Agar Gel Precipitation Test Research Articles

Examination of presence of specific antibodies against avian influenza virus in some species of wild birds

Infections caused by the avian influenza virus have been known for a long time and they are present, to a smaller or greater extent, in both extensive and intensive poultry production in many parts of the world. Epidemiological investigations have established a definite significance of the population of wild birds in maintaining and spreading this infection. Avian influenza is a zoonosis, and the virus has a great potential for causing mortality in humans, in particular its subtypes H5 and H7, which is why it has lately been provoking much attention among scientists and experts, as well as the general public. The objective of the work was to catch a certain number of wild birds in several locations in the Republic of Serbia, to identify them, and to collect samples of their blood serum for the determination of specific antibodies against the avian influenza virus. Birds were caught in ten locations in a manner that was safe for the birds themselves, as well as for the staff that did the catching. The birds were caught in especially produced nets, and in some cases in special traps. The caught wild birds were identified using the methods described in reference literature. All the names of the wild birds were coordinated with the valid Serbian nomenclature of European birds, prepared by prominent ornithologist and bird lover Milan Ružić. Following catching and identification, blood samples were taken from the birds from the wing vein (in bigger birds) or from the leg vein (in smaller birds). In taking blood samples from wild birds, all the principles of asepsis and antisepsis were followed in order to prevent any possibility of infection. After that, the birds were returned to their natural habitat, to the same locations in which they were caught. Serums were isolated from the taken blood samples and they were stored at -20oC until the final examinations. A total of 46 species of wild birds were identified among a total of 259 birds from which 259 samples of blood serum were isolated. The following were used for the detection of the presence of specific antibodies against the avian influenza virus in blood serum samples of wild birds: agar gel precipitation (AGP), the hemagglutination inhibition test (HI) for subtypes H5 and H7, the cELISA test with antigen for the A type avian infleunza virus, and the cELISA test with antigen for subtype H5 of the avian influenza virus. Due to the fact that about 360 different species of wild birds live in the Republic of Serbia, the number of 46 identified species covered by these investigations account for 12.77% of the total number of bird species present in Serbia, which is considered a good sample. Specific antibodies against the A type avian influenza virus were established in serum samples of only 9 of the 259 birds covered by examinations using the cELISA test. Of the 46 identified wild bird species, 6 belonged to birds that live exclusively in water habitats and are considered a reservoir of the avian influenza virus (white stork, mallard, mute swan, common pochard, common goldeneye, and Eurasian coot). Among the listed species, particular attention was drawn to 4 species of wild birds of the order Anseriformes and the family Anatidae (mallard, mute swan, common pochard, common goldeneye) of which there were 30 birds among the total of 259 examined. In the 30 blood serum samples of the listed bird species, specific antibodies against the A type avian influenza virus were established in 9 (30%) serum samples using cELISA. Specific antibodies against the avian influenza virus subtype H5 were established in 3 serum samples of mute swans (one serum sample originated from a mute swan which was tagged in Poland) and in one blood serum sample of a common pochard, or a total of 4 (13.33%) serum samples, using the hemagglutination inhibition test. Specific antibodies against the avian inluenza virus subtype H7 were established in 3 (10%) blood serum samples, in two serum samples from mallards and one sample from a mute swan, using the hemagglutination inhibition test. Specific antibodies against the avian influenza virus type A were not established in any examined bird species using the AGP test. In the population of wild bird species in the Republic of Serbia covered by these investigations, specific antibodies against the avian influenza virus were established only in serum samples of birds of the family Anatidae. Specific antibodies against the avian influenza virus type A established in 3 (6.52%) species of wild birds, and against subtypes H5 and H7 in 2 (4.34%) of the total of 46 examined species. The sensitivity of the cELISA test for the avian influenza virus subtype H5 and the hemagglutination inhibition test for subtype H5 amounted to 100%.

Evaluation of a competitive ELISA for antibody detection against avian influenza virus

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.

