Behavioural effects of the common brain-infecting parasite Pseudoloma neurophilia in laboratory zebrafish (Danio rerio).

Research conducted on model organisms may be biased due to undetected pathogen infections. Recently, screening studies discovered high prevalence of the microsporidium Pseudoloma neurophilia in zebrafish (Danio rerio) facilities. This spore-forming unicellular parasite aggregates in brain regions associated with motor function and anxiety, and despite its high occurrence little is known about how sub-clinical infection affects behaviour. Here, we assessed how P. neurophilia infection alters the zebrafish´s response to four commonly used neurobehavioral tests, namely: mirror biting, open field, light/dark preference and social preference, used to quantify aggression, exploration, anxiety, and sociability. Although sociability and aggression remained unaltered, infected hosts exhibited reduced activity, elevated rates of freezing behaviour, and sex-specific effects on exploration. These results indicate that caution is warranted in the interpretation of zebrafish behaviour, particularly since in most cases infection status is unknown. This highlights the importance of comprehensive monitoring procedures to detect sub-clinical infections in laboratory animals.

The first study on opportunistic intestinal microsporidiosis in IBD patients receiving immunosuppressive medications in Iran.

Microsporida are known as opportunistic unicellular organisms and have recently been reclassified as fungi that have been frequently reported from patients with congenital and acquired immunity failure disorders, worldwide. However, use of immunosuppressive medications in inflammatory bowel disease (IBD) patients significantly decreases overall immunity, and increases their susceptibility to opportunistic infections. Totally, 71 stool samples were collected from IBD patients consisted of 69 ulcerative colitis (UC) patients and two Crohn's disease (CD) patients. All patients had taken immunosuppressive and/or immunomodulator drugs for at least 3 weeks. DNA was extracted from all stool samples and Nested PCR was performed using genus-specific primers based on small subunit ribosomal RNA (SSU rRNA) gene. Fisher's Exact Test was applied to evaluate statistical association between microsporidia infection and sex, age and types of IBD. Mean of age ± s.d., women and men percentage of the attended patients were 36·17 ± 11·93, 60·6%, and 39·4%, respectively. A 440-bp fragment of SSU rRNA gene attributed to Enterocytozoon bieneusi was amplified from 12·7% of IBD patients. No Encephalitozoon DNA was detected in the samples. No microsporidia-positive sample was found in CD patients. Fisher's Exact Test showed that there was no statistically significant correlation between intestinal microsporidiosis and age, sex, and IBD types with P values: 0·389, 1·00, and 1·00, respectively. This study has shown IBD patients undergoing immunosuppressive/immunomodulators medications, which may be susceptible to intestinal microsporida infection. E. bieneusi is the commonest intestinal microsporidan reported from IBD patients.

Unusual fluorescent granulomas and myonecrosis in Danio rerio infected by the microsporidian pathogen Pseudoloma neurophilia.

Abstract Zebrafish are a powerful model organism to study disease. Like other animal models, Danio rerio colonies are at risk of pathogenic infection. Microsporidia, a group of intracellular fungus-like parasites, are one potential threat. Microsporidian spores germinate and spread causing pathological changes in the central nervous system, skeletal muscle, and other anatomic sites. Infection can impair breeding, cause other morbidities, and ultimately be lethal. Previously, detecting microsporidia in zebrafish has required sacrificing animals for histopathologic analysis or microscopic examination of fresh tissues. Here, we show that fish with microsporidial infection often have autofluorescent nodules, and we demonstrate infectious spread from nodule-bearing fish to healthy D. rerio. Histologic analyses revealed that fluorescent nodules are granulomatous lesions composed of spores, degenerating muscle, and inflammatory cells. Additional histologic staining verified that microsporidia were present, specifically, Pseudoloma neurophilia. Polymerase chain reaction (PCR)-based testing confirmed the presence of P. neurophilia. Further PCR testing excluded infection by another common zebrafish microsporidial parasite, Pleistophora hyphessobryconis. Collectively, these studies show that P. neurophilia can induce skeletal muscle granulomas in D. rerio, a previously unknown finding. Moreover, since granulomas autofluoresce, microscopic screening for P. neurophilia infection is feasible in live fish, avoiding the need to sacrifice fish for surveillance for this pathogen.

[Should the microsporidian spores be treated as dormant stages?].

