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M W Shirley

Publications and source records attributed to M W Shirley.

At least 19 recordsLinked to original sources
PubMed2006

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Amino Acid Sequence↗
PubMed2006

Genetic identification of antigens protective against coccidia.

The Eimeria species, causative agents of the disease coccidiosis, are genetically complex protozoan parasites endemic in livestock. Drug resistance remains commonplace among the Eimeria, and alternatives to chemotherapeutic control are being sought. Vaccines based upon live formulations of parasites are effective, but production costs are high, stimulating demand for a recombinant subunit vaccine. The identity of antigens suitable for inclusion in such vaccines remains elusive. Selection of immunoprotective antigens of the Eimeria species as vaccine candidates based upon recognition by the host immune system has been unsuccessful, obscured by the considerable number of molecules that are immunogenic but not immunoprotective. This is a common problem which characterizes work with most eukaryotic parasites. The identification of a selective criterion to directly access genetic loci that encode immunoprotective antigens of Eimeria maxima using a mapping strategy based upon parasite genetics, immune selection and DNA fingerprinting promises to revolutionize the process of antigen discovery. Linkage analyses of DNA markers amplified from populations of recombinant parasites defined by an ability to escape parent-specific deleterious selection by strain-specific immunity and chemotherapy has revealed four discrete regions within the E. maxima genome linked to escape from a protective immune response. These regions now form the basis of detailed study to identify antigens as candidates for inclusion in future vaccination strategies.

Animals↗
PubMed2005

Guidelines for evaluating the efficacy and safety of live anticoccidial vaccines, and obtaining approval for their use in chickens and turkeys.

These guidelines are intended to aid those engaged in poultry research in the design, implementation and interpretation of laboratory, floor-pen and field studies for the assessment of the efficacy and safety of live anticoccidial vaccines for immunization of chickens and turkeys against Eimeria species. In addition to efficacy and safety requirements, manufacture, quality control and licensing considerations are discussed. The guidelines do not address subunit vaccines comprising non-viable material, but many of the principles described will be relevant to such vaccines if they are developed in the future. Guidelines are available in some countries for avian vaccines of bacterial or viral origin but specific standards for anticoccidial vaccines in poultry have not, as far as we know, been produced. Information is provided on general requirements of registration authorities (based upon regulations applicable in the European Union and the USA) for obtaining marketing authorizations for vaccines. These guidelines may assist poultry specialists in providing specific information for administrators involved in the decision-making process leading to registration of new vaccines, and are intended to facilitate the worldwide adoption of consistent, standard procedures.

Animals↗
PubMed2004

EtCRK2, a cyclin-dependent kinase gene expressed during the sexual and asexual phases of the Eimeria tenella life cycle.

EtCRK2, a cyclin-dependent kinase from the coccidian parasite, Eimeria tenella is closely related to eukaryotic cyclin-dependent kinases that regulate progression of the cell cycle and to several cyclin-dependent kinases identified in the Apicomplexa. Northern blot analyses revealed that EtCRK2 is transcribed during both asexual (first-generation schizogony) and sexual (oocyst sporulation) replicative phases of the parasite life cycle. In addition, it appears to be transcriptionally regulated during meiosis. Recombinant EtCRK2 produced in Escherichia coli has kinase activity which is significantly stimulated by the addition of vertebrate cyclin A. This cyclin-dependent kinase may play a significant role in regulating critical cell cycle events during both asexual proliferation and sexual development of the parasite.

Amino Acid Sequence↗
PubMed2004

Integrating genetics and genomics to identify new leads for the control of Eimeria spp.

