Detection of Chlamydophila by Multiplex PCR

 

 Detection of Chlamydophila by Multiplex Polymerase Chain Reaction

 

 

PUDJIATMOKO, DVM., Ph.D

Veterinary Drug Assay Laboratory
(Balai Besar Pengujian Mutu dan Sertifikasi Obat hewan)
Gunung sindur, Bogor 16340

ABSTRACT

Molecular identification of 6 species of family Chlamydiaceae carried out by nested polymerase chain reaction (PCR) with species-specific primers derived from 16S rRNA gene was investigated. The 16SA and 16SB primers were used for amplification of 1,473 bp 16S rRNA gene fragments of 25 strains of Chlamydiaceae in the first PCR. A set of family-specific primer and 5 sets of species-specific primer were used for amplification of fragments of 16S rRNA gene in the nested PCR. The family-specific primers amplified all the 16S rRNA gene of Chlamvdophila spp. used. The species­specific primers were shown to be specific for each species. Multiplex PCR calTied out using 3 primers (2 sense primers and I antisense primer) in a single tube was applicable for detection of Chlamydophila felis, Chlamydophila caviae and Chlamydophila psittaci. C. psittaci-specific primers could be used for detection of Chlamydophila DNA extracted from 1 throat swab of human, 1 liver specimen of a parakeet and 2 fecal specimens of crows. C. felis ­specific primers could be used for detection of Chlamydophila DNA extracted from 4 throat swabs of humans and 2 conjunctival swabs of cats.

Key words: Chlamydiaceae, Chlamydophila, multiplex PCR

ABSTRAK

Identifikasi molekul 6 spesies famili Chlamydiaceae dengan cara nested polymerase chain reaction (PCR) telah dilakukan dengan menggunakan primer spesifik-spesies berasal dari gen 16S rRNA. Primer 16SA dan 16SB digunakan untuk mengamplifikasi 1.473 bp gen 16S rRNA dari 25 galur Chlamydiaceae pada PCR pertama. Satu set primer spesiflk-famili dan 5 set primer spesiflk-spesies digunakan untuk mengampliflkasi sebagian gen 16S rRNA pada nested PCR. Plimer spesiflk-famili dapat mengamplifikasi semua gen 16S rRNA dari Chlamlydophila spp. yang digunakan. Primer spesifik spesies menunjukkan spesifik untuk setiap spesies. PCR multiplek menggunakan 3 primer (2 primer sense clan I primer antisense) dalam satu tabung dapat diaplikasikan untuk mendeteksi Chlamydophila felis, Chlamydophila caviae dan Chlamydophila psittaci. Primer spesifik-C. psittaci dapat digunakan untuk mendeteksi DNA Chlamydophila yang diekstraksi clari I swab tenggorokan manusia, I spesimen hati burung pal'kit, clan 2 spesimen feses gagak. Primer spesifik-C. felis dapat digunakan untuk mendeteksi DNA Chlamydophila yang diekstraksi dari 4 swab tenggorokan manusia dan 2 swab selaput kelopak mata kucing.

Kata kunci : Chlamydiaceae, Chlamydophila, PCR multipleks

INTRODUCTION

Chlamydophila causes a wide variety of diseases in animals and humans (Fukushiand Hirai, 1992; Fukushi and Hirai, 1993a; Grayston et al., 1989; Moulder et al., 1984). The genetic diversity of Chlamydophila is well recognized by chromosomal DNA finger printings (Fukushi and Hirai, 1989), restriction fragment length polymorphism (RFLP) of rRNA gene loci (Fukushi and Hirai, 1993b), RFLP of ompA and groEL genes (Fukushi and Hirai, 1994), and random amplification of polymorphic DNA (RAPD) analysis (Pudjiatmoko et at., 1996). Chlamydophila classified into several genetic groups based on the ompA gene sequence (Kaltenboeck et at., 1993). The previous phylogenetic analysis of the genus Chlamydophila based on 16S rRNA gene sequences showed 6 genetic groups (Pudjiatmoko et aI., 1997). In 1999 the genus Chlamydophila was classified into 6 species: Chlamydophila psittaci, C. felis, C. caviae, C. abortus, C. pecorum and C. pneumoniae (Everett et aI., 1999). A practical method for genotyping Chlamydophila is not available yet.

Typing of Chlamydophila isolates has traditionally been done by immunological methods including fluorescent antibody and enzyme immunoassays (Fukushi and Hirai, 1989; Stamm et at., 1988; Storz and Krauss, 1985; Thomas et aI, 1990). Recently, m9lecular methods using gene amplification, LCR, and DNA sequencing have been employed for identification of isolates (Campbell et aI., 1993; Domeika et aI., 1994; Gaydos et aI., 1992; Holland et aI., 1990; Kaltenboeck et aI., 1991; Kaltenboeck et al., 1993; Kaltenboeck and Storz, 1992; Mahony et aI., 1993). This report describes a simple method of genetic typing of Chlamydophila by PCR using species-specific primer sets. Multiplex PCR carried out using 3 primers (2 sense primers and 1 antisense primer) in a single tube was applicable for detection of C. felis, C. caviae and C. psittaci. Furthermore, this method was applied for PCR and genetic typing of Chlamydophila spp. from clinical samples of human and animal.

