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The detection of K88, K99 fimbrial antigen and enterotoxin genes of Escherichia coli isolated from piglets and calves with diarrhoea in Indonesia ., Supar
Indonesian Journal of Animal and Veterinary Sciences Vol 2, No 1 (1996)
Publisher : Indonesian Animal Sciences Society

Enterotoxigenic Escherichia coli (ETEC) strains cause diarrhoeal disease in piglets and calves in Indonesia. These strains possess two virulence factors namely attachment and enterotoxin antigens . These factors could be detected phenotypically and genetically. Haemolytic Escherichia coli (E coli) isolates possessing K88 fimbrial antigen associated with 0-group 108 and 149. They were positive for K88 gene and demonstrated their ability to produce heat labile enterotoxin (LT) and genetically were all positive for LT gene . Seventeen isolates ofE coli K88 which associated with 0-group 149 were positive forSTb gene, other O-serotypes were negative . Ten isolates of Ecoli K88 which associated with 0-group 108 possessed K88, K99, LT and STa genes, but negative for STb gene . However, phenotypically the K99 antigen and STa toxin were not expressed under laboratory conditions, the reason was not well understood . E. coli K99 strains isolated from calves wit h diarrhoea were all associated with 0-group 9 and produced STa toxin when tested by suckling mousse bioassay. The E. coli K99 calf isolates were all hybridized with K99 and STa gene only . It is likely that K99 gene is associated with STa gene . The DNA hybridization technique is more convenience to be used for confirmation diagnosis of colibacillosis, however, not all veterinary laboratories could perform these tests . Â Keywords: Excherichia coli, probe, gene detection, virulence determinant

The application of enterotoxigenic Escherichia coli (ETEC) K99, F41 polyvalent vaccine in pregnant dairy cattle to control neonatal colibacillosis and mortality of calves ., Supar; ., Kusmiyati; Poerwadikarta, B
Indonesian Journal of Animal and Veterinary Sciences Vol 3, No 1 (1998)
Publisher : Indonesian Animal Sciences Society

Enterotoxigenic Escherichia coli (ETEC) strains possessing either K99, F41 or K99F41 are responsible for causing neonatal diarrhoea and mortality of calves and difficult to control using antimicrobial drugs. A whole cell ETEC vaccine containing fimbrial antigens of polyvalent strains based on field serotypes was produced . The efficacy of ETEC vaccine used to control neonatal colibacillosis of dairy calves was studied in experimental animals and field trials. Five pregnant dairy cow were used for experimental study. Three animals were injected subcutaneously with 5 ml vaccine at 6 weeks and again 2 weeks before expected date of calving, others were left unvaccinated as control. Two calves born from vaccinated cows were given colostrum and milk from their own mothers. A calf born from vaccinated cow was not given colostrum, but milk from other vaccinated cow at day 8 . Three day old calves receiving colostrum of vaccinated cows were challenged with 2 ml either ETEC K99 or F41 suspension containing 108 colony forming units per ml did not show clinical signs of diarrhoea and their body weight increased progressively. Whereas, a calf born from unvaccinated group was challenged with ETEC K99 developed clinical sign of diarrhoea at 15 hours later and died at 8 days post-inoculation . A calf born from unvaccinated cow was challenged with ETEC F41 developed watery diarrhoea, it did not die, but its body weight relatively did not increase. The use of two doses ofpolyvalent ETEC vaccine at late gestation gave protection to the suckling offspring against challenged . Under farm conditions, dams vaccination with 2 doses of polyvalent ETEC vaccine 6 week and 2 weeks before expected date of calving reduced the calf mortality from average of 13% per months to 0.7%. It was concluded that dams vaccination with polyvalent ETEC containing K99 and F41 fimbrial antigens gave protection to their suckling offsprings or through consuming their colostrum or milk against homologous ETEC infection. Â Keywords: Calf, colibacillosis, ETEC, dams vaccination

Development of fowl cholera vaccine: I. Protection of Pasteurella multocida local isolate vaccine against challenge of homologous and heterologous strains. ., Supar; Setiadi, Yudi; ., Djaenuri; Kurniasih, Nina; Poerwadhikarta, Bhakti; ., Sjafei
Indonesian Journal of Animal and Veterinary Sciences Vol 6, No 1 (2001)
Publisher : Indonesian Animal Sciences Society

