BSSP 2000 Spring Meeting University of Birmingham 17-19 April 2000 - Invited Papers
Plenary Talk
The Role of Protozoa in Aquatic Food Webs.
Hartmut Arndt, University of Cologne, Zoological Institute, Department of General Ecology and Limnology, D-50923 Koeln (Cologne), Germany.
Though Barry entitled my talk with "A Millennial Overview", I will only try to illustrate the changes regarding our knowledge on the role of heterotrophic protists for the last century. Most examples will come from heterotrophic flagellates, my favourite group. Stimulated by developments in other fields of biology and ecology in the beginning of the century most recent discoveries in protozoology were mainly possible due to the availability of new techniques. These methodological possibilities are still developing and further insights are expected from the wide application of new molecular biological techniques in the near future. Protists offer fantastic possibilities to investigate basic questions of general ecology. It was not just by chance that Gause used ciliates as his laboratory animals. However, only recently there is a renaissance of protists as laboratory organisms of general ecologists. This is due to the fact that ecologists have shown that protozoans are not only the most abundant animals on the earth but they are also the most productive animals on our planet. The biodiversity of the various protozoan groups is as different as their functions in aquatic ecosystems. I will try to review some of these major functions.
Amoebae
Morphotypes of amoebae and iconographic keys as a new tool for identification of these protists in ecological studies.
Alexey V. Smirnov Department of Invertebrate Zoology, Fac. of Biology & Soil Sciences, St.Petersburg State University, Russia.
Gymnamoebae (naked amoebae) is a non-systematic group of protists with dynamic, polymorphic body shape. It is a source of endless troubles for scientists studying biodiversity, ecology and systematics of protists. Experience of nearly three centuries of amoebae studies shows that classical morphology - the background of classical systematics, created and developed for and aimed at the description of static objects - is almost powerless when applied to dynamic objects like gymnamoebae. This is clearly reflected in the current situation with gymnamoebae studies. Absence of a descriptive morphological language for dynamic objects forces us to search for other ways to characterise amoebae. However any way must still utilize morphological distinction, as it remains the primary tool to recognise active biodiversity in natural habitats and currently can hardly be replaced with non-morphological methods.
One of the basic problems in amoebae study is species characterisation and construction of keys. Experience of recent 30 years indicates that pure LM systematics of gymnamoebae may work at higher levels of identification only, but is not reliable for species distinction. LM/EM systematics works well at the level of genera and species, but it is laborous and in fact replaces identification of an organism with the identification of a clone (or strain). All attempts to construct classical, dichotomic keys based on both EM and LM features failed or resulted in artificial, descriptive dichotomy, especially at higher levels of identification. Perhaps we should not try to apply classical approaches to the characterisation of species and key organisation for these protists, but to search for non-traditional ones in related fields of science.
Locomotive forms of amoebae have several more or less recognisable features, like general outlines, pseudopodial pattern, subpseudopodia, uroidal structures, dorsal folds. Combinations of these features are realised in the existing morphological diversity. Trying to perform overall analysis of the locomotive forms of amoebae we may find that the diversity is high, but not endless. Only few of hundreds of possible combinations are realised. A total of 20 morphotypes of amoeboid protists, encompassing all known species diversity of amoebae, are suggested. Basing on these morphotypes - general visual appearances of amoebae - it is possible to construct an iconographic key to gymnamoebae, which allows us to identify these protists using more natural way of the comparison of visual appearances of the cells rather than the hardly formalisable "key features" at higher levels of identification. Morphotypes do not replace traditional LM/EM methods, which are still required for reliable species identification. They allow to effectively determine the range of possible species for every examined specimen, which is very important in field ecological studies. Technical difficulties of construction of such key, which suggest an extensive use of images, graphical elements and cross-references may be overcome by usingthe hypertext document structure. Special construction of the interface allows to provide much more information in "3-dimensional" structure of the key than classical approaches. A prototype of the key is available at http://now.ifmo.ru/amoebae.htm.