 

MATERIALS AND METHODS

Microorganisms

Chlamydophila strains used in this study included 5 strains (Frt-Hu/Cal 10, Hu/Borg, Prk/6BCT, Prk/ Daruma, Prt/GCPI) of C. psittaci, 1 strain (Gp/Ie) of C. caviae, 7 strains (Fe/Pnl, Fe/ 145, Fe/Cello, Fe/B 166, Fe/C 164, Fe/C429, Fe/C454) of C. felis, 5 strains (Bo/ESST, Bo/Maeda, Bo/Shizuoka, Koala (type 2), Ov/IPA) of C. pecorum, 1 strain (TW/83T) of C. pneumoniae and 6 strains (A/Har-l T, B/TW-5/0T, C/ TW-3/0T, DfUW-3/Cx, EfUW-5/Cx, L2/434/Bu) of C. trachomatis (Pudjiatmoko et aI., 1997). To confirm the specificity of PCR, the following agents were tested: Staphylococcus aureus, Streptococcus pneumoniae (Gifu 8766 strain), Bordetella bronchoseptica (Gifu 1127 strain), Mycoplasma pneumoniae, Escherichia coli (C600 strain), Haemophilus influenzae (Gifu 3191 strain), Klebsiella pneumoniae (Gifu 2929 strain), Leginella pneumophila (SL94-1 strain), Leginella pneumophila (SL94-2 strain), Coxiella burnetii (Nine Mile ATCC YR 615 strain), Orientia tsutsugamushi (Gilliam Strain), Orientia tsutsugamushi (Karp strain) and Orientia tsutsugamushi (Kato strain). These strains were kindly provided by Dr. T. Ezaki of Gifu University, Japan and Dr. A. Tamura of Niigata College of Pharmacy, Japan.

Clinical samples

One hundred and forty throat swabs were collected from junior high school students with respiratory diseases. Eleven conjunctival swabs were collected from cats suffering conjunctivitis at the animal hospital of Gifu University and Tokyo Metropolitan Institute. Thirty-three fecal samples from crows and ten liver tissue samples from parakeets with systemic infections were also tested.

Preparation of chlamydial DNA

Procedures for preparation of Chlamydophila DNA have been described previously (Fukushi and Hirai, 1989). Extraction of chlamydial DNA from throat swabs, sera and fecal samples was performed according to the protocol of Higuchi (Higuchi, 1989). Briefly, all the samples were suspended in 1 ml of PBS in a 1.5 ml Eppendorf micro centrifuge tube, and centrifuged at 13,000 x g for 20 sec. The pellet was resuspended in 1 ml of washing buffer (10 mM Tris­HCI (pH 8.3), 50 mM KCI, 1.5 mM MgC12), and centrifuged at 13,000 x g for 20 sec. The pellet was resuspended in 1 ml lysis buffer (0.32 M sucrose, 10 mM tris-HCI (pH 7.5), 1 % Triton X-lOO), and centrifuged at 13,000 x g for 20 sec. DNA was released from the pellet by adding PCR buffer with nonionic detergents (50 mM KCI, 10 mM Tris-HCI (pH 8.3), 2.5 mM MgCI2, 0.1 mg/mi gelatin, 0.45 % NP40 and 0.45 % Tween 20), and proteinase K (120 pg/ml) to the washed pellet. The mixture was incubated for 1 h at 55° C, boiled for 10 min, and then chilled on ice.

Nucleotide primers

For amplifying chlaniydial 16S rRNA gene, a pair of primer 16SA-16SB, was constructed from the 16S rRNA sequences of the family Chlamydiaceae (Pudjiatmoko et at., 1997). Internal primers were prepared from the 16S rRNA gene from the C. felis, C. psittaci, C. pecorum, C. pneumoniae and C. trachomatis for the nested PCR. The primers were designated as ChFeA-ChFeB, ChPsA-ChPsB, ChPeA-ChPeB, ChPnA-ChPnB, and ChTrA-ChTrB primers for the C. felis, C. psittaci, C. pecorum, C. pneumoniae and C. trachomatis, respectively (Table 1). The combination of ChFeA-ChPsA-ChPsB primers was used in a one-step reaction carried out in a single tube for genotyping of C. caviae, C. felis and C. psittaci. The length of the DNA fragments generated in the PCRs are shown in Table 2.

