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Production and purification of streptavidin with higher biotin-binding activity Simson Tarigan; Sumarningsih . .
Jurnal Ilmu Ternak dan Veteriner Vol 19, No 3 (2014): SEPTEMBER 2014
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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.

Effects of Actinobacillus pleuropneumoniae cytotoxins on generation of oxygen radicals by porcine neutrophils Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 4, No 1 (1999): MARCH 1999
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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

Purification of neuraminidase from Influenza virus subtype H5N1 Simson Tarigan; Risa Indryani; Darminto .; Jagoda Ignjatovic
Jurnal Ilmu Ternak dan Veteriner Vol 14, No 1 (2009): MARCH 2009
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Influenza-virus neuraminidase plays vital role in the survival of the organisms. Vaccination of animals with this glycoprotein confers immune responses so that enable it to protect the animals from incoming infection. Supplementation of conventional vaccines with this glycoprotein increases the protection and longevity of the vaccine. Purified neuraminidase can also be used to develop serological tests for differentiation of serologically positive animals due to infection or to vaccination. In this study purification of neuraminidase from influenza virus subtype H5N1 was described. Triton x-100 and Octyl β-D-glucopyranoside were used to extract and diluted the glycoprotein membrane. The enzymatic activity of the neuraminidase was assayed using a fluorochrome substrate, 4-methylumbelliferyl-a-D-N-acetyl neuraminic acid, which was found to be simple, sensitive and suitable for the purification purpose. The neuraminidase was absorbed selectively on an oxamic-acid agarose column. The purity of neuraminidase eluted from this affinity column was high. A higher purity of the neuraminidase was obtained by further separation with gel filtration on Superdex-200. The purified neuraminidase was enzymatically active and did not contain any detectable haemagglutinin, either by haemagglutination assay or by monospecific antibodies raised against H5N1 hemagglutinin. The purified neuraminidase was recognized strongly by antibodies raised against an internal but only weakly by that against C-terminal regions of the neuraminidase protein of H5N1-influenza virus. The purified neuraminidase was in tetrameric forms but dissociated into monomeric form on reducing condition, or mostly dimeric form on non-reducing SDS-PAGE. Key Words: Neuraminidase, Influenza, H5N1, Methylumbelliferyl, Oxamic-acid

Protective value of immune responses developed in goats vaccinated with insoluble proteins from Sarcoptes Scabiei Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 10, No 2 (2005): JUNE 2005
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Vaccines developed from certain membrane proteins lining the lumen of arthropod’s gut have been demonstrated effective in the control of some arthropod ectoparasites. A similar approach could also be applied to Sarcoptes scabiei since this parasite also ingests its host immunoglobulins. To evaluate immune protection of the membrane proteins, insoluble mite proteins were fractionated by successive treatment in the solutions of 1.14 M NaCl, 2% SB 3-14 Zwitterion detergent, 6 M urea, 6 M guanidine-HCl and 5% SDS. Five groups of goats (6 or 7 goats per group) were immunised respectively with the protein fractions. Vaccination was performed 6 times, each with a dosage of 250 μg proteins, and 3 week intervals between vaccination. Group 6 (7 goats) received PBS and adjuvant only, and served as an unvaccinated control. One week after the last vaccination, all goats were challenged with 2000 live mites on the auricles. The development of lesions were examined at 1 day, 2 days, and then every week from week 1 to 8. All animals were bled and weighed every week, and at the end of the experiment, skin scrapings were collected to determine the mite burden. Antibody responses induced by vaccination and challenge were examined by ELISA and Western blotting. This experiment showed that vaccination with the insoluble-protein fractions resulted in the development of high level of specific antibodies but the responses did not have any protective value. The severity of lesions and mite burden in the vaccinated animals were not different from those in the unvaccinated control. Key Words: Sarcoptes scabiei, Insoluble Protein, Goat, Vaccination

Vaccination of goats with fresh extract from Sarcoptes scabiei confers partial protective immunity Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 11, No 2 (2006): JUNE 2006
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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

