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Category: Fungi and Fungal Pathogenesis
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Microsporidia are increasingly recognized as important parasites of all members of the animal kingdom, causing serious life-threatening diseases in humans and important economic losses in the fish and agriculture industries. This new book, the first since 1976 to address all aspects of microsporidia and microsporidiosis, provides biologists, physicians, and research scientists with a complete, modern view of this increasingly important group of parasites. Written by the foremost experts in the field, The Microsporidia and Microsporidiosis addresses structure, molecular biology, pathology, clinical disease in humans and animals, diagnosis, and treatment. It is copiously illustrated, with many color figures, and includes the latest methodologies for the study and diagnosis of microsporidia, as well as in vitro cultivation methods. Comprehensively referenced, the volume includes sources not available on MEDline.
Electronic Only, 553 pages, glossary, index.
The polar filament is the defining character of the microsporidia, and its function has attracted the attention of many investigators. Microsporidian species have been reported to infect nearly all of the invertebrate phyla, including such unicellular organisms as ciliates and gregarines, myxozoans, cnidarians, platyhelminths, nematodes, rotifers, annelids, molluscs, bryozoans, and arthropods, as well as all five classes of vertebrates; the greatest numbers of species infect arthropods and fish. Microsporidia are obligate intracellular parasites of eukaryotes, the transmittable stage being a resistant spore which is usually small, possesses a thick wall, and contains a characteristic polar tube apparatus. Glugea anomala, which causes subcutaneous cysts in fish (e.g., stickleback), was the first microsporidian parasite recognized to cause disease in vertebrates. Subsequently, other species such as Pleistophora spp and Nucleospora (Enterocytozoon) salmonis were implicated in sporadic but serious disease outbreaks among fish populations. These outbreaks have had an important economic impact on tropical freshwater fish, commercial fish farming, and the sport fishing industry. Once it became clear that human microsporidiosis was largely a disease of immuno-incompetent individuals, particularly those with AIDS, additional species were recognized that also caused disseminated infection. In addition, protozoan thymidylic synthetase and dihydrofolic acid reductase, which are present in protozoa as separate epitopes on the same protein, are found as separate proteins in the microsporidia as they are in yeast.
Classification of microsporidia is based on life cycle and structural characters. This chapter describes the structure and application of specific techniques, which helps to determine an organism is a microsporidian. The cell was interpreted to be a uninucleate gamete destined for fusion with another such cell to restore by plasmogamy the diplokaryotic state of a meront. When the cells are covered by the electron-dense material, they are classified as sporonts, the initial stage of sporogony. Whether Encephalitozoon intestinalis rightfully belongs to the genus Encephalitozoon or should be retained in its original genus, Septata. The genus Encephalitozoon is monomorphic, and single nuclei occur in all life cycle stages. Especially in the case of microsporidia from water-dwelling invertebrate hosts, the spores bear ornamentations or mucous layers on their surfaces that can be used as taxonomic characters. The size of spores is an important structural character, but its exact recording is difficult, especially in microsporidia with very small spores. Measuring spores on photographs, made with a carefully calibrated microscope and photographic enlarger, is a suitable alternative if a special eyepiece or image analyzer is not available.
This chapter reviews the morphological features and development of the microsporidia. The general life cycle pattern of the microsporidia can be divided into three phases: the infective or environmental phase, the proliferative phase, and the sporogony or spore forming phase. The chapter discusses the host-parasite interface during parasite development. Microsporidia with host-produced interfacial envelopes are those that are separated from the hyaloplasm by host-produced membranes during proliferation through spore formation. Microsporidia having indirect contact between host- and parasite-produced interfacial envelopes are separated from the hyaloplasm by membranes and/or secretions produced by both the host and the parasite. The proliferative phase has been referred to as schizogonic and merogonic by some authors, however, different authors have assigned different types of nuclear activity to the terms merogony and schizogony with respect to the microsporidia. Recognizing the diversity of polaroplast morphology and organization in various species of microsporidia, Larsson described five types of polaroplast structural arrangements. In addition to environmental spores, some microsporidia produce autoinfective spores which become activated and germinate within the same host in which they were just produced. A few microsporidia of the genus Amblyospora have two host cycles with morphologically different spores in each and different development in the males and females of the same host species.