PERAN ANTIBODI KUNING TELUR (IgY) SEBAGAI OPSONIN UNTUK PENCEGAHAN SERANGAN MUTAN STREPTOCOCCUS SEROTIPE D (STREPTOCOCCUS SOBRINUS)

The aim of this study was to explore the role of serotype d Mutan Streptococcus (Streptococcus sobrinus) spesific immunoglobulin Y (IgY-Ss) as opsonin against the same strain. The eggs were collected from Single Comb Brown Leghorn which has been immunized with Streptococcus sobrinus. Agar gel precipitation test was applied to detect IgY-Ss in serum and egg. Egg containing IgY-Ss was collected and extracted by PEG-Amonium sulphate and purified using fast protein liquid chromatography. The purity of IgY-Ss was determined by UV spectrophotometer. Molecular weight was established by SDS-PAGE (sodium dedocyl sulphate-poly acrilamide gel electrophoresis). Biological activities of IgY-Ss as opsonin was determined by phagocytosis assay. Phagositic activity of macrophages was not increased by preincubation of both S. sobrinus (107 CFU/ml) and 100 μg of IgY-S, however the phagositic capacity was increased from 1.6 bacterial cell/ macrophag to 5.17 bacterial cell/ macrophag. These finding suggest that IgY-Ss obtained from hens immunized with S. sobrinus provide an alternative to prevent S. sobrinus infection.

Isolation and identification of bovine herpes virus -1 (BHV-1) from semen of foreign breeds bulls

Bovine herpes virus-1 (BHV-1) was isolated from bull semen by inoculation onto chorioallantoic membrane of specific pathogen free eggs. The isolated virus was identified by agar gel precipitation test, Dot ELISA, pock reduction and neutralization test, as well as by histopathology. The isolated virus was propagated on Madin Darby Bovine Kidney cells and identified by polymerase chain reaction. In sero-survey for BHV-1 antibodies on 1091 collected serum samples 188 sera showed clear precipitation lines by AGPT

Seroprevalence of low pathogenic avian influenza (H9N2) and associated risk factors in the Gyeonggi-do of Korea during 2005-2006

Between November 2005 and March 2006, a total of 253 poultry flocks in the Gyeonggi-do of Korea were examined for seroprevalence against avian influenza (AI) using a hemagglutination inhibition (HI) test and an agar gel precipitation test. No low pathogenic avian influenza (LPAI) virus was isolated from 47 seropositive flocks that lacked clinical signs during sampling. The unadjusted percentage of seroprevalence rates of layer and broiler flocks were not significantly different, i.e., 26% (25/96) and 23% (22/97), respectively. The HI titer of the layers (mean = 89) was higher than the broilers (mean = 36; p < 0.001). A cross-sectional study was conducted for the seroprevalence of LPAI in the layers. Of 7 risk factors, farms employing one or more workers had a higher seropositive prevalence as compared to farms without hired employees (adjusted prevalence OR = 11.5, p = 0.031). Layer flocks older than 400 d had higher seropositivity than flocks younger than 300 d (OR = 4.9, p = 0.017). The farmers recognized at least one of the clinical signs in seropositive flocks, such as decreased egg production, respiratory syndromes, and increased mortality (OR = 2.3, p = 0.082). In a matched case-control study, 20 pairs of case and control flocks matched for type of flock, hired employees, age, and flock size were compared. Frequent cleansing with disinfectants was associated with a decreased risk of seropositivity (OR = 0.2, p = 0.022). Although there was a low statistical association, using a foot disinfectant when entering the building led to a decreased rate of seropositivity (OR = 0.3, p = 0.105).

Prevalence of contagious bovine pleuropneumonia in Borno State, Nigeria

Epidemiological situation of contagious bovine pleuropneumoia (CBPP) over a ten (10) year period in Borno State of Nigeria was examined. During this period, 1,178 out of 355,745 cattle examined at postmortem had lesions suggestive of CBPP giving an overall prevalence rate of 0.33%. Highest prevalence rate of 0.77% was recorded in 1988 while lowest prevalence rate of 0.08% was recorded in 1994. Total vaccination coverage during the same period was 2,344,520 with highest vaccination coverage of 488,054 (10.8%) in 1988 and the lowest coverage of 66,398 (1.5%) in 1997. A total of 37 outbreaks of CBPP were reported during the period under review with highest outbreak (8) occurring in 1990. Seasonal prevalence of the disease was found to be highest in rainy season (0.20%) than the dry season (0.13%) but was not statistically significant (P>0.05). In a related development, a total of 3,875 cattle were examined between January and July 1998 in an attempt to find the incidence of CBPP using agar gel precipitation test (AGPT). Out of 57 suspected lungs, 41 (1.1%) were positive for AGPT. The disease is still endemic in Borno State and vaccination coverage was grossly inadequate. Keywords: CBPP, cattle, AGPT, prevalence, vaccination Nigerian Veterinary Journal Vol. 27 (2) 2006: pp. 14-22

Application of Polymerase Chain Reaction Fingerprinting to Differentiate Ornithobacterium Rhinotracheale Isolates

