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Polymerase chain reaction optimization for the detection of Pasteurella multocida B:2, the causative agent of Haemorrhagic septicaemia Natalia, Lily; Priadi, Adin
Indonesian Journal of Animal and Veterinary Sciences Vol 6, No 4 (2001)
Publisher : Indonesian Animal Sciences Society

Specific detection of Pasteurella multocida type B:2 by polymerase chain reaction (PCR), using a set of DNA primers wasoptimised. Effects of the addition of ethylene diamine tetra acetic acid (EDTA) to the sample preparation, Escherichia coli contamination and the number of P. multocida on the PCR product was assessed. The PCR test was compared to the standard bacteriological method for the detection of P. multocida B:2 in tonsillar swab samples collected from slaughter houses of various regions in Indonesia. Addition of 100 mM EDTA-saline to P. multocida B:2 spiked tonsillar swab samples inhibits the production 350 base pairs (bp) PCR product. The inhibitory effect of the EDT A can be eliminated by three times washing with deionised water. The PCR can detect P. multocida as low as I organism and contanimation of 100 CFU of E. coli does not effect the PCR result. The results show that the DNA primers for P. multocida B:2 is sensitive and specific. The inhibitory effect of EDTA in PCR samples can be eliminated by washings. Keywords: PCR, EDT A, Pasteurella multocida B:2

Protection of a live Pasteurella multocida B:3,4 vaccine against haemorrhagic septicaemia in cattle Priadi, Adin; Natalia, Lily
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 1 (2002)
Publisher : Indonesian Animal Sciences Society

Cross protection conferred by a live Pasteurella multocida B:3,4 vaccine to infection by P. multocida B:2, the haemorrhagis septicaemia causing bacteria in cattle was investigated. Intranasal aerogenic immunization and subcutaneous injection of the live vaccine were applied to groups I and II of 5 Bali cattle respectively. Another group (III) of 5 cattle were vaccinated with standard oli adjuvant killed vaccine intramuscularly. Cattle were observed for clinical signs and body temperatures were measured. Sera were collected monthly for 12 month and kept at -200C for further testing by ELISA. No adverse sign was observed at cattle of groups I and II after vaccination with the live vaccine. Both intranasal and subcutaneous vaccination of live vaccine showed a similar serological response which started at month-5, peaked at month-(6-7) after vaccination and still sustained at the level above positive cut-off (88 ELISA Unit) at the end of observation month-12. Cattle vaccinated with killed adjuvanted vaccine responded earlier, peaked at 5-6 month after vaccination and declined steadily till the end of investigation. At 6 and 12 months after vaccination catlle were challenged with P. multocida B:2. All vaccinated cattle challenged at 6 months (C-1) and 12 (C-2) months after vaccination survived and showed no clinical signs. Body temperatures of all vaccinated cattle were normal and ranged from 38.10C to 39.10C and 38.50C to 39.50C for cattle chalenged at C-1 and C-2 respectively. However, there was 1 cattle of group I at C-1 showed an initial increase of body temperature to 400C and decreased to normal at 42 hours after challenge. One catlle of group II had a body temperature of 40.70C detected at 5 hours post C-2 and reached a normal temperature at hour-11. Both unvaccinated cattle at C-1 and C-2 died and had body temperatures of 41.40C and 41.10C respectively at the time of death. This investigation shows that live vaccine P. multocida B:3,4 is safe and can protect cattle from haemorrhagic septicaemia for at least 12 months. This vaccine is promising to be used to replace oil adjuvanted killed bacterin for haemorrhagic secticaemia. Key words: Live aerosol vaccine, protection, haemorrhagic septicaemia, cattle

Glasserâs disease in swine in Batam Island, Riau Province Priadi, Adin; Natalia, L; Poernomo, S
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 4 (2004)
Publisher : Indonesian Animal Sciences Society