The recognition of microsporidia as opportunistic pathogens in humans has led to increased interest in their molecular biology. Much of the recent work has focused on determination of the nucleotide sequences for rRNA genes. These sequences have been used in the development of diagnostic tools for species identification. In addition, such rRNA gene sequences have facilitated the development of a molecular phylogeny of these organisms. Given the increasing evidence of the importance of the microsporidia as both human and agricultural pathogens, recent work has focused on the identification of microsporidial genes that could serve as potential therapeutic targets. Prior to the advent of comparative analysis of molecular data, the classification of eukaryotes was based on morphological, ecological, and physiological characteristics. Speculation based on this information is problematic because the homology among such characters is often not discernible and characters that could be considered were thought to be either shared or derived depending on the underlying hypothesis used in construction of the phylogeny. A much different view of the phylogenetic placement of the Microspora has been suggested based on analysis of β-tubulin genes. The molecular data present an excellent means of identifying a species and provide an excellent data set for proposing evolutionary relatedness through phylogenetic analysis. Molecular techniques for the diagnosis of microsporidiosis appear to have high sensitivity and specificity. These methods have proven extremely useful both in the identification of animal models as well as in investigations of the epidemiology of microsporidia pathogenic to humans.
The microsporidia are a large group of highly specialized obligate intracellular protozoan parasites. Disturbances in the biochemical composition of tissues infected by intracellular parasites are of interest because such infections often significantly alter the electrolyte, carbohydrate, protein, and free amino acid pools of host cells. Until recently, biochemical investigations of the metabolic processes of the microsporidia have suffered because of insufficient numbers of the different parasite stages and inadequate methods for the in vitro cultivation and maintenance of these organisms. The authors have observed the disappearance of glycogen granules in the host cell cytoplasm during the early stages of parasite development without any concomitant change in the size or quantity of lipid droplets. The microsporidian species Spraguea lophii is a model for investigating externally mediated signal transduction and subsequent activation of the internal signal pathway for triggering of a missile cell, the microsporidian spore. Microsporidian meront stages appear to have actin-myosin and kinesin-associated molecular motors. This chapter focuses on keratin filaments found in two domains: (i) within the spore stage, the microsporidian sporophorous vesicle, and (ii) keratin in the host cell cytoplasm domain but situated at its interface with the parasite. Recently, however, some success has been achieved in isolating and maintaining meronts and discharged sporoplasms in extracellular support medium. Developing a simple in vitro model will be useful for many subsequent biochemical analyses.
The spores of microsporidia possess a unique, highly specialized structure, the polar tube, which is used to inject the parasite from the spore into a new host cell. Several theories have been proposed regarding the method by which the sporoplasm exits the spore and on the function of the polar filament or tube in this process. Electron-dense, particulate material fills the center of the filament. Weidner proposed that this material was unpolymerized polar tube protein (PTP). On the basis of ultrastructural observations, the eversion of the polar tube has been likened to a tube sliding within a tube. This chapter presents details on spore activation and discharge. When sporoblasts form, each one contains five to six coils of the preformed polar filament with the anchoring disk positioned at the anterior end. The major amino acids coded by the Encephalitozoon hellem and Encephalitozoon cuniculi PTP genes were proline and glycine. Application of the techniques of modern biology has resulted in the identification of several PTPs although the interactions and functional significance of these proteins remains to be determined.
This chapter describes the host-parasite relationships in animals with microsporidiosis and how they relate to microsporidian infections in humans. The majority of immunologically competent hosts who became infected with microsporidia (e.g., rabbits and mice) developed chronic and persistent infections with few clinical signs of disease. At least three levels or stages of defense mechanisms are expressed to prevent or control infection with most microorganisms. These are innate resistance, early induced responses, and adapted immune responses. The majority of the microsporidian spores appeared to be destroyed after phagocytosis, although some microsporidia taken up by phagocytosis had extruded their polar filaments into the cytoplasm of the macrophage. Microsporidia that infect mammals often persist in the face of innate resistance, early induced response, and adapted immune response mechanisms. The clinical manifestations of microsporidiosis in animals, however, have been highly predictive of the disease in humans, and a competent immune system has been shown to be important in preventing lethal disease. A balanced host-parasite relationship developed in many animals with microsporidiosis that displayed few clinical signs of disease yet carried persistent infections. A better understanding of the immune responses that establish a balanced host-parasite relationship in animals will likely prove beneficial in understanding the immunology of microsporidiosis in humans and in developing immunotherapeutic and chemotherapeutic strategies that can be applied to microsporidiosis.
