detection of canine distemper virus (cdv) using reverse ...

The Proceeding of the 1st Congress of South East Asia Veterinary School Association IPB ICC, Bogor-Indonesia, July 20-22 2010

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DETECTION OF CANINE DISTEMPER VIRUS (CDV) USING REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR)

Diah Iskandriati1, Andrea Sajuthi2,3, Endang Yuliastuti3, Willy Praira1, Cucu K. Sajuthi3,

Joko Pamungkas1,4 and Dondin Sajuthi1,2,4*

1Primate Research Center, Bogor Agricultural University, Bogor, Indonesia

2Department of Biology, University of San Fransisco, USA

324 Hours Veterinary Clinic of Dr. Cucu K. Sajuthi, Jakarta, Indonesia

4Faculty of Veterinary Medicine, Bogor Agricultural University, Bogor, Indonesia

*[email protected]

Keywords: Canine Distemper Virus (CDV), RT-PCR, Diagnosis, Canidae

Introduction

Canine distemper virus (CDV) is the etiological agent of one important viral disease of Canidae. It occurs worldwide, highly contagious disease in young dogs with high morbidity and mortality. CDV is an enveloped negative-strand RNA virus classified under the genus Morbillivirus within the family of Paramyxoviridae. CDV infection can result in subclinical infection, gastrointestinal signs and/or respiratory signs, frequently with central nervous system (CNS) involvement.

Diagnosis of canine distemper (CD) in acute or subacute form had been done usually based on clinical signs and history in unvaccinated puppies. Due to the unpredictable and variable course of distemper e.g. length of viremia, organ manifestation and lack of immune responses, the final diagnosis for most animals remain uncertain. Serological diagnosis might be accomplished through detection of anti-CDV IgM, but it still pose as a problem in vaccinated dogs within 3 weeks post vaccination. In addition, diagnosis could be made through isolation of the virus in continous cell lines such as CRFK and MDCK cells. However, virus isolation takes several days to weeks as well as very laborious methods.

In this study the comparison between clinical symptom of distemper, serological assay to CDV protein and RT-PCR was conducted to get better understanding of CD diagnosis. Materials and Methods Dog specimens. Feces, saliva and nasal discharges were collected from 22 dogs with clinical symptom to CDV infection at Dr. Cucu Kartini’s veterinary clinic in Jakarta. Serological test to detect the presence of CDV protein was done in all dogs using dip-stick test from VetAll Laboratories, Korea. Samples were diluted in 1 mL of PBS buffer and filtered through a 0.45um filter. RNA Isolation. 140 uL of the filtered samples were placed into 1.5 mL centrifuge tubes and then used for RNA extraction. Total RNA was prepared using the Qiagen QIAamp Viral RNA Mini Kit following manufacturer’s instructions. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Nested PCR. Primers specific to nucleocapsid (NP) gene of CDV Onderstepoort strain was adapted from Kim et al (2001). 50 uL of One Step Reverse Transcriptase reagent (Qiagen) was added to 10 pmol/uL each of the primers and RNA template. cDNA was synthesized through incubation at 50°C for 30 min and the enzyme was denatured at 94°C for 15 min using GeneAmp 9700 Thermal Cycler followed by 30 cycles at 94°C for 30 sec, 54°C for 30 sec, and 72°C for 1 min then ended with final extension at 72°C for 10 min. PCR products were run in 2% agarose and visualized using GelDoc (BioRad, USA). In nested PCR, 1 uL of first PCR amplicon was used as template and amplified with the inner primers pair (Kim et al, 2001). PCR amplification was carried out in 30 sequential cycles at 94°C for 30 sec, 54°C for 30 sec, and 72°C for 1 min. Results and Discussion

Five hundred and forty nine base-pair (bp) fragment of NP gene can be amplified from feces, saliva and nasal discharge in samples of infected dogs. However, in most of the samples the result were inconclusive, thus the nested PCR was carried out and the 419 bp fragment was successfully amplified (Fig.1). CDV RNA can be isolated from all fecal samples from positive animals but not from all saliva and nasal discharge samples, suggesting that animal were still shedding the virus in the digestive tract. Interestingly, CDV RNA was also found in feces from animal (ID# 19) with distemper encephalitis symptom. Unfortunately no serological assay was done in this animal. PCR assay could detect viral RNA presence in two individuals in the absence of the CDV protein and vice versa in one individual (Table 1). This funding suggested that the serological assay was sensitive enough to detect the presence of viral protein in samples of infected dogs. However, confirmation test such as PCR should be done in negative samples.

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Table 1 Serological and PCR assay to Canine Distemper Virus

Animal ID

Serological test results

PCR results

Animal ID


PCR results

Animal ID


PCR results

1 + + 8 + + 15 - - 2 + + 9 + - 16 - - 3 + + 10 - + 17 - - 4 + + 11 - - 18 + + 5 + + 12 - - 19 NT + 6 + + 13 - + 20 - - 7 + + 14 - - 21 + + 22 - -

NT: not tested

Fig.1. PCR results to NP CDV (L) DNA ladder 100 bp (1) reagent control (2) CDV vaccine (3) ID# 3

Feces, (F), (4) ID#7 F, (5) ID#6 nasal discharge (ND), (6) ID#4 ND, (7) ID#5 ND, (8) ID#3 ND, (9) CDV vaccine (10) ID#2 F, (11) ID#1 F, (12) reagent control

Conclusion

Beside clinical signs, both serological tests to CDV protein and PCR assay for CDV RNA can be used as a confirmatory diagnosis for canine distemper in infected dogs. In this study the presence of CDV RNA in the feces can be found in all infected dogs even the one with encephalitis distemper. References Frisk, A.L., König, M., Moritz, A., Baumgärtner, W. Detection of canine distemper virus nucleoprotein RNA

by reverse transcription-PCR using serum, whole blood, and cerebrospinal fluid from dogs with distemper. J Clin Microbiol 1999, 37, 3634-3643.

Kim, Y.H., Cho, K.W., Youn, H.Y., Yoo, H.S., Han, H.R. Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR. J Vet Sci. 2001, 2, 59-63.

Lan NT, Yamaguchi R, Kien TT, Hirai, T, Hidaka Y and Nam NH. First isolation and characterization of Canine Distemper Virus in Vietnam with the Immunohistochemical Examination of the Dog. J Vet Med Sci. 2009, 71(2), 155-162


161

DETECTION OF HOTSPOTS AREA IN MALARIA SURVEILLANCE IN BANGKA DISTRICT, INDONESIA

Etih Sudarnika1, Mirnawati Sudarwanto1, Asep Saefuddin1, Umi Cahyaningsih1, Upik Kesumawati

Hadi1, Rita Kusriastuti2, Jodi Vanden Eng3, William A. Hawley4

1Faculty of Veterinary Medicine, Institut Pertanian Bogor, Jl. Agatis Kampus IPB Darmaga Bogor 16680,

West Java, Indonesia 2Directorat of Zoonosis, General of Disease Control and Environment Health, Ministry of Health of

Indonesia, Jakarta, Indonesia 3Centers for Disease Control and Prevention, Atlanta, Georgia, USA

4United Nations Children’s Fund, Jakarta, Indonesia

Keywords: hot spots, malaria, prospective space-time scan statistics, spatial statistics, surveillance Introduction

Indonesia is one of the malaria endemic countries. According to malaria endemicity map of Indonesia year 2007, it is estimated 45% of Indonesia population is living in high risk of malaria contagion area. One of the endemic areas in Indonesia is Bangka District in Bangka Belitung Province. Bangka District is categorized as medium level for Malaria endemicity with represented by annual malaria incidence (AMI) of 29.3 of 1000 people in 2007 (Ministry of Health, 2008). Surveillance activity is one of the important events to eradicate and control malaria. From this surveillance activity it is expected could establish a preventive action in a quick response especially in certain malaria hotspot area at a certain time to control the diseases. Methods

