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M W Lightowlers

Publications and source records attributed to M W Lightowlers.

At least 19 recordsLinked to original sources

Genetic variation within Taenia multiceps in Sardinia, Western Mediterranean (Italy).

Investigations were undertaken on Taenia multiceps to determine if genetic variation was present within the parasites of Sardinia (Italy). Forty samples were obtained from various locations of Sardinia and deoxyribonucleic acid (DNA) was extracted. Polymerase chain reaction (PCR) was performed on NADH dehydrogenase I (ND1) and cytochrome c subunit 1 (CO1) mitochondrial genes and amplicons were then sequenced and aligned with Bioedit software. Pairwise comparison between the ND1 sequences of the T. multiceps isolates showed differences ranging from 1.27 to 2.54% using an isolate obtained from Wales as an outgroup, while COI sequences showed within the samples coming from Sardinia a lesser degree of variability, ranging from 0.22 to 0.67%. Considering the two genes, it was possible to define at least three specific genetic variants in Sardinian samples, which we have termed Tm1, Tm2, and Tm3. This is the first description of genetic variability in T. multiceps. Further investigations will be required to understand to what extent the genetic variability described in this paper would be reflected also in phenotypic differences.

Animals↗

Antibody responses and epitope specificities to the Taenia solium cysticercosis vaccines TSOL18 and TSOL45-1A.

Taenia solium is a cestode parasite that causes cysticercosis in humans and pigs. This study examined the antibody responses in pigs immunized with the TSOL18 and TSOL45-1A recombinant vaccines against T. solium cysticercosis. Immunization with these proteins induced specific, complement-fixing antibodies against the recombinant antigens that are believed to be associated with vaccine-induced protection against T. solium infection. Sera from immunized pigs were used to define the linear B-cell epitopes of TSOL18 and TSOL45-1A. Prominent reactivity was revealed to one linear epitope on TSOL18 and two linear epitopes on TSOL45-1A. These, and oncosphere antigens from other taeniid cestodes, contain a protein sequence motif suggesting that they may show a tertiary structure similar to the fibronectin type III domain (FnIII). Comparison of the location of linear antigenic epitopes in TSOL18 and TSOL45-1A within the proposed FnIII structure to those within related cestode vaccine antigens reveals conservation in the positioning of the epitopes between oncosphere antigens from different taeniid species.

Amino Acid Sequence↗

Molecular characterization of Echinococcus granulosus strains in Sardinia.

Investigations were undertaken to determine the genotypes of the parasite Echinococcus granulosus that were present in livestock animals on the island of Sardinia. Liver, lung, and spleen samples were obtained from 770 sheep, 229 cattle, and 277 pigs slaughtered in Sardinia between January 2003 and April 2005, and the number and fertility of hydatid cysts were determined. Protoscoleces and/or germinal layer were collected from individual cysts, DNA was extracted from 91 samples, and polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods were used for identification of the strain genotype for each sample (G1, G5, G6/G7). Fragments of the mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase I were sequenced. Hydatid disease prevalence of 75.3, 41.5, and 9.4% were found in the organs collected from sheep, cattle, and pigs, respectively. Molecular analysis showed that 89 of 91 ovine, bovine, and swine cysts belonged to the G1 genotype (common sheep strain) of E. granulosus. Parasite isolates from two pigs were identified to belong to the G7 genotype (pig strain). Our results confirm the high prevalence of E. granulosus infection in livestock animals in Sardinia and reveal the presence of at least two parasite genotypes in Sardinia.

Animals↗

Effect of cyclosporin A on the survival and ultrastructure of Echinococcus granulosus protoscoleces in vitro.

