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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

Ingestion of host immunoglobulin by Sarcoptes scabiei Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 10, No 1 (2005): MARCH 2005
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Scabies is one of the most important diseases in human and veterinary medicine. The available control measures that rely on acaricides are unsustainable, costly and environmentally unfriendly. Vaccination which is supposedly the most attractive alternative control, is sustainable, potentially cheap and environmentally friendly. Recent development in protein biochemistry and recombinant technology have facilitated the development of anti-parasite vaccine which in the past was impossible. One prerequisite for the anti-parasite-vaccine development is that the parasite has to ingest its host immunoglobulin. This study, therefore, was designed to determine whether Sarcoptes scabiei, a non blood-feeding parasite that resides on the avascular cornified layer of the skin, ingest its host immunoglobulin. Sections of routinely processed mites and skin from a mangy goat were probed with peroxidase-conjugated-anti-goat IgG and the immune complex was visualised with diaminobenzidine solution. To determine whether the ingested IgG was still intact or had been fragmented by the proteolytic enzymes, immunoblotting analysis of SDS-PAGE- fractionated proteins extracted from washed mites was performed. Quantification of IgG was done byan Elisa using purified goat IgG as control. This study showed that IgG in the mites confined to the mite’s gut only, and only a fraction of mite population ingested the IgG. The ingested IgG, as shown by immunoblot analysis, was mostly still intact. This study indicates that development of anti-scabies vaccines is reasonable. Key Words: Sarcoptes scabiei, Immunoglobulin

Identification and characterisation of heat-stable allergens from Sarcoptes scabiei Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 11, No 1 (2006): MARCH 2006
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Animals or human recovered from Sarcoptes scabiei infestation acquired protective immunity against reinfestation. The protective immunity is considered to be associated with a type-1-hypersensitivity reaction against allergens instigated by the mites during infestation. It is assumed that these allergens have the potential to be used as the main component of an anti-scabies vaccine. The purpose of this study is to identify and characterise the sarcoptic allergens. For this purpose, 645 mg of mites, collected from mangy goats, were homogenised in PBS to prepare soluble mite proteins. Fractionation of proteins was initially performed on a Q-sepharose column but the results were unsatisfactory. Consequently, SDS PAGE was used as an alternative. Proteins from the gel were transferred onto a nitrocellulose membrane. The membrane was cut into strips so each strip contained proteins with molecular weights of ³ 90, 80-90, 70-80, 60-70, 50-60, 40-50, 30-40, 25-30, 20-25, 15-20 and 10-15 kDa, respectively. The heat stability of the allergens was determined by heating the suspension at 60ºC for 60 minutes, whereas their dialysability was evaluated using a 10-kDa-cut-off ultramembrane. The activity of the allergens was assayed by an intradermal test on sensitised goats. This study showed that mite protein extract was very potent allergens since mite extract containing as little as 1 ng mite proteins still caused an obvious hypersensitive reaction. The mite extract contained heat-stable, dialysable and non-dialysable allergens. All fractions recovered from a Q-sepharose column contained allergens with almost equal potency. Fractionation with the SDS-PAGE revealed that the allergens had molecular weights of 35 and <10 kDa. The former allergen is assumed to be a member of group 10 allergens, whereas the later belong to haptenic allergens. Kata Kunci: Sarcoptes Scabiei, Allergens, Heat-Stable, Group 10, Hapten

Actinobacillus pleuropneumoniae cytotoxins on size, granularity and viability of porcine neutrophils Simson Tarigan
Jurnal Ilmu Ternak dan Veteriner Vol 3, No 3 (1998)
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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

Characterisation of enzymatic activities of H5N1 influenza virus Simson Tarigan; Risa Indriani; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 12, No 2 (2007): JUNE 2007
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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

Production and purification of Bacillus anthracis protective antigen Simson Tarigan; Rahmat S Adji; Lily Natalia
Jurnal Ilmu Ternak dan Veteriner Vol 10, No 3 (2005): SEPTEMBER 2005
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Protective antigen (PA) plays crucial roles in the pathogenicity and virulence of Bacillus anthracis. Animals or human immunised with the protein acquire a complete protection against the disease. In addition to vaccine, PA can also be developed into a sensitive diagnostic test for anthrax. The purpose of this study was to produce PA using a culture medium easily obtained, and to develop a simple and effective technique for purification of the protein. To produce PA, B. anthracis Sterne 34F2 strain was first grown on blood agar, then bacterial colonies were suspended and incubated for 2 hours in RPMI-1640 supplemented with NaHCO3 and Tris. Protein components in the culture supernatant were separated consecutively with Phenyl sepharose, Qsepharose and Superdex-200 columns. This order was used in order to simplify and speed up the purification process. The PA contained in the fractions was detected by a dot blot or an ELISA using commercial PA specific antibody. The PA was absorbed strongly by the phenyl sepharose whereas other proteins were absorbed weakly or not absorbed at all. When these PA-containing fractions were loaded into Q-sepharose column, PA was absorbed considerably weaker than contaminated proteins. Although the level of purity obtained from the Q-sepharose column was satisfactory, further separation on Superdex produced an even higher purity. However, on SDS-PAGE analysis, the purified PA was seen as a two-band protein (54.7 and 29.2 kDa) because of nicked proteolysis. On an immunoblot assay, only the 54.7 band was recognised by the PA-specific antibody. Despite the nick proteolysis, the PA purified in this study was considered to retain its biological activities. Key Words: Bacillus anthracis, Protective Antigen, Protein Purification

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

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

The Role of Point-of-Care Test to Control Highly Pathogenic Avian Influenza in Indonesia Simson Tarigan
WARTAZOA, Indonesian Bulletin of Animal and Veterinary Sciences Vol 26, No 1 (2016): MARCH 2016
Publisher : Indonesian Center for Animal Research and Development

Rapid diagnosis followed by stamping out protocol has proven to be the most effective means of eradication of Highly Pathogenic Avian Influenza (HPAI) H5N1. The standard of AI tests are PCR and virus isolation, however their results often take times to initiate rapid eradication protocol. For that reason, a point-of-care (POC) test which is a rapid test used at the location of outbreak (where mortality and morbidity of poultry occur), to help field officer for identification of AI. This paper describes several techniques for detection of HPAI H5N1 virus, POC test and principal mechanism of lateral flow immunoassay which has been generally used in POC test. At present, POC test for HPAI H5N1 rather low sensitivity and expensive, therefore, further research is needed to improve its sensitivity of the tool.

Subclinical Infection by Avian Influenza H5N1 Virus in Vaccinated Poultry Simson Tarigan
WARTAZOA, Indonesian Bulletin of Animal and Veterinary Sciences Vol 25, No 2 (2015): JUNE 2015
Publisher : Indonesian Center for Animal Research and Development

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

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

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

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