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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

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. Â Vaccine Â for scabies, Â as Â forÂ other Â ectoparasitic Â diseases, Â includes subunit vaccine Â developed Â from Â 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. Â 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

Diagnostic tool comprises one of the vital components in the control ofÂ 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 agaroseÂ 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 ofÂ 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

The purpose of this study was to compare macroscopic dermatopathology in naÃ¯ve 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 (naÃ¯ve). 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 naÃ¯ve 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 naÃ¯ve 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. Â Key words: Sarcoptes scabiei var. caprae, sensitisation, protective immunity, immediate hypersensitive

Purification of neuraminidase from Influenza virus subtype H5N1 Tarigan, Simson; Indryani, Risa; ., Darminto; Ignjatovic, Jagoda
Indonesian Journal of Animal and Veterinary Sciences Vol 14, No 1 (2009)
Publisher : Indonesian Animal Sciences Society

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 Î²-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.Â 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

Histopathological changes in naive and sensitised goats caused by Sarcoptes scabiei infestation Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 8, No 2 (2003)
Publisher : Indonesian Animal Sciences Society

The purpose of this study is to compare the histopathological changes in naÃ¯ve 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 naÃ¯ve. 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 Gramâs when indicated. Lesions in the naÃ¯ve 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 naÃ¯ve 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 naÃ¯ve 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. Â Key words: Sarcoptes scabiei, naÃ¯ve, sensitised, histopathology, eosinophils, cutaneous anaphylaxis, protective immunity

Antibody response in naÃ¯ve and sensitised goats infested by Sarcoptes scabiei Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 4 (2004)
Publisher : Indonesian Animal Sciences Society

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. Â Key words: Sarcoptes scabiei, antibody, goats

Production and purification of Bacillus anthracis protective antigen Tarigan, Simson; Adji, Rahmat S; Natalia, Lily
Indonesian Journal of Animal and Veterinary Sciences Vol 10, No 3 (2005)
Publisher : Indonesian Animal Sciences Society

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. Â Â Key Words: Bacillus anthracis, Protective Antigen, Protein Purification

Protective value of immune responses developed in goats vaccinated with insoluble proteins from Sarcoptes Scabiei Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 10, No 2 (2005)
Publisher : Indonesian Animal Sciences Society

Vaccines developed from certain membrane proteins lining the lumen of arthropodâs 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 Î¼g 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. Â Â Key Words: Sarcoptes scabiei, Insoluble Protein, Goat, Vaccination

Ingestion of host immunoglobulin by Sarcoptes scabiei Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 10, No 1 (2005)
Publisher : Indonesian Animal Sciences Society

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 miteâs 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. Â Â Key Words: Sarcoptes scabiei, Immunoglobulin

Identification and characterisation of heat-stable allergens from Sarcoptes scabiei Tarigan, Simson
Indonesian Journal of Animal and Veterinary Sciences Vol 11, No 1 (2006)
Publisher : Indonesian Animal Sciences Society

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 Â³ 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 60ÂºC 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

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