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R S Tozer - One of the best experts on this subject based on the ideXlab platform.

  • trapping old world screw worm fly Chrysomya bezziana villeneuve diptera calliphoridae in papua new guinea including the coastal border with torres strait
    Australian Journal of Entomology, 2013
    Co-Authors: Philip Spradbery, R S Tozer
    Abstract:

    Trapping of Old World screw-worm fly, Chrysomya bezziana Villeneuve (Calliphoridae), using a single sticky trap baited with swormlure was undertaken at 20 locations along the southern coastline of Papua New Guinea bordering the Torres Strait on two occasions in 1981. The traps were operated for 6.5 and 12.5 days, respectively, with total trap catches of 9 male and 95 female screw-worm fly. The mean daily trap catches were 0.23 and 0.30 C. bezziana flies for the two occasions despite nine of the trap locations failing to detect any screw-worm flies. The results of screw-worm fly trapping and/or sentinel cattle studies in several other locations in Papua New Guinea are also presented for comparison, with mean fly trap catches ranging from 0.04 to 0.23 and egg masses from 0.55 to 1.12 per sentinel per day. Comparisons with similar studies in Malaysia indicate that the C. bezziana populations along the Torres Strait border are relatively high with serious biosecurity implications for Australia.

  • field assessment of synthetic attractants and traps for the old world screw worm fly Chrysomya bezziana
    Veterinary Parasitology, 2012
    Co-Authors: Rudolf Urech, J. P. Spradbery, R S Tozer, P E Green, Geoffrey W Brown, D G Mayer, Tack Y Kan
    Abstract:

    Abstract The performance of newly developed trapping systems for the Old World screw-worm fly, Chrysomya bezziana has been determined in field trials on cattle farms in Malaysia. The efficacy of non-sticky traps and new attractants to trap C. bezziana and non-target flies was compared with the standard sticky trap and Swormlure. The optimal trap was a modified LuciTrap ® with a new attractant mixture, Bezzilure-2. The LuciTrap/Bezzilure-2 caught on average 3.1 times more C. bezziana than the sticky trap with Swormlure ( P C. bezziana against Chrysomya megacephala and Chrysomya rufifacies with factors of 5.9 and 6.4, respectively. The LuciTrap also discriminates with factors of 90 and 3.6 against Hemipyrellia sp. and sarcophagid flesh flies respectively, compared to the sticky trap. The LuciTrap/Bezzilure-2 system is recommended for screwworm fly surveillance as it is more attractive and selective towards C. bezziana and provides flies of better quality for identification than the sticky trap.

  • dispersal of the old world screw worm fly Chrysomya bezziana
    Medical and Veterinary Entomology, 1995
    Co-Authors: J. P. Spradbery, R J Mahon, R Morton, R S Tozer
    Abstract:

    . Dispersal of the Old World screw-worm fly, Chrysomya bezziana Villeneuve, was studied in Papua New Guinea by releasing radio-isotope labelled, laboratory-reared flies and collecting their labelled egg masses from sentinel cattle. A log-linear model was developed to describe recapture rate. Distance was found to dominate the model and was represented by a bilinear (‘broken-stick’) term as log-distance. Further terms in the model such as attractiveness of the site (estimated from the number of non-labelled egg masses), the season of the year and a time trend were statistically significant but of minor importance. From the model, the median distance females dispersed before depositing an egg mass was 10.8 km. The maximum distance from the release site that egg masses were recovered was 100 km. The dispersal ability of C. bezziana is discussed in terms of its impact on the prospects of eradicating this species using SIRM if an outbreak occurred in Australia.

  • the efficacy of insecticides against the screw worm fly Chrysomya bezziana
    Australian Veterinary Journal, 1991
    Co-Authors: J. P. Spradbery, R S Tozer, A A Pound
    Abstract:

    A number of insecticides used for ectoparasite control in the livestock industry were screened for their efficacy against larvae of the screw-worm fly, Chrysomya bezziana, using in vivo and laboratory tests. Proprietary screw-worm fly treatments (after exposure to outdoor conditions for up to 10 days) were also tested against eggs and adults of C bezziana. Three of these were also evaluated on naturally acquired screw-worm infestations. Residual protection was generally of short duration. Among the organophosphorus compounds, the most effective formulations contained relatively high concentrations (3 to 4% al) of coumaphos, 2.5% fenchlorphos or low concentrations (0.05 to 0.5% al) of diazinon, chlorfenvinphos and fenthion methyl. Two chlorinated hydrocarbon insecticides containing 3% lindane and 5% dieldrin were very effective but are now prohibited for use in Australia. Preparations had serious deficiencies when used under field conditions, especially for treating large, deepseated myiases for which systemic insecticides are recommended. A comparison of methods demonstrated that a laboratory test could supersede live animal experimentation, at least for the initial screening of potential insecticides.