Pasteurella multocida locally isolated from chicken and ducks (BCC 299, BCC 2331, DY1, DY2, 12TG, 15TG) andimported strains (BCC 1359, 1362; HEDDLESTON group 1 and 6 respectively) had been tested for its pathogenicity in theprevious study. The aims of this experiment were to study the preparation of local isolate pasteurellosis vaccines and to determine the protective effect of that vaccines in chicken against the highly pathogenic local isolates of P. multocida. Killed monovalent, bivalent and polyvalent pasteurellosis vaccines were prepared and each was adjunvanted with aluminum hydroxide gel at a final concentration of 1.5% and the cell concentration was equal to the No 10 of MacFarland tube standard. Each of the vaccine prepared was used to vaccinated on a group of six week old of layer chicken (8 per group). Each chicken was subcutaneously injected with 0.2 ml of vaccine, four weeks later each was boostered with similar vaccine with the same dose. Two weeks after giving the boostered vaccine each group of chicken were challenged, half with life bacterium of P. Multocida BCC 2331 and other with DY2. Any chick which survive after challenge was designated as protected by vaccination. Before vaccination 1 ml of blood was drawn from each of chicken and then two weeks apart up to challenge. Serum from each sample was separated and kept in deep freeze until tested by enzyme-linked immunosorbent assay (ELISA). Chicken vaccinated with killed whole cell P. multocida vaccines of monovalent (BCC 2331 or DY2) and bivalent (BCC 2331 + DY2) were protected against challenge with live bacterium of either BCC 2331 or DY2 at rate 67-100%. There was no protection in chicken vaccinated with either BCC 299, DY1, 12TG, 15TG, BCC 1359, or 1362 killed vaccine. Similarly no protection of chicken vaccinated with either DY1 + BCC299, 12TG + 15TG or BCC 1359 + BCC 1362 bivalent vaccines. The protection rate of the polyvalent local isolate vaccine was at average 50-75%. All vaccinated chicken showed the presence of antibody responses againsted the extract cell and whole cell antigens of either P. multocida BCC 2331 or DY2 local isolate as detected by ELISA. The antibody responses from vaccinated chicken against extra cellular antigens prepared from broth cultures of BCC 2331 and DY2 were detected only from vaccinated chicken with vaccine containing killed antigen of BCC 2331 and/or DY2 isolate. It is likely, the local isolate of P. multocida BCC 2331 and DY2 would be benefit for producing inactive fowl cholera vaccine use in Indonesia, but the protective antigen that enhances immune protection should be determined by means of immunoblotting techniques. Â Key words: Pasteurella multocida, fowl cholera, vaccine, protection

The development of fowl cholera vaccine: II. Pathogenicity and vaccine protection of Pasteurella multocida local isolates in experimental ducks ., Supar; Setiadi, Yudi; ., Djaenuri; Kurniasih, Nina; Poerwadhikarta, B; ., Sjafei
Indonesian Journal of Animal and Veterinary Sciences Vol 6, No 2 (2001)
Publisher : Indonesian Animal Sciences Society

Pasteurellosis or fowl cholera in ducks occurs sporadically along the year in many high duck population areas of Java and other parts of Indonesia. Some isolates of Pasteurella multocida from ducks and chicken are kept at the BALITVET culture collection. The aims of this research were to evaluate the pathogenicity of local isolates and imported strains of P. multocida and to study the pasteurellosis local isolate vaccine and protection assay in ducks. Two imported strains of P. multocida (BCC 1359, BCC 1362) and 6 local isolates (BCC 299, BCC 2331, DY1, DY2, 12TG, 15TG) were used in this study. In the pathogenicity assays the imported strains and local isolates were activated in mice and in duct and then in brain hearth infusion broth containing 5% normal sheep serum. Each of broth culture was diluted, each dilution (102 and 104) of strains or isolates was injected intraperitoneally into a group of normal ducks. Antigen for vaccine, each was produced in sheep blood (5%) agar. Cells were harvested and killed with 0.1% formalin. Monovalent, bivalent, and polyvalent vaccines were prepared, at concentration equal to the Macfarland standard tube No 10, and each was adjuvanted with alhydrogel at final concentration of 1.5%. Each vaccine type was injected into a group of 10 week old ducks (8 animals per group), with 0.2 ml/injection. Four weeks later each animal in group were boostered with the same vaccine, dose, route as the previous injection. Before vaccination each animal was bleed through wing vena, then every two weeks, serum was collected and stored at -20oC. Two weeks after boostered, three days after the last blood sample collection, half animal of each group were challenged intraperitonelly with the BCC 2331 and half with DY2 live broth culture. The pathogenicity assays showed the only BCC 2331 and DY2 killed the experimental ducks, the other did not. The animals vaccinated with either BCC 2331, Â DY2 or bivalent (BCC 2331+DY2) vaccines were protected with either life bacterial challenged of either BCC 2331 or DY2 local isolates. It is likely, P. multocida BCC 2331 and DY2 isolates can be used for pasteurellosis candidate vaccine used in Indonesia, but it still needs more studies in the immunological of protective antigens. Â Key words: Pasteurella multocida, fowl cholera, ducks, vaccine, protection

