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Marshall W Lightowlers

Publications and source records attributed to Marshall W Lightowlers.

18 recordsLinked to original sources
PubMed2006

Taenia solium and Taenia ovis: stage-specific expression of the vaccine antigen genes, TSOL18, TSOL16, and homologues, in oncospheres.

Recombinant antigens that have been cloned from Taenia solium and Taenia ovis have been shown to be highly effective when used as vaccines against cysticercosis in the intermediate hosts. This study investigated the presence of mRNA encoding the TSOL18 and TSOL16 antigens in different life-cycle stages of T. solium, and their related homologues in T. ovis. Reverse transcription-PCR and Southern blotting demonstrated that the antigens are stage-specifically expressed in the oncosphere. The apparent absence of expression of TSOL18 in the metacestode life-cycle stage suggests that the vaccine based on this antigen targets exclusively the early stages in the development of the parasite.

Animals↗
PubMed2006

Echinococcus multilocularis: developmental stage-specific expression of Antigen B 8-kDa-subunits.

Antigen B (AgB) initially found in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa. Four genes encoding these 8-kDa-subunits have been identified from E. granulosus. In this study we isolated five genes encoding 8-kDa-subunits of AgB from Echinococcus multilocularis. Sequence comparison of isolated cDNA clones demonstrated that one of these five clones was completely identical to EmAgB8/1 which had been isolated previously by our group, and three of them were 94.5, 90.8, and 91.9% homologous to E. granulosus antigen B 8-kDa subunit genes, EgAgB8/2, EgAgB8/3, and EgAgB8/4, respectively. The remaining clone shared 51-58% homology with the nucleotide sequences of AgB genes. Gene-specific RT-PCR and Western blot analyses revealed that these genes were expressed in a developmentally regulated manner in E. multilocularis vesicles, protoscoleces, and immature adult worms. Possible functions of different expression manners are also discussed.

Amino Acid Sequence↗
PubMed2006

In vitro oncosphere-killing assays to determine immunity to the larvae of Taenia pisiformis, Taenia ovis, Taenia saginata, and Taenia solium.

Taeniid cestodes infect humans and livestock, causing considerable morbidity and mortality, as well as economic loss. Substantial progress has been made toward the production of recombinant vaccines against cysticercosis in livestock animals. Further development of these vaccines would be aided if a reliable in vitro test were available to measure host-protective immune responses in vaccinated animals. Here, we describe in vitro oncosphere-killing assays for the quantification of host-protective serum antibodies against Taenia pisiformis, Taenia ovis, Taenia saginata, and Taenia solium in rabbits, sheep, cattle, and pigs, respectively. Activated oncospheres of T. pisiformis, T. ovis, T. saginata, and T. solium were incubated in vitro in culture medium, test serum, and a source of complement, and oncosphere killing was assessed after 10 days of culture. In vitro oncosphere killing reflected the presence of specific antibody, and the oncosphere-killing assay typically indicated immunity to the homologous parasite that had been determined in vivo. This study describes the first reliable oncosphere-killing assays for T. pisiformis, T. ovis, T. saginata, and T. solium. These assays will be used for further research into the optimization of recombinant vaccines against cysticercosis.

Animals↗
PubMed2005

Recent advances in characterization of Echinococcus antigen B.

Antigen B (AgB) in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa and a highly immunogenic major antigen in echinococcal infection. The antigen is comprised of a group of subunit monomers of approximately 8 kDa in molecular size. Recent studies have revealed that the E. granulosus AgB (EgAgB) shows a high degree of genetic variability and the genes encoding the EgAgB 8-kDa subunit monomers that have been identified to date could be grouped into four clades, corresponding to the genes EgAgB8/1, EgAgB8/2, EgAgB8/3 and EgAgB8/4. It has been suggested that the recombinant EgAgB8/2 (rEgAgB8/2) provides better performance in serodiagnosis of human cystic echinococcosis (CE) than does the recombinant EgAgB8/1 (rEgAgB8/1). The EgAgB has been identified as a protease inhibitor with an ability to inhibit recruitment of neutrophils and exploit activation of T helper cells by eliciting a non-protective Th2 cell response, predominantly in patients with progressive CE. Recently it has been revealed that AgB also exists in the cyst fluid of Echinococcus multilocularis. Five different cDNAs encoding the EgAgB homologues have been identified in vesicles, protoscoleces and/or immature adult worms of E. multilocularis and named as EmAgB8/1, EmAgB8/2, EmAgB8/3, EmAgB8/4 and EmAgB8/5. These genes appeared to be expressed in a developmentally regulated manner in the parasite life cycle. This review focuses on recent advances in molecular biological and immunological characterization of AgB from both of E. granulosus and E. multilocularis.

