australia, United Kingdom, Canada, Japan, Malaysia, Taiwan, China, and Singapore. The IUVTH in collaboration with IVMA and Indonesia Animal Hospital Association (ARSHI) also host the Indonesian Animal Hospital and Clinic Expo (INAHEX) every two years. Our hospital host the 1st INAHEX in 2017 and 2nd INAHEX in 2019 with a line-up of local and international speakers, who are experts in their fields. This expo, not only become the platform of information and ideas sharing among colleagues but also allows numerous exhibitors to display the newest innovations in veterinary products and services, and holds scientific sessions and workshops as well. Furthermore, Indonesia has a large number of special interest groups, also known as non-territorial organizations, which provide opportunities for members with shared

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 interests or expertise to develop their skills and knowledge within a specific area. These groups provide relevantly and focused continuing professional development, networking and social activities as well. For example, the Indonesian Associaton of Traditional Chinese Veterinary Medicine (IATCVM) who annually collaborates with our hospital to co-host the 3rd and 4th session of Small Animal Acupuncture Class in Indonesia from Chi Institute of Traditional Chinese Veterinary Medicine (Florida, USA) since 2015. Speakers and students from at least 9 countries (USA, Hong Kong, Singapore, Malaysia, Indonesia, Philippines, India, Dubai, and Australia) had come and learned together in our hospital to pursue the Certified Veterinary Acupuncture degree. As for 2020, the IUVTH together with IPB International Certified Training (IICT) is planning to holds 20 on-site CPD programs with several topics but not limited to, such as ultrasonography, abdominal and thoracic radiology, rehabilitation medicine, exotic pet medicine, hematology, gastrointestinal endoscopy, anesthesia, soft tissue surgery, veterinary entrepreneurship, emergency medicine, equine reproduction, dermatology, feline internal medicine, small animal acupuncture, zoo and wildlife animal medicine, and training for veterinary technician. Besides, the IUVTH also plans to hold an online course with the main target is the colleagues who work in a remote area, with financial restrictions and great distance from the education center. This is also one of the hospital roles in helping disseminate veterinary knowledge nation-wide. We are currently seeking speakers both from within and outside the country to participate in our CPD activities as speakers or instructors, in both online and on-site courses this year. To support its function as an education center for both students and veterinarians, the IUVTH has set up two centers namely, CPD training center and Diagnostic Imaging Center (DIC). The CPD training center is co-manage by both IUVTH and IICT, as an on-site location for seminar and workshop, meanwhile, the DIC is managed by IUVTH, IICT, and several privates companies. The DIC itself not only has the function as a learning center for diagnostic imaging for our students but also has the function as a referral radiology cases. We received and help to interpret the radiograph from veterinarians nationwide. The DIC is supervised by one Diplomate in Asian College of Veterinary Internal Medicine (DAiCVIM, Specialist in Cardiology), radiology lecturers, five hospital veterinarians, and four graduate students (three Ph.D. candidates, 1 master candidate). With those many CPD activities, both faculty and hospital not only facilitate the continuing education for veterinarians and alumni, but also provides an opportunity for co-assistant students, graduate students, and hospital veterinarians themselves to be directly involved in those events. They can learn and gain experience, interact with colleagues from various fields of the veterinary profession and updating their knowledge and veterinary skills as well. Through CPD activities, both hospital and faculty have the opportunity to expand the connections and relationships with various institutions from many countries. Through this symposium, we hope to have a good fortune to collaborate with Japan and Indonesia veterinary schools in both education and research activities in the future.

Keywords: continuing professional development, veterinary teaching hospital, IPB university

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-5

REPRODUCTIVE PHYSIOLOGY AND MANAGEMENT FOR DOMESTIC ANIMALS

Hiroya KADOKAWA* Join Graduate School of Veterinary Medicine, Yamaguchi University, Yamaguchi- shi, JAPAN, *[email protected]

Abstract Background Reproduction is the most important limiting factor in all of the animal industries. If reproduction is not good, number of young animals will be shortage, and female animals can not become pregnant which is necessary for lactation of mammals. While needs of animal protein is increasing in middle class people in various countries including Asian countries, speed of increasing animal numbers are still very low due to difficulties of reproduction. Objective The previous studies and concepts are not enough to clarify important mechanisms and to develop new tools in order to improve reproduction in food animals. We have tried to discover new physiological mechanism to suppress or stimulate reproductive performance from the views of management, neuroendocrinology, new hormone and receptors, nutritional factors affecting functions of brain, pituitary and reproductive tracts. This presentation will introduce the recent scientific activities of our group. Examples of our recent scientific results Lipid raft containg GnRH receptor and newly discovered receptors in bovine gonadotrophs We have developed a unique antibody to extracellular region of GnRH receptor (anti-GnRHR). Utilizing the anti-GnRHR, we clarified that GnRH receptor is located on the special region of plasma membrane namely “lipid raft”. Utilzing the antibody and next generation sequence etc., we have discovered that the gonadotroph lipid raft contain also new receptors. Now, utilzing this seed, we are challenging to develop new tools to solve infertility in animals. Newly discovered estrogen receptor GPR30 to induce infertility by suppressing LH secretion Recently, we have discovered that a new estradiol receptor, GPR30, is present in the plasma membrane of bovine gonadotrope. GPR30 activated by estradiol rapidly (within a few minutes) suppress LH secretion without affecting the gene expression, thus, nongenomic mechanism. GPR30 can be activated by various estrogen-like compounds produced by fungi. Therefore, this may ne the mechanism of infertility in poor farmers!! Molecular mechanisms to suppress oviduct functions in lean and obese cows Obese and lean heifers produce fewer excellent-grade embryos than normal heifers due to unknown mechanisms (Kadokawa et al. 2008 ). We have recently clarified that 2 proteins expressed in bovine oviducts, GMCSF and MIF, are the important key proteins for this phenomenon. Oviducts are not the “Dark Continent”, and we should launch the expedition!! Molecular mechanisms to suppress oviduct-uterus functions in cows after aging

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 We have recently also discoverd a ovarian molecule to stimulate oviduct-uterus function, namely AMH. Bovine oviduct-uterus express the AMH receptors, and furthermore, the ligand-receptor mechasnism may be suppressed after aging to induce infertility. Conclusions We have discovered new physiological mechanisms to control reproductive functions in domestic animals. Then, we have found new risk factor (e.g. mycoestrogens, and obesity) and important hints to stimulate reproductive functions (e.g. new receptors, and managing oviduct-uteus function). We should begin the international collaboration between Indonesian universities and Kadokawa’s group in Yamaguchi university in Japan. Especially, we hope to invite excellent Indonesian students to our doctor course. Further information of our group are available in the following site: http://web.cc.yamaguchi-u.ac.jp/~hiroya/

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-6

CYTOTOXIC EFFECTS OF MENTHOL

Kenji Takahashi, Toshio Ohta* Department of Veterinary Pharmacology, Faculty of Agriculture, Tottori University, 680-8553, Japan *[email protected]

Abstract Background and Objective Menthol, a cooling monoterpene derived from a peppermint, is used as an essential oil for various usages. It functions as an agonist of a transient receptor potential (TRP) melastatin 8 (TRPM8) and TRP ankyrin 1 (TRPA1) channels. Both channels are related to various diseases including a lung injury as nociceptors. However, the cytotoxic action of menthol is not fully understood. In the present study, we investigated the effects of menthol on the viability of a lung cancer epithelial cell line, A549. Method/approach The cell viability was measured by using a WST-8 reagent and a trypan blue dye exclusion test. The apoptotic cells were detected by annexin V binding flowcytometrically. The expressions of both channels were detected by using RT-PCR 2+ 2+ and western blotting. The intracellular Ca concentration ([Ca ]i) was measured by using fluorometric Ca2+-imaging system. The activity of Ca2+-ATPase was determined by using a colorimetric assay based on the molybdenum blue method. The mitochondrial membrane potential and the production of reactive oxygen species (ROS) were detected by using each fluorescent reagent. Results The expressions of TRPM8 and TRPA1 were detected at both levels of mRNAs and 2+ proteins in A549. Menthol at 3 mM induced [Ca ]i increases which were not attenuated by the removal of extracellular Ca2+ and both TRP channel inhibitors but inhibited by an ER Ca2+ pump inhibitor. The activity of Ca2+-ATPase was suppressed by menthol. Menthol-induced cytotoxic action was not attenuated by intracellular Ca2+ chelation. The mitochondria membrane potential was decreased by menthol. Apoptosis occurred after the application of menthol. Although menthol generated the ROS, an antioxidant failed to inhibit menthol-induced apoptosis. Conclusions These results indicate that menthol induces the cytotoxicity at relative higher concentration in A549. Menthol-induced apoptosis due to mitochondrial dysfunction was independent on the ROS generation. Although menthol mobilized Ca2+ from intracellular stores, its cytotoxic effect is not related to the activation of TRP channels 2+ and the increase of [Ca ]i. It maybe provides that menthol is a useful lead compound for anti-cancer drugs.

Keywords: Menthol, Monoterpene, TRP channel, Calcium ion, Apoptosis

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-7

CHANGE OBSERVATION CONDISTION OF RABBIT ORGANS IN INTERVALS DIFFERENT TIME (POST INTERVAL MORTEM)

Albiruni Haryo, Miranti Verdania, Mimin Wulandari. Lutfiana Pratiwi, Vilinda Maya Pathology Anatomy Laboratory, Faculty of Veterinary Medicine Brawijaya University

Abstract Postmortem interval (PMI) is one important method that needs to be done to help identify the time of death in forensic science. Post Mortem Interval refers to the time interval between the discovery or examination of a corpse with the time of death. The direction and the possibility of time can be used to determine the likelihood of happening in more detail and direction. The importance of PMI's actions in veterinary science is related to cases of animal abuse in society, which involve the role of humans both intentionally and unintentionally resulting in death. In this study the method used for determining PMI estimation is postmortem evaluation of physiological and physical changes such as the distribution and amount of livor mortis, rigor mortis, changes in body temperature, and the level of body damage. Four rabbits were used in this study, which were buried, and were observed at certain intervals, with a maximum observation distance of one week. Abnormal organs are observed, collected, and processed and histotechnically. With the results that this study proves that there is a change in the post-death process that can be called a Post-Mortem change, by measuring the time interval in the process of death.

Keywords: Death, Post Mortem Interval, Rabbit

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-8

THE STUDY OF CALF WEIGHT ON THE ESTROUS POSTPARTUM (EEP) PERIOD ON PERANAKAN ONGOLE (PO) CATTLE

Agung Budiyanto1*, Erif Maha Nugraha Setyawan1, Soedarmanto Indarjulianto2 Jumaryoto3 Topas Wicaksono Priyo Jr3 1Department of Reproduction and Obstetric, 2Department of Internal Medicine, 3Post Graduate of Sain Veteriner, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta *Corresponding author: [email protected]

Abstract The long period of estrous postpartum (EEP) of Indonesia cattle is one of reproductive management problem. Recently, length of EEP Indonesia more than standart. This problem will make the costly management budget. And influence will make the decrease of economic benefit at the rural Indonesian breeding sector. The types of cattle that are often raised in rural Indonesia are Cross Ongole (PO), Simmental Cross Ongole (SimPO) and Limousine Cross Ongole (LimPO). Reproductive efficiency can be seen from good reproductive performance. Several factor has been known that affect on the EEP, such as, breed, age, body condition but there is no data yet about the effect of calf weight to the EEP on PO cattle. This study was used 16 breed Cross Ongole (PO) cattle, aged 2-15 years, body condition score 2.5-3.5, and at least already calving once, The have no reproductive diseases and have normally birth. The data of Estrous postpartum was got from the anamnesis with the owner. Calf weight have checked were used the scales in kilogram soon after birth. Thr results showed that there were no difference between groups of calf weight to estrous postpartum on PO cattle (P>0.5) statistically. Further study have to do to know several factors that influenced on EEP.

Keywords: cattle, PO, estrous postpartum, calf weight

Background One sector of economic contributors in rural Indonesia is the cattle breeding sector. The types of cattle that used to at Indonesia are Cross Ongole (PO), Simmental Cross Ongole (SimPO) and Limousine Cross Ongole (LimPO). What influences the success of beef cattle farming is reproductive efficiency and average daily. Reproductive efficiency can be seen from good reproductive performance. Reproductive performance parameters are Service per conception, calving interval and estrous postpartum. Estrus postpartum is one condition of the return of functions of the female reproductive organs and channels after childbirth (Christoffor and Baliarti, 2008). This EEP periode in Indonesia still very long, caused by several factors. After giving birth the cow enters the puerpurium, at that time there is uterine involution, endometrial regeneration, and

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 then estrus after breeding (Partodiharjo, 1987). We haven’t found any information or data about affect calf weight to the estrous postpartum period on PO cattle. Objective This research aim to obtain new information about the effect of calf weight to the estrous postpartum on PO cattle. Materials and Methods The sample consists of 16 PO cattle that are kept on the local’s farm in District Sleman Region Special of Yogyakarta, given the same feed, live in the same village, and the criteria material is cow, aged is 2-15 years old, at least already calving once, don’t have reproductive diseases, normally birth. Estrous postpartum were used the data from anamnesis with the owner of cattle. Calf weight check were used by scales in kilogram after birth.

Results Table 1. Data on calf weight and estrous postpartum at PO cattle. No Average of Calf Weight (kg) Periode of Estrous postpartum (day) 1 <25 86,4 2 26-30 90,2 3 >31 85,5

These data showed that there were not significantly difference of calf weight and estrous postpartum on PO cattle (P>0.5). The period between birth and first estrus after calving in beef cattle varies between 45 - 104 days. Calf weight did not provide a negative correlation with estrous postpartum but estrus first after giving birth is strongly influenced by the presence of calves that suckle the mother (Christoffor and Baliarti, 2008). Calf weaning for too long will cause a delay in ovarian activity in the mother so that postpartus will be extended (Rusdi et al., 2016). Another factors were speculated have influenced on the length of EEP were mother condition, produce milk, age the management postpartum, nutrition, time suckling (Montiel et al., 2005; Rhodes et al., 2002; Laflammae et al., 1992) Conclusion There is no influence of calf weight on the period of EEP of Indonesia cattle. References Christoffor W. T. H. M., dan Baliarti, E. 2008. Kinerja Reproduksi Induk Sapi Silangan Simmental Peranakan Ongole dan Sapi Peranakan Ongole Periode Postpartum. Sains Peternakan Vol. 6: 45-53 Laflammae, L. F., Connor, M. L. 1992. Effect of Postpartum Nutrition and Cow Body Condition at Parturition on Subsequent Performance of Beef Cattle. Canadian Journal of Animal Science. Vol 72: 843-851 Montiel, F., Ahuja, C. 2005. Body Condition and Suckling as Factors Influencing the Duration of Pospartum Anestrus in Cattle: A Review. Animal Reproduction Science. Vol. 85: 1-26. Partodihardjo, S. 1987. Ilmu Reproduksi Hewan. Penerbit: Mutiara, Jakarta: Penerbit Angkasa

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Rhodes, F. M., McDougall. S., Burke, C. R., Verkerk, G. A., Macmillan, K. L. 2002. Invited Review: Treatment Of Cows With An Extended Postpartum Anestrous Interval. Journal Dairy Science. Vol 86 1876-1894 Rusdi, B, Hartono, M, dan Suharyati, S. 2016. Calving Interval pada Sapi Bali di Kabupaten Pringsewu. Jurnal Ilmiah Peternakan Terpadu Vol. 4(4):

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-9

EXTRACT OF ARTOCARPUS ALTILIS LEAVES IMPROVED KIDNEY FUNCTION OF STREPTOKINASE INDUCED ACUTE GLOMERULONEPHRITIS MICE BASED ON BUN AND CREATININE LEVEL

Safitri Ummahatul Mukminah1), Djoko Winarso1), Fajar Shodiq Permata1)* Faculty of Veterinary Medicine, Universitas Brawijaya, 2nd UB Puncak Dieng Eksklusif, Kalisongo, Dau, Malang, Indonesia 615151 Corresponding author: [email protected]

Abstract Acute Glomerulonephritis is a complex immune disorder that impair kidney function. Streptokinase could induce the animal model of the disease. Blood Urea Nitrogen (BUN) and Creatinine levels of the blood are an indicator of the kidney function. Leaves of Artocarpus altilis has flavonoid to neutralize the effect of streptokinase. The aim of this study was examining the impact of leaves extract of Artocarpus altilis to decrease the level of BUN and creatinine in as acute glomerulonephritis mice (Mus musculus) model that induced by streptokinase. Twenty mice were divided into five groups consist of negative control, positive control group, and three treatment groups used the extract with different doses, 16,8 mg/day, 25,2 mg/day, and 33,6 mg/day respectively. The treatment is orally for 14 days. Single-dose of streptokinase as 2500 IU was delivered intramuscular. Serum from blood was collected on day 15th. BUN and creatinine level was measured using Biosystem Autoanalyzer tool. The data were analyzed statistically using One Way ANOVA continued Tukey test with α = 0,05. The results were that the extract leaves of Artocarpus altilis decreased significantly (p<0.05) of the BUN level in all groups and became similar (p≥0.05) with negative groups. The creatinine level had become the same with negative groups in dose 16,8 mg/day. However, the creatinine trend was increasing, followed by elevation of treatments. The conclusion was the dose of 16.8 mg/day of the extract of Artocarpus altilis leaves improved kidney function in Streptokinase induced acute glomerulonephritis model of mice.

Keywords: acute glomerulonephritis, Artocarpus altilis, blood urea nitrogen, creatinine, streptokinase.

Introduction The acute glomerulonephritis case is many in the cats. The acute glomerulonephritis related to complex immune reactions1. Leaves of Artocarpus altilis has potency diuretic2 and contains flavonoid as an antioxidant3. Streptokinase could as induction agent to make animal models of glomerunolenephritis with a complex immune reaction4. The reaction induces the acute inflammation which release many free radical in kidney and decreases glomerular filtration rate (GFR). The disruption of GFR would impact the elevation of BUN and blood creatinine levels. BUN and

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 creatinine is a common indicator of kidney function. The extract of Artocarpus altilis leaves has diuretic and antioxidant capacities that would improve the kidney function for acute glomerulonephritis disorder based on BUN and blood creatinine levels. Objective The objective of the research was to examine the impact of the extract of Artocarpus altilis leaves to repair the kidney function in Streptokinase induced acute glomerulonephritis in mice model based on blood BUN and creatinine model. Methods Experimental design As many twenty mice (Balb/C, 30-40 gram, male) divide 5 groups which consist of negative control groups (no induction and treatment), positive control groups (streptokinase induced acute glomerulonephritis), and three treatment groups with different dose (16,8 mg/day, 25,2 mg/day, and 33,6 mg/day). The research was approved for Ethical clearance by the Research Ethics Committee of Universitas Brawijaya with certificate number 810-KEP-UB. Extraction process Simplicia of Artocarpus altilis leaves were extracted using maceration with ethanol 70% for 2 days (48 hours) then continued evaporation in 60-70oC for 5 hours in evaporation machine followed 80oC in a water bath for one hour. The phytochemistry qualitative test conducted to detect flavonoid in the extract and the result was positive. The extraction process and the flavonoid test were conducted in UPT Materia Medica, Batu. Preparation of the animal model Acute glomerulonephritis was modeled by Streptokinase induction. The dose of Streptokinase was 2500 IU per mouse delivered intramuscularly by a single dose5. Streptokinase was diluted by aqua pro injection to administrated in mice. Administration of extract The extract of Artocarpus altilis leaves was administreted with 3 different doses to 3 groups, respectively, there were 16,8 mg/day, 25,2 mg/day, and 33,6 mg/day, based on conversion diuretic dose (400, 600, 800 mg/kg BW) of Artocarpus altilis in the rat with modification2. The extract diluted by aquadest to give orally by gastic gavage per day until 14th days. Blood collection and serum isolation Mice blood was collected by intracardial and placed in plain vacutainer, and serum was isolated by centrifugation 3000 rpm 5 minutes. Measurement of BUN and blood Creatinine level BUN and blood Creatinine levels were measured from serum using Autoanalyzer Biosystem in the Laboratory of Clinical Pathology, Medical Faculty, UB. Bun levels were measured using a urease-salicylate method6. The principle of the methods is urea of serum would react with urease and salicylate; then, the reaction is measured by spectrophotometer. The creatinine levels were calculated by the Jaffe method as calorimetry which there are a reaction creatinine and picric acid in base condition to form creatinine-picric complex as magenta color7. Data analysis BUN and blood creatinine levels were analyzed statistically. One Way ANOVA followed the Tukey test with a significance level of 95% comparing among groups.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Result Results of the research were the significant reduction (p<0.05) of BUN level after treatment with the extract in all doses (Table 1), but the blood creatinine level decreased significantly in dose 16,8 mg/ day and 25,2 mg/day but third group showed a significant increase of creatinine (Table 2). Table 1. BUN levels among groups Groups Means of BUN level (mg/dL) ± SD Negative control groups 19,77 ± 1,00a Positive control groups 29,27 ± 0,54b Dose 1 (16,8 mg/day) 21,05 ± 3,52a Dose 2 (25,2 mg/day) 21,27 ± 1,70a Dose 3 (33,6 mg/day) 23,07 ± 2,30a Note: The different uppercase notation indicated the significantly different (p<0.05)

Table 2. Blood creatinine levels among groups Groups Means of blood creatinine level (mg/dL) ± SD Negative control groups 0,33 ± 0,05a Positive control groups 0,75 ± 0,06c Dose 1 (16,8 mg/day) 0,48 ± 0,10ab Dose 2 (25,2 mg/day) 0,50± 0,14ab Dose 3 (33,6 mg/day) 0,65±0,13bc Note: The different uppercase notation indicated the significantly different (p<0.05)