Glasserâs disease or Haemophilus parasuis in swine causes a considerable economic losses. This disease decreases farm production due to high mortality. In a field investigation, H. parasuis serotype 12 was isolated from the lung of a ten week old post weaning pig suffering from pneumonia in Bulan island, Riau Province. The isolation of H. parasuis in a pig herd showing increasing mortality is the first reported in Indonesia. Antibiotic sensitivity test using disc diffusion methods, showed that the isolate was sensitive to bacitracin, baytril, erythromycin and was resistance to neomycin, kanamycin, doxycyclin, ampicillin and sulphamethoxazol-trimethoprim. Vaccination in weaned piglet using commercial inactivated vaccine was monitored using Enzyme-linked immunosorbent assay (ELISA). Crude extract of culture H. parasuis serotype 12 was used as the ELISA coating antigen. There was no significant immune response detected by ELISA 3 months after vaccination. Â Key words: Glasserâs disease, swine, drug sensitivity, ELISA

Utilization of probiotics for controlling clostridial necrotic enteritis in broiler chickens Natalia, Lily; Priadi, Adin
Indonesian Journal of Animal and Veterinary Sciences Vol 10, No 1 (2005)
Publisher : Indonesian Animal Sciences Society

Clostridial necrotic enteritis (CNE) is a common disease among rapidly growing broiler chickens. The purpose of this trial was to study the utilisation of probiotics in controlling experimental CNE in broiler chickens. Chicken normal gut bacterial flora (mucosal starter culture selective/MCS) was used as a competitive exclusion treatment in broiler chicken and its influence to the occurence of clostridial necrotic enteritis were observed. The study comprised of 4 broiler cages treatments of probiotics (2 different dose of MCS, commercial probiotic, 1 cage untreated as control). Probiotics were given orally upon arrival. All groups were given live coccidial vaccine (as predisposing factor for CNE) and challenged with 108 Clostridium perfringens tipe A and C spores on day 10 and 12. The results showed that the probiotics could reduced the incidence and severity of CNE after challenge and improved the performance of chickens treated. Untreated group showed 40% of the mortality due to CNE, and 30% of the chicken showed subclinical necrotic enteritis (SNE). Â Â Key Words: Clostridial Necrotic Enteritis, Probiotics, Broiler Chickeni

Infection of Ornithobacterium rhinotracheale (ORT) in chickens in Indonesia Priadi, Adin; Natalia, Lily
Indonesian Journal of Animal and Veterinary Sciences Vol 11, No 1 (2006)
Publisher : Indonesian Animal Sciences Society

Ornithobacterium rhinotracheale is a bacterium identified as a new species in 1994 and generally associated with respiratory distress in chickens. From 214 of sinus swabs, tracheal swabs, lungs, airsac, liver heart blood samples and yolk sacs of chickens suffered from respiratory distresses, 6 isolates of O. rhinotracheale were isolated. These isolates were obtained from tracheal swabs of broiler chickens aged between 28-35 days old and broiler breeder of 32 weeks old. Upon incubated on blood agar for 48 hours at 37oC in a 5% CO2 atmosphere, round, convect and grey colonies with diametres of 1-2 mm were observed. The bacteria were pleomorphic, Gram negative rods, negative catalase and positive oxidase. Biochemically, the bacteria did not change potassium nitrate, tryptophan, glucose, arginine, urea, esculin, gelatine, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenyl-acetate in API 20 NE system but Î² âgalactosidase was produced. In the API 20 NE system, the isolates were identified as 0020004, 0060004, 0020104 codes. Tracheitis, air sacculitis, pneumonia and cheesy air sacs were pathological changes generally found in chickens infected with O. rhinotracheale. Trachea is the most important organ for the isolation of O. rhinotracheale. Key Words: Ornithobacterium rhinotracheale, Infection, Chicken, Indonesia

Phage typing and sensitivity test to antibiotics of Salmonella enteritidis isolates from Indonesia Poernomo, Sri; Priadi, Adin; Natalia, Lily
Indonesian Journal of Animal and Veterinary Sciences Vol 11, No 2 (2006)
Publisher : Indonesian Animal Sciences Society