Eimerian parasites display a biologically interesting range of phenotypic variation. In addition to a wide spectrum of drug-resistance phenotypes that are expressed similarly by many other parasites, the Eimeria spp. present some unique phenotypes. For example, unique lines of Eimeria spp. include those selected for growth in the chorioallantoic membrane of the embryonating hens egg or for faster growth (precocious development) in the mature host. The many laboratory-derived egg-adapted or precocious lines also share a phenotype of a marked attenuation of virulence, the basis of which is different as a consequence of the in ovo or in vivo selection procedures used. Of current interest is the fact that some wild-type populations of Eimeria maxima are characterized by an ability to induce protective immunity that is strain-specific. The molecular basis of phenotypes that define Eimeria spp. is now increasingly amenable to investigation, both through technical improvements in genetic linkage studies and the availability of a comprehensive genome sequence for the caecal parasite E. tenella. The most exciting phenotype in the context of vaccination and the development of new vaccines is the trait of strain-specific immunity associated with E. maxima. Recent work in this laboratory has shown that infection of two inbred lines of White Leghorn chickens with the W strain of E. maxima leads to complete protection to challenge with the homologous parasite, but to complete escape of the heterologous H strain, i.e. the W strain induces an exquisitely strain-specific protective immune response with respect to the H strain. This dichotomy of survival in the face of immune-mediated killing has been examined further and, notably, mating between a drug-resistant W strain and a drug-sensitive H strain leads to recombination between the genetic loci responsible for the specificity of protective immunity and resistance to the anticoccidial drug robenidine. Such a finding opens the way forward for genetic mapping of the loci responsible for the induction of protective immunity and integration with the genome sequencing efforts.

Animals↗
PubMed2003

Amplified fragment length polymorphism analyses of Eimeria spp.: an improved process for genetic studies of recombinant parasites.

Application of the amplified fragment length polymorphism (AFLP) technique to genetic mapping studies requires high quality DNA as a template. In the case of Eimeria spp., this has previously been in the form of chromosomal DNA obtained from purified sporozoites recovered from large numbers of oocysts (generally up to 2 x 10(8)). In order for the AFLP technique to be more easily applied to studies on the genetics of Eimeria maxima, for which only smaller numbers of oocysts are available, a simplified, more efficient method for the recovery of genomic DNA from small numbers of oocysts was developed. Our new method should also be useful for genetic analyses of other coccidial parasites and for the recovery of AFLP-quality DNA from other pathogens.

Animals↗
PubMed2003

The Houghton strain of Eimeria tenella: a review of the type strain selected for genome sequencing.

A landmark decision was taken in 2002 by the Biotechnology and Biological Sciences Research Council (BBSRC) in the UK to fund the genome sequencing of an eimerian (apicomplexan) parasite from the fowl. The project is a joint collaboration between the Institute for Animal Health (IAH) at Compton, UK and the Wellcome Trust Sanger Institute, Cambridge, UK (see http://www.sanger.ac.uk/Projects/E_tenella/ and http://www.iah.bbsrc.ac.uk/eimeria/). The species chosen, Eimeria tenella, is the best known member of the genus and the Houghton (H) strain is one of the most widely studied. The H strain was isolated in 1949 in the UK. It was maintained initially at the Houghton Poultry Research Station (HPRS), Houghton, and, following closure of that laboratory in 1992, thereafter at the IAH in Compton. At various times the parasite has been provided to other institutions and research groups carrying out coccidiosis research in the fowl. The H strain has been utilised in many fundamental studies on the eimerian life cycle and its relationship with the host. It has also been used to investigate the nature of drug resistance, and to derive attenuated lines-one of which, a "precocious line", is a component of a multivalent live attenuated coccidiosis vaccine (Paracox vaccine, Schering-Plough Animal Health). In this article, some immunological, epidemiological, genetic, and chemotherapeutic investigations with the H strain are reviewed, a summary is provided of the biological characteristics of the parasite and some of the core methods used to prepare purified extracellular life cycle stages for experimental studies or passage are given.

Animals↗
PubMed2002

Sustainable coccidiosis control in poultry production: the role of live vaccines.

The development of new methods of administering coccidiosis vaccines has facilitated their use in the hatchery and thereby improved prospects for the economic vaccination of broilers. The acquisition of protective immunity to Eimeria species is boosted by further exposure to infection after vaccination. Factors that affect the reproductive efficiency of non-attenuated and attenuated vaccines are considered and the key role that oocyst production plays in establishing and maintaining uniform immunity in a flock of chickens is discussed. In addition to immunisation, a possible advantage to the application of certain vaccines is that their use could repopulate poultry houses with drug-sensitive organisms. Theoretical rotation programmes in which the use of drugs is alternated with that of vaccines are described. Variability of the cross-protective immune response between strains of the same species should be considered during vaccine development and subsequent use. The significance of less common species of Eimeria, not included in all vaccines, also needs to be assessed. An important consideration is the occurrence of pathogens other than Eimeria (such as the bacterium Clostridium) in flocks given coccidiosis vaccines and the methods by which they might be controlled. More research is required into the relationship between bacterial and viral infections of poultry and coccidiosis vaccination. Vaccines need to be developed that are simple to apply and cost effective for use in areas of the world where small-scale poultry production is commonplace. In the near future it is likely that more live vaccines based upon oocysts derived from attenuated strains of Eimeria will be developed but in the longer term vaccines will be based on the selective presentation to the host of specific molecules that can induce protective immunity. This achievement will require significant investment from the private and public sectors, and, if successful, will facilitate the sustainable control of coccidiosis in poultry production.