The kidney function showed repair better after the fourteen days administration of the extract of Artocarpus altilis leaves based on BUN and blood creatinine levels. The reduction of BUN and blood creatinine levels showed that GFR was improvement. This effect proved that Artocarpus altilis has the potency to treat acute glomerulonephritis. The healing ability of Artocarpus altilis is because of the flavonoid compound of leaves3. The flavonoid as an antioxidant could neutralize to abundant of free radical due to inflammation in the kidney. Artocarpus altilis has potency diuretic2 to increase GFR so that it repairs the glomerulus8. The third dose showed the elevation of blood creatinine. It caused by induction of Streptokinase which administrated intramusculary. Streptokinase is an enzyme from Streptococcus bacteria which has mechanism to change plasminogen to plasmin then form complex immune9 to damage the muscle. The necrosis of muscle release creatinine. This phenomenon was the side effect of administration streptokinase via intramuscular. Conclusion The conclusion of the research was the extract of Artocarpus altilis leaves has therapy potency to improved the kidney function in the streptokinase induced acute glomerulonephritis in mice model based on BUN and blood creatinine level. The best dose from the result was 16,8 mg/day. References 1. Rossi F, Aresu L, Martini V, et al. Immune-complex glomerulonephritis in cats: a retrospective study based on clinico-pathological data, histopathology and

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 ultrastructural features. BMC Vet Res. 2019;15(1):303. doi:10.1186/s12917-019- 2046-y 2. Windarsih. KEMAMPUAN DIURETIK EKSTRAK ETANOL BUAH SUKUN.pdf. 2017. http://eprints.ums.ac.id/51393/13/NASKAH%20PUBLIKASI%20perpus%20pusa t.pdf. Accessed February 1, 2020. 3. Kusuma AT, Adelah A, Abidin Z, Najib A. Determination of Flavonoid Content of Ethyl Acetate Extract of Breadfruit Leaves (Artocarpus altilis). 1(1):7. 4. Nordstrand A, Norgren M, Ferretti JJ, Holm SE. Streptokinase as a Mediator of Acute Post-Streptococcal Glomerulonephritis in an Experimental Mouse Model. Infection and Immunity. 1998;66(1):315-321. doi:10.1128/IAI.66.1.315-321.1998 5. Ahmad HM. Pengaruh Ekstrak Etanol Daun Kemangi (Ocimum Gratissimum L.) Pada Mencit Model Glomerulonefritis Akut (Gna) Hasil Induksi Streptokinase Terhadap Kadar Ifnγ-Gr1 Limpa Dan Gambaran Histopatologi Hepar. August 2018. http://repository.ub.ac.id/13895/. Accessed February 1, 2020. 6. Taylor AJ, Vadgama P. Analytical Reviews in Clinical Biochemistry: The Estimation of Urea. Annals of Clinical Biochemistry: An international journal of biochemistry and laboratory medicine. 1992;29(3):245-264. doi:10.1177/000456329202900301 7. Küme T, Sağlam B, Ergon C, Sisman AR. Evaluation and comparison of Abbott Jaffe and enzymatic creatinine methods: Could the old method meet the new requirements? J Clin Lab Anal. 2018;32(1):e22168. doi:10.1002/jcla.22168 8. A S SS, Ravindran A, Rai M, Kateel R, A S S. EVALUATION OF DIURETIC ACTIVITY OF ETHANOLIC EXTRACT OF ARTOCARPUS HETEROPHYLLUS SEEDS IN ALBINO WISTAR RATS. Int J Res Ayurveda Pharm. 2016;7(2):202-206. doi:10.7897/2277-4343.07287 9. Huish S, Thelwell C, Longstaff C. Activity Regulation by Fibrinogen and Fibrin of Streptokinase from Streptococcus Pyogenes. Brissette CA, ed. PLoS ONE. 2017;12(1):e0170936. doi:10.1371/journal.pone.0170936 Acknowledgment The authors thank Mrs. Safitri Ummahatul Mukminah for the dedication conducted research. The authors thank to UPT Materia Medica Batu for extraction process and Laboratory of Clinical Pathology, Medical Faculty for BUN and creatinine measurement

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-10

PREVALENCE AND ASSOCIATED RISK FACTOR OF FELINE LOWER URINARY TRACT DISEASE IN CATS ATTENDING PRIMARY CARE VETERINARY PRACTICES IN YOGYAKARTA

A. Nururrozi1*, S. Indarjulianto1, Yanuartono1, S. Hartati1, H. Wuryastuty1, Yuriadi1, I. Widiyono1, I. Tjahajati1, H. Purnamaningsih1, G.T. Mulyati1, and S. Raharjo1 1Departement Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Jl.Fauna No 2, Karangmalang, Yogyakarta Tel : +62-274-560862, Fax +62-274-560861 Corresponding author : [email protected]

Abstract Feline lower urinary tract disease (FLUTD) is one of the most common disease in Yogyakarta’s cats population. This article reports a cohort study performing on cats diagnosed with FLUTD. The medical data recorded in Veterinary Clinic, Department Internal Medicine, Universitas Gadjah Mada from 2017 to 2019 were obtained in order to conduct this study. The clinical signs were evaluated and accompanied with the results of laboratory test in a population of cats showing symptoms hematuria, stranguria, pollakiuria, dysuria, and periuria. Medical history of all the patients were analyzed descriptively. Cats with a documented physical examination, urinalysis, urine culture, and ultrasound examination were included. The incidence of FLUTD was obtained from 285 patients during last three years from the Veterinary Clinic of Universitas Gadjah Mada. The most frequent FLUTD diagnosed di Yogyakarta including FIC, UTI, urolithiasis, urethral plug, and neoplasia, were 63.9%, 21.1%, 9.5%, 4.9%, 0.7% respectively. This study showed that FLUTD cases is related with sex and aged. The cats with FIC and UP were amongst the youngest animals in the analyzed group, and the risk of urinary tract infection and neoplasia increased with age, as the highest age-average was noted in neoplasia. The insidence FLUTD is much more higher in male cats (82.1%) than in female (17.9%).

Keywords: FLUTD, prevalence, sex, age, Yogyakarta

Introduction Feline lower urinary tract disease (FLUTD) is a common reason for cat owners to seek veterinary care (Dorsch et al., 2014). Etiologies of FLUTD include feline idiopathic cystitis (FIC), urethral obstructions, urinary tract neoplasia, and urinary tract infection (Hostutler et al., 2005; Gerber et al., 2005). The data obtained for this study corresponds complaints (signalment) from the owner commonly observed signs of hematuria, urinary straining (stranguria), inappropriate urination (periuria), and dysuria (Gerber et al., 2005; Forrester and Towell, 2015). FLUTD is a wide term that involves many disorders, especially, idiopathic cystitis, it is proved that between 55% and 63% of the cats with FLUTD are concluded to suffer from FIC (Dorsch et al., 2014; Forrester

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 and Towell, 2015). The other common causes of FLUTD are urinary calculi, urethral plugs and urinary tract infections. Neoplasia are categorized to be the less common causes of FLUTD (Gerber et al., 2005). FLUTD occurs on both the sexes, meanwhile it is rare in females and common in male cats, due to the anatomy of penile urethra (Lekchroensuk et al. 2001; Hostutler et al. 2005). FLUTD were diagnosed accurately based on medical history as well as results of imaging and laboratory tests (Gunn-Moore, 2003). Castration and spaying is believed considered to increase of the risk FLUTD because of the association with inhibition of urethral growth, induction of weight gain and a sedentary lifestyle. Previous studies indicate that young cats under ten years are more risky of developing FIC and urolithiasis, whereas geriatric cats are more likely to develop neoplasia and UTIs (Lekchroensuk et al. 2001; Gunn-Moore, 2003; Hostutler et al. 2005). Common breeds involved in FLUTD cases are such as Persian, Himalayan and Russian Blue (Dorsch et al., 2014). These breeds have been considered predisposed to urolith formation as the cause of obstructive disease (Lekchroensuk et al. 2001). Objective The epidemiology data in FLUTD cases have not been reported in Yogyakarta. The purpose of this cohort study was to evaluate the insidence, causes, and risk factor associated in lower urinary tract disease found in Yogyakarta. Methods A total number of 285 cats were selected from the Veterinary Clinic, Department Internal Medicine, Universitas Gadjah Mada, Indonesia, during a period of three years (2017-2019). Cats with clinical signs of hematuria, stranguria, pollakiuria, periuria and dysuria was used in this research. Only cats with a documented physical examination, a complete urinalysis (urine specific gravity, urine dipstick, and sediment) and urine culture performed at initial presentation as well as ultrasound and/or radiographs on presentation were included (Lamberger et al., 2011). Urinalysis of the patients were required to determine the urinary abnormalities, urine biochemistry, sediment evaluation, and type of calculi examination (Lamberger et al., 2011). Ultrasonography was performed on the patients to describe the presence or absence of calculi in the urinary tract and the sign of inflammation. Blood examination was conducted to determine the status of urea in the blood and also the kidney function of the patients. The complete physicochemical analyses of urine samples include determination of glucose, bilirubin, ketone bodies and hemoglobin content, pH, specific gravity of urine, and the protein test conducted (Lamberger et al., 2011). Samples collected from patients diagnosed with multiple changes observed in bladder and urinary tract epithelial cells based on ultrasound scans were considered as suspect samples. The urine from suspect samples were tested cytologically and microbiological culturing. Urine samples were centrifuged and the sediment was evaluated for the presence of erythrocytes and white blood cells. Crystal formations were observed and evaluated. Microbiological tests were culture suspect samples obtained by catheterization, on Mannitol Salt Agar, MacConkey Agar and 5% sheep blood agar, then incubated at 37oC for 24 hours (Lister et al., 2009; Dokuzeylul et al., 2015).

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 The studied patients were categorized into feline idiopathic cystitis, urolithiasis, urethral plug, urinary tract infection, and neoplasia according to their respective diagnoses. UTI is diagnosed by significant bacterial growth from urine samples (Dorsch et al., 2014; Dokuzeylul et al., 2015). Urethral plug is diagnosed by detection of plugs in urethral obstruction. Urolith is diagnoses using ultrasonography or x-ray (Dorsch et al., 2014). Neoplasia is diagnoses by ultrasonography to identify mass lesion. FIC is a diagnosis of exclusion, must be performed to rule out other specific causes (Forrester and Towell, 2015). Results The prevalence of FLUTD cases was recorded from 285 patients in the year of 2017-2019 from the Veterinary Clinic of Universitas Gadjah Mada, Indonesia. Precise medical records including clinical examinations, ultrasound test, blood and urine tests, were obtained from the clinic. The signalment from the owners provided information on the animal’s behavior during urination. The examined population consisted of 234 males (82.1%) and 51 females (17.9%). A total of 97.2% patients with urinary tract infection and 100% of cats with neoplasms were male. The prevalence of other diseases was higher in males, and all patients with neoplasia were males. The average age of cats with FLUTD was 2.4 years. Urethral plugs was observed in the youngest cats (0.8- 1 years), and neoplasia was noted in the older cats. The studied population shows that cats with urinary tract infection and neoplasia were significantly older than cats with feline idiopathic cystitis, urethral plug, and urolithiasis. The age and sex proportions of the five groups diagnosed with FLUTD in the studied population of 285 cats are, presented in Table 1.

Table 1. The age and sex proportions of the five groups diagnosed with feline lower urinary tract disease in the studied population of 285 cats All cats FIC UTI UP Urolithiasis Neoplasia Total 285 (100) 182 (63.9) 60 (21.1) 14 (4.9) 27 (9.5) 2 (0.7) (0.2-7) (0.6-7) (0.3-5.5) (0.8-1) (0.2-6) (6.5-9) Age (year) 2.4 2 3.1 0.9 1.8 7.8 Males (%) 82.1 77.5 97.2 92.8 67.6 100 Females (%) 17.9 22.5 2.8 7.2 32.4 0.0 FIC = feline idiopathic cystitis, UTI = urinary tract infections, UP = urethral plugs This study describes of feline idiopathic cystitis, urinary tract disease, urolithiasis, urethral plug, and neoplasia found in a population of 285 Indonesian cats. This study reports that feline idiopathic cystitis is the most common underlying cause of FLUTD similar to that in Polish cats (Lew-Kojrys et al., 2017) and German cats (Dorsch et al., 2014). Prevalence observed in different countries by other researches showed feline idiopathic cystitis also having a high percentage (Lekcharoensuk et al., 2001; Gerber et al., 2005; Kruger et al., 2009). The prevalence of feline idiopathic cystitis and urinary tract infection in Indonesian cats is more significant than in Polish cats. The second most common cause of FLUTD reported in this study was urinary tract infection with a rate of 25.3%. Lekcharoensuk et al., (2001) reports major predisposing factors of feline UTIs are old age, perineal urethrostomy, low urine specific gravity, and previous catheterization history. The bacteria often found in feline UTI tests are Eschericihia coli, Enteroscoccus spp and Staphylococcus felis (Lister et al., 2009).

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Based on different researcher found that bacterial UTI in cats are generally seen in female cats. In our study however, male cats appeared to be diagnosed of UTI more than females. Bacterial UTI can occur in healthy male cats with indwelling urinary catheters whereby the risk of infection increases with duration of catheterization (Dokuzeylul et al., 2015). Urolithiasis was observed in 13% of cats with FLUTD. Based on the results of urine sediment analysis, stranguria and hematuria was noted in 97% and 57% of cases. The formation of crystal struvite and oxalates were observed in majority of urolithiasis cases. In this study, hematuria in cats with FLUTD was found to be 40% under microscopic analyses. Based on other researcher conducted by Kruger et al., (2009), 97% prevalence had hematuria found in the urine samples and later in the year 2011 by Saevik et al., 83% prevalence of hematuria. Previous studies reported that oxalate crystals and stones are more often found in Himalayan, Persian, Burmese cats, and Russian blue. Genetic factors may also contribute to increased risk of calcium oxalate urolith formation. In this study, a total of 128 patients were Persian cats with 60% identified urolithiasis (Lekcharoensuk et al., 2001; Lew-Kojrys et al., 2017). The second highest prevalence was seen in Domestic breeds with 58% in feline urinary tract infection. Most of the cats in our sample were indoor and outdoor cats who had higher risks of the infection prior to drinking water from flower bowls and drains with bacteria infestations (Dokuzeylul et al., 2015; Lew-Kojrys et al., 2017). Clinical symptoms showed that majority of the patients in a number of 129 cats (45.3%) suffered from stranguria, 34 cats (11.9%) suffered from pollakiuria, 17 cats (6.0%) suffered from dysuria and 9 cats (3.2%) suffered from periuria. Macroscopic hematuria was observed in 40.4% of the patients. The clinical features of the five groups diagnosed with FLUTD in the studied population of 285 cats are presented in Table 2.

Table 2. Clinical symptoms of all cats with feline lower urinary tract disease and cats diagnosed with five different conditions (n (%) of cats) All cats FIC UTI UP Urolithiasis Neoplasia Total 285 (100) 182 (63.9) 60 (21.1) 14 (4.9) 27 (9.5) 2 (0.7) Haematuria 115 (40.4) 66 (41.3) 24 (33.3) 2 (14.3) 21 (56.8) 2 (100) Stranguria 129 (45.3) 30 (18.8) 62 (86.1) 9 (64.3) 26 (97.3) 2 (100) Pollakiuria 34 (11.9) 18 (11.3) 1 (1.4) 0 (0.0) 15 (40.5) 0 (0.0) Dysuria 17 (6.0) 4 (2.5) 1 (1.4) 12 (85.7) 0 (0.0) 0 (0.0) Periuria 9 (3.2) 5 (3.1) 2 (2.8) 0 (0.0) 1 (2.7) 1 (50.0) FIC = feline idiopathic cystitis, UTI = urinary tract infections, UP = urethral plugs Haematuria was observed highest in feline idiopathic cystitis with 41% compared to other clinical signs. Stranguria was noted to be the highest occurrence in all patients, 86.1% patients were diagnosed urinary tract infections suffering from stranguria. In the studied population, 40.5% patients with urolithiasis showed clinical signs of pollakiuria and 2.7% showed periuria. The identified patients of urolithiasis were 13% patients, had oxalate stones and struvite stones observed under ultrasound scans. Patients

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 diagnosed with urinary tract infections and urethral plugs reached 25.3% and 4.9% respectively. The studied population comprised of 128 (44.9%) Persian, 101 (35.4%) domesticated, 22 (7.7%) mix bred, 10 (3.5%) Himalayan, 7 (2.5%) Angora, 4 (1.4%) British short hair, 8 (2.8%) Maine coons, and 5 (1.8%) unknown breeds of cats.

Table 3. The breed proportions of the five groups diagnosed with feline lower urinary tract disease in the studied population of 285 cats All cats FIC UTI UP Urolithiasis Neoplasia (n = 285) (n = 182) (n = 60) (n = 14) (n = 27) (n = 2) Persia 128 86 (53.8) 12 (16.7) 6 (42.9) 22 (59.5) 2 (100) (44.9) Domestic 101 44 (27.5) 42 (58.3) 3 (21.4) 12 (32.4) 0 (0.0) (35.4) Mix breed 22 (7.7) 10 (6.3) 10 (13.9) 2 (14.3) 2 (5.4) 0 (0.0) Himalayan 10 (3.5) 5 (3.1) 3 (4.2) 0 (0.0) 2 (5.4) 0 (0.0) Angora 7 (2.5) 4 (2.5) 2 (2.8) 1 (7.1) 0 (0.0) 0 (0.0) British 4 (1.4) 2 (1.3) 1 (1.4) 1 (7.1) 0 (0.0) 0 (0.0) short hair Maine coon 8 (2.8) 5 (3.1) 1 (1.4) 1 (7.1) 1 (2.7) 0 (0.0) Unknown 5 (1.8) 4 (2.5) 1 (1.4) 0 (0.0) 0 (0.0) 0 (0.0) breed FIC = feline idiopathic cystitis, UTI = urinary tract infections, UP = urethral plugs In the studied population, 128 (44.9%) were Persian breeds. The studied population showed the high prevalence of Persian cats in feline idiopathic cystitis and urolithiasis. Domestic cats had a prevalence of 101 patients in total, with a number of 58.3% being the highest record in urinary tract infection. The prevalence of 50% of Himalayan breed cats were diagnosed with feline idiopathic cystitis. Conclusions FIC is the most common diagnostic in cats with FLUTD in Yogyakarta. The prevalence of UTI is higher than that reported in Europe or the USA. The incidence rate of FLUTD cases in Yogyakarta is related to age and sex. Older cats more often show symptoms of UTI and neoplasia, while young cats more often show symptoms of FIC and UP. The prevalence of male cats experiencing FLUTD in Yogyakarta is much higher than female cats. The result of this study are similar with previous research in other country. Acknowledgment We express our gratitude to Universitas Gadjah Mada by supporting this research by Research Grant "Research Capacity Enhancement for Young Lecturers of UGM" according to the Job Implementation Assignment Letter Number 3905 / UN1 / DITLIT / DIT-LIT / LT / 2019 dated May 8, 2019 from Directorate of Research, Universitas Gadjah Mada References Dokuzeylül, B., Kahraman, B., Bayrakal, A., Siğirci, B., Çelik, B., Ikiz, S., Kayar, A. and OR, M. (2015). Bacterial species isolated from cats with lower urinary tract infection and their susceptibilities to cefovecin. Irish Veterinary Journal, 68(1), p.2

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Dorsch R, Remer C, Sauter-Louis C, Hartmann K (2014): Feline lower urinary tract disease in a German cat population. A retrospective analysis of demographic data, causes and clinical signs. Tieraerztliche Praxis Ausgabe K: Kleintiere/Heimtiere 42, 231-239. Forrester SD, Towell TL (2015): Feline idiopathic cystitis, Veterinary Clinics of North America: Small Animal Practice 45, 783-806. Gerber, B., Boretti, F., Kley, S., Laluha, P., Muller, C., Sieber, N., Unterer, S., Wenger, M., Fluckiger, M., Glaus, T. and Reusch, C. (2005). Evaluation of clinical signs and causes of lower urinary tract disease in European cats. Journal of Small Animal Practice, 46(12), pp.571-577 Gunn-Moore D (2003): Feline lower urinary tract disease. Journal of Feline Medicine and Surgery 5, 133-138. Hostutler R, Chew DJ and DiBartola SP (2005). Recent concepts in feline lower urinary tract disease. Veterinary Clinics of North America Small Animal Practice, 35(1): 147-170 Kruger JM, Osborne CA, Lulich JP (2009): Changing paradigms of feline idiopathic cystitis. Veterinary Clinics of North America: Small Animal Practice 39, 15-40. Lekcharoensuk C, Osborne CA, Lulich JP (2001): Epidemiologic study of risk factors for lower urinary tract diseases in cats. Journal of the American Veterinary Medical Association 218, 1429-1435. Lemberger SI, Deeg CA, Hauck SM, Amann B, Hirmer S, Hartmann K, Dorsch R (2011): Comparison of urine protein profiles in cats without urinary tract disease and cats with idiopathic cystitis, bacterial urinary tract infection, or urolithiasis. American Journal of Veterinary Research 72, 1407-1415. Lew-Kojrys S, Mikulska-Skupien E, Snarksa A, Krystkiewicz W and Pomianowski A (2017): Evaluation of clinical signs and causes of lower urinary tract disease in Polish cats. Journal of Veterinarni Medicina, 62, 2017 (07):386-393. Lister A (2009). Occult bacterial lower urinary tract infections in cats—Urinalysis and culture findings. Vet Microbiol, 136:130. Saevik BK, Trangerud C, Ottesen N, Sorum H, Eggertsdottin AV (2011): Causes of lower urinary tract disease in Norwegian cats. Journal of Feline Medicine and Surgery 13, 410-417.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-11

PHARMACOKINETIC OF TYLOSIN AND ENROFLOXACIN COMBINATION FOLLOWING ORAL ADMINISTRATION IN BROILER

Ida Fitriana1*, Agustina Dwi Wijayanti1, Dwi Cahyo Budi Setiawan1, Anggi Muhtar Pratama1, and Sugiyono2 1Departement of Pharmacology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Jl. Fauna No.2 Karangmalang, Sleman, Yogyakarta, Indonesia, 2PT. Tekad Mandiri Citra, Jl. Kawaluyaan Raya, Jatisari, Buahbatu, Bandung, Jawa Barat *Corresponding’s E-mail: [email protected]

Abstract Tylosin and enrofloxacin in the broiler body will experience a pharmacokinetic phase including absorption, distribution, metabolism, and excretion processes. This study aims to describe the pharmacokinetics of tylosin and enrofloxacin in broiler chicken plasma after administration of pharmaceutical veterinary formulation, orally, with high performance liquid chromatography (HPLC). Forty-five broilers were kept in postal cages of experimental animals starting from the DOC, given antibiotic-free feed and drinking water ad libitum. After the 21th days, forty (40) broilers were given a pharmaceutical veterinary formulation of tylosin and enrofloxacin with a concentration of 1g/2L of drinking water as much as 1 mL, orally. Five broilers were not given a pharmaceutical veterinary formulation of tylosin and enrofloxacin and were used as controls (considered to be 0th minute), only given drinking water. Blood sampling was also carried out on forty broilers treated with tylosin and enrofloxacin which were grouped according to the sampling time, which was at 0.5; 1; 2; 4; 8; 16; 24; 48; and 72 hours after drug administration. Analysis of the concentration of tylosin and enrofloxacin in plasma samples was carried out using HPLC. Data analysis was to obtain the values of the pharmacokinetic parameters were the maximum concentration (Cmax), maximum time (Tmax), Volume of distributions (Vd), Clearence (Cl), elimination half-time (T½e), and Area Under Curve (AUC) with non-compartment models. These pharmacokinetic parameters are Cmax of tylosin and enrofloxacin in plasma by 1.32 and 17.15g/mL, Tmax with a of 24 and 16 hours, AUC of tylosin and enrofloxacin in plasma was 4,296.40 and 67,124.16µg/mL, respectively. The longest T½e of enrofloxacin in plasma was 277.18 hours. Clearance of tylosin and enrofloxacin were 0.09 and 0.006 mL/min/kg with a Vd of 104.95 and 143.01L/kg.