Genomic DNA restriction endonuclease from Pasteurella multocida isolated from Indonesia, katha strain and reference strains and analysed by PFGE ., Supar
Indonesian Journal of Animal and Veterinary Sciences Vol 8, No 3 (2003)
Publisher : Indonesian Animal Sciences Society

Pasteurella multocida strains are the causative disease agents of wide range of domestic and wild animals in Indonesia. The most important serotypes are associated with Hemorrhagic septicaemic (HS) diseases in cattle and buffaloes, cholera in ducks and chickens. The HS disease associated with P. multocia in large ruminants in Indonesia is controled by killed whole cell vaccines produced by the use of P. multocida Katha strains. There is no discriminatory data of the molecular biology technique has been applied to investigate P. multocida isolates from different geographic locations in Indonesia. The purpose of this studies were to observe the genetic diversity among P. multocida isolated from various geograpic locations and compared with Katha vaccine strain and other reference strains. A total samples of 38 isolates and strains of P. multocida were analysed by means of pulsed-field gel electrophoresis (PFGE). Each sample was grown in nutrient broth, cells were separeted by centrifugation. Whole cell pellet was mixed with agarose and then prepared agarose plugs. The genomic DNA of each sample was digested in situ (plug) with either restriction endonuclease of ApaI and/or BamHI. The digested genomic DNA of each sample was analysed by PFGE, the genomic DNA restricted profile of each sample was compared with others. The use of ApaI restriction endonuclease digestion and analysed by PFGE, demonstrated that 34 out of 38 P. multocia samples could be differentiated into 16 ApaI types, whereas based on the BamHI digestion of these samples were differentiated into 20 BamHI types. Genomic DNA restriction pattern of Indonesian P. multocida isolates originated from cattle and buffaloes associated with haemorrhagic septicaemic diseases demonstrated different pattern to those of vaccine Katha strain, poultry strains as well as the reference strains currenly kept at Balitvet Culture Collection (BCC) unit. Two P. multocida isolates derived from ducks with cholera disease showed similar pattern support the previous findings which exhibited similar pathogecity and vaccine protection. The majority of HS causing P. multocida isolates from some provinces in Indonesia belong to ApaI type 1 (7 isolates), six of these isolates belong to BamHI type 1. The BamHI restriction endonuclease digestion demonstrated higher discriminatory than that of ApaI. These restrection endonucleases digestion combined with PFGE analysis were effective for molecullar defferentiation of P. multocida strains and could be applied to other bacterial veterinary pathogens as well as for local isolate vaccine sellection. Â Key words: Pasteurella multocida, restriction endonuclease analysis, genomic DNA, PFGE

Development of alpha hemolytic verotoxigenic Escherichia coli vaccine: Verotoxic antibody responses in experimental animals, mice, rabbits and dairy cows ., Supar; Peorwadikarta, B; Kurniasih, Nina; ., Djaenuri
Indonesian Journal of Animal and Veterinary Sciences Vol 4, No 1 (1999)
Publisher : Indonesian Animal Sciences Society