Salmonella enteritidis (SE) is frequently implicated in disease outbreaks such as human food poisoning. Phage typing have been proved to be a valuable and sensitive tool in the control of SE infections. The ability of phage to distinguish varieties among apparently identical serotypes led to the development and acceptance of phage typing as a significant epidemiological procedure. To determine the epidemiological pattern of SE, phage typing of 53 SE isolated from various sources in Indonesia during 1991â1999, has been conducted using 16 typing phages of phage typing scheme of SE obtained from the International Collaborating Center for Enteric Phage typing, Central Public Health Laboratory, Colindale, UK. The lyse blood isosensitest was then used to test the sensitivity of the Salmonella isolates to antibiotics. The phage typing results obtained that of 53 Salmonella isolates there were one S. infantis, one S. berta, and 46 SE phage type 4, 2 SE phage type 7 (from chicken and water), 1 SE phage type 6 (from chicken) and 2 SE phage type 1 (from chicken). SE phage type 4 isolates comprised of 2 isolates from human, 19 isolates from chicken (young and adult), 17 isolates from day old chicks, 4 isolates from fluff, 2 isolates from chicken meat, 1 isolate from poultry farm water, 1 isolate from dog organ. These findings indicated that contaminated chicken appeared to be the sources of human and dog for SE infection. The results of sensitivity test of the isolates to antibiotics showed that most of the Salmonella isolates from Indonesia were resistant to the antibiotics tested. Key Words: Salmonella Enteritidis, Phage typing, Sensitivity test, Indonesia, Chicken

Development of enzyme-linked immunosorbent assay for detecting Ornithobacterium rhinotracheale (ORT) infection in chicken Priadi, Adin; Natalia, Lily
Indonesian Journal of Animal and Veterinary Sciences Vol 11, No 3 (2006)
Publisher : Indonesian Animal Sciences Society

Ornithobacterium rhinotracheale (ORT) has been recognized in chicken in Indonesia and incriminated as a possible additional causative agent in respiratory disease complex. An enzyme-linked immunosorbent assay (ELISA) has been developed for the seroepidemiological study of ORT infection in chickens. Ten weeks old chickens are injected with 0.5 ml of killed O.Â rhinotracheale emulsified in Freunds complete adjuvant at a concentration of 109 CFU/ml. Hyperimmune sera and non-reactive control sera were used to standardized the ELISA for ORT infection. Optimum condition for the ORT ELISA was antigen dilution 1/800, serum dilution 1/100 and 1/4000 conjugate dilution. Optical density cut-off point was determined by using 31 serum samples from 2 broiler farms. Cut-off for negative serum was 0.27 (mean + 3 standard deviation). With these optima, 187 chicken sera from broiler, layer and broiler breeder farms were collected and screened. Seroconvertions were detected from broiler and layer farms in Magelang district, Central Java (Bojong I, Paremono, Bojong II, Keblukan) and a broiler breeder farm in West Java. The seraconvertion were 0, 10, 94, 88 and 100 percents respectively. These figures show that the prevalence of O. rhinotracheale infection in chicken in layer and breeder farms were very high. Key Words: Ornithobacterium rhinotracheale (ORT), ELISA, Chicken

Clostridial necrotic enteritis in chicken associated with growth rate depression Priadi, Adin; Natalia, Lily
Indonesian Journal of Animal and Veterinary Sciences Vol 13, No 1 (2008)
Publisher : Indonesian Animal Sciences Society

Clostridium perfringens (C. perfringens) is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen causing necrotic enteritis. C. perfringens only causes necrotic enteritis when it transforms from non-toxin producing type to toxin producing type. The alpha toxin, (phospholipase C) is believed to be a key to the occurrence of Clostridial necrotic enteritis (CNE). The best known predisposing factor is mucosal damage, caused by coccidiosis that damages the intestinal lining, making the gut susceptible to infections including C. perfringens. The purpose of this study was to observe the chicken performance in experimental CNE and field cases of CNE. Diagnosis of CNE were made by latex agglutination test, isolation and identification of the agent. Pathological and histopathological changes were also observed. Experimentally, NE could be reproduced when Eimeria sp and C. perfringens spores are inoculated in chicken. Signs of an NE are wet litter and diarrhea, and an increase in mortality is not often obvious. The depression of growth rate and feed efficiency of chicken become noticeable by week 5 because of damage to the intestine and the subsequent reduction in digestion and absorption of food. Subclinical form of CNE was also frequently found in the field, leading to significant decreases in performance. Chicken gut samples examinations revealed that subclinical form of CNE causes damage to the intestinal mucosa caused by C. perfringens leads to decreased digestion and absorption, increased feed conversion ratio and reduced weight gain. Dual infection with C. perfringens and Eimeria sp. was frequently found in field. The results of these studies provide evidence for C. perfringens as a causative bacteria for growth depression. Key Words: Clostridial Necrotic Enteritis, Chicken, Growth Depression