The taxonomy of vahlkampfiid amoebae: new insights arising from ribosomal DNA analyses.
Johan De Jonckheere, Scientific Institute of Public Health - Louis Pasteur, Brussels and Susan Brown, Culture Collection of Algae and Protozoa, Centre for Ecology and Hydrology, Windermere.
In common with other amoeba lineages, the current classification scheme for the family Vahlkampfiidae is based on morphological and ultrastructural characters. Due to their relative lack of distinguishing features, it has been difficult to determine evolutionary relationships between vahlkampfiid amoebae, to identify new isolates and to determine the degree of difference which indicates an intergenic or intragenic relationship between species.
Identification of vahlkampfiid species is particularly important because of the ubiquitous distribution of the pathogen Naegleria fowleri, which is morphologically indistinguishable from non-pathogenic Naegleria species. A range of biochemical and molecular biology methods have been used to detect the pathogen in environmental samples. As a consequence, many more Naegleria species have been differentiated. We present how ribosomal DNA sequence comparisons have differentiated 17 new Naegleria species over the last 17 years and the consequential need to modify the original, phenotype-based, definition of the genus.
The only other multi-species vahlkampfiid genus, Vahlkampfia, has also been investigated. This genus was defined by two negative characters and, in contrast to the genus Naegleria, Vahlkampfia species are morphologically diverse. We found that Vahlkampfia species do not form a discrete cluster in ribosomal DNA-based phylogenetic trees; some species appear to be closely-related to other genera. Consequently, we propose a new classification scheme for these species.
Testate protozoa: major contributors to deep-sea biodiversity?
Andrew J. Gooday Southampton Oceanography Centre, Empress Dock, European Way, Southampton, SO14 3 ZH, UK.
The ocean floor is the most expansive environment on earth and also the least well studied. During the 1960s, our understanding of the communities living in this remote habitat was revolutionized by the discovery that they are locally highly diverse. Since then, much effort has been devoted to documenting the scale of deep-sea biodiversity and clarifying the mechanisms, which help to maintain it. The metazoan macrofauna has been the main focus of attention and less is known about protozoan and meiofaunal diversity. Our knowledge of testate protozoa, however, has improved considerably during recent years. Several groups, particularly the foraminifera, probably play an important ecological role in deep-sea ecosystems and locally are highly diverse. Well over 100 morphospecies of smaller foraminifera (meiofaunal size range; i.e. 32-500µm) may be present in a single small core sample. Larger foraminifera (macrofaunal size range; i.e. >300 or 500µm) are also rich in species. Local foraminiferal diversity is probably at least comparable to that of nematodes (meiofauna) and polychaetes (macrofauna) and probably reflects the ecological versatility of these highly successful protozoans. Many species are undescribed and belong to delicate soft-shelled taxa , which have been overlooked routinely in deep-sea studies.
Other kinds of testate amoebae live in the deep sea. The best known are the xenophyophores which can reach spectacular dimensions (15cm or more). In addition, gromiids (Order Filosea) have been recognised recently in bathyal habitats. Xenophyophores are much less diverse than foraminifera (less than 10 species typically occur together at a single site) and only one deep-sea gromiid species has been described so far. However, these very large protozoans are abundant, and probably ecologically important, in eutrophic areas of the deep sea.
Environmental Relations of the Amoebo-Flagellate Tetramitus rostratus.
Stephen Coupe and Humphrey G Smith, Environmental Sciences, Coventry University, Coventry, CV1 5FB
Tetramitus rostratus is a cosmopolitan amoebo-flagellate with three clearly defined physiological forms, amoeba, flagellate and cyst. The effect of five different temperature regimes on T. rostratus was assessed in terms of its growth rates, cardinal temperatures and encystment responses.
Growth was seen at all temperatures between 4 and 30°C with optimum growth at 25°C. Extrapolated minimum and maximum temperatures were -3°C and 32°C. T. rostratus may be described as a facultative psychrotroph with the ability to grow at low temperatures. Encystment was seen at all temperatures with a peak encystment of 29% at 4°C; temperature was thought not to be an exclusive trigger for encystment at the regimes studied. Effective growth at low temperatures, flagellate dispersal and an encystment capability are advantageous in soils which experience extremes of temperature, moisture and nutrient availability.
