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The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several... more
The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several pharmacological agents. One of the drugs, reserpine, binds very tightly to the transporter and remains bound even after solubilization, a finding that has proven useful for purification of the transporter from bovine adrenal medulla in a fully functional state. The sequences of 26 N-terminal amino acids and of an additional 7-amino acid internal peptide are presented. Antibodies against a synthetic peptide based on the above sequences immunoprecipitate the transporter, confirming the conclusion that the peptide sequence is derived from bovine VAT. To our knowledge, documentation of sequences of vesicular neurotransmitter transporters has not been presented previously. In addition, the sequences obtained are highly homologous to the predicted sequence...
Research Interests: Biochemistry, Biology, Medicine, Multidisciplinary, Neuropeptides, and 15 moreSequence alignment, Amines, Animals, Drug Resistance, Peptides, Glycoproteins, Membrane transport proteins, Cattle, Model System, Transporter, Amino Acid Sequence, Adrenal Medulla, Molecular Sequence Data, Adrenal glands, and Chinese hamster ovary cells
Amoebic gill disease can be experimentally induced by the exposure of salmonids to Neoparamoeba spp. freshly isolated from infected fish, while cultured amoebae are non-infective. Results from our previous work suggested that one key... more
Amoebic gill disease can be experimentally induced by the exposure of salmonids to Neoparamoeba spp. freshly isolated from infected fish, while cultured amoebae are non-infective. Results from our previous work suggested that one key difference between infectious and non-infectious Neoparamoeba were the highly glycosylated molecules in the glycocalyx. To characterise these surface glycans or glycoproteins we used a monoclonal antibody (mAb 44C12) specific to a surface molecule unique to infective parasites. This mAb recognised a carbohydrate epitope on a high molecular weight antigen (HMWA) that make up 15-19% of the total protein in a soluble extract of infectious parasites. The HMWA consisted of at least four glycoprotein subunits of molecular weight (MW) greater than 150 kDa that form disulfide-linked complexes of MW greater than 600 kDa. Chemical deglycosylation yielded at least four protein bands of approximate MW 46, 34, 28 and 18 kDA. While a similar HMWA complex was present in non-infective parasites, the glycoprotein subunits were of lower MW and exhibited differences in glycosylation. The four glycoproteins subunits recognised by mAb 44C12 were resistant to degradation by PNGase F, PNGase A, O-glycosidase plus β-1, 4-galactosidase, β-N-acetylglucosaminidase and neuraminidase. The major monosaccharides in the HMWA from infectious parasites were rhamnose, fucose, galactose, and mannose while sialic acids were absent. The carbohydrate portion constituted more than 90% of the total weight of the HMWA from infectious Neoparamoeba spp. Preliminary results indicate that immunisation of salmon with HMWA does not lead to protection against challenge infection; rather it may even have an immunosuppressive effect.
Research Interests: Microbiology, Zoology, Fisheries, Biology, Fish Diseases, and 15 moreMedicine, Animals, Atlantic Salmon, Fisheries Sciences, Glycoproteins, Salmo salar, Veterinary Sciences, Molecular weight, Sialic Acid, Monoclonal Antibody, Fetuin, Amoebozoa, Immunoblotting, High Molecular Weight, and Total protein
Amoebic gill disease (AGD) is a severely debilitating disease, which mainly affects the salmonid industry. It causes high production losses worldwide, including Tasmania, where it is the main health problem in farmed Atlantic salmon.... more
Amoebic gill disease (AGD) is a severely debilitating disease, which mainly affects the salmonid industry. It causes high production losses worldwide, including Tasmania, where it is the main health problem in farmed Atlantic salmon. Without the use of control procedures such as fresh water bathing and/or by maintaining fish population densities at a level commensurate with proper hygiene, this disease can often lead to the death of over 50% of infected salmon. AGD is caused by the parasitic amoeba, Neoparamoeba perurans , which binds to and inhabits the gill epithelium of growing fish. Pathology is associated with severe epithelial hyperplasia, fusion of gill lamellae, lowering of oxygen tension and pH of the blood and eventual death of the fish. In order to understand this disease process, research has been carried out, to study the immune response of fish to primary and secondary infections, the genetic basis of resistance to infection as well as how the parasite binds to the hos...
