[PARASITES OF THE COMMON ROACH (RUTILUS RUTILUS L.) UNDER THE IMPACT OF INDUSTRIAL POLLUTION OF A LAKE].

The consequences of man-induced transformation of Lake Kostomukshskoe (tailings dump) related to increased mineralization and entry of highly dispersed dredge material not typical for northern lakes were studied using roach parasites as the example. It was found that the roach parasite fauna has been losing rare and scant parasite species of different taxonomic groups, mainly helminthes with an indirect life cycle. Common and dominant species of myxosporidians, monogeneans, trematodes of genera Diplostomum and Tylodelphys have been preserved. The chances of survival are higher for those parasites with direct life cycle. Also the parasite species whose larvae can actively penetrate the host have been survived. The species Ligula intestinalis, Philometra rishta, Pseudocapillaria tomentosa had reported very rare. Fish are infected with these parasites by feeding of Copepoda and oligochaetes.

[Identification of mosquito-parasitic microsporidia, Amblyospora rugosa and Trichoctosporea pygopellita (Microsporidia: Amblysporidae), from Acanthocyclops venustus and Acanthocyclops reductus (Copepoda: Cyclopidae), based on small subunit rDNA analysis].

Identical small subunit rDNA sequences were obtained for microsporidia Amblyospora rugosa from blood-sucking mosquitoes larvae Ochlerotatus cantans, O. cataphylla and copepods Acanthocyclops venustus, as well as for Trichoctosporea pygopellita from mosquitoes larvae Ochlerotatus cyprius, O. excrucians and copepods Acanthocyclops reductus. The data on molecular phylogeny and ecological researches show that in Siberia mosquito-parasitic microsporidia of the genera Amblyospora and Trichoctosporea have complex life cycle involving likely intermediate hosts, Acanthocyclops copepods. Life cycle of parasites is synchronized with phenology of their hosts. The phylogenetic analyses shows, that genus Trichoctosporea should be transferred from the family Thelohaniidae to the family Amblyosporidae.

Converging seasonal prevalence dynamics in experimental epidemics.

BACKGROUND: Regular seasonal changes in prevalence of infectious diseases are often observed in nature, but the mechanisms are rarely understood. Empirical tests aiming at a better understanding of seasonal prevalence patterns are not feasible for most diseases and thus are widely lacking. Here, we set out to study experimentally the seasonal prevalence in an aquatic host-parasite system. The microsporidian parasite Hamiltosporidium tvärminnensis exhibits pronounced seasonality in natural rock pool populations of its host, Daphnia magna with a regular increase of prevalence during summer and a decrease during winter. An earlier study was, however, unable to test if different starting conditions (initial prevalence) influence the dynamics of the disease in the long term. Here, we aim at testing how the starting prevalence affects the regular prevalence changes over a 4-year period in experimental populations. RESULTS: In an outdoor experiment, populations were set up to include the extremes of the prevalence spectrum observed in natural populations: 5% initial prevalence mimicking a newly invading parasite, 100% mimicking a rock pool population founded by infected hosts only, and 50% prevalence which is commonly observed in natural populations in spring. The parasite exhibited similar prevalence changes in all treatments, but seasonal patterns in the 100% treatment differed significantly from those in the 5% and 50% treatments. Populations started with 5% and 50% prevalence exhibited strong and regular seasonality already in the first year. In contrast, the amplitude of changes in the 100% treatment was low throughout the experiment demonstrating the long-lasting effect of initial conditions on prevalence dynamics. CONCLUSIONS: Our study shows that the time needed to approach the seasonal changes in prevalence depends strongly on the initial prevalence. Because individual D. magna populations in this rock pool metapopulation are mostly short lived, only few populations might ever reach a point where the initial conditions are not visible anymore.

Evolutionary strategies and adaptations for survival between mosquito-parasitic microsporidia and their intermediate copepod hosts: a comparative examination of Amblyospora connecticus and Hyalinocysta chapmani (Microsporidia: Amblyosporidae).

