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The isolation of Gurnbiro virus from larvae and darkling Ivelles (Carcinops pumilin) Parede, Lies; Indriani, Risa; ., Sukarsih
Indonesian Journal of Animal and Veterinary Sciences Vol 2, No 1 (1996)
Publisher : Indonesian Animal Sciences Society

Gumboro (infectious bursal disease, IBD) virus was isolated from darkling beetles (Carrinaps pumilin) and their larvae in a commercial pulletchicken farm with repeated outbreaks incidence of Gumboro disease in Tangertng, West Java. In addition, these over populated beetles and their larvae were suspected to be infected and then shed the virus or acted as vectors. Isolation was done by repeated passages of virus using chicken embryo fibroblast cells as prime media, which then showed the evidence of cylop: ihic effecis. The isolation was followed by antigen detection by means of ELISA test. Â Key words: Gumboro disease, infectious bursal disease, darkling beetle, Carcinops punulin Â

Identification of volatile compounds from myiasis wounds and its responsesfor Chrysomya bezziana Wardhana, April H; ., Sukarsih; Urech, R
Indonesian Journal of Animal and Veterinary Sciences Vol 10, No 1 (2005)
Publisher : Indonesian Animal Sciences Society

Development of attractant for screwworm fly was required in myiasis control on livestock. The purpose of this study is to identify of volatile compounds from myiasis wound infested with Chrysomya bezziana larvae including to assess their responses in both cage and room assays. Both Friesian-Holstein heifer (FH) (animal 1) and Bali cattle (animal 2) were used as myiasis model. The artificial wounds (8-10 cm) were conducted on the rump of both animals and infested with about 200 eggs of C.bezziana. Odours from the infested wound were collected on day 1 and 3 for animal1 and day 3 and 5 for animal 2, post C. bezziana larvae infestation. Two different collection devices were used: firstly, absorption onto Tenax kept in steel tubes, whichwas attached to a collected bowl. The volatile organic compounds were collected from the wound and the surrounding animal hide by flowins the air through the inlet and outlet. Secondly, a solid phase micro extraction (SPME) device was inserted into bowl with passive (no air flow) odour collection. Gass chromatography/mass spectrometry was used to identify volatile compounds from wound. The compounds of the wound on animal 1, collected on day 1, produced only minor quantities of compounds (nonanal, decanal, hexanal and heptanal). Minor components such as DMDS and DMTS were only detected on day 3. The compounds of the wound on animal 2 was more varied and had a peculiar strike-like smell on day 3 and 5. They included indole, phenol, acetone, various sulfides (DMS, DMDS, DMTS), alcohols (butanol, 3-methylbutanol), aldehydes and acids. These compounds were selected and formulated into attractant (B92) then tested in both cage and room assays using SL-2 as control. Respond of flies was analyzed by ANOVA 5% (cage assay) and T test 5% (room assay). The result showed that the fly response to B92 was very low compared to SL-2 in cage assays (P<0.05). The addition of B92 to SL-2 could not increase the catch of flies in the cage assays (SL-2+B92=10:1; 10:3), there was no difference between SL-2 and B92/SL-2 in room assay, the fly response still low (P>0.05). Â Â Key Words: Volatile Compound, Myiasis, Wound, Chrysomya bezziana

Technique development of attractant test for Chrysomya bezziana in laboratory and semi-field conditions Wardhana, April H; ., Sukarsih
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 1 (2004)
Publisher : Indonesian Animal Sciences Society

Swormlure (SL-2), synthetic attractant for the New World Screwworm Fly (NWSF), Cochliomya hominivorax, have been developed and used in the America. The effectiveness of swormlure in attracting the Old World Screwworm Fly (OWSF), Chrysomya bezziana is not well defined. The aim of the study was to provide suitable condition of the attractant in trapping the higher number at the OWSF in laboratory (cage assay) and semi-field (room assay) conditions. The cage assay to screen responses olfactory stimuli of OWSF was developed to asses the fly responses to lights, exhaust fan (on or off), the fliesâ physiological status and whether there was any bias between cages or trap positions. Modifications were made to provide suitable physical and environmental conditions for candidate attractant. These included darkening all windows with paper, the construction of support for the fly cages and installation of additional lights centred above the fly cages. The room assay was used as an intermediate step between the cage assay and the field experiment. The number of entered flies into the trap indicated flies respond to SL-2. The data of cage assay was analysed by ANOVA and data of room assay was analysed by T test (5%). The results showed that standard experimental conditions for the cage assay: two lights above the cages on and the central lights off, covering fluorescent lights with oil paper, the jar trap positions on the centre line parallel to the lights and exhaust fan was turned off (no air flow) during the session but was turned on in between sessions to reduce the odour from SL-2 in laboratory (p>0.05). The standard experimental conditions for the room assay used four fluorescents tubes, exhaust fan turned off during the replicates but turned on after replicated 3 and 6 for 15 minutes. Yellow half-size sticky was used as standard target (p>0.05). Â Key words: Swormlure, SL-2, attractant, Chrysomya bezziana

