Kloning gen virulen Streptococcus agalactiae sebagai bahan dasar vaksin rekombinan

ABSTRACT Streptococcus agalactiae has emerged as an important pathogen that affects Nile tilapia in Indonesia aquaculture. Vaccination is one of the most effective tools for enhancing host defense and protecting fish from pathogens. DNA vaccine is a third generation of vaccines based on the gene encoding a vaccine antigen rather than the antigen itself. Mga is DNA-binding protein that activates expression of several important virulence gene, including those encoding M protein (emm), C5a peptidase (SCPA) and mga. The goals of this study were to isolate and molecular characterize the mga gene of local isolate of S. agalactiae to support the development of DNA vaccine. Local bacterial strain was isolated from Nile tilapia  farming in West Java, Indonesia. Bacterial identification was accomplished by PCR, using 16S rRNA primers, which revealed the 1,500 bp PCR product. Mga gene isolation was accomplished by PCR using mga gene S. agalactiae SAF and SAR- specific primers, which revealed the 1,494 bp PCR product. Mga gene was cloned into pGEM T-easy and sequenced using M13 primers. SalI and NotI restriction enzymes were used to digest the pGEM T-easy vector containing mga gene. Mga gene was cloned into pMBA containing beta actin promoter of Japanese medaka. The 16S rRNA sequence analyses confirmed that the local bacteria was 97% similarity with S. agalactiae strain 15-92MPnew. The nucleotide sequence analyses confirmed that the clones were contained 98% similarity with M protein mga  S. agalactiae. The mga gene  controlled by MBA promoter has constructed successfully as a candidate of DNA vaccine to against S. agalactiae infection in Nile tilapia. Keywords: DNA Vaccine, Streptococcus agalactiae, mga gene, Oreochromis niloticus, recombinant DNA  ABSTRAK Streptococcus agalactiae merupakan patogen penting yang mempengaruhi budidaya ikan nila di Indonesia. Vaksinasi merupakan salah satu metode yang paling efektif untuk meningkatkan pertahanan dan melindungi ikan dari patogen. Vaksin DNA adalah vaksin generasi ketiga yang mengandung gen penyandi antigen vaksin. Mga adalah protein DNA-binding yang mengaktifkan ekspresi beberapa gen virulensi, termasuk M protein (emm), C5a peptidase (SCPA) dan mga. Tujuan dari penelitian ini adalah untuk mengisolasi dan karakterisasi secara molekuler gen mga dari isolat lokal S. agalactiae untuk mendukung pengembangan vaksin DNA. Identifikasi bakteri dilakukan dengan PCR, menggunakan primer 16S rRNA dengan produk PCR 1.500 bp. Isolasi gen mga dilakukan dengan metode PCR menggunakan primer SAF dan SAR dengan ukuran target 1.494 bp. Gen mga dikloning ke vektor pGEMT–easy dan disekuensing menggunakan primer M13.  Enzim Sal I dan Not I digunakan untuk memotong gen mga dari vektor pGEMT- easy, selanjutnya gen mga dikloning ke vektor pMBA yang mengandung promoter beta-aktin ikan medaka Jepang. Berdasarkan analisis menggunakan gen 16S rRNA diperoleh bahwa sampel memiliki kesamaan 97% sebagai S. agalactiae. Analisis sekuen nukleotida menunjukkan bahwa klon mengandung gen mga dengan 98% kesamaan dengan M protein mga S. agalactiae. Konstruksi mga gene yang dikendalikan oleh promoter MBA telah berhasil dibuat dan ini merupakan kandidat vaksin DNA untuk mengendalikan infeksi S. agalactiae pada ikan nila. Kata kunci: Vaksin DNA, Streptococcus agalactiae, gen mga, Oreochromis niloticus, DNA rekombinan