Spores of bacteria, fungi, microsporidia and other protists are traditionally treated as dormant stages, intended to the long-term survival in the environment and to activation of parasitic forms during the infestation of a new host. However, in the process of examination of insect microsporidia at the molecular cellular levels and also at the level of organisms and populations, we came to a conclusion that spores are very active developmental stages with the entire potential directed to the rapid and successful infestation of new hosts during contact with the later. The work summarizes the original data demonstrating (1) the necessity of the rapid activation of microsporidian spores during host contact, (2) hopelessness of the long retaining of viability by spores of many microsporidia in the environment after leaving host organism; and (3) specific accumulation of metabolic ferments in "dormant" spores, but not in actively proliferating prespore developmental stages. On the basis of these data we conclude that microsporidian spores tend to shorten the period when they stay outside host organism to the maximal degree. The probability of host infestation within the limited time period increases due to diverse modes of transmission of pathogens, accumulation of maximally possible volume of infective spores, and the rapid mobilization of the extrusion apparatus.

Association of Ovavesicula popilliae (Microsporida: Ovavesiculidae) with winter mortality of larvae and reduced fecundity of female Japanese beetles (Coleoptera: Scarabaeidae).

Populations of Japanese beetle at sites in Michigan where Ovavesicula popilliae (Andreadis) was introduced in 1999 and 2000 were compared with nearby control sites from fall of 2005 through spring of 2008. Percent infection by O. popilliae and winter mortality of Japanese beetle were determined by sampling larvae in October and April from 12 golf holes on six courses in southeast Michigan and eight holes on four courses in southwest Michigan. Adult Japanese beetles were also collected from these golf courses in July and August of 2007 to determine the impact of O. popilliae-infection on egg development in females. In southeast Michigan, O. popilliae appeared to spread rapidly from the 100 m(2) plots where it was previously introduced to surrounding golf course holes between 2000 and 2006. However, data from southwest Michigan suggests that O. popilliae had already been introduced into the area. Regression analysis of data from all 20 golf course holes gives a significant relationship between percent infection of larvae with O. popilliae and winter mortality of Japanese beetle. Mean winter mortality of larvae around golf course holes where <10% were infected with O. popilliae was 24.7% compared with 41.7% mortality where 10-30% were infected, and 72.0% mortality where >30% were infected. Females infected with O. popilliae contained 50% fewer mature eggs than uninfected females. In addition, females from golf courses where all of the fairways and roughs were treated annually with imidacloprid contained 48% fewer mature eggs than females from golf courses where insecticides were only used on the fairways or not at all.

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.

Interaction of an entomopathogen with an insect social form: an epizootic of Thelohania solenopsae (Microsporidia) in a population of the red imported fire ant, Solenopsis invicta.

This is the first report of Thelohania solenopsae infections in monogyne (single-queen) Solenopsis invicta colonies in the field. In a 0.2-ha plot near Baton Rouge, Louisiana, inter-colony prevalence was 63% infection in June, 1999, when the population was 100% monogyne. In February, 2000, 21% of 33 monogyne and 90% of 10 polygyne colonies were infected. By May, 2001, the polygyne colonies had disappeared and only one of 34 monogyne colonies was infected, the final detection of T. solenopsae in the plot. Colony size did not differ significantly among the four types (monogyne versus polygynexinfected versus uninfected).

Invasion success of Fibrillanosema crangonycis, n.sp., n.g.: a novel vertically transmitted microsporidian parasite from the invasive amphipod host Crangonyx pseudogracilis.

Parasitism is known to be an important factor in determining the success of biological invasions. Here we examine Crangonyx pseudogracilis, a North American amphipod invasive in the United Kingdom and describe a novel microsporidium, Fibrillanosema crangonycis n.sp., n.g. The primary site of infection is the female gonad and the parasite is transovarially transmitted to the eggs. PCR screening reveals a female bias in the distribution of parasites (96.6% of females, N=29; 22.2% of males, N=27), which is indicative of host sex ratio distortion. The morphological and molecular characterisations of this new microsporidium place it outside all currently established genera. On the basis of these differences, we erect the new genus Fibrillanosema n.g. While F. crangonycis is morphologically identical to uncharacterised microsporidia from populations of North American amphipods, it is distinct from microsporidia found in European populations of amphipods. These data support the hypothesis that vertically transmitted parasites may be selectively retained during invasion events. Furthermore where vertical transmission is combined with host sex ratio distortion these parasites may directly enhance host invasion success through increased rates of population growth.

