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Detection of antibody responses by using haemagglutination inhibiton test and the protection titer of avian influenza virus H5N1 subtype Risa Indriani; N.L.P.I Dharmayanti; A Wiyono; Darminto .; L Parede
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 3 (2004): SEPTEMBER 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Study on the detection of antibody responses using haemagglutination inhibition (HI) test and the protection titer to Avian influenza (AI) virus H5N1 subtype local isolate has been conducted at the Research Institute for Veterinary Science (RIVS). A total number of 50 village chicken (10 chicken served as un-injected controls) and 30 quail were injected intramuscularly with inactivated virus of AI H5N1 subtype local isolate. Serum samples were collected 3 weeks after injection and were tested using haemagglutination inhibition tests. The correlation between antibody titer and its protection to AI virus H5N1 local isolate were measured by challenging the birds with AI virus H5N1 local isolate The HI test was then used to determine field serum samples. A total number of 48 village chicken from three (3) Districts (Bekasi, Tangerang and Bogor) and 96 quails from two (2) farms in District of Sukabumi which were all vaccinated with commercial AI adjuvant vaccine were sampled. The study revealed that village chicken and quails showed antibody responses after 3 weeks vaccination and that titer of ≥ 3 log 2 was able to protect chicken and quails when they were challenged with local isolate virus. Based on this result, village chicken field samples from Districts of Tangerang, Bekasi and Bogor showed antibody titer which will protect 50, 100 and 85% of the flocks respectively. While quail field samples from Farm I and Farm II in District of Sukabumi showed antibody titer which will protect 60-100% and 0-80% of the flocks respectively. It is concluded that the study has successfully measured antibody titer to AI virus H5N1 subtype which protect village chicken and quails from local isolate virus challenge so that the results will be used to analyze field serum samples after vaccination program to eradicate AI from Indonesia. Key words: Antibody responses, haemagglutination inhibition test, protection titer, AI virus H5N1subtype

Lateral vaccination against Newcastle disease in broilers : Effect of ratio and density Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 1, No 3 (1995)
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Ratio (50%, 33% or 20% of directly vaccinated and in-contact vaccinated birds) and density (5 birds, 10 birds or 15 birds per square meter) for the effectiveness of lateral transmission of in-contact vaccination against Newcastle disease (ND) were evaluated in this study . The antibody patterns and the protection against challenge virus were used as criteria. Generally, antibody responses induced by direct vaccination showed higher titres compared to those induced by the in-contact vaccination, but at two week after the second vaccination, the differences were not significant. At the in-contact vaccinated birds, no significant difference (P>0 .05) was observed in the pattern of antibody development by ratio . However, group of vaccinated birds with the ratio of 20% tended to have lower protection . The results of the evaluation of density demonstrated that there was no effect of density (P>0 .05) to the pattern of antibody development, although the higher density seemed to have the higher protection. However, the density of 15 birds/m2 increasing the susceptibility to the other diseases. Based on the data obtained in this research, it could be concluded that (1) the optimal ratio for the effective lateral transmissibility is 33%, and (2) the optimal density for the effective lateral transmissibility is 10 birds/m2.

Antibody response and protection of inactivated-local isolate vaccine for infectious bronchitis in laying chicken Risa Indriani; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 6, No 2 (2001): JUNE 2001
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. IBV infection consists of many serotypes and can only be controlled by vaccination. An effective IB vaccine should be prepared from local isolates, due to the antigenic variation among serotypes. The aims of this research were to develop inactivated IB vaccine derived from IBV local isolate and to determine the efficacy of that vaccine in layer flocks. Five layer chicken groups were used in this experiments, group I was vaccinated with commercial IBV live vaccine thrice, group II was vaccinated with commercial IBV live vaccine once and repeated with inactivated local IBV isolate twice, group III was vaccinated with commercial IBV live vaccine once and repeated with commercial inactivated twice, group IV was vaccinated with IBV live vaccine once, and group V was not vaccinated. After the chickens reached at a stable egg production they were challenged with IBV local isolates. Antibody responses were examined by means of haemagglutination hibitition (HI) test and HI titres were expressed as log2 of the reciprocal of the highest dilution of serum causing inhibition of a log2 HA titre of 2. The mean titres of antibody responses of chicken in group I, II, III, IV, and V was 4.9 ± 0.87, 6.8 ± 0.97, 7.7 ± 0.46, 2.9 ± 0.94, and 2.0 ± 1.67 respectively. The levels of protection against challenges were determined by viral isolation, this in group I, II, III, IV, and V was 63, 73, 60, 50, and 0% respectively. Clinical symptom of egg quality was slightly reduced in group I, IV, and V and it were unchanged in group II and III. Group II gave better in number of egg production than the other groups. The results indicated that the IBV inactivated localisolate vaccine gave high titres of antibody and higher protection rates than that of commercial IBV inactivated vaccine. Inaddition, IBV local isolate vaccinated group prevented from declining egg production after challenged with IBV local isolate. Key words: Infectious bronchitis, layer, antibody titre, vaccine, challenge virus