Data of malaria cases was collected from laboratory log book at all health centers in Bangka District, in the period of June June 2007 until July 2008. Definition of malaria case was who tested positive by examination of Plasmodium parasites in the laboratory. Clinical examination and dignontic rapid test are not recorded as a case. If in one community health center work area was found a case of malaria which originated from other community health center work area, then the case was put in the list of patient domicile address. Geographical coordinates of health centers were recorded by GPS (Global Positioning System). Demography data was obtained from BPS (National Bureau of Statistics) and Bappeda of Bangka District. It was assumed that all Bangka residents were in risk of malaria contagion. Geospatial analysis used was prospective space-time scan statistics developed by Kulldorff, 1997. The data were assumed spreading with Poisson distribution. Data were analyzed using SatScan version 7.0. Mapping was done using ArcGIS version 9.3.1 (ESRI, Redlands, CA, USA). Results and Discussion

Malaria incidence (annual parasites incidence/API) varied according to work area of Community Health Center and observation period. During the research time, the overall malaria incidence in Bangka District was 1.78%. The highest incidence rate was in Sinar Baru Community Health Center which had 3.45%, followed by Kenanga Community Health Center with 3.14%. Both Community Health Center were having work area surrounded by beaches, which was a potential area for Anopheles mosquito. Surveillance result during observation period was spatially presented in a monthly malaria incidence rate for each community health center shown in Figure 1. A darker color gradation is showing increment of a higher malaria incidence rate. From Figure1, it was shown that in initial stage of research Belinyu Community Health Center was having the highest malaria incidence rate, but then gradually decreasing towards the ending of observation period. On the other hand, Bakam area was having low malaria incidence rate at initial stage, but then increasing towards the ending of observation period.

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Figure 1 Monthly malaria incidence rate of each community health center in Bangka District.

Using the prospective space-time scan statistics, for observation period of June 1st, 2007 to July 31st, 2008, time aggregation length was 1 month, maximum spatial cluster size was 50% of population at risk, and maximum temporal cluster size was 50% of study period, results were shown in Table 1.

Table 1 Detection of recently emerging clusters of malaria in Bangka

Most likely

cluster Cluster period Cases Expected Relative

risk p

Primary cluster: Sinar Baru 2008/1/1 -

2008/7/31 187 56.76 3.455 0.001

Secondary cluster:

Kenanga 2008/1/1 - 2008/7/31

133 53.05 2.581 0.001

Bakam 2008/3/1 - 2008/7/31

93 68.12 1.378 0.145

From Table 1, it was shown that the first emerging cluster was Sinar Baru Community Health

Center with cluster period January 1st, 2008 to July 31st, 2008 with relative risk value of 3.455; the second emerging cluster was Kenanga Community Health Center with 133 case and relative risk value of 2.581; the next emerging cluster was Bakam Community Health Center but showing no significant p values. Conclusion

The most likely emerging cluster is Sinar Baru with time frame during January 2008 until July 2008. The secondary emerging clusters are Kenanga with time frame during January 2008 until July 2008 and Bakam with time frame during March 2008 and July 2008, but not significant for Bakam. Sinar Baru and Kenanga were the areas which need a further investigation and priority in the control. Acknowledgements

We thank the people of Bangka District, District Health Service officers, and their local authorities for their excellent cooperation. We would also like to thank Ministry of Health, Republic of Indonesia for the support, Center for Disease Control and Prevention (CDC) and UNICEF for financial support. Lastly, words of thanks for all the team members. References [MOH RI] Ministry of Health, Republic of Indonesia. 2008. Indonesian Health Profile. Jakarta: MOH RI. Kulldorff M. 1997. A Spatial Scan Statistic. Communs. Statist. Med., 26. 1481-1496.


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BIOSECURITY MEASURES ON BROILER FARMS IN SUBANG, WEST JAVA INDONESIA

Etih Sudarnika1, Y Ridwan1, AZ Ilyas 1, C Basri 1, DW Lukman1, T. Sunartatie1, BA Wibowo 1, A Sugama2, P Hermans3, AJ Nell 4

1 Department of Animal Infectious Diseases, Faculty of Veterinary Medicine, Bogor Agricultural University

2 District Livestock Services, Subang District, West Java, Indonesia 3Central Veterinary Institute, part of Wageningen University Research Centre, Lelystad, The Netherlands

4Centre for Development Innovation, Wageningen University and Research Centre, Wageningen, The Netherlands

Keywords: biosecurity, sector 3, broiler farm

Introduction

Poultry farms can be categorized into four sectors according to a classification system developed by the FAO (FAO, 2004). Sector 3 and 4 farms, by definition, have lower levels of biosecurity than farms belonging to sector 1 and 2. Therefore, poultry farms in sector 3 and 4 have a higher potential risk for acquiring and transmitting disease, including Highly Pathogenic Avian Influenza (HPAI). As part of a larger collaborative multi-intervention pilot project in Cipunagara Sub-district of Subang District, the Bogor Agricultural University, the Livestock Services of Subang and the Indonesian-Dutch Partnership on HPAI control are designing interventions to raise biosecurity levels of sector 3 farms in this sub-district. However, relatively little is known about the present biosecurity levels on these farms. The aim of this study therefore was to provide information on the present biosecurity measures which are in place on sector 3 farms in Cipunagara and to measure farmers’ attitudes and opinions towards biosecurity improvements. Methods

A cross-sectional survey was carried out from January 18th until January 24th, 2010. The target population was sector 3 poultry farms in Cipunagara Subdistrict, Subang District, West Java. There were 25 sector 3 poultry farms in Cipunagara at the time of the survey which were all broiler farms. It was decided to sample the entire target population of 25 farms. Data were collected by means of a questionnaire which was modified from an existing questionnaire developed by the Australian Centre for International Agricultural Research (ACIAR). Respondents were farm owners or farm managers. The questionnaire was administered during a face-to-face interview and contained questions about existing biosecurity measures, farm infrastructure, farm management, poultry health and productivity as well as farmer’s knowledge of biosecurity and their opinion about the ease of implementation of biosecurity measures. Questions pertaining to biosecurity could be grouped into the three principles of biosecurity as defined by the FAO (FAO, 2008), namely traffic control (2 questions), sanitation (15 questions) and isolation (23 questions). The proportion of the biosecurity measures which were applied on each farm out of the total number of biosecurity measures addressed by the questionnaire (40 items) was calculated and expressed as a percentage. Farmers’ opinions about the ease of implementation of biosecurity measures was scored on a scale of 1 to 3 with 1 being not very easy and 3 being very easy to implement. The proportion of farmers’ opinions falling in scores 1, 2 or 3 for a variety of biosecurity measures was then calculated. Data were analyzed using Microsoft Office Excel 2007. Results and Discussion

The proportion of biosecurity measures which were applied by poultry farms in sector 3 in Cipunagara Subdistrict out of the total of 40 biosecurity measures addressed by the questionnaire is presented in Table 1.

Table 1 shows that the level of biosecurity on poultry farms in sector 3 is still far from optimal. Out of the total of 40 biosecurity items which were addressed in the questionnaire, a maximum of 23 (57.5%) items were present on one farm. An average of 16.88 (42.2%) biosecurity items were present on the surveyed farms. The least number of biosecurity measures which were present on the farms were related to traffic control. Only one farm (4%) had a sign instructing visitors to report before entering the farm and not a single farm had a visitor registration system in the form of a log book. The highest number of biosecurity measures which were present on the farms were related to isolation (segregation) with an average of 18.4 (46.1%) items and a maximum of 26 (65.2%) items being present.