Escherichia coli isolates showing alpha hemolytic on blood agar were found in calves with bloody diarrhoea from Bandung, Sukabumi and Bogor. Three E. coli isolates designated as (B34c, B909, B910) were pathogenic for mice and selected for vaccine candidates. Crude supernatant of tryptic soy broth (TSB) cultures precipitated with saturated ammoniumsulphate caused cytopathic effect on vero cell line. For vaccine preparation the isolates were grown on tryptic soy agar (TSA). Whole cell inactive vaccine containing equal proportion of each isolate was prepared and emulsified in aluminum hydroxide gel at a final concentration of 1.5% and cell concentration equal to the tube number 10 of MacFarland standard. Mice and rabbits were injected subcutaneously of monovalent vaccine with the dose of 0.2 ml and 1 ml respectively. Four weeks later mice or rabbits were boostered with the same dose. Before injection each animal was bleed, subsequently every two weeks period up to 4 weeks after the second injection. The sera were separated and kept at -20oC up to serological assay with an enzyme linked immunoassay (ELISA) was done. Vaccinated mice with VTEC were challenged with homologous isolate did not die, where as 60% of unvaccinated mice died within 2 days post challenged. Pregnant cows were given 2 doses of three valent vaccine 6 weeks and again 2 weeks before expected date of calving. Calves born were given colostrum and milk of the own mother. The three day old calf was challenged with three isolates of verotoxigenic E. coli studied. Calves born from vaccinated cows did not show diarrhoea at post challenged, whereas calf from unvaccinated cow demonstrated bloody diarrhoea at post challenged. From the experimental animals (mice and calves) demonstrated the presence of protection against challenged. The antiverotoxic antibody probably involved in important role of immunoprotection. These were supported by the presence of high titer anti verotoxic antibody in serum of experimental animals (mice and cows), as well as in experimental rabbits, but none in the unvaccinated animals. Â Key words: E. coli, alpha hemolytic, verotoxic, dysentery, vaccine

The pathogenesis of Pasteurella multocida local isolates in mice and chicken ., Supar; Setiadi, Yudi; ., Djaenuri; Kurniasih, Nina; Poerwadhikarta, Bhakti
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 1 (2000)
Publisher : Indonesian Animal Sciences Society

Avian cholera or fowl cholera is a bacterial disease caused by Pasteurella multocida strain of serogroup A, has been recognized as important disease in domestic poultry such as ducks and chicken. P. multocida strains derived from overseas and local isolates are stored as freeze dried and kept at the Research hlstitute for Veterinary Science (BALITVET) culture collection (BCC). Some of those bacteria are still alive and can be used as vaccine candidates. Each strain or isolate was activated in brain heart infusion broth containing foetal calf serum and incubated at 37Â°C then it was identitied by biochemical reactions. Field surveys Were conducted in Central Java and South Kalimantan to observe fowl cholera problems and sample collections for isolation of pathogens. Of the 14 of Pasteurella multocida strains or isolates from BCC, II strains (9 imported 2 local isolates) were still alive. In addition to this 2 isolates trom chicken and duck were viable. Seven out of 9 imported strains killed mice within 3 x 24 hours, similarly for the local isolates (BCC 299, 2331, DYI, DY2, 12TG, 15TG). However, the only BCC 2331 and DY2 isolates were able to kill two week old chicken witIlin 6 x 24 hours post inoculation. From this experiment indicated that the P. multocida local isolates (BCC 2,331 and DY2) are more pathogenic than that of imported strains. Two strains of imported P. multocida BCC 2331, 1362 and 6 local isolates (BCC 299, 2331, DYI, DY2, 12TG and 15TG would be selected for mono- and polyvalent vaccine candidates in the following experiments and the highly patogenic BCC 2331 and DY2 isolates would be used to challenge the vaccinated animals. Â Key words: Pasteurella multocida local isolate, pathogenicity, vaccine against fowl cholera candidate

Studies on piglet diarrhoea associated with enterotoxigenic escherichia coli and its control by vaccination Supar .
Hemera Zoa Vol. 77 No. 2 (1995): Jurnal Hemera Zoa
Publisher : Hemera Zoa

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Piglet neonatal diarrhoea occured in all the piggeries studied in Indonesia. Field studies demonstrated the prevalence of diarrhoea in all piggeries were at range from 13.4% to 43.7% with and average 24.7%. The majority of piglets with diarrhoea were in the first 2 weeks of life. Mortality of piglets were at rates between 12.2% to 31.6% with an average 17.9%. The distribution of piglet mortality preceded by diarrhoea recorded in an intensive study in a piggery G for a seven week period showed a high positive correlation with diarrhoea (r2 = 0.79; P