Two species of feminizing microsporidian parasite coexist in populations of Gammarus duebeni.

The amphipod crustacean Gammarus duebeni hosts two species of vertically transmitted microsporidian parasites, Nosema granulosis and Microsporidium sp. A. Here it is demonstrated that these co-occurring parasite species both cause infected females to produce female-biased broods. A survey of European G. duebeni populations demonstrates that these two parasites co-occur in six of 10 populations. These findings contrast with the theoretical prediction that two vertically transmitted feminizing parasites should not coexist in a panmictic population of susceptible hosts at equilibrium. Possible explanations for the co-occurrence of the two feminizing microsporidia in G. duebeni include the recent invasion of a new parasite, horizontal transmission of one or both parasites and the spread of alleles for resistance to the dominant parasite in host populations.

Test of synergistic interaction between infection and inbreeding in Daphnia magna.

It has been proposed that parasitic infections increase selection against inbred genotypes. We tested this hypothesis experimentally using pairs of selfed and outcrossed sibling lines of the freshwater crustacean Daphnia magna, which can be maintained clonally. We studied the performance of selfed relative to outcrossed sibling clones during repeated pairwise clonal competition in the presence and absence of two species of microsporidian parasites. In 13 of the 14 pairs, the selfed clones did worse than the outcrossed ones in the control treatment, but the presence of either parasite did not result in an overall increase in this difference. Rather, it decreased the performance of the selfed relative to the outcrossed sibling in some pairs and increased it in others. Moreover, the two parasite species did not have the same effect in a given pair. This indicates that, contrary to the hypothesis that parasites generally lead to a decreased performance of inbred genotypes, their effect may depend on the genetic background of the host as well as on the parasite species, and suggests that inbreeding can lead to reduced or increased resistance to parasites. Our findings also indicate that there is variation for specific resistance to different species of parasites in the meta-population from which the hosts for this study were obtained.

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.

Mouse antibody response to a microsporidian parasite following inoculation with a gene coding for parasite ribosomal RNA.

This study found that a plasmid construct encoding the small-subunit ribosomal RNA (SSUrRNA) of the microsporidian Microgemma caulleryi generates a humoral response upon intramuscular inoculation in mice. The plasmid used was pCMV, following preliminary trials indicating efficient beta-galactosidase gene expression in mouse muscle cells transfected with pCMV/beta-Gal. The antibodies produced after inoculation with pCMV/SSUDNA recognized parasite spore antigens and reached maximum levels at 30 days postinoculation, subsequently remaining stable for at least 120 days. Due to the highly conserved sequence of the SSUrDNA in different microsporidian species, these results open up interesting prospects for broad-spectrum vaccination.

Ultrastructural description of the spore maturation stages of the clam parasite Minchinia tapetis (Vilela, 1951) (Haplosporida: Haplosporidiidae).

The fine structure of maturing spores of a haplosporidian parasite found in the gill, mantle and foot tissues of Ruditapes decussatus L. (Mollusca, Bivalvia), a species of commercial importance in Portugal, is described. When observed free in suspension, immature spores exhibit one or two epispore cytoplasmic extensions (ECE) which constitute a projection of a portion of the exosporoplasm, sometimes without ultrastructural organisation, surrounded by the plasmalemma. Free spores observed by light microscopy (LM) after 3-5 days of incubation in filtered sea-water exhibit no ECE attached to the spore wall. The mature spore is ovoid to ellipsoid, operculate, uninucleate and measures c. 4.8 microm long and c. 3.9 microm wide. The spore shape and size and the identity of the host living in the same geographical region suggest that this species is the same as previously described using LM observations as Haplosporidium tapetis Vilela, 1951 and later transferred to Minchinia Labbé, 1896.

Microsporidiosis in mammals.

Microsporidia are small, single-celled, obligately intracellular parasites that have caused significant agricultural losses and interference with biomedical research. Interest in the microsporidia is growing, as these organisms are recognized as agents of opportunistic infections in persons with AIDS and in organ transplant recipients. Microsporidiosis is also being recognized in children and travelers, and furthermore, concern exists about the potential of zoonotic and waterborne transmission of microsporidia to humans. This article reviews the basic biology and epidemiology of microsporidiosis in mammals.

[Microsporidian spores in the stool specimens of toddlers, with or without diarrhea, from Tucumán, Argentina]. / Esporas de microsporidios en las heces de niños de corta edad, con y sin diarrea, de Tucumán, Argentina.