PCR amplification of clinical samples

Chlamydopila 16S rRNA genes were amplified by using a pair of 16SA and 16SB primers in the first PCR. The PCR was performed with 5 ul of throat swab, fecal, cell culture samples or chromosomal DNA of Chlamydopila extracts in a total volume of 50 ul. Five ul of the first PCR product was submitted to the nested PCR using a pair of family- and species-specific DNA primer. The final reaction mixture contained 40 pmol of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.0-2.0 mM MgCI2, 200 pM each of dATP, dCTP, dGTP, and dTTP, 0.01 % gelatin, and 2.5 units of Taq polymerase (Takara Shuzo Co., Ltd., Kyoto, Japan) in 50 ul. Concentration of MgCl2 was 1.0 mM for ChPsA-ChPsB, ChFeA-CbPsB and ChTrA-ChTrB primers, 1.5 mM for 16SA-16SB, ChA-ChB, and ChPnA-ChPnB primers, and 2.0 mM for ChPeA­ChPeB primers. The components and procedure for the multiplex PCR were described above except 2 sense primers for C. felis (ChFeA) and C. psittaci (ChPsA) and an antisense primer for C. psittaci (ChPsB) was used in the method. The thermal cycle amplification program for the first PCR was as follows: initial melting at 94°C for 3 min and then 35 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min and the last extension for 7 min follow by holding at 4°C. The program for nested PCR was as follows: initial melting at 94°C for 3 min and then 35 cycles with denaturation at 94°C for 30 see, annealing at 55°C for 30 sec and extension at 72°C for 2 min and the last extension for 7 min follow by holding at 4 C. Five ul PCR products were electrophoresed in 2 % agarose gel. DNA was stained with ethidium bromide and visualized by UV light.

Table 2. Specificity of peR test of 5 species-spesific primer sets of Chlamydophila

Isolation of Chlamydophila

Clinical specimens were ground to make a 10 % emulsion in SPG containing 500 ug/ml streptomycin and Kanamycin. The mixtures were clarified by centrifugation. The supernatant fluid was inoculated into yolk sac of specific pathogen-free embryonated chicken eggs that had been incubated at 37°C for 6­7 days. The inoculated eggs were incubated at 37°C for 3 - 7 days, and yolk sac smears were microscopically examined for viable Chlamydiae after staining by the Gimenez method (Schachter, 1988).

RESULTS

Specificity of PCR amplification

To evaluate the specificity of our primer sets, 25 Chlamydophila and 2 Gram-negative and 7 Gram­positive bacterial and 4 Rickettsial strains were tested in this study. In the first PCR, the 16S rRNA gene fragment of all Chlamydophila strains was amplified by the family-specific primer set of 16SA-16SB, and the size of PCR product was about 1470 bp (Figure 1). In the second PCR, the 16S rRNA gene fragment of all Chlamydophila DNA was examined by amplification with the family specific-primer set, ChA-ChB. The size of PCR product was about 390 bp (Figure 1). A DNA product of approximately 820 bp in size was observed with C. pneumoniae DNA with the primer set ChPnA-ChPnB. DNA products with approximately 200 bp and 250 bp were observed with the C. feils and C. psittaci DNA with the primer sets of ChFeA-ChFeB and ChPsA-ChPsB, respectively (Figure 2). DNA products with approximately 420 bp and 160 bp were observed with C. pecorum and C. trachomatis DNA with the primer sets of ChPeA-ChPeB and ChTrA-ChTrB, respectively (Figure 3). The 16S rRNA gene fragments of C. caviae were amplified by primer sets of ChFeA­ChFeB and ChPsA-ChPsB, or ChFeA-ChPsB. The 16S rRNA gene fragment of C. felis was also amplified by primer set of ChFeA-ChPsB. Thus the species specific sets were specific for each species except guinea pig (Table 2). All 2 Gram-positive and 7 Gram-negative bacterial and 4 Rickettsial strains tested yielded no PCR product with all primer pairs used in both the first and second PCR.

Multiplex PCR

To simplify PCR reaction, co-amplification was carried out by simultaneous use of three primers (ChFeA, ChPsA and ChPsB) ina single tube. In this reaction, the ChFeA-ChPsB primer pair produced about 410 bp of the fragment of , C. felis strains, while ChPsA-ChPsB primer pair produced about 250 bp fragment of C. psittaci strains (Figure 4). These primer pairs produced two previously identified fragments with C. caviae strain. However, Chlamydophila strains from other genetic groups did not produce any PCR products.

Sensitivity of PCR amplification

The sensitivity of the PCR method developed in the present work was evaluated by testing serial dilution of purified DNA from 25 Chlamydophila strains. In the first PCR, PCR signals from 0.5 pg to 50 ug of template DNA could be detected in 50 ul aliquots of reaction. When the first PCR products were submitted to nested PCR, detectable levels were from 5 fg to 50 ng of the original template Chlamydophila DNA (Figure 4).

Direct detection of Chlamydophila from clinical specimens

After the specificity and sensitivity of the PCR method were determined, the evaluation of direct detection and genotyping of Chlamydophila from clinical samples was done simultaneously. Chlamydophila organisms were detected by multiplex PCR in 1 liver specimen from 10 parakeets, 2 conjunctival swabs from 11 cats, 2 fecal specimens from 33 crows, and 5 throat swabs from 140 humans. PCR results in parakeets and cats were confirmed by isolation. Isolation was not attempted with crow and human specimens. The species o~ these ten positive PCR amplified DNAs were determined to be a C. psittaci from parakeet, two C. psittaci from crows, a C. psittaci from human, a C. felis from cat, a C. felis from parakeet, and four C. felis from humans. The genotype of isolates from parakeets and cats confirmed the PCR typing results.