Keywords: Pharmacokinetics, enrofloxacin, tylosin, plasma broiler, HPLC

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO1-12

LEAD AND CADMIUM CONTAMINATION IN BLOOD CATTLE WHICH IS SLAUGHTERED AT THE TRADITIONAL SLAUGHTERHOUSE

I K. Berata*, I W. Sudira, I M. Kardena, I.B.O. Winaya Faculty of Veterianry Medicine Udayana University Corresponding author : [email protected]

Abstract The lead (Pb) and cadmium (Cd) are widely studied because if exposed they will greatly threaten the health of animals and humans. Air, land and water environmental pollution is suspected as a source of animal and human contamination. Cattle raised in urban landfills are found to be exposed to lead in the blood and internal organs, but a small portion are reported to be exposed to heavy metals cadmium. Among cattle that are kept in landfills, it is suspected that many are slaughtered at traditional slaughterhouses, so it is interesting to study about the presence of lead and cadmium in cattle slaughtered at traditional slaughterhouses. A total of 100 cattle were taken aseptically before being cut. Blood samples were examined for the content of lead and cadmium in the analytical laboratory of Udayana University. Examination of heavy metal content was carried out using the atomic absorption spectrophotometry (AAS) method. The results obtained were 4 samples (4%) positive containing lead and 3 samples (3%) containing cadmium. In contrast to the results of research on cattle raised at a landfill in Suwung Denpasar, 72% positive data were obtained from exposure to lead in their blood, but none were exposed to cadmium. The conclusion is still heavy metal contamination of lead and cadmium as much as 4% and 3% in cattle slaughtered at traditional slaughterhouses, respectively.

Keywords: lead, cadmium, traditional slaughterhouse

Introduction Lead (Pb) and cadmium heavy metals are very dangerous for animal and human health. The effects of Pb poisoning on animals and humans are hepatotoxic, nephrotoxic, genotoxicity and neurotoxicity (Sharma, et al, 2014). Lead poisoning in children is reported to cause growth disorders, hyperactivity and decreased cognitive power. Cognitive disorders reported by the young endothelial barrier in the brain are very easily penetrated by the lead element to replace Ca so that communication between the astrocytes and the endothelium is broken (Brochin et al. 2008). Cadmium toxicity, among others, damages the organ systems of the body, such as the urinary system, the respiratory system (lungs), the circulatory system (blood and heart), damage to the reproductive system, nervous system, and can even result in bone fragility (Jaishankar et al, 2014). Cattle as a source of animal protein, many are kept in places that are not suitable, including in landfills. From the results of the study found the presence of lead heavy metal contamination in the blood and tissue of cattle that are kept in rubbish dumps in the city of Denpasar (Berata et al 2016; Berata et al., 2017). It is suspected that cattle that are kept in landfills and other improper places tend to be slaughtered in traditional slaughterhouses.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Objective The objective of this study was to determine the presence of contamination of lead and cadmium in the blood of cattle slaughtered at traditional slaughterhouses Methods Measurement of the presence of heavy metals was carried out by the atomic absorption spectrophotometry (AAS) method (Sikiric et al 2003) at the Analytical Laboratory of Udayana University Results From 100 blood samples was found 4 samples (4%) were positive containing lead and 3 samples (3%) positively contained cadmium. Of the 4 positive samples containing lead, 2 of them were also positive containing cadmium. Examination results are presented in Table 1. From these results it shows that there are still cattle originating from improper maintenance systems, even worse than the results of inspections on cattle that are kept in a Denpasar municipal landfill, because no cadmium contamination has been found ( Berata et al., 2016).

Table 1. Levels of the lead and cadmium in blood samples Content of Pb (ppm) 6,96 0 4,61 9,85 3,56 Content of Cd (ppm) 4,09 17, 64 0 0 1,42

Conclusion Cattle slaughtered at traditional slaughterhouses were found to have 4% and 3% heavy metal lead and cadmium contamination. Beef consumers can get health if they continually consume tainted beef, so it is important to supervise traditional slaughterhouses.

References Berata, IK., Ni Nyoman Werdi Susari, NNW., Kardena, IM., Ariana, INT. 2016. Cemaran Timah Hitam dalam Darah Sapi Bali yang Dipelihara di Tempat Pembuangan Akhir Kota Denpasar J.Vet 17 (4): 641-646 Berata, IK, Susari, NNW., Kardena, IM., Winaya, IBO., Manuaba, IBP. 2017. Comparison of lead contamination in innards and muscle tissues of Bali cattle reared in Suwung Landfll. Bali Med.J. 6(1): 147-149 Brochin1, R., Leone, S2, Phillips1, D., , Shepard1 N, Zisa1, D & Angerio, A, 2008. The Cellular Effect of Lead Poisoning and Its Clinical Picture. The Georgetown Undergraduate Journal of Health Sciences 5(2) 1-8 Jaishankar M, Tseten T, Anbalagan N, Mathew BB, Beeregowda KN. 2014. Toxicity, mechanism and health effects of some heavy metals. Interdiscip Toxicol. 7(2):60- 72. Sharma, B., Singh, S. and Siddiqi, NJ. 2014. Review Article: Biomedical Implications of Heavy Metals Induced Imbalances in Redox Systems . Hindawi Publishing Corporation.BioMed. Research International. Vol 2014, Article ID 640754, 26 pages Sikiric, M. Brajenovic N. Pavlovic I. Havranek JL. Plavljanic, N. 2003. Determination of metals in cow's milk by flame atomic absorption spectrophotometry. Czech J.Anim. Sci. 48(11): 481–486. World Health Organization (WHO). 2010. Action Is Needed on Chemicals of Major Public Health Concern. Public Health Environmental. Terdapat pada: https://www.who.int/ipcs/ assessment/public_health/chemicals_phc/en/. Diakses pada 1 Oktober 2019

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Acknowledgment Authors thank to the rector of Udayana University through the head of the research institute and community service of Udayana University, for funding this research.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO2-1

EPIDEMIOLOGICAL SURVEY AND ELUCIDATION OF THE PATHOGENIC MECHANISM OF ZOONOTIC VIRUSES

Hayasaka D*, Shimoda H and Takano A Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University

Abstract Our study themes are epidemiological studies, development of diagnosis, and prevention and treatment methods for viral infectious diseases including arthropod- borne and zoonotic viruses among various animals. Since companion animals share their life space with human, there are huge effects against human and from human as zoonotic diseases. Also, since they have some chance to contact with wild animals, there are infectious diseases that transmit between wild animals and companion animals. Our research is based on the investigation of host-parasite relationship between natural host and pathogens to prevent us from the emerging and re-emerging infectious diseases. Our target is focused on arthropod-borne and wild mammalian- derived viruses. We also have been working on exploration of novel viruses from various species of animals, ticks and mosquitoes. In addition, we are ongoing to elucidate the pathogenic mechanism of virus infections such as encephalitis and hemorrhagic viruses using a mouse model.

Keywords: viruses, zoonosis, tick-borne diseases, mouse model

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO2-2

ADHESION OF TOXOPLASMA GONDII TACHYZOITE-INFECTED VEHCLE LEUKOCYTES TO CAPILLARY ENDOTHERIAL CELLS TRIGGERES TIMELY PARASITE EGRESSION

M Baba, T Batanova, K Kitoh, Y Takashima* Dep. Vet. Parasitol., Gifu University, Japan *Corresponding author: [email protected]

Abstract Intracellular pathogens have numerous strategies for effective dissemination within the host. Many intracellular pathogens first infect leukocytes, which they use as a vehicle to transport them to target organs. Once at the target organ, intracellular parasite Toxoplasma gondii can cross the capillary wall in extracellular form by infecting endothelial cells. However, after egression from leukocytes, extracellular parasites face the risk of host immune attack. In this study, observation of infected mouse organs, using a method that renders tissue transparent, revealed that adhesion of tachyzoite-infected leukocytes to endothelial cells triggers immediate egression of the parasite.

Keywords: Toxoplasma gondii, adhesion, endothelial cell

Introduction T. gondii is an obligate intracellular parasite responsible for congenital infections, abortion and opportunistic diseases in immunodeficient individuals. It has been reported that T. gondii tachyzoites infect leukocytes in the lamina propria extravascular space[1, 2] and that T. gondii-infected leukocytes in the general circulation transport the tachyzoites to the peripheral organs [3-6]. However, it is still unknown how T. gondii transit from the infected leukocytes in the general circulation to solid organs. Objective To reveal the mechanism, of transition of T. gondii from leukocytes in the blood stream to the solid organs. Methods In vivo Leukocytes of a GFP-expressing transgenic mouse were infected with transgenic T. gondii tachyzoites expressing a red fluorescent protein, DsRed Express. The infected leukocytes were injected into the tail vain of wild-type mice. After a few hours, organs of the recipient mice were harvested, made transparent, and observed using confocal laser scanning microscopy (Fig).

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020

In vitro Tachyzoite-infected leukocytes from the GFP mouse were added to a monolayer of lung endothelial cells and incubated for 30 min at 37 °C in a 5% CO2 incubator. As controls tachyzoite-infected leukocytes from the GFP mouse were also cultured alone or with an endothelial cell monolayer separated by 0.4-µm mesh. And then, egression of tachyzoites from the leukocytes was monitored. Results 1. Tachyzoite-infected leukocytes remain in the lungs We injected tachyzoite-infected leukocytes (infection rate: 19.7–40.4%) into the tail vein of mice and visualized the flow of these leukocytes into the solid organs, lung and liver. Thirty minutes after injection, the infected leukocytes had reached the lung and liver. At this moment, the infection rate of leukocytes that remained in the lung was statistically higher than that of the injected leukocyte suspension. Thirty minutes after injection, over half of the infected leukocytes in the lung appeared elongated in shape. The frequency of detection of elongated cells among the total number of infected cells increased with time. By contrast, most of the uninfected leukocytes maintained a round shape throughout the observation period. It indicated that tachyzoite-infected leukocytes adhere to solid tissue and remain in the lung more effectively than non- infected leukocytes. 2. Egression of T. gondii tachyzoites from leukocytes In the present study, to elucidate the timing of tachyzoite egression from leukocytes in the lung, tachyzoite-infected leukocytes were injected into the tail vein and changes in the number of intraleukocytic and extraleukocytic tachyzoites over a time course were evaluated in the lung. Extraleukocytic tachyzoites were removed from the the leukocyte suspension at the time of injection. However, a few extracellular tachyzoites were observed in the lung 30 min after injection and the extracellular tachyzoite number increased during the 4-h observation period. These results suggested that tachyzoites within leukocytes that remained in the lung rapidly egressed before proliferation. We hypothesized that the attachment of tachyzoite-infected leukocytes to endothelial cells triggers egression of tachyzoites. Therefore, we isolated mouse lung endothelial cells and co-cultured them with tachyzoite-infected leukocytes. As observed in the lung, tachyzoite-infected leukocytes in the co-culture system also attached to lung endothelial cells more effectively than non-infected leukocytes. However, when tachyzoite- infected leukocytes were cultured alone or separated from endothelial cells by a 0.4- µm mesh, the appearance of extraleukocytic tachyzoites was reduced. These results indicated that tachyzoite egression from leukocytes was required for direct attachment

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 of infected leukocytes to lung endothelial cells. Furthermore, secreted soluble factors from lung endothelial cells did not contribute to parasite egression. 3. Contribution of CD162, expressed by endothelial cells, to tachyzoite egression We confirmed that there was no statistically significant difference in the total number of attaching leukocytes between freshly isolated and passaged endothelial cells. However, egression of tachyzoites from the infected leukocytes was observed only when the vehicle leukocytes attached to the freshly isolated cells. These results indicated that the endothelial cells lost the ability to selectively attach to tachyzoite- infected vehicle leukocytes and trigger tachyzoite egress, within only two to three passages. Therefore, we compared the expression level of 88 genes coding for cell adhesion-related proteins between the freshly isolated and three times passaged endothelial cells and found that expression level of the gene encoding selectin-P ligand (Selplg), also known as CD162 significantly decreased throughout the three passages of the endothelial cells. To examine whether CD162 molecules on the surface of endothelial cells induce tachyzoite egression from leukocytes, the freshly isolated lung endothelial cells were treated with anti-CD162 antibody. The antibody treatment of the freshly isolated endothelial cells drastically decreased the number of egressed tachyzoites from the co-cultured tachyzoites-infected leukocytes. This indicated that CD162 molecules on the surface of the lung endothelial cells contribute to tachyzoite egress from the attaching leukocytes. Conclusions Ttachyzoite-infected leukocytes in the general circulation remain in the lung by effective adhesion to lung endothelial cells and that this adhesion triggers tachyzoite egression from the vehicle leukocyte. CD162 molecules expressed on the endothelial cells were found to contribute to egression. This signal enables the parasite to time egression from its vehicle leukocyte to coincide with arrival at a target organ, minimizing the opportunity for immune attack during the transition from a vehicle leukocyte to capillary endothelial cells. References 1. Buzoni-Gatel, D. et al. Murine ileitis after intracellular parasite infection is controlled by TGF-beta-producing intraepithelial lymphocytes. Gastroenterology 120, 914–924 (2001). 2. Luangsay, S. et al. CCR5 mediates specific migration of Toxoplasma gondii-primed CD8 lymphocytes to inflammatory intestinal epithelial cells. Gastroenterology 125, 491–500 (2003). 3. Courret, N. et al. CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii. Blood 107, 309–316 (2006). 4. Unno, A. et al. Dissemination of extracellular and intracellular Toxoplasma gondii tachyzoites in the blood flow. Parasitol Int 57, 515–518 5. Norose, K., Naoi, K., Fang, H. & Yano, A. In vivo study of toxoplasmic parasitemia using interferon-gamma-deficient mice: absolute cell number of leukocytes, parasite load and cell susceptibility. Parasitol Int 57, 447–453 (2008). 6. Konradt, C. et al. Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system. Nature microbial. 15;1:16001. (2016).

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Acknowledgment This work was supported by grants from JSPS KAKENHI, including a Grant-in- Aid for Scientific Research (B) , 15H04592.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO2-3

PREVALENCE OF TREMATODOSIS IN BALI CATTLE IN WEST TIMOR

A. Winarso1*, M.G.M. Jo2, Y.R. Nadja2, D.T. Dhiu2, M. Cantona2, L.D. Amleni2, S.J. Koanak2, Y.V. Nawa2, A.N. Daki2, J.F.T.L. Pora2 1Departement of Animal Diseases and Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Nusa Cendana, Kupang, East Nusa Tenggara, Indonesia 2 Veterinary Internship Program, Faculty of Veterinary Medicine, Universitas Nusa Cendana, Kupang, East Nusa Tenggara, Indonesia *Corresponding author: [email protected]

Abstract Trematodes are common and major gastrointestinal parasite in cattle farms around the world. The epidemiological data are important to develop control measures. There is lack of trematodiasis data report in West Timor. This study aims to determine the prevalence of trematode infection in bali cattle in semi-arid area of West Timor. A cross-sectional study was conducted in September 2019 involving a total of 30 heads of adult bali cattle in Oeba Slaughterhouse of Kupang City. Stools samples was collected for coproscopy by multilevel filtration. Results showed that bali cattle in West Timor are infected with Fasciola sp (13.33%) and amphistome (6.67%).

Keywords: Bos sondaicus, coproscopy, Fasciola, Paramphistomum, semi-arid tropics

Introduction East Nusa Tenggara Province is one of the livestock producing provinces in Indonesia. Beef cattle is the main commodity. By semi-intensive grazing system, bali cattle are prone to exposure to diseases, including parasitic diseases. In addition, by the traditional farming management, the infection would become serious in impact even though unrecognized by the farmer in developing country. Trematodes are common and major gastrointestinal parasite in cattle farms around the world (Fromsa et al. 2011). They are causing massive economic losses by weight loss, reduced carcass and meat production and quality, reduced milk yield, organ condemnation, and treatment cost (Khoramian et al. 2014; Jaja et al. 2017). Several cases also ended up with fatality. Some of parasites also remain a great risk for public health as considered zoonotic. The epidemiological data are important to develop control measures. There is lack of trematodiasis data report in West Timor. A few reports are available, but they are focusing on fascioliasis by post-mortem examination without amphistome infection data. Objective This study aims to determine the prevalence of trematode infection in bali cattle in semi-arid area of West Timor. Method/Approach This cross-sectional study was conducted in September 2019, involving a total of 30 heads of adult bali cattle in Oeba Slaughterhouse of Kupang City. The animals in the slaughterhouse were from greater Kupang and neighboring regencies in Western Timor, so it was considered as ideal spot to collect the samples. The fecal samples were collected by rectal palpation, stored in a cooling box, and were brought to Laboratory

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 of Parasitology, FVM Universitas Nusa Cendana for examination. For confirming trematode eggs presentation, we used multilevel filtration mesh (mesh size: 400 µm, 100 µm, and 45 µm). Two grams of feces was put in a glass and added with 20ml of tap water. The fecal suspense then was passed through the multilevel filter. The sediment resided in last filter (mesh size: 45 µm) was put in petri dish, added with 2ml water and 2 drops of methylene blue, examined under stereo microscope by 4x objective magnification. Identification based on trematode egg morphology according to Zajac and Conboy (2012). Examination results then tabulated and presented as descriptive statistic. Results and Discussion There are two known species of Fasciola infecting domestic animals and human, i.e.: Fasciola hepatica and F. gigantica (Pfukenyi et al. 2005). F. gigantica was the only species occurred in Indonesia (Purwaningsih et al. 2012) and most part of tropics region in the world (Degheidy and Al-Malki 2012; Usip et al. 2014). Paramphistomum is the most common amphistome in ruminants (Aragaw & Tilahun 2018), although Indonesia have Orthocoelium indonesiense (Eduardo 1980).