Surgical treatment of human hydatidsosis involves the use of various scolicidal agents to kill infective Echinococcus granulosus protoscoleces that may disseminate into the peritoneal cavity during surgery and potentially re-infect the patient. Currently, no scolicidal agent is completely effective in killing intracystic protoscoleces in humans. Cyclosporin A (CsA) has previously been found to be lethal for E. granulosus protoscoleces in vitro. In this study, we further assessed the effectiveness of CsA as a scolicidal agent by testing the toxic effect of CsA at higher doses over various time-periods. Experiments were performed on activated and unactivated protoscoleces cultured in nutrient medium or sheep hydatid cyst fluid. All activated protoscoleces were killed following culture in 100 microg/ml of CsA for 3 days and 50 or 20 microg/ml for 5 days. The lethal effect of CsA on unactivated protoscoleces varied but reached 100% over 15 days in culture with 100 or 50 microg/ml of CsA. Pulse treatment of protoscoleces with 50, 20 or 10 microg/ml of CsA for 5 min or 72 h killed all parasites by day 10 and day 5 respectively. Untreated protoscoleces remained greater than 95 % viable for the duration of experiments. Changes in protoscolex ultrastructure induced by treatment with 10 microg/ml of CsA over 10 days in in vitro culture was assessed by TEM. Protoscolex alterations observed in treated parasites included an increase in cellular vacuolization, swelling of mitochondria, rounding of cells, damage to the tegument, decrease in glycogen, a breakdown of the extracellular matrix and an increase in lipid globules. The untreated protoscoleces, by comparison, had few changes during the 10-day culture period with the exception of large amounts of extracellular glycogen observed in the protoscoleces at culture days 7 and 10. From these results, CsA is clearly an effective scolicidal agent in vitro that may have potential application as a new therapeutic agent in the treatment of human hydatid disease.

Animals↗

Immunity and vaccine control of Echinococcus granulosus infection in animal intermediate hosts.

Much progress has been made with characterisation of the EG95 vaccine which can be used to prevent hydatid infection in animal intermediate hosts of Echinococcus granulosus. The vaccine comprises a single recombinant oncosphere antigen and the adjuvant Quil A. It induces complement-fixing antibodies that kill the invading oncosphere early in an infection. In the majority of vaccinated animals, no hydatid cysts occur following a challenge infection. However, a small number of viable cysts may occur in some vaccinated animals. The vaccine has proved effective in vaccine trials carried out in sheep in New Zealand, Australia, Argentina, Chile and China as well as in goats and cattle. Investigations of the genetic diversity of the gene encoding EG95 have identified no unequivocal variation within the G1 strain parasites; however DNA sequence diversity within the EG95 family of genes has been found in G6/G7 parasites. GMP production scale-up of the vaccine has been undertaken in New Zealand and China and it is expected that the vaccine will be become available through these sources for implementation as part of hydatid control programs worldwide.

Animals↗

Vaccination for the prevention of cysticercosis.

Several species of taeniid cestode parasites cause cysticercosis in their intermediate hosts. The most important species is Taenia solium, which infects pigs as the natural animal intermediate host but also may infect humans as intermediate hosts, leading to the disease known as neurocysticercosis. T. solium has been identified as a potentially eradicable disease and increasing attention is being placed on efforts to control transmission of the parasite. One option to assist with control of the disease is to prevent infection occurring in pigs by vaccination, thereby breaking the parasite's life-cycle and removing the source of infection for humans. Several approaches are being examined towards development of vaccines against T. solium, one of which is the application of recombinant oncosphere antigens. Two different oncosphere antigens, designated TSOL18 and TSOL45, have been evaluated, each of which has been shown to induce complete or near complete protection against experimental challenge infection in four separate vaccine trials in pigs. Investigations have begun towards characterising various aspects of this vaccine before undertaking controlled field trials. The TSOL18/TSOL45 vaccine has the potential to make a substantial contribution to the control and, potentially, the eradication of human neurocysticercosis.

Animals↗

Molecular and genetic characterisation of the host-protective oncosphere antigens of taeniid cestode parasites.

Highly effective recombinant vaccines have been developed against Taenia ovis infection in sheep, Taenia saginata infection in cattle, Taenia solium infection in pigs, Echinococcus granulosus and Echinococcus multilocularis infections in a variety of intermediate host species. These vaccines have been based on the identification and expression in Escherichia coli of antigens derived from the oncosphere life cycle stage, contained within the parasites' eggs. Investigation of the molecular aspects of these proteins and the genes encoding them have revealed a number of common features, including the presence of a predicted secretory signal sequence, and one or two copies of a fibronectin type III domain, each encoded by separate exons within the associated gene. Evidence has been obtained to confirm glycosylation of some of these antigens. Ongoing investigations will shed light on the biological roles played by the proteins within the parasites and the mechanism by which they make the parasites vulnerable to vaccine-induced immune responses.