R J Mahon - One of the best experts on this subject based on the ideXlab platform.

  • identification of the geographical source of adults of the old world screw worm fly Chrysomya bezziana villeneuve diptera calliphoridae by multivariate analysis of cuticular hydrocarbons
    Comparative Biochemistry and Physiology B, 1998
    Co-Authors: W V Brown, J. P. Spradbery, R Morton, Michael J Lacey, R J Mahon
    Abstract:

    Abstract The cuticular hydrocarbon compositions of male and female Chrysomya bezziana from different locations are qualitatively similar but show quantitative differences. The qualitative similarity and lack of marked quantitative differences over its whole range is consistent with C. bezziana being a single species. This accords with the results of studies on hybridization, morphology, allozymes, and chromosomes. Canonical variate analysis of quantitative differences has enabled grouping of locations by geographical regions. Components insensitive to age and sex have been identified that would permit the location by geographical region of a fly of unknown age and sex. However, greater precision is obtained if the sex of the fly be known.

  • dispersal of the old world screw worm fly Chrysomya bezziana
    Medical and Veterinary Entomology, 1995
    Co-Authors: J. P. Spradbery, R J Mahon, R Morton, R S Tozer
    Abstract:

    . Dispersal of the Old World screw-worm fly, Chrysomya bezziana Villeneuve, was studied in Papua New Guinea by releasing radio-isotope labelled, laboratory-reared flies and collecting their labelled egg masses from sentinel cattle. A log-linear model was developed to describe recapture rate. Distance was found to dominate the model and was represented by a bilinear (‘broken-stick’) term as log-distance. Further terms in the model such as attractiveness of the site (estimated from the number of non-labelled egg masses), the season of the year and a time trend were statistically significant but of minor importance. From the model, the median distance females dispersed before depositing an egg mass was 10.8 km. The maximum distance from the release site that egg masses were recovered was 100 km. The dispersal ability of C. bezziana is discussed in terms of its impact on the prospects of eradicating this species using SIRM if an outbreak occurred in Australia.

  • cytogenetic variation in natural populations of the old world screwworm fly Chrysomya bezziana diptera calliphoridae
    Genome, 1994
    Co-Authors: D G Bedo, J. P. Spradbery, R J Mahon
    Abstract:

    The existence of sibling species in the Old World screwworm fly Chrysomya bezziana would raise serious problems in eradicating this pest if it entered Australia. Cytogenetic variation in C. bezziana was investigated by analyzing pupal trichogen polytene chromosomes. Natural populations of C. bezziana spanning its range from southern Africa to Papua New Guinea were examined as well as hybrids between a New Guinea laboratory strain and natural populations. No evidence of sibling species was found. All populations exhibited the same basic banding pattern as the standard sequence established from a Papua New Guinea strain. Extensive asynapsis of chromosome homologues was found in some hybrid crosses and was therefore measured in all populations and hybrids to detect systematic variation. Asynapsis levels in most hybrids could not be statistically distinguished from those present in the parent populations except for crosses between populations at the ends of the range. This result does not permit asynapsis levels to be used in establishing the origin of introduced flies by estimating their distance from known populations. One inversion polymorphism and six band polymorphisms spread over three chromosomes were analyzed. Populations in each sampled region had characteristic combinations of band polymorphisms. This may offer a diagnostic method for determining the origin of flies accidentally introduced to Australia.

  • genetic variation in the old world screw worm fly Chrysomya bezziana diptera calliphoridae
    Bulletin of Entomological Research, 1991
    Co-Authors: K L Strong, R J Mahon
    Abstract:

    The Old World screw-worm fly, Chrysomya bezziana Villeneuve, occurs in Africa, the Middle East, Malaysia, Indonesia and Papua New Guinea, but it is not found in Australia. Introduction into Australia from any of these source areas would threaten the viability of much of the grazing industry in the northern part of the continent. Proposed control by the sterile insect release method (SIRM) would be compromised by the existence of sibling species within C. bezziana. This study examines the degree of genetic differentiation throughout the extensive range of the fly to assess if the degree of geographic differentiation indicates the existence of sibling species and, allows identification of the source of any introduced flies. Electrophoretic analysis of 23 loci from samples collected in southern Africa, the Middle East, Malaysia, Indonesia, and Papua New Guinea revealed 11 polymorphic loci. Overall, populations show remarkably little divergence given the geographic distribution of sample sites. None of the populations sampled were fixed for alternative electromorphs. There is no evidence from this study for the presence of sibling species within C. bezziana.

