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Clostridial necrotic enteritis: field cases and the role of vaccination against coccidiosis to the incidence in broiler chicken Natalia, Lily; Priadi, A
Indonesian Journal of Animal and Veterinary Sciences Vol 8, No 1 (2003)
Publisher : Indonesian Animal Sciences Society

A study of necrotic enteritis in broiler chicken was carried out in West Java. Isolates of highly toxigenic C. perfringens of type C were isolated from field cases of necrotic enteritis which most frequently occured in chicken with vaccine-inducedcoccidial lesions. Experimental study in broiler chicken was conducted to determine the predisporing factor for necroric enteritis. Three possible factors were evaluated, those were feeding with high animal protein ingreadients, vaccination with live attenuated anticoccidial vaccine, and feeding with spores of C. perfringens type A and C. The occurrence of intestinal coocidial leions in chicks vaccinated with life attenuated anticoccidial vaccine was demonstrated. The highest mortality and the significant intestinal lesions of chickens were observed in group treated with live attenuated anticoccidial vaccine and C. perfringens type A and C spores (P<0.01). From the experiment, it was found that vaccine induced-coccidial lesions and C. perfringens type A and C were the predisposing factors of necrotic enteritis. These results suggest that the concurrent infection with coccidia and C. perfringens has a synergistic effect on mortality and intestinal lesions in necrotic enteritis. Â Key words : Clostridial necrotic enteritis, broiler chicken, coccidiosis

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

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

Rapid identification of Bacillus anthracis by cell wall and capsule components direct fluorescent antibody assay Natalia, Lily; Adji, Rahmat Setya
Indonesian Journal of Animal and Veterinary Sciences Vol 13, No 2 (2008)
Publisher : Indonesian Animal Sciences Society

During the outbreak of anthrax, early diagnosis is critical for effective treatment. Numerous attempts have been made to design antigen based detection tests and to rapidly identify truly anthrax specific antigens for B. anthracis. In Indonesia, standard identification of B. anthracis relies on a combination of time consuming steps including bacterial culture and Ascoli precipitin test, which can take several days to provide a diagnosis. In this study, two component (cell wall and capsule) direct fluorescent antibody assay (DFA) were developed to rapidly identify and to directly detect capsulated B. anthracis. The component used in cell wall DFA (CW-DFA) assay is polysaccharide-peptidoglycan complex, which was prepared from B. anthracis culture by cell lysis, guanidine and sodium dodecyl sulphate (SDS) extraction. The component used in capsule DFA (CAP-DFA) is poly-D-glutamic acid (PGA) which were prepared by extraction of B. anthracis capsule. Component of polysaccharide-peptidoglycan complex and PGA conjugated with hemocyanin were then used as immunogen for immunizing rabbits using Freundâs complete/incomplete adjuvant. The hyperimmune sera were then collected, purified and conjugated to Fluorecent Iso Thiocyanate (FITC). B. anthracis isolates and non B. anthracis isolates were tested by the CW-DFA and CAP-DFA Assays. B. cereus, B. subtilis, other Bacillus sp. and other Gram positive rod bacteria were negative, while capsulated B anthracis gave positive results. The two component (CW DFA and CAP-DFA) assay are specific rapid confirmatory test for capsulated B. anthracis. Key Words: Bacillus anthracis, Cell Wall and Capsule Direct Fluorescent Antibody Assay

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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