Development of specific PCR assays for the detection of Cryptocaryon irritans

Cryptocaryon irritans is one of the most important protozoan pathogens of marine fish, causing the “white spot” disease and posing a significant problem to marine aquaculture. In the present study, a C. irritans-specific reverse primer (S15) was designed based on the published sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of C. irritans and used together with the conserved forward primer P1 to develop a specific polymerase chain reaction (PCR) assay for direct, rapid, and specific detection of C. irritans. The specificity of these primers was tested with both closely and distantly related ciliates (Pseudokeroronpsis rubra, Pseudokeroronpsis carnae, Euplotes sp. 1, Ichthyophthirius multifiliis, Pseudourostyla cristata, and Paramecium caudaium), and only C. irritans was detected and no product was amplified from any other ciliates examined in this study using the specific primer set P1-S15. The specific PCR assay was able to detect as low as 45 pg of C. irritans DNA and a nested PCR assay using two primer sets (P1/NC2, P1/S15) increased the sensitivity, allowing the detection of a single C. irritans. The species-specific PCR assays should provide useful tools for the diagnosis, prevention, and molecular epidemiological investigations of C. irritans infection in marine fish

Genome of the facultative scuticociliatosis pathogen Pseudocohnilembus persalinus provides insight into its virulence through horizontal gene transfer

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Appearance and pathogenicity of ovarian parasite Marteilioides chungmuensis in the farmed Pacific oysters, Crassostrea gigas, in Korea

The ovarian parasite Marteilioides chungmuensis that infects the ovaries of Pacific oyster Crassostrea gigas has increased in frequency in farmed oysters on the southern coast of Korean peninsula since the early 1990s. The appearance and pathogenicity of the ovarian parasite in the farmed oyster in Jinhae Bay, Korea, were investigated in 1996 and 1997. Infection by M. chungmuensis was highest during spawning (from June to August) and gonadal regenerating season of the oysters (from September to October), with prevalences ranging from 13.3 to 57.1% in 1996 and from 28.6 to 61.5% in 1997, respectively. The surveyed oysters showed signs of recovery from the infection after October. Glycogen levels were considerably lower in M. chungmuensis-infected oysters those that of uninfected oysters. A rapid accumulation of glycogen was observed in uninfected oysters together with the gonadal regeneration after the summer spawning. By contrast, no increase in glycogen content was found in infected oysters until the end of the investigation. Lipid levels were slightly higher in the infected oysters than in the uninfected oysters. Serum protein concentrations were significantly lower in the infected oysters than in the uninfected oysters. Also, the increase of serum protein concentration after the summer spawning was apparent in the uninfected oysters but not in the infected oysters. These results indicate that the infections by M. chungmuensis may have an adverse impact on metabolic recovery after spawning of the oysters.open1112sciescopu

Hierarchically Self-Organized Monolithic Nanoporous Membrane for Excellent Virus Enrichment

Enrichment of viruses is essential for making high dose viral stocks for vaccines and virus-related research. Since the widely used ultracentrifugation for concentrating viral stock requires ultra-high speed rotation, it easily destroys the activity of some viruses, for instance, hepatitis c virus (HCV), which has a fragile structure and low virus titer. We introduce a novel method to concentrate HCV virus in stock by using a hierarchically self-organized monolithic nanoporous membrane made by stepwise anodization. The pores at the top part of the membrane have very regular sizes that are suitable for the perfect filtration of the virus particles in the stock. On the other hand, the remaining part has large pores that maintain high flux and mechanical strength of the membrane under the high pressure (up to 10 bar). The enrichment efficiency of HCV in crude stocks by using the membrane became over 91%, which is four times higher than that (similar to 22%) obtained by conventionally used centrifugation. A very high efficiency results from the perfect filtration and no damage to the virion particles during the enrichment process, whereas significant damage to the HCV occurs during centrifugation. The hierarchically self-organized monolithic nanoporous membrane could be effectively employed for concentrating various fragile viruses in stocks, for instance, rabies virus and human immunodeficiency virus in addition to HCV virus.X1154sciescopu

An Important Role of alpha-Hemolysin in Extracellular Vesicles on the Development of Atopic Dermatitis Induced by Staphylococcus aureus

Skin barrier disruption and dermal inflammation are key phenotypes of atopic dermatitis (AD). Staphylococcus aureus secretes extracellular vesicles (EVs), which are involved in AD pathogenesis. Here, we evaluated the role of EVs-associated alpha-hemolysin derived from S. aureus in AD pathogenesis. alpha-hemolysin production from S. aureus was detected using western blot analyses. The cytotoxic activity of alpha-hemolysin on HaCaT keratinocytes was evaluated by measuring cell viability after treating cells with soluble and EVs-associated alpha-hemolysin. To determine the type of cell death, HaCaT keratinocytes were stained with annexin V and 7-AAD. The in vivo effects of alpha-hemolysin were evaluated by application of soluble and EV-associated alpha-hemolysin on the mouse skin. The present study showed that increased alpha-hemolysin was produced by S. aureus colonized on AD patients compared to healthy subjects. alpha-hemolysin production was also related to AD severity. In addition, EV-associated alpha-hemolysin was more cytotoxic to HaCaT keratinocytes than soluble alpha-hemolysin, and alpha-hemolysin-negative EVs did not induce keratinocyte death. EV-associated alpha-hemolysin induced necrosis, but soluble alpha-hemolysin induced apoptosis of keratinocytes. In vivo, skin barrier disruption and epidermal hyperplasia were induced by soluble and EV-associated alpha-hemolysin. However, AD-like dermal inflammation was only caused by EV-associated alpha-hemolysin. Moreover, neither skin barrier disruption nor AD-like skin inflammation was induced by alpha-hemolysin-negative EVs. Taken together, alpha-Hemolysin secreted from S. aureus, particularly the EV-associated form, induces both skin barrier disruption and AD-like skin inflammation, suggesting that EV-associated alpha-hemolysin is a novel diagnostic and therapeutic target for the control of AD.open111618sciescopu