Peter Willadsen - One of the best experts on this subject based on the ideXlab platform.

  • biochemical and molecular characterization of serine proteases from larvae of Chrysomya bezziana the old world screwworm fly
    Insect Biochemistry and Molecular Biology, 2001
    Co-Authors: Sri Muharsini, Brian Paul Dalrymple, Tony Vuocolo, S E Hamilton, Peter Willadsen, Gene Wijffels
    Abstract:

    The diversity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an example of concerted evolution of a trypsin gene in Chrysomya bezziana.

  • vaccination against the old world screwworm fly Chrysomya bezziana
    Parasite Immunology, 2000
    Co-Authors: Sutijono Partoutomo, George Riding, Gene Wijffels, Edi Satria, C H Eisemann, Peter Willadsen
    Abstract:

    Chrysomya bezziana is an endemic pest of livestock or a threat to livestock production in large areas of Africa, the Middle East, southern and south-east Asia and Australia. Its control is difficult. The feasibility of vaccinating against this pest has now been explored. In-vitro and in-vivo assays have been established. Using these assays, it has been shown that first instar larvae, third instar peritrophic membrane and cardia are all sources of material able to induce immunological reactions in sheep which lead to significant reductions in larval growth. In-vitro assays following vaccination with peritrophic membrane also show larval mortality. Taken together, these effects lead to an 82% reduction in the weight of recovered larvae in vitro and 45% reduction in vivo. Preliminary evidence suggests that the mechanism of protection may be complex.

  • an investigation of the feasibility of vaccinating against the old world screwworm fly Chrysomya bezziana
    Jurnal Ilmu Ternak dan Veteriner, 2000
    Co-Authors: Peter Willadsen, Sutijono Partoutomo
    Abstract:

    The problem of the Old World Screwworm fly Chrysomya bezziana and the limitations of current methods of control are discussed briefly. Any attempt to investigate the feasibility of vaccinating against the fly, as a novel control technology, demands the establishment of methods for fly culture, the production of sources of antigens and the development of assay techniques suitable for the assessment of vaccination effects. This must be coupled to a strategy for vaccine development. This strategy is described, as a prelude to a series of papers evaluating the feasibility of vaccination in detail. Key words: Chrysomya bezziana , screwworm fly, vaccine

  • vaccination trials in sheep against Chrysomya bezziana larvae using the recombinant peritrophin antigens cb15 cb42 and cb48
    Jurnal Ilmu Ternak dan Veteriner, 2000
    Co-Authors: Sujitno Partoutomo, Gene Wijffels, Tony Voucoloco, Peter Willadsen
    Abstract:

    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. Key words: Chrysomya bezziana , recombinant antigen, peritrophin, vaccination

  • Vaccination trials in sheep against Chrysomya bezziana larvae using the recombinant peritrophin antigens Cb15, Cb42 and Cb48
    Pusat Penelitian dan Pengembangan Peternakan, 2000
    Co-Authors: Sujitno Partoutomo, Gene Wijffels, Tony Voucoloco, Peter Willadsen
    Abstract:

    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

J. P. Spradbery - One of the best experts on this subject based on the ideXlab platform.

  • field assessment of synthetic attractants and traps for the old world screw worm fly Chrysomya bezziana
    Veterinary Parasitology, 2012
    Co-Authors: Rudolf Urech, J. P. Spradbery, R S Tozer, P E Green, Geoffrey W Brown, D G Mayer, Tack Y Kan
    Abstract:

    Abstract The performance of newly developed trapping systems for the Old World screw-worm fly, Chrysomya bezziana has been determined in field trials on cattle farms in Malaysia. The efficacy of non-sticky traps and new attractants to trap C. bezziana and non-target flies was compared with the standard sticky trap and Swormlure. The optimal trap was a modified LuciTrap ® with a new attractant mixture, Bezzilure-2. The LuciTrap/Bezzilure-2 caught on average 3.1 times more C. bezziana than the sticky trap with Swormlure ( P C. bezziana against Chrysomya megacephala and Chrysomya rufifacies with factors of 5.9 and 6.4, respectively. The LuciTrap also discriminates with factors of 90 and 3.6 against Hemipyrellia sp. and sarcophagid flesh flies respectively, compared to the sticky trap. The LuciTrap/Bezzilure-2 system is recommended for screwworm fly surveillance as it is more attractive and selective towards C. bezziana and provides flies of better quality for identification than the sticky trap.