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Table 1 The number and percentage of biosecurity practices applied at poultry farms in sector 3 in Cipunagara, Subang, out of the total number of biosecurity measures addressed by the questionnaire (n=40)

Principles of biosecurity

Number of items

Minimum Average Maximum

Traffic Control 2 0 (0.0%)

0.04 (2.0%)

1 50.0%

Sanitation 15 4 (26.7%)

6.56 (43.7%)

9 (60.0%)

Isolation 23 7 (30.0%)

10.28 (45.0%)

14 (61.0%)

Over all 40 12 (30.0%)

16.88 (42.2%)

23 (57.5%)

Twenty four farmers (96%) separated sick birds from healthy birds and burned or buried dead

birds, whereas all 25 farms indicated that they never allowed unsold poultry to return from the market to the farm and 17 (68%) farms practiced an all in all out system. Nevertheless, none of the farms controlled the access of wild birds, rodents, or insects into the poultry shed or had strict measures to keep other poultry or domestic animals away from their flock. Most farms (18 farms; 72%) did not have fences, 20 (80%) farms did not have gates, 21 (84%) farms did not have locks on gates, 21 (84%) farms did not have warning signs in front of the farms and sheds, and not a single farm had a hygiene barrier in front of sheds. In terms of sanitation measures, almost all (21 farms; 84%) provided hand washing facilities for workers, cleaned (with water and soap) and disinfected the sheds regularly after finishing the production cycle, and 17 (68%) farms changed the litter at least two times in a cycle. All of the farms sourced their water either from a well or from the tap. Nevertheless, other hygiene facilities were lacking such as just 12 from 32 (37.5%) sheds had foot baths in front of the sheds, just 1 (4%) farm had special clothes and 7 (28%) farms had boots to wear while handling the poultry, and just 4 (12.5%) sheds had warning signs for workers or visitors reminding them of proper hygienic standards. In addition, there was open water on 13 (52%) of the farms which can be an attractant for wild fowl, insects and rodents.

The biosecurity measures which all farmers considered most easy to implement were burning of dead chickens, burying dead chickens and removing the manure from the house. In contrast, the biosecurity measure which the majority of farmers (84%) found not very easy to implement was showering at the main entrance. Conclusion

The majority of farms in this survey were independent farms with limited or no supervision by contract companies and/or government. This could partly explain their mediocre biosecurity status. However, the fact that the majority of farmers considered almost all of the addressed biosecurity measures very easy to implement, suggests that the deficiencies in biosecurity which were found in this survey are more a result of a perceived lack of urgency than because of structural or financial constraints. This leaves room for an educational and supervisory role for the local district livestock services. Acknowledgements

We thank the broiler farmers which took part in this survey and the Subang District Livestock Service Officers for their excellent cooperation. We would also like to thank all team members who assisted in the collection and the processing of the data. References [FAO, 2004] Recommendations on the Prevention, Control and Eradication of Highly Pathogenic Avian

Influenza in Asia. FAO Position Paper, September 2004. Rome, Italy [FAO, 2008] Biosecurity for Highly Pathogenic Avian Influenza. Issues and Options. Rome, Italy


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PRODUCTION AND ISOLATION OF IgY ANTI-Fasciola gigantica EXCRETORY SECRETORY ANTIGEN FROM YOLK EGGS

Fadjar Satrija1, Sri Murtini2, Yusuf Ridwan1, Elok B. Retnani1

1Division of Parasitology and Medical Entomology, 2Division of Medical Microbiology, Department of Animal Diseases and Veterinary Public Health, Faculty of Veterinary Medicine, Bogor Agricultural

University, Jl. Agatis Kampus IPB Darmaga-Bogor, Indonesia

Keywords : Fasciola gigantica, ES antigen, IgY, Yolk egg Introduction

Liver fluke (Fasciola gigantica) is one of the most economically important parasite of ruminants in Indonesia. Conventional coprological examination is only able to detect liver fluke infection as early as 6 week post infection (p.i.) when the worm reached its maturity. In this stage the infection may already cause pathological changes, and shedding trematode eggs to pasture. Therefore it is an urgent need to develop a diagnostic tool that able to detect early stage of liver fluke infection. Objective of the present study was to raise polyclonal antibody (IgY) in chicken yolk egg to detect F. gigantica excretory-secretory (ES) antigen.

Materials and Methods

Antigen production Adult liver flukes were collected from liver of a buffalo slaughtered at Bekasi Slaughter House.

Fasciola gigantica excretory/secretory antigen was prepared by a similar method described by Estuningsih et al (2004). The liver flukes were washed four times at room temperature with 0.9% sodium chloride to remove all contaminants. The worms were subsequently incubated for 4-6 hours at 37oC in RPMI 1640 medium supplemented with 100 IU penicillin and 100 µg streptomicin 100 µg per ml of the medium. After incubation the medium was centrifuged (1,500 g for 10 minutes at 4 oC), The supernatant was collected and its protein concentration was measured using Bradford method. The antigen was store in aliquots at -20 oC until used.

Polyclonal antibody production Polyclonal antibody was raised in four 30 weeks-old Loghmann Brown chicken layers. The chicken

were each immunized with 150 µg F. gigantica excretory/secretory (E/S) antigen through intravenous injection, and then subcutaneous injection of the E/S antigen in Freund’s complete adjuvant (1:1) one week later. This was followed by two boosts at three week intervals with same doses of the E/S antigen in Freund’s incomplete adjuvant (1:1). Presence of polyclonal antibody in blood was monitored by Agar gel precipitation test (AGPT) on serum samples collected three week after the last booster onwards. After a positive reaction was found in serum sample, all eggs laid from that day were stored in refrigerator to be used in IgY purification.

The IgY anti F. gigantica excretory/secretory (E/S) were purified from yolk eggs using IgY antibody purification kit (EGGstract® IgY Purification System, PROMEGA). Capability of polyclonal antibodies in the yolk eggs and purified IgY fractions to detect F. gigantica (E/S) antigen was determined using Agar gel precipitation test (AGPT). The polyclonal antibody was also tested against E/S antigens of other ruminant helminthes, i.e. Paramphistomum cervi (Trematode), Moniezia sp (Cestode), and Haemonchus contortus (Nematode). Results and Discussion

Antibody against F. gigantica ES antigen was detected in the chicken sera from week 8, whereas antibody in the yolk appeared from week 9. Appearance of the specific antibody to F. gigantica ES antigen in present experiment was four weeks faster than those recorded by Estuningsih (2006) in rabbit.

Both IgY in yolk eggs and The purified IgY showed positive reaction to ES antigens derived from F. gigantica recovered from buffalo and sheep indicating that the polyclonal antibody can detect liver fluke from both host species (Figure 1). No cross reaction was found between IgY anti F. gigantica with ES antigens of other ruminant helminthes, i.e. Paramphistomum cervi (Trematode), Moniezia sp (Cestode), and Haemonchus contortus (Nematode), suggesting high specificity of the polyclonal antibody (IgY) to F. gigantica E/S antigen.

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Figure 1. Agar gel precipitation test (AGPT) shows precipitation lines between antibodi (IgY) anti ES F. gigantica in yolk eggs (A), or purified IgY (B) and F. gigantica E/S antigen of adult liver fluke worms from buffalo (2) and sheep (3). No precipitation lines appear between the IgY antibodies and E/S antigens of Paramphistomum sp (4), Moniezia sp (5), H. contortus (5), -Bunostomum sp (6). Control PBS (1)

Conclusion

The polyclonal antibody (IgY) anti F. gigantica E/S showed high specificity to the antigen therefore potential to be used as capture antibody in ELISA for detection of F. gigantica coproantigen.

References Estuningsih SE, Widjajanti S, Adiwinata G, Piedrahita D. 2004. Detection of coproantigens by sandwich

ELISA in sheep experimentally infected with Fasciola gigantica. Trop. Biomed. Supplement : 51-56. Estuningsih SE. 2006. Diagnosa infeksi Fasciola gigantica pada sapi dengan uji capture-ELISA untuk

deteksi antigen dalam feses. Jurnal Ilmu Ternak dan Veteriner. 11: 229-234.