Animals↗
PubMed2001

Successful VATS ligation of a large anomalous branch producing IMA steal syndrome after MIDCAB.

The occurrence of a flow "steal phenomenon" from a large branch of the internal mammary artery (IMA) is rare and its pathogenesis is still controversial. We describe a case of a patent large anomalous left IMA branch which produced recurrent angina 3 years post MIDCAB. Transcatheter coil obliteration of the vessel produced symptomatic relief. However, six months later, chest pains recurred in association with unwinding of the coil and recannulization of the collateral vessel. Successful ligation of the culprit branch using a VATS approach is presented.

Angina Pectoris↗
PubMed2001

Modelling host cell availability and the crowding effect in Eimeria infections.

Within-host mathematical models of Eimeria maxima and Eimeria praecox infections of the chicken are presented and used to investigate the role of host cell availability as a possible determinant of the so-called 'crowding effect'; whereby the fecundity of the parasites decreases as infectious dose increases. Assumptions about the number of available host cells, the average lifespan of these cells and the age structure within the host-cell population were made and mathematical models were constructed and combined with experimental data to test whether these conditions could reproduce the crowding effect in the two species. Experimental data demonstrated that crowding during in vivo infections was apparent following very low infectious doses, but none of the models could adequately reproduce crowding at the same doses while maintaining realistic estimates of the dynamics of the enterocyte pool. However, both the size and lifespan of the enterocyte pool were demonstrated to have substantial effects on the fecundity of the infections, particularly at higher doses. These data indicate that host cell availability cannot be solely responsible for the crowding effect. Alternative factors such as the influence of the primary immune response to the parasite may also be explored using within-host models and other applications of these models are discussed.

Animals↗
PubMed2000

The genome of Eimeria spp., with special reference to Eimeria tenella--a coccidium from the chicken.

Eimeria spp. contain at least four genomes. The nuclear genome is best studied in the avian species Eimeria tenella and comprises about 60 Mbp DNA contained within ca. 14 chromosomes; other avian and lupine species appear to possess a nuclear genome of similar size. In addition, sequence data and hybridisation studies have provided direct evidence for extrachromosomal mitochondrial and plastid DNA genomes, and double-stranded RNA segments have also been described. The unique phenotype of "precocious" development that characterises some selected lines of Eimeria spp. not only provides the basis for the first generation of live attenuated vaccines, but offers a significant entrée into studies on the regulation of an apicomplexan life-cycle. With a view to identifying loci implicated in the trait of precocious development, a genetic linkage map of the genome of E. tenella is being constructed in this laboratory from analyses of the inheritance of over 400 polymorphic DNA markers in the progeny of a cross between complementary drug-resistant and precocious parents. Other projects that impinge directly or indirectly on the genome and/or genetics of Eimeria spp. are currently in progress in several laboratories, and include the derivation of expressed sequence tag data and the development of ancillary technologies such as transfection techniques. No large-scale genomic DNA sequencing projects have been reported.

Animals↗
PubMed2000

A genetic linkage map of the apicomplexan protozoan parasite Eimeria tenella.