The development of an âin vivo assay techniqueâ as a tool for measuring protective immune responses of vaccine against myiasis in sheep Partoutomo, S.; ., Sukarsih; Satria, E.; Eisemann, C.H.
Indonesian Journal of Animal and Veterinary Sciences Vol 3, No 4 (1998)
Publisher : Indonesian Animal Sciences Society

An âin vivo assay techniqueâ is urgently needed for measuring protective immune effects of a myiasis vaccine in sheep. Such a technique is being developed simultaneously with the development of a vaccine against myiasis caused by the screwworm fly Chrysomya bezziana under a collaborative project undertaken by Balitvet, ITB and CSIRO (Australia) and funded by ACIAR. Experiments were conducted in naive sheep. C. bezziana larvae were allowed to develop on abraded skin in aluminium rings which had been attached to the sheep by means of a glue (Aibon) on the day prior to infection. Rings were arranged on clipped areas close to the mid line of the sheepâs back, two rings on the right side and two rings on the left. Four trials were performed, involving studies on the effects of including wet sponges in the rings to maintain humidity (Trial 1); the effects of sponge and blended meat as counting and transferring media during infection (Trial 2); the effects of the repellants citronella, eucalyptus oil and neem extract in assisting the recovery of larvae (Trial 3); and the effects of the reducing the infective dose from 50 to 25 1st instar larvae/ring and using a fine brush for counting and transferring larvae instead of using a forceps as in the previous groups (Trial 4) on the larval recovery rates (LRR). The results indicated that the inclusion of wet sponges in the rings, the use of sponge and blended meat as counting and transferring media during infection, and the application of repellants all increased the LRR to some extent; however, variations among individual rings remained high. On the other hand, the reduction of infective dose of larvae from 50 to 25 1st instar larvae/ring and using a fine brush for counting and transferring larvae sharply increased the LRR while substantially decreasing the coefficient variations. Key words : Myiasis, Chrysomya bezziana, larval recovery rate

Development of myiasis vaccine: In vitro detection of immunoprotective responses of peritrophic membrane protein, first instar larva Ll supernatant and pellet antigen of fly Chrysomyia bezziana in sheep ., Sukarsih; Partoutomo, S; Satria, E; Eisemann, C.H; Willadsen, P
Indonesian Journal of Animal and Veterinary Sciences Vol 4, No 3 (1999)
Publisher : Indonesian Animal Sciences Society

Myiasis control by means of individual treatment of animals which are mainly rised extensively is time consumed and expensive. The alternative way to control this disease by vaccination is considered effective and economically accepted. However the expected vaccine is now still being developed under a collaborative project between CSIRO, Inter-University Centre on Biotechnology-ITB and Research Institute for Veterinary Science and funded by ACIAR. There are several antigens have been identified as vaccine candidates and an in vitro bioassay technique has been developed for assessing the immunoresponses of vaccine in sheep. Three antigens were used for vaccines in this study, these included protein peritrophic membrane (PM), soluble extract (SE) and pellet extract (PE) of 1st instar larvae of Chrysomya bezziana. Twenty four experimental sheep were divided into 4 groups of 6 animals, 3 groups of animals were injected with PM, SE and PE vaccines with the dose rate of 0.5 g PM/head, 0.8 g PE/head and 4.2 ml LE/head respectively, and the other one group was injected with 4 ml PBS/head as a control group. Vaccination with the same dose was repeated 4 weeks after the 1st vaccination as a booster, and 2 weeks after the booster the sheep were challenged with live larvae, 3 days after challenge animals were killed. Sera were collected at the day of vaccination, 4 weeks after vaccination, 2 weeks after booster, and 3 days after challenge. An in vitro bioassay technique was conducted by culturing 1st instar larvae on five media containing sera collected from each experimental animal. The effects of sera on cultivated larvae were assessed by means of larval weight and larval mortality rate. The results indicated that the growth rate and survival of cultivated larvae in media containing anti-PM sera were significantly lower (P<0.01) compared to the larvae cultivated on media with sera on the day of vaccination. The larval weight depression by anti- PM sera collected at 3 days after challenge was 65% of that larvae cultivated on media with sera collected on the day of vaccination. Anti-PM sera depressed the growth rate and survival of larvae significantly greater (P<0.05) than that of anti-PE or anti-LE sera. It is concluded that PM has the best immunoresponses and as the candidate of choice for myiasis vaccine. Â Key words : In vitro bioassay, myiasis, immunoresponses, Chrysomya bezziana