  • identification of the geographical source of adults of the old world screw worm fly Chrysomya bezziana villeneuve diptera calliphoridae by multivariate analysis of cuticular hydrocarbons
    Comparative Biochemistry and Physiology B, 1998
    Co-Authors: W V Brown, J. P. Spradbery, R Morton, Michael J Lacey, R J Mahon
    Abstract:

    Abstract The cuticular hydrocarbon compositions of male and female Chrysomya bezziana from different locations are qualitatively similar but show quantitative differences. The qualitative similarity and lack of marked quantitative differences over its whole range is consistent with C. bezziana being a single species. This accords with the results of studies on hybridization, morphology, allozymes, and chromosomes. Canonical variate analysis of quantitative differences has enabled grouping of locations by geographical regions. Components insensitive to age and sex have been identified that would permit the location by geographical region of a fly of unknown age and sex. However, greater precision is obtained if the sex of the fly be known.

  • dispersal of the old world screw worm fly Chrysomya bezziana
    Medical and Veterinary Entomology, 1995
    Co-Authors: J. P. Spradbery, R J Mahon, R Morton, R S Tozer
    Abstract:

    . Dispersal of the Old World screw-worm fly, Chrysomya bezziana Villeneuve, was studied in Papua New Guinea by releasing radio-isotope labelled, laboratory-reared flies and collecting their labelled egg masses from sentinel cattle. A log-linear model was developed to describe recapture rate. Distance was found to dominate the model and was represented by a bilinear (‘broken-stick’) term as log-distance. Further terms in the model such as attractiveness of the site (estimated from the number of non-labelled egg masses), the season of the year and a time trend were statistically significant but of minor importance. From the model, the median distance females dispersed before depositing an egg mass was 10.8 km. The maximum distance from the release site that egg masses were recovered was 100 km. The dispersal ability of C. bezziana is discussed in terms of its impact on the prospects of eradicating this species using SIRM if an outbreak occurred in Australia.

  • cytogenetic variation in natural populations of the old world screwworm fly Chrysomya bezziana diptera calliphoridae
    Genome, 1994
    Co-Authors: D G Bedo, J. P. Spradbery, R J Mahon
    Abstract:

    The existence of sibling species in the Old World screwworm fly Chrysomya bezziana would raise serious problems in eradicating this pest if it entered Australia. Cytogenetic variation in C. bezziana was investigated by analyzing pupal trichogen polytene chromosomes. Natural populations of C. bezziana spanning its range from southern Africa to Papua New Guinea were examined as well as hybrids between a New Guinea laboratory strain and natural populations. No evidence of sibling species was found. All populations exhibited the same basic banding pattern as the standard sequence established from a Papua New Guinea strain. Extensive asynapsis of chromosome homologues was found in some hybrid crosses and was therefore measured in all populations and hybrids to detect systematic variation. Asynapsis levels in most hybrids could not be statistically distinguished from those present in the parent populations except for crosses between populations at the ends of the range. This result does not permit asynapsis levels to be used in establishing the origin of introduced flies by estimating their distance from known populations. One inversion polymorphism and six band polymorphisms spread over three chromosomes were analyzed. Populations in each sampled region had characteristic combinations of band polymorphisms. This may offer a diagnostic method for determining the origin of flies accidentally introduced to Australia.