Table 1. The prevalence of single and mix infection with trematode in bali cattle (n=30) Trematodes egg encountered Number positive (% Cumulative Prevalence) frequency Fasciola sp. 4 (13.33% 13.33% Amphistome 2 (6.67%) 20.00% Co-infection 0 (0.00%) 20.00% Free 24 (80.00%) 100.00%

Infection with Fasciola sp. and amphistome were found in this study (see Table 1). The overall prevalence for fasciolosis and infection with amphistome were 13.33% and 6.67%, respectively. No co-infection was detected in this study. Cumulative prevalence of both trematode infection was 20.00%. The prevalence of fascioliasis found in this study is slightly lower than previously reported by post-mortem examination in slaughterhouse (17.19%) (Damayanti et al. 2019). In Jogjakarta, a wet tropics area of Indonesia, trematodiasis was reported in higher prevalence (50%), with 48% for fascioliasis and 47% for paramphistomiasis (Rinca et al. 2019). We propose that these differences are due to climatic difference. Greater risk of fascioliasis occurred in areas having greater annual rainfall. High rainfall areas are suitable for snails’ niche and favorable for pre-parasitic stage development of trematodes. High temperature, low humidity and direct sunlight exposure in semi-arid area reduce the viability of metacercariae. (Pfukenyi et al. 2005). Animals get high exposure in the dry season as the receding water uncovers encysted metacercariae in the bottom parts of herbage (Chongmobmi & Panda 2018). Bali cattle grazed in paddy field after the rice harvest period in West Timor have greater metacercariae exposure risk, since the bottom part of rice plants are submerged in water during wet season. As we use qualitative method in this study, we did not observe the density of trematode egg in the feces. But, according Aragaw and Tilahun (2018), moderate infections with trematodes and chronic fasciolosis may lead to poor body condition (Aragaw & Tilahun 2018). Conclusions

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 The prevalence of trematodosis in Bali cattle in West Timor were 20.00% (13.33% for Fasciola and 6.67% for amphistome). Since trematode are of animal health and welfare importance, this finding suggests that there is still considerable economic loss in cattle production. Since Fasciola is important in foodborne zoonotic diseases and enhanced in travel medicine, the province government should more pay attention in promoting tourism in East Nusa Tenggara. Acknowledgements This study was supported by Faculty of Veterinary Medicine Universitas Nusa Cendana, UPT RPH and Dinas Pertanian Kota Kupang. References Aragaw K, Tilahun H. 2018. Coprological study of trematode infections and associated host risk factors in cattle during the dry season in and around Bahir Dar, northwest Ethiopia. Vet. Anim. Sci. 7: #100041. Chongmobmi M, Panda SM. 2018. Bovine Gastrointestinal Trematodosis In Nigeria: A Review. IOSR-JAVS. 11 (11): 08-19. DOI: 10.9790/2380-1111020819. Damayanti LPE, Almet J, Detha AIR. 2019. Deteksi dan prevalensi fasciolosis pada sapi bali di Rumah Potong Hewan ( RPH ) Oeba Kota Kupang. J. Vet. Nusant. 2 (1): 13–18. Degheidy NS, Al-Malki JS. 2012. Epidemiological studies of fasciolosis in human and animals at Taif, Saudi Arabia. World Appl. Sci. J. 19 (8): 1099–1104. Eduardo SL. 1980. Orthocoelium indonesiense, a new species of amphistome from ruminants in Indonesia. Systematic Parasitology. 1: 203-210. Fromsa A, Meharenet B, Mekibib B. 2011. Major Trematode Infections of Cattle Slaughtered at JimmaMunicipality Abattoir and the Occurrence of the Intermediate Hosts in Selected Water Bodies of the Zone. J. Anim. Vet. Adv. 10(12): 1592-1597. Jaja IF, Mushonga B, Green E, Muchenje V. 2017. Seasonal prevalence, body condition score and risk factors of bovine fasciolosis in South Africa. Vet. Anim. Sci., 4 (6): 1–7. Khoramian H, Arbabi M, Osqoi MM, Delavari M, Hooshyar H, Asgari M. 2014. Prevalence of ruminants fascioliasis and their economic effects in Kashan, center of Iran. Asian Pac. J. Trop. Biomed., 4 (11): 918–922. Pfukenyi DM, Monrad J, Mukaratirwa S. 2005. Epidemiology of trematode infections in cattle in Zimbabwe: a review. J. South Afr. Vet. Assoc. 76(1): 9–17. Rinca KF, Prastowo J, Widodo DP, Nugraheni YR. 2019. Trematodiasis occurrence in cattle along the Progo River, Yogyakarta, Indonesia. Vet. World. 12(4): 593-597. Usip LPE, Ibanga ES, Edoho HJ, Amadi EC, Utah E. 2014. Prevalence of Fascioliasis and the economic loss of condemned liver due to Fasciola infection in Cattle slaughtered at three abattoirs in Eket Urban , Akwa Ibom State of Nigeria. Glo. Adv. Res. J. Food. Sci. Technol. 3(3): 54–75. Zajac AM, Conboy GA. 2012. Veterinary Clinical Parasitology, 8th Ed. West Sussex: Willey-Blackwell.

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SCREENING OF SPARROWS (Passer domesticus) FOR Salmonella sp INFECTION IN YOGYAKARTA WITH MOLECULAR AND CULTIVICATION METHOD

Okti H1*, Marla A1, Sidna A1, And Tri U1 1Department of Microbiology, Faculty of Veterinary Medicine, Universitas Gadjah Mada *Corresponding author: [email protected]*

Abstract Salmonella sp is one of important enteric pathogens that has the ability to infect a variety of both sdomestic and wild animal species. Salmonella sp is responsible for about 850/100.000 reported cases of human gastroenteritis each year in Indonesia. Therefore, the current study was aim to know the prevalence of Sparrows as vectors of Salmonella sp in Yogyakarta and to compare cultivation methods with polymerase chain reaction (PCR) for the detection of Salmonella sp. A total of 30 fecal dropping samples were collected from Sparrows. Salmonella sp was cultivated by conventional and polymerase chain reaction (PCR) method. DNA was extracted from the faecal samples. The ability of PCR to amplify a Salmonella-specific DNA product (769-bp sequence covering the Salmonella invA genes) was examined. In this studies, we found that Sparrows as reservoir for Salmonella sp, and that Salmonella infection is expressed as carriage with no obvious disease manifestations. The prevalance of Salmonella sp in Sparrows was 3,3% and there was no significant difference between cultivation method and PCR for the detection of Salmonella sp. The study indicate that Sparrows can spread zoonotic enteric pathogens and this necessitates monitoring the epidemiologic status of these pathogens among birds and humans and applying the appropriate intervention measures to prevent the transmission of Salmonellosis.

Keywords: Salmonella sp, invA genes, Sparrows

Introduction Salmonellosis is an important zoonotic infection in domestic and wild animals species. Salmonella sp is spreaded through contact with infected droppings and aerosol exposure to bacteria-contaminated feces (Sareyyupoglu et al., 2007). Salmonella sp is responsible for about 850/100.000 reported cases of human gastroenteritis each year in Indonesia. Sparrows are a type of wild birds included in the order Passeriformes recorded as a reservoir of various zoonotic diseases caused by bacteria (Boseret et al., 2013). Diagnosis of Salmonella can detected by culture of feces, blood, spleen, liver, and intestinal contents. In addition, very small numbers of viable organisms present in the feces may fail to grow in artificial laboratory media (Schrank et al., 2001; Feeder et al., 2001). More recently, molecular techniques like polymerase chain reaction (PCR) were developed and used to detect Salmonella in different clinical materials (Oliviera et al., 2002; Whyte et al., 2002). Molecular testing has been most successful in areas for which conventional microbiologic techniques do not exist, are too slow, or are too expensive (Sharma & Carlson, 2000).

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Objective Study was aim to know the prevalence of Sparrows as vectors of Salmonella sp in Yogyakarta and to compare cultivation methods with polymerase chain reaction (PCR) for the detection of Salmonella sp Methods This study used 30 sparrows obtained from bird hunters in the Yogyakarta area. Fresh stool samples were collected in BHI media and cultivication on MacConkey Agar (MCA).This study has the ethical requirements for research in experimental animals from the Integrated Research and Testing Laboratory (LPPT) of Gadjah Mada University with certificate number: 00033/04 / LPPT / VI / 2019. DNA extraction using DNA extraction kit (Favorgen, Biotech Corp). Salmonella sp detection was carried out using invA F primers (5 '- CGGTGGTTTTAAGCGTACTCTT-3') and invA R (5 '- CGAATATGCTCCACAAGGTTA-3') with the InvA gene target (769bp). The PCR reaction was carried out under initial denaturation conditions at a temperature of 94ºC for 2 minutes, followed by 38 cycles consisting of denaturation at a temperature of 94ºC for 20 seconds, anneling at 60ºC for 1 minute, and extension at 72ºC for 1 minute. Results Salmonella sp detection was carried out using invA genes. According to Malorny et al., (2003) the invA gene became an international standard for detecting Salmonella sp at the genus level using clinical samples such as feces. Based on PCR pooling results obtained positive results on pooling 2 (Figure 1.) Each fecal swab sample at pooling 2 performed PCR using invA genes showed one positive sample of Salmonella sp (3.3%). Sparrows as reservoir for Salmonella sp, and that Salmonella infection is expressed as carriage with no obvious disease manifestations.

769 bp 769 bp

Figure 1. Agarose gel electrophoresis of the polymerase chain reaction products amplifying invA gene of Salmonella sp. M=marker; 1=negative control; 2-16: samples

The percentage of Salmonella sp in sparrows in Yogyakarta is higher when compared to previous studies in various countries except the UK which has a percentage of Salmonella sp in wild birds of 1.6% (Lawson et al., 2014). Research conducted by Afema & Sisco (2016) found that the percentage of Salmonella sp in birds was 4.1%, this is higher than the percentage of Salmonella in Yogyakarta. The same thing was revealed by Milan et al., (2004) who found the prevalence of Salmonella sp in wild

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 birds was 7.8%, higher than the prevalence of Salmonella in sparrows in Yogyakarta. Both cultivication method and direct PCR didn’t have significantly different (Table-1).

Table-1. Direct PCR and cultivication of Salmonella sp infection in Sparrows (30 samples) Laboratoric Diagnosis Result Cultivication (Non fermented 2a Lactose) PCR positif (769bp) 1b a,b Same coloumn with different superscript are not significantly different (p> 0,05).

Conclusion The prevalance of Salmonella sp in Sparrows was 3,3% and there was no significant difference between cultivation method and PCR for the detection of Salmonella sp. Sparrows can spread zoonotic enteric pathogens and this necessitates monitoring the epidemiologic status of these pathogens among birds and humans and applying the appropriate intervention measures to prevent the transmission of Salmonellosis References Afema, J.A. and Sischo, W.M. 2016. Salmonella in Wild Birds Utilizing Protected and Human Impacted Habitats, Uganda. EcoHealth 13, 558–569 Boseret, G., Losson,B., Mainil, J.G., Thiry, G., Saegerman, C.2013. Zoonoses in pet birds: review and perspectives. Veterinary Research 2013, 44:36 Feder, I., J. C. Nietfeld, J. Galland, T. Yeary, J. M. Sargeant, R. Oberst, M. L. Tamplin, and J. B. Luchansky. 2001. Comparison of cultivation and PCR-hybridization for detection of Salmonella in porcine fecal and water samples. J. Clin. Microbiol. 39:2477–2484. Lawson, B., Pinna, E., Horton, R.A., Macgregor, S.K., John, S.K.,Chantrey, J., Duff, P.J., Kirkwood, J.K., Simpson, V.R., Robinson, R.A., Wain, J., Cunningham, A.A. 2014. Epidemiological Evidence That Garden Birds Are a Source of Human Salmonellosis in England and Wales. Plos one 9 (2) Malorny, B., Hoorfar, J., Hugas, M., Heuvelink, A., Fach, P., Ellerbroek, L., Bunge, C., Dorn, C. and Helmut, R. 2003. Inter laboratory diagnostic accuracy of a Salmonella specific PCRbased method. Int. J. Food Microbiol., 89: 241-249 Millan, J., Aduriz, G., Moreno, B., Juste, R.A. & Barral, M. 2014. Salmonella isolates from wild birds and mammals in the Basque Country (Spain). Rev. sci. tech. Off. int. Epiz., 2004, 23 (3), 905-911 Oliveira, S. D., L. R. Santos, D. M. T. Schuch, A. B. Silva, C. T. P. Salle, and C. W. Canal. 2002. Detection and identification of salmonellas from poultry-related samples by PCR. Vet. Microbiol. 87:25–35. Schrank, I. S., M. A. Z. Mores, J. L. A. Costa, A. P. G. Frazzon, R. Soncini, A. Schrank, M. H. Vainstein, and S. C. Silva. 2001. Influence of enrichment media and application of a PCR based method to detect Salmonella in poultry industry products and clinical samples. Vet. Microbiol. 82:45–53. Sharma, V. K., and S. A. Carlson. 2000. Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture. Appl. Environ. Microbiol. 66:5472–5476.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Sareyyupoglu, B., Celik Ok, A., Cantekin, Z., Yardımcı, H., Akan, M. and Akcay, A. 2007. Polymerase Chain Reaction Detection of Salmonella spp. in Fecal Samples of Pet Birds. Avian Diseases, 52(1):163-167. Whyte, P., K. McGill, J. D. Collins, and E. Gormley. 2002. The prevalence and PCR detection of Salmonella contamination in raw poultry. Vet. Microbiol. 89:53–60.

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WHAT TRANSMITS HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS TO POULTRY HOUSE CHICKENS?

Tsuyoshi Yamaguchi*, Tatsufumi Usui, Yukiko Tomioka and Toshihiro Itoh Avian Zoonosis Research Center, Tottori University, Tottori, Japan [email protected]

Abstract Background Highly pathogenic avian influenza virus (HPAIV) infections have occurred in poultry farms and wild birds during the winter migratory season of ducks and other waterfowl in Japan. These migratory waterfowl are believed to play a critical role in the distribution of HPAIV in Japan. However, it is still unclear how and by what route the virus is transmitted. Objective Determine how and by what route the virus is transmitted to chickens in poultry house. Methods Motion-activated cameras were set in poultry farms to determine the wild animals entered into poultry houses. Subsequently, experimental infections using 5 black rats and 3 weasels were conducted. These animals were intra-nasally inoculated with HPAIV subtype H5N1 under anesthesia. Clinical signs, body weight and body temperature were observed for 14 days post-inoculation (dpi) to understand their susceptibility against HPAIV infection. Results and Discussion The photos showed that there were many black rats in most of the poultry houses and confirmed their direct or indirect contacts with chickens. Furthermore, in many poultry farms, even those with windowless houses, the photos also showed that Mustelids, such as weasels and martens, and cats frequently entered poultry houses. Furthermore, the experimental infections using black rats and weasels showed that HPAIV was isolated from oral swabs of 3 black rats at 5 dpi and each of the weasels at 5 to 7 dpi. One weasel died at 9 dpi but the other animals did not show any obvious clinical symptoms. These findings showed that peri-domestic wild mammals mentioned above can be infected and shed the virus from the mouth for several days, and therefore can be an infectious source of HPAIV for poultry. Additional evidence pointing to weasels is that they have a larger home range than black rats, prey on wild birds and tend to prefer waterside habitats. Conclusions Although HPAIV natural infections have so far not been confirmed in rats or Mustelidae including weasels in Japan, our study suggests that especially the weasel can play an important role in connecting the infected waterfowl to poultry house chickens. Keywords: HPAIV, chicken, weasel, rat, transmission

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ISOLATION AND CHARACTERIZATION OF HERAMATSU VIRUS (REOVIRIDAE: ORBIVIRUS) FROM A BAT (MINIOPTERUS FULIGINOSUS) IN JAPAN

Supriyono1, Hiroshi Shimoda1, Ryusei Kuwata1,2, Ai Takano1, Keita Noguchi1, Kenzo Yonemitsu1, Shohei Minami1, Ngo Thuy Bao Tran1, Yudai Kuroda1, Kango Tatemoto 1, Milagros Virhuez Mendoza1, Daisuke Hayasaka1, Ken Maeda1,3* 1) Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University 2) Faculty of Veterinary Medicine, Okayama University of Science 3) Department of Veterinary Science, National Institute of Infectious Diseases, Japan

Abstract Introduction Bats are mammalian that harbor some serious pathogens, Nipah virus, SARS coronavirus and so on. Investigation of pathogens in bats in Japan may be valuable to assess the risk of emerging infectious diseases originated from bats. In this study, possible arbovirus, Heramatsu virus, was isolated from a bat and characterized. Material and Methods Eastern bent-wing bats (Miniopterus fuliginosus) were captured in Wakayama prefecture, Japan, in 2016. Virus isolation was attempted from brains using two mammalian cell lines, Vero and BHK-21. Next generation sequencing was performed to identify the isolated virus. Viral growth kinetics of isolated virus was determined in 15 mammalian cell lines, human (HEK-293T, Huh-7), green monkey (Vero), mice (Neuro-2a), hamster (BHK-21, HmLu-1), porcine (CPK), dog (DH-82), cat (Fcwf-4), bats (YubFKT1, YubFKT2, DEMKT1, FBKT, BKT P122), cattle (MDBK) and 3 mosquitoes cell lines (Ae. Ae, C6/36, CTR). Detection of antibody against isolated virus was performed by virus neutralization (VN) test among wild boars (n=121), Sika deer (n=182) and wild monkeys (n=110) in Yamaguchi prefecture and raccoons (n=88) in Wakayama prefecture. Results and Discussion We successfully isolated one virus from bats brain. Phylogenetic analysis revealed that the virus belongs to Reoviridae, Orbivirus and was closely related with Heramatsu virus (HRMV) (66.2%-98.7% identity). The virus could grow in HEK-293T, BHK-21, Huh-7, Vero, HmLu-1, Neuro-2a, Fcwf-4, YubFKT1, YubFKT2, DEMKT1, Ae. Ae, C6/36, and CTR, and two cell lines of them, HEK 293T and BHK-21 cells, supported the growth well. VN test showed that sika deer (6/182), wild boar (8/121) and monkey (37/110) were positive for VN antibody against the virus, while wild raccoons in Wakayama prefecture did not possess any detectable antibody. Interestingly, Sika deer (1:40-1:640) and wild boar (1:80-1:640) had higher VN titer than the other animals. Conclusion HRMV was reported to be isolated from blood of an Eastern long-fingered bat (Myotis macrodactylus) in Kagoshima prefecture, Japan 1965. Our results also

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 suggested that Myotis bats seem to be origin of HRMV, and wild boar and Sika deer may play an important role in the transmission cycle of HRMV. In addition, HRMV seems to be an arbovirus, because most orbiviruses are arboviruses. Further examination of HRMV will be required for risk assessment in human and animals.

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DETECTION OF ARCANOBACTERIUM PLURANIMALIUM IN MUSKOX USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) ASSAY TARGETING THE PLA GENE

Siti Gusti Ningrum1*, Osama Sammra2, Christoph Lämmler2, Amir Abdulmawjood3 1Faculty of Veterinary Medicine, Wijaya Kusuma Surabaya University, Surabaya, 60225, Indonesia 2Institut für Pharmakologie und Toxicologie, Justus-Liebig-Universität Giessen, Schubertstr. 81, 35392 Giessen, Germany 3Institut für Lebensmittelqualität und-sicherheit, Stiftung Tierärztliche Hochschule Hannover, 30173 Hannover, Germany *Corresponding author: [email protected]

Abstract Arcanobacterium pluranimalium is a new bacterial pathogen species of the genus Arcanobacterium. In this study, loop-mediated ishotermal amplification (LAMP) based in gene pla encoding pluranimaliumlysin was used for detecting this novel species isolated from muskox. Compared with sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla, the LAMP method exhibited is simpler and might allow low cost identification of this bacteria.

Keywords: Arcanobacterium pluranimalium - muskox - 16S rDNA - loop-mediated ishotermal amplification - pla

Introduction Arcanobacterium pluranimalium is a new species of the genus Arcanobacterium which was initially isolated from a dead harbor porpoise and a dead fallow dear (Lawson et al. 2001). The identification of A. pluranimalium could be performed genotypically for highly specific identification of this species. Sequencing of 16s rDNA has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories (Woo et al. 2008). More recently, Balbutskaya et al. (2014) also described pla as bacterial target gene for sequencing. Nowadays, loop-mediated isothermal amplification (LAMP) could be an alternative for identification of this species as previously described by Abdulmawjood et al. (2015) Objective The present study was designed to investigate an A. pluranimalium strain isolated from muskox using LAMP assay targeting the pluranimaliumlysin encoding gene pla. Methods The isolate investigated in the present study was obtained from a 19-year-old female muskox from Zoo of Cologne, Germany, which, after suffer from severe weakness, was euthanized in January 2015. Postmortem analysis revealed a diffuse catarrhal

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 gastroenteritis, a stomatitis profunda, a diffuse fatty liver, and arthrosis of tarsal and carpal joints. Bacterial investigations yielded the isolation of A. pluranimalium and several other bacteria from the liver in low number and the isolation of aerobic bacilli, α-hemolytic streptococci and Micrococcus sp. from the intestine, also in low numbers. The reference strain used as positive control was obtained from Justus Liebig University bacterial collection. 16s rDNA and the pluranimaliumlysin encoding gene pla were sequenced as described by Balbutskaya et al. (2014). The presence of gene pla was determined with a previously described loop-mediated isothermal amplification (LAMP) assay (Abdulmawjood et al. 2015). This was performed using a heat block and subsequent detection of the LAMP product in an agarose gel and by using a real-time fluorometer (Genie II®, Optigene, UK). Results The bacterial strain investigated in the present study could be identified genotypically as A. pluranimalium. Sequencing 16S rDNA and pluranimaliumlysin encoding gene pla revealed a sequence identity of 99.9% and 99.6% to the respective sequences of type strain A. pluranimalium DSM 13483. The species-specific gene pla of A. pluranimalium 230/15 of the present study could also successfully be detected by using a LAMP assay. References Abdulmawjood A., Wickhorst J., Sammra O., Lämmler C., Foster G., Wragg P.N,. Prenger-Berninghoff E., Klein G. 2015. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium. Mol Cell Probes. 29: 468-472. Balbutskaya A., Sammra O., Nagib S., Hijazin M., Alber J., Lämmler C., Foster G., Erhard M., Wragg P.N., Abdulmawjood A., Prenger-Berninghoff E. 2014. Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla. Vet Microbiol. 168: 428-431. Lawson P.A., Falsen E., Foster G., Eriksson E., Weiss N., Collins M.D. (2001): Arcanobacterium pluranimalium sp. nov., isolated from porpoise and deer. Int J Syst Evol Microbiol. 51: 55-59. Woo P.C.Y., Lau, S.K.P., Teng, J.L.L., Tse, H. and Yuen, K.Y., 2008. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clinical Microbiology and Infection. 14(10):908-934.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO2-8

PATHOLOGICAL CHANGES OF LIVER AND SMALL INTESTINE IN CHICKEN EMBRYO AFTER INOCULATED WITH VIRULENT ISOLATE OF NEWCASTLE DISEASE VIRUS

I Gede Hendra Prasetya Wicaksana1, Anak Agung Ayu Mirah Adi2, I Made Kardena2 1. Graduate Student of Veterinary Medicine Udayana University, 2. Pathology Laboratory-Faculty of Veterinary Medicine Udayana University. Email: [email protected]

Abstract Newcastle disease is a pathogenic viral disease in poultry which is infectious and can cause massive economic losses. To understand the pathogenesis and the distribution pattern of the virus in the tissues, pathological observation was evaluated using newly virulent isolate Newcastle disease virus (NDV) that was inoculated in embryonated chicken eggs. As many as seven embryonic chicken eggs aged 11 days and specific antibody negative against Newcastle disease, divided into two categories: inoculated with phosphate buffer saline and inoculated with isolates. Then the allantois fluid was tested using hemagglutination assay and hemagglutination inhibition tests to prove the infection serologically. The livers and small intestines of the inoculated products were collected and followed with the process of histopathological preparation using Hematoxylin and Eosin (HE) stain. The pathological analysis showed that all organs had necrosis, hemorrhages, inflammation, and congestion. However, necrosis, hemorrhages, and inflammation that were observed in liver occurred at 60%, 40% and 60% of the samples, respectively. Our finding provided valuable insight into the pathology of a virulent isolate of NDV which is dominated by blood circulation disorders with necrosis and inflammation in the chicken’s embryos and have important implication for the future studies.