Establishment and maintenance of a colony of the Old World Screwworm fly Chrysomya bezziana at Balitvet in Bogor, West Java, Indonesia ., Sukarsih; Partoutomo, Sujitno; Tozer, Robert; Satria, Edy; Wijeffels, Gene; Riding, George
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 3 (2000)
Publisher : Indonesian Animal Sciences Society

A colony of the Old World Screwworm fly, Chrysomya bezziana, has been established and maintained at Balai Penelitian Veteriner, Bogor, West Java, Indonesia since 1994. Culture on a convenient, defined diet able to sustain long term reproducibility of insect performance is described. The long-term viability of the colony has been further assured with an effective and reliable culturing procedure, with the genetic diversity and robustness of the colony being maintained through regular infusions of wild-type field isolates. The colony has been a reliable source of larval material used for the isolation and identification of protective antigens in the ACIAR funded Screwworm Fly Vaccination Project. Larvae have been used in the development and routine application of assays employed to evaluate vaccine efficacy. The colony continues to be a valuable resource for both screwworm fly vaccine and other new research includi the evaluation of screwworm fly attractants and lures. Â Key words: Chrysomya bezziana, culture, colony, larvae, vaccination

Fractionation, identification and vaccination efficacy of native antigens from the screwworm fly, Chrysomya bezziana Riding, George; Muharsini, Sri; Pearson, Roger; ., Sukarsih; Satria, Edy; Wijffels, Gene; Willadseni, Petter
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 3 (2000)
Publisher : Indonesian Animal Sciences Society

sources of potential protective antigens from the sheep blowfly Lucilia cuprina. Their importance in the screwworm fly Chrysomya bezziana has now been investigated. Purified serine proteases from Chrysomya bezziana were tested for their potential as vaccine antigens in sheep, efficacy being assessed by in vitro and in vivo assays with larval Chrysomya bezziana. No effect of vaccination was observed by the in vitro assay. However, in the in vivo challenge, larval weights were diminished in the vaccinated sheep, although larval recoveries increased marginally. Vaccination with Chrysomya bezziana peritrophic membrane does induce an effective immune response against the parasite resulting in a significant reduction in larval growth and considerable larval mortality in the in vitro assay. Sequential fractionation of the peritrophic membrane with various surfactants and chaotrophic agents of increasing solubilisation capacity resulted in the separation of discrete groups of proteins. The groups Â of fractionated proteins were tested in a vaccination trial in sheep with vaccine efficacy assessed by in vitro assays. The urea extract, guanidine-HCl extract and SDS soluble fraction each induced significant levels of protection against Chrysomya bezziana larvae but the effects were poorer than those obtained from vaccination with whole, native peritrophic membrane. Several major proteins selected from the three most protective fractions were purified by SDS polyacrylamide gel electrophoresis. Since insufficient quantities of these proteins were available for vaccination trials, they were either sequenced directly from the N-terminus or subjected to endoproteinase Lys-C digestion, followed by peptide purification and amino acid sequencing. This gave the information necessary for the expression of several of these Â roteins as recombinants in a form suitable for vaccination studies. Â Key words: Chrysomya bezziana, peritrophic membrane, vaccination, amino acid sequence, serine protease

Vaccination trials in sheep against Chrysomya bezziana larvae using the recombinant peritrophin antigens Cb15, Cb42 and Cb48 ., Sukarsih; Partoutomo, Sujitno; Wijffels, Gene; Voucoloco, Tony; Willadsen, Peter
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 3 (2000)
Publisher : Indonesian Animal Sciences Society

Recombinant forms of a number of peritrophic membrane proteins from the screwworm fly Chrysomya bezziana have been assessed in vitro and in vivo for their efficacy as antigens in vaccination against the tissue-invasive, larval form of the parasite. The proteins included Cb15 and Cb42 expressed in Escherichia coli and Cb48 expressed in both Escherichia coli and Pichia pastoris. In all cases, the in vitro assays of larval growth on serum from vaccinated sheep failed to show inhibition of larval weight gain or any detrimental effect on larval survival relative to controls. Chrysomya bezziana Cb48 has a significant degree of sequence identity with the antigen PM48 from Lucilia cuprina. Feeding Lucilia cuprina larvae on antisera to Cb48 induced a small but statistically significant reduction in weight gain, as does feeding on antisera to PM48. In vivo, larvae feeding on sheep vaccinated with Escherichia coli-expressed Cb15 and Cb42 and Pichia pastoris-expressed Cb48 showed marginally greater weight gain and survival which was equal to or greater than that on non-vaccinated sheep. The significance of these observations is discussed. Â Key words: Chrysomya bezziana, recombinant antigen, peritrophin, vaccination