The epizootiology, transmission dynamics, and survival strategies employed by two mosquito-parasitic microsporidia that utilize copepods as intermediate hosts are examined in relation to the biological attributes of their hosts and the environments in which they inhabit. Amblyospora connecticus Andreadis, 1988, a parasite of Ochlerotatus cantator (Coquillett) and Acanthocyclops vernalis (Fischer) is found in an unstable salt marsh environment that is subject to periodic flooding and drying. Both hosts have distinct non-overlapping generations. A. connecticus exhibits a well-defined seasonal transmission cycle that relies heavily on maternal-mediated transovarial transmission by female O. cantator during the summer, and horizontal transmission via the copepod host during the spring (copepod to mosquito) and fall (mosquito to copepod). Its survival strategies include: delayed virulence, low pathogenicity and high tissue specificity that allow for transstadial transmission of horizontally acquired infections and maximum spore production, reliance on living hosts throughout most of its life cycle with overwintering in the copepod, polymorphic development that is well synchronized with host physiology, and production and dissemination of infectious spores that are coincident with the seasonal occurrence of susceptible stages in each host. Hyalinocysta chapmani Hazard et Oldacre, 1975, a parasite of Culiseta melanura (Coquillett) and Orthocyclops modestus (Herrick) is found in a comparatively stable, subterranean habitat that is inundated with water throughout the year. Copepods are omnipresent and C. melanura has overlapping broods. H. chapmani is maintained in a continuous cycle of horizontal transmission between each host throughout the summer and fall but lacks a developmental sequence leading to transovarial transmission in the mosquito host. It relies on living hosts for most of its life cycle and overwinters in diapausing mosquito larvae. Transstadial transmission does not occur and there is no dimorphic development in the mosquito host. The spatial and temporal overlap of both mosquito and copepod hosts during the summer and fall affords abundant opportunity for continuous horizontal transmission and increases the likelihood that H. chapmani will find a target host, thus negating the need for a transovarial route. It is hypothesized that natural selection has favoured the production of meiospores in larval female mosquitoes rather than congenital transfer of infection to progeny via ovarian infection as a strategy for achieving greater transmission success. Analysis of the molecular phylogeny data suggest that (1) transovarial transmission and the developmental sequence leading to ovarian infection have been secondarily lost in H. chapmani, as they occur in all other closely related genera, (2) the ancestral state included complex life cycles involving transovarial transmission and an intermediate host, and (3) mosquito-parasitic microsporidia are adjusting their life cycles to accommodate host ecological conditions.

Review of the sequential development of Loma salmonae (Microsporidia) based on experimental infections of rainbow trout (Oncorhynchus mykiss) and Chinook salmon (O. tshawytscha).

Loma salmonae is a common gill parasite of salmonids, and essentially all species in the genus Oncorhynchus are susceptible. Infections occur in both fresh and salt water. Loma salmonae is directly transmissible by ingestion of spores or infected tissue. The parasite infects the wall of blood vessels of various organs, but the gill is the primary site of infection. Initial infection occurs in the intestine, and xenomas are easily detected in the gills by standard histology at 4-6 wk post-exposure. A few presporogonic stages of the parasite are found in the heart endothelium prior to xenoma formation in the gills. Ultrastructure studies of early infections demonstrated that wandering blood cells transport the meronts to the gills, and that merogony occurs in pillar cells and other cells underlying the gill endothelium. Xenomas develop in these cells, resulting in hypertrophied host cells filled with spores. Xenomas ultimately rupture, and are associated with severe inflammation in which free spores are found in macrophages. The parasites are most pathogenic during this phase of the infection, resulting in severe vasculitis and clinical disease. Both rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus ishawytscha) recover from infections, but free spores persist in kidney and spleen phagocytes for many months after xenomas are absent in Chinook salmon. Fish that have recovered from the infection show strong immunity against the parasite, lasting up to 1 year. Fish are susceptible to infection by other routes of exposure by spores; co-habitation, anal gavage, and intramuscular, intraperitoneal and intravascular injection. Autoinfection probably occurs following release of spores in blood vessels after xenomas rupture. The optimal temperature for L. salmonae infections is 15-17 degrees C, with a permissive range of 11-20 degrees C.

Diagnostic pathology of microsporidiosis.

Microsporidia are ubiquitous spore-forming parasites that are important worldwide pathogens in the HIV/AIDS pandemic. They are also increasingly being seen in HIV(-) individuals. Infection has been documented in almost every tissue and organ in the body and in a broad spectrum of cell types, including epithelial, mesenchymal, and neural. Microsporidia elicit a wide range of pathology, e.g., inflammation and cell death, and symptoms, e.g., shortness of breath, sinusitis, and diarrhea with wasting. Untreated, microsporidiosis has been documented as a cause of death.

Epizootiology of Hyalinocysta chapmani (Microsporidia: Thelohaniidae) infections in field populations of Culiseta melanura (Diptera: Culicidae) and Orthocyclops modestus (Copepoda: Cyclopidae): a three-year investigation.