This chapter describes the clinical and pathogenic features of microsporidiosis. It mainly focuses on intestinal disease. Most reports of intestinal microsporidiosis have involved human immunodeficiency virus (HIV)-infected individuals. Although the majority of cases have been diagnosed in homosexual males, diagnoses also have been made in heterosexual women and in children. Enterocytozoon bieneusi has been identified in patients with other immune deficiencies, as well as in immuno competent individuals who are asymptomatic or have a self-limited diarrheal illness. Microsporidia were originally believed to cause intestinal disease on the basis of their identification in abnormal small intestinal mucosa of severely immunosuppressed AIDS patients with chronic diarrhea and weight loss. The cardinal feature of intestinal microsporidiosis is injury to the small intestinal epithelium, leading to malabsorption. The various stages in the life cycle of E. bieneusi and Encephalitozoon intestinalis have been characterized by transmission electron microscopy (TEM) in several laboratories. Intestinal microsporidiosis affects the topography of the small intestinal surface. E. bieneusi is most often observed in the upper third of the villus and not in the crypt. There is no acute inflammation. Two agents, fumagillin and albendazole, have been shown to have activity against microsporidia both in vitro and in vivo. In contrast to E. bieneusi, E. intestinalis has a uniformly excellent response to albendazole therapy.
One of the difficulties in understanding the epidemiology of ocular microsporidiosis is the constantly changing nomenclature and the difficulty in speciation. The clinical and histopathologic appearance of ocular microsporidiosis can be divided into two distinct patterns of infection, one involving the deep corneal stroma and seen in immunocompetent patients, and the other a superficial epithelial keratopathy infection seen in immunodeficient individuals. Both deep stromal and superficial epithelial keratitides due to ocular microsporidiosis are rare but must be considered in situations where these entities do not respond to conventional therapy. Ocular microsporidiosis has emerged as the cause of bilateral keratoconjunctivitis in immunosuppressed individuals. The differential diagnosis of microsporidial infection causing stromal disease is broader; diagnosis is unlikely based on clinical examination. Microsporidia are sufficiently unique to be classified as a separate phylum. The early light microscopy (LM) findings of ocular microsporidiosis were instrumental in localizing and recognizing these organisms in other tissues. Both isolated, deep stromal keratitis and superficial epithelial keratitis caused by microsporidia are rare. However, superficial keratitis is important, as it may be the initial manifestation of systemic microsporidia infection. Therapy, either topical or systemic, can be visually and pathologically evaluated with minimal morbidity.
The most robust and widely practicable technique for the diagnosis of microsporidial infection is light microscopic morphological demonstration of the organisms themselves. Evaluation of patients with suspected microsporidiosis should begin with light microscopic examination of stool specimens and urine or cytological examination of other body fluids. The most common clinical finding of ocular microsporidiosis is keratoconjunctivitis. In patients with suspected ocular microsporidiosis, urine and respiratory secretions also should be examined for microsporidia. In patients with ocular microsporidiosis, urine and respiratory secretions should also be examined for the presence of microsporidial spores. Serologic assays (including carbon immunoassay [CIA], IFAT, enzyme-linked immunosorbent assay [ELISA], and Western blot [WB]) have been applied in detecting specific IgG antibodies directed to Encephalitozoon intestinalis spores in humans and to Encephalitozoon cuniculi spores in humans and several animal species. Aside from studies on ocular microsporidiosis in presumably otherwise healthy persons, detailed histopathologic investigation of human microsporidial infection has been performed only in immunodeficient individuals. One clue that microsporidiosis may be present is based on the observation that infected epithelial cells containing mature spores are often shed intact into the lumen from the underlying basement membrane. Upper and lower respiratory tract infections due to microsporidia are associated almost exclusively with disseminated disease produced by all three members of the genus Encephalitozoon. Microsporidia have been found in almost every organ system. Molecular techniques have been used to compare microsporidial isolates obtained from humans and animals in order to study possible sources of infection and reservoir hosts.