Animals↗

Vaccination against cestode parasites: anti-helminth vaccines that work and why.

Highly effective recombinant vaccines have been developed against the helminth parasites Taenia ovis, Taenia saginata and Echinococcus granulosus. These vaccines indicate that it is possible to achieve a reliable, high level of protection against a complex metazoan parasite using defined recombinant antigens. However, the effectiveness of the vaccines against the taeniid cestodes stands in contrast to the more limited successes which characterise attempts to develop vaccines against other platyhelminth or nematode parasites. This review examines the features of the host-parasite relationships among the taeniid cestodes which have formed the basis for vaccine development. Particular consideration is given to the methodologies that have been used in making the cestode vaccines that might be of interest to researchers working on vaccination against other helminths. In developing the cestode vaccines, antigens from the parasites' infective larval stage contained within the egg (oncosphere) were identified as having the potential to induce high levels of protection in vaccinated hosts. A series of vaccination trials with antigen fractions, and associated immunological analyses, identified individual protective antigens or fractions. These were cloned from cDNA and the recombinant proteins expressed in Escherichia coli. This strategy was independently successful in developing vaccines against T. ovis and E. granulosus. Identification of protective antigens for these species enabled rapid identification, cloning and expression of their homologues in related species and thereby the development of effective vaccines against T. saginata, E. multilocularis and, more recently, T. solium. The T. saginata vaccine provides an excellent example of the use of two antigen components, each of which were not protective when used individually, but when combined they induce a reliable, high level of protection. One important contributing factor to the success of vaccine development for the taeniid cestodes was the concentration on studies seeking to identify native host-protective antigens, before the adoption of recombinant methodologies. The cestode vaccines are being developed towards practical (commercial) application. The high level of efficacy of the vaccines against T. solium cysticercosis and hydatid disease suggests that they would be effective also if used directly in humans.

Animals↗

Progress in control of hydatidosis using vaccination--a review of formulation and delivery of the vaccine and recommendations for practical use in control programmes.

A vaccine to protect sheep, goats, and bovines against hydatid disease caused by the cysts of Echinococcus granulosus is prepared as a recombinant fusion protein expressed in Escherichia coli. Solubilised inclusion bodies are injected, together with Quil A, subcutaneously on two occasions 1 month or more apart, and induce protection against infection which lasts for at least 12 months. A third injection given 6-12 months after the second injection induces a high and long-lasting protection against artificial or natural challenge infections. This review describes work carried out on the formulation, safety and efficacy of the vaccine under laboratory and field conditions, using artificial or natural challenges with E. granulosus eggs, followed by necropsy. Hydatid control programmes based on regular treatment of all dogs with the correct dose of a highly-efficient anthelmintic have sometimes not been successful in Continental environments. Access to dogs is difficult in summer because of the distances to summer pastures, and is often impossible in winter because of snow. A control program using strategic twice-yearly anthelmintic treatment of dogs is likely to be successful provided grazing animals are vaccinated as well. Vaccination as a control tool only requires the veterinarians to visit two times a year, and while the veterinarian is present, the dogs can be treated with anthelmintic for little additional cost. One visit should take place after the autumn kill of animals for winter consumption, and this is a good time to vaccinate animals born in the summer, and also all other animals while they are healthy and immunologically responsive. The other visit should take place in the spring, at which time animals born during winter can be vaccinated. Although a single immunization has been shown to induce a useful degree of protection, where possible it is best to give two initial injections, 1 month apart. If it is possible for veterinarians to stay in the field for 2 months in November/December and March/April, in order to give the two injections, a more rapid onset of full protective immunity will initially be achieved than if the injections are given 6 months apart. A large-scale safety and efficacy trial involving 50,000 and 100,000 lambs in Qinghai and Xinjiang Provinces of China has taken place. Results have confirmed safety and efficacy. In most countries, prevalence of infection increases with age. The vaccine has no effect on established cysts, and therefore, in order to prevent the biomass of Echinococcus spp. from increasing, it might be an effective strategy to begin a control programme by vaccinating all animals. Because many of the older stock will already be infected, they will remain a source of infection for dogs for the average lifetime of the stock. Dogs will still be able to be infected from the older stock, and will continue to infect humans. We advocate that a vaccination programme be accompanied by education about hydatid disease, and anthelmintic treatment of dogs in late autumn and early spring.