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SummaryWe have extracted a protein of 14 kDa from purified oocyst walls of several Eimeria species. Polyclonal antibodies were raised in rats against the 14 kDa proteins of E. acervulina and E. tenella. On immunoblots these antisera... more
SummaryWe have extracted a protein of 14 kDa from purified oocyst walls of several Eimeria species. Polyclonal antibodies were raised in rats against the 14 kDa proteins of E. acervulina and E. tenella. On immunoblots these antisera reacted in a highly specific manner with the homologous 14 kDa antigens, but not with heterologous antigens. In addition, specific binding of the two antisera to oocyst wall fragments of E. acervulina and E. tenella was demonstrated by immunofluorescence. Partial amino-terminal sequences comprising 20 amino acid residues were obtained from the 14 kDa oocyst wall proteins of E. acervulina and E. tenella. They are characterized by an abundance of amino acids containing hydroxyl groups in their side chains (serine, tyrosine, threonine). Binding of the oocyst wall protein of E. tenella by peanut agglutinin indicates the presence of O-linked carbohydrates.
Research Interests: Biochemistry, Parasitology, Biology, Medicine, Sequence Analysis, and 15 moreAnimals, Lectins, Glycosylation, Eimeria, Antiserum, Rats, Polyclonal Antibodies, Amino Acid Profile, Veterinary Sciences, Molecular weight, Protozoan Proteins, Amino Acid Sequence, Cell Wall, Molecular Sequence Data, and Eimeria tenella
Infection of breeding hens with Eimeria maxima induces production of parasite-specific antibodies which are transferred, via the egg yolk, to hatchling chicks. These antibodies (immunoglobulin G) are highly protective, mediating up to a... more
Infection of breeding hens with Eimeria maxima induces production of parasite-specific antibodies which are transferred, via the egg yolk, to hatchling chicks. These antibodies (immunoglobulin G) are highly protective, mediating up to a 97% reduction in oocyst excretion in challenged hatchlings. However, the degree of maternally derived immunity transferred by the hens to their offspring declines with increasing time after infection of the hens. This decline in immunity is directly related to declining immunoglobulin G titers. However, sera from highly protected hatchlings recognize only a very few E. maxima proteins on Western blots (immunoblots). In particular, a 230-kDa protein band is outstanding for its association with maternally derived immunity to E. maxima in hatchlings. This band was excised from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) preparative gel of crude merozoite protein extract. The SDS-PAGE cutout was emulsified in Freund's adjuv...
Research Interests: Biology, Virology, Medicine, Biological Sciences, Female, and 15 moreAnimals, Infection, Male, Eimeria, Antibody, Chickens, Standard Error, Coccidiosis, Economic Loss, Protozoan Proteins, Enzyme Linked Immunosorbent Assay, immunoglobulin G, Broiler breeder, Chick embryo, and Medical and Health Sciences
Eimeria maxima gametocytes contain two major antigens with molecular masses of 56 and 82 kilodaltons (kDa) which are recognized by convalescent sera from immune chickens. Preparations enriched in these two antigens were used to immunize... more
Eimeria maxima gametocytes contain two major antigens with molecular masses of 56 and 82 kilodaltons (kDa) which are recognized by convalescent sera from immune chickens. Preparations enriched in these two antigens were used to immunize mice, and several monoclonal antibodies which specifically reacted with the 56-kDa antigen were produced. One of these monoclonal antibodies of the immunoglobulin M subclass, along with immune chicken sera raised against affinity-purified 56- and 82-kDa antigens, was used to passively immunize chicks. On the basis of the parameter of total oocyst output, it was found that these antibodies provided partial protection (40 to 50% inhibition) against E. maxima challenge infections.
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The parasitic protozoan Leishmania mexicana amazonensis has two developmental stages: a motile flagellated promastigote stage and a sessile intracellular amastigote stage. In our previous work, cells of the promastigote stage were found... more
The parasitic protozoan Leishmania mexicana amazonensis has two developmental stages: a motile flagellated promastigote stage and a sessile intracellular amastigote stage. In our previous work, cells of the promastigote stage were found to synthesize more tubulin protein than those of the amastigote stage. Here, tubulin mRNAs in these leishmanias were analyzed. Based on dot blot hybridization between total leishmanial RNA and tubulin-specific cDNA probes derived from chicken brain, amastigotes and promastigotes were found to have approximately equal amounts of alpha- and beta-tubulin mRNAs. RNA blotting of leishmanial RNA, using chicken tubulin cDNA probes, showed that amastigotes and promastigotes both gave a single mRNA species of 2100 nucleotides for alpha-tubulin in roughly similar quantities. However, such analysis for beta-tubulin revealed mainly a single mRNA species of 3600 nucleotides for amastigotes and three species of 2800, 3600, and 4400 nucleotides for promastigotes, t...