Ornithobacterium rhinotracheale (ORT) is an infectious respiratory pathogen of chickens, turkeys, and wild birds. There are 18 serotypes of ORT reported worldwide. In this study, enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction and random amplified polymorphic DNA assay with Universal M13 primer-based fingerprinting techniques were investigated for their ability to differentiate ORT isolates. The authors examined 50 field isolates and 8 reference strains of ORT for their genetic differences. The fingerprint patterns were compared with serotyping results of ORT by the agar gel precipitation test. M13 fingerprinting revealed different patterns for 6 reference serotypes of ORT that were tested, namely, C, D, E, I, J, and K. Ornithobacterium rhinotracheale reference serotypes A and F yielded indistinguishable fingerprints with M13 fingerprinting. The ERIC 1R technique discerned only 5 of the 8 reference serotypes of ORT. Distinct fingerprints were also found within the ORT serotypes with both techniques. From 58 isolates of ORT that were fingerprinted belonging to 8 ORT serotypes, 10 different fingerprints were obtained with M13 fingerprinting and 6 different fingerprints were obtained with ERIC 1R fingerprinting. M13 fingerprinting technique was found to be more discriminative in differentiating ORT isolates than the ERIC 1R fingerprinting technique. These results suggest that fingerprinting techniques may be a more discerning tool for characterizing ORT isolates than the serological test using the agar gel precipitation test. This fingerprinting technique could potentially be a valuable tool in identifying an isolate from a clinical outbreak of ORT infection for development of an autogenous vaccine.

Molecular characterization of field isolates and vaccine strains of infectious bursal disease virus

The present investigation was conducted to study the genetic heterogenicity and molecular polymorphism among the field isolates and vaccine strains of infectious bursal disease virus (IBDV). Samples of bursa of Fabricius from 15 suspected outbreaks of infectious bursal disease (IBD) were subjected to agar gel precipitation test (AGPT), virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) combined with restriction fragment length polymorphism (RFLP). Nine out of 15 samples were found positive in AGPT while 14 were found positive both by virus isolation and RT-PCR. PCR amplified 474bp fragment from the variable region of VP2. Sac I, Stu I, Alu I, Ssp I and Mbo I restriction enzymes were used for characterization of all the 14 IBDV isolates and four reference vaccine strains. Sac I, Stu I, Alu I and Ssp I could differentiate classical virulent IBD (cvIBD) vaccine virus strains from very virulent IBD (vvIBD) field isolates by their varying restriction patterns. Based on above results two field isolates (VPL and VMK) were placed in cvIBD virus group and 12 field isolates were placed in vvIBD virus group. Virus neutralisation test (VNT) using rabbit raised Georgia strain anti-serum, however, could not differentiate between cvIBD virus and vvIBD virus. It was concluded that RT-PCR combined with RFLP assay using restriction enzymes Sac I, Stu I, Alu I and Ssp I can be used for rapid differentiation and classification of field isolates of IBDV.

Analyses of the results of different test systems in the 2005 global proficiency testing schemes for infectious bursal disease virus and Newcastle disease virus antibody detection in chicken serum

The results of global proficiency testing schemes (PTS) for serological tests to detect antibodies against infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) in chicken serum, in which 125 and 120 laboratories, respectively, participated from Africa, Asia, Europe, Central and South America, were used to analyse the performances of different antibody test systems such as virus neutralization tests, haemagglutination inhibition tests, enzyme-linked immunosorbent assays and agar gel precipitation tests. All laboratories were asked to carry out their routine diagnostic tests for the detection of IBDV and NDV antibodies as usual. This global ring trial provided a large amount of data on variation within and between laboratories and test systems used worldwide. The data showed that the variation between the quantitative test results of different laboratories (R between) using the IBDV virus neutralization tests and the NDV haemagglutination inhibition test was higher (about double) compared with the variation within commercial enzyme-linked immunosorbent assay systems. Although both tests are often referred to and used as the “gold standard” in experimental and scientific studies, official procedures and for the validation of tests, this study shows that there is an urgent need for a global implementation of recommended test procedures and/or the inclusion of international reference sera in these studies.

Isolation of Actinobacillus pleuropneumoniae Serovar 15-Like Strain from a Field Case of Porcine Pleuropneumonia in Japan

An Actinobacillus pleuropneumoniae strain isolated from a field case of porcine pleuropneumonia in Japan, was closely related to a reference strain of serovar 15, which is a newly proposed serovar according to an analysis of field isolates originating from Australia. The isolate had biological and biochemical properties consistent with A. pleuropneumoniae biovar 1, and reacted strongly to a rabbit antiserum raised against a reference strain of serovar 15 in an agar gel precipitation test. The nucleotide sequence of a hyper variable region in the 16S RNA gene of the isolate was identical to that of the reference strain of serovar 15. The isolate possessed A. pleuropneumoniae-RTX toxin (Apx) II, III, and IV genes, consistent with serovar 15. Its virulence in mice was lower than that of ApxI-bearing strains but higher than that of other ApxIII-bearing strains. This is the first report describing the isolation of A. pleuropneumoniae serovar 15-like strain from a country or region other than Australia.

