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Vaccination of goats with fresh extract from Sarcoptes scabiei confers partial protective immunity Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 11, No 2 (2006)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (104.165 KB) | DOI: 10.14334/jitv.v11i2.519

Abstract

Protective immunity has been known to develop in animals infested with Sarcoptes scabiei. However, our previous attempt to induce protective immunity in goats by vaccination with fractions of soluble or insoluble mite proteins had been unsuccessful. Degradation or denaturation of protective antigens occurred during vaccine preparation was suggested as one possible cause of the failure. In this study, mite proteins that used to immunise animals were prepared rapidly in order to prevent protein degradation or denaturation. About 150 mg of freshly isolated mites were rapidly homogenised, centrifuged then separated into supernatant and pellet fractions. Twenty-eight goats were allocated equally into 4 groups. Group-1 goats were vaccinated with the whole mite homogenate supernatant, group 2 with the supernatant, group 3 with the pellet, and group 4 with PBS (unvaccinated control). Vaccination was conducted three times, with three-week intervals between vaccinations, using Quil A as adjuvant, and each vaccination using fresh mite homogenates. One week after the last vaccination, all animals were challenged with approximately 2000 live mites. The severity of lesions, scored from 0 (no lesions) to 5 (>75% infested auricle affected), were determined one day, two days, then every week after challenge. Mite challenge caused the development of skin lesions in all animals. No significant differences between vaccinated and unvaccinated animals were observed in regards to the severity of lesions. However, the mite densities in vaccinated animals were significantly lower (P=0.015) than those in unvaccinated control. This study indicates that the protective antigens of S. scabiei are liable to degradation or denaturation and exist in a very low concentration or have vary low antigenicity. This implies isolation of the protective antigens by the conventional approach, fracionation of the whole mite proteins and testing each fractions in vaccination trials, is seemingly inappropriate for S. scabiei. Key Words: Sarcoptes scabiei, Vaccination, Fresh Homogenate, Partial Immunity
Characterisation of enzymatic activities of H5N1 influenza virus Tarigan, Simson; Indriani, Risa; ., Darminto
Indonesian Journal of Animal and Veterinary Sciences Vol 12, No 2 (2007)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (195.159 KB) | DOI: 10.14334/jitv.v12i2.554

Abstract

One of the two glycoproteins projected from the surface of the influenza virus is identified as neuraminidase. This enzyme enables the virus to spread in the host, and therefore it plays vital roles in the viral pathogenicity. From the viewpoint of disease control, neuraminidase is used as the target for the development of anti-flu drugs, and for the development of diagnostic test to differentiate infected from vaccinated animals (DIVA). Since the roles of the enzyme are very important, information regarding the characteristics and the procedure to measure its activity, which is the purpose of this study, is essential. The optimum incubation time of the neuraminidase-substrate (fetuin) reaction and the optimum pH of the buffer were determined. The stability of the enzyme against heating, supplementation or chelating of calcium ion, and b-propiolactone treatment were analysed. This study showed that neuraminidase from H5N1-influenza virus was, in regards to the characteristics investigated in this study, was comparable to that from Clostridium perfringens. The optimum incubation time for the viral and Clostridial neuraminidases were 60 and 30 minutes, respectively; whereas, the optimum pH for both neuraminidase was 6-7. At pH 8, both neuraminidase were inactive. Supplementation of calcium ion tended to increase activity but chelating of the cation did not have any observable effects. Treatment with 0.2% b-propiolactone for 6 hours reduced the activity, whereas heating at 60°C for 60 minutes abolished all activity. Since inactivation by b-propiolactone is partially only, neuraminidase assay could be performed safely in ordinary laboratories using b-propiolactone-treated-influenza virus, rather than the life virus. The thermolabile nature of the enzyme will complicate any attempt to purify the enzyme. Key Words: H5N1, Neuraminidase, Stability, Thiobarbituric Assay
Purification and production of monospecific antibody to the hemagglutinin from Subtype H5N1 influenza virus Tarigan, Simson; Indriani, R; Hewajuli, D
Indonesian Journal of Animal and Veterinary Sciences Vol 15, No 4 (2010)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (455.929 KB) | DOI: 10.14334/jitv.v15i4.670