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The histidine-rich protein (HRP) of the avian malaria parasite Plasmodium lophurae contains 70% histidine. It is found in dense cytoplasmic granules and during the erythrocytic cycle it accumulates to represent 10% of the dry weight of... more
The histidine-rich protein (HRP) of the avian malaria parasite Plasmodium lophurae contains 70% histidine. It is found in dense cytoplasmic granules and during the erythrocytic cycle it accumulates to represent 10% of the dry weight of the parasite. In the present work the HRP mRNA was studied by in vitro translation and by the use of a polyhistidine oligonucleotide probe. The HRP mRNA contains 2,000-2,100 nucleotides encoding a protein with an apparent molecular weight of 50,000. In addition a HRP of molecular weight 35,000-40,000 is also produced in vitro, probably as a result of proteolytic cleavage of the molecular weight 50,000 polypeptide which corresponds to in vivo labeled and purified HRP. The HRP represents a much larger proportion of the in vitro products synthesized in the homologous cell-free system compared to the rabbit reticulocyte system, and it reflects more closely the pattern of protein synthesis seen in vivo. In addition, HRP mRNA is more abundant in polysomes i...
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Infection of breeding hens with Eimeria maxima induces production of Eimeria-specific IgG antibodies which are transferred to hatchlings via the egg yolk and confer a high degree of maternal immunity against homologous challenge and... more
Infection of breeding hens with Eimeria maxima induces production of Eimeria-specific IgG antibodies which are transferred to hatchlings via the egg yolk and confer a high degree of maternal immunity against homologous challenge and partial immunity to infection with another important species, Eimeria tenella. As an example, in an experiment using hatchlings from eggs collected between days 28 and 39 after infection of the hens with 20 000 sporulated E. maxima oocysts, control chicks (challenged with 100 sporulated oocysts) excreted 6·8±1·2 million (mean±s.e., n = 10) or 5·8±1·2 million (n = 8) oocysts of E. maxima or E. tenella, respectively, compared to 0·9±0·4 million (n = 5) E. maxima oocysts or 2·2±0·4 million (n = 9) E. tenella oocysts excreted by hatchlings of infected hens. This represents an 87% reduction in oocyst excretion with regard to E. maxima and a 62% reduction in oocyst excretion with regard to E. tenella in the progeny of the infected hens. In another experiment, ...
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Transmission-blocking immunity may have great potential for use in the control of diseases caused by apicomplexan parasites. In this review I will describe our work on the application of transmission-blocking immunity to the control of... more
Transmission-blocking immunity may have great potential for use in the control of diseases caused by apicomplexan parasites. In this review I will describe our work on the application of transmission-blocking immunity to the control of the Eimeria parasite and compare our results to those working on transmission-blocking immunity against Cryptosporidium and Plasmodium. Eimeria causes the disease known as coccidiosis in domestic animals. Coccidiosis is particularly problematic in the chicken industry, mainly due to the crowded rearing conditions under which chicks are raised. In our work we identified, isolated and characterized 3 major gametocyte antigens (230 kDa, 82 kDa and 56/54 kDa) of Eimeria maxima. We used these native glycoproteins to immunize laying hens that, via the egg yolk, provide large amounts of transmission-blocking maternal antibodies to offspring chicks. We demonstrated that hatchlings from immunized hens shed 60-80% fewer oocysts (i.e. the infective stage of the life-cycle of Eimeria) than those from control hens. Such a reduction in oocyst output acts to significantly reduce parasite numbers in the litter of chicks raised in floor pens. This reduction in oocyst output is comparable to that seen using the most effective coccidiostat drugs and is probably sufficient to control coccidiosis under field conditions. Based on our results together with those of other groups working on transmission-blocking immunity against Cryptosporidium and Plasmodium, it appears that this immunological approach holds great promise for the control of apicomplexan parasites that cause diseases in both animals and man.
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Eimeria maxima gametocytes were isolated from infected chicken intestinal tissue by treatment with hyaluronidase and subsequent filtration through polymon filters. The isolated gametocytes were analyzed by microscopical and biochemical... more
Eimeria maxima gametocytes were isolated from infected chicken intestinal tissue by treatment with hyaluronidase and subsequent filtration through polymon filters. The isolated gametocytes were analyzed by microscopical and biochemical methods and shown to be highly enriched. The antigenicity of the gametocytes was analyzed in mice, rabbits, and chickens by ELISA and indirect immunofluorescence. Contrary to published results, we have found gametocytes to be highly immunogenic in all animals tested.