DISCUSSION

The PCR using primers targeted to the 16S rRNA gene were developed for direct identification of Chlamydophila spp. in the present study. The specificity of the amplifications was confirmed by sequencing of DNA from PCR amplified products and isolates from clinical samples. Using a primer set for the family Chlamydiaceae in combination with five primer sets for the existing species C. caviae, C. felis, C. psittaci, C. pecorum, C. pneumoniae, and C. trachomatis, each designated chlamydial species was able to be differentiated. The method was applied for detection and direct typing of Chlamydophila for clinical samples from humans and animals. The results of the study demonstrated that this method may be useful for laboratory diagnosis.

The sensitivity of our method that detect 1 to 50 IFU or 5 to 50 fg is equivalent to other method that use the MOMP gene (Campbell et al., 1992; Valassina et al., 1995), l6S rRNA gene (Gaydos et al., 1992) and plasmid (Valassina et al., 1995). The PCR with species-specific primer pairs could identified species of Chlamydophila. However, no amplification was observed with other bacteria in both of the PCR. The results indicated that the PCR methods revealed specific to detect family Chlamydiaceae and identify genetic groups of Chlamydophila.

Currently 3 genetic groups are recognized for C. Caviae, C. felis and C. psittaci (Pudjiatmoko et al., 1997; Everett et aI., 1999). Differentiation ofthese 3 genetic groups requires 6 primers (3 sense and 3 antisense primers) using 3 sets of tubes. In method of this study, PCR was carried out using three primers (two sense primers and an antisense primer) in a single tube for both detect and identify species C. caviae, C. felis, and C. psittaci simultaneously. This multiplex PCR method uses less reagent, time and labor. The multiplex PCR technique appears to be suitable method for a rapid detection and genotyping of Chlamydophila isolates.

No case of feline chlamydiosis in human has been reported in Japan. It is also rare in other countries. A human case of feline pneumonitis was reported in 1969 (Schachter et al., 1969). Our study found 4 out of 140 high school children have feline pneumonitis. This result indicate that transmission of feline pneumonitis from cats to human is not rare and infection is often subclinical.

ACKNOWLEDGMENT

This study was supported by grants-in-aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture.

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Special SOM AMAF Agustus 2009

 

 Laporan Special SOM AMAF 11 – 13 Agustus 2009 di Ho Chi Minh City

 

 

I. PENDAHULUAN

Pertemuan Special Senior Officials’ Meeting of the 30th Meeting of the ASEAN Ministers on Agriculture and Forestry (Special SOM-30th AMAF) telah diselenggarakan di Ho Chi Minh City, Vietnam, 11-13 Agustus 2009. Pertemuan dipimpin oleh Dr. Le Van Minh, Director-General of International Cooperation Department, Ministry of Agriculture and Rural Development of Vietnam dan dihadiri oleh seluruh negara anggota ASEAN, wakil dari Asian Development Bank (ADB) serta Sekretariat ASEAN. Delegasi RI dipimpin oleh Dr. Tachrir Fathoni, Kepala Badan Litbang Kehutanan dengan anggota delri yang terdiri dari unsur-unsur Deptan, Dephut, DKP dan Deplu.

II. JALANNYA DAN HASIL-HASIL PERTEMUAN

Roadmap of ASEAN Community

Pertemuan menggarisbawahi usulan dari ASEAN Task Force on Codex (ATCF) dan ASEAN Sectoral Working Group on Livestock (ASWGL) bahwa pengembangan Good Animal Husbandry Practices dan ASEAN Good Hygiene Practices sebagaimana tercantum dalam AEC Blueprint, tidak dapat berdiri sendiri melainkan harus dikoordinasikan dengan badan sektoral lainnya yang terkait, termasuk ASEAN Expert Group on Food Safety (AEGFS).

ASEAN Integrated Food Security (AIFS) Framework and Strategic Plan of Action on Food Security (SPA-FS)

Sekretariat ASEAN menyampaikan perkembangan mengenai implementasi AIFS Framework and SPA. Sejumlah kegiatan yang telah dilaksanakan sejauh ini, antara lain: pembuatan video ASEAN and World Food Security, penyelenggaraan 2009 Roundtable Conference on East Asia Food Security Cooperation Strategy, 21-22 April 2009 di Beijing, serta ASEAN-FAO Regional Conference on Food Security, 27-28 Mei 2009 di Bangkok.

Dalam kesempatan ini, perwakilan ADB, Mr. Katsuji Matsunami, menyampaikan presentasi proposal berjudul ADB Support for ASEAN Integrated Food Security. Proposal ini memiliki 3 (tiga) fokus, yaitu mendukung pengembangan ASEAN Plus Three Emergency Rice Reserve (APTERR), mengembangkan pasar beras internasional yang stabil serta memperkuat sistem informasi ketahanan pangan.

Pertemuan pada prinsipnya dapat menyetujui proposal ADB tersebut, namun meminta agar dalam tahap finalisasinya juga dikaitkan dengan inisiatif yang telah ada sebelumnya, yaitu: East Asia Emergency Rice Reserve (EAERR) dan ASEAN Food Security Information System (AFSIS). Selain itu, mengingat beras merupakan komoditas yang sensitif secara politis, maka ADB diminta untuk dapat menyampaikan beberapa alternatif opsi dukungan pengembangan ketahanan pangan agar SOM-AMAF dapat memutuskan opsi mana yang paling dapat diterima oleh seluruh negara ASEAN pada pertemuan Prep-SOM – 31st AMAF mendatang di bulan Oktober 2009.