Isolation and characterization of virus of highly pathogenic avian influenza H5 subtype of chicken from outbreaks in Indonesia Agus Wiyono; R Indriani; N.L.P.I Dhamaryanti; R Damayanti; L Parede; T Syafriati; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 1 (2004): MARCH 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

A study on the isolation and characterization of Highly Pathogenic Avian Influenza of chicken from outbreaks in Indonesia was conducted at Indonesian Research Institute for Veterinary Science. Outbreaks of avian disease had been reported in Indonesia since August 2003 affecting commercial layer, broiler, quail, and ostrich and also native chicken with showing clinical signs such as cyanosis of wattle and comb, nasal discharges and hypersalivation, subcutaneous ptechiae on foot and leg, diarre and sudden high mortality. The aim of this study is to isolate and characterize the causal agent of the disease. Samples of serum, feather follicle, tracheal swab, as well as organs of proventriculus, intestine, caecal tonsil, trachea and lungs were collected from infected animals. Serum samples were tested haemaglutination/haemaglutination inhibition to Newcastle Disease and Egg Drop Syndrome viruses. Isolation of virus of the causal agent of the outbreak was conducted from samples of feather follicle, tracheal swab, and organs using 11 days old specific pathogen free (SPF) embryonated eggs. The isolated viruses were then characterised by agar gel precipitation test using swine influenza reference antisera, by haemaglutination inhibition using H1 to H15 reference antisera, and by electron microscope examination. The pathogenicity of the viruses was confirmed by intravenous pathogenicity index test and its culture in Chicken Embryo Fibroblast primary cell culture without addition of trypsin. The study revealed that the causative agent of the outbreaks of avian disease in Indonesia was avian influenza H5 subtype virus based upon serological tests, virus isolation and characterization using swine influenza reference antisera, and electron microscope examination. While subtyping of the viruses using H1 to H15 reference antisera suggested that the virus is very likely to be an avian influenza H5N1 subtype virus. The pathogenicity test confirmed that the viruses are highly pathogenic to experimental animals. It is concluded that the causative agent of the outbreaks of avian disease in Indonesia was avian influenza H5 subtype virus. The result has been the basis of further study such as development serological tests and vaccine production. The decission of Indonesian Government to conduct vaccination program using homolog vaccine in order to control the disease is regarded as the correct choice. However, it should be accompanied by conducting surveillance and monitoring of the disease as well as the possibility of mutation of virus. The program should be coordinated nationally. Key words: Virus isolation, characterization, chicken, outbreak, highly pathogenic avian influenza (HPAI), H5 subtype, Indonesia

Identification of avian influenza virus of Indonesian isolates by reverse transcriptase polymerase chain reaction (RT-PCR) method N.L.P.I. Dharmayanti; R Damayanti; A Wiyono; R Indriani; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 2 (2004): JUNE 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

An outbreak of avian influenza in Indonesia was reported at the first time at the beginning of September 2003 causing high mortality among poultry population especially commercial layer chicken farms in Java, Sumatra and Bali islands. From the outbreaks highly pathogenic avian infuenza viruses have been isolated and characterized by rapid, HA, HI and AGP tests. However, these isolates are still needed to be further molecularly characterized. The aim of this study is to identify by further subtyping the avian viruses by means of RT-PCR using Matrix, H7 and H5 primers. The study reveals that the RT-PCR using Matrix primer amplified a 200-300 basepairs (bp) Jawa Timur isolates were collected from East Java, while Jawa Barat isolates were from West Java. The RT-PCR using H7 primers did not amplify any product, while H5 primer amplified a 500-600 bp product from the isolates. It is concluded that the outbreak of poultry disease in East and West Java was caused by an avian influenza H5 subtype. Key words: Identification, avian influenza virus, RT-PCR, H5 subtype

Serotype variation among infectious bronchitis viral isolates taken from several areas of Java Risa Indriani; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 4 (2000): DECEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Infectious bronchitis (IB) is an acute highly contagious viral respiratory disease of poultry caused by virus belongs to the family of Coronaviridae. The virus consist of many serotypes with low level of cross-protectivity among serotypes. Field data showed that the outbreaks of IB were frequently reported in chicken flocks, although vaccinations against the disease have been practiced. Hence, the study on serotype relationship among isolates of the viruses is essentially required. The aim of this study was to isolate and characterize IB viruses from chicken flocks in some areas of Java. Isolation of the virus was carried out in nine-day old embrionated chicken eggs and identified by means of agar gel precipitation (AGP) tests against standard antisera to IB virus. The serotypes of the IB viral isolates were determined by cross-neutralization tests in nine day old embryonated chicken eggs using r value derived from homologous and heterologous serum titres as criteria. This study obtained 12 IB viral isolates which were identified on the basis of the ability to cause lesions in chicken embryos and positive to agar gel presipitation test against standard positive antiserum to the virus. Based on the cross-neutralization tests in embryonated chicken eggs, isolate I.9 was formed to have relationship closed to Mass-41 serotype, while I.2, I. 3, and I.7 isolates were closely to the serotype of Con-46. Virus isolates (I.5, I.14, I.24, and I.25) were decided to have no serotype relationships to either Mass-41 or Con-46 serotype. Since the I.5, I.14, I.24 and I.25 isolates were not neutralized by antisera against the previous identified local infectious bronchitis viral isolates, and that were considered to be distinct serotype to the previously identified local IB viral isolates. Key words: Infectious bronchitis, virus, embryonated egg, cross neutralization test.