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A MACROSCOPIC STUDIES ON INHIBITORY EFFECT OF ETHANOL EXTRACT OF CURCUMA ZEDOARIA (BERG.) ROSCOE. RHIZOMES ON TUMORIGENESIS OF MAMMARY GLAND

WHICH INDUCED BY N-METIL-N-NITROSOUREA IN RABBIT

Gunanti1, Bambang Pontjo Priosoeryanto2, Ietje Wintarsih3, Ros Sumarny4, Janto Dwi Haryadi

1Division of Surgery and Radiology, Faculty of Veterinary Medicine, Bogor Agricultural University 2Division of Pathology, Faculty of Veterinary Medicine, Bogor Agricultural University 3Division of Pharmacy, Faculty of Veterinary Medicine, Bogor Agricultural University

4Division of Pharmacology, Faculty of Pharmacy, Pancasila University, Jakarta 3Student of Faculty of Veterinary Medicine, Bogor Agricultural University

Introduction

The incidence of mammary tumors commonly found in dog (Wilson 2006). So long as treatment is directed to the improvement of animal health (Wilson 2006). Another step that can achieved by surgical removal of tumors, radiation and chemotherapy, but these measures have some weaknesses. (Wilson 2006). Indonesia has many plants that can utilized for treatment. One of the natural materials used to inhibit tumor growth is temu putih (Curcuma zedoaria (Berg.) Roscoe) (Syukur & Hernani 2002). To evaluate the efficacy of temu putih in inhibiting tumor cell growth in research done with rhizomes ethanol extract of temu putih on rabbit induced with chemical carcinogen n-methyl-n-nitrosourea. The effectiveness of tumor inhibition by ethanol extract of temu putih can be evaluate by the observation of macroscopic tumor growth in mammary gland induced with n-methyl-n-nitrosourea (MNU).

Materials and Methods

Rabbits were used divided into three groups, each group consisted of three rabbits. Group I rabbit only induced with tumor. Group II rabbits induced tumors and treated with ethanol extract of temu putih. Group III rabbits induced tumors and treated with curcumin. Preparation of ethanol extract of rhizome temu putih (extraction) begins with the process of maceration. Maserat which have been obtained and then evaporated. The result of evaporation used for tumor therapy on this study.

Tumor induction was done in mammary gland number two on both sides (intra mammary), three times in the first, second and third week. MNU doses were used 50µg/kg body weight. Ethanol extract of temu putih was dissolved in propylene glycol. Ethanol extract of temu putih and curcumin given by using stomach tube, it was given every day during therapy (four weeks). Temu putih was given on dose of 250mg/kg body weigth (Murwanti et al. 2004), while curcumin was given on dose 60mg/kg body weight. Tumor growth was observed by measuring tumor diameter. Measurements was done once a week for four weeks of treatment. The measurement was done by first marking the gland tumors mammary, then measure the diameter of the circle using a ruler. Data obtained mammary gland diameter calculated and averaged. Then compared among the treatments.

Results and Discussion

In the positive control group which performed only with MNU without inhibiting either done by using the ethanol extract of temu putih or using curcumin. Mammary gland diameter in the positive control group tumors was increased in the second week, increased again in the third week and decreased in the fourth week, this happens because induction of MNU conducted for three times, in the first week, second week and third week. Mammary gland diameter measured on the positive control group can be used as a reference for comparison to investigate the inhibition that occurred in the group treated with ethanol extract of rhizome white Intersection or with curcumin administration (Figure 1).

Figure 1 Diameter mammary glands of rabbits induced n-methyl-n-nitrosourea after four weeks. Information: Diameter gland mammary group of rabbits was only induced by tumors (a), groups of rabbits induced tumors and treated with curcumin (b), tumor-induced rabbit group and treated with ethanol extract of temu putih (c).

a b c

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Figure 2 shows the average diameter of mammary glands of rabbits induced by MNU are measured each week in each treatment group. Inhibition of tumor formation can be seen in the group treated with curcumin. Mammary gland diameter measured in the groups given curcumin had a smaller average than those, which were not given any therapy. Based on research that has been done curcumin had inhibitory effect tumor formation. Curcumin has been investigated to suppress proliferation of cancer cells through a mechanism to induce apoptosis (Murwanti et al. 2004).

In the group treated with ethanol extract of temu putih show the differences in tumor size when compared to untreated group. Diameters measured in the group treated with ethanol extract of temu putih lower than the untreated group. The ethanol extract of temu putih contain curcuminoids retrieval, essential oils and polysaccharides. Curcuminoids include curcumin (Murwanti et al. 2004).

Figure 2 Comparison of the average diameter of glands mammary rabbits each treatment group after MNU

induced every week.

In the group treated with ethanol extract of temu putih has a higher average when compared with the group given curcumin, except in the fourth week of treatment group met the white rhizome ethanol extract has a lower average tumor diameter compared with curcumin group. This occurred because the substance contained in the ethanol extract of temu putih, one of which is curcumin. Percentage of curcumin contained in the ethanol extract of rhizome white Intersection lower than curcumin, which is extracted from curcuma, so that tumor growth inhibition caused by curcumin was greater when compared with tumor growth inhibition by ethanol extract of temu putih.

Conclusion

From all the results of tests carried out by macroscopic against tumors growing in diameter, it can be concluded that the activity of ethanol extract of rhizome white retrieval can inhibit tumor growth.

References

Murwanti R, Edy M, N and Susi Arief AK. 2004. Effects of Ethanol Extracts Rhizome Temu Putih

(Curcuma zedoaria ROSC.) Against Lung Tumor Growth in Mice Phase Females Post Initiation Induced by benzo [a] pyrene. Indonesian Pharmaceutical Magazine, 15 (1), 7-12.

Wilson LM. 2006. Growth Disorders Prolifrasi, and differentiation cell. In Depth: The concept of Clinical Pathophysiology Disease Processes. 6th ed. U. Brahm Pendit, translators. Huriawati Hartanto, editor. Jakarta: EGC. Translation of: pathophysiology: Clinical Concepts of Desease Prosseses.


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THE EFFECT OF ZEDOARIAE RHIZOME ETHANOL EXTRACT (CURCUMA ZEDOARIA (BERG.) ROSCOE) TOWARD CLINICAL SIGNS OF PRE AND POST SURGERY IN TUMOR INDUCED RABBIT

Gunanti1, Rini Madyastuti Purwono2, Heryudianto Vibowo3,

Bambang Pontjo Priosoeryanto4, Ros Sumarni5

1Division of Surgery and Radiology, Faculty of Veterinary Medicine, Bogor Agricultural University 2Division of Pharmacy, Faculty of Veterinary Medicine, Bogor Agricultural University

3Student of Faculty of Veterinary Medicine, Bogor Agricultural University 4Division of Pathology, Faculty of Veterinary Medicine, Bogor Agricultural University

5Faculty of Pharmacy, Pancasila University

Introduction

The tumor is a degenerative disease that can infect humans and animals. Treatment is generally done, both on humans and animals is to use chemical drugs that also give side effect. To anticipate the medication has side effects and expensive treatments, human began to develop an alternative treatment. Treatment is by using materials of plant origin which have medicinal properties. One of the plants commonly used drugs or has a property is a plant from a family gathering, one of them is white Intersection. White Intersection has antioxidant properties that can withstand the rate of growth of cancer cells, substance antiinflammation and increase the amount of red blood cells. One animal that is widely used as laboratory animals are rabbits. Economic considerations, size, ease of control and blood sampling and preparation makes the rabbit is used as animal models for tumor research and testing of medicinal products. Also rabbit has the so that appropriate for use in an investigation. Besides rabbits also had a relatively mammari gland size larger than mice and rats so that the tumor is expected to be more obvious and larger.

Materials and methods

Tumor induction mammari was performed on the second mammary gland right and left. Inoculation is done by way intramammari directed perpendicular to the mammari gland. Inoculation is done every once a week for three times. Injected dose taken is 50 µg/kg. Clinical examinations (body temperature, heart and respiratory frequency) carried out every once a week for nine weeks of treatment. The action taken is preoperative clinical observations such as counting the frequency of breathing, heart frequency and body temperature. Operations carried out by performing a mastectomy and ovariohisterektomi. Mastectomy surgery performed incision ellipse encircled tumor tumor, prepared from the surrounding tissue, then the tumor was taken (Bubenik 2006). Last sutured with cat gut size 4/0 to the network and the skin using silk thread size 3/0. For ovariohisterektomi operations conducted with the appointment of the ovary and uterus through the posterior median laparotomy (Gunanti 2001). The incision carried posteriorly along umbilikal ± 5 cm (Bubenik 2006). Animals reared and given antibiotics Novamox G ® at a dose of 50mg/ 50kg body weight by intra-muscular for five days. Bandages changed every day. On the seventh day the wound was dry and the skin stitches are opened. Results and Discussion

The observation of body temperature showed no significant difference between group A with group B and C in the pre surgery (p <0.05) and post surgery (p <0.05), and still within the normal range 38.5-40.0oC (Carpenter 2003). Results are shown in Figure 1 shows that in group B and C show the state of pre temperature operation that is higher than post-surgery. Post operative observations of temperature in group B and C are not significantly different from group A (P> 0.05). Cardiac frequency observations reveal a significant difference between groups B and C with group A pre surgery (P <0.05) although still within the normal range 130-325x/minute (Carpenter 2003). Post surgery showed no significant difference between groups B and C with group A (P <0.05). Figure 2 shows that the groups B and C showed a decline compared to the heart frequency during the pre surgery. In the figure 3 shows no significant difference in respiratory frequency between groups C and A (P <0.05) whereas in group B showed no significant difference with group A (P <0.05) but there was an increase compared with group A. Post operative respiratory frequency shown in Figure 3 indicating the absence of significant differences between groups B and C than group A (P <0.05). Figure 4 shows the group B and C, respiratory frequency decreased during post operative than during the pre surgery.