Progress of Country Initiatives for Programmes or Projects on Food, Agriculture and Forestry By ASEAN Member States

Indonesia telah menyampaikan agar 2 (dua) inisiatif proposal yang diajukan Indonesia, yaitu: Indonesia’s Initiative on the ASEAN Networking for Agriculture Vulnerability to Climate Change (agenda item 4.1) dan Indonesia’s Initiative on ASEAN Strategy in Addressing the Impact of Climate Change on Agriculture, Fisheries and Forestry (agenda item 4.2) digabungkan menjadi satu inisiatif baru yang terfokus pada strategi adaptasi pertanian dan pengembangan sumber daya terbarukan.

Pertemuan juga menerima laporan perkembangan dari beberapa inisiatif lainnya, yaitu: i) Malaysia’s Initiative on the Establishment of a Network for Pesticide Regulatory Database dan APHCN, ii) Philippines Initiative on ASEAN IPM Network, iii) Singapore Initiative on ASEAN GMF Testing Network dan AARNET, serta iv) Thailand’s Initiative on ASEAN Food Safety Network, ASEAN Rapid Alert System for Food and Feed, dan Collaboration of ASEAN R&D in Sericulture.

Consideration of the Report of the 16th Meeting of the Joint Committee (JC) on ASEAN Cooperation in Agriculture and Forest Products Promotion Scheme, 14-16 July 2009, Siem Reap, Cambodia

Pertemuan menyepakati draft MoU on ASEAN Cooperation in Agriculture and Forest Products Promotion Scheme (lampiran 1) setelah membahas beberapa perubahan. Pertama, Indonesia telah mengusulkan agar judul MoU perlu ditambahkan kata ‘fishery’, namun judul MoU yang disepakati tidak memasukkan fishery, dengan pertimbangan bahwa ‘agriculture’ sudah mencakup livestock dan fishery. Selanjutnya, Malaysia menarik kembali usulan untuk menambahkan istilah ‘secrecy’ dalam chapter IX karena dianggap cukup menggunakan ‘confidentiality’. Selain itu, pertemuan juga menyetujui rekomendasi pertemuan ASOF ke-12 untuk menghapuskan National Focal Point on Promotion of Forest Product agar tidak terjadi duplikasi, memperhatikan bahwa dalam kerangka ASOF telah dibentuk ASEAN Experts Group on Forest Product Development yang memiliki tujuan, agenda dan program kerja yang sama.

Selain itu, disepakati pula bahwa draft MOU on ASEAN Cooperation in Agriculture and Forest Products Promotion Scheme, komoditi forest products akan ditangani oleh ASEAN Forest Products Industry Club (AFPIC). Indonesia mengusulkan Direktorat Jenderal Bina Produksi Kehutanan (BPK), Dephut sebagai national coordinator dan focal point untuk Forest Products. Selanjutnya, ASEAN NFPWG on Forest Products ditangani oleh AFPIC-Malaysia.

Cooperation in Food

Thailand selaku tuan rumah pertemuan ke-29 ASEAN Food Security Reserve Board (AFSRB) menginformasikan bahwa penyelenggaraannya akan dilaksanakan pada akhir tahun 2009. Sehubungan dengan hal ini, pertemuan mengimbau Thailand agar jadwal dan tempat penyelenggaraan pertemuan ke-29 ASFRB dapat disampaikan kepada seluruh negara anggota pada kesempatan pertama.

Pertemuan juga menerima laporan dan rekomendasi Brunei Darussalam (Chairman AWG on Halal Food) dan Lao PDR (Chairman ATF on Codex).

Consideration of the Report of the 16th Meeting of the ASEAN Sectoral Working Group on Crops (ASWGC), 20-22 May 2009, Manila, Philippines

Pertemuan dapat menerima laporan Philippina selaku Chairman ASWGC dan sepakat untuk merekomendasikan beberapa dokumen untuk disahkan dalam AMAF mendatang, yaitu :
List of 7 ASEAN MRLs for 5 pesticides: carbendazim (grapes and oranges), chlorpyrifos (longans and litchi), phosalone (durian), ethion (pummelo) and deltamethrin (chilli pepper)

ASEAN Standards for Young Coconut, Banana, Garlic and Shallot
The establishment of Expert Working Group on ASEAN GAP (EWG-ASEAN GAP)

Consideration of the Report of the 17th Meeting of the ASEAN Sectoral Working Group on Livestock (ASWGL), 6-8 May 2009, Yogyakarta, Indonesia

Indonesia melaporkan hasil-hasil dari pertemuan tersebut, antara lain kemajuan proyek-proyek kerjasama seperti Regularization of Products and Utilization of Animal Vaccines, Promotion of International Trade in Livestock and Livestock Products, serta Strengthening of Animal Disease Control Programme among ASEAN Member States. Pertemuan menerima laporan Indonesia selaku Chairman ASWGL. Selanjutnya pertemuan sepakat untuk merekomendasikan dua dokumen untuk disahkan pada AMAF mendatang, yaitu :
Accreditation of the Veterinary Biologics Assay Division (VBAD) Pakchong, Thailand
ASEAN Criteria for Accreditation of Milk Processing Establishment (setelah dilakukan revisi terlebih dahulu oleh Malaysia).