Keywords: pathology, chicken embryo, virulent isolate, Newcastle disease virus

Introduction Newcastle disease (ND) is a viral disease caused by virulent strains of ND virus (NDV) with a high mortality rate that affecting many of avian species. This virus is reported to be endemic and causes epizootic outbreak in domestic poultry (Miller et al., 2010). In South-East Asia, the disease is also endemic, including in Indonesia (Adi et al.,2010). Newcastle disease virus does not only infect adult chickens, but it also infects the embryo (Swayne et al., 2013). Bwala et al. 2012, reported by using immunohistochemistry stain, some NDVs have a tropism in the oviduct that possibility lead to the virus transmit directly into the embryo. Despite the ND is one of notable disease in the poultry, there is only a few studies on the pathology of NDV that infected the chicken embryo, especially from field isolate case in Indonesia. Objective This research aimed to determine pathological observation and the lesion distribution pattern in the liver and the small intestine of the chicken embryo due to inoculation of the newly virulent isolate Tabanan-1/ARP/2017.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Methods Seven embryonic chicken eggs (ECE) with specific antibody negative (SAN) against NDV characteristic from white leghorn chicken aged eleven days were randomly distributed into two experimental groups, consisted of five embryos that inoculated with 0.2 ml suspension containing of 27 Hemagglutinin Unit from virulent isolate, NDV Tabanan 1/ARP/2017 (Adi et al., 2019) as the infected group; whereas two embryos were used as a control group which inoculated with 0.2 ml phosphate- buffered saline (PBS). Both of them inoculated via allantois route. The ECEs then incubated and observed every 12 hours until the embryos seen dead and the allantois fluid was collected and tested by using Hemagglutinin assay (HA) and Hemagglutinin inhibition (HI) test to confirm NDV serologically. Tissue samples of the embryos were collected from the liver, and small intestine to create histopathology preparation using HE stain. All tissues were observed under a light microscope, and the lesions were recorded. Result Histopathological examination in all organs of the infected group revealed necrosis, hemorrhage, inflammation, and congestion (Figure 1). Moreover, necrosis, hemorrhage, and inflammation in liver also found in 60%, 40% and 60% samples in this research respectively (Table 1). The other lesions were found in all (100%) of the sample. The small intestine has more severe damaged in the tissue than in the liver. On the other hand, there was no lesion found in the control group.

Table 1. Prominent lesion of chicken embryos tissues after infected by NDV isolateTabanan-1/AK/2017 Group Organ Lesion Control (a/b) * Infected (a/b) * Liver necrosis 0/2 3/5 hemorrhage 0/2 2/5 inflammation 0/2 3/5 congestion 0/2 5/5 Small intestine necrosis 0/2 5/5 hemorrhage 0/2 5/5 inflammation 0/2 5/5 congestion 0/2 5/5

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020

a b

c d

Figure 1. Histopathology of the organ that collected from the chicken embryo. a Liver control group (Bar 30 µm). b Congestion and mononuclear cells infiltration can be observed in liver from the infected group (Bar 30 µm). c Small intestine from the control group. d Small intestine of the infected group can be seen debris cell from necrotic villi of the intestine (arrowhead), congestion (arrow) and diffused mononuclear cells infiltration (Bar 30 µm). HE stains

Conclusion The newly isolated Tabanan-1/ARP/2017 in chicken embryo can cause lesion which is dominated by blood circulation disorder with necrosis and inflammation that was in livers and small intestines. References Adi AAAM, Astawa NM, Putra KSA, Hayashi Y, Matsutomo Y. 2010. Isolation and characterization of Pathogenic Newcastle disease virus from a Nature Case in Indonesia. J. Vet. Med. Sci 72(3): 313-319 Adi AAAM, Astawa I N M, Putra I GAA. 2019. The efficacy of BEI-inactivated vaccines of Gianyar-1/AK/2014 virulent strain in protecting chickens against Tabanan-1/ARP/2017 virulent NDV isolate. Vet World 12 (6): 758-764 Bwala DG, Clift S, Duncan NM, Bisschop SP, Oludayo FF. 2012. Determination of the distribution of lentogenic vaccine and virulent Newcastle disease virus antigen in the oviduct of SPF and commercial hen using immunohistochemistry. Res Vet Sci 93: 520–528 Miller P, Decanini E, Afonso C. 2010. Newcastle disease: Evolution of genotypes and the related diagnostic challenges. Infection, Genetics and Evolution 10(1): 26-35

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Swayne DE, Glisson JR, Mc Dougald LR, Nolan LK, Suarez DL, Nair P. 2013. Diseases of Poultry, Thirteenth edition. Wiley-Blackwell. Ames

Acknowledgment The virus inoculation, serological test and embryonic chicken eggs that used in this study were carried out in Virology Laboratory of Animal Disease Investigation Center (DIC) Denpasar, Bali. The authors wish to thank DIC Director for providing the facilities.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO2-9

SOCIO-CULTURAL PRACTICES IN BALI ARE POTENTIALLY ASSOCIATED WITH CLINICAL CASES OF ACUTE ENCEPHALITIS SYNDROME RELATED TO JAPANESE ENCEPHALITIS INFECTION

I Made Kardena1, 2*, Anak Agung Ayu Mirah Adi1, Michael Laurence2, Mark O’ Dea2, Mieghan Bruce2 1Faculty of Veterinary Medicine, Udayana University, Bali - Indonesia 2College of Veterinary Medicine, Murdoch University, Australia, *a postgraduate student Email: [email protected]

Abstract The high cases of acute encephalitis syndrome as the clinical sign of Japanese encephalitis infection in Bali for the last 3 years have led to the Indonesian government applied national program vaccination against JEV that firstly conducted in this province in March 2018. Even though vaccination in humans has been applied, no herd immunity is generated. Therefore, other related factors have to be considered and evaluated to have more understanding on the disease transmission in order to minimize the disease cycle and transmission. This review is conducted to assess socio-cultural practices of the Balinese in regard to the risk of Japanese encephalitis infection in Bali. Recent and related published articles have been reviewed and compiled to assess and describe the potential socio-cultural factors in relation to the cases occurrence in the island. Data from local related authorized officers have also been used to convey the recent situation of the cases. Balinese local practices for agricultural system called Subak and the traditional ceremony Tumpek Kandang are potentially related to the development and wide distribution of local rice paddy fields, common practice in pig farming that provides high density of pig population, and the abundance of the mosquito vector as the potential factors in relation to the cases of acute syndrome encephalitis related to the Japanese encephalitis infection in the area. Further studies related to the JE transmission and cycle, including factors associated with the occurrence of the disease linked to humans, animals and the vector are needed so that more understanding and knowledge on the disease in the area can be achieved in order to reduce the risk of the disease transmission.

Keywords: Bali, Japanese encephalitis, potential associated factors, socio-cultural practices.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO2-10

CORRELATION BETWEEN BODY LENGTH AND BODY WEIGHT AGAINST UTERINE LENGTH AND TESTICULAR WEIGHT OF SUNDA PORCUPINE FROM CENTRAL JAVA

Yosua Ka1*, Irma P2, Teguh B2* 1Department of Reproduction and Obstetrics, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia; 2Department of Anatomy, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia *Corresponding author: [email protected]

Abstract Sunda porcupine or Hystrix javanica is an endemic animal in Indonesia. This species can be found only in Java, Bali, Sumbawa, Flores, Lombok, Madura, and Tonahdjampea island, Indonesia. Their population in nature decreased due to hunt and change of habitats. The data about the reproductive organ of male and female Sunda porcupine will become necessary in the future. Correlation between body length and body weight against uterine length and testicular weight of Sunda porcupine were never reported before. We did a study since 2013 to collect as much data about this species. In this paper, we assessed the relationship between body length against uterine length from 17 female Sunda porcupine and body weight against testicular weight from 17 male Sunda porcupine. The samples were obtained from slaughterhouses that supply porcupine meat for extreme culinary restaurant in Tawangmangu and Ngawi, Central Java Province. The body length and body weight were measured after the animals were slaughtered. The uterine length was measured from the cervix to near the ovary and the testicular weight was measured in a pair. The data were analyzed statistically with Ms.Excel software. Based on our analysis, the correlation coefficient (r) of body length and uterus length of female Sunda porcupine was r=0,755691. This showed that there was a strong correlation between body length and uterus length. The linear graph showed the upward sloping line that indicated a positive correlation. It means if the body length increased, the uterus length will increase too. The regression coefficient showed R2=0,5711. It indicated that the body length will affect the uterus length of about 57,11%. Regression equation was y=0,3077x-13,79. The correlation coefficient (r) of body weight and testicular weight of male Sunda porcupine was r=0,708277. This showed that there was a strong correlation too between body weight and testicular weight. The linear graph showed the upward sloping line that indicated a positive correlation. It means if the bodyweight increased, the testicular weight will increase too. The regression coefficient showed R2=0,5017. It indicated that the bodyweight will affect testicular weight of about 50,17%. Regression equation was y=3,2634x-16,837. Reproductive organ size is one of the factors that can be used to judge reproductive status in animals. The founding about the strong positive correlation between body length and body weight against the reproductive organ of Sunda porcupine will be useful in the future to support the conservation efforts of this species. However, a study to determine mature and immature Sunda porcupine based on reproductive organ size needs to be done furthermore. Keywords: Sunda porcupine, body length, body weight, uterine length, testicular weight

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Introduction Sunda porcupine or Hystrix javanica is an endemic animal in Indonesia (Aplin, 2016). This species can be found only in Java, Bali, Sumbawa, Flores, Lombok, Madura, and Tonahdjampea island, Indonesia (Van Weers 1979, 1983; Woods and Kilpatrick 2005). The habitat and ecology of Sunda porcupine are forests of subtropical/tropical moist lowland and shrubland of subtropical/tropical moist. In 2016, the International Union for Conservation of Natural Resources (IUCN) still classified this species into Least Concern which means the population in nature is still abundant. However, Indonesia Government has classified Sunda porcupine into a protected animal since 2018. Their population in nature decreases due to hunt and change of habitats. The data about the reproductive organ of male and female Sunda porcupine will become necessary in the future. Puberty or sexual maturation in females is marked by the production of ova by the follicles, the development of the reproductive tract, and the desire for mating. While in males is marked by the desire to mate and the ability to produce sperm (Anonim, 2020). Classification of mature and immature animals based on the primary and secondary reproductive organs size has been demonstrated by many researchers in various species (Springer, 1960; Bass et al., 1973; Jones and Geen, 1977; Natanson and Cailliet, 1986). Reproductive organ size can be determined by the size of other organs. In some species, reproductive organ size correlates with body size. However, the correlation between body length and body weight against uterine length and testicular weight of Sunda porcupine were never reported before. Objective This study was conducted to analyze the correlation between body length and body weight against uterine length and testicular weight of Sunda porcupine from Tawangmangu and Ngawi Districts, Central Java Province, Indonesia. Method Samples were obtained from slaughterhouses in Tawangmangu and Ngawi District from 2013 until 2018. Seventeen female Sunda porcupines and seventeen male Sunda porcupines were used in this study. Body length was measured from nose to the end of the tail, while uterus length was measured from the cervix to the near ovary. Bodyweight and a pair of testes with its epididymis were measured with a weigher. The data were analyzed statistically with Ms. Excel program software. Results The correlation coefficient of body length and uterus length of female Sunda porcupine was r=0,755691. The linear graph showed the upward sloping line (Figure 1). The determination coefficient was R2=0,5711. These results showed that there was a positive correlation between body length and uterus length of female Sunda porcupine. Based on the coefficient interval table, the level of relation was categorized as strong (Table 1). This means if the body length was increased then the uterus length will increase too and vice versa. The body length affects the uterus length of about 57,11%.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020

Correlation: body length and uterus length

20 y = 0.3077x - 13.79 15 R² = 0.5711 10 Body Length X Uterus Length 5 Linear (Body Length X 0 Uterus Length) Uterus Length (cm) Length Uterus 50 60 70 80 90 100 Body Length (cm)

Figure 1. Linear Graph of Correlation: body length and uterus length. The correlation coefficient of body weight and testicular weight of male Sunda porcupine was r=0,708277. The linear graph showed the upward sloping line (Figure 2). The determination coefficient was R2=0,5017. These results showed that there was a positive correlation between body weight and testicular weight of male Sunda porcupine. Based on the coefficient interval table, the level of relation was categorized as strong (Table 1). This means if the body weight was increased then the testicular weight will increase too and vice versa. Bodyweight affects the testicular weight of about 50,17%. Correlation: body weight and testicular weight

37 y = 3.2634x - 16.837 27 R² = 0.5017 Body Weight X 17 Testicular Weight 7 Linear (Body Weight X -3 Testicular Weight)

3 5 7 9 11 13 Testicular Weight (gr) Weight Testicular Body Weight (kg)

Figure 2. Linear Graph of Correlation: body weight and testicular weight.

There is a relation between growth and reproduction in animals that reach a determinate size, such as birds and mammals (Hamel and Côté, 2009). In the female, body size can also positively correlate with fecundity (Green and Rothstein, 1991). Flassy (2007) reported that Gray Tree Kangaroo (Dendrologus inustus) become mature after they reach 500-800 mm body length. Besides, reproductive organ length has a positive correlation too with body length and body mass (Koibur et al., 2011). In the male, testis size relative to body size is widely used as a measure of reproductive investment and is thought to be under strong sexual selection (Hosken and Ward, 2001; Byrne et al., 2002). Mi et al. (2012) reported that testis weight showed a positive relationship with body weight in Rhacophorus omeimontis.

Table 1. Coefficient interval table and level of relation.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Coefficient Interval Level of Relation 0,000-0,199 Very Low 0,200-0,399 Low 0,400-0,599 Average 0,600-0,799 Strong 0,800-1,000 Very Strong

Conclusion The body length of female Sunda porcupine has a positive correlation with uterus length, while bodyweight of male Sunda porcupine has a positive correlation with testicular weight.

References Anonim. 2020. Introduction to Animal Reproduction. In cooperation with the agricultural education department and the college of agricultural, food and natural resources, the University of Missouri-Columbia. https://dese.mo.gov/sites/default/files/aged-Animal-Repro-Student-Ref..pdf. Downloaded on 10 January 2020. Aplin, K. 2016. Hystrix javanica. The IUCN Red List of Threatened Species 2016: e.T10752A22231749.https://dx.doi.org/10.2305/IUCN.UK.2016- 2.RLTS.T10752A22231749.en. Downloaded on 10 January 2020. Bass, A.J., D’Aubrey, J.D., Kistnasamy, N. 1973. Sharks of the east coast of southern Africa. I. The genus Carcharhinus (Carcharhinidae). Oceanographic Research Institute (Durban) Investigational Report. 33:168. Byrne, P.G., Roberts, J.D., Simmons, L.W. 2002. Sperm competition selects for increased testes mass in Australian frogs. J Evol Biol. 15:347-355. Flassy, D.A. 2007. Fauna Tanah Kita untuk Pengenalan di Sekolah dan Pengetahuan Umum. Balai Pustaka. Jakarta. Green, W.C.H., Rothstein, A. 1991. Trade-offs between growth and reproduction in female bison. Oecologia. 86:521-527. Hamel, S., Côté, S.D. 2009. Foraging decisions in a capital breeder: trade-offs between mass gain and lactation. Oecologia. 161:421-432. Hosken, D.J., Ward, P.I. 2001. Experimental evidence for testis size evolution via sperm competition. Ecol Lett. 4:10-13. Jones, B.C., Geen, G.H. 1977. Reproduction and embryonic development of spiny dogfish (Squalus acanthias) in the Strait of Georgia, British Columbia. J Fish Res Board Can. 34:1286-1292. Koibur, J.F., Kustono, Widayati, D.T. 2011. The characteristics and female reproductive organ of gray tree kangaroo (Dendrolagus inustus) in papua. Buletin Peternakan. 35(1):17-23. Mi, Z.P., Liao, W.B., Jin, L., Lou, S.L., Cheng, J., Wu, H. 2012. Testis Asymmetry and Sperm Length in Rhacophorus omeimontis. ZOOLOGICAL SCIENCE. 29:000- 000. Natanson, L.J., Cailliet, G.M. 1986. Reproduction and development of the Pacific angel shark, Squatina californica, off Santa Barbara, California. Copeia. 1986:987-994. Springer, S. 1960. Natural history of the sandbar shark, Eulamia milberti. Fish Bull. 61:1-38.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 van Weers, D.J. 1979. Notes on southeast Asian porcupines (Hystricidae, Rodentia) IV. On the taxonomy of the subgenus Acanthion F. Cuvier. Beaufortia. 29(356):215- 272. Van Weers, D.J. 1983. Specific distinction in Old World porcupines. Zoologische Garten. 53:226-232. Woods, C.A.,Kilpatrick, C.W. 2005. Infraorder Hystricognathi. In: Wilson, D.E. and Reeder, D.M. (eds), Mammal Species of the World, pp. 1538-1599. The Johns Hopkins University Press, Baltimore, MD, USA.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO2-11

THE CORRELATION OF BODY LENGTH WITH TOTAL INTESTINE LENGTH OF SUNDA PORCUPINE

Irma P1, Yosua Ka2 And Teguh B1 1Department of Anatomy, Faculty of Veterinary Medicine, Universitas Gadjah Mada 2Department of Reproduction and Obstetric, Faculty of Veterinary Medicine, Universitas Gadjah Mada *Corresponding author: [email protected]

Abtract Sunda porcupine is belonging to endemic species in Indonesia. The hunting of sunda porcupine is commonly popular to providing meat needs as culinary icon in Tawangmangu Sub-district. However, Indonesian Ministry of Forestry and Environment had declared the regulation about Sunda porcupine protection to maintain the population. This study was conducted to investigate the correlation of body length with total intestine length of Sunda porcupine. Body length and intestine length were measured from 8 Sunda porcupine from Tawangmangu Sub-district, Central Java, Indonesia. Body length and intestine length were measured by using tape mater (cm). Morphometric analyze result showed there was very low negative correlation between body length and total intestine length. On the other hand, there was strong positive correlation between caecum length and total intestine length. In conclusion, when the body length increased, the total intestine length decrease and when the caecum length increased, the total intestine length increase.

Keywords: Sunda porcupine, body length, intestine length, correlation, morphometric.

Introduction Sunda porcupine (Hystrix javanica) is belonging to Rodentia order, which the body covered by quills (Roze, 1989). This species is endemic animal which is distributed in Java, Bali, Nusa Tenggara, and Sulawesi (Weers, 1979). Sunda porcupine’s meat is a culinary icon. (Sukiya, 2005; Norsuhana et al, 2012) and the quills have aestethic value (Inayah, 2016) in Tawangmangu sub-district. An increase of Sunda porcupine’s hunting lead to decline of population (Chung et al, 2016). Indonesian ministry of environment and forestry was declared that Sunda porcupine is one of protected species, however, International Union for Conservation of Nature (IUCN) still classify Sunda porcupine in least concern group. Sunda porcupine’s organ system documentation has been conducted and it has been a part of Sunda porcupine research sequences since 2013. However, morphometric information about Sunda porcupine has not been reported yet. Morphometric data is important as forensic reference to measure individual body size estimation in various age and sex. The morphometric information could become the scientific information base in conservation implementation. The increase of Sunda porcupine’s population in captive breeding had been promoted. Feed requirement is needed to maintenance the existence of Sunda porcupine. Diet type and feeding habit affect to digestion and absorption of nutrient and directly affect to body growth and development (Jarmanto et al., 2014). Information about digestive system, particularly morphology and

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 morphometry are needed to provide the appropriate feed for Sunda porcupine body growth. Objective This study was conducted to investigate the correlation of body length with total intestine length of Sunda porcupine Methods This study conducted by using eight Sunda porcupine from Tawangmangu sub- district, Central Java. The study was approved by animal ethic committee of Universitas Gadjah Mada (326/KEC-LPPT/IX/2015). Body length and intestine length were measured by using tape meter (cm). Body length was measured from the tip of Sunda porcupine’s nose to proximal base of tail. The total intestine length was measured from the pyloric sphincter to ileocaecal junction (small intestine) and caecum to rectum (large intestine). Correlation between two variables were analyzed by Pearson correlation and Linear regression test method by using Microsoft excel 2016 for Windows 10. Results The measurement value of body length and total intestine length were used for Pearson analysis. Correlation coefficient value (r) between Sunda porcupine’s body length and total intestine length was -0,007. According to Pearson analysis interpretation, r value indicated the correlation between two variables were very low. Then, r value between Sunda porcupine’s caecum length and total intestine length was 0,658. The r values indicated there was strong correlation between caecum length and total intestine length. Linear regression analysis was conducted to examine the association between two variables. Figure 1 showed the linear graph of body length and total intestine length. The downward line of body length and total intestine length indicate the negative correlation. It meant, when body length increased, the total intestine length decrease. On the other hand, figure 2 showed the linear graph of caecum length and total intestine length. The upward line indicated the positive correlation between caecum length and total intestine length which it meant, when the caecum length increased, the total intestine length increase. According to linear analysis, the body length affected to the total intestine length 1,54% (R2 = 0,0154). This value indicated the increase of body length, affect to the decrease of total intestine length. Then, the caecum length affected to the total intestine length 38,62% (R2 = 0,3862). This value indicated the increase of caecum length, affect to the increase of total intestine length. The equation is also obtained from linear regression analysis method. y = -4,2976x1 + 598,09 y= 21,548x2 + 481,79 y = Total intestine length; x1 = body length; x2 = caecum length The equations could be applied to estimate the total length if only the data of body length or caecum length which is available. Intestine is susceptible with eaten diet. The adaptability of intestine with the type of diet affect to nutrient absorption and body growth. Previous study reported that the type of diet affected to intestine length. There is a positive correlation among intestine length, age and diet type (Al-Dabbagh et al., 1987). Correct diet selection based on the age in the captive breeding is important to reach the maximum nutrient absorption for body growth. On the other hand, caecum is the first part of large intestine, located in the caudal abdomen. In rats and mice, caecum mainly located on the left caudal abdomen (Uysal e al., 2016). The location of caecum

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 which is easy to find, is enable to estimate the total intestine length only by caecum measurement. The association between body length and total intestine length 750

700 y = -4.2976x + 598.09 650 R² = 0.0154

600 Body length x total 550 intestine length Linear (Body length x

500 total intestine length) Total intestine Totalintestine length(cm) 450

400 47 60 60 62 63 65 73 74 Body length (cm)

Figure 1. Linear graph of body length and total intestine length. The line indicated there was very low negative correlation.

