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Studies on the susceptibility of ostriches (Struthio camelus) to the Indonesian velogenic strain of Newcastle disease virus Darminto .; Sjamsul Bahri
Jurnal Ilmu Ternak dan Veteriner Vol 2, No 4 (1998)
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Susceptibility of ostriches (Struthio camelus) to the Indonesian velogenic strain of Newcastle disease virus (NDV) was evaluated by artificial infection . Twelve - 5 to 6 week old ostriches were divided into 3 groups each containing 4 birds . The first group was inoculated through respiratory system by dropping directly the virus solution into the nostrils, while the second group was inoculated through digestive system by dropping directly the virus solution into the oesophagus, with the dose of infection 106ELDSo (50%-embryo lethal dose) per bird . Meanwhile, the third group was treated as uninfected control . All infected birds developed antibody responses, but only two inoculated birds from the first group and two inoculated birds from the second group developed clinical signs of Newcastle disease (ND), with no specific pathological alterations . Infected birds, either sicks or healthy, excreted the challenge viruses through the respiratory system and still be detected up to the end of this experiment, ie . 15 days post-inoculation . The challenge viruses can be re-isolated from the brain, trachea, lungs, heart, liver, spleen, kidneys, small intestine, cecal-tonsil, and proventriculus of the infected birds . This study concludes that: (1) the ostriches are susceptible to the infection of the Indonesian velogenic strain ofNDV; (2) all infected birds developed immune responses, but only half of them develops el jtigi aj i disease ; (3) the infected birds excreted the challenge viruses for a considerable long time which may play role as the Mginiseti.ce ofinfectron the other healthy ostriches ; and (4) the challenge viruses can be re-isolated from various organs of the birds . . Keywords : Newcastle disease vir4, 9strich, immune response, artificial infection

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

Inactive vaccine derived from velogenic strain of local Newcastle disease virus . Darminto .; P Ronohardjo
Jurnal Ilmu Ternak dan Veteriner Vol 2, No 1 (1996)
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The objective of this research is to evaluate an application of an inactive Newcastle disease (ND) vaccine derived from velogenic strain of local Newcastle disease virus (NDV). In this research . the Ira strain of velogenic ND virus was grown in specific pathogen free (SPF) eggs and then was inactivated by formalin at a final concentration of 1 :1,000 at 4°C. The inactive antigen was then emulsified with an oil adjuvant or aluminium hydroxide gel before being administered for vaccination in layers and compared to a commercial inactive ND vaccine . Results indicated that application of these inactivated ND vaccines for booster vaccination following vaccination with an active lentogenic ND virus in pullets nearly producing eggs, resulted in high antibody titre which persisted for considerable long period of time and capable of protecting layers from sick of ND and from reducing egg production . Hence, it could be concluded that the inactivated vaccine emulsified in either oil-adjuvant (lanolin-paraffin) or aluminium hydroxide gel were considered to be highly immunogenic and capable of protecting layers from sick of ND and from reducing egg production Keywords : Newcastle disease, inactive vaccine, oil adjuvant . aluminium hydroxide, immunogenicity, protection

Comparison of sequences of hypervariable region (HVR) subunit S-1 gene of field isolate I-37 infectious bronchitis virus with Connecticut serotype N.L.P Indi Dharmayanti; Risa Indriani; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 8, No 2 (2003): JUNE 2003
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Infectious Bronchitis is a contagious and acute respiratory disease in chickens caused by infectious bronchitis virus (IBV).Antigenic differences in IBV are associated with changes in the sequence of the spike glycoprotein (S). The subunit S1 which demonstrates more sequence variability than S-2 have been identified as hypervariable region (HVR-1 and 2). There were several IB virus field isolates included I-37 have been identified in Indonesia by serum neutralization method. However, gene sequence variation in HVR subunit S-1 had not yet been identified. Isolate I-37 was close to the serotype Connecticut 46 (Conn 46). The aim of this study is to identify sequence variation of HVR subunit S-1 gene of isolate I-37 produced by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and sequencing. Several procedures were carried out in the study including virus titration, propagation and was concentrated from the allantoic fluid infected with IBV. Then, RNA was extracted for RTPCR. urther the product was sequnced and its homology with IBV references from GenBank was compared by GenMac version 8.0. Result showed that isolate I-37 produced 515 bp of amplification product. Isolate I-37 and Conn 46 are same serotype, yet their HVR subunit S-1 nucleotides and amino acids (protein) differ by 6.9% and 15.6% respectively. It might be concluded that isolate I-37 was variant of Conn 46. Key words: Sequences variation, IBV, I-37 field isolate, HVR subunit S-1 gene