Inactivation of infectious bursal disease virus by binary ethylenimine and formalin

In this experiment conducted to study the inactivation dynamics of infectious bursal disease virus (IBDV) by binary ethylenimine (BEI) in comparison with formalin, IBDV was isolated from the bursa of infected chickens and its confirmation was done by agar gel precipitation test. Viral suspensions were subjected to inactivation with BEI and formalin for pre-set time intervals. BEI was employed at concentrations of 0.001 and 0.002 mol/L while formalin was used at 0.1% and 0.2%. Sampling was done at 6, 12, 24, 36 and 48 h of incubation and samples were tested for their inactivation status in 9-day-old embryonated eggs and 3-week-old broiler chickens. IBDV was completely inactivated by 0.001 and 0.002 mol/L BEI after 36 h of incubation at 37 degrees C, whereas formalin at 0.1% and 0.2% concentrations inactivated IBDV in 24 h.

Isolation of Lumpy skin disease virus form naturally infected cattle previously vaccinated with live attenuated sheep poxvirus vaccine

Lumpy skin disease virus (LSDV) was isolated, from naturally infected cattle that have a history of previous vaccination with live attenuated sheep pox virus (SPV) vaccine. The virus was isolated on chorio-allantoic membrane (CAM) of specific pathogen free (SPF) embryonated chicken eggs (ECE) and identified by agar gel precipitation test (AGPT) and neutralization test using specific hyperimmune serum against LSDV and SPV. Characteristic intracytoplasmic inclusion bodies was detected in trypsenized cell of infected CAM stained with H&E. Laboratory studies for characterization of isolated LSDV revealed that it was stable at a wide range of pH, but it was inactivated by exposure to 56 0C for 15 minutes. Treatment of isolated LSDV with lipid solvents (20% ethyle ether and chloroform) reduced the virus titer 3.2 and 4.4 log respectively after 24 hrs at 4 0C .On cross neutralization testcomplete neutralization of isolated LSDV was obtained with both reference LSDV and SPV antisera. Cattle vaccinated with live attenuated SPV vaccine under experimental condition found to be protected against natural field infection with LSDV.

Serological and Virological Studies of Newcastle Disease and Avian Influenza in Slaughter-Age Ostriches (Struthio camelus) in Japan

Serum samples from 191 ostriches (Struthio camelus) in Japan were tested for antibodies to Newcastle disease virus (NDV) and avian influenza virus (AIV). Twenty-two (12%) contained NDV-specific neutralizing antibodies by a virus-neutralization (VN) test without vaccination. Antibodies to AIV were not detected in the any sera by an agar gel precipitation test. Seven serum samples that had vaccinated with live NDV by eye drop were all positive by the VN test at 1 month post vaccination. A haemagglutination inhibition (HI) test for NDV seemed not to be suitable for ostriches because of non-specific agglutination of chicken red blood cells. No haemagglutinating viruses were isolated. This is the first report on detection of antibodies against NDV in ostriches in Japan.

Passive immunization using purified IgYs against infectious bursal disease of chickens in Pakistan

Infectious bursal disease (IBD) is an acute and highly contagious disease of young chickens caused by Birnavirus. Mortality of infected birds can be best prevented if injected with antibodies. The present study was an attempt to raise specific hyper-immune polyclonal antibodies against IBD virus in Pakistan. Commercial layers divided into four groups were injected with IBD vaccine subcutaneously according to four different treatment regimens. Eggs were collected daily and antibodies were purified from yolk with dextran sulphate. Titers of antibodies in serum and yolk were evaluated with enzyme linked immunosorbant assay and agar gel precipitation test. Antibody titers were significantly higher in yolk than serum. Eggs collected at 28 days post-vaccination had maximum antibody titers. Of treatment regimens, T3 was found to be most effective for hyperimmunization. Lyophilized antibodies stored at 4℃ did not lose their activity till the end of experiment. IBD virus infected birds were injected with purified antibodies which induced 92% recovery as compared to control birds. The study implicates that the purified antibodies may be useful as a therapeutic agent to cure IBD infected birds.

