Articles

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

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

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

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
Use of Polymerase Chain Reaction Enzyme Linked Oligonucleotide Sorbent Assay (Pcr-Elosa) for Detection of Disease Agents Tarigan, Simson
WARTAZOA. Indonesian Bulletin of Animal and Veterinary Sciences Vol 21, No 1 (2011): MARCH 2011
Publisher : Indonesian Center for Animal Research and Development

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (252.898 KB) | DOI: 10.14334/wartazoa.v21i1.949

Abstract

Diagnostic tool comprises one of the vital components in the control of  infectious diseases. One of the most common techniques in the diagnosis of infectious disease currently available is the polymerase chain reaction (PCR) because this technique is very sensitive, specific, and rapid. This technique requires an adjunct technique to indicate the formation of the right reaction product. Agarose gel electrophoresis has been the most common technique to visualise the PCR product or amplicon. Enzyme linked oligonucleotide sorbent assay (ELOSA) is an alternative technique which is more sensitive and gives more important identity of the amplicon. This technique can be more than 100 times as sensitive as a gel agarose  electrophoresis, and very specific since confirmation of the amplicon is carried out by DNA hibridisation. The capacity of the ELOSA can also be extended to the detection of disease-causal agent at subtype level, or detection of mutation at particular location in a gene. Since the equipment used for ELOSA is similar to that for ELISA (enzyme linked immunosorbent assay), a large number of samples can be accomplished rapidly. As in ELISA, a number of variation can be made in ELOSA depend on the requirement. Nucleotide can be immobilised on the microwell plate either by passive adsorbtion, by first conjugation of  nucleotide with biotin then immobilisation on streptavidin-coated microwell plate, or immobilisaion by covalent bonding. The PCR and ELOSA can be performed at separate or in a single tube by first immobilising the PCR primers on the surface of microwell plates. Key words: PCR amplicon, agarose electrophoresis, oligonucleotide immobilisation, DNA hybridisation
Antibody response in naïve and sensitised goats infested by Sarcoptes scabiei Tarigan, Simson
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 4 (2004): DECEMBER 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (547.021 KB) | DOI: 10.14334/jitv.v9i4.436

Abstract

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
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  
Scabies Vaccine is Required, but Difficult to be Made Tarigan, Simson
Indonesian Bulletin of Animal and Veterinary Sciences Vol 17, No 1 (2007)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (88.068 KB) | DOI: 10.14334/wartazoa.v17i1.889

Abstract

Sarcoptes scabiei, the mite causing scabies, infests human and at least 40 species of animals. The losses associated with the disease as a public health burden and economic losses are enormous because its prevalence is very high. The current available control by treating individuals diagnosed to have the disease is both ineffective and unpractical. Besides, dissatisfaction with the pharmacological control is escalating due to the development of resistance in the mites and rejection by consumers for animals products contaminated with drug residues. Vaccination is considered to be most the attractive alternative control although the availability of vaccine is still a long way off. Control of scabies by vaccination is considered to be feasible since animals recovered from the disease posses protective immunity against mite reinfestation. In addition, despite the fact that the mites reside not deeper than the unvascularised stratum corneum and they are not blood sucking parasites, they do ingest their host immunoglobulin.  Vaccine  for scabies,  as  for  other  ectoparasitic  diseases,  includes subunit vaccine  developed  from  mite protective antigen produced by recombinant technology. Identification of sarcoptic protective antigen which comprise the first step in the vaccine development impede by the lability and low abundance of the protective antigen, and the difficulty in obtaining sufficient amount of mites. Identification of sarcoptic protective antigen by conventional biochemical technique, although the technique has been successful for other parasites, has been unsatisfactory for S. scabiei. Identifying the protective antigen just among proteins having vital functions in the survival of mites and accessible by the effector arms of the host immune system seems to be a more feasible alternative. The allergens and membrane proteins lining the digestive tract of the mites seem to fulfil the criteria.   Key words: Sarcoptes scabiei, protective antigen, scabies vaccine
Use of Polymerase Chain Reaction Enzyme Linked Oligonucleotide Sorbent Assay (Pcr-Elosa) for Detection of Disease Agents Tarigan, Simson
Indonesian Bulletin of Animal and Veterinary Sciences Vol 21, No 1 (2011)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (252.898 KB) | DOI: 10.14334/wartazoa.v21i1.949

Abstract

Diagnostic tool comprises one of the vital components in the control of  infectious diseases. One of the most common techniques in the diagnosis of infectious disease currently available is the polymerase chain reaction (PCR) because this technique is very sensitive, specific, and rapid. This technique requires an adjunct technique to indicate the formation of the right reaction product. Agarose gel electrophoresis has been the most common technique to visualise the PCR product or amplicon. Enzyme linked oligonucleotide sorbent assay (ELOSA) is an alternative technique which is more sensitive and gives more important identity of the amplicon. This technique can be more than 100 times as sensitive as a gel agarose  electrophoresis, and very specific since confirmation of the amplicon is carried out by DNA hibridisation. The capacity of the ELOSA can also be extended to the detection of disease-causal agent at subtype level, or detection of mutation at particular location in a gene. Since the equipment used for ELOSA is similar to that for ELISA (enzyme linked immunosorbent assay), a large number of samples can be accomplished rapidly. As in ELISA, a number of variation can be made in ELOSA depend on the requirement. Nucleotide can be immobilised on the microwell plate either by passive adsorbtion, by first conjugation of  nucleotide with biotin then immobilisation on streptavidin-coated microwell plate, or immobilisaion by covalent bonding. The PCR and ELOSA can be performed at separate or in a single tube by first immobilising the PCR primers on the surface of microwell plates. Key words: PCR amplicon, agarose electrophoresis, oligonucleotide immobilisation, DNA hybridisation
Dermatopathology of Caprine Scabies and Protective Immunity in Sensitised Goats Against Sarcoptes scabiei Reinfestation Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 4 (2002)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (228.643 KB) | DOI: 10.14334/jitv.v7i4.303

Abstract

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