Apicomplexan protozoan parasites have complex life cycles that involve phases of asexual and sexual reproduction. Some genera have intermediate insect hosts, for example, Plasmodium spp. (the cause of malaria), but related genera such as Eimeria spp. (causative agents of coccidiosis in poultry) have a direct life cycle occurring in only a single host. Mechanisms that regulate the life cycles of apicomplexan parasites are unknown, but the intracellular growth of avian Eimeria spp. is easily shortened by serial selection for the first parasites to complete the transition from asexual to sexual reproduction (to yield so-called precocious lines). To investigate the genetic basis of such an abbreviated life cycle, we have used the species E. tenella and analyzed the inheritance of 443 polymorphic DNA markers in 22 recombinant cloned progeny derived from a cross between parents that had selectable phenotypes of precocious development or resistance to an anticoccidial drug. The markers were placed in 16 linkage groups (which defined 12 chromosomes) and a further 57 unlinked groups. Two linkage groups showed an association (P =.0105) with the traits of precocious development or drug-resistance and were mapped to chromosome 2 (ca 1.2 Mbp) and chromosome 1 (ca 1.0 Mbp), respectively. The map provides a framework for further studies on the identification of genetic loci implicated in the regulation of the life cycle of an important protozoan parasite and a representative of a major taxonomic group. [A table with the segregation data is available as an online supplement at http://www.genome.org.]

Animals↗
PubMed1999

A survey of genes in Eimeria tenella merozoites by EST sequencing.

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.

Animals↗
PubMed1999

PCR identification of chicken Eimeria: a simplified read-out.

A polymerase chain reaction (PCR) assay, based on the amplification of internal transcribed spacer 1 (ITS1) regions of ribosomal DNA, was developed for the chicken coccidian species Eimeria maxima, E. mitis and E. praecox. Thus, taking into account our previous work, a complete set of ITS1-based, species-specific primers for the detection and discrimination of all seven Eimeria species that infect the domestic fowl is now available. ITS1 primers for each of these seven species of Eimeria were also used as capture probes in a paper chromatography assay (PACHA). The addition of PACHA to the PCR assay provided a faster, more simplified read-out compared to staining of amplified bands in an agarose gel with ethidium bromide.

Journal Article↗
PubMed1998

Characterisation of a Cryptosporidium parvum-specific cDNA clone and detection of parasite DNA in mucosal scrapings of infected mice.

A cDNA library was constructed using total RNA extracted from oocysts and sporozoites of the protozoan parasite Cryptosporidium parvum. The expression library was screened with an anti-C. parvum antiserum and a clone, Cp3.4, with a 2043 bp insert, was extracted. Southern blot analysis demonstrated a single copy gene that was located on a 1.6 Mb chromosome. The gene was found to be C. parvum specific as Cp3.4 did not cross-hybridise with chromosomal DNA from three other apicomplexan parasites. The cDNA encodes a polypeptide with a predicted membrane helix at its C-terminal end which is flanked by stretches of acidic amino acids. Overall, the polypeptide has a low isoelectric point (pI) of 3.94. A total of 21 glycine/proline-rich octapeptides were identified which represented variations of a consensus sequence. The function of this protein is yet unknown. Using Cp3.4-specific PCR primers, this C. parvum gene could be amplified from as little as 0.8 pg of purified parasite DNA in a single polymerase chain reaction. Less than 0.1 ng of DNA from the ileum mucosa of immunosuppressed adult mice that had been infected with C. parvum oocysts was required to detect the parasites. In non-immunosuppressed mice that were infected and which did not shed oocysts in numbers detectable by acid-fast staining, parasite development could be detected in 25 ng of total mucosa DNA. This PCR approach may be a valuable technique for the detection of parasite infections in situations where conventional staining methods fail, such as chronic, low-grade infections or the detection of parasites in potential reservoir hosts.

Amino Acid Sequence↗
PubMed1998

Mapping the genome of Eimeria tenella: use of a yeast artificial chromosome library as the basis for a physical map.

Efforts to derive a physical map of the genome of Eimeria tenella are being made to complement genetic mapping and to assist in the production of an integrated map. A large insert library of DNA from the Houghton (H) reference strain of E. tenella has been constructed in yeast artificial chromosomes (YACs). The library contains a tenfold E. tenella genome equivalent consisting of 3177 clones arrayed in 96-well microtitre plates and gridded on nylon membranes at 1728 clones/86 cm2. Size fractionation of a random sample of 185 YACs revealed an average insert size of approximately 170 kb. Hybridisation of four E. tenella single-copy probes to the gridded colony filters produced around four or more positive clones. A probe representing the whole of the 1.2-Mb chromosome 2, excised from a preparative gel following pulsed-field gel electrophoresis, revealed between 80 and 100 positive clones.

Animals↗