ISOLASI DAN KARAKTERISASI BIOLOGIK VIRUS NEWCASTLE DISEASE

Tujuan dari penelitian ini adalah memperoleh informasi infeksi dan sifat-sifat biologis virus Newcastle disease (ND) pada ayam ras maupunayam buras. Sejumlah 529 sampel usap kloaka dikoleksi dari pasar-pasar tradisional dan peternakan rakyat di daerah Bogor dan Tangerang. Sebanyak 20 sampel terdeteksi positif melalui real time reverse transcription-polymerase chain reaction (RRT-PCR) sedangkan dengan isolasi virus terdeteksi positif sebanyak 11 sampel. Selanjutnya empat isolat representasi lokasi dipilih untuk karakterisasi patogenisitas dengan metode mean death time (MDT) pada telur ayam berembrio (TAB) specific pathogen free (SPF), dan karakterisasi antigenisitas menggunakan metode haemagglutination inhibition (HI) dan virus netralisasi (VN) untuk melihat keragaman antar isolat virus. Uji MDT memperlihatkan dua isolat(kode isolat II dan XIII) adalah mesogenik, satu isolat (kode isolate VIII) termasuk lentogenik, dan satu isolat (kode isolat TW) bersifat velogenic. Tiga dari empat isolat menunjukkan hubungan antigenik dengan virus ND strain Komarov dan G7 (genotipe 7) tetapi tidak dengan strain referensi Lasota, sementara isolat velogenic (kode TW) yang berasal dari ayam kampung menunjukkan reaksi silang tinggi dengan antiserum terhadap tiga strain referensi di atas. Sampel-sampel yang diambil di lapang baik itu berasal dari pasar dan peternakan rakyat meskipun secara klinis tidak memperlihatkan gejala sakit dapat diisolasi virus ND dengan karakteristik yang beragam

Fecal Lipid Content, Serum Lipid Profile, and Intra-Abdominal Fat Accumulation in Normal Rats Supplemented with Rice Bran Oil

This study aimed to investigate the mechanism of rice bran oil on altering lipid absorption and blood lipid level in normal rat. Male Sprague Dawley rat age 3 months old, weighted 250–300 g were grouped into three groups: control (aquabidest 1 ml), orlistat (2.16 mg / 200 g body weight), and rice bran oil (1.04 mg γ-oryzanol / 200 g body weight). The intervention was given through oral gavage, daily for 28 days. Indicators observed were growth performance, total cholesterol and serum triglyceride levels, lipid levels in feces, and accumulation of intra-abdominal fat. The results showed that the treatment did not significantly affect body weight gain. Fecal lipid levels of orlistat, rice bran oil and control group respectively were 0.19g; 0.17g and 0.13 g (p<0.05), while the percentage of indigestible lipids for orlistat, control and rice bran oil group were 26%, 17% and 13% respectively (p<0.05). Total cholesterol and serum triglyceride levels in rice bran oil group were significantly lower than controls. Rice bran oil did not significantly affect the percentage of total intra-abdominal fat and the weight of the heart and kidney (p>0.05). The intervention of rice bran oil was shown to reduce blood cholesterol and triglyceride levels in normal mice and did not accumulate intra-abdominal fat. The results suggest that rice bran oil might have an effect on blood lipid regulation but not by preventing lipid absorption

AQ-9 Identification of Sumateran Wild Boar Meat (Sus scrofa vittatus) by Restriction Fragment Length Polymorphism (RFLP) Analysis of Cytochrome b Gene

Sumateran wild boars have been super abundant in Sumateran forest. In Indonesia, this wildlife condition has led to the exploitation for commercial purpose. The high number of Sumateran wild boars population increases wild boar hunting resulting in an abundant availability of wild boar meat in the food market with extremely cheap price. The macroscopic similarity of wild boar meat and beef has prompted the local people to abuse this situation by selling wild boar meat in traditional market as beef. Based on annual record from Cilegon Class II Quarantine Office in 2014, there were nine smuggling cases or a total of 21.556 kg of wild boar meat smuggling effort that were prevented by Cilegon Quarantine officers. The number of food safety concerns related to smuggling of wild boar or counterfeiting beef with wild boar is a very detrimental condition for consumers, especially consumers in traditional markets.The checking of genuineness or validity of food products is an important effort to protect people from consuming unhealthy food and to indicate whether the food is halal or not. Studies of meat detection should be continuously developed as an effort to protect consumers. Genetic method is the most specific and sensitive method to check food ingredients authenticity by detecting the presence of genetic material or deoxyribonucleic acid (DNA). It results from the specific character of the structure of DNA particles and the possibility of using the information included in them. The most frequent loci used for species identificationin phylogenetics and biodiversity studies are mitochondrial cytochrome b (cyt b).Genetic method is the most specific and sensitive tool for analyzing the authenticity of food ingredients in a molecular level by means of detecting the presence of genetic material or deoxyribonucleic acid (DNA). One of the various methods could be used to detect genetic material is polymerase chain reaction (PCR). Specifically, one of such method frequently used in food industry to observe animal derived product fabrication is PCR restriction fragment length polymorphism (RFLP). PCR-RFLP is based on the comparison of the bands profile generated after certain enzymes digest the DNA target. PCR-RFLP is appropriate for meat testing due its ability in exploiting sequence variation in designated DNA region that allows species differentiation even from closely related species through DNA fragment restrictions selected by suitable restriction enzyme. PCR-RFLP is advantageous since it is simple, cheaper, and easier to be adjusted for routine big-scale studies such as surveillance program