Animals↗

Vaccines for prevention of cysticercosis.

Neurocysticercosis due to Taenia solium infection is an important cause of human morbidity and mortality. Despite the availability of effective anthelmintics, the disease remains prevalent in many parts of the world and there is a need for new and improved measures for control of the infection. An effective vaccine to prevent infection in pigs, the parasite's natural intermediate host, would be a valuable new option to assist with T. solium control. Several approaches are being used currently towards the development of a T. solium vaccine and these approaches are reviewed briefly, with emphasis on the use of recombinant oncosphere antigens. Highly effective vaccines have been developed against cysticercosis in sheep and cattle caused by Taenia ovis and Taenia saginata, respectively. This success has encouraged the adoption of a similar strategy for T. solium. The recent finding that one oncosphere antigen, TSOL18, can induce complete protection against T. solium infection in pigs, highlights the potential for development of a practical vaccine. A vision is proposed for the development of a safe, effective, inexpensive vaccine for pigs, which can be administered in an edible form. Through an international collaborative effort, research is progressing towards the realisation of such a vaccine and its use to reduce the global burden of neurocysticercosis.

Animals↗

Anti-parasitic effect of cyclosporin A on Echinococcus granulosus and characterization of the associated cyclophilin protein.

Cyclophilins are a family of proteins found ubiquitously in eukaryotes, many of which bind to the immunosuppressive drug cyclosporin A (CsA). CsA has been found to have anti-parasitic effects against a variety of helminth and protozoan parasites and this activity could be mediated via cyclophilin. In this study we characterize a full length cyclophilin gene from Echinococcus granulosus, the associated natural gene and expression pattern, and investigate the functional properties of the recombinant E. granulosus cyclophilin protein. In addition, the effects of CsA were investigated on E. granulosus protoscoleces in in vitro culture. The full length E. granulosus cyclophilin cDNA encodes a protein of 20 kDa and is encoded by a single gene (EGCyP-1) comprising 2 exons separated by a 31 bp intron. The gene is expressed constitutively in all E. granulosus life-cycle stages examined. Recombinant E. granulosus cyclophilin (egCyP-l) exhibited functional enzyme activity as an isomerase. Treatment of in vitro cultures of E. granulosus protoscoleces with CsA was found to be lethal to the parasites. No protoscoleces survived treatment with 10 microg/ml of CsA over 7 culture days, as determined by observing motility and the uptake of toluidine blue dye. Untreated protoscoleces remained viable for the duration of experiments. The survival of protoscoleces was CsA dose dependent. A concentration of 10 microg/ml CsA was 100% lethal while doses of 8 microg/ml and 5 microg/ml resulted in 82% and 32% killing, respectively, after 7 days in culture. The anti-parasitic activity of CsA may have the potential to be developed as a new therapeutic agent for treatment of cystic hydatidosis in humans.

Amino Acid Sequence↗

Vaccination against hydatid disease.

Hydatid disease is a parasitic infection that causes widespread human morbidity and mortality. Livestock animals, particularly sheep, are involved in the parasite's lifecycle. A defined antigen vaccine has been developed which can prevent hydatid infection in sheep. The vaccine has been shown to be highly effective in animal trials, with almost complete immunity persisting for more than a year after vaccination. Use of the vaccine in livestock may decrease transmission of the parasite and, indirectly, reduce the incidence of infections in humans. In some regions animal vaccination or other hydatid control measures are unlikely to be applicable. In these areas, direct vaccination of humans against hydatid infection may be the only practical option for disease prevention. The extraordinary effectiveness of the hydatid vaccine in the parasite's natural animal hosts singles this vaccine out as having perhaps the greatest potential for development of the first effective human vaccine against a parasitic disease.

Animals↗

Vaccines against cysticercosis and hydatidosis.