Acanthamoeba
Simon Kilvington Department of Microbiology & Immunology, Medical Sciences Building, PO Box 138, University Road, Leicester LE1 9HN, tel: +44 (0)116 252 2950, fax: +44 (0)116 252 5030.
Acanthamoeba is a genus of small free-living amoeba characterised by a trophozoite and cyst stage. The organism is common in the environment and can be isolated from virtually all soil and aquatic sites. Keratitis caused by Acanthamoeba is a rare but sight-threatening infection most commonly seen in contact lens wearers. Poor hygiene practices such as rinsing or storing lenses in tap water and defaulting on lens disinfection procedures are recognised risk factors in acquiring the disease. Acanthamoeba have also been shown to support the intracellular growth and survival of human pathogenic bacteria. Current research interests include: the epidemiology of acanthamoeba keratitis particularly in relation to domestic tap water; the efficacy of contact lens disinfectants and potential therapeutic agents; the application of molecular DNA typing methods in the identification and differentiation of acanthamoeba keratitis strains; the role of Acanthamoeba is the ecology and pathogenicity of the bacteria Legionella pneumophila, Listeria monocytogenes and Burkholderia cepacia. The findings of these studies will be summarised.
Amoeboid movement: an overview.
Terry M. Preston, Biology Department, University College London, Gower Street, London WC1E 6BT.
The locomotion of amoebae requires an actin-based cytoskeleton. This is extremely dynamic undergoing constant reorganisation as the cell moves through its own length. In the case of Naegleria this may only take 20 seconds; for Acanthamoeba 1-2 minutes. The presence of a gel-like cortex enclosing a less viscous endoplasm is characteristic of amoebae. Considering the most simple model, based on a limax amoeba, movement occurs by the anterior protrusion of a single pseudopodium at a time. A key feature of cytoplasmic organisation is the cortical gel tube. During locomotion this grows anteriorly by recruitment of material from the endoplasm. Simultaneously the tube disassembles at the uroid, its constituents returning to the more fluid endoplasm. Growth and breakdown need to occur at similar rates. There has to be a propulsive mechanism driving the endoplasm forwards to balance loss & recruitment of the gel tube building blocks. Effective motility requires the gel tube to be stabilised by anchorage, via the cell membrane to the substratum. It follows that the process of formation anteriorly and the breakdown posteriorly of cell-substratum adhesions must be integrated into the cyclic growth of the cortical tube. Some of the questions emerging from this model will be addressed.
Gliding motility versus crawling: a cost benefit analysis.
Conrad King, Dept. of Biology, University College, Gower Street, London WC1E 6BT
Within the Protozoa 2 types of substratum -dependent motility can be recognised:
- Crawling movement in which the cell shape is changing as locomotion procedes.
Amoebae provide good examples of this and will be illustrated using Acanthamoeba and Naegleria . Naegleria amoebae (20mm long) can move at a speed of about 1mm sec-1 so a minimalist model would suggest the cytoskeleton would turnover once every 20 seconds. A maximum value for recorded amoeboid movement is in the range of 1-2mm sec -1 ( excluding Amoeba!). There is large scale deformation of the ventral surface to accomodate the required cell-substratum adhesion. - Gliding movement in which the cell shape remains constant.
Apicomplexan protozoans provide good examples of gliding with the gregarine Porospora gigantea achieving rates of 40-50mm sec-1. Eimeria tenella sporozoites (length 12mm) can move a cell length in less than a second. Using reflexion interference microscopy there was no evidence for flattening on the ventral surface during gliding . A cell surface motor system could demonstrated using latex beads.
These 2 strategies will be appraised in the context of biological function.
Vaccines against Protozoa
Vaccination against malaria.
Stephen Phillips. Infection and Immunity, University of Glasgow, Glasgow, G12 8QQ.