Technique development of attractant test for Chrysomya bezziana in laboratory and semi-field conditions April H Wardhana; Sukarsih .
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 1 (2004): MARCH 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Swormlure (SL-2), synthetic attractant for the New World Screwworm Fly (NWSF), Cochliomya hominivorax, have been developed and used in the America. The effectiveness of swormlure in attracting the Old World Screwworm Fly (OWSF), Chrysomya bezziana is not well defined. The aim of the study was to provide suitable condition of the attractant in trapping the higher number at the OWSF in laboratory (cage assay) and semi-field (room assay) conditions. The cage assay to screen responses olfactory stimuli of OWSF was developed to asses the fly responses to lights, exhaust fan (on or off), the flies’ physiological status and whether there was any bias between cages or trap positions. Modifications were made to provide suitable physical and environmental conditions for candidate attractant. These included darkening all windows with paper, the construction of support for the fly cages and installation of additional lights centred above the fly cages. The room assay was used as an intermediate step between the cage assay and the field experiment. The number of entered flies into the trap indicated flies respond to SL-2. The data of cage assay was analysed by ANOVA and data of room assay was analysed by T test (5%). The results showed that standard experimental conditions for the cage assay: two lights above the cages on and the central lights off, covering fluorescent lights with oil paper, the jar trap positions on the centre line parallel to the lights and exhaust fan was turned off (no air flow) during the session but was turned on in between sessions to reduce the odour from SL-2 in laboratory (p>0.05). The standard experimental conditions for the room assay used four fluorescents tubes, exhaust fan turned off during the replicates but turned on after replicated 3 and 6 for 15 minutes. Yellow half-size sticky was used as standard target (p>0.05). Key words: Swormlure, SL-2, attractant, Chrysomya bezziana

Development of myiasis vaccine: In vitro detection of immunoprotective responses of peritrophic membrane protein, first instar larva Ll supernatant and pellet antigen of fly Chrysomyia bezziana in sheep Sukarsih .; S Partoutomo; E Satria; C.H Eisemann; P Willadsen
Jurnal Ilmu Ternak dan Veteriner Vol 4, No 3 (1999): SEPTEMBER 1999
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Myiasis control by means of individual treatment of animals which are mainly rised extensively is time consumed and expensive. The alternative way to control this disease by vaccination is considered effective and economically accepted. However the expected vaccine is now still being developed under a collaborative project between CSIRO, Inter-University Centre on Biotechnology-ITB and Research Institute for Veterinary Science and funded by ACIAR. There are several antigens have been identified as vaccine candidates and an in vitro bioassay technique has been developed for assessing the immunoresponses of vaccine in sheep. Three antigens were used for vaccines in this study, these included protein peritrophic membrane (PM), soluble extract (SE) and pellet extract (PE) of 1st instar larvae of Chrysomya bezziana. Twenty four experimental sheep were divided into 4 groups of 6 animals, 3 groups of animals were injected with PM, SE and PE vaccines with the dose rate of 0.5 g PM/head, 0.8 g PE/head and 4.2 ml LE/head respectively, and the other one group was injected with 4 ml PBS/head as a control group. Vaccination with the same dose was repeated 4 weeks after the 1st vaccination as a booster, and 2 weeks after the booster the sheep were challenged with live larvae, 3 days after challenge animals were killed. Sera were collected at the day of vaccination, 4 weeks after vaccination, 2 weeks after booster, and 3 days after challenge. An in vitro bioassay technique was conducted by culturing 1st instar larvae on five media containing sera collected from each experimental animal. The effects of sera on cultivated larvae were assessed by means of larval weight and larval mortality rate. The results indicated that the growth rate and survival of cultivated larvae in media containing anti-PM sera were significantly lower (P<0.01) compared to the larvae cultivated on media with sera on the day of vaccination. The larval weight depression by anti- PM sera collected at 3 days after challenge was 65% of that larvae cultivated on media with sera collected on the day of vaccination. Anti-PM sera depressed the growth rate and survival of larvae significantly greater (P<0.05) than that of anti-PE or anti-LE sera. It is concluded that PM has the best immunoresponses and as the candidate of choice for myiasis vaccine. Key words : In vitro bioassay, myiasis, immunoresponses, Chrysomya bezziana

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