Abstract

The purpose of this study was to purify the hemagglutinin from H5N1 virus and to generate monospecific antibody appropriate for production of sensitive and specific immunoassay for H5N1 avian influenza. For this purpose, a local isolate H5N1 virus (A/Ck/West Java/Hamd/2006) was propagated in chicken embryos. The viral pellet was dissolved in a Triton-X-100 solution, undissolved viral particles were pelleted by ultracentrifuge, and the supernatant containing viral surface glycoproteins (Hemagglutinin and neuraminidase) was collected. The neuraminidase in the supernatant was absorbed by passing the supernatant through an Oxamic-acid-superose column. After dialyzing extensively, the filtrate was further fractionated with an anion exchange chromatography (Q-sepharose) column. Proteins adsorbed by the column were eluted stepwisely with 0.10, 0.25, 0.25 and 0.75 M NaCl in 20 mM Tris, ph 8. Hemagglutinin (H5) was found to be eluted from the column with the 0.5 M NaCl elution buffer. The purified H5 was free from other viral proteins based on immunoassays using commercial antibodies to H5N1 nucleoprotein and neuraminidase. When used as ELISA’s coating antigen, the purified H5 proved to be sensitive and specific for hemagglutinin H5. Cross reactions with other type-A-influenza virus, H6, H7 dan H9, were negligibly low. For the production of monospecific antiserum, the purified H5 was separated with SDS-PAGE, the band containing the H5 monomer was cut out , homogenised and injected into rabbits. The antiserum was capable of detecting the presence of inactivated H5N1 virus in a very dilute suspension, with a detection limit of 0.04 heagglutination (HA) unit. The purified hemagglutinin and the serum raised against it should be useful for developing specific, sensitive and affordable immunoassay for H5N1 avian influenza. Key Words: H5N1, Hemagglutinin, Triton, Oxamic-acid Sepharose, Q sepharose, ELISA
Production and purification of streptavidin with higher biotin-binding activity Tarigan, Simson; ., Sumarningsih .
Indonesian Journal of Animal and Veterinary Sciences Vol 19, No 3 (2014)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14334/jitv.v19i3.1086

Abstract

The objective of this study was to develop practical, efficient method for production, purification and assay of  binding activity of streptavidin. Streptomyces avidinii was first propagated on agar plates, the bacterial cells on the agar were scrapped and suspended in a defined synthetic media (4.4 ml/cm2). After 7 days agitation on a rotary shaker (200 rpm/min) at room temprature (≈28°C), the bacterial cells in the culture were pelleted. The culture supernatant was concentrated to 1/62 original volume with 75% saturation ammonium sulphate. After intensive dialysis against ammonium carbonate buffer pH 11, the suspension was loaded into an iminobiotin agarose column chromatography. The adsorbed protein (streptavidin) was eluted with sodium acetate buffer, pH 4, and the eluate was concentrated with an ultrafiltration divice and suspended in PBS. The strepatavidin-binding activity was  assayed by a competitive ELISA, a competition between streptavidin in the sample and the HRP-streptavidin conjugate for the biotin (biotinyl IgG) immobilised on wells of a microtitre plate. The detection limit of this assay measured 0.16 µg/ml streptavidin. The method developed in this study produced 160 µg/ml streptavidin in the culture supernatant. After concentration with the ammonium sulphate, the streptavidin concentration increased to 4 mg/ml (69% recovery). At the final step of purification, streptavidin with 10 mg/ml concentration was obtained. The purity of the streptavidin was higher (95%) with a recovary of 19%. The purified streptavidin in this study appeared as a dimer core streotavidin on SDS PAGE and its binding activity was twice as high as that of a commercial one. Key Words: Streptavidin, Streptomyces avidinii, Ammonium Sulphate, Iminobiotin Agarose, Binding Activity
Actinobacillus pleuropneumoniae cytotoxins on size, granularity and viability of porcine neutrophils Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 3, No 3 (1998)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1064.805 KB) | DOI: 10.14334/jitv.v3i3.116

Abstract

Cytotoxins produced by Actinobacillus pleuropneumoniae are supposed to play major roles in bacterial pathogenicity and virulence. To gain better understanding in the mechanism of the pathogenicity, cytotoxic activities of the toxins on porcine neutrophils were investigated in vitro. Changes in cell size, granularity and viability were examined with a flow cytometer. Cell size and granularity correlate with forward light scatter and right angle light scatter, respectively; whereas, cell viability corresponds with fluorescent intensity of cells stained with propidium iodide . At low concentrations (dilutions between 1/10 and 1/100 of bacterial culture supernatants),  the cytotoxins induced severe swelling and degranulation of neutrophils; whereas, at higher concentrations (dilutions of 51/10 bacterial culture supernatants), the cytotoxins caused rapid cell death. There was no significant difference in cytotoxic activities of Cyooxins derived from various serotypes (serotypes 1, 2, 3, 5 and 7  ) of A. pleuropneumoniae . Morphologically, the cytotoxin-treated neutrophils stained with Giemsa showed profound changes. Neutrophils treated with low dosages of Cyooxins became swollen with spherical nuclei . Higher concentration of cytotoxins study indicates strongly that important mechanism in the caused vactiolation of cytoplasts, enlargement or disintegration of nuclei . This in vitro intoxication of neutrophils by cytotoxins produced by A. pleuropneumoniae comprises anpathogenicity of the bacteria.   Key words : Actitiobacilluspleuropneumoniae, cytotoxin, neutrophils, pig, flow cytometry
Effects of Actinobacillus pleuropneumoniae cytotoxins on generation of oxygen radicals by porcine neutrophils Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 4, No 1 (1999)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (161.106 KB) | DOI: 10.14334/jitv.v4i1.136