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Figure 1 The mean body temperature of rabbits before surgery (1-5 weeks) and after surgery (6-9 weeks)

in each treatment group. the ethanol extract of zedoary and curcumin has the activities as an anti-inflammatory (Murwanti et al. 2004). Curcumin which is also contained in the ethanol extract of zedoary retrieval works by inhibiting cyclooxygenase enzymes, which then inhibits the formation of acid prostanoid arachidonat. Prostanoid which inhibited the formation of prostaglandins that will lower the set point temperature in the hypothalamus would decrease (Yoshioka et al. 1998). Also according to Murwanti et al. (2004) Intersection of white have activities to inhibit the biosynthesis of leukotrienes and inhibit the action of the enzyme 5-lipooksigenasi arachidonat. Although there was a decrease from group B and C to group A, the ethanol extract of zedoary and curcumin can maintain body temperature within normal range (38.5-40.0oC (Carpenter 2003)). Curcumin and zedoary ethanol extract also has an effect to inhibit the enzyme prostaglandin synthetase, biosynthesis of leukotrienes and the action of the enzyme 5-lipooksigenase arachidonat that will reduce the formation of prostaglandins that have an effect on the decrease of cardiac frequency (Murwanti et al. 2004). Prostaglandins inhibition would lower the set point temperature on the hypothalamus (Yoshioka et al. 1 998). Simultaneously with the decrease in body temperature then the respiratory frequency will come down. The decrease seen in the figure 4 shows that administration of curcumin and zedoary ethanol extract can maintain the frequency of breathing in the normal scale of 30-60 breaths / min. (Carpenter 2003).

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Conclusion From the research that has been done can be concluded that ethanol extract of rhizome white

Intersection able to maintain body temperature, heart and respiratory frequency in the normal range during the pre and post operation on MNU-induced rabbits. References Bubenik LJ. 2006. Small Animal Surgery Nursing. Di dalam: McCurnin MC, Bassert JM, editor. Clinical

Textbook for Veterinery Technician. , St. Louis: Elsevier Saunders. Carpenter JW. 2003. Lagomorpha. Di dalam: Miller F, editor. Zoo and Wild Animal Medicine. Ed ke-5. ,

St. Louis: Saunders. hal 410-419 Gunanti. 2001. Metode Laparotomi dan Gambaran Persembuhan Pasca Bedah Reproduksi untuk Koleksi

Oosit dalam Upaya Produksi Embrio In vitro pada Kucing Lokal (Felis domestica) [disertasi]. Guyton AC, Hall JE. 1996. Textbook of Medical Physiology. Philadelphia: W. B. Saunders Company. Murwanti R, Meiyanto E, Nurrochmad A dan Kristina SA. 2004. Efek ekstrak etanol rimpang temu putih

(Curcuma zedoaria Rosc.) terhadap pertumbuhan tumor baru fase post inisiasi pada mencit betina diinduksi Benzo[a]piren. Majalah Farmasi Indonesia, 15 (1), 7-12

Yoshioka T, Fujii E, Endo M, Hohsho H, Shibuya H dan Uraki T. 1998. Antiinflammatory potency of dehydrocurdione, a zedoary-derived sesquiterpene. Inflamm Res, 47(12): 476-481.


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THE PRESENCE OF NUCLEAR MATERIALS DURING MEIOTIC MATURATION OF RECIPIENT OOCYTES AFFECT DEVELOPMENTAL ABILITY OF SOMATIC CELL NUCLEAR TRANSFER

EMBRYOS IN PIGS.

M. Fahrudin1, K. Kikuchi2, NWK karja3, , M. Ozawa2, T. Somfai2, M. Nakai2, J. Noguchi2, K. Ohnuma2, H. Kaneko2 and T. Nagai4

1Department of Anatomy Physiology and Pharmacology, Faculty of Veterinary Medicine, Bogor

Agricultural University, Bogor 16680, Indonesia 2Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba,

Ibaraki 305-8602, Japan 3 Department of Clinic Reproduction and Pathology, , Faculty of Veterinary Medicine, Bogor Agricultural

University, Bogor 16680, Indonesia 2Department of Research Planning and Coordination, National Institute of Livestock and Grassland

Science, Tsukuba, Ibaraki, 305-0901, Japan

Introduction

In somatic cell nuclear transfer (SCNT) maturation promoting factor (MPF) is believed to be one of the factors involved with nuclear envelope breakdown and chromatin condensation of the transferred nucleus. Although MPF activity is high both in metaphase-I or -II oocytes (M-I and M-II, respectively), only M-II oocytes have been used exclusively as recipient cytoplasts in SCNT. In this study, we examined the effect of nucleus removal during meiotic maturation on the ability of cytoplast to support development of reconstructed embryos to the blastocyst stage after fusion with cumulus cells. Materials and Methods Collection of Follicular Oocytes and In Vitro Maturation

Collection and maturation of porcine oocytes were carried out as reported previously (1). Briefly, porcine ovaries obtained from a slaughterhouse were transported to the laboratory at 37�C in Dulbecco‘’s phosphate-buffered saline (PBS; Nissui Pharmaceutical Co. Ltd., Tokyo). Follicles were sliced with a surgical blade in TCM-199 (with Hanks’ salts) supplemented with 5% (v/v) fetal bovine serum (FBS; Gibco, Invitrogen Corp., Grand Island, NY), 20 mM HEPES, 100 IU/ml penicillin G potassium and 0.1 mg/ml streptomycin sulfate, and the cumulus oocyte complexes (COCs) were collected. Following briefly washing in a modified NCSU-37, approximately 50 COCs were cultured for 20-22 h in 500 µl of the maturation medium, a modified NCSU-37 solution (2) containing 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 50 µM ß-mercaptoethanol, 1 mM dibutyryl cAMP (dbcAMP), 10 IU/ml eCG (PMS, Nihon Zenyaku Kogyo, Koriyama, Japan), 10 IU/ml hCG (Puberogen, Sankyo, Tokyo, Japan) and antibiotics, in four-well dishes (Nalge Nunc International, Denmark). They were then cultured in the maturation medium without dbcAMP and hormones for another 26-28 h. Maturation culture was carried out at 39�C under 5% O2 with CO2 and N2 adjusted to 5% and 90%, respectively. Preparation of Recipient Cytoplasts

Metaphase-I and –II oocytes obtained at 30 and 48h of IVM culture were enucleated with the gradient centrifugation method (3). In brief, a group of 100 cumulus-free oocytes were centrifuged in 1.5 ml microcentrifuge tube at 13,000 × g for 9 min in TCM-199 to stratify the cytoplasm. The zona pellucida was then removed by an exposure to 0.5% pronase in TCM-199 for 2-3 min, followed by gently pipetting to remove remnant zona pellucida. A group of 50 zona-free oocytes were then layered on a 300 µl discontinuous gradient (100 µl of 45, 30, and 7.5% (v/v), respectively) of Percoll (Amersham Biochem) in TCM-199 supplemented with 5 µg/ml cytochalasin B. These gradients were then centrifuged at 5,000 g for 4 sec. Oocyte cytoplamic fragments (OCFs) over 60 �m in diameter were selected and were stained with 5 µg/ml Hoechst 33342 (Calbiochem) and then examined under an epifluorescence microscope to selected enucleated fragments (cytoplasts). Embryo Reconstruction and In Vitro Culture

Donor somatic cells were obtained from primarily culture of cumulus cells in wells of four-well dishes in 400 µl Dulbecco’s modified Eagle medium/F12 (Gibco, Invitrogen Corp.) supplemented with 5% FBS. Following harvested the cumulus cells with standard trypsinization, the cumulus cells were seeded into the middle of four-well dishes at very low concentration (until somatic cells layered singlely).

