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6. The cytotoxicity and molecular mechanisms of the Clostridium perfringens NetB toxin

Author

ISLAM, AKM Azharul, NAKATANI, Mitsuki, NAKAJIMA, Takayuki, KOHDA, Tomoko, and MUKAMOTO, Masafumi

Subjects
Full Paper, Clostridium perfringens, Bacterial Toxins, necrotic enteritis, Enteritis, Cell Line, Japan, necrotic enteritis toxin B-like, Clostridium Infections, cytotoxicity, Animals, Public Health, Chickens, Injections, Intraperitoneal, Poultry Diseases
Abstract

The necrotic enteritis toxin B-like (NetB) toxin secreted by Clostridium perfringens is a key virulence agent in the pathogenesis of avian necrotic enteritis, a disease that causes significant economic loss to the poultry industry worldwide. NetB was purified from Clostridium perfringens type G (CNEOP004) that was isolated from chickens with necrotic enteritis in Japan. EC50 of this purified NetB toward chicken liver-derived LMH cells was 0.63 µg/ml. In vivo pathogenicity of NetB to chicks produced characteristic lesions of necrotic enteritis. Analysis of the localization of the NetB monomer and oligomer molecules on LMH cells showed that both molecules of the toxin were localized in non-lipid raft regions. Moreover, removal of cholesterol with the cholesterol depletion assay carried out in LMH cells detected both oligomers and monomers of the NetB molecule. These data suggest that the NetB toxin may recognize membrane molecules different from cholesterol in non-raft region. Furthermore, NetB-binding molecules on LMH cell membranes using the toxin overlay assay with immunoblotting showed that protein molecules of different molecular sizes were bound to NetB on non-lipid raft fractions. Further studies are necessary to characterize these protein molecules to examine their specific association with NetB binding and oligomerization.

Published
2020

14. Hypochlorous acid solution is a potent antiviral agent against SARS‐CoV‐2.

Author

Hatanaka, Noritoshi, Yasugi, Mayo, Sato, Tomoko, Mukamoto, Masafumi, and Yamasaki, Shinji

Subjects
ANTIVIRAL agents, HYPOCHLORITES, SARS-CoV-2, ACID solutions
Abstract

Aim: A novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) suddenly appeared in Wuhan, China, and has caused pandemic. In this study, we evaluated antiviral activity of purified hypochlorous acid (HClO) against coronaviruses such as SARS‐CoV‐2 and transmissible gastroenteritis virus (TGEV) responsible for pig diseases. Materials and Results: In a suspension test, 28.1 ppm HClO solution inactivated SARS‐CoV‐2 in phosphate‐buffered saline with the reduction of 104 of 50% tissue culture infectious dose per ml (TCID50 per ml) within 10 s. When its concentration increased to 59.4 ppm, the virus titre decreased to below the detection limit (reduction of 5 logs TCID50) within 10 s even in the presence of 0.1% foetal bovine serum. In a carrier test, incubation with 125 ppm HClO solution for 10 min or 250 ppm for 5 min inactivated SARS‐CoV‐2 by more than 4 logs TCID50 per ml or below the detection limit. Because the titre of TGEV was 10‐fold higher, TGEV was used for SARS‐CoV‐2 in a suspension test. As expected, 56.3 ppm HClO solution inactivated TGEV by 6 logs TCID50 within 30 s. Conclusions: In a carrier test, 125 ppm HClO solution for 10 min incubation is adequate to inactivate 4 logs TCID50 per ml of SARS‐CoV‐2 or more while in a suspension test 56.3 ppm HClO is adequate to inactivate 5 logs TCID50 per ml of SARS‐CoV‐2 when incubated for only 10 s regardless of presence or absence of organic matter. Significance and Impact of the Study: Effectiveness of HClO solution against SARS‐CoV‐2 was demonstrated by both suspension and carrier tests. HClO solution inactivated SARS‐CoV‐2 by 5 logs TCID50 within 10 s. HClO solution has several advantages such as none toxicity, none irritation to skin and none flammable. Thus, HClO solution can be used as a disinfectant for SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

Published
2022
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24. Genotyping and characterisation of the secretory lipolytic enzymes of Malassezia pachydermatis isolates collected from dogs

Author

Teramoto, Hideshi, Kumeda, Yuko, Yokoigawa, Kumio, Hosomi, Koji, Kozaki, Shunji, Mukamoto, Masafumi, Kohda, Tomoko, Teramoto, Hideshi, Kumeda, Yuko, Yokoigawa, Kumio, Hosomi, Koji, Kozaki, Shunji, Mukamoto, Masafumi, and Kohda, Tomoko

Abstract

Introduction: Malassezia species are commensals of normal skin microbial flora of humans and animals. These may become pathogenic under certain conditions such as those associated with atopic dermatitis or otitis externa in dogs. Material and methods: Isolates of Malassezia pachydermatis were obtained from 27 dogs with healthy external ears and 32 dogs with otitis externa. Isolates were characterised on the basis of their first internal transcribed spacer (ITS) and internal spacer 1 (IGS1) sequences. Their extracellular lipase and phospholipase activity were also analysed. Three types of phospholipase inhibitor were used to identify the subclasses of phospholipase associated with otitis externa. Results: The clinical isolates were classified into three ITS and three IGS1 sequence types. No significant differences in pathogenicity were detected among the ITS or IGS1 genotypes, and all of the isolates exhibited similar levels of lipase activity. The isolates derived from the dogs with otitis externa showed significantly higher phospholipase activity than those obtained from the dogs with healthy external ears. A phospholipase D inhibitor reduced the phospholipase activity of the isolates obtained from the dogs with otitis externa. Conclusions: This study did not show any significant differences in pathogenicity among the ITS or IGS1 genotypes but does suggest that phospholipase D might be one of the virulence factors involved in the inflammation of the external ear caused by M. pachydermatis.