  • the efficacy of insecticides against the screw worm fly Chrysomya bezziana
    Australian Veterinary Journal, 1991
    Co-Authors: J. P. Spradbery, R S Tozer, A A Pound
    Abstract:

    A number of insecticides used for ectoparasite control in the livestock industry were screened for their efficacy against larvae of the screw-worm fly, Chrysomya bezziana, using in vivo and laboratory tests. Proprietary screw-worm fly treatments (after exposure to outdoor conditions for up to 10 days) were also tested against eggs and adults of C bezziana. Three of these were also evaluated on naturally acquired screw-worm infestations. Residual protection was generally of short duration. Among the organophosphorus compounds, the most effective formulations contained relatively high concentrations (3 to 4% al) of coumaphos, 2.5% fenchlorphos or low concentrations (0.05 to 0.5% al) of diazinon, chlorfenvinphos and fenthion methyl. Two chlorinated hydrocarbon insecticides containing 3% lindane and 5% dieldrin were very effective but are now prohibited for use in Australia. Preparations had serious deficiencies when used under field conditions, especially for treating large, deepseated myiases for which systemic insecticides are recommended. A comparison of methods demonstrated that a laboratory test could supersede live animal experimentation, at least for the initial screening of potential insecticides.

Gene Wijffels - One of the best experts on this subject based on the ideXlab platform.

  • biochemical and molecular characterization of serine proteases from larvae of Chrysomya bezziana the old world screwworm fly
    Insect Biochemistry and Molecular Biology, 2001
    Co-Authors: Sri Muharsini, Brian Paul Dalrymple, Tony Vuocolo, S E Hamilton, Peter Willadsen, Gene Wijffels
    Abstract:

    The diversity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an example of concerted evolution of a trypsin gene in Chrysomya bezziana.

  • vaccination against the old world screwworm fly Chrysomya bezziana
    Parasite Immunology, 2000
    Co-Authors: Sutijono Partoutomo, George Riding, Gene Wijffels, Edi Satria, C H Eisemann, Peter Willadsen
    Abstract:

    Chrysomya bezziana is an endemic pest of livestock or a threat to livestock production in large areas of Africa, the Middle East, southern and south-east Asia and Australia. Its control is difficult. The feasibility of vaccinating against this pest has now been explored. In-vitro and in-vivo assays have been established. Using these assays, it has been shown that first instar larvae, third instar peritrophic membrane and cardia are all sources of material able to induce immunological reactions in sheep which lead to significant reductions in larval growth. In-vitro assays following vaccination with peritrophic membrane also show larval mortality. Taken together, these effects lead to an 82% reduction in the weight of recovered larvae in vitro and 45% reduction in vivo. Preliminary evidence suggests that the mechanism of protection may be complex.

  • fractionation identification and vaccination efficacy of native antigens from the screwworm fly Chrysomya bezziana
    Jurnal Ilmu Ternak dan Veteriner, 2000
    Co-Authors: George Riding, Sri Muharsini, Roger Pearson, Edy Satria, Gene Wijffels, Petter Willadseni
    Abstract:

    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  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  roteins as recombinants in a form suitable for vaccination studies. Key words: Chrysomya bezziana, peritrophic membrane, vaccination, amino acid sequence, serine protease

  • purification of recombinant peritrophic membrane proteins of the old world screwworm fly Chrysomya bezziana
    Jurnal Ilmu Ternak dan Veteriner, 2000
    Co-Authors: Roger D Pearson, Sri Muharsini, Gene Wijffels, Tony Voucoloco
    Abstract:

    To evaluate the feasibility of vaccinating sheep against the Old World Screwworm fly Chrysomya bezziana several recombinant peritrophin proteins were expressed in either a denatured form in Escherichia coli or a native-like form in Pichia pastoris cultures. Purification of the hexaHis tagged proteins was achieved by immobilized metal affinity chromatography. Proteins purified under reducing conditions were refolded using a glutathione shuffle procedure. Purification of a glutathione-Stransferase fusion protein was attempted using glutathione affinity chromatography in conjunction with anion exchange chromatography. The authenticity of the expressed proteins was verified by amino terminal amino acid sequencing. Carbohydrate analysis using biotinylated lectins revealed that Cb-peritrophin-48 expressed in Pichia pastoris was glycosylated with high mannose-type sugars. Four of the purified recombinant proteins were used to evaluate their protective immunogenicity in sheep against Chrysomya bezziana strike. Key words: Screwworm fly, Chrysomya bezziana, recombinant proteins, immobilized metal affinity chromatography

  • vaccination trials in sheep against Chrysomya bezziana larvae using the recombinant peritrophin antigens cb15 cb42 and cb48
    Jurnal Ilmu Ternak dan Veteriner, 2000
    Co-Authors: Sujitno Partoutomo, Gene Wijffels, Tony Voucoloco, Peter Willadsen
    Abstract:

    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. Key words: Chrysomya bezziana , recombinant antigen, peritrophin, vaccination