Antibody response in naïve and sensitised goats infested by Sarcoptes scabiei Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 4 (2004): DECEMBER 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The purpose of this study was to characterize the IgG and IgE antibody responses in goats infested repeatedly with Sarcoptes scabiei. Ten goats purchased from scabies-free farms were infested with 2000 live mites on the auricles. Fifty days after the initial infestation, the goats were treated with ivermectin. After being completely recovered, the goats were reinfested then treated again at 50 days post infestation. Blood samples were collected at the time of the first infestation, then every 10 days afterwards for 270 days. Seroconversion for IgG took place after 30 days following the first infestation, whereas the maximum level of the specific IgG antibodies occurred after 50 days. Immunoblot analysis identified a number of antigens (Mr 180, 135, 43 and 38 KDa) that recognised by the IgG at 10 days and continuously recognised throughout the course of the multiple infestations. Being consistently recognised, those antigens should be essential in the development immunological diagnostic tests for scabies. The levels of scabies-specific IgE antibodies increased slowly during the first infestation and rapidly dropped following treatment of the animals with ivermectin. In the second and third infestations, however, the reaginic antibodies rose rapidly and with a grater level. On immunoblot analysis, at least 10 antigens (Mr 130, 72, 64, 58, 48, 44, 41, 39, 27 and 25 KDa) were observed to be recognised by the IgE present in the sera from scabies-infested animals. Since IgE response is considered to play a major role in the immune protection, those allergens, therefore, could be used as the main component of an anti-scabies vaccine. Key words: Sarcoptes scabiei, antibody, goats

Purification and production of monospecific antibody to the hemagglutinin from Subtype H5N1 influenza virus Simson Tarigan; R Indriani; D Hewajuli
Jurnal Ilmu Ternak dan Veteriner Vol 15, No 4 (2010): DECEMBER 2010
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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

Circulating H5N1 virus among native chicken living around commercial layer farms Simson Tarigan; Risa Indriani; J. Ignjatovic
Jurnal Ilmu Ternak dan Veteriner Vol 20, No 3 (2015): SEPTEMBER 2015
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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.

Dermatopathology of Caprine Scabies and Protective Immunity in Sensitised Goats Against Sarcoptes scabiei Reinfestation Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 7, No 4 (2002): DECEMBER 2002
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The purpose of this study was to compare macroscopic dermatopathology in naïve and sensitised goats, and to assess protective immunity possessed by sensitised goats against Sarcoptes scabiei challenge. Eighteen goats were allocated evenly into 3 groups; group 1 sensitised with the mite twice, group 2 once and group 3 was not sensitised (naïve). Sensitisation was done by infesting goats with the mites on the auricle and infestation was allowed to progress for 7 weeks, then the goats were treated with Ivermectin to obtain complete recovery. After sensitisation, all sensitised and naïve goats were infested with the mites on the auricles. Infestation in the sensitised goat caused severe immediate hypersensitivity that resulted in severe peracute pustular dermatitis. After one week, however, the lesion waned slowly. At 7 weeks post infestation, the remnant of lesion could only be perceived by palpation on the primary site of infestation as a mild papular dermatitis. Infestation on the naïve goats, in contrast, produced slowly progressing lesions which at 7-week post infestation, it ended up with severe crusted scabies affecting almost the whole skin. Antigens responsible for the immediate hypersensitivity which are supposedly contained in the mite secretions or excretions are immunologically protective but unlikely to have the capacity to induce a complete protection against mite challenge in immunised animals. This notion is based on the fact obtained from this study that goats sensitised twice did not possess a higher immune protection against mite challenge than goats sensitised once. Key words: Sarcoptes scabiei var. caprae, sensitisation, protective immunity, immediate hypersensitive

Histopathological changes in naive and sensitised goats caused by Sarcoptes scabiei infestation Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 8, No 2 (2003): JUNE 2003
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The purpose of this study is to compare the histopathological changes in naïve and sensitised goats caused by Sarcoptes scabiei infestation. Thirty goats were allocated evenly into 5 groups. Groups 1, 2 and 3 goats were sensitised once, twice and thrice, respectively; whereas groups 4 and 5 were left unsensitised or naïve. Sensitisation was done by infesting the animals with the mite, then 7 week afterwards the animals were completely cured from the mange. After the sensitisation, all, except group 5, goats were infested on both auricles each with approximately 2000 life mites. Biopsies were collected from each group at 2 day then at weekly intervals from 1 to 7 weeks following infestation. The samples were routinely processed, paraffin blocked, and tissue sections were stained with Haematoxyllin and Eosin (H & E), Giemsa, Carbol Chromatrope, and Gram’s when indicated. Lesions in the naïve goats developed progressively characterised by thick parakeratotic crusts honeycombed with tunnels containing large number of mites. Lesions in sensitised goats, which were different qualitatively to those in naïve goats, developed rapidly characterised by copious amount of serocellular exudates in and on the surface of the epidermis, and marked oedema and cell infiltrations in the dermis. Dermal infiltration by eosinophils, which was rare in naïve goats, was apparently an important feature in the sensitised goats. Lesions developed in the sensitised goats were interpreted to be the manifestation of cutaneous anaphylaxis. Resistance or protective immunity against mite reinfestation developed in the sensitised goats is supposedly attributed to this anaphylactic responses. Key words: Sarcoptes scabiei, naïve, sensitised, histopathology, eosinophils, cutaneous anaphylaxis, protective immunity

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