Uji Patogenisitas Zoospora Kapang Lagenidium giganteum terhadap Larva Instar-2 Nyamuk Aedes aegypti Skala Laboratorium (PATHOGENICITY TEST OF ZOOSPORA LAGENIDIUM GIGANTEUM FUNGI AGAINST AEDES AEGYPTI LARVAE 2nd UNDER LABORATORY CONDITION)

Dengue Haemorrhagic fever (DHF) is one of fearsome diseases in society. Incidence of the disease isincreasing. Dengue fever is caused by dengue virus and transmitted by Aedes aegypti mosquito vector.Various chemical controls have been conducted to prevent the spread of the disease, but active contents ofthe chemical controlling substances are suspected causing many negative effect, in environment, such asvector resistance, death of non target living creatures, and environmental contamination.This researchobjective was to find an alternative solution in order to control the dengue vector by using entomopathogenicfungi as biological control agent. This research was conducted by isolation and identification of fungiinfecting mosquito larvae. Macroscopic observation revealed that one of the nine isolation products wasLagenidium giganteum. The effectiveness test in laboratory showed the zoospore LD50 to Ae.aegypti larvaeof instar 2nd was 2,35 x 106 zoospore/ml, while the LD95 value was 1,35 x 107 zoospore/ml. The oosporeeffectiveness test showed LD50 was 6,7 x 102 oospore/ml and LD95 was 1,94 x 103 oospore/ml. Using LPCBdye and blue tolouidin 2,5%, the infection mechanism of L.giganteum fungi in Ae.aegypti mosquito larvawas detected. The research is concluded that the entomophatogen fungi L. giganteum was very prospectiveto be used as a biological control agent against vector of DHF

Efektivitas Larutan Desinfektan Dalam Menginaktivasi Virus Avian Influenza pada Bulu Unggas

Avian Influenza (AI) virus  is pathogenic agent that can spread from one area to another area through the transportation of infected animals or their products such as feathers. This research was aimed to inactivated AI virus with 775 ppm sodium hypochlorite and 0.1% chloroxylenol to examine the difference of treatment by time (day) for inactivation AI virus on the feathers. AI virus isolate A/chicken/sidrap/ 07160336‐2/2016 used in this research was obtained from Balai Besar Veteriner Maros. The treatment of disinfectants were performed on the first day (the day of disinfectan solutions were prepared), the third day and the seventh day by soaked the feathers in disinfectant solution for 10 minutes. The effectiveness of disinfectans were evaluated by inactivation index. The result show that the average of inactivation index of 775 ppm of sodium hypochlorit was 4.17 for the first day, 5.17 for the third day, and 4.20 for the seventh day, while the average of inactivation index of 0.1% chloroxylenol was 5.50 for the first day, 6.43 for the third day, and 5.77 for the seventh day. Our result indicated that the sodium hypochlorit and chloroxylenol were effective for inactivation of AI virus. The 0.1% of chloroxylenol was more effective for inactivation AI virus than 775 ppm of sodium hypochlorit, whilst the most effective duration for the treatment is the three days

Deteksi Coxiella burnetii Penyebab Q fever pada Sapi, Domba dan Kambing di Bogor dan Bali (DETECTION OF COXELLA BURNETII, THE CAUSAL AGENT OF Q FEVER

A study to detect Coxiella burnetii, an intracellular bacterium causing Q fever in human and livestock animals, was carried out in several ruminants in Bogor and Bali. The methods used for the detection was Nested-Polymerase Chain Reaction (Nested-PCR). Two pairs of primers, the first (OMP1 and OMP2) and the second (OMP3 and OMP4) were used to detect the genomic sequences and the conserved specific sequences of Coxiella burnetii, respectively. Organ samples such as liver and lung from 410 livestock ruminants, consisting of cattle (245 samples), sheep (105 samples) and goats (60 samples) were collected from several slaughter houses in Bogor and Bali. As many as 15 (6.12%) out of 245 cattle, 6 (5.71%) out of 105 sheep and none from goat were infected by Coxiella burnetii. Interestingly, 3 out of 15 infected cattle were Bali cattle. The results clearly indicate that Q fever is likely to be widespread among ruminant animals in Indonesia