The recombinant vaccines that have been developed against cysticercosis and hydatidosis in sheep and cattle are remarkable for their effectiveness and are prominent as examples of the very few non-living vaccines against parasitic diseases. Their development has been through practical application of molecular parasitology, utilising immunochemical techniques in antigen identification and recombinant DNA methods in antigen production. This brief overview discusses the contribution of molecular techniques to the successful development of recombinant vaccines against Taenia ovis, Taenia saginata and Echinococcus granulosus as well as the immunological and genomic studies that have arisen from their development.

Animals↗

The comparative efficacy of CTLA-4 and L-selectin targeted DNA vaccines in mice and sheep.

The access of antigens to antigen presenting cells (APCs) appears to be a rate-limiting step in the generation of immune responses to DNA vaccines. The cytotoxic T lymphocyte antigen 4 (CTLA-4) and L-selectin represent attractive ligands for use in the targeting of antigen to APCs and lymph nodes. CTLA-4 binds with high affinity to the B7 membrane antigen on APCs, while L-selectin functions as a lymphocyte homing marker and binds to CD34 on the surface of high endothelial venule cells. DNA vaccines encoding human immunoglobulin (HIg), fused to either CTLA-4 or L-selectin, have been shown to generate up to 10,000-fold higher anti-HIg antibody responses than DNA vaccines encoding HIg alone. In this study, the ability of CTLA-4 or L-selectin mediated targeting to enhance the humoral immune response to an alternate vaccine antigen was investigated. DNA vaccines encoding CTLA-4-HIg and L-selectin-HIg fused to the host-protective 45W antigen from Taenia ovis were constructed. In BALB/c mice, the L-selectin targeted vaccine did not improve either the magnitude or speed of antibody responses of vaccinated mice. In contrast, the CTLA-4 targeted DNA vaccine generated 45W-specific antibody responses which were up to 30-fold higher than those achieved with non-targeted DNA vaccination. The kinetic of the antibody response generated following CTLA-4 targeted DNA vaccination was also significantly faster than that achieved with non-targeted DNA vaccination, or with adjuvanted protein vaccination. Vaccination of outbred sheep with DNA vaccines expressing either murine or ovine CTLA-4 targeted antigen failed to enhance immune responses. These findings indicate that CTLA-4 targeting may find application in the improvement of DNA vaccines, but requires further development for applications in large animal species.

Abatacept↗

The human IgG3 hinge mediates the formation of antigen dimers that enhance humoral immune responses to DNA immunisation.

A series of plasmid DNA constructs containing the 45W antigen gene from Taenia ovis were used to investigate the impact of antigen dimerisation on the humoral immune response to genetic immunisation. Genes encoding dimeric 45W were generated via fusion to the hinge region of human IgG3 (hIg). This region was selected because it is compact and contains 11 inter-chain disulphide-bridges. The DNA encoding the IgG3 hinge contains four exons, with the last three exons being repeats and possibly superfluous. Plasmids containing the 45W gene linked to exons 1-2, 1-3 or 1-4 of the hIgG3 hinge, were compared to a control plasmid containing a form of the 45W gene which encodes secreted, monomeric 45W protein. Western blot analysis was used to investigate the formation of the fusion-proteins in transfected Cos-7 cells. The full-length fusion construct expressed predominantly dimeric forms of the fusion-protein, while truncation of the hinge region decreased the abundance of dimeric fusion-protein and increased the proportion monomeric fusion antigen. In immunised BALB/c mice, 45W-specific antibody titres were increased 3 to 4-fold via fusion to the full-length hinge region, whereas the truncated constructs were similar to the control. IgG subclass analysis indicated that all mice generated predominantly IgG1, IgG2a and IgG2b antibodies. Therefore, these results suggest that the efficient formation of dimeric antigen, via fusion to the full-length hinge of human IgG3, can increase the immunogenicity of expressed antigens without altering the form of the immune response elicited by DNA immunisation.

Animals↗

The immune response to a DNA vaccine can be modulated by co-delivery of cytokine genes using a DNA prime-protein boost strategy.