Cooperation in Fisheries

Consideration of the 17th Meeting of the ASEAN Sectoral Working Group on Fisheries (ASWGFi), 3-5 Juni 2009, Quang Nam, Vietnam

Menanggapi diberlakukannya EC Regulation No 1005/2008 to prevent, deter and eliminate illegal, unreported and uregulated (IUU) Fishing, yang akan diberlakukan pada 1 Januari 2010, telah disusun ASEAN Common Position on EC Regulation on IUU Fishing Catch Certification Scheme (lampiran 2) yang meminta penerapan EC Regulation tersebut diterapkan berdasarkan phase-based approach dan ditunda masa berlakunya.

Indonesia menyampaikan posisi untuk tidak mendukung ASEAN Common Position dimaksud, sebaliknya mendukung sepenuhnya EC Regulation untuk tetap diberlakukan 1 Januari 2010. Selain itu, Kamboja juga menyatakan bahwa sektor perikanannya belum siap untuk menerapkan kebijakan EC tersebut sehingga tidak mendukung ASEAN Common Position. Di sisi lain, delapan Negara ASEAN lainnya menyatakan setuju dengan ASEAN common position tersebut, terutama mengenai phase-based approach.

Memperhatikan adanya tiga kepentingan yang berbeda, Sekretariat ASEAN menawarkan dua opsi yang perlu dipertimbangkan yaitu tetap mengadopsi ASEAN Common Position dengan skema ASEAN minus Indonesia dan Kamboja atau pendekatan bilateral masing-masing negara ASEAN dengan pihak Uni Eropa. Hal ini belum mencapai konsensus dan akan ditindaklanjuti Sekretariat ASEAN dengan focal point perikanan Negara-negara ASEAN.

Cooperation in Forestry

Pertemuan dapat menerima laporan Myanmar terhadap hasil 12th Meeting of the ASEAN Senior Officials on Forestry (ASOF), 25-27 Juni 2009 di Nay Pyi Taw, Myanmar dan mengusulkan agar 2 (dua) dokumen penting, yaitu: ASEAN Criteria and Indicators for Legality of Timber dan ASEAN Guidelines on Phased Approach to Forest Certification dapat disetujui oleh AMAF.

Indonesia menyampaikan bahwa ASEAN Position Paper on REDD Plus sedang dalam penyelesaian di bawah koordinasi Indonesia (ARRN-FCC) dan mengusulkan agar menjadi bahan untuk dibahas dalam pertemuan 31st AMAF mendatang.

ASEAN Plus Three Cooperation in Food Agriculture and Forestry Sector

Thailand selaku Lead Country untuk East Asia Emergency Rice Reserve (EAERR) Pilot Project dan ASEAN Food Security Information System (AFSIS) menyampaikan laporannya. Terkait EAERR, dilaporkan bahwa pada 11th Project Steering Committee (PSC) yang berlangsung pada 10-11 Februari 2009 di Siem Reap, Kamboja telah diusulkan untuk mengembangkan EAERR menjadi ASEAN Plus Three Emergency Rice Reserve (APTERR) dan mengkaitkannya dengan ASEAN Integrated Food Security Policy Framework and its Strategic Action Plan of Action sebagai salah satu pilar dalam kerangka implementasinya. Hingga saat ini, draft APTERR Agreement hampir dapat disepakati, kecuali terkait pasal mengenai origin of rice, dikarenakan Thailand, Jepang dan Korsel masih memiliki keberatan.

Sementara itu, terkait dengan AFSIS, disampaikan bahwa the 1st Phase Project telah berakhir pada tahun 2007. Selanjutnya, pertemuan AMAF+3 telah menyetujui implemetasi 2nd Phase Project untuk periode 2008-2012. Elemen-elemen tambahan dari 2nd Phase ini ialah Early Warning Information, Agricultural Commodity Outlook dan Mutual Technical Cooperation.

Cooperation in Food, Agriculture and Forestry with Dialogue Partners, International Organizations and, Other Third Parties

ASEAN-India

Mengingat Indonesia telah menarik proposal Indonesia’s Initiative on ASEAN Networking for Agriculture Vulnerability to Climate Change yang sebelumnya akan dibiayai oleh ASEAN-India Fund serta menggantinya dengan proposal baru, maka perubahan ini akan disampaikan terlebih dahulu oleh Sekretariat ASEAN kepada pihak India.

ASEAN-Korea

Indonesia menyampaikan bahwa ASEAN Sekretariat telah mengirimkan surat undangan untuk Pertemuan ke-1 Ad-Hoc Working Group on the establishment of Asian Forest Cooperation Organization (AFoCO). Pertemuan ini akan diselenggarakan pada 24-26 Agustus 2009 di Jakarta.