Animals↗
PubMed2005

Vaccines against cysticercosis and hydatidosis: foundations in taeniid cestode immunology.

Recombinant oncosphere antigens have been used in the development of effective vaccines for the prevention of cysticercosis caused by Taenia ovis, Taenia saginata, Taenia solium and hydatid disease caused by Echinococcus granulosus. These vaccines were developed utilizing information gathered from numerous researchers over some 60 years which had established many of the principals concerning immunity to taeniid cestodes in their intermediate hosts. Australian scientists, or scientists with close Australian connections, made seminal early contributions to the understanding of cestode immunology that provided the foundations for more recent application of recombinant DNA methods and the development of practical vaccines. Here, some particular contributions to the field are highlighted from Drs. Michael Gemmell, Michael Rickard, David Heath and Graham Mitchell together with a précis of the recent progress in vaccine development, particularly for prevention of cysticercosis due to T. solium.

Animals↗
PubMed2005

The effect of antigen targeting sequences on antibody responses to hepatitis E virus DNA vaccines in rats and sheep.

Expression of the capsid (PORF2) protein of hepatitis E virus (HEV) in mammalian cells results in heterogeneous intracellular processing with a mixture of stable and rapidly degraded forms, which might be expected to influence immune responses to DNA immunisation. Plasmids encoding the N-terminal 22 or 50 amino acids of PORF2 (Sig1 or Sig3, respectively) fused at the N-terminus of the ORF2.1 antigen of HEV (amino acids 394-660 of PORF2) were examined for processing in vitro and antibody responses in vivo, in both rats and sheep. Unmodified ORF2.1 is an unstable cytosolic protein and Sig1-ORF2.1 is a stable membrane-associated protein, whereas Sig3-ORF2.1 demonstrated heterogeneous processing analogous to that of full-length PORF2. After DNA immunisation, Sig1-ORF2.1 demonstrated a 30-fold enhancement of antibody responses in rats compared to untargeted ORF2.1, increasing to more than 200-fold after boosting with recombinant protein, but was ineffective in sheep. In contrast, Sig3-ORF2.1 did not give a significant effect in rats, but demonstrated 4-5-fold enhancement of antibody responses in sheep, and this enhancement was maintained after boosting with recombinant protein. These results suggest that Sig3 in particular may have promise as a targeting molecule for DNA vaccines in large animals.

Amino Acid Sequence↗
PubMed2005

Hydatid disease: vaccinology and development of the EG95 recombinant vaccine.

Hydatid disease is a zoonotic parasitic disease that is distributed widely around the world and causes substantial human morbidity and mortality, particularly in developing countries. Reduction of human hydatid disease using anthelmintics, together with changes in human lifestyle and animal management practices, have been unsuccessful in some developing countries where the disease still persists. Substantial progress has been made towards developing a practical, recombinant vaccine in sheep, to interrupt the lifecycle of Echinococcus granulosus and to prevent subsequent transmission from dogs to humans. This review focuses on the scientific advances in the development of a recombinant vaccine for hydatid disease and the remaining challenges facing the widespread use of the vaccine for control of hydatid disease in endemic areas.

Animals↗
PubMed2005

Vaccination of pigs to control human neurocysticercosis.

Taenia solium taeniasis/cysticercosis is a zoonotic disease complex in which the pig is an obligate intermediate host. The infection is widespread, particularly in the developing world, and neurocysticercosis is a major cause of human neurologic disease where the parasite is endemic. Despite easy availability, effective anti-parasitic drugs have not been deployed effectively to control disease transmission. We have investigated a vaccine strategy to prevent parasite infection of the pig intermediate host. Such a strategy would interrupt the parasite's life cycle and eliminate the source of infection for humans. Two recombinant antigens selected from the parasite oncosphere life cycle stage were tested in vaccination trials in pigs that were challenged orally with Taenia solium eggs. Both antigens were highly effective in protecting the pigs against infection with the parasite (98.6% and 99.9% protection, respectively). No viable cysts were found in eight pigs vaccinated with one of the antigens. A recombinant subunit vaccine based on oncosphere antigens has the potential to improve the available control measures for T. solium and thereby reduce or eliminate neurocysticercosis.

Animals↗
PubMed2004

Cysticercosis/taeniasis in Asia and the Pacific.