A large-scale DNA vaccination trial was performed in sheep to investigate whether co-delivery of the cytokine genes IL-4, IL-5, IL-15, GM-CSF or IFN-gamma could modulate the immune response generated to an antigen, in a DNA prime-recombinant protein boost regime. Vaccination with the recombinant EG95 protein has been shown to induce protection in sheep from Echinococcus granulosus infection, the causative agent of hydatid disease. Here we demonstrate that vaccination with DNA encoding EG95 effectively primed the humoral response, as judged by high IgG anti-EG95 titres detected one-week after a boost with the recombinant protein. However, by two weeks after protein-boost the titres in the control group had reached levels similar to the groups primed with EG95 DNA. Priming with two doses of DNA vaccine followed by boosting with recombinant protein induced a predominantly IgG1 response. In contrast, priming and boosting with the protein vaccine generated a strong IgG2 response. Co-delivery of the EG95 DNA vaccine with DNA encoding GM-CSF enhanced the antibody titre to EG95 while co-delivery of IFN-gamma or IL-4 encoding DNA appeared to reduce the ability of the DNA vaccine to prime an IgG antibody response. This study has demonstrated the efficacy of the co-delivery of cytokines to modulate immune responses generated in a DNA prime-protein boost strategy.

Animals↗

Alternative splicing and sequence diversity of transcripts from the oncosphere stage of Taenia solium with homology to the 45W antigen of Taenia ovis.

Genes and transcripts which show homology to the host-protective 45W antigen of Taenia ovis have been cloned from the human parasite Taenia solium. The T. solium genes cloned in this study (TSO45) show conserved genomic structural features which are also features of the T. ovis 45W gene family. The TSO45 genes consist of a four exon and three intron structure. Eight TSO45 transcripts, encoded by at least five genes, were cloned from T. solium oncospheres and comparison of their DNA sequence indicates that some transcripts have arisen by alternative splicing, the first demonstration of exon inclusion/exon skipping in cestodes. Alternative splicing occurred with respect to both exons II and III with three splice variants identified from the TSO45-1 gene and two splice variants from TSO45-5. The proteins encoded by this family of genes contain putative N-linked glycosylation sites, an amino terminal secretory signal, a hydrophobic carboxy terminal sequence characteristic of GPI-anchored proteins and fibronectin type III motifs. These features are common to their T. ovis and Taenia saginata homologues. The similarities of the TSO45 genes cloned in this study with genes encoding host-protective antigens of T. ovis and T. saginata indicates that the encoded T. solium proteins are quite possibly antigenic and have potential use as a vaccine to prevent T. solium infection in the parasite's intermediate host. In this respect, the generation of sequence diversity and hence potential antigenic diversity through alternative splicing of TSO45 genes may have implications for the use of these proteins in vaccines against T. solium cysticercosis.

Alternative Splicing↗

A gene family expressing a host-protective antigen of Echinococcus granulosus.

Echinococcus granulosus causes cystic hydatidosis in humans. A recombinant antigen vaccine has been developed, for use in the parasite's natural animal intermediate hosts, that may provide a new tool for control of hydatid disease transmission. The antigen, designated EG95, is encoded by a cDNA the features of which indicate it to be an incomplete copy of the associated mRNA. Characterisation of the gene(s) encoding the antigen was undertaken in order to enable subsequent study of genetic variability in the gene and associated protein in different parasite isolates. Southern hybridisation studies of E. granulosus genomic DNA probed with the eg95 cDNA revealed that the gene belonged to a gene family. DNA sequence analysis of cloned genomic fragments indicated that the gene family consists of at least seven members, one of which is a pseudogene. The gene having identity with the eg95 cDNA was cloned and sequenced, and the full length mRNA characterised. Genomic sequence and structure of the eg95 gene family members are highly conserved with respect to the gene encoding EG95. Four eg95-related genes are predicted to express an identical EG95 protein and all four were shown to be expressed in the oncosphere life-cycle stage. The full length EG95 protein has a predicted molecular mass of 16.9 kDa, secretory signal sequence, carboxy-terminal glycosylphosphatidylinositol hydrophobic anchor motif and a fibronectin type III domain. PCR amplification conditions were established which allow gene-specific characterisation of the eg95 gene in E. granulosus isolates from different host species and geographical locations.

5' Untranslated Regions↗