Three enucleated-OCFs were aggregated with a single somatic cell in phytohemaglutinin solution (300 µg/ml) dissolved in PBS. Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 µsec, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 µsec at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant reconstructed embryos were incubated in culture medium containing 5 µg/ml cytochalasin B for

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at least 2 h. And then the embryos were briefly washed with NCSU-37, transferred to WOW culture system (4), and cultured in glucose-free NCSU-37 containing 4 mg/ml BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Day 0 to Day 2 (the day of nuclear transfer is designated to be Day 0), and then from Day 2 to Day 7 they were cultured in NCSU-37 supplemented with 5.55 mM D-glucose and 5% FBS. Experimental Design

In Group A, the couplets (cytoplasts and cumulus cell) were fused immediately after enucleation at 30 h after IVM and then activated approximately at 48 h after IVM. In group B, enucleated OCFs at 30 h after IVM were further cultured until 48 h of IVM then were fused with cumulus cell and activated 1 h after fusion. As a control group, enucleated OCF obtained from 48-h in vitro matured were fused with cumulus cells and activated 1 h after fusion. To access the effect onset of enucleation on the remodeling of somatic cell nucleus, at 1, 10 and 24 h after activation, the reconstructed embryos were mounted on a slide glass and stained with 1% aceto-orcein and then examined under a phase contrast microscope. Results

The rates of embryos underwent premature chromosome condensation (PCC) in Group B was significantly lower than those in Group A (51/69; 73.9% vs 76/76; 100% and 24/76; 31.6% vs 45/91; 49.5% for 1 h and 10 h after activation, respectively), whereas some of them had pseudo-pronuclei. By 24 h after activation there was no detectable differences in the rates cleavage (2/70; 2.9% vs 2/61; 3.3%), however the rates were significantly lower than that of the control group (23/90; 25.6%, Chi-square, P<0.05). Following in vitro culture for 7-D, none of the embryos in Group B developed to the blastocyst stage. However, a few proportions of the embryos (2/117; 1.7%) in Group A developed to the blastocyst stage, however, the rate was significantly lower than that of the control group (10/112; 8.9%; Chi-square, P=0.03). These results reflected that occurrence of PCC alone did not reflected the efficiency of remodeling and subsequent development of transferred somatic cell nuclei to the blastocyst stage. Conclusions

These results suggest that a specific nucleus-associate factor(s) that seems to be essential for the successful remodeling of the transferred nucleus and the development of SCNT embryos to the blastocyst stage might be changed following oocytes enucleation.

. References Kikuchi et al., Biol Reprod 2002;66: 1033-1041. Petters RM and Wells KD. J Reprod Fertil Suppl 1993;48: 61-73. Fahrudin et al.,Cloning and Stem Cell 2007;9:216-228 Vajta et al., Biol Reprod 2003;68: 571-578.


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SEQUENCE GASTROGRAPHY TECHNIQUE IN CAMPBELL’S DWARF HAMSTER (Phodopus campbelli)

M.F. Ulum,1 D. Noviana,1 J.D. Haryadi,2 R. Maulida,2 S.T. Dachlan,2 H. Vibowo.2

1Division of Veterinary Surgery and Radiology, Faculty of Veterinery Medicine, Bogor Agricultural

University. Bogor 16680, Indonesia 2Faculty of Veterinary Medicine Student, Bogor Agricultural University. Bogor 16680, Indonesia

E-mail: [email protected]

Keywords: Barium Sulfat (BaSO4), Campbell’s dwarf hamster, Gastrography, OmnipaqueTM(Iohexol), Phodopus campbelli.

Introduction

Hamster are rodents in the family Cricetidae characterized by large cheek pouches, short tail and thick bodies. Several species of hamster are kept as pets or for research purposes. There are two types of hamster, the Syrian hamster and dwarf hamster. Campbelli hamster (Phodopus campbelli) is one of the dwarf hamster that become famous as a pet (Goodman 2002).

The stomach is a musculoglandular organ that connects the esophagus and duodenum (Barber & Mahaffey 1998). Hamster has a distinctly compartmentalized stomach consisting of two parts, the forestomach (pars cardiaca) and the glandular stomach (pars pylorica). The parts are divided by a muscular-like sphincter (Goodman 2002; O’Malley 2005). The total dimension of the stomach are approximately 3.5x2cm (O’Malley 2005). Ingesta within the stomach may obscure some lesions or stimulate other lesions and thus create false-negative or false-positive results. Therefore, under ideal conditions, routine radiographic examination of the stomach should be performed on an animal that has been fasted for 12 to 24 hours. Survey radiographs may be sufficient to diagnose some gastric abnormalities. If contrast studies are deemed necessary, survey radiography of stomach should always precede contrast studies (Barber & Mahaffey 1998).

Material and Method

Animal laboratory and Anaesthesia. Three month old years with average ±44 grams body weight of two campbell’s dwarf hamster (Phodopus campbelli) male and female was anaesthezied with eter to handled and restrain, ketamin-HCl 50 mg/kg body weight (APVMA Approval No.: 51188/50ML/0505, TROY LABORATORIES PTY LIMITED, Australia) and xylazin-HCl 5 mg/kg body weight (APVMA Approval No.: 38654/50ML/0398, TROY LABORATORIES PTY LIMITED, Australia). Ketamin-HCl and xylazin-HCl on one syringe and then given parenteral intraperitonial route.

Gastrography Tehnique. An campbell’s dwarf hamster receive positive contrast agent Barium Sulphate (BaSO4, ) 30%w/w dose 10mL/kg body weight and another’s hamster receive positive contrast agent iohexol (OmnipaqoeTM) dose 10mL/kg body weight given peroraly with modificated-feeding tube using IV catheter 16G without needle (IV Cannula OD=1.7mm, L=45.0mm, F=180ml/min, IVENA JMS Singapore PTE LTD) thorough stomach.

Radiographic Tehnique. The radiographic image taken using diagnostic portable x-ray (Diagnostic X-Ray Unit Model VR 1020 No. Seri 021974, Made in Japan MA Medical Corp.), cassette 24x30 cm (Intensifying screen type JPI GREEN 400 B 8E1233, Korea). X-ray diagnostic setting on 1.6mAs, 50kVp and 100cm film focus distance (FFD). The first radiographic imaging was taken without the contrast agent and then sequence radiograph was taken in 15, 30, 45, 60 minutes and 24 hours with the contrast agent. The radiographic view in this study is dorsoventral (DV), ventrodorsal (VD), right requmbency (L lateral) and left requmbency (R lateral). Result and Discussion

Survey radiographs with contrast agent are useful to diagnose some gastric abnormalities on shape and functional of organ. Barber & Mahaffey 1998 was explained the various of radiographic techniques on small animal there are conventional barium sulfate gastrography, lo-volume gastrography, double-contrast gastrography, pneumogastrography, gastrography using idionated contrast media, and gastric emptying studies using barium-food mixtures. In this study were used gastrography with BaSO4 and iohexol contrast agent to compare radiographic image of stomach organ.

For complete evaluation of the stomach, four conventional views may be necessary: the ventrodorsal view with the animal in dorsal recumbency, the dorsoventral view with the animal in ventral recumbency, the right recumbent lateral view, and the left recumbent lateral view. Oblique views may occasionally be of value to isolate or project certain areas of the stomach, such as the pylorus (Barber & Mahaffey 1998). Complete evaluation with BaSO4 and iohexol difference because the BaSO4 precipitate

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o fold of stomach mucosa. But iohexol contrast agent immediately cover overall of stomach shape. There are occur because the iohexol is liquid that easier to distribute on the stomach organ.