Three taeniid tapeworms infect humans in Asia and the Pacific: Taenia solim, Taenia saginata, and Taenia asiatica. Although there is continuing debate about the definition of a new species, phylogenetic analyses of these parasites have provided multiple lines of evidence that T. asiatica is an independent species and the sister species of T. saginata. Here we review briefly the morphology, pathology, molecular biology, distribution and control options of taeniasis/cysticercosis in Asia and the Pacific and comment on the potential role which dogs may play in the transmission of T. solium. Special attention is focused on Indonesia: taeniasis caused by T. asiatica in North Sumatra, taeniasis/cysticercosis of T. solium and taeniasis of T. saginata in Bali, and taeniasis/cysticercosis of T. solium in Papua (formerly Irian Jaya). Issues relating to the spread of taeniasis/cysticercosis caused by T. solium in Papua New Guinea are highlighted, since serological evidence suggests that cysticercosis occurs among the local residents. The use of modern techniques for detection of taeniasis in humans and cysticercosis in humans, pigs and dogs, with the possible adoption of new control measures will provide a better understanding of the epidemiology of taeniasis/cysticercosis in Asia and the Pacific and lead to improved control of zoonotic and simultaneously meat-borne disease transmission.

Animals↗
PubMed2004

Induction of protection against porcine cysticercosis by vaccination with recombinant oncosphere antigens.

Two recombinant Taenia solium oncosphere antigens, designated TSOL18 and TSOL45-1A, were investigated as vaccines to prevent transmission of the zoonotic disease cysticercosis through pigs. Both antigens were effective in inducing very high levels of protection (up to 100%) in three independent vaccine trials in pigs against experimental challenge infection with T. solium eggs, which were undertaken in Mexico and Cameroon. This is the highest level of protection that has been achieved against T. solium infection in pigs by vaccination with a defined antigen. TSOL18 and TSOL45-1A provide the basis for development of a highly effective practical vaccine that could assist in the control and, potentially, the eradication of human neurocysticercosis.

Animals↗
PubMed2004

Molecular cloning, expression, and serological evaluation of an 8-kilodalton subunit of antigen B from Echinococcus multilocularis.

Full-length cDNA and genomic DNA encoding an 8-kDa subunit of antigen B from Echinococcus multilocularis (designated EmAgB8/1) were isolated from an E. multilocularis metacestode cDNA library and a protoscolex genomic DNA library, respectively. The open reading frame of the cDNA clone encodes a polypeptide comprising 85 amino acids with a 20-amino-acid NH(2)-terminal signal sequence, which was confirmed following N-terminal sequencing of the native antigen. Reverse transcription-PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E. multilocularis. The gene consists of two exons (encoding the signal sequence and mature protein) separated by a 91-bp intron. The mature form was expressed in Escherichia coli, and its antigenic reactivity was compared with that of a counterpart, an 8-kDa subunit of antigen B from Echinococcus granulosus (EgAgB8/1) by Western blotting and enzyme-linked immunosorbent assay (ELISA) with serum samples from patients confirmed to have cystic echinococcosis (CE) and alveolar echinococcosis (AE). Recombinant EmAgB8/1 showed positive reactions in Western blots with 81.3% (65 of 80) of serum samples from CE patients and 40.6% (26 of 64) of serum samples from AE patients, while recombinant EgAgB8/1 showed positive reactions with 86% (43 of 50) and 42% (19 of 45) of the serum samples from these CE and AE patients, respectively. By the ELISA, both EmAgB8/1 and EgAgB8/1 exhibited similar positive reactions with 88% (44 of 50) of serum samples from CE patients and 37.8% (17 of 45) serum samples from AE patients. Statistical analysis revealed that the sensitivity of EmAgB8/1 was comparable to that of EgAgB8/1 for the serodiagnosis of echinococcal diseases. There was no cross-reaction with sera from patients with cysticercosis, which often cross-react when native antigens are used for serodiagnosis.

Amino Acid Sequence↗
PubMed2003

Molecular cloning of genes encoding oncosphere proteins reveals conservation of modular protein structure in cestode antigens.

Recombinant oncosphere antigens have been found to be remarkably effective when used as vaccines against cysticercosis and hydatid disease. Comparison of the structural features of these proteins and their associated genes suggest common features between antigens. Here molecular cloning is used to complete comparisons of Taenia solium, Taenia saginata, Taenia ovis, Echinococcus granulosus and Echinococcus multilocularis oncosphere antigens and genes. The exon/intron structure of genes cloned from T. solium and T. ovis genomic DNA (tsol16 and to16, respectively) in this study was found to be highly conserved. Two closely related tsol16 genes were cloned from the T. solium genome. Their corresponding transcripts were cloned from T. solium oncospheres and a comparison of their deduced amino-acid sequence with that of the protein encoded by to16 indicates that these proteins are the most highly conserved oncosphere proteins identified so far. Cloning of another gene from T. solium (designated tsol18) and comparison with the homologous gene of T. saginata (tsa18) also revealed substantial conservation of gene structure. Comparisons of the genes cloned in this study with genes encoding oncosphere antigens from other taeniid cestodes identified striking conservation of exon structure. The highly conserved regions of the genes encode a putative secretory signal and fibronectin type III domain in each of the oncosphere proteins. The location of exon boundaries in relation to protein features identifies a clear modular structure among all members of these oncosphere antigens. Identification of structural conservation of genes encoding antigenic proteins across several taeniid species suggests that the encoded proteins play important roles in host infection and parasite survival.