Abstract

Cytotoxins produced by Actinobacillus pleuropneumoniae (App) suggested to be the most important pathogenic and virulent factors for this organism. However, the mechanisms on how the cytotoxins contribute to the disease process remain unclear. The purpose of this study is to investigate the effect of the cytotoxins on the oxidative-burst metabolism of porcine neutrophils. In this study, neutrophils were firstly loaded with an oxidative probe dichlorofluorescin diacetate (DCFHDA) then expose to cytotoxins. Cells producing oxygen radicals emitted fluorescence and its intensity was measured with a FACScan flow cytometer. All cytotoxins derived from either App serotypes producing ApxI and ApxII, App serotypes producing ApxII only, or App serotypes producing ApxII and ApxIII were capable of stimulating neutrophils for oxygen-radical generation. However, compared with phorbol myristate acetate (PMA), App cytotoxins were much weaker as stimulants for oxygen radicals. In addition, Apx preparation stimulated an oxidative-burst metabolism of neutrophils at a low, narrow range of Apx doses. At higher doses, the toxins inhibit the oxidative burst metabolism. The effects of cytotoxins produced by App during infection on recruited neutrophils into the lungs are assumed to be comparable to those observed in this in vitro study. Neutrophils, and other host cells, adjacent to the bacteria become lysis due to high toxin concentration, whereas those at some distance to the bacteria produce oxygen radicals which in turn cause tissue damage or necrosis.   Key words: Actinobacillus pleuropneumoniae, cytotoxin, Apx, neutrophils, pig, oxygen radical, flow cytometry  
Subclinical Infection by Avian Influenza H5N1 Virus in Vaccinated Poultry Tarigan, Simson
WARTAZOA. Indonesian Bulletin of Animal and Veterinary Sciences Vol 25, No 2 (2015): JUNE 2015
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (207.372 KB) | DOI: 10.14334/wartazoa.v25i2.1144

Abstract

Highly Pathogenic Avian Influenza (HPAI) H5N1 is endemic in Indonesia especially in unvaccinated sector-4 poultry. Considering that vaccination against influenza viruses does not induce sterilizing immunity and the source of infection is prevalent around the vaccinated farms, infection in the commercial layers and breeders may be common. Because infection in vaccinated birds is usually subclinical, its presence is unnoticable. The virus in such farms may be circulated persistently and become the source of infection to the surrounding areas. The test, Differentiation Infected from Vaccinated Animals (DIVA) that can be used to identify subclinically infected farms is not available yet in Indonesia. Observation on sentinel chicken among vaccinated birds is a sensitive and accurate method but unsafe for HPAI. The DIVA method based on heterologous neuraminidase has been successfully used in Italy, but it is difficult to be applied in Indonesia. The DIVA method based on Ectodomain protein M2 virus Influenza (M2e) uses antibody against M2e as infection marker and does not limit the subtype of vaccine used. This method is potential to be used in Indonesia because the M2e is very conserved across all avian influenza viruses and has high proportion of post-infected seroconverted birds. Key words: H5N1, DIVA test, heterologous neuraminidase, M2e, vaccination
The reliability of DIVA test based on M2e peptide exceed those based on HA2 or NS1 peptides Tarigan, Simson; Sumarningsih, . .; Ignjatovic, J.
Indonesian Journal of Animal and Veterinary Sciences Vol 20, No 2 (2015): JUNE 2015
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (524.763 KB) | DOI: 10.14334/jitv.v20i2.1167