Other Matters

Terkait dengan usulan untuk pengembangan dan implementasi ASEAN Multi-Sectoral Framework on Climate Change and Food Security (AFCC-FS), delegasi Kamboja mempertanyakan judul yang menggunakan istilah ‘food security’, mengingat pada pertemuan AMAF, disepakati untuk mengkaji dampak perubahan iklim terhadap ‘agriculture’, bukan hanya ketahanan pangan. Kamboja juga menilai bahwa istilah ‘multi-sectoral’ tidak perlu dicantumkan dalam judul. Sekretariat ASEAN menanggapi bahwa usulan Kamboja akan dibahas dalam workshop mendatang. Selain itu, Indonesia telah mengusulkan agar ASEAN memiliki common position dalam pertemuan COP-15 yang akan datang di Copenhagen, Denmark.

Terkait Strategic Plan of Action (SPA) for the ASEAN Cooperation in Food, Agriculture and Forestry (2005-2010), sejumlah action lines-nya saat ini telah tercakup dalam AEC Blueprint serta ASCC Blueprint. Oleh karenanya perlu dipertimbangkan untuk mengkonsolidasikan isi dari masing-masing dokumen tersebut.

Rangkaian pertemuan Thirty-First Meeting of the ASEAN Ministers on Agriculture and Forestry (31st AMAF) dan the 9th Meeting of the ASEAN Ministers on Agriculture and Forestry Plus Three (9th AMAF Plus Three) akan diselenggarakan pada 31 Oktober-5 November 2009, di Jerudong, Brunei Darussalam.

III. PENGAMATAN

Sesuai dengan harapan negara-negara anggota ASEAN, Indonesia diminta menjadi prime mover dalam rangka antisipasi dan mitigasi dampak perubahan iklim pada sektor pertanian, kehutanan dan perikanan. Untuk itu, pada pertemuan-pertemuan berikutnya, Indonesia harus siap memimpin dalam aktivitas ASEAN tersebut, termasuk mencari peluang pendanaannya bersama-sama dengan Sekretariat ASEAN.
Agar Indonesia turut memikirkan sikap bersama yang diambil ASEAN terhadap EC Regulation on IUU Fishing tanpa mengorbankan kepentingan Indonesia yang mendukung penerapan EC Regulation tersebut mulai tanggal 1 Januari 2010.

Pelajar Indonesia Juara Olimpiade Biologi

 

 Tiga Pelajar Indonesia Raih Juara International Olimpiade Internasional ke 20 di Tsukuba, Jepang

 

 

Indonesia raih 1 emas 1 perak dan 1 perunggu dalam kejuaraan Olimpiade Biologi Internasional ke 20 ( the 20th INTERNATIONAL BIOLOGY OLYMPIAD) di Tsukuba Jepang – July 12-19, 2009IBO ke 20 tahun 2009 telah diselenggarakan di Tsukuba, Jepang. Acara Olimpiade Biologi ini bertepatan dengan perayaan 200 tahun Charles Darwin serta 150 tahun publikasi “The Origin of Spesies”.

Acara IBO tahun ini diikuti oleh delegasi dari 56 negara dengan jumlah siswa 221 orang, ditambah 4 negara sebagai observer. Acara pembukaan hari Senin, 13 Juli 2009 dihadiri oleh Prince & Princess Akishimo sebagai Honorary President IBO ke 20. Chairperson Organizing Committee IBO ke 20, Dr. Hirro Imura dari Universitas Tsukuba mengawali acara pembukaan, dilanjutkan oleh sambutan dari Chairman IBO, Dr. Poonpipoe Kasemsap (Koordinator Tim IBO Thailand). Kemudian Menteri Pendidikan, Kultur, Sains & Teknologi Jepang, Mr. Ryu Shinoya; Chairman Japan Science Foundation, Dr. Akito Arima dan President University of Tsukuba, Dr. Nobuhiro Tamada. Prince & Princess Akishimo yang menghadiri acara pembukaan sejak 30 menit sebelum dimulainya acara juga memberikan sambutan sekaligus membuka acara IBO ke 20.

Prince & Princess Jepang terus mengikuti acara hingga melakukan ramah tamah dan bersantap siang bersama dengan para peserta. Empat (4) siswa IBO ke 20 yang mewakili Indonesia adalah: Anugerah Erlaut, siswa kelas XII, SMA Kharisma Bangsa, Tangerang. Pada OSN 2007 di Surabaya meraih Medali Emas dan mendapatkan The Best Teori. Pada IBO ke 19 tahun 2008 di India, Anugerah memperoleh Medali Perak.

Irfan Haris, siswa kelas X, SMAN 1 Pringsewu, Lampung. Pada OSN 2008 di Makasar meraih Medali Perak. Tahun 2007, Irfan mengikuti IJSO di Taiwan dan memperoleh Medali Perak.
Danang Crysnanto, siswa kelas XI, SMAN 1 Wonogiri. Pada OSN 2008 di Makasar meraih Medali Perunggu.

Elbert Wijaya, siswa kelas XII, SMANK 1 Penabur Jakarta, Pada OSN 2008 di Makasar meraih Medali Perak.Ke empat siswa di dampingi Pembina Tim IBO Indonesia, yaitu: Dr. Agus Dana Permana; Dr. Maelita R. Moeis; Dr. Devi Nandita Choesin; dan Dr. Iriawati dari SITH – ITB, Dr. Sucipto Hariyanto dari Biologi UNAIR, serta Ir. Gunardi Sihhatmanahadi dari DEPDIKNAS.