Ancaman terhadap Masuknya Virus Penyakit Mulut dan Kuku melalui Daging Ilegal di Perbatasan Darat Indonesia-Malaysia

Penelitian ini bertujuan untuk menganalisis keberadaan daging ilegal di perbatasan darat Indonesia-Malaysia sebagai ancaman risiko masuknya virus PMK ke wilayah Indonesia. Data primer diambil menggunakan teknik pengumpulan pendapat pakar dengan kuisioner, wawancara mendalam (in-depth interview) dan pengamatan langsung di lapang. Data sekunder diperoleh dari publikasi ilmiah dan tulisan atau data yang tidak dipublikasi (statistik, dokumen dan laporan dari instansi berwenang). Penentuan responden secara purposive sampling. Hasil studi menunjukkan bahwa daging ilegal diperkirakan berasal dari berbagai negara termasuk dari negara/zona yang berstatus endemis PMK seperti Semenanjung Malaysia, Thailand, India dan negara/zona yang dinyatakan tidak diketahui oleh responden. Jenis daging ilegal yang masuk ke Entikong berisiko sebagai sumber infeksi PMK seperti daging beku bertulang tanpa limfoglandula dan jeroan beku tanpa limfoglandula. Berdasarkan jalur dan frekuensi pengangkutan, perkiraan volume pemasukan daging ilegal menunjukkan kemungkinan daging masih bisa lolos melalui jalur non-kendaraan. Kondisi-kondisi tersebut mengindikasikan bahwa pemasukan daging ilegal dapat sebagai ancaman risiko masuknya virus PMK ke Indonesia khususnya di perbatasan darat Indonesia-Malaysia, Entikong. Perlu dilakukan upaya pencegahan untuk mengurangi ancaman risiko yaitu dengan melakukan pengawasan yang lebih ketat di pintu-pintu pemasukan dengan koordinasi lintas instansi di perbatasan untuk bersama-sama mencegah pemasukan daging ilegal

Identification of Gene Resistance to Avian Influenza Virus (Mx Gene) among Wild Waterbirds

The Mx gene is an antiviral gene used to determine the resistance or the susceptibility to different types of viruses, including the Avian Influenza (AI) virus subtype H5N1. The AI virus subtype H5N1 infection in chickens causes Mx gene polymorphism. The Mx+ gene shows resistant to the AI virus subtype H5N1, whereas the Mx- gene shows signs of susceptible. The objective of this research was to detect the Mx gene in wild aquatic birds using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method with the primer pairs F2 and NE-R2/R and the RsaI restriction enzyme. DNA samples were obtained from eight species of wild waterbirds with positive and negative exposure to the AI virus subtype H5N1. DNA amplification results showed that the Mx gene in wild aquatic birds is found in a 100 bp fragment, which is the same as the Mx gene found in chickens. However, unlike chickens, the Mx gene in wild aquatic birds did not show any polymorphism. This study proves that Mx- based resistance to AI virus subtype H5N1 in different in wild birds than in chickens. Keywords: Mx gene, wild waterbirds, exposure, AI virus subtype H5N1, resistance   Keywords: Mx gene, wild waterbirds, exposure, AI virus subtype H5N1, resistance&nbsp

Identification of Gene Resistance to Avian InfluenzaVirus (Mx Gene) among Wild Waterbirds

The Mx gene is an antiviral gene used to determine the resistance or the susceptibility to different types of viruses, including the Avian Influenza (AI) virus subtype H5N1. The AI virus subtype H5N1 infection in chickens causes Mx gene polymorphism. The Mx+ gene shows resistant to the AIvirus subtype H5N1, whereas the Mx-gene shows signs of susceptible. The objective of thisresearch was to detect the Mxgene in wild aquatic birds using the Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) method with the primer pairs F2 and NE-R2/R and the RsaI restriction enzyme. DNA samples were obtained from eight species of wild waterbirds with positive and negative exposure to the AI virus subtype H5N1. DNA amplification results showed that the Mxgene in wild aquatic birds is found in a 100 bp fragment, which is the same as the Mx gene found in chickens. However, unlike chickens, the Mxgene in wild aquatic birds did not show any polymorphism. This study proves that Mx- based resistance to AI virus subtype H5N1 in different in wild birds than in chickens