The association between caecum length and total intestine length 750

700 y = 21.548x + 481.79 R² = 0.3882 650

600 Caecum length x total 550 intestine length Linear (Caecum length x

500 total intestine length) Total intestine Totalintestine length(cm) 450

400 16 17.5 19 21 22 23 28 28.5 Caecum length (cm)

Figure 2. Linear graph of caecum length and total intestine length. The line indicated there was strong positive correlation.

Conclusion Very low and negative correlation between body length and total intestine length indicate, when the body length increase, the total intestine length decrease. Then, strong

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 and positive correlation between caecum length and total intestine length indicate, when the caecum length increase, the total intestine length increase. References Al-Dabbagh, K.Y., Jiad, J.H., Wheed, I.N., 1987. The influence of diet on the intestine length of the White-cheeked Bulbul. Ornis Scandinavica 18 (2): 150-152 Chung Y.F., Lim N.T.L., Shunari M., Wang D.J., Chan S.K.L. 2016. Records of the Malayan porcupine, Hystrix brachyura (Mammalia: Rodentia: Hystricidae) in Singapore. Nature in Singapore 9: 63-68 Inayah N. 2016. Potensi pengembangan landak (Hystrix sp) sebagai produk komersial. Fauna Indonesia 15: 37-43. [Indonesian] Jarmanto, Yusfiati, Elvira, P. 2014. Morpometric digestive tract of ikan parang-parang (Chirocentrus dorub Forsskal 1775) from Bengkalis Sea, Riau Province. JOM FMIFA. 1(2): 464-471 Norsuhana A.H., Shukor M.N., Aminah A. 2012. Perceptions on captive malayan porcupine (Hystrix brachyura) Meat by Malaysian urban consumers. Health Environ J. 3 (1): 67-78. Roze U. 1989. The North American Porcupine. Smithsonian Institution Press. Washington. 40-52. Sukiya. 2005. Biologi Vertebrata . Universitas Negeri Malang Press. Malang. 14-20. Uysal, M., Gul, S.S., Karaman, S., Tas, U., Sapmaz, H. I., Uysal, F., Aytekin, K., Tumer, M. K., 2016. Caecum location in laboratory rats and mice: an anatomical and radiological study. Laboratory Animals: 1-10 Weers, D. J. V. 1979. Notes on Southeast Asian Porcupines (Hystricidae, Rodentia) IV. On the taxonomy of the subgenus Acanthion F. Cuvier, 1823 with notes on the other taxa of the family. Beautifortia. 29(356): 215–272.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 SO2-12

A NOVEL SPECIFIC ROLE OF GLYCOPROTEIN C IN HERPES SIMPLEX VIRUS INFECTIVITY

Tri Komala Sari1,2*, Katrina A. Gianopulos3, Seth M. Schneider3, Darin J. Weed2, Suzzane M. Pritchard2, And Anthony V. Nicola2 1Faculty of Veterinary Medicine, Udayana University, Bali, Indonesia 1Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, USA 3School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, USA *Correspondence: [email protected] Note: This proceeding is part of a manuscript entitled “Herpes Simplex Virus Glycoprotein C Regulates Low pH Entry”, which now has been accepted for publication in American Society for Microbiology Journal mSphere https://www.biorxiv.org/content/10.1101/858472v1.full

Abstract Herpes simplex viruses (HSVs) cause significant morbidity and mortality in humans worldwide. Herpesviruses mediate entry by a multi-component, virus-encoded machinery. Herpesviruses enter cells by endosomal low pH and pH-neutral mechanisms in a cell-specific manner. HSV mediates cell entry via envelope glycoproteins gB, gD, and the heterodimer gH/gL regardless of pH or endocytosis requirements. HSV envelope proteins that function selectively in a given entry pathway have been elusive. Here we demonstrate that gC regulates cell entry and infection by a low pH pathway. Conformational changes in the core herpesviral fusogen gB are critical for membrane fusion. The presence of gC conferred a higher pH threshold to acid-induced antigenic changes in gB. Thus, gC may selectively facilitate low pH entry by regulating conformational changes in the fusion protein gB. We propose that gC modulates the HSV fusion machinery during entry into pathophysiologically relevant cells, such as human epidermal keratinocytes.

Keywords: herpesviruses, herpes simplex virus, viral entry, viral glycoproteins

Introduction The prototype alphaherpesvirus, herpes simplex virus, utilizes an acidic endosomal pathway to enter epithelial cells and a pH-independent, direct penetration pathway to enter neuronal cells (1,2). The cellular triggers of herpesvirus entry, including intracompartmental pH, remain incompletely understood. HSV entry requires a host cell receptor that binds to viral glycoprotein D, such as nectin-1 (3-5), which is present on epithelial cells and in neuronal synapses (6, 7), but additional virus-host interactions are likely critical for entry. HSV particles contain at least 12 different virus-encoded envelope proteins. HSV entry into all cells requires gB, gD, and gH/gL. However, the majority of the remaining viral envelope proteins are not thought to be required for

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 entry via either low pH or pH-neutral routes (8). Envelope proteins specific for a given HSV entry pathway have not been identified. Glycoprotein B is conserved among herpesviruses and is a member of the class III fusion protein family. Unlike other class III fusion proteins such as VSV G and baculovirus gp64, herpesviral gB alone is not sufficient for fusion and requires additional viral proteins, most commonly gH/gL (9). Activation and regulation of the fusion function of gB are incompletely understood. The gH/gL complex is thought to positively regulate gB (10-12). HSV-1 gB undergoes conformational changes during fusion and entry (13, 14). Low pH specifically induces reversible changes in gB domains I and V, which comprise a functional region containing hydrophobic fusion loops (13). Acid-triggered changes in specific gB epitopes correlate with fusion activity: (i) HSV particles entering by endocytosis have reduced reactivity with gB domain I antibody H126, and elevation of endosomal pH blocks this change (13), (ii) irreversible acid-triggered changes in the H126 epitope coincide with irreversible acid-inactivation of HSV fusion and entry (15), and (iii) a hyperfusogenic form of gB has reduced reactivity with domain I and domain V antibodies, similar to low pH-treated gB (16). Thus, the acidic milieu of endosomes may serve as a host cell trigger of gB function. HSV-1 gC, a 511 amino acid, type I integral membrane glycoprotein, mediates HSV-1 attachment to host cell surface glycosaminoglycans. This interaction is not essential for HSV entry (17-19). Here we report that gC regulates low pH viral entry independent of its known role in cell attachment. We demonstrate that gC facilitates low-pH-induced antigenic changes in gB and that gC enhances the ability of HSV to enter and infect cells by a low-pH pathway. We propose a model in which gC modulates HSV entry mediated by gB, gD and gH/gL into physiologically relevant cell types, such as human keratinocytes. Results HSV-1 gC facilitates entry and infectivity of cells that support a low pH entry mechanism The contribution of gC to entry of HSV-1 by a low pH pathway was evaluated. HSV entry into CHO- nectin-1 cells and human keratinocytes proceeds via a low pH endocytic pathway and is well-characterized (1, 2). Here we used HSV-1 KOS strain with the gC gene deleted (HSV-1 ΔgC2-3) and a repaired version of this virus containing the wildtype gC gene (HSV-1 gC2-3R) (20).The efficiency of ΔgC infection of CHO-nectin-1 cells and primary human keratinocytes (HEKa) was compared to Vero and IMR-32 cells, which support pH-neutral entry via penetration at the plasma membrane (21, 22). To control for attachment, virus was first added to cells at 4 ̊C for 1 hr. Following a shift to 37 ̊C for 6 h, viral antigen-positive cell staining was quantitated as a fraction of attached virus. The efficiency of HSV-1gCR entry into each cell type was similar under the conditions tested. In contrast, entry of HSV-1 ΔgC into CHO-nectin-1 and HEKa cells was ~50% and 35% less efficient than into Vero and IMR-32 cells, respectively. To probe further the role of gC in low pH entry, we monitored the kinetics of intracellular transport of HSV-1. Virus was attached to cells at 4 ̊C for 1 h. Cultures were then shifted to 37 ̊C, and at different times post-infection (p.i.), the remaining extracellular virions were inactivated, and cells were lysed. The detection of infectious HSV in cell lysates reflects enveloped HSV present within cellular endocytic compartments. As expected,

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 HSV uptake into vesicles was rapid. At 10 min p.i. in CHO-nectin-1 cells, there was a peak of intracellular, infectious gCR virus. Following endocytic uptake, HSV fuses rapidly with the endosomal membrane and releases its capsid into the cytosol (23). This was reflected in the sharp decrease in infectious enveloped gCR recovered by 20 min p.i. Interestingly, ~50% of infectious, intracellular ΔgC was recovered as late as 40 min p.i., suggesting a delay in HSV fusion with endocytic compartments in the absence of gC. Analysis of HSV-1 gCR and ΔgC entry into HEKa cells yielded similar results. gC positively regulates low pH-induced antigenic changes in gB to begin to delineate the mechanism underlying gC’s role in low pH entry, the effect of gC on low pH-triggered antigenic changes in gB was assessed. The prefusion conformation of gB in the virion envelope undergoes low pH- triggered changes in gB domains I, V (13), and II (this study). These changes are at least partially reversible (13, 24-26) and are thought to be important for membrane fusion (12, 13, 25). Domain I of gB contains internal hydrophobic fusion loops that are critical for membrane fusion (20, 21). ΔgC or gCR virions were treated with a range of mildly acidic pHs and blotted immediately to nitrocellulose membrane. Blots were probed at neutral pH with 9 monoclonal antibodies (MAbs) to distinct epitopes in gB, and antibody reactivity was detected Domain I MAb H126 had reduced reactivity with virion gB that had been treated with low pH, regardless of the presence of gC. Importantly, in the absence of gC, the pH at which changes in the accessibility of the H126 gB epitope occurred was reduced by at least 0.6 pH units. Similarly, the pH of antigenic change in the gB Domain II epitope H1838 was decreased in the absence of gC. H1838 reactivity with gB in the ΔgC virus was particularly resistant to low pH; treatment with pH 5.0, the lowest tested, still resulted in >50% reactivity, suggesting that gC alters the pH of antigenic change in this gB epitope by > 0.7 pH units. Acidic pH triggers specific, not global, changes in gB conformation, as low pH does not induce changes in the SS10 (domain IV) or H1817 (domain VI) epitopes of gB (13). gC did not alter their susceptibility to pH. Domain V MAbs SS106 and SS144 had a > 50% reduction in reactivity with virion gB following pH 5.0 treatment, although reduction of reactivity in gC is less pronounced. In the absence of gC, the pH threshold for gB conformational changes, particularly in domain I and II is lower by 0.4-0.7 pH units. As a comparison, variants and mutants of influenza HA exhibit a ~ 0.2-0.6 shift in the pH of both conformational change and fusion (27, 28). The H126 epitope in the fusion domain of gB might be particularly important for the pH-activation of fusion (15). In the absence of gC, a more acidic pH is required to trigger changes in the accessibility of the H126 epitope. Acknowledgments We are grateful to Curtis Brandt, Gary Cohen, Roselyn Eisenberg, David Johnson, Lenore Pereira, and Patricia Spear for providing reagents. This study was supported by National Institutes of Health (NIH) grant R01 AI119159 (A.V.N) and NIH Training Grant T32 GM008336 (T.K., D.J.W., and K.A.G.). References 1. Nicola AV, Hou J, Major EO, Straus SE. 2005. Herpes simplex virus type 1 enters human epidermal keratinocytes, but not neurons, via a pH-dependent endocytic pathway. Journal of virology 79:7609-7616.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 2. Nicola AV, McEvoy AM, Straus SE. 2003. Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells. Journal of virology 77:5324-5332. 3. Geraghty RJ, Krummenacher C, Cohen GH, Eisenberg RJ, Spear PG. 1998. Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 280:1618-1620. 4. Spear PG, Eisenberg RJ, Cohen GH. 2000. Three classes of cell surface receptors for alphaherpesvirus entry. Virology 275:1-8. 5. Campadelli-Fiume G, Cocchi F, Menotti L, Lopez M. 2000. The novel receptors that mediate the entry of herpes simplex viruses and animal alphaherpesviruses into cells. Rev Med Virol 10:305-319. 6. Mizoguchi A, Nakanishi H, Kimura K, Matsubara K, Ozaki-Kuroda K, Katata T, Honda T, Kiyohara Y, Heo K, Higashi M, Tsutsumi T, Sonoda S, Ide C, Takai Y. 2002. Nectin: an adhesion molecule involved in formation of synapses. J Cell Biol 156:555-565. 7. Galen B, Cheshenko N, Tuyama A, Ramratnam B, Herold BC. 2006. Access to nectin favors herpes simplex virus infection at the apical surface of polarized human epithelial cells. J Virol 80:12209-12218. 8. Komala Sari T, Pritchard SM, Cunha CW, Wudiri GA, Laws EI, Aguilar HC, Taus NS, Nicola AV. 2013. Contributions of herpes simplex virus 1 envelope proteins to entry by endocytosis. Journal of virology 87:13922-13926. 9. Baquero E, Albertini AA, Gaudin Y. 2015. Recent mechanistic and structural insights on class III viral fusion glycoproteins. Curr Opin Struct Biol 33:52-60. 10. Chowdary TK, Cairns TM, Atanasiu D, Cohen GH, Eisenberg RJ, Heldwein EE. 2010. Crystal structure of the conserved herpesvirus fusion regulator complex gH- gL. Nat Struct Mol Biol 17:882-888. 11. Atanasiu D, Whitbeck JC, Cairns TM, Reilly B, Cohen GH, Eisenberg RJ. 2007. Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion. Proc Natl Acad Sci U S A 104:18718-18723. 12. Avitabile E, Forghieri C, Campadelli-Fiume G. 2007. Complexes between herpes simplex virus glycoproteins gD, gB, and gH detected in cells by complementation of split enhanced green fluorescent protein. J Virol 81:11532-11537. 13. Dollery SJ, Delboy MG, Nicola AV. 2010. Low pH-induced conformational change in herpes simplex virus glycoprotein B. Journal of virology 84:3759-3766. 14. Gallagher JR, Atanasiu D, Saw WT, Paradisgarten MJ, Whitbeck JC, Eisenberg RJ, Cohen GH. 2014. Functional fluorescent protein insertions in herpes simplex virus gB report on gB conformation before and after execution of membrane fusion. PLoS Pathog 10:e1004373. 15. Weed DJ, Pritchard SM, Gonzalez F, Aguilar HC, Nicola AV. 2017. Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry. J Virol 91. 16. Roller DG, Dollery SJ, Doyle JL, Nicola AV. 2008. Structure-function analysis of herpes simplex virus glycoprotein B with fusion-from-without activity. Virology 382:207-216.

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 17. Herold BC, WuDunn D, Soltys N, Spear PG. 1991. Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. Journal of virology 65:1090-1098. 18. WuDunn D, Spear PG. 1989. Initial interaction of herpes simplex virus with cells is binding to heparin sulfate. J Virol 63:52-58. 19. Shukla D, Spear PG. 2001. Herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry. J Clin Invest 108:503-510. 20. Herold BC, Visalli RJ, Susmarski N, Brandt CR, Spear PG. 1994. Glycoprotein C- independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B. The Journal of general virology 75 ( Pt 6):1211-1222. 21. Koyama AH, Uchida T. 1987. The mode of entry of herpes simplex virus type 1 into Vero cells. Microbiology and immunology 31:123-130. 22. Wittels M, Spear PG. 1991. Penetration of cells by herpes simplex virus does not require a low pH-dependent endocytic pathway. Virus research 18:271-290. 23. Nicola AV, Straus SE. 2004. Cellular and viral requirements for rapid endocytic entry of herpes simplex virus. Journal of virology 78:7508-7517. 24. Siekavizza-Robles CR, Dollery SJ, Nicola AV. 2010. Reversible conformational change in herpes simplex virus glycoprotein B with fusion-from-without activity is triggered by mildly acidic pH. Virology journal 7:352. 25. Gaudin Y. 2000. Reversibility in fusion protein conformational changes. The intriguing case of rhabdovirus- induced membrane fusion. Subcell Biochem 34:379-408. 26. Gaudin Y, Tuffereau C, Segretain D, Knossow M, Flamand A. 1991. Reversible conformational changes and fusion activity of rabies virus glycoprotein. J Virol 65:4853-4859. 27. Daniels RS, Downie JC, Hay AJ, Knossow M, Skehel JJ, Wang ML, Wiley DC. 1985. Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell 40:431-439. 28. Doms RW, Gething MJ, Henneberry J, White J, Helenius A. 1986. Variant influenza virus hemagglutinin that induces fusion at elevated pH. J Virol 57:603- 613.

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POSTER PRESENTATION

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IDENTIFICATION OF HOST FACTORS IN Francisella tularensis INFECTION

Sonoko Matsumoto*, Takashi Shimizu, Kenta Watanabe, Masahisa Watarai The Joint Faculty of Veterinary Medicine, Yamaguchi University *Corresponding author: [email protected]

Abstract Francisella tularensis is intracellular bacterium and causes zoonotic disease tularemia. F. tularensis is highly pathogenic and classified as a category A bioterrorism agent. Although some pathogenic factors of F. tularensis have been identified, important factors of host cells related to Francisella infection remain unclear. In this study, we screened the inhibitor library of eukaryotic cell signaling and selected inhibitors that inhibited or enhanced intracellular growth of F. tularensis. Among them, we focused on 3 inhibitors (Paclitaxel, Cucurbitacin I, and SB 218078). Paclitaxel, an inhibitor of β-tubulin depolymerization, slightly inhibited intracellular growth of F. tularensis. Cucurbitacin I, an inhibitor of janus kinase-2 (Jak2), inhibited infection of F. tularensis through suppression of phagocytosis of host cells. SB 218078, an inhibitor of check point kinase-1 in cell cycle, enhanced the intracellular growth. These results indicate that microtubules, Jak2 pathway, cell cycle check point, and phagocytosis of host cell involve in F. tularensis infection.

Keywords: Francisella, Tularemia, Jak2/STAT, Tubulin, Check point.

Introduction and objective F. tularensis, a pathogenic intracellular bacterium causes zoonotic disease tularemia. F. tularensis has wide range of host and lifecycle of F. tularensis is maintained in wild animals such as rabbits and rodents and arthropods such as tick. F. tularensis infects to human through contact with wild animals or bite of arthropods. Tularemia has a high mortality and F. tularensis is classified as a category A bioterrorism agent. Although some pathogenic factors of F. tularensis such as type VI secretion system have been identified, important factors of host cells during Francisella infection are poorly understood. Method/approach Less pathogenic F. tularensis, F. tularensis subsp. novicida (F. novicida) was used as a surrogate for pathogenic F. tularensis subsp. tularensis. GFP expressing F. novicida was infected to murine macrophage cell line J774 treated with inhibitor library containing 331 compounds at MOI=0.3 and fluorescence

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 of GFP was measured using plate reader at 24 h post infection. Phagocytic activity of J774 cell was measured using GFP conjugated glass beads. Results Among 331 inhibitors, 70 inhibitors decreased, 112 inhibitors increased bacterial number (GFP fluorescence intensity) in J774 cells. In this study we focused on 2 inhibitors (paclitaxel, cucurbitacin I) decreasing F. novicida infection, and 1 inhibitor (SB 218078) increasing the infection. The growth of F. novicida in culture medium with these inhibitors was not affected. Intracellular growth of F. novicida in J774 cells treated with inhibitors were examined by plate counting and confocal microscopy observation. Although paclitaxel slightly decreased the intracellular growth of F. novicida, cucurbitacin I significantly decreased the intracellular growth. In contrast, SB218078 increased the intracellular growth of F. novicida. To evaluate the phagocytic activity of inhibitor-treated J774 cells, GFP-conjugated glass-beads were added to J774 cells. Cucurbitacin I reduced the uptake of glass-beads, while other inhibitors showed no effect. Conclusions Paclitaxel, an inhibitor of β-tubulin depolymerization, and cucurbitacin I, an inhibitor of janus kinase-2 (Jak2), inhibited intracellular growth of F. tularensis. SB 218078, an inhibitor of check point kineae-1 in cell cycle, enhanced the intracellular growth. In addition, cucurbitacin I reduced the phagocytic activity of host cells. These results indicate that microtubules, Jak2 pathway, cell cycle check point, and phagocytic activity of host cell contribute to the pathogenicity of F. tularensis.