The epizootiology, transmission dynamics and survival strategies employed by the microsporidium Hyalinocysta chapmani were examined in field populations of its primary mosquito host, Culiseta melanura and its intermediate copepod host, Orthocyclops modestus over a three-year period in an aquatic subterranean habitat. H. chapmani was enzootic and was maintained in a continuous cycle of horizontal transmission between each host. There were three distinct periods during the summer and fall when developing mosquito larvae acquired infections; each was preceded by or coincident with the detection of infected copepods. Results were corroborated in laboratory bioassays, wherein transmission was achieved in mosquito larvae that were reared in water and sediment samples taken from the site during the same time periods. The highest infection rates, ranging from 60% to 48%, were repeatedly observed during the first six weeks of larval development. These were coincident with the most sustained collections of infected copepods obtained during the year and highest levels of infection achieved in the laboratory transmission studies. The high prevalence rates of lethal infection observed in larval populations of C. melanura at this site are among the highest recorded for any mosquito-parasitic microsporidium and clearly suggest that H. chapmani is an important natural enemy of C. melanura. H. chapmani appears to overwinter in diapausing mosquito larvae but may also persist in copepods. The absence of vertical transmission in the life cycle of H. chapmani and the sole reliance on horizontal transmission via an intermediate host are unique survival strategies not seen among other mosquito-parasitic microsporidia. The epizootiological data suggest that this transmission strategy is a function of the biological attributes of the hosts and the comparatively stable environment in which they inhabit. The subterranean habitat is inundated with water throughout the year; copepods are omnipresent and C. melanura has overlapping broods. The spatial and temporal overlap of both hosts affords abundant opportunity for continuous horizontal transmission and increases the likelihood that H. chapmani will find a target host. It is hypothesized that natural selection has favored the production of meiospores in female host mosquitoes rather than congenital transfer of infection to progeny via ovarian infection as a strategy for achieving greater transmission success.

Brachiola algerae spore membrane systems, their activity during extrusion, and a new structural entity, the multilayered interlaced network, associated with the polar tube and the sporoplasm.

The microsporidial genus, Brachiola, contains three species: the type species Brachiola vesicularum (identified from an AIDS patient) and two species transferred from the genus Nosema, becoming Brachiola connori and Brachiola algerae. A developmental feature of the genus Brachiola is the "thickened" plasmalemma from sporoplasm through sporoblast stage. The sporoplasm has been reported to have a thick plasmalemma at 1-h postextrusion. The purpose of this investigation was to observe B. algerae spores before, during and after germination to determine if the plasmalemma is thick at the point of extrusion and if not, when and how it forms. New understandings regarding the polar filament position inside the spore, places it outside the sporoplasm proper with the sporoplasm limiting membrane invaginations surrounding it. These invaginations, present a possible location for aquaporins. The multilayered interlaced network (MIN), a new organelle (possibly of Golgi origin from the sporoblast), was observed inside the spore and sporoplasm; it formed an attachment to the end of the extruded polar tube and contributed to the thickening of the sporoplasm plasmalemma. A thin "unit limiting membrane", present on the sporoplasm at the time of extrusion, is connected to the MIN by many cross-connections forming the "thick blistered" surface by 30 min-postextrusion.

Localization of the initial developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss).

The intracellular microsporidian parasite Loma salmonae affects salmonids of the genus Oncorhynchus and is a significant cause of economic losses in pen-reared Chinook salmon (O. tshawytscha) in British Columbia. Loma salmonae infection is easily recognized by the xenomas that form in the gills, but early stages of infection are difficult to detect in histologic sections. In situ hybridization (ISH), using an L. salmonae-specific digoxigenin-labeled single-stranded DNA probe, was used to detect the parasite during the early stages of infection. Loma salmonae was detected in the gut mucosal epithelium as early as 24 hours postexposure (PE), and it localized in the lamina propria of the intestine within 24 hours of infection. After the parasite was detected in the lamina propria, dividing merogonic stages in infected cells in the heart were detected by ISH as early as 2 days PE, providing the first evidence of parasitaemia and hematogenous distribution of this parasite in infected blood cells. The parasites inside the infected cells appeared to be undergoing merogony as they passed through the heart, indicating that proliferation may start at the site of infection, before the parasite arrives to the gills for their final developmental phase. This is the first time that L. salmonae passage through the intestinal wall and migration to the heart has been visualized; however, the identity of the cells harboring the parasite has yet to be determined.

The human isolate of Brachiola algerae (Phylum Microspora): development in SCID mice and description of its fine structure features.

