• Parasites, Hosts and Diseases
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• v.41 no.4
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• pp.189-196
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• 2003

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of $55^{\circ}C$. Phenylmethylsulfonylfluoride and 4-(2-Aminoethyl)-benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.

• Parasites, Hosts and Diseases
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• v.42 no.3
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• pp.93-119
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• 2004

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal anti-body reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 188 small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.

• Parasites, Hosts and Diseases
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• v.44 no.4 s.140
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• pp.321-330
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• 2006

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Pretense has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castelianii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.

• Parasites, Hosts and Diseases
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• v.39 no.2
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• pp.161-170
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• 2001

We conducted both the small subunit ribosomal DNA (SSU rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and mitochondrial (mt) DNA RFLP analyses for a genetic characterization of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Twenty-three strains of Acanthamoeba from the American Type Culture Collection and twelve clinical isolates from Korean patients were used as reference strains. Thirty-nine isolates from contact lens storage cases were classified into seven types (KA/LS1 , KA/LS2, KA/LS4, KA/LS5, KA/LS7 KA/LS18, KA/LS31). Four types (KA/LS1 , KA/LS2, KA/LS5, KA/LS18) including 33 isolates were regarded as A. castellanii complex by riboprints. KA/LS1 type was the most predominant (51.3%) in the present survey area, followed by KA/LS2 (20.9%), and KA/LSS (7.7%) types. Amoebae of KA/LS1 type had the same mtDNA RFLP and riboprint patterns as KA/E2 and KA/E12 strains, clinical isolates from Korean keratitis patients. Amoebae of KA/LS2 type had the identical mtDNA RFLP patterns with A. castellanii Ma strain, a corneal isolate from an American patient as amoebae of KA/LS5 type, with KA/E3 and KA/E8 strains from other Korean keratitis patients. Amoebae of KA/LS 18 type had identical patterns with JAC/E1, an ocular isolate from a Japanese patient. Three types , which remain unidentified at species level, were not corresponded with any clinical isolate in their mtDNA RFLP and riboprint patterns. Out of 39 isolates analyzed in this study, mtDNA RFLP and riboprint patterns of 33 isolates (84.6%) were identical to already known clinical isolates, and therefore, they may be regarded as potentially keratopathogenic. These results suggest that contact lens wearers in Seoul should pay more attention to hygienic maintenance of contact lens storage cases for the prevention of Acanthamoeba keratitis.

• Journal of fish pathology
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• v.34 no.2
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• pp.185-199
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• 2021

Vibriosis is an important septicemic bacterial disease that affects a variety of commercial fish species, including cultured Dicentrarchus labrax. Nanotechnology has become an important modern tool for fish diseases prevention. Furthermore, nanomaterials have the ability to prevent and treat fish diseases. The current study was aimed to identify the causative agent of massive mortality of D. labrax commercial farm in Alexandria, Egypt. Experimental infection and the median lethal dose (LD50) of pathogenic isolate were assessed. Also, the effect of ginger nanoparticles (GNPs) and Sacchromyces cerevisiae as feed additives for prevention of vibriosis in D. labrax was carried out. Similarly, the tissue immunstimulant genes, IL-1β and TLR2 were measured in the spleen of feeding groups. The clinical signs of naturally diseased D. labrax showed corneal opacity and paleness of gills with excessive mucous secretion. The post-mortem abnormalities were severe hemorrhage and adhesion of internal organs. After bacteriological isolation and identification, the causative agent of mortality in the current study was Vibrio alginolyticus. The LD₅₀ of V. alginolyticus was 1.5×10^5.4 CFU/ml. The experimentally infected D. labrax showed ulceration, exophthalmia and skin hemorrhages. The post-mortem findings of the experimentally infected D. labrax revealed internal hemorrhage, spleen darkness and paleness of liver. There is no mortality and 100% RPS in groups fed GNPs then injected with V. alginolyticus, in those fed a combination of GNPs and S. cerevisiae and a group fed normal diet then injected with physiological saline (control negative), respectively. Contrarily, there was 10% mortality and 87.5 RPS in the group fed S. cerevisae then injected with V. alginolyticus. On the other hand, the control positive group showed 79% mortality. The spleen IL-1β and TLR2 immunostimulant genes were significantly increased in groups of fish fed GNNP, S. cerevisiae and a combination of GNPs and S. cerevisiae, respectively compared to control group. The highest stimulation of those immunostimulant genes was found in the group fed a combination of GNPs and S. cerevisiae, while fish fed S. cerevisiae had the lowest level. Dietary combination of GNPs and S. cerevisiae was shown to be efficient in preventing of vibriosis, with greatest stimulation of spleen IL-1β and TLR2 immunostimulant genes.