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MOLECULAR DETECTION OF MOMP GENE OF Chlamydia spp. FROM WILD BIRD’S FECAL SAMPLE IN YOGYAKARTA

Marla Anggita*, Okti Herawati, Widya Asmara, A.E.T.H. Wahyuni, Tri Untari, Michael Haryadi Wibowo, Sidna Artanto Department of Microbiology, Faculty of Veterinary Medicine Universitas Gadjah Mada, Yogyakarta 55281 *Corresponding author: [email protected]*

Abstract Indonesia is a rich country in terms of biodiversity. There are less than 7.000 species of fauna found in Indonesia and 1.600 of them are birds. Chlamydia spp. is an obligate intracellular bacterium that causes avian chlamydiosis. Chlamydia infects birds of family Passeridae, Psittachiformes and Columbidae. In birds, Chlamydia spp. can be found in the respiratory tract, and transmitted directly to other birds through the nasal fluid. Chlamydia can be transmitted from birds to humans, and the main source comes from bird feces. This study aims to detect the MOMP gene of Chlamydia spp. from the wild bird’s fecal sample in Yogyakarta using PCR. DNA was extracted from 30 samples of bird’s feces. The extracted DNA then amplified by PCR using primers CPR and CPF. The amplicons were electrophorized to see the visualization of DNA on the agarose gel and displayed an amplification product of 850 bp. The results showed that 70% (21 of 30) of the samples were detected positive of the MOMP gene. This indicates that wild bird’s feces can be a source of the spread of Chlamydia spp. to humans, birds, and other animals, although the birds did not show any sign of infection. Further research is needed to find out the Chlamydia species in wild birds in Indonesia, especially in the Yogyakarta region. Keywords: Chlamydia spp., wild bird, PCR

Introduction Chlamydia spp. is an obligate intracellular bacterium that can be transmitted from animals to humans. Transmission occurs due to direct contact with birds infected with Chlamydia spp. (Harkinezhad et al., 2009). Transmission of the disease occurs between animals through excretion from feces and nasal fluid (Gerlach, 1999). Direct transmission through aerosols from nasal fluid and feces is the most common transmission in birds (Grimes, 1994). Humans who have a higher risk are people who have direct contact with bird’s feces, including the owners, medical workers at the zoo or pet shops. Cases of Chlamydia transmission from animal to human were recorded in 26 cases and 19 (73%) of them were transmitted by birds (Moroney et al., 1998). Birds that are susceptible to bacterial infections are Psittaciformes and Columbiformes (Krawiec et al., 2015). Detection based on clinical symptoms and serological testing alone is difficult to determine Chlamydia infection in animals. PCR detection is needed to find out animals infected by

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 Chlamydia. The animals infected can be treated so they do not become a source of zoonotic transmission. Objective This study focused to detect Chlamydia spp. from fecal sample of the wild bird in Yogyakarta using PCR. Method Animals used in this study were approved by the Ethical Clearance Committee from Universitas Gadjah Mada, Indonesia with Certificate Number: 00033/04/LPPT/VI/2019. Wild birds used in this study were obtained from the bird hunter in Yogyakarta. Fresh fecal samples were collected from thirty Wild birds from the Yogyakarta area. Genomic DNA was extracted using Stool DNA Isolation Mini Kit (Favorgen, Biotech Corp). Primers used to detect the MOMP gene of the Chlamydia family are CPF (5’-GCAAGACACTCCTCAAAGCC-3’) as forward and CPR (5’- CCTTCCCACATAGTGCCATC-3’) as reverse. The PCR product was electrophorized to see the amplified DNA band. Electrophoresis was performed at 100V for 30 minutes. The gel then visualized under UV light to see the DNA band. Result Twenty-one out of thirty (70%) of the fecal samples were positive of the MOMP gene using PCR. Analyze of PCR product in agarose gel showed the product of 850 bps (Figure 1).

Figure 1. Electrophoresis gel showed PCR products of 850 bps (arrow), M: marker, K(-): negative control, respectively, lines 1, 7, 8, 10 -19: MOMP gene-positive samples.

The primers used in this study to detect the MOMP gene of Chlamydia spp are CPF (5’-GCAAGACACTCCTCAAAGCC-3’) and CPR (5’- CCTTCCCACATAGTGCCATC-3’). Major outer membrane protein (MOMP) is a component of bacterial membranes that function as structural proteins in Chlamydia. MOMP is currently being developed as an antigenic component in making a Chlamydia vaccine, replacing the use of all components of Chlamydia bacteria (Phillips et al., 2019). In humans, Chlamydia psittaci is the cause of psittacosis, which infected the

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 respiratory tract and can be transmitted through contact with infected birds. The family of Chlamydiaceae is an etiological agent of various important diseases in animals. Bacteria from this family not only infect birds but also infect mammals, some types of reptiles and amphibians. The positive sample of the MOMP gene in initial PCR optimization showed product results between 800-900 bps. This study detected the MOMP gene in fecal samples of birds although the birds showed no clinical symptoms of Chlamydia infection. The result showed that the wild birds can be the carrier of Chlamydia spp in the body and secreted through feces. These results also showed the possibility that Chlamydia infection in birds is rarely observed, and the appearance of clinical symptoms is likely due to close contact with the source of transmission of the disease (feces). We were detecting the MOMP gene of Chlamydia spp. as many as 70% of the total fecal samples (21 of 30) using conventional PCR methods. According to the study conducted by Doosti and Arshi (2011), they succeeded in detecting Chlamydia psittaci in pigeon fecal samples by 14,3% (64 of 445) and Krawiec et al (2015) detected 7,3% (27 of 369) from fecal samples of various wild birds including those from Psittachiformes and Passeridae are positive of Chlamydia spp. using Real-Time PCR. Conclusion This study showed the prevalence of MOMP positive gene from the fecal sample of wild birds in Yogyakarta is 70% (21 of 30). The wild bird can act as carriers by spreading the bacteria through feces without showing any clinical symptoms. Further research on the species of Chlamydia in wild birds that are abundant in Indonesia, especially in Yogyakarta region is needed to determine variations in the types of Chlamydia that can be carried by wild birds. References Doosti, A and Arshi, A. 2011. Determination of the Prevalence of Chlamydia psittaci by PCR in Iranian Pigeons. International Journal of Biology, 3(4): 79-82. Gerlach, H. (1999). Chlamydia. In: Avian medicine: Principles and Application.. HBD International Inc., Delray Beach, Florida. pp. 984-996. Grimes, J.E. (1994). Avian chlamydiosis. In: Handbook of Zoonoses, 2nd ed. CRC Press, Boca Raton, Florida. pp. 389-402. Harkinezhad, T., Geens, T., dan Vanrompany, D. 2009. Chlamydophila psittaci Infection in Birds: A Review with emphasis on zonotic consequences. Vet Microbiology 135 (1): 68-77 Krawiec, M., Piasecki, T., dan Wieliczko, A. 2015. Prevalance of Chlamydia psittaci and other Chlamydia Species in Wild Birds in Poland. Vector Borne and Zoonotic Diseases Vol 15 No 11: 652-655 Moroney, J.F., Guevara, R., Iverson, C. 1998. Detecton of Chlamydiosis in a shipment of pet bird, eading to recognition of an outbreak of clinically mild psittacosis in humans. Clin Infect. Dis 26(6):1425-1429

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COMBINATION OF LIPID BUBBLE AND ULTRASOUND IRRADIATION ENHANCES ANTI-TUMOR EFFECT OF LIPOSOMAL DOXORUBICIN IN VIVO

Inoru Yokoe1,2, Kazuo Maruyama3, Tomohiro Osaki2* 1The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan 2Joint Department of Veterinary Clinical Medicine, Faculty of Agriculture, Tottori University, Tottori, Japan 3Lab. of Theranostics, Faculty of Pharma-Science, Teikyo University, Tokyo, Japan *Corresponding author: [email protected]

Abstract Background: Lipid bubble (LB) is a microbubble agent which consists of phospholipids as an outer shell and perfluoropropane as inner gas. It has been reported that the combination of LB and ultrasound (US) irradiation can deliver therapeutic agents like drugs and genes to the irradiated tissue efficiently. Thus, this combination has been attracting attention as a promising novel drug delivery system (DDS). In the field of cancer treatment, liposomal doxorubicin (doxil) is known as a DDS anti-cancer drug which accumulates in tumor tissue. In this study, we investigated whether the combination of LB and US irradiation can improve doxil delivery to tumor tissue and enhance its anti-tumor effect. Materials and methods: Tumor-bearing mice were made by subcutaneous inoculation of EMT6 (mouse breast cancer cells) into the back of BALB/c mice. In experiment 1, FITC-labeled dextran/LB mixture or doxil/LB mixture was injected into the tail vein. Immediately after injection, the tumor tissue was irradiated with US (1 MHz, 1-3 W/cm2, 1 min). One hour later, the tumor tissue was excised. The fluorescence intensity of extraction liquid of the tumor tissues was measured, and the weight of each drug in the tumor tissue was calculated. In experiment 2, immediately after intravenous administration of the doxil/LB mixture to the tumor-bearing mice, the tumor tissue was irradiated with US (1 MHz, 2 W/cm2, 1 min). This treatment was performed three times at day 0, 2, and 4. The tumor volume was measured every other day until day 22. Results and conclusion: In experiment 1, the combination of LB and US irradiation increased the weight of FITC-labeled dextran and doxil in tumor tissue. In experiment 2, tumor growth was suppressed the most in the doxil, LB and US combination group compared to the other groups. In conclusion, it was considered that the combination of LB and US irradiation improved the delivery of doxil into tumor tissue and enhanced the anti-tumor effect. We plan to investigate mechanisms of drug delivery by the combination of LB and US irradiation in vitro. We also plan to move on to a clinical trial of cancer treatment using LB, US and doxil in veterinary medicine. Keywords: lipid bubble, ultrasound, drug delivery, liposomal doxorubicin, anti- cancer effect

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THE DIARRHEA SYMPTOM IN CALVES INFECTED BY ESCHERICHIA COLI K99 AND RECEIVED SUSPENSION OF IGG ANTI ESCHERICHIA COLI K99

Arief P Mihardi 1)*, Anita Esfandiari 1), Sus D Widhyari 1), Sri Murtini 2), Retno Wulansari 1), Leni Maylina 1,3) 1) Veterinary Internal Medicine Divison, Department of Clinic Reproduction and Pathology, Faculty of Veterinary Medicine, IPB University, Bogor, Indonesia 2) Medical Microbiology Division, Department of Animal Diseases and Veterinary Health, Faculty of Veterinary Medicine, IPB University, Bogor, Indonesia 3) Postgraduated Student, Yamaguchi University, Japan. *Corresponding author. E-mail: [email protected] / [email protected]

Abstract Collibacillosis is disease cause Escherichia coli (E. Coli) infecion that usually infected calves. The one of clinical sign E. coli infection in calves is diarrhea. This Experiment was conducted to observation diarrhea in calves were infected by E. coli K99 and received suspention of IgG anti E. coli K99. This Experiment used twelve calves that were devided into three groups, consist of four calves each group. The groups is Negative Control/NC (were not infected and no treatment); Possitive Control/PC (were infected by E. coli K99); and Suspension/SP (were infected by E. coli K99 and received suspension of IgG anti E. coli K 99). The E. coli K99 was administered orally with dosage of 5 x 1010 cfu/mL/calf. The suspension of IgG anti E. coli K99 were given orally with dosage of 1.76 gram IgG anti E. coli K 99/calf/day which is given at one hour before, four hours and nine hours after infection in the first day and twice a day for the next day until two days. The diarrhea observation were done at 0, 12, 24, 48, 72, and 168 hours after E. coli infection. The observation showed that diarrhea in PC group started at 12 hours after infection until finished observation. The diarrhea in SP group were started at 24 hours after infection until finished observation, but the faeces consistency were semisolid at 72 hours until finished observation. The suspension of IgG anti E. coli K99 may reduce diarrhea symptom in calves were caused by E. coli K99.

Keywords: Calves, diarrhea, Escherichia coli K99, suspension of IgG anti E. coli K99

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COMPARISON OF THREE CULTURE MEDIA IN MAINTAINING CORNEAL EPITHELIAL CELLS DERIVED FROM FOUR ANIMAL SPECIES IN-VITRO

M Kawasaki1, N Ito2, Y Okamoto3* 1 The United Graduate School of Veterinary Science, Yamaguchi University, Japan; 2 Tottori University Veterinary Medical Center, Tottori University, Japan; 3 Joint Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Japan Corresponding author: [email protected]*

Abstract Introduction: Techniques to culture corneal epithelial cells derived from animal species which are popular in veterinary ophthalmology have not been well described. Identifying species-specific optimal culture conditions would allow the use of these cells in various ophthalmic studies such as cytotoxicity assays and investigations of corneal pathophysiology unique to a particular species. Objective: To evaluate usefulness of three culture media in maintaining corneal epithelial cells derived from four animal species: porcine, canine, feline, and equine. Methods: The cells were obtained from animals which were euthanized for the reasons unrelated to the study. The cells were cultured and passaged on collagen gel with one of the three media: laboratory-mixed media with serum (A) and without serum (B), and a commercially available pre-mixed medium without serum (C). Morphology and proliferative capacity of the cells in each medium were evaluated. Results: Good cell morphology and the highest proliferative capacity were achieved in the medium A with porcine and canine cells, and in the medium B with feline and equine cells. Both cell morphology and proliferative capacity were poorly maintained in the medium C with the cells of all four species. Conclusion: The results demonstrated varying growth characteristics of corneal epithelial cells among the four species under the same culture conditions. Designing a culture environment tailored for each species would greatly help maintain cell viability more effectively and efficiently. The present study would have implications on management of corneal epithelial cells in-vitro sourced from the four animal species.

Keywords: corneal epithelial cell culture, porcine, canine, feline, equine

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MORPHOMETRIC ANALYSIS OF THE INTESTINE OF DOMESTIC QUAILS (COTURNIX COTURNIX JAPONICA) TREATED WITH DIFFERENT LEVELS OF CLAM SHELL FLOUR OF PLACUNA PLACENTA

Agus Widodo*, Lita Rakhma Yustinasari, Eka Pramyrtha Hestianah Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia *Corresponding author: [email protected]

Abstract

Twenty female quails aged 10 weeks were used and accommodated in laying cages for a further 4 weeks. We used a randomized complete design, in which basal feed was supplemented with different levels of clam shell flour of Placuna placenta: 0% (P0 as control), 2.0% (P1), 3.0% (P2), and 4.0% (P3). The birds were weighed, killed, and samples were collected from the duodenum, jejunum, and ileum then submitted to the histological routine. The sections obtained were stained in hematoxylin & eosin. For morphometric analysis, 20 villi and 20 crypts of each segment of the small intestine were measured in order to determine the height and area of the villi, as well as the depth of the crypts. The results showed that although the integrity of the small intestine was maintained in all the birds treated with the different clam shell flour of Placuna placenta levels, a clam shell flour of Placuna placenta level of 3.0% showed the most promise, as the levels of 0% and 2% did not cause any alteration in the intestinal tract. Furthermore, a clam shell flour of Placuna placenta level of 4.0% led to a significant reduction in the height of the villosities, and in consequence reduced the digestive and absorptive capabilities. Basal feed mixed with 3.0% clam shell flour of Placuna placenta is effective as a natural feed supplement (calcium source) for domestic quails.

Keywords: Placuna placenta, feed supplement, Coturnix coturnix japonica, small intestine, quail

Introduction The waste of clam shell of Placuna placenta can be utilize as byproducts optimally because it's high calcium content can be used as a natural source of calcium (Agustini et al., 2011). According to Nelson (1999), calcium deficiency causes poor egg shell quality, atrophy, less bony development of the chick, and mortality at the end of incubation. These health problems will affect growth and cause deficiency of absorption of food in the small intestine (Ye et al., 2006). The properties of the small intestine of birds are related to the histological characteristics of the mucosa in the different segments. There are several factors which influence the digestive and absorptive processes, among them the structure of the epithelium of the small intestine of birds, which have an impact on the somatic development and egg production of the animals. Moreover, most digestive and absorptive processes occur in the small intestine, and the breakdown of the nutrients is made in the lumen of the duodenum, with the help of digestive enzymes (Macari et al., 1994). Calcium absorption occurs actively in all the segments of the small intestine, especially in the duodenum and jejunum. The transport mechanisms for calcium in the wall of the enterocyte are associated with the presence of a transporting protein present

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 in the microvilli [calcium-binding protein (CaBP)]. Another process for calcium intake is provided by the ionic calcium conduction channels. In this connection, the calcium intake of the organism is related to the movement of calcium through the enterocyte membrane or through the intercellular spaces (Bronner, 1987). Objective This study was designed to check how different levels of clam shell flour of Placuna placenta (CSFP) effect the intestinal mucosa of domestic quails (Coturnix coturnix japonica). Method/Approach Basal feed was quail layer feed with ~17% protein content. It was mixed with different levels of clam shell flour of Placuna placenta (CSFP). The following concentrations were analyzed: P0 as control (Basal Feed + 0% CSFP), P1 (Basal Feed + 2.0% CSFP), P2 (Basal Feed + 3.0% CSFP), P3 (Basal Feed + 4.0% CSFP). Twenty female domestic quails (Coturnix coturnix japonica) aged 8 weeks were divided into four groups of five birds and accommodated in four laying cages. Acclimatization was conducted for two weeks. Each group was maintained for four weeks. After exactly four week treatment, the quails were weighed and sacrificed by dislocation of the neck. The duodenum, jejunum and ileum of the quails were fixed in 10% formalin, embedded in paraffin wax, sectioned at 5 µm thickness, and stained with Hematoxylin and Eosin. In order to determine the height of the villi, area, and depth of the crypts, 20 villi and 20 crypts were measured on each segment for all four groups. The crypts were measured from their base upwards to the region of transition between crypt and villus, where the epithelial layer of each hemi-crypt continues with the epithelium of the villus. The morphometric measurements were made using light microscopy (100× magnification). The length and width of villi were measured using a computer with the Image Raster program. The area of villi (AV) (µm2) was calculated using the following formula: (villi basal width + villi apical width) / (villi apical width x villi height). Measurement of crypt depth (CD) was observed in five different fields of view in one histological slide. Calculations were done on five fields of view on each crypt. The data were analyzed statistically by analysis of variance (ANOVA) followed by least significant difference and Duncan’s test at the 5% confidence level. Results The analysis of the duodenum morphology of domestic quails at day 28 treated with CSFP (Table 1) showed that length villi (LV), crypts depth (CD) and area villi (AV) duodenum has not significant differences than control group. Our investigation of jejunum morphology showed that P2 (Basal Feed + 3.0% CSFP) treatment of the length villi (LV) were significantly longer than the others. On the other hand, the crypt depth (CD) in the jejunum was not significantly different, for any CSFP-treated group, from that of the control (p<0.05). The area villi (AV) of the P2 treatment were different significantly between other treatments. Ileum morphology was different significantly for P2 (Basal Feed + 3.0% CSFP) treatment of the leght villi (LV) between the control and any of the treatment groups. Area villi (AV) and the crypts depth (CD) in the ileum were not significantly different than control and other treatment groups. Macari et al. (1994) found that the mucosa of the intestinal tract showed a decrease in the height of the villus and the crypt depth, from the duodenum towards the ileum. We observed that the birds which were administered 3.0% dietary calcium showed a significant increase in height of the jejunum and ileum villi in comparison with the birds that received 4.0 %. Discordant results were shown by Smith et al. (1990), who observed a variation in the structure of the intestinal mucosa in chickens selected for growth (or lack of it), noting that though the selected birds revealed no alteration in the height of the

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 duodenum villi they showed a 20-fold increase in the area of the villi in this segment. Such results lead us to believe that although integrity of the gastrointestinal tract was observed in all the birds treated with different calcium levels, the level of 3.0% demonstrated an more adequate adaptable effect of calcium on the intestinal mucosa in comparison with the birds which received 4.0%, which caused a significant diminution in the height of the villi, showing that an excess of calcium, probably, effects behavior of the intestinal mucosa. It is important to emphasize that there is an individual and specific level of calcium absorption in each bone, it being a hereditary factor related to the phenotypic characteristics of the domestic quail (Coturnix coturnix japonica).

Table 1. Morphometric analysis of the intestine of domestic quails (coturnix coturnix japonica) treated with different levels of clam shell flour of Placuna placenta Parameter Basal feed mixed Basal feed mixed Basal feed mixed Basal feed mixed without CSFP with 2.0% CSFP with 3.0% CSFP with 4.0% CSFP (P0) (P1) (P2) (P3) Duodenum length villi 893.94±21.96a 871.46±23.61a 862.23±35.81a 858.01±33.30a crypts depth 136.35±5.87a 129.59±5.28a 134.27±6.40a 133.81±3.93a Area villi 71727.60±4482.72a 72277.60±4287.18a 71117.60±6188.08a 71969.40±5512.58a Jejunum length villi 642.39±32.30b 616.53±6.71b 714.28±13.12c 540.15±27.10a crypts depth 99.86±10.23a 98.52±10.14a 104.51±11.42ab 103.97±20.43ab Area villi 32548.60±2011.65a 32167.40±2466.84a 39038.80±1507.26b 31870.00±1505.70a Ileum legth villi 426.97±21.51a 442.19±19.09a 503.62±13.86b 423.71±16.55a crypts depth 71.72±2.87a 71.96±2.11a 71.13±3.50a 70.47±3.95a Area villi 25796.80±1664.62a 25686.60±3072.25a 25625.20±2348.91a 24660.80±2133.89a

Conclusions Administration of a clam shell flour of Placuna placenta mixed with basal feed influences feed efficiency of domestic quails. Treatment with the 3.0% clam shell flour of Placuna placenta mixed with basal feed increased the length of villi of jejunum and ileum, and the area villi in the jejunum of domestic quails compared with the untreated control. References Agustini, T.W., A.S Fahri, I. Widowato, A. Sarwono. 2011. Pemanfaatan limbah cangkang kerang simping (Amusium pleuronectes) dalam pembuatan cookies kaya kalsium. Jurnal Pengolahan Hasil Perikanan Indonesia, 14(1): 8-13. Bronner, F. (1987) Intestinal Calcium Absorbtion: Mechanisms and Applications. Journal of Nutrition 117, 1347–1352. Macari, M., L. R. Furlan, and E. Gonzales. 1994. Fisiologia Aviaria Aplicada a Frangos de Corte. Jaboticabal: FUNEP/UNESP, pp. 187-97, 269-270 Nelson, F. E. 1999. Calcium and Phosphorus requirement for the breeding Coturnix quail. Poult. Sci., (43): 1346. Smith, M.W., M.A. Mitchell, and M. A. Peacoch. 1990. Effects of genetic selection on growth rate and intestinal structure in the domestic fowl (Gallus domesticus). Comp. Biochem. Physiol. 97A, 57-63. Ye C.X., Y.J Liu, L.X. Tian., K.S. Mai., Z.Y. Du., H.J. Yang., dan J. Niu. 2006. Effect of dietary calcium and phosphorus on growth, feed efficiency, mineral content and body composition of juvenile grouper, Epinephelus coioides. Journal of Aquaculture, 255: 263-271.