Epidemiology of Japanese–B– encephalitis infection in pigs in Riau and North Sumatera Provinces Indrawati Sendow; Tatty Syafriati; Upik Kesumawati Hadi; Martin Malole; Susi Soviana; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 8, No 1 (2003): MARCH 2003
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Epidemiology study on Japanese-B-Encephalitis (JE) was conducted in Riau and North Sumatera Provinces. A total of 190 pig sera from Riau Province and 164 pig sera from North Sumatera were tested using competitive ELISA (C-ELISA) to detect antibodies against JE virus. Insect collection was also conducted using several methods near pig farms in those provinces and identified into species to gain more information on its role to distribute JE infection. Serological results indicated that 70% pig in Sumatera and 94% pig in Riau had antibodies against JE virus. The highest prevalence of reaktor was detected in pig of more than 4 months age in both Provinces. The results of insect collection showed that Culex tritaeniorchynchus and Culex quinquefasciatus were the most dominant species in both provinces. Based on serological testing, indicated that JE virus infected pig in Sumatera and Riau Provinces, and higher reactor was obtained in older pig. Culex tritaeniorchynchus and Culex quinquefasciatus were the dominant insect species in both provinces, hence those species had a possibility to play an important role of JE transmission. Key words: JE, pigs, serology, insects

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

Development of a nested PCR for detection of Bovine herpesvirus-1 (BHV-1) in bovine nasal secretion and semen Muharam Saepulloh; R.M Abdul Adjid; I Wayan T. Wibawan; Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 13, No 2 (2008): JUNE 2008
Publisher : Indonesian Center for Animal Research and Development (ICARD)

A nested polymerase chain reaction (nPCR) assay for detection of Bovine herpesvirus-1 (BHV-1) in bovine semen and nasal secretions was successfully developed. The nested Polymerase Chain Reaction was based on external and internal primers from the viral gD glycoprotein gene. This nPCR assay was 1000-fold more sensitive than using PCR external primer. The nested PCR has a detection limit as low as 5 ag/ml pure BHV-1 DNA and 100,75 TCID50/500 mL BHV-1 infected cells. On the other hand, PCR using external primer had detection limit of about 5 fg/ml pure BHV-1 DNA. Specificity studies showed that nPCR could only detect BHV-1, whereas BHV-4, PRV, PI-3 and BRSV can not be detected. In addition, nPCR was also capable in detecting BHV-1 in nasal secretion samples from animal without clinical signs. A total of 405 samples consisted of 381 nasal secretion and 24 fresh semen samples have been tested with the nPCR. The result revealed that from 381 nasal secretion samples tested, 14 samples showed to be positive (3.68%), consisting of 13 out of 294 (4.42%) nasal secretion samples collected from Pangalengan West Java, and 1 out of 87 (1.54%) samples collected from Bogor. Furthermore, 2 out of 11 (18.18%) extended semen samples collected from Bogor and 2 out of 13 (15.38%) fresh semen samples collected from Pasuruan also showed to be positive. In addition, the nPCR was faster and easier to perform than the standard viral isolation test. It is concluded that, the nPCR can be used as test of choice for routine diagnosis of BHV-1. Key Words: Nested PCR, BHV-1, Semen, Glycoprotein D Gene, TCID50

In-contact vaccination against Newcastle disease in village chickens : a comparative analysis between laboratory and field trials Darminto .
Jurnal Ilmu Ternak dan Veteriner Vol 1, No 2 (1995)
Publisher : Indonesian Center for Animal Research and Development (ICARD)

A total of 4,977 sera from a sentinel cattle in West and East Nusa Tenggara were tested for antibody against BHV-1, the causal agent of infectious bovine rhinotracheitis (IBR). These sera were collected between June 1990 and June 1993, and were tested by using serum neutralization test (SNT). Out of these sera, 3,713 were suitable for IBR SNT. A total of 349 sera (10.4%) reacted. IBR reactors were more prevalent in East Nusa Tenggara (NTT) than in West Nusa Tenggara (NTB) . Based on this survey, it is concluded that antibodies against IBR virus are present among cattle in East and West Nusa Tenggara .

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