In the present study, a high percentage of Japanese anglerfish, Lophius litulon (Jordan, 1902), contained a microsporidian infection of the nervous tissues. Xenomas were removed and prepared for standard wax histology and transmission electron microscopy (TEM). DNA extractions were performed on parasite spores and used in PCR and sequencing reactions. Fresh spores measured 3.4 × 1.8 µm and were uniform in size with no dimorphism observed. TEM confirmed that only a single developmental cycle and a single spore form were present. Small subunit (SSU) rDNA sequences were >99.5% similar to those of Spraguea lophii (Doflein, 1898) and Glugea americanus (Takvorian et Cali, 1986) from the European and American Lophius spp. respectively. The microsporidian from the nervous tissue of L. litulon undoubtedly belongs in the genus Spraguea Sprague et Vávra, 1976 and the authors suggest a revision to the generic description of Spraguea to include monomorphic forms and the transfer of Glugea americanus to Spraguea americana comb. n. Since no major differences in ultrastructure or SSU rDNA sequence data exist between Spraguea americana and the microsporidian from the Japanese anglerfish, they evidently belong to the same species. This report of Spraguea americana is the first report of a Spraguea species from L. litulon and indeed from the Pacific water mass.
The structure of the human microsporidium found by Yachnis and colleagues in two AIDS patients (Am. J. Clin. Pathol. 106: 535-43, 1996) (hereafter referred to as HMY) was investigated by light and transmission electron microscopy and compared with Thelohania apodemi Doby, Jeannes et Raoult, 1963, a microsporidian of small rodents. The fine structure of the HMY was found to be similar to that of Trachipleistophora hominis Hollister, Canning, Weidner, Field, Kench et Marriott, 1996. Characteristic is the presence of a thick layer of electron dense material on the outer lace of the meront plasmalemma, which is maintained during the whole life cycle and which later persists as an electron dense coat on the sporophorous vesicle (SPOV). However, HMY is distinguished from T. hominis during sporogony, as two types of SPOV and spores are formed in HMY. One type of SPOV contains thick-wallcd spores (usually 8 or more in number) with anisofilar polar filaments of 7 + 2 pattem, while the other type contains only two thin-walled spores with a smaller number (3-5) of isofilar polar filament coils. The HMY differs from T. apodemi which also forms SPOV with 8 spores inside, but the spores of which are larger in size and have 9 + 2 polar filament pattern.
Microsporidia are a cause of emerging and opportunistic infections in humans and animals. Although two drugs are currently being used to treat microsporidiosis, concerns exist that albendazole is only selective for inhibiting some species of microsporidia that infect mammals, and fumagillin appears to have been found to be toxic. During a limited sequence survey of the Vittaforma corneae (syn. Nosema corneum Shadduck, Meccoli, Davis et Font, 1990) genome, a partial gene encoding for the ParC topoisomerase IV subunit was identified. The purpose of this set of studies was to determine if fluoroquinolones, which target topoisomerase IV, exert activity against Encephalitozoon intestinalis (syn. Septata intestinalis Cali, Kotler et Orenstein, 1993) and V. corneae in vitro, and whether these compounds could prolong survival of V. corneae-infected athymic mice. Fifteen fluoroquinolones were tested. Of these, norfloxacin and ofloxacin inhibited E. intestinalis replication by more than 70% compared with non-treated control cultures, while gatifloxacin, lomefloxacin, moxifloxacin, and nalidixic acid (sodium salt) inhibited both E. intestinalis and V. corneae by at least 60% at concentrations not toxic to the host cells. These drugs were tested in vivo also, where gatifloxacin, lomefloxacin, norfloxacin, and ofloxacin prolonged survival of V. corneae-infected athymic mice (P < 0.05), whereas moxifloxacin and nalidixic acid failed to prolong survival. Therefore, these results support continued studies for evaluating the efficacy of the fluoroquinolones for treating microsporidiosis and for characterizing the target(s) of these fluoroquinolones in the microsporidia.
The production of three cytokines, interferon gamma (IFN-y), interleukin 10 (1L-10) and interleukin 12 (IL-12), was measured after intraperitoneal infection of immunocompetent Balb/c mice and immunodeficient SCID mice with the microsporidian, Encephalitozoon cuniculi Levaditi, Nicolau ct Schoen, 1923. High levels of IFN-y were detected in ex vivo cultures of peritoneal exudate cells (PEC) of Balb/c mice, a lower, but earlier IFN-y response was observed in PEC from SCID mice. The early 1L-10 response was detected in ex vivo cultures of splenocytes from Balb/c but not from SCID mice, explaining a delay in the IFN-y response in Balb/c mice. IL-12 was detected in PEC cultures from SCID mice, indicating an alternative pathway of IFN-y production by NK. cells stimulated by IL-12 derived from macrophages.
The survival of Encephalitozoon cuniculi Lcvaditi, Nicolau et Schoen, 1923 spores suspended in distilled water and exposed at defined temperatures was investigated. Infectivity of E. cuniculi spores was tested by inoculation of SCID mice. There was no marked loss of infectivity of spores stored at 4°C for two years or frozen at -12°C and -24°C for 1, 8, and 24 h. Although there was a remarkable loss of infectivity, spores remained infective after freezing at -70°C for 1 and 8 h. Heating at 60°C and 70°C for 5 min and 1 min, respectively, rendered the microsporidia non-infective. These findings demonstrate that E. cuniculi spores suspended in water can survive freezing temperatures but lost infectivity in water that reached a temperature of 60°C at 5 min.