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ACTIVATION OF Α2 ADRENERGIC RECEPTORS INHIBITS TRPV1 ACTIVITIES RESULTING IN ANALGESIA

Y. Matsushita1, M. Manabe2, N. Kitamura12*, I. Shibuya12 1Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, 2Laboratory of Veterinary Physiology., Faculty of. Agriculture., Tottori University *Corresponding author: [email protected]

Abstract Most animals have endogenous mechanisms of analgesia. In the central nervous system, the descending antinociceptive system (DAS) is well known as a primary system of the pain relief and noradrenaline (NA) is one of the most important neurotransmitters in the DAS. NA activates an α2 adrenergic receptor expressed in central terminals of primary sensory neurons in the dorsal horn of the spinal cord and inhibits the release of excitatory neurotransmitters from synaptic terminals of primary neurons. Transient receptor potential vanilloid 1 (TRPV1), one of the polymodal non- selective cation channels, is mainly expressed in the primary nociceptive neurons and acts as a sensor of various types of noxious stimuli, such as heat, acid and vanilloids including capsaicin. Although it is well known that an adrenergic system and TRPV1 contribute to pain modulation, the functional association between them was unclear. In this study, we examined effects of activation of adrenergic receptors on TRPV1 activities in vivo and in vitro experiments. At first, we investigated effects of NA on TRPV1 activation by capsaicin using isolated rat dorsal root ganglion (DRG) neuronsin the whole-cell voltage-clamp recording. Capsaicin evoked inward cationic currents though TRPV1 and NA inhibited them significantly. Adrenergic receptors are classified into α1, α2 and β subtypes and NA activates all of the subtypes of adrenergic receptors. To investigate which adrenergic receptor subtype contributes to the inhibition of capsaicin -induced TRPV1 currents in DRG neurons, we examined effects of specific antagonists of α1, α2 and β receptors on the inhibitory action of NA and effects of specific agonists of them on the TRPV1 currents. Clonidine, an α2 receptor agonist, inhibited TRPV1 currents and yohimbine, an α2 receptor antagonist, reversed the inhibition of TRPV1 currents by NA and clonidine. Since it was clarified that the activation of α2 receptors inhibits TRPV1 activities in isolated DRG neurons, we investigated effects of clonidine on several kinds of pain behaviors of rats. Pain behaviors induced by capsaicin, heat and formalin were inhibited by clonidine. These results suggest that the activation of α2 adrenergic receptors inhibits TRPV1 activities at the peripheral nerve level of primary nociceptive neurons, resulting in analgesia.

Keywords: pain, TRPV1, α2 adrenergic receptor, DRG neuron

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EVALUATION OF HEMATOLOGICAL PROFILES IN THE POLICE DOGS INFECTED BY Ehrlichia canis THAT GIVEN TREATMENT COMBINATION

Leni Maylina1*, Ratyan Tri Widowati2, Febri Fadhli Zumaro2, Alyssa2, Agus Wijaya1, Sus Derthi Widhyari1, Rini Madyastuti3, Anita Esfandiari1, Retno Wulansari1, Arief Purwo Mihardi1, Jeanni Dumayanti4 1Division of Internal Medicine, Department of Clinic, Reproduction and Pathology, Faculty of Veterinary Medicine, Bogor Agricultural University (FVM-IPB University) 2Undergraduated students, FVM-IPB University 3Division of Pharmacy, Department of Clinic, Reproduction and Pathology, FVM-IPB University 4Clinic of DITPOLSATWA Kelapa Dua Depok *corresponding author: [email protected]

Abstract Introduction Ehrlichiosis, the disease is causes by Ehrlichia canis, a gram negative obligate intracellular bacteria. Ehrlichia canis infection causes anemia, leukopenia, and thrombocytopenia in dogs. Generally, the treatment method of ehrlichiosis by symptomatic based on the clinical finding. Objectives This study evaluates the hematology profiles (erythrocyte, leucocyte and thrombocyte) of five dogs that infected by Ehrlichia canis that treated using a combination of drugs. Methods Five dogs that positive of Ehrlichia canis by 4DX Snap Test® were chosen without discrimination by sex, breed, age, and abilities. They were administration by treatment using a drug combination of antibiotics (doxycycline 10 mg/kg), antiinflammation (dexamethasone 1,25 mg/kg), blood supplement (Iron Dextran 1,5 ml, Sangobion® one capsule, and Hematopan® 1,5 ml) and vitamin (Biodin® 1,5 ml). Blood analysis using hematology analyzer device, VetScan HM5®. Observed parameters include erythrocyte parameters (erythrocyte count, hematocrit rate, hemoglobin value, and erythrocyte index); leucocyte parameters (leukocyte count and leukocyte cell type such as monocyte, lymphocyte, neutrophil, basophil, and eosinophil); and thrombocyte profiles (platelet count, PCT, MPV, and PDW). The parameters was examined before (H-0), during (H-21), and after (H-42) treatment using a combination of drugs. Results The result of this study shows that the combination of drugs showed not significant changes on erythrocyte profiles. Value of Erythrocyte, hemoglobin, and hematocrit is in under normal value and increase on H-2. Erythrocyte index shown most dogs have normocytic normochromic anemia (60%), while the other dogs have microcytic hypochromic anemia (40%). The evaluation showed that the administration of drug

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 combination not significant change the parameter value of leukocyte in the dogs that infected by Ehrlichia canis before, during, and after treatment. The result of thrombocyte parameters revealed that dogs in this study have thrombocytopenia and a slight increase in PLT, PCT, MPV while the administration of drugs (H-21) but tend to be decreased in PCT, MPV, and PDW after the administration of drugs (H-42). Conclusion Therefore, a treatment with a combination of drugs is sufficient to treat ehrlichiosis.

Keywords: dogs, Ehrlichia canis, hematology profiles, treatment combination

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DESIGN OF FEED DEVELOPMENT TECHNOLOGY IN PUMICE SOIL IN NORTH LOMBOK TO REALIZE NTB BSS

Lalu Unsunnidhal1 1Faculty of Veterinary Medicine, Pendidikan Mandalika University, Mataram 83125, West Nusa Tenggara Province, Indonesia *Corresponding author : [email protected]

Abstract

West Nusa Tenggara has a dry land which is a soil based on pumice soil. On the other hand, the need for forage by ruminant animals is running low, especially in the dry season. Therefore, we need a design technology for the development of feed plants in pumice soil in North Lombok to realize NTB BSS (Bumi One Million Cow).The purpose of this activity is to determine the chemical composition of hay (a mixture of sorghum and straw from legume) with the design technology used in the field.This study used a plot experiment divided into randomized block design with three replications. This experiment consists of two factors. The two factors were arranged factorially, resulting in 15 treatment combinations for each test. Each experimental unit is processed into hay and then the chemical composition is determined. Then the data obtained, processed using analysis of variance on the basis of a randomized block design factorial pattern of 3 X 5 and if there are significant differences, followed by Duncan's multiple range test.The results showed that the use of dry land in the village of Akar-Akar as a place for planting sorghum plants with M2P4 and M3P1 technology designs produced hay with crude fiber and crude protein content that was very better than straw from sorghum grown in fertile land.

Keywords: Hay, Technology Design, Pumice Soil

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DETECTION OF HEAVY METAL LEAD (PB) IN SEPAT SIAM FISH (TRICHOPODUS PECTORALIS) MEAT IN LAKE TEMPE, SOUTH SULAWESI

A. Ayu Nur Ramadhani1, Sitti Arifah1, Erdi Purwanto2, Sharifuddin Bin Andy Omar3, Zainal Abidin Kholilullah1* 1Study Program of Veterinary Medicine, Faculty of Medicine, Hasanuddin University, Jl. Perintis Kemerdekaan KM.10, Tamalanrea Indah, Makassar, South Sulawesi 90245 2Maros Veterinary Centre, Jl. Dr. Ratulangi, Allepolea, Lau District, Maros Regency, South Sulawesi 90514 3Faculty of Marines and Fisheries Sciences, Hasanuddin University, Jl. Perintis Kemerdekaan KM.10, Tamalanrea Indah, Makassar, South Sulawesi 90245

Abstract Trichopodus pectoralis is a type of freshwater fish member of the gurami tribe found in Lake Tempe. This fish can be found in the central part of Lake Tempe which is known as the Pacok Balanda area. In the fisheries sector, pollution can occur in aquatic organisms due to heavy metals that pollute the waters. Meat is one part of fish that cannot be separated from the influence of heavy metal pollution that will cause toxic effects if it is found in the body of living things and exceeds its threshold. This study aims to determine the lead heavy metal deposit (Pb) in conjoined sepat fish (T. pectoralis) in Lake Tempe. This research was conducted in April - May 2019 at the Laboratory of Animal Food Chemistry, Department of Nutrition and Animal Feed of the Faculty of Animal Husbandry, Hasanuddin University, using a sample of nine T. pectoralis fish that had a body length of 12-18.3 cm with an average 15.32 ± 1.75 cm and body weights 28 - 82 g with an average of 54.55 ± 18.02 g. Measurement of Pb levels in fish meat using the Atomic Absorption Spectrophotometer (AAS) test method. The results showed the presence of Pb heavy metal deposits in T. pectoralis fish meat in Lake Tempe with levels ranging from 0.55 - 14.45 mg.kg-1. The conclusion of this research is the Pb content in T. pectoralis fish meat in Lake Tempe which is above the standard quality limit of metal contamination.

Keywords: Pollutants, Tempe lakes, timber, sire, Trichopodus pectoralis

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DIVERSE MORPHOTYPES OF TRYPANOSOMES IN FROGS AND TOADS FROM SURABAYA AND LOMBOK ISLAND, INDONESIA

Imron Rosyadi1,*, Muchammad Yunus2, Kholik3, Hiroshi Sato1* 1Joint Graduate School of Veterinary Medicine, Yamaguchi University 2Faculty of Veterinary Medicine, Airlangga University 3Faculty of Veterinary Medicine, Pendidikan Mandalika University *Corresponding author: [email protected]; [email protected]

Abstract Background: Species identification of trypanosomes in anurans (frogs and toads) is hampered in depth by difficulties in microscopic detection of bloodstream trypomastigotes due to extremely low levels of parasitemia, as well as evident pleomorphism of found trypomastigotes even in the same blood smear prepared from a single host individual, or the same host species. It is uncertain whether a single trypanosome species shows variable morphotypes, or superinfection of different species explains occurrence of multiple morphotypes in the same smear. Brazilian researchers (Ferreira et al. 2007, 2008) could not find any closer genetic relationships between trypanosomes of the similar morphotype, but between trypanosomes isolated from phylogenetically related anuran hosts. This may indicate a possible convergence in morphology of anuran trypanosomes evolved in phylogenetically distant hosts or in geographically remote areas. We are challenging taxonomic problems of anuran trypanosomes by examining frogs and toads collected in Indonesia where few studies have been conducted on anuran trypanosomes. Methods: In Surabaya, 20 crab-eating frogs (Fejervarya cancrivora), 17 Asian common toads (Duttaphyrynus melanostictus), and 18 flower pot toads (Kaloula baleata) were collected in February 9, 2019. On Lombok Is., 33 crab-eating frogs and 2 Asian common toads were collected on August 8, 2019. Cardiac blood was smeared on glass, and stained with Giemsa solution for microscopic observation. A few drops of the blood were kept on Whatman™ FTA™ Classical Card (GE Healthcare UK) for DNA analyses in the laboratory (partial 18S rDNA and gGAPDH nucleotide sequencing by the nested PCR). Results: Intensive microscopic observation found 17.6%—45.5% positive anurans of different groups: 7 (35.0%) and 15 (45.5%) crab-eating frogs in Surabaya and Lombok, respectively; 3 (17.6%) Asian common toads; and 6 (33.3%) flower pot toads in Surabaya. Of 31 trypanosome-positive blood smears, 21 (67.7%) contained four or less trypanosomes, demonstrating low levels of parasitemia (maximum 34 trypomastigotes/slide). Detected trypomastigotes were classified into 11 morphotypes (Fig. 1), and 2—5 different morphotypes occurred in blood smears containing 7 or more trypanosomes/slide. These morphotypes exhibited morphological resemblances to T. rotatorium, T. chattoni, T.ranarum, T.neveulemarie and T. tsunezomiyatai. Successful sequencing of three 18S rDNA fragments amplified by the nest PCR (1st round PCR

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International Symposium in Veterinary Science: Strengthening The Collaboration Between Indonesian and Japan Veterinary School, Bali Indonesia February 13th 2020 using TRY927F/TRP927R, and 2nd PCR using SSU561F/SSU561R according to Noyes et al. 1999) indicated that Indonesian isolates of anuran trypanosomes were distinct from aforementioned Trypanosoma spp. One sequence was closest to Trypanosoma sp. isolated from Asian house geckoes (MN629907, MN629900) and sandflies (MK603813, MK603816) from Thailand or Pakistan (AB520638). Perspectives: Occurrence of multiple morphotypes in a sample, or high frequency of pleomorphism of trypomastigotes have hampered taxonomic works on anuran trypanosomes. By extensive molecular genetic analyses of blood samples containing different morphotypes of trypomastigotes, as well as intensive microscopic observation, we are challenging taxonomic complications of anuran trypanosomes. Rich species diversity of anuran hosts for trypanosomes in Indonesia may give us an additional chance to understand geographical dispersion and speciation of anuran trypanosomes. Keyword: anuran trypanosome, morphotype, phylogeny, 18S ribosomal DNA gene (18S rDNA), Indonesia

M1 M2 M3 M4

M5 M6 M7 M8

M9 M10 M10 M11

Fig. 1. Morphotypes of anuran trypanosomes found in the present study (M1, T. rotatorium-like; M4, T. tsunezomiyatai-like; M5, T. chattoni-like; M8: T. neveulemarie-like; and M11: T. ranarum-like).

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PHYLOGENETIC ANALYSES OF SOME INTESTINAL FLUKES OF FROGS COLLECTED IN SURABAYA AND LOMBOK IS., INDONESIA, AND MATI, MINDANAO IS., PHILLIPINES

M. Yunus1,*, Kholik2,**, K. Kamimura3, L. A. Jimenez4, And H. Sato3,*** 1Faculty of Veterinary Medicine, Airlangga University 2Faculty of Veterinary Medicine, Nusa Tenggara Barat University 3Joint Faculty of Veterinary Medicine, Yamaguchi University 4Director, Regional ICRM Center, Davao Oriental State University * [email protected]; ** [email protected]; *** sato7dp4@yamaguchi- u.ac.jp

Abstract Phylogenetic study was conducted on some representative intestinal flukes to clarify their taxonomic positions, which has been studied mainly based on morphological criteria to date. Host frog species were determined referring to nucleotide sequences of mitochondrial 12S rRNA gene according to Kotaki et al. (2010). Collected flukes from the intestine were killed by hot water, and fixed in 10% neutral-buffered formalin (for morphological observation) and 70% ethanol (for molecular genetic analysis). 1) Glypthelmins staffordi Tubangui 1928: This species was collected from the intestine of the crab-eating frog, Fejervarya cancrivora (Gravenhorst, 1829), at 100% prevalence (20/20) in Surabaya, and at 51.5% prevalence (17/33) on Lombok Is. These frogs were collected on Feb., 9, 2019 and Aug. 8, 2019, respectively. 2) Diplodiscus magnus Srivastava, 1934: This species was collected from the intestine of the crab-eating frog at 50.0% prevalence (10/20) in Surabaya, and at 60.6% prevalence (20/33) on Lombok Is. These frogs were collected on Feb., 9, 2019 and Aug. 8, 2019, respectively. 3) Mesocoelium meggitti Bhalerao, 1927: This species was collected from the intestine of the flower pot toad, Kaloula baleata (Müller in Oort et Müller, 1833), in Surabaya at 5.6% prevalence (1/18) on Feb. 12, 2019. 4) Mesocoelium burti Fernando, 1933: This species was collected from the intestine of the giant marine frog, Rhinella marina (Linnaeus, 1758), in Mati on Mindanao Is., Philippines, at 42.9% prevalence (3/7). In the present study, morphologically-confirmed fluke specimens (four species mentioned above) were for the first time subjected to nucleotide sequencing of the ribosomal RNA genes (rDNA) over 5,800-bp length, including the 18S rDNA, internal transcribed spacer (ITS) I and II regions, 5.8S rDNA, and 28S rDNA to assess the usability of each rDNA region for fluke molecular taxonomy.

Keywords: frog, trematode, phylogeny, ribosomal RNA gene (rDNA), taxonomy

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ANTIMICROBIAL RESISTANCE OF PASTEURELLA MULTOCIDA ISOLATED FROM STRAY DOG IN MANDALIKA DOG SHELTER TO VARIOUS ANTIBIOTICS

Yumi Sartika1, Zulkarnaen1, Rahmawati2, Kholik1,3* 1 Faculty of Veterinary Medicine, Pendidikan Mandalika University, Mataram 83125, West Nusa Tenggara Province, Indonesia 2Laboratory of Public Health and Calibration, Province of Nusa Tenggara Barat. 3 Doctoral Program, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia Corresponding author: [email protected]*

Abstract Background Pasteurella species cause numerous endemic and epizootic diseases. Pasteurella multocida is a common commensal or opportunistic pathogen found in the upper respiratory tracts of Dogs. Dogs can be one of the potential reservoirs of antimicrobial resistance (AMR) bacteria that can be transmitted to humans on Kuta Beach as a tourist destination. Mandalika dogs shelter is a stray dog shelter in the special economic zone of Kuta Beach on Lombok Island. Objective The purpose of this research is to describe the resistance of Pasteurella isolated from stray dogs to various antibiotics in Mandalika dogs shelter Method Samples were taken with sterile cotton swabs from the nose of five (5) stray dogs. The samples were dipped in sterile Brain Heart Infusion Broth (BHI) and immediately they took to the Laboratory of Public Health and Calibration, Province of Nusa Tenggara Barat. they were incubated overnight in an incubator. After 24 hours the samples were inoculated in Blood Agar Plate under sterile conditions. The colonies of Pasteurella multocida were purified and characterized by standard Gram staining and biochemical methods. The Pasteurella multocida isolates were cultured using Muller- Hinton agar and incubated at 37°C for 24 h. The pure isolates of P. multocida were determined using the disk diffusion of four antibiotics (Ciprofloxacin, Tetracycline, Penicillin G, and Clindamycin) for antimicrobial susceptibility according to The Clinical Laboratory Standards Institute (CLSI) recommendations. Results The result showed that Pasteurella multocida was resistance to Penicillin G with 23 mm in diameter of zone of inhibition but P. multocida was sensitive to Ciprofloxacin, Tetracycline, and Clindamycin with 38 mm, 22 mm, and 24 mm in diameter of zone of inhibition, respectively based on CLSI interpretation. Conclusions Based on the zone of inhibition of the disk diffusion method for antimicrobial susceptibility testing, Pasteurella multocida isolated from stray dogs only resistance to Penicillin G.

Keywords: Antimicrobial resistance, Various Antibiotic, Pasteurella multocida

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BLOOD UREA NITROGEN (BUN) AND CREATININ VALUES OF MUS MUSCULUS GIVEN BY COMPOUND FOUR SPECIES OF ZINGIBERACEAE

Anggi Muhtar Pratama, Agustina Dwi Wijayanti, Dwi Cahyo Budi Setiawan, Ida Fitriana, Aria Ika Septana

Department of Pharmacology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta

Corresponden author: [email protected]

Abstract Lots of zingiberaceae family plants that are used as herbal plants. Empirically it has also been believed that people can cure various types of diseases. Four species of the zingiberaceae family that are widely used are Temulawak (Curcuma xanthorrhiza), Turmeric (Curcuma longa), Kencur (Kaempferia galanga), and Ginger (Zingiber officinale). At present there is not much information about the combination of the four ingredients, for that we need information about the safety of the use of these herbal ingredients for the body, especially on the function of the vital organs of the kidney. Total of 15 Mus musculus male, body weight 20-25 grams and aged 2 months were divided into 5 groups. Negative control group (K-) with aquades administration, positive control group (K+) by giving stimuno® in accordance with the rules of use, treatment group 1 (P1) by giving the compound of 250 mg/kgBW, treatment group 2 (P2) by giving the compound of 500 mg/kgBW, treatment group 3 (P3) by giving the compound of 1000 mg/kgBW. Animals are treated every day, then on the seven day after the treatment, plasma is taken to be observed kidney function (BUN and Creatinin values). The value of BUN (mg/dL) in (K-) 19.30±1.32, (K+) 17.47±1.29, (P1) 20.87±2.70, (P2) 18.49±0.43, (P3) 19.42±2.29 and value of Creatinin (mg/dL) in (K-) 0.50±0.10, (K+) 0.53±0.12, (P1) 0.57±0.15, (P2) 0.37±0.06, (P3) 0.33±0.12. There was an increase Creatinin value in the K-, K+, and P1 group. The concluded that the dosage of 250 mg/kgBW, 500 mg/kgBW, and 1000 mg/kgBW does not cause changes in value of BUN, but dosage of 250 mg/kgBW increase value of Creatinin. Further research still needs to be done with different dosage variations to get a safe dose to consume these herbs every day.

Keywords: BUN, Creatinin, Mus musculus, Zingiberaceae

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