Research Interests: Microbiology, RNA, Filtration, Experimental parasitology, Mice, and 15 moreAnimals, Immunization, Eimeria, Isolation, Fluorescent Antibody Technique, POULTRY DISEASES, Chickens, Coccidiosis, Veterinary Sciences, Rabbits, Hour, Bovine Serum Albumin, Enzyme Linked Immunosorbent Assay, Immunoglobulin, and Micrometer
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... be sufficiently nE to pevent the dzmatc incease h oow numkE wen n pout houw hter between ... to develop and, in some cases, birds remained susceptible to infection by all three species for at ... However, as long as the num bers of... more
... be sufficiently nE to pevent the dzmatc incease h oow numkE wen n pout houw hter between ... to develop and, in some cases, birds remained susceptible to infection by all three species for at ... However, as long as the num bers of parasites remained low, there appeared to be no ...
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Infection of chickens with Eimeria maxima induces the production of parasite-specific antisera which can be used passively to protect naive chickens against infection. Globulin fractions of these antisera can also be used passively to... more
Infection of chickens with Eimeria maxima induces the production of parasite-specific antisera which can be used passively to protect naive chickens against infection. Globulin fractions of these antisera can also be used passively to protect chickens. Similarly, intramuscular injection of soybean lectin affinity purified gametocyte antigens of E. maxima in Freund's Complete Adjuvant induces production of antibodies which are maternally transferred and thereby protect hatchlings against E. maxima. ELISA analyses of serum pools having varying protective capacities revealed good correlations between passive protection and levels of anti-unsporulated oocyst, anti-sporulated oocyst, anti-merozoite and anti-gametocyte antibodies. Western blotting demonstrated that the sera mainly recognized a number of high molecular weight antigens in all developmental stages and that the intensity of the reactions reflected the degree of protection induced by the sera. Sera from birds immunized with gametocyte antigens also recognized high molecular weight antigens from all the developmental stages, with banding patterns remarkably similar to those observed for sera from infected birds. Taken together, these results indicate that antibodies can protect against infection with E. maxima and these antibodies may recognize and act against asexual and/or sexual stages of the parasite.
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Amoebic gill disease, the main disease of concern to the salmon industry in Tasmania, is caused by the amoeba, Neoparamoeba spp. Experimental infection can only be induced by exposure to wild-type (WT) parasites isolated from the gills of... more
Amoebic gill disease, the main disease of concern to the salmon industry in Tasmania, is caused by the amoeba, Neoparamoeba spp. Experimental infection can only be induced by exposure to wild-type (WT) parasites isolated from the gills of infected fish, as cultured amoebae are non-infective. To characterize the surface antigens of WT parasites, we produced monoclonal antibodies (mAbs) using subtractive immunization. Mice inoculated with non-infective parasites were treated with cyclophosphamide, to deplete reactive lymphocytes, and then immunized with different antigen preparations from infective parasites. When whole parasites were used for boosting, the percentage of WT unique mAbs was very high (86%) as was the percentage of mAbs specific for carbohydrate epitopes (89%). When deglycosylated membranes were used, the numbers of mAbs specific for non-carbohydrate epitopes did not increase, but the total number of WT unique mAbs was reduced (86-40%). Using an untreated membrane preparation, the total number of mAbs to surface molecules was very high, but all recognized carbohydrate epitopes. The total number of mAbs recognizing carbohydrate epitopes on the surface of the WT parasites was 97%, suggesting that the dominant epitopes on the surface molecules unique to WT parasites are carbohydrate in nature.
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Eimeria maxima gametocytes contain two major antigens with molecular masses of 56 and 82 kilodaltons (kDa) which are recognized by convalescent sera from immune chickens. Preparations enriched in these two antigens were used to immunize... more
Eimeria maxima gametocytes contain two major antigens with molecular masses of 56 and 82 kilodaltons (kDa) which are recognized by convalescent sera from immune chickens. Preparations enriched in these two antigens were used to immunize mice, and several monoclonal antibodies which specifically reacted with the 56-kDa antigen were produced. One of these monoclonal antibodies of the immunoglobulin M subclass, along with immune chicken sera raised against affinity-purified 56- and 82-kDa antigens, was used to passively immunize chicks. On the basis of the parameter of total oocyst output, it was found that these antibodies provided partial protection (40 to 50% inhibition) against E. maxima challenge infections.