Setelah pembukaan, sekitar pukul 14.00 waktu setempat para pembina yang bertindak sebagai Juri mulai melakukan diskusi dan penterjemahan 4 set soal Test Praktikum, yaitu : 1) Anatomi Hewan dan Tumbuhan : Anatomi ulat sutera (Bombix mori), bunga dan buah Vigna angularis (sejenis tanaman kacang); 2) Biokimia dan Biologi Molekuler : Penetuan aktifitas enzim asam fosfatase yang dilihat berdasarkan reaksi fosfatase dari perubahan absorbansi karena perubahan konsentrasi yang diukur dengan spektrofotometer; 3) Genetika : Karakter berbagai lalat buah yang sangat sering digunakan sebagai studi Genetika, serta pigmen matanya yang dilihat dengan cara kromatografi dan analisis proteinnya dengan elktroforesis; 4) Fisiologi Sel : Bentuk dan jumlah sel ragi yang berproliferasi, serta mekanisme pergerakan regenerasi alga uniseluler. Seluruh Test Praktikum dilakukan di Department of Biological Sciences, Universitas Tsukuba.

Para juri dari Indonesia selesai dengan menterjemahkan soal pada pukl 03.00. Selasa, 14-7-2009 Test Praktikum dimulai pukul 09.00 dan selesai pukul 17.00. Setiap bidang test harus diselesaikan oleh para siswa selama 90 menit.Para siswa diberi kesempatan istirahat dengan melakukan tour ke Pusat Penelitian Roket Jepang, Science Center, serta salah satu Kuil yang menjadi World Heritage, yaitu Nikko Toshugu. Selama para siswa melakukan tour, para Juri menterjemahkan 2 set soal Test Teori.

Diskusi dan penterjemahan soal berlangsung hari Rabu, 15-7-2009 dan diselesaikan selama sekitar 14 jam, walaupun ada beberapa delegasi yang bekerja hingga 18 jam.Kamis, 16-7-2009, seluruh siswa melakukan Test Teori di Universitas Tsukuba selama 4,5 jam dengan diselingi istirahat 1 jam untuk makan siang.Setelah melakukan koreksi hasil pemeriksaan Panitia setempat, para Juri melakukan moderasi dan berdiskusi mengenai penilaian serta jumlah medali yang akan diberikan.

Acara Penutupan IBO ke 20 dilakukan pada hari Sabtu, 18-7-2009, diawali dengan spesial seminar yang disampaikan oleh Prof. Dr. Makoto Asashima, salah satu ahli Biologi Perkembangan di dunia. Pada pukul 16.00 mulai diumumkan para pemenang yang meraih medali perunggu, perak dan emas. Dengan bangga, Tim IBO Indonesia 2009 berhasil mempersembahkan 1 Medali Emas yang diperoleh Anugerah Erlaut, 1 Medali Perak dari Irfan Haris dan 1 Medali Perunggu dari Elbert Wijaya. Hasil tersebut merupakan kerja keras para putra terbaik Indonesia, walaupun tidak lepas dari bantuan para pembina, pengajar, asisten, tutor, dan guru mereka, serta Departemen Pendidikan Nasional yang senantiasa membuat Program Olimpiade Internasional di bidang Biologi, Kimia, Fisika, Matematika, Komputer dan Astronomi berjalan dengan lancar.

Untuk mempersiapkan peserta IBO ke 21 tahun 2010 yang akan dilaksanakan di Korea Selatan, pada awal Agustus 2009, Depdiknas akan melakukan seleksi melakui ajang Olimpiade Sains Nasional yang akan diselenggarakan di Jakarta dari tanggal 3 – 9 Agustus 2009.

Koordinator

Tim IBO Indonesia

Dr. Agus Dana Permana

SITH – ITB

Kabar dari Pengurus IKAMAJA Jawa Timur

Tulisan di bawah ini surat dari Ikamaja Jawa Timur:

Assalamu'alaikum War. Wab.
Salam kangen pada Bapak, semoga Bapak dalam lindungan Allah SWT, kami atas nama pengurus Ikamaja Jawa Timur mengucapkan terima kasih yang tak terhingga pada Bapak, perlu Bapak ketahui bahwa anggota IKAMAJA di Jawa Timur telah melakukan aktivitas dibidang masing-masing apa yang telah dipelajari selama di Induk Semang, kami ingin selalu berkomunikasi dengan Bapak dengan e-mail apabila bapak berkenan agar bapak bisa mengetahui kegiatan teman-teman Ikamaja di Jawa Timur, dan tidak putus tali silaturahmi, kami nantinya juga ingin mengetahui alamat e-mail atau facebook dari Induk semang kami dan salam buat semuanya, terutama kepada Bapak. Dan ini program yang telah di laksanakan atas nama organisasi Ikamaja di Jawa Timur yaitu setiap 3 bulan sekali kita koordinasi semua anggota, untuk pendanaan kita telah melaksanakan penghijauan yang bekerja sama dengan Perhutani Kanwil Jawa Timur dengan program sejuta pohon, semoga ini bermanfaat untuk kita semua. dan banyak lagi program yang lain. Demikian, atas diterimanya dari kami semoga bermanfaat.
Wassalamu’alaikum War. Wab.
Pengurus Ikamaja Jatim