The application of enterotoxigenic Escherichia coli (ETEC) K99, F41 polyvalent vaccine in pregnant dairy cattle to control neonatal colibacillosis and mortality of calves Supar .; Kusmiyati .; B Poerwadikarta
Jurnal Ilmu Ternak dan Veteriner Vol 3, No 1 (1998)
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Enterotoxigenic Escherichia coli (ETEC) strains possessing either K99, F41 or K99F41 are responsible for causing neonatal diarrhoea and mortality of calves and difficult to control using antimicrobial drugs. A whole cell ETEC vaccine containing fimbrial antigens of polyvalent strains based on field serotypes was produced . The efficacy of ETEC vaccine used to control neonatal colibacillosis of dairy calves was studied in experimental animals and field trials. Five pregnant dairy cow were used for experimental study. Three animals were injected subcutaneously with 5 ml vaccine at 6 weeks and again 2 weeks before expected date of calving, others were left unvaccinated as control. Two calves born from vaccinated cows were given colostrum and milk from their own mothers. A calf born from vaccinated cow was not given colostrum, but milk from other vaccinated cow at day 8 . Three day old calves receiving colostrum of vaccinated cows were challenged with 2 ml either ETEC K99 or F41 suspension containing 108 colony forming units per ml did not show clinical signs of diarrhoea and their body weight increased progressively. Whereas, a calf born from unvaccinated group was challenged with ETEC K99 developed clinical sign of diarrhoea at 15 hours later and died at 8 days post-inoculation . A calf born from unvaccinated cow was challenged with ETEC F41 developed watery diarrhoea, it did not die, but its body weight relatively did not increase. The use of two doses ofpolyvalent ETEC vaccine at late gestation gave protection to the suckling offspring against challenged . Under farm conditions, dams vaccination with 2 doses of polyvalent ETEC vaccine 6 week and 2 weeks before expected date of calving reduced the calf mortality from average of 13% per months to 0.7%. It was concluded that dams vaccination with polyvalent ETEC containing K99 and F41 fimbrial antigens gave protection to their suckling offsprings or through consuming their colostrum or milk against homologous ETEC infection. Keywords: Calf, colibacillosis, ETEC, dams vaccination

The pathogenesis of Pasteurella multocida local isolates in mice and chicken Supar .; Yudi Setiadi; Djaenuri .; Nina Kurniasih; Bhakti Poerwadhikarta
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 1 (2000): MARCH 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Avian cholera or fowl cholera is a bacterial disease caused by Pasteurella multocida strain of serogroup A, has been recognized as important disease in domestic poultry such as ducks and chicken. P. multocida strains derived from overseas and local isolates are stored as freeze dried and kept at the Research hlstitute for Veterinary Science (BALITVET) culture collection (BCC). Some of those bacteria are still alive and can be used as vaccine candidates. Each strain or isolate was activated in brain heart infusion broth containing foetal calf serum and incubated at 37°C then it was identitied by biochemical reactions. Field surveys Were conducted in Central Java and South Kalimantan to observe fowl cholera problems and sample collections for isolation of pathogens. Of the 14 of Pasteurella multocida strains or isolates from BCC, II strains (9 imported 2 local isolates) were still alive. In addition to this 2 isolates trom chicken and duck were viable. Seven out of 9 imported strains killed mice within 3 x 24 hours, similarly for the local isolates (BCC 299, 2331, DYI, DY2, 12TG, 15TG). However, the only BCC 2331 and DY2 isolates were able to kill two week old chicken witIlin 6 x 24 hours post inoculation. From this experiment indicated that the P. multocida local isolates (BCC 2,331 and DY2) are more pathogenic than that of imported strains. Two strains of imported P. multocida BCC 2331, 1362 and 6 local isolates (BCC 299, 2331, DYI, DY2, 12TG and 15TG would be selected for mono- and polyvalent vaccine candidates in the following experiments and the highly patogenic BCC 2331 and DY2 isolates would be used to challenge the vaccinated animals. Key words: Pasteurella multocida local isolate, pathogenicity, vaccine against fowl cholera candidate

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