An investigation has been carried out from September 1995 to December 1997 to search for microsporidian spores in the stool specimens of 344 toddlers aged 1 to 24 months, hospitalized at a pediatric institution in Tucumán. They were classified in two groups: I, made up of 222 children suffering from severe diarrheas, and II by 122 affected by different pathologies, except gastroenteritis. The detection of microsporidia was done by light microscopy in smears of stained stool specimens by using the Weber modified Kokoskin method. Copro-parasitological and coprobacteriological studies were also carried out and the nutritional status of each child was determined. In group I, microsporidia were found in 12/122 cases (7.2%), 4/68 belong to eutrophic children (5.9%), and 12/137 to undernourished children (8.8%); 8/16 positives were found to be related with other enteropatogenics. In group II, microsporidia were detected in 10/122 (8.2%), 4/47 in eutrophic children (8.5%), 4/54 in undernourished children (7.4%) and without data in two cases. They were related with other enteropatogenics in 5/10 positives. Tucumán can be estimated as an area with a low rate of HIV infection in toddlers, then it can be estimated that the studied sample was essentially HIV negative. The occurrence of microsporidia was important and did not show significant differences between toddlers with or without diarrhea, eutrophic or undernourished children.

Vairimorpha imperfecta n.sp., a microsporidian exhibiting an abortive octosporous sporogony in Plutella xylostella L. (Lepidoptera: Yponomeutidae).

The microsporidian genus Nosema is characterized by development in direct control with host cell cytoplasm, diplokaryotic nuclei throughout development and disporous sporogony. The genus Vairimorpha exhibits the same features plus an octoporous sporogony producing uninucleate spores in a sporophorous vesicle. A microsporidium from diamondback moth, Plutella xylostella, falls between Nosema and Vairimorpha in that it initiates but fails to complete the octosporous sequence in this host. The name Vairimorpha imperfecta n.sp. is proposed. Merogony is mainly by formation of buds from multinucleate meronts, the buds remaining attached in chains. Diplokaryotic spores measure 4.3 x 2.0 microns (fresh) and have 15.5 coils of the polar tube in 1 rank. The octosporous sporogony is aborted owing to irregular formation of nuclear spindles, incomplete cytoplasmic fission and bizarre deposition of electron-dense episporontal secretions. Phylogenetic analyses of the sequences of the small subunit rRNA genes of V. imperfecta and of several Nosema and Vairimorpha spp. place V. imperfecta in a clade with Nosema spp. from Lepidoptera rather than in the clade containing the more typical species of Vairimorpha. It is suggested that the ancestors of the Vairimorpha/Nosema complex of species exhibited both disporous and octosporous sporogonies, as does the type species of Vairimorpha, Vairimorpha necatrix. It would follow that true Nosema spp. have lost the ability to express an octosporous sequence and that V. imperfecta is in the process of losing it. It is proposed that the genera Nosema and Vairimorpha be placed in the same family Nosematidae Labbé 1899, rather than in separate families and orders as at present.

Esporas de microsporidios en las heces de niños de corta edad, con y sin diarrea, de Tucumán, Argentina / Microsporidian spores in the stool specimens of toddlers, with or without diarrhea, from Tucumßn, Argentina

An investigation has been carried out from September 1995 to December 1997 to search for microsporidian spores in the stool specimens of 344 toddlers aged 1 to 24 months, hospitalized at a pediatric institution in Tucumßn. They were classified in two groups: I, made up of 222 children suffering from severe diarrheas, and II by 122 affected by different pathologies, except gastroenteritis. The detection of microsporidia was done by light microscopy in smears of stained stool specimens by using the Weber modified Kokoskin method. Copro-parasitological and coprobacteriological studies were also carried out and the nutritional status of each child was determined. In group I, microsporidia were found in 12/122 cases (7.2), 4/68 belong to eutrophic children (5.9), and 12/137 to undernourished children (8.8); 8/16 positives were found to be related with other enteropatogenics. In group II, microsporidia were detected in 10/122 (8.2), 4/47 in eutrophic children (8.5), 4/54 in undernourished children (7.4) and without data in two cases. They were related with other enteropatogenics in 5/10 positives. Tucumßn can be estimated as an area with a low rate of HIV infection in toddlers, then it can be estimated that the studied sample was essentially HIV negative. The occurrence of microsporidia was important and did not show significant differences between toddlers with or without diarrhea, eutrophic or undernourished children.(AU)