The use of filter paper as a transport device for serology of Pasteurella multocida infection : Analysis and comparison ofprotein composition of filter paper extract and serum Natalia, Lily; Priadi, Adin
Indonesian Journal of Animal and Veterinary Sciences Vol 3, No 3 (1998)
Publisher : Indonesian Animal Sciences Society

Two methods for collecting blood specimens for measuring antibody to Pasteurella multocida were compared. Blood was collected on filter-paper strips, air-dried and stored at 4Â°C along with paired samples collected by venepumeture . Analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the protein composition of filter paper extract and serum was similar. Both samples had common proteins of 67, 52-58 and 27 kDa. However, there are two proteins bands of 14 and 30 kDa that were only found in, filter-paper extract. Westernblot analysis also showed that samples from both sampling techniques reacted to P. multocida proteins of 43 kDa. Samples from experimental and field animals were also collected by the two techniques and assayed by enzyme-linked immunosorbent assay (ELISA) for P. multocida antibodies . The agreement between samples from experimental animals and the field using ELISA was analyzed . Samples from experimental animals, showed a very high correlation (r = 0.931) in ELISA results among samples collected by the two techniques. However, the correlation was lower (r = 0.799) in samples collected from the field. Cost analysis showed that filter-paper collection technique was 100 times more economical compared to venepuncture technique. It was concluded that eluates of whole blood dried on filter paper can be used as an alternative to sera in ELISA for measuring antibodies to P. multocida. Â Key words : Pasteurella multocida, serological tests, filter paper

Protection of inactive intranasal Ã¡ntrax vaccine to Bacillus anthracis infection Priadi, Adin; Natalia, Lily; Adji, Rahmat S.
Indonesian Journal of Animal and Veterinary Sciences Vol 15, No 2 (2010)
Publisher : Indonesian Animal Sciences Society

Ãnthrax is an endemic zoonotic disease distributed in many parts of Indonesia. Although vaccination program has been implemented in many areas, cases are still frequently reported. Farmers are reluctant to vaccinate their livestock since spore vaccine used in the field often cause side effects and death of the animals. To overcome this problem, an inactive vaccine composes of Bacillus anthracis toxins, cell wall and capsule subunits was developed. B. anthracis Sterne strain (34F2) was selected to produce toxins and cell walls. Local Bacillus anthracis isolated from Citaringgul was used to produce capsule as the Polymerase Chain Reaction (PCR) revealed that this isolate poses cap gene encoding for capsule. Two vaccines compose of 15 Î¼g toxoid, 30 Î¼g of capsule, 15 Î¼g of cell wall and 30 Î¼g toxoid, 60 Î¼g of capsule, 15 Î¼g of cell walls were designated as vaccine I and vaccine II respectively. For each experiment, 10 mice were nasally immunized by placing 5 Î¼l of vaccine into each nare 3 times at 2-week intervals. A group of 10 mice were unvaccinated and used as control. Blood was collected fortnightly to monitor antibody responses. All mice were challenged with 2 x 105 B. anthracis Sterne spores injected subcutaneously two weeks after the last vaccination. Two weeks after vaccination of antibodies to B. anthracis toxin, capsule and cell wall were detected in dot-blot assay. Mice that were immunised intranasally with chitosan adjuvanted vaccine developed high IgG responses in sera as detected by ELISA, and the response was dose dependent. Vaccine II gave better response than vaccine I. Vaccine I and II protected mice from challenge at a rate of 60 and 80% respectively. This results showed that intranasal B. anthracis vaccine composes of toxin, capsule and cell wall with chitosan as an adjuvant gave a good protection against B. anthracis Sterne spores challenge in mice. Key Words: Inactive Intranasal Ãntrax Vaccine, Protection, Bacillus anthracis, Mice

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