The concentration of the barium mixture varies relative to its intended use from 30% to 60% (w/w) (Barber & Mahaffey 1998). In this studies were used 30% (w/w) and distended stomach and intestine occurred after 24 hour. Stomach shape, fold of mucosa and motility of stomach seen sequence by BaSO4 contrast.

Orally administered iohexol is very poorly absorbed from the normal gastrointestinal tract. Only 0.1 to 0.5 percent of the oral dose was excreted by the kidneys and produces good visualization of the gastrointestinal tract. Iohexol is particularly useful when barium sulfate is contraindicated as in patients with suspected bowel perforation or those where aspiration of contrast medium is a possibility (Medicine online 2005). The stomach seen overall shape covered by iohexol and no distended intestine occurred after 24 hour.

(A) plain (B) 15 minute (C) 30 minute (D) 45 minute (E) 60 minute Picture 1. Dorsoventral sequence view radiographic image of female campbell’s dwarf hamster. A. Plain radiographic image without contrast agent. The stomach is difficult to differences from the other abdominal organs. B. Radiographic image taken 15 minute after receive positive contrast agent (BaSO4). The fore stomach wall shape seen radiopaque covered by the contrast agent. C and D. The radiographic image taken 30 and 45 minutes later after receive BaSO4. The mid stomach shape beginning seen cover by the BaSO4. E. Radiographic image taken 60 minute later. The full mid stomach shape seen cover by the BaSO4.

(A) plain (B) 15 minute (C) 30 minute (D) 45 minute (E) 60 minute Picture 2. Dorsoventral sequence view radiographic image of male campbell’s dwarf hamster. A. Plain radiographic image without contrast. The stomach seen vague with a radiolucent appear were know as stomach gases. B and C. Radiographic image taken 15 and 30 minutes after receive positive contras agent (iohexol). The whole stomach was seen radiopaque with a stomach gases seen radiolucent. D and E. Radiographic image was taken 45 and 60 minute later. The whole stomach shape seen more radiopaque than several minute before.

(A) plain (B) 15 minute (C) 30 minute (D) 45 minute (E) 60 minute

Picture 3. Left requmbency/right lateral sequence view radiographic image of female campbell’s dwarf hamster. A. Plain image without contrast agent. The stomach is difficult to differences from the other abdominal organs. B. Radiographic image taken 15 minute after receive positive contrast agent (BaSO4). The fore stomach wall shape seen radiopaque covered by the contrast agent. C and D. Radiographic image taken 30 and 45 minutes later after receive BaSO4. The mid stomach wall (fold) shape beginning seen covered by the BaSO4. E. Radiographic image taken 60 and 75 minutes after contrast given. The full mid stomach shape seen cover by the BaSO4. The BaSO4 accumulate and seen more radiopaque than several minute before on fore stomach.


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(A) plain (B) 15 minute (C) 30 minute (D) 45 minute (E) 60 minute Picture 4. Left requmbency/right lateral sequence view radiographic image of female campbell’s dwarf hamster. A. Plain image without contrast agent. The stomach shape is difficult to differences from the other abdominal organs but for this case seen more gases on stomach (radiolucent). B and C. Radiographic image taken 15 and 30 minutes after receive positive contras agent (iohexol). The whole stomach was seen radiopaque with a stomach gases seen radiolucent. D and E. Radiographic image was taken 45 and 60 minute later. The whole stomach shape immediately seen more radiopaque than several minute before.

(A) DV of BaSO4 (B) VD of

BaSO4 (C) DV of Iohexol

(D) VD of Iohexol

Picture 5. Dorsoventral and ventrodorsal view of radiographic image taken after 24 hour. A and B. The stomach and intestine seen distended with more gases. Little BaSO4 were run over to cover the wall and more gases distended on intestine.Dorsoventral and ventrodorsal view radiographic image of female and male campbell’s dwarf hamster. C and D. The feces seen radiopaque in large intestine were spare of contras agent (iohexol). No distended intestine with gases accumulation.

(A) L lateral of

BaSO4 (B) R lateral of

BaSO4 (C) L lateral of

Iohexol (D) R lateral of

Iohexol Picture 6. Dorsoventral and ventrodorsal view of radiographic image taken after 24 hour. A and B. The stomach and intestine seen distended with more gases. Little BaSO4 were run over to cover the wall and more gases distended on intestine.Dorsoventral and ventrodorsal view radiographic image of female and male campbell’s dwarf hamster. C and D. The feces seen radiopaque in large intestine were spare of contras agent (iohexol). No distended intestine with gases accumulation occur.

Conclusion

Useful gastrogphy using BaSO4 better than iohexol to view sequence of stomach shape, fold of mucosa and motility than iohexol but that have contraindicated with distended intestine. Furthermore that iohexol is useful to view overall immediately to seen the shape of stomach organ and secure if suspected perforation diagnose occur. References Barbers DL, Mahaffey MB. 1998. The Stomach. in: Textbook of Veterinary Diagnostic Radiology. 3rd

edition. Thrall DE, editor. Pennsylvania: WB Saunders Company. Goodman G. 2002. Hamster. On : Meredith, A dan Redrobe, editor. BSAVA Manual of Exotic Pets Ed4th:

UK: British Small Animal Veterinary Association (BSAVA). Medicine Online. 2005. OMNIPAQUETM (Iohexol) Injection 240 300 350.

http://www.medicineonline.com/drugs/o/2754/OMNIPAQUE-iohexol-Injection-240-300-350.html. GE Healthcare Inc. Pricenton, NJ 08540.

O’Malley B. 2005. Clinical Anatomy and Physiology of Exotic Species. Elsevier Saunders: Germany.


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CASE STUDY: SURGICAL APPROACH TO REMOVE SUBCUTANEOUS MASS TUMORS IN A CAMPBELL’S DWARF HAMSTER (PHODOPUS CAMPBELLI)

M.F. Ulum,(1) S.N. Handayani,(2) Y. Riza,(2) R. Asryyuni,(2) E. Handharyani.(3)

1Division of Veterinary Surgery and Radiology, Faculty of Veterinary Medicine,

Bogor Agricultural University 2Faculty of Veterinary Medicine Student, Bogor Agricultural University

3Division of Veterinary Pathology, Faculty of Veterinary Medicine, Bogor Agricultural University E-mail: [email protected]

Key words: Campbell’s dwarf hamster, Phodopus campbelli, Subcutaneous mass tumor, Surgery, Radiography

Case. A campbell’s dwarf hamster (Phodopus campbelli) with 1,3 years old have swollen at lateral

left and right subcutaneous mass of abdomen region. That swollen becomes greater in 2 last month later. The size of left mass is ± 1.5 cm, right mass is ± 2 cm with soft consistency and easy to move. The temporary diagnose is benign mass tumor and the campmbell’s dwarf hamster were dissection to remove subcutaneous mass.

Anaesthesia. Inhalant anaestheticum (ether) is used to handling and restraint easier. General anesthesia in the first surgery used Zoletil (VIRBAC Animal Health, 1ère avenue, L.I.D, Carros France) dose 20mg/kg bw (Mason, 1997) and xylazin-HCl (TROY Laboratories PTY Limited, Australia) dose 5mg/kg bw (Mason, 1997) in one syringe by IP injection. The second surgery used general anesthesia with ketamin-HCl anaesthetic (TROY Laboratories PTY Limited, Australia) dose 50mg/kg (Mason, 1997) and xylazin-HCl dose 5mg/kg bw in one syringe by IP injection route.

The general anesthesia is better than local anesthesia to minimize stress in small animals (Gaertner et al 2008). Anesthetic agent choice in the first surgery is a zoletil and xylazine combination. Murray et al 2000 explain that zoletil (tiletamin and zolazepam combination) not recommended to used on surgical procedure in small rodent. That cause prolonged recovery and become severe hypothermia and hypercausia. Zoletil and xylazine-HCl combination make good onset time, but have prolonged on recovery time (Kohl et al 2007 and Mason 1997). In this case, onset of anesthesia about 41s and duration of anesthesia about 7hr 49min to recovery.

