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Pathogenesis of Haemorrhagic Septicaemia (HS) in cattle and buffalo: clinical signs, pathological changes, reisolation and detection of Pasteurella multocida using culture medium and Polymerase Chain Reaction (PCR} Priadi, Adin; Natalia, Lily
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 1 (2000)
Publisher : Indonesian Animal Sciences Society

In the study of the pathogenesis of Haemorrhagic Septicaemia (HS), one cattle and one buffalo were infected subcutaneously with a dose of 4 x 108 colony forming units of Pasteurella multocida B:2 in the neck region. The post infection clinical findings were observed. During this observation period, bacterial isolation was carried out from heparinised blood and nasal swabs. The buffalo succumbed 2 hours earlier than the cattle.111e post mortem pathological changes in cattle and buffalo were similar but the lesions most severe in the buffalo. The prominent changes were observed in the lungs and bronchi of both animals. Bacterial reisolation and Polymerase Chain Reaction (PCR) for P. multocida were carried out trom various samples kept at room temperature without any preservative for 15, 35 and 59 hours after the death of the animals. After 59 hours, heavily contaminated samples were found in all organs except bone marrow. Reisolation of P. multocida trom these samples was difficult, however, the organism can still be identified by PCRTo improve the viability of Pasteurella multocida and reducing the growth of contaminants, transport medium containing selective antibiotics was developed. Amikacin and Gentamicin were good selective antibiotics to suppress other contaminating organisms. Â Key words: Pathogenesis, Pasteurella multocida B:2, cattle and buffalo, selective medium, PCR

Enteritis necroticans pada ayam broiler akibat infeksi sekunder clostridium perfringeens Tipe A Agus Setiyono; Rachmat Nabib; Gatut Ashadi; Aisjah Girinda; Lily Natalia
Hemera Zoa Vol. 76 No. 1 (1993): Jurnal Hemera Zoa
Publisher : Hemera Zoa

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The experiment was conducted to determine the relationship between conccidiosis vaccination and secondary infection of Clostridium perfringens type-A as a cause of Enteritis Necroticans in broiler chickens.The completely randomized design was used in this experiment. In this study 102 Arbor Acres strain unsexed chicken were randomly assigned to 8 treatment groups with 12 chicks each. Feed and water were given ad libitum.Treatment factors in this experiment were coccidiostat (Salinomycin 6%), coccidiosis vaccine and Clostridium perfringens type-A isolate, and their combinations.The anatomy pathology (AP) and histopathology (HP) figures of the chicken intestines were evaluated.Histopathology results indicated that the relationship between coccidiosis vaccination and secondary infection of Clostridium perfringens type-A was highly significant (p < 0.01) but the anatomy pathology figures showed that the relationship was not significant (p < 0.05).

Development of enzyme-linked immunosorbent assay for detecting Ornithobacterium rhinotracheale (ORT) infection in chicken Adin Priadi; Lily Natalia
Jurnal Ilmu Ternak dan Veteriner Vol 11, No 3 (2006): SEPTEMBER 2006
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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. rhinotracheale emulsified in Freund's 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

Protection of inactive intranasal ántrax vaccine to Bacillus anthracis infection Adin Priadi; Lily Natalia; Rahmat S. Adji
Jurnal Ilmu Ternak dan Veteriner Vol 15, No 2 (2010): JUNE 2010
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Ánthrax 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 μg toxoid, 30 μg of capsule, 15 μg of cell wall and 30 μg toxoid, 60 μg of capsule, 15 μg of cell walls were designated as vaccine I and vaccine II respectively. For each experiment, 10 mice were nasally immunized by placing 5 μl 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 Ántrax Vaccine, Protection, Bacillus anthracis, Mice

Protection of a live Pasteurella multocida B:3,4 vaccine against haemorrhagic septicaemia in cattle Adin Priadi; Lily Natalia
Jurnal Ilmu Ternak dan Veteriner Vol 7, No 1 (2002): MARCH 2002
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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

Utilization of probiotics for controlling clostridial necrotic enteritis in broiler chickens Lily Natalia; Adin Priadi
Jurnal Ilmu Ternak dan Veteriner Vol 10, No 1 (2005): MARCH 2005
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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). Key Words: Clostridial Necrotic Enteritis, Probiotics, Broiler Chickeni

Pathogenesis of Haemorrhagic Septicaemia (HS) in cattle and buffalo: clinical signs, pathological changes, reisolation and detection of Pasteurella multocida using culture medium and Polymerase Chain Reaction (PCR} Adin Priadi; Lily Natalia
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 1 (2000): MARCH 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

In the study of the pathogenesis of Haemorrhagic Septicaemia (HS), one cattle and one buffalo were infected subcutaneously with a dose of 4 x 108 colony forming units of Pasteurella multocida B:2 in the neck region. The post infection clinical findings were observed. During this observation period, bacterial isolation was carried out from heparinised blood and nasal swabs. The buffalo succumbed 2 hours earlier than the cattle.111e post mortem pathological changes in cattle and buffalo were similar but the lesions most severe in the buffalo. The prominent changes were observed in the lungs and bronchi of both animals. Bacterial reisolation and Polymerase Chain Reaction (PCR) for P. multocida were carried out trom various samples kept at room temperature without any preservative for 15, 35 and 59 hours after the death of the animals. After 59 hours, heavily contaminated samples were found in all organs except bone marrow. Reisolation of P. multocida trom these samples was difficult, however, the organism can still be identified by PCRTo improve the viability of Pasteurella multocida and reducing the growth of contaminants, transport medium containing selective antibiotics was developed. Amikacin and Gentamicin were good selective antibiotics to suppress other contaminating organisms. Key words: Pathogenesis, Pasteurella multocida B:2, cattle and buffalo, selective medium, PCR

Rapid identification of Bacillus anthracis by cell wall and capsule components direct fluorescent antibody assay Lily Natalia; Rahmat Setya Adji
Jurnal Ilmu Ternak dan Veteriner Vol 13, No 2 (2008): JUNE 2008
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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 Freund’s 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

Polymerase chain reaction optimization for the detection of Pasteurella multocida B:2, the causative agent of Haemorrhagic septicaemia Lily Natalia; Adin Priadi
Jurnal Ilmu Ternak dan Veteriner Vol 6, No 4 (2001): DECEMBER 2001
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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

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 Lily Natalia; Adin Priadi
Jurnal Ilmu Ternak dan Veteriner Vol 3, No 3 (1998)
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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 4°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. Key words : Pasteurella multocida, serological tests, filter paper

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