Development of a One-Step Strip Test for the Diagnosis of Chicken Infectious Bursal Disease

A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 x 10(4) ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1-2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.

Reverse Transcriptase-Polymerase Chain Reaction to Detect Avian Encephalomyelitis Virus

A reverse transcriptase-polymerase chain reaction (RT-PCR) was developed and optimized for the detection of avian encephalomyelitis virus (AEV). A pair of primers was prepared based on the VP2 gene of the structural protein P1 region of the AEV genome. An avian encephalomyelitis virus-specific 619-base pair cDNA product was amplified by these primers from five reference/field strains of AEVs but not from 10 other avian pathogenic viruses and bacteria. The RT-PCR assay developed in this study was found to be sensitive and specific with as little as 10 pg of avian encephalomyelitis virus RNA detected using gel electrophoresis. Furthermore, AEV-RT-PCR was able to detect AE virus from chicken embryo brain at 3 days postinoculation as compared with the AE agar gel precipitation test (AGP), which required up to 11 days of incubation in the embryos.

A rapid assay for detecting antibody against leucocytozoonosis in chickens with a latex agglutination test using recombinant R7 antigen

A method for detecting antibody against leucocytozoonosis in chickens by latex agglutination (LA) was developed using latex beads coated with recombinant R7 (rR7), an outer membrane antigen of second-generation schizonts of Leucocytozoon caulleryi. Compared with the agar gel precipitation test, which is widely used in Japan, LA could detect antibody induced by L. caulleryi infection with greater sensitivity. No agglutination was detected with sera from specific pathogen free or layer chickens that had not been vaccinated with oil-adjuvanted rR7 antigen, or infected with L. caulleryi, nor with sera from chickens inoculated with other pathogens, establishing the specificity of the assay. The LA test was also able to be used to quantify serum antibody induced by vaccination, which is not possible with the agar gel precipitation test. This study has shown that LA using the rR7 antigen is a simple, quick, and useful method for detecting antibody against L. caulleryi.

Comparison of agal gel precipitation test (AGPT) and enzyme linked immunosorbent assay (ELISA) in the detection of infectious bursal disease virus (IBDV) antibody in village chickens ın Oyo State, Nigeria

The use of agar gel precipitation test (AGPT) and enzyme linked immunosorbent assay (ELISA) in assaying for the presence of infectious bursal disease (IBD) virus antibody in village chickens in Oyo State, Nigeria, was compared. Out of 400 sera subjected to ELISA, 360 (90%) samples where positive for IBD virus (Serotype 1) antibody with a titre range of 3,441 to 22,444 whereas, by the AGPT, out of 392 sera tested, 248 (63.3%) samples were positive with a titre range of 0log2 (neat) to 4log2. This study has correctly presented the seroprevalence of IBD in village chickens in this environment and has further confirmed the superior sensitivity of the ELISA technique. Nigerian Veterinary Journal Vol.25(1) 2004: 26-29

An epizootic of equine influenza in Upper Egypt in 2000

This study describes an epizootic of respiratory tract disease caused by influenza virus infection in a large population of equines in Luxor and Aswan, Upper Egypt, during the winter of 2000. The epizootic started in January and the infection rate reached its peak in February before gradually decreasing until the end of April, 2000. Horses, donkeys and mules of all ages and both sexes were affected. Free movement of the infected equines and direct contact between the animals at markets facilitated the rapid spread of the disease. The cause of the epizootic was established by use of serological testing and the identification of the influenza virus in nasal secretions. Egg inoculation and the haemagglutination test were used to detect the influenza virus. Both haemagglutination inhibition (HI) and agar gel precipitation tests were performed to identify the isolated influenza virus using reference antisera against A/Equi-1 (H7N7) and A/Equi-2 (H3N8). Antibodies against the equine influenza virus were demonstrated in 416 (95.6%) out of 435 collected sera using the HI test. High rectal temperature, inappetence, conjunctivitis, redness of nasal mucosa, a serous to mucopurulent nasal discharge and a harsh dry cough were the most common clinical manifestations. Stress factors, such as using equines for heavy transportation and drawing, precipitated the onset of the disease, intensified the clinical syndrome, delayed recovery and facilitated secondary bacterial infection. The present study suggested that the absence of a vaccination programme against equine influenza was one of the principal causes of the spread of infection during this outbreak. In conclusion, the implementation of a national equine influenza vaccination programme, using an effective updated vaccine, is essential in Egypt.

Isolation and characterization of virus of highly pathogenic avian influenza H5 subtype of chicken from outbreaks in Indonesia

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