Isolation in culture should always be attempted even when a presumptive diagnosis has been made. This is particularly important for the purpose of establishing a bank of isolates to be used for antigenic, molecular, and biochemical analyses. The first attempt to culture microsporidia was made in 1937 by Trager, who was partially successful in establishing Nosema bombycis infection of a cell culture developed from the ovarian tube lining cells of the silkworm (Bombyx mori). Cultures of Encephalitozoon cuniculi were initiated in several different ways: by adding infected tissue explants to cultured cells, by allowing infected cells in explanted cells to grow, by allowing germination of spores in the presence of cells and thereafter infecting cells, and by scraping infected cells from infected cultures and adding them to fresh cell cultures. The first microsporidian isolated from a human, Vittaforma corneae (Nosema corneum) was cultured from a corneal biopsy that was shipped to the laboratory overnight in Hanks' balanced salt solution (HBSS). Only a few attempts have been made to isolate microsporidia into culture from feces because enteric bacteria and yeast usually overgrow the rich culture medium that is used, which impedes isolation of the fastidious microsporidia, especially Enterocytozoon bieneusi. Inoculated cell cultures should be examined frequently with an inverted microscope preferably equipped with phase-contrast or differential interference-contrast optics. One great advantage of obtaining spores from in vitro cultures is the absence of bacterial and fungal contaminants.
Encephalitozoon cuniculi was first reported in rabbits and laboratory rodents in the early 1920s, and this species remains the most frequently observed organism in nonhuman vertebrates. Serological surveys reported prevalence rates as high as 80% in hamsters, 30% in rats, and 85% in guinea pigs. This chapter describes microsporidial infections in a variety of domestic mammalian hosts such as mice, rats, guinea pigs and hamsters, and wild mammalian hosts such as rodents and rabbits. It discusses a few cases of naturally occurring microsporidiosis that have been reported in nonhuman primates. The chapter also talks about microsporidial infections in dometic animal hosts and wild carnivores. Until recently, the species of microsporidia in avian tissues had not been determined, and no parasite species had been categorized as an avian pathogen. Only a few cases of naturally occurring microsporidiosis, including several different parasite genera, are reported in amphibian or reptile hosts.
Microsporidia of fishes are widely distributed by both host species and geographic location. Whereas most fish microsporidia are host specific, at least at the genus level, a few show broad host specificity. This chapter provides a general overview of economic importance, immunology, and treatment, and reviews the most important genera. Pleistophora hyphessobryconis Shaperclaus, 1941, well known to fish hobbyists as the source of neon tetra disease, is one of the most common parasites of aquarium fishes. Presently, hostparasite relationships of fish microsporidia can be broadly classified into two groups: those associated with xenoma formation (e.g., Glugea) and those associated with non-xenoma forming microsporidia (e.g., Nucleospora). Species of Glugea are among the most intensively studied fish microsporidia. They infect the submucosal intestinal cells of a variety of wild and cultured fish. Glugea spp., like most fish microsporidia, are transmitted directly via ingestion. In recent years there has been increased research interest in fish microsporidia along with an increase in the economic importance of microsporidia in fish culture.
This chapter highlights the biological and life cycle features of entomogenous microsporidia and provides some basic information on their taxonomic distribution. The development of all insect-parasitic microsporidia is restricted to the cytoplasm of the host cell. The development of all insect-parasitic microsporidia is restricted to the cytoplasm of the host cell. Specialized relationships between microsporidia and the host at the cellular level have been termed xenomas. Weiser distinguished two main types of xenomas in insects, syncytial and neoplastic. The chapter presents the major and minor pathways of transmission for insect microsporidia. Alternatively, the criteria used to establish the genera of microsporidia from aquatic insects may not truly reflect phylogenetic diversity but adaptations to specific habitats and host systems. A section lists a few genera for which insects are not the type hosts. This is because many species have been reported to occur in insects (such as Thelohania, Pleistophora) or because there are possible links to insect microsporidia (such as Trichotuzetia). Finally, the chapter provides diagnostic information which is primarily restricted to the features of sporulation and the spore, with the addition of distinguishing life cycle characteristics when available. The type host and species are given followed by comments on distribution and other matters deemed of importance.
This chapter focuses on the epidemiology of human microsporidiosis. The coverage of the chapter includes prevalence and geographic distribution, case demographics and populations at risk, and five potential modes of transmission: zoonotic infections, waterborne transmission, airborne transmission, respiratory tract acquisition and direct person-to-person transmission. Surveys for antibodies to microsporidia in human sera have focused exclusively on human exposure to Encephalitozoon species. Although highly active antiretroviral therapy is a promising treatment regimen for prevention of microsporidiosis in patients with AIDS, long-term follow-up is clearly needed to assess whether these patients are actually free of the intracellular stages of microsporidia or are temporarily in remission with an inactive infection.
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