Published
2015

25. Effect of Muramyl Dipeptide on Phagocyte Functions in Rainbow Trout (Oncorhynchus mykiss)

Author

HIROTA, Yoshikatsu, MUKAMOTO, Masafumi, BABA, Tsuyoshi, KODAMA, Hiroshi, and AZUMA, Ichiro

Subjects
endocrine system, animal structures, urogenital system, animal diseases, digestive system
Abstract

application/pdf, The activation of head kidney phagocytes was studied in rainbow trout injected with an immunopotentiator muramyl dipeptide (MDP). Phagocytes showed dose-dependent migratory response to zymosan-treated rainbow trout serum in chemotaxis assay using Nuclepore membrane. Phagocytes of MDP-injected fish showed higher degree of chemotactic response than those from control fish, Phagocytic activity against hydrophilic plastic beads was enhanced when zymosan-treated trout serum was added to cultures. The activity increased after injection of MDP to trout. Increased level of superoxide generation was observed in phagocytes from MDP-injected fish compared with those from control fish when the cells were stimulated either with phorbol 12-myristate 13-acetate, Aeromonas salmonicida cell lysate or opsonized zymosan in cytochrome C reduction test., Bulletin of the University of Osaka Prefecture. Ser. B, Agriculture and life sciences. 1993, 45, p.67-74

Published
1993

27. Draft Genome Sequence of Clostridium botulinum Type B Strain Osaka05, Isolated from an Infant Patient with Botulism in Japan

Author

Sakaguchi, Yoshihiko, primary, Hosomi, Koji, additional, Uchiyama, Jumpei, additional, Ogura, Yoshitoshi, additional, Umeda, Kaoru, additional, Sakaguchi, Masakiyo, additional, Kohda, Tomoko, additional, Mukamoto, Masafumi, additional, Misawa, Naoaki, additional, Matsuzaki, Shigenobu, additional, Hayashi, Tetsuya, additional, and Kozaki, Shunji, additional

Published
2014
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28. Pathogenicity of botulinum C·D mosaic toxins in animals

Author

Kohda Tomoko, Kozaki Shunji, Mukamoto Masafumi, and Nakamura Keiji

Subjects
Toxin, Biology, Toxicology, medicine.disease_cause, medicine.disease, Virology, Microbiology, medicine, Neurotoxin, Clostridium botulinum, Avian botulism, Botulism, Typing, Primer (molecular biology), Gene
Abstract

Clostridium botulinum types C and D are related to avian and mammalian botulism. Immunological cross-reactions between the two neurotoxins (BoNTs/C and D) remain to be elucidated. In avian botulism, we found that the isolates in Japan produced a toxin that was neutralized by both type C and D antitoxins. Analyses of the nucleotide sequences of several strains demonstrated that the BoNT gene comprises two-thirds of BoNT/C and one-third of the BoNT/D gene, suggesting that the avian isolates produce the mosaic of BoNT/C and BoNT/D. The isolate harboring C/D mosaic toxin gene may be characteristic of subtype C α , which was considered to produce C1 and D toxic factors. In type D, some strains (such as strain South African) produced a D/C mosaic toxin. In Japan, bovine botulism has occurred sporadically since 1999. We genetically and immunologically examined the BoNT of bovine botulism-related isolates. Eventually all bovine isolates produced the D/C mosaic neurotoxin, showing that it exhibits the highest lethal activity in mice compared with those of other types of BoNTs. We prepared four sets of primers to differentiate the genes for the mosaic and authentic forms. PCR with the primer sets was useful for typing mosaic toxins. These results indicate that the C·D mosaic neurotoxins are probably a pathogenic agent causing certain forms of avian and bovine botulism.

Published
2008

39. A new BoNT/B subtype detected from infant botulism in Japan

Author

Seto Yoshiyuki, Kohda Tomoko, Kozaki Shunji, Mukamoto Masafumi, and Umeda Kaoru

Subjects
Clostridium botulinum type B, Infant Botulism, Multiplex polymerase chain reaction, Genotype, Pulsed-field gel electrophoresis, Biology, Toxicology, Virology, Genotyping, Subtyping, Southern blot, Microbiology
Abstract

Clostridium botulinum type B has recently been isolated from food-borne and infant botulism cases. The BoNT/B in these cases was divided into two main subtypes, B1 and B2, based on analysis of the amino acid sequences. The genetic properties of the C. botulinum type B strains in relation to infant botulism in Japan (strains 111, Osaka05 and Osaka06) were analyzed by sequencing of the bont/B and nontoxic component genes (ha70, ha17, ha33, p21 and ntnh), PFGE genotyping and southern blot hybridization with bont/B probe. Strain Osaka05 produced particular BoNT/B, which were grouped to a novel variant on phylogenetic analysis. The substitutions of amino acid residues were concentrated in the heavy chain, especially in the HCC subdomain. Strains 111 and Osaka06 were classified as subtype B2. The nontoxic component gene arrangements within these subtypes were identical to each other. Multiplex PCR method for BoNT/B subtyping (B1, B2 and Osaka05) was newly established based on bont/B nucleotide sequences variability. Three isolates from infant botulism in Japan were identified as different genotypes based on Sma I-digested PFGE. The BoNT/B encoding gene was shown to be located on large plasmids (approximately 150 or 260–280 kbp) for various subtype strains including isolates from infant botulism in Japan using Southern blot hybridization after nondigested PFGE with bont/B probe.

Published
2008

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