Animals↗
PubMed2003

Evaluation of use of recombinant Em18 and affinity-purified Em18 for serological differentiation of alveolar echinococcosis from cystic echinococcosis and other parasitic infections.

To further evaluate recombinant Em18 antigen (rEm18) for immunodiagnosis of human alveolar echinococcosis, 208 serum samples were examined by enzyme-linked immunosorbent assay (ELISA). To comparatively assess the results of rEm18-ELISA, ELISA and immunoblot analysis with two affinity-purified native antigens were also performed with 45 selected serum samples. The results indicate that rEm18 is highly useful for serodiagnosis.

Animals↗
PubMed2002

Evaluation of three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus.

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.

Animals↗
PubMed2002

Usefulness of hydatid cyst fluid of Echinococcus granulosus developed in mice with secondary infection for serodiagnosis of cystic Echinococcosis in humans.

The aim of this work was to assess the usefulness of hydatid cyst fluid (HCF) of Echinococcus granulosus, obtained from mice experimentally infected with hydatid cyst tissue homogenates, for the serodiagnosis of cystic echinococcosis (CE) in humans. The sensitivity and specificity of HCF obtained from mice for the detection of immunoglobulin G (IgG) antibodies in the sera of CE patients were compared with those of HCF from sheep and/or from a human CE patient by using immunoblotting (IB) and an enzyme-linked immunosorbent assay (ELISA). HCFs obtained from three different host species all were highly useful for immunoblotting, and sera from 19 (95%) of 20 CE patients equally recognized the antigen B subunit (approximately 8 kDa). HCF from mice showed a cross-reaction with 9 of 20 alveolar echinococcosis (AE) sera (45%), whereas HCFs from two other host species cross-reacted with 14 of the AE sera (70%). Although 2 (10%) of 20 sera from neurocysticercosis (NCC) patients were false positive with HCF from both sheep and humans, none of these sera showed a positive reaction with HCF from mouse origin. ELISAs with HCFs from both mouse and sheep origins detected all 20 CE and AE sera; however, these ELISAs showed 45% (9 of 20) and 60% (12 of 20) false-positive reactions with 20 NCC sera, respectively. The presence of nonspecific human IgG in HCF obtained from a CE patient prevented us from applying it to the ELISA. HCF of E. granulosus, obtained from laboratory mice with a secondary infection with hydatid cyst tissue homogenates, appears to be highly useful for the serodiagnosis of CE in humans and may be useful in domestic animals.

Animals↗
PubMed2002

Molecular cloning of a vaccine antigen against infection with the larval stage of Echinococcus multilocularis.

Alveolar and cystic hydatidosis are caused by infection with the larval stages of Echinococcus multilocularis and Echinococcus granulosus, respectively. A host-protective antigen has been identified in E. granulosus. Here we identify the presence of a closely related protein in E. multilocularis, characterize and express a cDNA encoding the antigen (designated EM95), determine the structure of the em95 gene, and demonstrate that the EM95 recombinant protein can be used to induce significant levels of protection against challenge infection with E. multilocularis eggs in mice.

Adjuvants, Immunologic↗
PubMedDate not supplied

Echinococcus granulosus: oncosphere-specific transcription of genes encoding a host-protective antigen.

A recombinant antigen vaccine has been developed which is effective in preventing the hydatid parasite, Echinococcus granulosus from infecting its animal intermediate hosts. The vaccine antigen, designated EG95, is expressed by a cDNA cloned from E. granulosus oncosphere mRNA. The gene encoding EG95 belongs to a small gene family where six of the members are transcribed in the oncosphere. Conditions were established which allowed specific RT-PCR amplification of mRNA for each gene family member and these conditions were applied to determine transcription patterns for each gene family member in gravid adult worms, oncospheres, and protoscoleces. The four eg95 gene family members which encode an identical EG95 protein, were transcribed only in the oncosphere. In contrast, two gene family members that encode variant EG95 proteins did not have transcription patterns confined to the oncosphere. These findings suggest a common biological role for four genes in the gene family and a separate role for the other, more variant gene family members.

Animals↗