Development of inactivated-local isolate vaccine for infectious bronchitis Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 4, No 2 (1999): JUNE 1999
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Infectious bronchitis (IB) is an acute highly contagious viral respiratory disease of poultry caused by coronavirus. The disease causes high mortality in young chicks, reduce body weight gain in broilers and remarkable drop in egg production. IB can only be controlled by vaccination, but due to the antigenic variation among serotypes of IB viruses, the effective IB vaccine should be prepared from local isolates. The aim of this research is to develop inactivated IB vaccine derived from local IB isolates. Local isolates of IB viruses designated as I-37, I-269 and PTS-III were propagated respectively in specific pathogen free (SPF) chicken eggs, the viruses then were inactivated by formaline at final concentration of 1:1,000. Subsequently, the inactivated viruses were mixed and emulsified in oil emulsion adjuvant with sorbitant mono-oleic as an emulsifier. The vaccine then was tested for its safety, potency and efficacy in broiler chickens. Birds inoculated twice with a two-week interval by inactivated vaccine did not show any adverse reaction, either systemic or local reaction. The inoculated birds developed antibody responses with high titre, while antibody of the control birds remain negative. In addition, efficacy test which was conducted in broilers demonstrated that birds vaccinated by live-commercial vaccine and boosted three weeks later by Balitvet inactivated vaccine showed high level of antibody production which provided high level of protection against challenged virus (76% against I-37, 92% against I-269 and 68% against PTS-III challenge viruses). From this study, it can be concluded that inactivated local IB vaccine is considered to be safe, potent and efficacious. The vaccine stimulates high titre of antibody responses, which provide high level of protection against challenged viruses. Key words: Infectious bronchitis, virus, broiler chicken, antibody, inactivated vaccine

Detection of avian influenza virus H5N1 subtype in organs of chicken affected by higly pathogenic avian infuenza in East and West Java by using immunohistochemical technique R Damayanti; N.L.P.I Dharmayanti; R Indriani; A Wiyono; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 3 (2004): SEPTEMBER 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The study was conducted to detect antigen H5N1 of highly pathogenic Avian Influenza (HPAI) virus in various farms in East and West Java. The immunohistochemical technique was applied due to Hematoxilin-eosin (H&E) staining was impossible to visualize the antigen in tissue. Immunohistochemical staining was applied for some visceral organs collected from the areas where the outbreaks occurred in September-October 2003. The specimens were processed as histopathological paraffin blocks using standard method. The blocks that were suspected to have antigen H5N1 were cut and rabbit antisera to H5N1 produced from the local isolate was applied as the primary antibody. Biotinylated secondary antibody and avidin biotin peroxidase from a commercial kit were administered. The antigen present in the tissues were visualized by adding a substrate called Amino Ethyl Carbazole (AEC) resulting in reddish brown colour. This immunostaining proved to be accurate and reliably quick method to detect H5N1 antigen present in the avian tissues. In conclusion, the outbreak of bird flu was caused by H5N1 strain and the antigen could be found in wattles, combs, brain, trachea, lungs, heart, proventriculus, liver, spleen, kidney and ovary. Key words: Highly pathogenic Avian Influenza (HPAI), chicken, H5N1, outbreak, immunohistochemistry

The development of an Enzyme Linked Immunosorbent Assay for detecting Injectious laryngotrachitis viral antibodies in chicken serum Risa Indriani; R.M Abdul Adjid; Darminto .; Helmy Hamid
Jurnal Ilmu Ternak dan Veteriner Vol 7, No 2 (2002): JUNE 2002
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The aim of this study was to develop an Enzyme-linked immunosorbent assay (ELISA) for detection of antibody against gallid herpes virus, the causal agent of infectious laryngotracheitis (ILT) in chicken. Its application in experimental chicken under laboratory condition was also evaluated. Results showed that ELISA for ILT was developed successfully with sensitivity and specificity was 98% and 97,14% respectively. The ELISA was able to determine the dynamic of antibodies respond in experimental chickens following vaccination and artificial infection with ILT virus. It was concluded that this ELISA offers a simple, sensitive and specific antibody assay for detection of antibodies against ILT virus in chickens arising from vaccination or infection. Key words: ELISA, antibody, chicken, Infectious laryngotrachitis

