Morphological and molecular studies of Vairimorpha necatrix BM, a new strain of the microsporidium V. necatrix (Microsporidia, Burenellidae) recorded in the silkworm, Bombyx mori.

Vairimorpha sp. BM (2012) is a recent isolate of the microsporidia from the silkworm in Shandong, China. The ultrastructure, tissue pathology and molecular characterization of this isolate is described in this study. This pathogenic fungus causes pebrine disease in silkworms which manifests as a systemic infection. Meanwhile, the silkworm eggs produced by the infected moths were examined using a microscope and PCR amplification. Neither spores nor the expected PCR band were observed, suggesting that no vertical transmission occurred in Bombyx mori. In addition, the ultrastructure of the isolate was studied by light microscopy and transmission electron microscopy. Two types of spores were observed: diplokaroytic spores with 13-17 coils of polar tubes and monokaryotic spores with less coils of polar tubes which could form octospores; however, no sporophorous vesicles were observed. Finally, phylogenetic analysis of the small subunit rRNA genes of Vairimorpha species showed that this isolate has a closer relationship to Vairimorpha necatrix than the other species studied. This result also is supported by phylogenetic analysis based on their actin genes, heat shock protein 70 (HSP70) and RNA polymerase II (RPB1). Based on the information gained during this study, we propose that this microsporidian species infecting B. mori should be given the name V. necatrix BM.

Hyperparasitism has wide-ranging implications for studies on the invertebrate phase of myxosporean (Myxozoa) life cycles.

All of the actinospore releasing oligochaetes collected in an environmental sample were found to be infected with the microsporidian Neoflabelliforma aurantiae n. gen. n. sp. Ultrastructural and phylogenetic studies on this microsporidian indicated similarities with Flabelliforma magnivora but not with the type species Flabelliforma montana, necessitating the formation of a new genus Neoflabelliforma and reassignment of F. magnivora as Neoflabelliforma magnivora n. comb. The development of N. aurantiae is described both parasitising the oligochaete worm and hyperparasitising the concurrent myxosporean infection. The effect of N. aurantiae on the myxosporeans was deleterious and progressive, eventually stopping all actinospore formation. Its discovery has the potential to impact on areas examining the phase of myxosporean life cycles in the invertebrate host, from transmission studies and epidemiology to re-evaluating the basic steps of intra-oligochaete development. Recent evidence has suggested that studies using invertebrate systems should consider possible adverse effects that co-infections can have on experimental outcomes. The discovery of N. aurantiae highlights the need for careful screening of experimental animals to help circumvent erroneous results.

Molecular diagnostic methods for detection of Thelohania contejeani (Microsporidia), the causative agent of porcelain disease in crayfish.

Diagnosis of Thelohania contejeani in the crayfish Astacus astacus is currently based on observation of gross clinical signs--opaque appearance of the abdomen and whitish colouration of the musculature--and confirmed by microscopic examination of histological sections of muscle. We have developed 2 molecular diagnostic methods for sensitive and rapid detection of porcelain disease in its early stages: PCR and loop-mediated isothermal amplification (LAMP). The PCR test utilises a primer based on the T. contejeani small subunit ssu ribosomal RNA (ssu rRNA) gene and amplified parasite DNA with high specificity and a detection limit of 10(-5) dilution. The LAMP assay involves incubation of the target DNA with a set of 6 primers and Bst DNA polymerase for 60 min at 65 degrees C in a water bath or heating block, followed by visualisation of the reaction products with the SYBR Green I stain; sensitivity of visual detection with SYBR Green I is equivalent to that with agarose gel electrophoresis. The LAMP assay can detect T. contejeani DNA to a dilution of 10(-7). The LAMP assay is 100 times more sensitive than the PCR test and is the method we recommend as an alternative to traditional means of diagnosing T. contejeani.

Keratitis caused by Trachipleistophora anthropopthera.

We report a case of intrastromal keratitis in a 42-year-old male with underlying human immunodeficiency virus-1 infection. Numerous microsporidial spores were found from corneal biopsy. Ultrastructural studies of corneal tissues revealed dimorphic sporophorous vesicles containing characteristic spores belonging to Trachipleistophora anthropopthera. Infection could be controlled by penetrating keratoplasty but not by topical fumagillin and systemic albendazole per se. This is the first report of human keratitis caused by this organism.