Then the second surgery used different combination, that is ketamin-HCl and xylazine-HCl. Ketamine-HCl is a generally anesthetic that leaped a bounds and a lack of cardiopulmonary depressant effect (Plumb, 2005). In this combination have duration about 60-90 minute with characteristic as a muscle relaxant (Murray et al 2000).

(A) (B)

Picture 1. The proportion of two abdominal subcutaneous tumor mass. A. Campbell’s dwarf hamster with two subcutaneous abdominal region tumor mass. B. The first surgery to removed the left subcutaneous tumor mass.

Minor Surgery. The minor surgery have been made in 2 stage. The first surgical approach

removed the left mass (picture 1) and upon the right mass after 2 month later (picture 2). This minor surgery technique to remove subcutaneous mass tumor was explained by Fossum 2002. The minor surgery beginning by cut the skin around the subcutaneous mass tumor after disinfectant by iodine tincture. After that, the mass tumor separated from the subcutan tissue and removed used scissor. The

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vascularization in the left mass is fewer than the right mass tumor. The suture technique in this surgery is a simple interrupted suture with a 13 cutting needle (size 13) and suture with catgut 4/0 absorbable chromic type. The macroscopic color of subcutaneous mass is dark red were the left region ± 1.5 cm and the right region ± 2 cm.

(A) (B)

Picture 2. The second surgery. A. The subcutaneous tumor mass on right region before surgery. B. The minor surgery to remove tumor mass.

Post operative care. Oxytetracyclin (VET-OXY LA, SANBE, Bandung Indonesia) by 30mg/kg bw

(Goodman, 2002) at once dose by IM route injection and chloramphenicol which treated by 50 mg/kg daily (Goodman, 2002), as a therapy after surgery for 5 day later.

Radiographic x-ray investigation. The radiographic image taken using a radiodiagnostic portable x-ray (Diagnostic X-Ray Unit Model VR 1020 No. Seri 021974, Made in Japan MA Medical Corp.), cassette 24x30 cm (Intensifying screen type JPI GREEN 400 B 8E1233, Korea). X-ray diagnostic setting on 1.6mAs, 50kVp and 100cm film focus distance (FFD). Gross Findings

The mass was firstly found subcutaneously at the left lateral abdominal region, white in color, firm in consistency, was 20x15x10 mm in sized. The second tumor was growth at right lateral abdominal region, white in color, has mildly hard in consistency, multilobulated, was 20x10x10 mm in sized.

(A) (B)

Picture 3. The abdominal subcutaneous mass tumor after dissection (surgery). A. The abdominal subcutaneous mass tumor on left region

B. The abdominal subcutaneous mass tumor on right region Histopathological Findings

First Tumor. The tumor cells are arranged in sheet, consisting of pleomorphic cells and some cells has spindle shaped. These cells scattered throughout this population are distinctive cells with large hyperchromatic nuclei. These cells can be mononuclear, multilobulated, or multinucleated, with low mitotic figures. The cytoplasm was eosinophilic. The diagnosis of the first tumor is Malignant Fibous Histiocytoma/MFH.

Second Tumor. Neoplastic cells are pleomorphic, arranged in storiform or solid sheet with collagenous materials found between cells. Most cells has short processes, and has round to ovoid nuclei with indistinct nucleoli. Some cells has vacuolated cytoplasms. These cells mixed with histiocytoid


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cells and an infiltrate of lymphocytes, plasma cells, neutrophils and occasionally eosinophils. Histioid cells are frequently karyomegalic or multinucleate, with nuclear atypia. In several part of this neoplasia, the new blood capillaries were found. And giant cells which have multinuclear were frequently found. The second case was also diagnosed as Malignant Fibrous Histiocytoma (MFH) and giant cell variant.

(A) (B)

Picture 4. Radiographic image of the campbell’s dwarf hamster. A. Ventrodorsal /dorso requmbency view. B. Left requmbency/right lateral view.

Discussion

The histologic features of storiform-pleomorphic variant of MFH are unique and are usually diagnostic. Dermal or subcutaneous MFH tends to be locally expansile (Meuten, 2002). Reports vary as to the metastatic potential of this neoplasm. Complete excision can be curative for solitary dermal or subcutaneous masses. There is no recognized successful treatment for multicentric MFH.

Recurrence subcutaneous mass occur on sternum region and flank region. The size of subcutaneous mass tumor about ± 1 cm. Radiographic image was performed to investigation the metastatic on lungs. The gross radiographic image show that no metastatic evidence on lungs. There is no opacity mass found on the lungs (picture 4).

References Fossum, TW. 2002. Small animal Surgery. 2nd edition. USA : Mosby An Affiliate of Elsevier Gaertner DJ, TM Hallman, FC Hankenson and MA Batchelder. 2008. Anaesthesia and Analgesia in

Laboratory Animals in Anesthesia and Analgesia in Rodents. 2nd edition. USA: Academic Press. p Goodman G. 2002. Hamster. On : Meredith, A dan Redrobe, editor. BSAVA Manual of Exotic Pets Ed4th:

UK: British Small Animal Veterinary Association (BSAVA).p Murray KA, Cinthia P and Garry LB. 2000. Laboratory Animals: Rodent Anesthesia & Analgesia. USA:

University of Washington. Plumb DC. 2005. Veterinary Drug Handbook 5th edition. USA: Blackwell Publishing.


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DRINKING HEALTHY WILD CIVET COFFEE (KOPI LUWAK)

M. Winugroho1, R. Hidayat2, and Atik Marijati1

1Indonesia Research Institute for Animal Production, Ciawi-Bogor, Indonesia. 2Department of Veterinary Science-IPB, Bogor, Indonesia.

Keywords: Wild civet coffee, physical, laboratory, cupping test, scoring system.

Introduction

Since the price is high, many fake wild civet coffee beans available in the markets. This includes ordinary coffee fermented by civet dung or coffee beans obtained by giving them to civets kept in cages. Another possibility, a mixture between ground civet coffee and ordinary coffee beans, roasted together. Mitrabhumi (http:///www.mitrabumi-coffee.2010) explains physically differences between civet versus ordinary coffees. Major civet coffee in the world is originated from Indonesia but practically no published paper on how to evaluate the beans quantitatively. In this paper, scoring system to check the authentic civet coffee is proposed. Testing the Originality of Wild Civet Coffee 1. Physical test/SEM. In Indonesia, physical characteristics normal coffee beans are evaluated using

SNI 01-2907-2008. In addition, color and hardiness scores, and microscopic pictures preferable by SEM are recommended. Based on SEM study, the surface of civet coffee beans are smoother.

2. Laboratory analysis. Proximate analysis, mainly protein and volatile fatty acid contents are assessed. Using electrophoresis, proteinase penetrating coffee beans causing storage protein breakdown (Marcone, 2004). The civet coffee has lower protein content.

3. Microbiology test. Bacteria counts are evaluated, a lower types and numbers in civet coffee is reported (Marcone, 2004).

4. Cupping test. In cupping test, bitter taste and volatile aroma are evaluated by coffee professional testers (Edwin Maolana, 2008. http://edwin.maolana.web.id?p=112).

PROPOSED SCORING SYSTEM FOR EVALUATING WILD CIVET COFFEE

(Proved wild civet coffee versus samples tested) Weighted Score Total Score (1,2,3,7,8,9)

1. Physical test/SEM a. SNI 01-2907-2008 10 b. SEM 10

2. Lab analysis a. Protein content 10 b. Volatile compounds 10

3. Microbiology test Bacterial counts 30

4. Cupping test (Edwin Maolana 2008) 30

Total Score 100 (…………….)

Conclusion

The higher the score the higher the originality of the wild civet coffee tested. Acknowledgements Special thanks to Mr Rudy Pesik for financial supports including providing professional cupping testers. References Edwin Maolana (2008). http://edwin.maolana.web.id?p=112. Marcone, M. (2004). Composition and properties of Indonesian palm civet coffee (Kopi Luwak) and

Ethiopian civet coffee. Food Res. Int. 37 (9):901-9 12), Department of Food Science, Ontario Agricultural College, Guelph, Ont., Canada.

Mitrabhumi. http:///www.mitrabumi-coffee.2010. SNI 01 -2907 -2008

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