Protective antibody titre against Newcastle disease in ostriches (Struthio camelus) Darminto .; Sjamsul Bahri; N. Suryana
Jurnal Ilmu Ternak dan Veteriner Vol 3, No 4 (1998)
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The aim of this study was to define an estimated antibody titre which was considered to be protective against Newcastle disease (ND) virus infection in ostriches. Eighteen young ostriches of 4 days of age were divided into two groups each containing 9 birds. The first group was unvaccinated and the second group was vaccinated against ND virus twice at 4 and 14 days of age. Antibody titres were monitored at 1, 14, 28, 42, 56, 70 and 85 days of age by haemagglutination inhibition (HI) test. All birds were then challenged with a velogenic strain of ND virus, Ita strain, at 42 days of age. The excretion of the challenge virus were monitored daily after challenge up to the end of this experiment. Several organs such as brain, trachea, lungs and spleen were collected from died birds for re-isolation of the challenged virus. Results indicated that all unvaccinated birds succumbed to the challenged virus, except one bird that survived challenged. In contrast to the unvaccinated birds, all vaccinated birds survived challenged, except two birds with low antibody titres succumbed challenged. All birds with antibody titres of 4 (HI-log2) or greater survived challenged. All challenged birds excreted the challenged virus through out their oropharyngs. Moreover, challenged virus can be successfully re-isolated from most organs of the died birds. This study concludes that : (a) the estimated protective titre against ND in ostriches is 4 (HI-log2), (b) the immune status for ostrich with antibody titre less the 4 (HI-log2) could not be defined, and (c) vaccination against Newcastle disease in ostriches could successfully prevent birds from sick and died of ND, but unable to prevent virus infection and unable to stop carrier status after infection. Key words : Newcastle disease, ostrich, antibody, protective titre

The clinico-pathological effects of chicken infected with highly pathogenic avian influenza in some farms located in East Java and West Java R Damayanti; NLP.I Dharmayanti; R Indriani; Achmad Selamet Wiyono; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 2 (2004): JUNE 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The study was conducted to investigate the clinico-pathological features of highly contagious disease occurred in chicken located in East and West Java during the outbreak in September-October 2003. Six farms located in Districts of Surabaya, Malang and Blitar of East Java had been visited. They were mainly commercial layer, breeder layer and breeder broiler, which the population was between 14.000, 80.000, and aged 17-70 weeks. Where as five farms in West Java (Districts of Bogor, Bekasi and their surrounding areas) were visited and consisted of commercial layer and breeder broiler, having population of 3000-16.000 and aged 11-53 weeks. Observation was made according to clinical, gross pathological and histopathological changes. Clinically, most of them had cyanotic wattle and comb and subcutaneous petechiation of non-feathered part of the legs. These were also seen in necropsy, accompanied by general circulatory disturbances in most organs: namely pectoral and thigh muscle, trachea, lungs, epicard, myocard, proventriculus, liver, kidney and ovary. In addition, the liver was congested, friable and necrotic in some parts. Histologically, hemorrhage and non suppurative inflammatory reaction were observed in the brain, skin (comb, wattle and non feathered leg), skeletal muscle, trachea, lung, heart, proventriculus, liver, kidney and ovary whereas vasculitis was found especially in the skin of the wattle and comb, brain and kidney. It is concluded that based on the clinicopathological findings the outbreak of poultry disease in East and West Java were attributed to highly pathogenic avian influenza. Key words: Highly pathogenic avian influenza (HPAI), chicken, clinico-pathology, outbreak, East Java, West Java

Genetic characteristic of protein membran of avian influenza viruses H5N1 subtype N.L.P Indi Dharmayanti; D.A Hewajuli; A Ratnawati; R Indriani; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 15, No 3 (2010): SEPTEMBER 2010
Publisher : Indonesian Center for Animal Research and Development (ICARD)

In 2006-2008 there were findings about the antigenic drift on AI virus due to vaccination and the AI H5N1 subtype viruses which was similar to H5N1 viruses in human. The findings indicated that the AI viruses continue and undergoing to mutate and try to adapt with their environment. The objective of this study was to characterize the mutation of recent AI viruses (2009) on the membran protein namely Hemagglutinin (HA), Neuraminidase (NA) and Matrix 2 (M2). In this study RT-PCR – sequencing methods and genetic analysis for the protein membran of AI viruses were used. Result revealed that there were specific mutation belong to AI 2009 viruses on HA and NA protein such as AI virus mutation in 2008 which was isolated from backyard chicken. The mutations were non synonimous and not caused by immunological pressure. Furthermore, M2 analysis indicated that the viruses were resistant to amantadine. Key Words: Mutation, AI Subtype H5N1 Viruses, Membran Protein

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