There are more cases of malaria than 30 years ago, the result of a combination of factors such as drug resistance, insecticide resistance, political unrest, migrations and lack of resources to devote to malaria control activities. A role for a malaria vaccine has been identified in combating the disease and the principal target is Plasmodium falciparum, the parasite responsible for the majority of the 2-3M deaths annually from the disease. The expectation is that, unlike most of the vaccines currently in use in humans, malaria vaccine(s) will be subunit vaccine(s). The malaria genome is thought to carry 5000-7000 genes and the challenge is to identify from these the major proteins capable of inducing a protective immune response.
Three vaccine targets are under investigation: pre-erythrocytic stages, asexual erythrocytic stages, and sexual stages (transmission blocking). It is possible to induce complete protection by immunization with irradiated sporozoites but this is impractical on a large scale. The challenge is to understand the immune mechanisms induced by the irradiated sporozoites and generate them with subunit vaccines. To date the most promising are a recombinant vaccine based on the circumsporozoite protein repeat region linked to a hepatitis B subunit with a novel adjuvant, and the so-called 'prime boost strategy' in which a DNA vaccine carrying pre-erythrocytic stage sequences is given first and is followed by a modified vaccinia virus carrying the same sequences.
A number of asexual stage antigens have been identified as vaccine candidates including merozoite surface antigen 1 (MSP1), apical membrane antigen and erythrocyte binding protein. The native MSP1 protein has given complete protection in monkeys with Freunds adjuvant, an adjuvant not used in people. One asexual stage vaccine, SPf66, devised by Manuel Patarroyo in Colombia, has been through extensive field trials with varying degrees of success but the best is still too low to warrant widescale use of this product.
Several sexual stage antigens are being targeted. One, Pfs25, is an ookinete surface antigen and recombinant forms of this antigen are showing good promise. Unfortunately immunity induced by this antigen will not be boosted by infection.
The review will start with a brief immunolgy lesson for the non-immunologists, a little background on vaccines currently in use in man, a resume of the perceived potential of the different forms of antimalarial vaccines and a discussion of the particular difficulties which have been and continue to be faced by researchers pursuing malaria vaccines.
The immunobiology and vaccine potential Leishmania mexicana cysteine proteinase.
Jim Alexander. Department of Immunology, University of Strathclyde, 31 Taylor Street, Glasgow, G4 ONR.
We have previously demonstrated that Leishmania mexicana cysteine proteinase-deficient mutants have reduced ability to infect mice. Moreover, they induce a strong Th1 response and have proved to be good vaccine candidates in some, though not all, mice strains. Our recent studies indicate that wild-type mice induce non-healing disease by inhibiting IL-12 production and depending on the site of infection inducing IL-4.
Cysteine proteinase-dependent mutants while apparently being able to initially infect mice in a manner similar to wild-type parasites have reduced ability to limit IL-12 production and lesions if formed heal.
As well as examining the vaccine potential of the cysteine proteinase mutants we are examining the potential of the recombinant cysteine proteinase to vaccinate mice against Leishmania mexicana.
Intestinal Coccidiosis: Immunoprotective Mechanisms and Vaccination.
Adrian L. Smith1 and Martin W. Shirley2 Divisions of Immunology1 and Molecular Biology2, Institute For Animal Health, Compton Laboratories, Compton, nr Newbury, Berkshire, RG20 7NN.
Intestinal coccidiosis is caused by infection with Eimeria spp (phylum Apicomplexa) and is an important disease of intensively reared livestock, particularly poultry. As demonstrated by the success of a live attenuated vaccine the immunological approach is a viable alternative to prophylactic drug treatment for the control of coccidiosis. However, due to a variety of reasons (including cost) a large proportion of the industry has yet to convert to vaccination. We are working toward the development of the next generation of vaccines that will impact the whole industry and we will discuss some of the practical difficulties and research requirements to achieve this goal.