Ocular, peroral, intraperitoneal, intramuscular, and subcutaneous inoculation of severe combined immunodeficient (SCID) mice with spores of the human isolate (CDC: V404) of Brachiola algerae (syn. Nosema algerae) (Phylum Microspora) revealed that the microsporidium develops in viscera of the immunodeficient mouse host, but only after the ocular administration of spores. It is hypothesized that the physico-chemical milieu of the conjunctiva and cornea helped to adapt the originally 'poikilothermic microsporidian' to the conditions within the homoiothermic organism. Ocular application of spores caused no clinical signs of disease at the application site. However, severe infection in the liver was found 60 days after infection, manifested as hepatosplenomegaly and multifocal miliary necroses and granulomas containing parasites. No microsporidia were found in any other tissues. Transmission electron microscopy revealed characteristic tubulovesicular 'secretory materials' on the plasma membrane of all developmental stages of B. algerae except sporoblasts and spores. These formations increase the parasite surface and allow more efficient metabolic communication of the parasite with the host cell. It is hypothesized that the presence of these structures is a factor helping the parasite to grow in a variety of hosts and tissues. Ultrastructural characters support the likelihood that B. algerae and B. vesicularum are conspecific, and that there exists a relationship between species of the genera Brachiola and Anncaliia.

Microsporidia and Candida spores: their discrimination by Calcofluor, trichrome-blue and methylene-blue combination staining.

Faeces of immunocompromised patients are often contaminated with the chitin-containing spores of microsporidia and Candida, which exclude the use of the chitin-specific fluorescent brightener Calcofluor white M2R for the identification of microsporidian spores. We developed a combination staining of Calcofluor white M2R with modified trichrome-blue staining and subsequent methylene-blue incubation which permits discrimination between these two types of spores. As a basis for diagnosis, a difference in the fluorescence pattern (365-440 nm) is combined with a difference in the light microscopic staining pattern. Under fluorescence conditions microsporidia spores have a spotted, brilliant white Calcofluor fluorescence and can easily be identified, while Candida spores show a reddish purple colour. Under the light microscope microsporidian spores show a light red colour with nonstained vacuole spots or strips in contrast to the yeast spores with their red-brown colour. This combination technique offers a highly specific means for the diagnosis of microsporidia spores in faeces.

Molecular characterization and taxonomy of a new species of Caudosporidae (Microsporidia) from black flies (diptera: simuliidae) with host-derived relationships of the North American caudosporids.

A new species of microsporidium, Caudospora palustris (Microsporidia: Caudosporidae), is described from 3 species of black flies (Cnephia ornithophilia and diploid and triploid cytospecies of Stegopterna mutata), bringing to 7 the total species of caudosporids recorded from North America. This new species of caudosporid is recorded from swamp streams of the Coastal Plain from New Jersey to Georgia, with single records from the New Jersey mountains and the Upper Peninsula of Michigan. Densities of patently infected larvae (up to 10,600/m2) and spore production (nearly 8x10(11)/m2) are the greatest recorded for any microsporidium of black flies. The ultrastructure of this new species is presented, along with the first molecular characterization for a microsporidium of black flies. The phylogenetic position of black fly microsporidia within the phylum Microsporidia is presented; however, the analysis does not support the inclusion of C. palustris in any clade. Key features of all North American caudosporids are provided, and possible evolutionary trajectories are proposed based on optimization of caudosporid species on the phylogeny of their 22 known host species, including 16 that represent new host species records.

Horizontal transmission of Amblyospora albifasciati García and Becnel, 1994 (Microsporidia: amblyosporidae), to a copepod intermediate host and the neotropical mosquito, Aedes albifasciatus (Macquart, 1837).

The life cycle of Amblyospora albifasciati is characterized by three sporulation sequences involving the definitive mosquito host and a copepod intermediate host. Meiospores of A. albifasciati were infectious per os to adult females of the copepod Mesocyclops annulatus. All developmental stages in the copepod had unpaired nuclei, with sporulation involving the formation of a sporontogenic interfacial envelope and the production of a second type of uninucleate spore. These spores, formed in the ovaries of M. annulatus, were large, pyriform, and measured 10.4 x 4.8 microm. They infected Aedes albifasciatus larvae when ingested to initiate a sequence that involves schizogony and gametogony and ends with plasmogamy and nuclear association to form diplokaryotic meronts. Oval binucleate spores (9.3 x 3.1 microm) are formed in the adult mosquito and are responsible for vertical transmission to the filial generation.

Production of monoclonal antibodies directed against the microsporidium Enterocytozoon bieneusi.

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories.

Microsporidian intrasporal sugars and their role in germination.