An experimental infection with the microsporidian Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 was studied using a model of immunocompetent BALB/c mice and immunodeficient SCID mice. The course of infection after intraperitoneal inoculation of E. cuniculi spores was evaluated using the presence of spores in peritoneal macrophages as a criterion. First significant decrease in the proportion of infected cells was recorded on day 9 post infection (p.i.) in BALB/c mice. From day 14 p.i. no spores were observed in macrophages from BALB/c mice, while the number of infected macrophages from SCID mice increased until the death of the mice. The natural killer (NK) cell activity of mouse splenocytes was compared with the production of interferon gamma (IFN-γ) by these cells. While in BALB/c mice NK activity peaked on days 9 and 14 p.i., in SCID mice the marked increase of NK activity was recorded close before death of mice, on day 21 p.i. in correlation with the production of IFN-γ. Production of specific antibodies was demonstrated from day 9 p.i. in sera from BALB/c mice. It is concluded that intraperitoneal infection of SCID mice with spores of E. cuniculi results in the marked increase in the number of peritoneal exudate cells and in the percentage of infected cells close before death of mice. Neither high activity of NK cells nor increased production of IFN-γ are sufficient for the recovery of SCID mice from an E. cuniculi infection.
The microsporidian Kabatana arthuri (Lom, Dyková et Shaharom, 1990) induced severe regressive changes in trunk muscles of Pangasius sutchi (Fowler) from Thailand. Necrotic changes developed in muscle fibres around the developmental stages and on the periphery of giant aggregates of spores. The main feature of the host defence reaction was the phagocytic activity of macrophages. Inflammatory reaction was only exceptionally observed. Spore-laden macrophages were found in various tissues and organs; their infiltration in epidermis including its outermost layers may effectively enhance the spread of infection while the hosts still live.
Hexokinase (HXK) is the first key enzyme in the glycolytic pathway and plays an extremely important role in energy metabolism. By searching the microsporidian database, we found a sequence (NBO_27g0008) of Nosema bombycis Nägali, 1857 with high similarity to hexokinase-2, and named it as NbHXK2. The NbHXK2 gene has 894 bp and encodes 297 amino acids with 34.241 kD molecular weight and 5.26 isoelectric point. NbHXK2 contains 31 phosphorylation sites and 4 potential N-glycosylation sites with signal peptides and no transmembrane domain. Multiple sequence alignment showed that NbHXK2 shares more than 40% amino acid identity with that of other microsporidia, and the homology with hexokinase-2 of Nosema tyriae Canning, Curry, Cheney, Lafranchi-Tristem, Kawakami, Hatakeyama, Iwano et Ishihara, 1999, Nosema pyrausta (Paillot, 1927) and Nosema ceranae Fries, Feng, da Silva, Slemenda et Pieniazek, 1996 was 89.17%, 87.82% and 69.86%, respectively. Phylogenetic analysis based on the amino acid sequence of hexokinase showed that all microsporidia cluster together in the same clade, and are far away from animals, plants and fungi, and that N. bombycis is closely related to N. tyriae; N. pyrausta; N. ceranae and Nosema apis Zander, 1909. Immunolocalisation with the prepared polyclonal antibody showed that NbHXK2 was mainly distributed in the cytoplasm and plasmalemma in proliferative, sporulation stage and mature spore of N. bombycis. qRT-PCR assay showed that the NbHXK2 expressed at higher level during spore germination and at early stage of proliferation. These results indicate that N. bombycis may use its own glycolytic pathways to supply energy for infection and development, especially germination and in the early stage of proliferation, and acquire energy from the host through certain ways as well.
Intestinal microsporidiosis was documented by detecting abundant slightly curved spores (2.9 x 1.2 pm) in the faeces of five of twelve skinks Mabuya perrotetii Duméril et Bibron, 1839 that originated from Ghana. Clinically, the microsporidiosis was characterized by decreased appetite, diarrhea, and weight loss. Histopathological changes consisted of villous atrophy, blunting of mucosa and flattening of individual epithelial cells in the large intestine. The ultrastructure of microsporidian spores was consistent with an Encephalitozoon species. The PCR-RFLP assay and the heleroduplex mobility shift analyses were used to verify that the skink microsporidian is a species of the genus Encephalitozoon Levaditi, Nicolau et Schoen, 1923 and indicate that this microsporidian is not E. hellem, E. intestinalis or a strain of E. cuniculi. The microsporidia in African skink represent an Encephalitozoon species morphologically identical to Encephalitozoon lacerine Canning, 1981.
Microsporidia are intracellular parasites of insects and other higher eukaryotes. The microsporidian Nosema philosamiae Talukdar, 1961 was isolated from the eri silkworm, Philosamia cynthia ricini Grote. In the present study, alpha- and beta-tubulin genes from N. philosamiae were characterized. The identity analysis of nucleotide and amino acid sequences indicated high similarity with species of Nosema Nägeli, 1857 sensu lato (nucleotide sequences, ≥ 96.0%; amino acid sequences, ≥ 99.0%). However, the tubulin genes of N. philosamiae share low sequence similarity with that of N. ceranae Fries, Feng, da Silva, Slemenda et Pieniazek, 1996 (strain BRL01) and a Nosema/Vairimorpha species. Phylogenies based on alpha-, beta- and combined alpha- plus beta-tubulin gene sequences showed that N. philosamiae, along with the true Nosema species, forms a separate clade with a high bootstrap value, with N. ceranae BRL01 forming a clade of its own. The results indicated that the alpha- and beta-tubulin sequences may be useful as a diagnostic tool to discriminate the true Nosema group from the Nosema/Vairimorpha group.