One important area is to determine the cell types and mechanisms of immunity to Eimeria spp. and we will draw upon some of our recent work with E. vermiformis to illustrate our knowledge of immune protection. Briefly, immunity is dependent upon TCR??+ T cells while the TCR??+ subset of T cells is required for prevention of immunopathology. During primary infection, CD4+ MHC class II-restricted T cells are the most important immune cell type that limit infection via an IFN?-dependent mechanism. Moreover, recent data suggest that all the effect of IFN? is direct and mediated by interaction with the IFN?R on the enterocyte. Other immune cells such as B cells and T cells restricted to ?2m-dependent, TAP 1-independent antigen presentation pathways play a supporting role during immune-mediated resistance to primary infection. In contrast, in the immune animal both MHC class I-restricted (CD8+) and MHC class II-restricted (CD4+) T cells mediate the host response to infection. Thus, we can target multiple types of T cell response when designing the next generation of vaccines against coccidiosis.
Toxoplasma gondii and Neospora caninum: immune responses and prospects for vaccination.
Elisabeth A. Innes, Moredun Research Institute, Pentlands Science Park, Edinburgh EH26 OPZ.
Toxoplasma gondii and Neospora caninum are two closely related protozoan parasites that cause significant disease in both man and animals. T. gondii is a very sucessful parasite capable of infecting all warm blooded animals, causing a wide spectrum of disease ranging from acute severe illness (often proving fatal) in animals such as marsupials and new world monkeys to animals such as sheep, goats and pigs where infection during pregnancy can cause severe disease of the foetus, through to cattle and horses who are very resistant to the disease.Toxoplasmosisis also a major concern to pregnant women who may transmit the disease to the developing foetus with devastating consequences. As T.gondii is such a ubiquitous parasite large numbers of the population are infected. This can pose problems inimmunosuppressed individuals where recrudescence of the parasite can also cause severe disease. Controlling the disease by vaccination is a real possibility as following a primary infection most individuals remain persistently infected for life and develop an effective protective immunity. The main problem areas in designing effective vaccines are: the identification of relevant antigens, relevant immune responses and methods to induce and maintain this responsiveness. In any host-parasite relationship it is unlikely that any one anti-parasite effector mechanism operates in isolation to effect host resistance. The net effect of any one component of the spectrum of immune responses induced by the various parasite life-cycle stages will either be; host protective, parasite protective, irrelevant or harmful to the host. The immune responses induced by a vaccine need not necessarily cover the whole spectrum of immune responses; the effect of a few key responses may tip the balance in favour of the host. Considerable work has been done examining the host immune response to T.gondii including the role of innate immune responses very early in infection involving NK cells and macrophages resulting in the production of the cytokine interferon gamma which is pivotal in host resistance. The pro-inflammatory cytokines are also important in the induction of the adaptive immune response involving Th1 CD4 T-cells and CD8 Tcells. Several immunodominant and stage-specific antigens have been identified and and some have been studied further as potential vaccine candidates. Clearly vaccines to prevent abortion or foetal damage are a high priority but we should also consider vaccines designed to reduce exposure to the parasite such as those used in cats to reduce oocyst shedding or in meat producing animals to reduce the numbers of cysts. A review of the current vaccine strategies against T.gondii will be given along with new approaches looking at delivery systems to induce appropriate immune responses using killed antigens and the use of genetic immunisation.
Neospora caninum is emerging as a major cause of abortion in cattle worldwide and is also known to cause disease in congentially infected dogs. There is no conclusive evidence, to date, that N.caninum can infect and cause disease in people. Less is known about the immune response to N.caninum but work looking at experimental infections in mice and cattle have shown the importance of pro-inflammatory cytokines and T-cells. Unlike the situation with T.gondii, cattle seem to develop only partial protective immunity following infection with N.caninum and evidence from field studies shows that cattle may have repeat abortions due to neosporosis although the numbers are low (less than 5%). The immune response to N.caninum will be reviewed along with strategies looking at vaccine development against this disease.
Finally, it is known that there are commonly recognised antigens between T.gondii and N.caninum. Is there any cross-protective immunity between these two parasites?