The hypothesis that spores of terrestrial and aquatic microsporidia differ in their utilization of sugars was tested by evaluating the sugars in germinated and ungerminated spores of several species in each category. The aquatic species tested were Vavraia culicis, Edhazardia aedis, and Nosema algerae and the terrestrial species were Vairimorpha necatrix, Nosema disstriae, Nosema apis, Vairimorpha lymantriae, and Nosema spp. from Spodoptera exigua and Plutella xylostella. The percentage germination varied between species, ranging between 40 and 92%. Total sugars (anthrone reactive) and reducing sugars (Nelson's test) remained unchanged through germination in the three terrestrial species tested; however, reducing sugars increased significantly in the aquatic species. High-performance liquid chromatography and gas chromatography revealed a preponderance of trehalose in all species and large quantities of sorbitol in all species except N. algerae and E. aedis. Other sugars were present in some species in much lower concentrations. After germination no changes in sugar content were observed in terrestrial species; however, all aquatic species lost trehalose with a concomitant increase in fructose and/or glucose concentrations. Increased osmotic potential from breakdown of trehalose has been postulated to induce germination of the aquatic species, but another explanation must be found for the terrestrial species.

Coevolutionary interactions between host life histories and parasite life cycles.

Several recent studies have discussed the interaction of host life-history traits and parasite life cycles. It has been observed that the life-history of a host often changes after infection by a parasite. In some cases, changes of host life-history traits reduce the costs of parasitism and can be interpreted as a form of resistance against the parasite. In other cases, changes of host life-history traits increase the parasite's transmission and can be interpreted as manipulation by the parasite. Alternatively, changes of host's life-history traits can also induce responses in the parasite's life cycle traits. After a brief review of recent studies, we treat in more detail the interaction between the microsporidian parasite Edhazardia aedis and its host, the mosquito Aedes aegypti. We consider the interactions between the host's life-history and parasite's life cycle that help shape the evolutionary ecology of their relationship. In particular, these interactions determine whether the parasite is benign and transmits vertically or is virulent and transmits horizontally.

Relevant criteria for detecting microsporidia in stool specimens.

By using different staining techniques, 479 stool specimens from 212 diarrheic patients with AIDS were examined for microsporidian spores. Calcofluor fluorescence staining of 119 specimens revealed fluorescent ovoid structures of microsporidian size. Staining of these samples according to the method of Weber et al. (R. Weber, R. T. Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin, and G. S. Visvesvara, N. Engl. J. Med. 326:161-166, 1992) with trichrome produced six specimens with pinkish spores containing the characteristic microsporidian belt-like structure. The 6 specimens were processed for transmission electron microscopy, as were another 21 specimens which did not present the belt-like structure after trichrome staining but which looked highly suspicious after fluorescence staining. In these 21 samples, only fungal spores and, particularly, bacterial Clostridium spores were demonstrated, whereas in the 6 samples diagnosed positive after trichrome staining, the existence of microsporidia could be verified by electron microscopy. Based on our observations, we propose that the belt-like structure seen with the Weber stains in microsporidian spores corresponds to structures existing in priming-stage spores. The results suggest that routine microscopical fecal diagnosis for microsporidian infection should include a screening by fluorescence staining and, subsequently, a confirmatory viewing of fluorescence-positive samples after trichrome staining.

Microsporidia of the genus Trachipleistophora--causative agents of human microsporidiosis: description of Trachipleistophora anthropophthera n. sp. (Protozoa: Microsporidia).

Trachipleistophora anthropophthera n. sp., was found at autopsy in the brain of one and in the brain, kidneys, pancreas, thyroid, parathyroid, heart, liver, spleen, lymph nodes, and bone marrow of a second patient with AIDS. The parasite is similar to the recently described T. hominis Hollister, Canning, Weidner, Field, Kench and Marriott, 1996, in having isolated nuclei, meronts with a thick layer of electron dense material on the outer face of their plasmalemma and sporogony during which spores are formed inside a thick-walled sporophorous vesicle. In contrast to T. hominis, this species is dimorphic as it forms two kinds of sporophorous vesicles and spores: Type I--round to oval polysporous sporophorous vesicle, 7-10 microns in size, usually with eight spores (3.7 x 2.0 microns), thick endospores, subterminal anchoring disc and anisofilar polar filaments forming seven thicker and two thinner terminal coils. This type of sporophorous vesicle is associated with 25-30 nm filaments extending into the host cell cytoplasm. Type II--smaller, bisporous sporophorous vesicle (4-5 x 2.2-2.5 microns) with two, nearly round, thin-walled spores, 2.2-2.5 x 1.8-2.0 microns in size, having 4-5 isofilar coils. No outside filamentous elements are associated with the bisporous sporophorous vesicle. Both types of sporophorous vesicles were common in the infected brain tissue and could be found within the same cell. The newly described species, together with T. hominis and previously reported Pleistophora-like parasites from human muscle, likely represent a group of closely related human microsporidia.