Abstract

One of the most important disadvantage of vaccination against avian influenza is that it cannot protect vaccinated birds against infection. When vaccinated poultry are heavily exposed to the virus, prolonged, unrecognised, subclinical infection may persist on the farm. The condition can only be serologically monitored by a DIVA (differentiation of infected from vaccinated animals) test, whereas conventional diagnostic tests cannot be used. The DIVA tests based on an antibody response following virus replication is the most appropriate approach. For H5N1 influenza such antibodies includes those to the M2e and NS1 proteins and an epitope on the HA2 subunit (HA_488-516). The purpose of this study was to compare the magnitude of the antibody  response in chickens vaccinated and infected with an H5N1 virus strain. For that purpose, sera collected from naïve, vaccinated and infected  birds, at 1, 2-3, ≥4 weeks post challenge were used. Antibodies were measured by ELISA using biotinylated synthetic peptides as coating antigens. The peptides used include four NS1 peptides corresponding to different regions of the NS1 protein and HA_488-516and M2e peptides. Peptides were coated onto microtitre plates either directly or via a streptavidin bridge. The results showed that vaccination did not cause antibody conversion to any of the peptides, where as challenged birds developed a high antibody response to M2e but, low response to the NS1 and HA2 peptides. Antibodies to the later peptides were detected only by the streptavidin-peptide ELISA. The ELISA based on NS1 or HA_488-516 peptides, therefore, are not reliable for use as DIVA test in H5N1 avian influenza virus infection. Key Words: M2e, NS1 Protein, HA2 Peptide, DIVA Test, H5N1
Circulating H5N1 virus among native chicken living around commercial layer farms Tarigan, Simson; Indriani, Risa; Ignjatovic, J.
Indonesian Journal of Animal and Veterinary Sciences Vol 20, No 3 (2015): SEPTEMBER 2015
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (510.948 KB) | DOI: 10.14334/jitv.v20i3.1190

Abstract

Soon after the application of vaccination programme against high pathogenic avian influenza H5N1 outbreak of the disease in breeder and commercial layer farms has diminished remarkably in West Java. This study aimed to investigate whether the H5N1 decline is related to the disappearance of source of infection around the farms. Serum samples were collected from 421 native chicken living around commercial layer farms in the Districs of Cianajur and Sukabumi, West Java in March-April 2014.  Antibodies to avian influenza virus (AIV) H5N1 were measured using haemaglutination inhibition (HI), ELISAs and immunoblotting that measured presence of antibodies to the haemagglutin of H5N1 strain, as well as the M2e and nucleoprotein (NP) of all avian influenza viruses. Based on the combined results, 8.6% of the native chickens were seropositive to AI virus based on one or more of serological tests. This study provided serological evidence that H5N1 virus was still circulating among native chicken living around commercial layer farms. Many positive sera were however positive for antibodies in one test only: 2.4%, 3.3% and 3.8% by HI test, M2e and NP ELISA, respectively. It could be speculated that the incongruity of the results is due to the fact that HI, MM2e ELISA and NP ELISA all measure different type of antibodies and the duration of these antibodies in serum following infection with H5N1 differ. The fact that H5N1 virus is still circulating around commercial layer farms infers that the commercial farms are still under threat and therefore vaccination and strict biosecurity are still needed. Key Words: H5N1, Native Chicken, Commercial Layer, Nucleoprotein, M2e, HI Test
PENERAPAN MODEL PEMBELAJARAN PROBLEM BASED LEARNING (PBL) DENGAN MENGGUNAKAN MACROMEDIA FLASH UNTUK MENINGKATKAN BERPIKIR KREATIF DAN HASIL BELAJAR SISWA PADA POKOK BAHASAN KELARUTAN DAN HASIL KALI KELARUTAN Tarigan, Simson; Manurung, Hisar; Situmorang, manihar
TABULARASA Vol 12, No 2 (2015): Jurnal TABULARASA
Publisher : TABULARASA

Show Abstract | Download Original | Original Source | Check in Google Scholar

Abstract

Penelitian ini bertujuan untuk: Hasil Studi pada produk kelarutan dan bahan kimia kelarutan diajarkan oleh model PBL dengan macromedia flash lebih tinggi daripada siswa diajarkan oleh PBL tanpa model media dan model DI, berpikir kreatif pada kelarutan bahan kimia dan produk kelarutan diajarkan oleh PBL model dengan macromedia flash lebih tinggi daripada siswa diajarkan oleh model PBL tanpa model media dan DI, hubungan antara berpikir kreatif dengan hasil belajar siswa diajarkan dengan model DI, hubungan antara berpikir kreatif dengan hasil belajar siswa yang diajarkan oleh model PBL tanpa media dan hubungan antara berpikir kreatif dengan hasil belajar siswa yang diajarkan oleh model PBL dengan Macromedia Flash. Metode penelitian adalah metode kuasi-eksperimental. Sampel adalah 3 kelas di 3 sekolah di Medan, yang ditentukan oleh random sampling terdiri dari percobaan 1 (Model DI), percobaan 2 (model PBL tanpa media yang) dan percobaan 3 (model PBL dengan Macromedia Flash). Hasil penelitian menunjukkan hasil penelitian eksperimental dengan 3 lebih tinggi dari percobaan 2 dan percobaan 1, berpikir kreatif dengan percobaan 3 lebih tinggi dari percobaan 2 dan percobaan 1, ada hubungan antara berpikir kreatif dengan menggunakan pembelajaran eksperimental hasil-hasil 1, ada hubungan antara berpikir kreatif dengan hasil belajar menggunakan percobaan 2, ada hubungan antara berpikir kreatif dengan hasil belajar menggunakan percobaan 3.