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論文題目位論文の要旨Studies on the antimicrobial effect of Mallotus japonicus on growth ofAeromonas hydrophila, A. salmonicida, Edwardsiella tarda and Vibrio
anguillarum（アカメガシワ Mallotus japonicus の Aeromonas hydrophila、 A. salmonicida、
Edwardsiella tarda および Vibrio anguillarum に対する抗菌効果について）

広島大学大学院生物圏科学研究科
生物資源科学専攻
学生番号 D180294
氏
名 Ebenezer Koranteng Appiah

Summary
The extensive use of antibiotic in aquaculture is primarily due to the growing cultivation offishes in limited culture environments. Large quantities of culture fish products are beingcultured in limited culture environments, and this predisposes them to a number of stressors
which increase their susceptibility to several disease infections and/or pathogens. However, asa measure to control their spread, several synthetic antimicrobials are being used and this causes
detrimental side effect to both culture animals and humans consumes them. So therefore, inorder to replace antibiotic use in aquaculture, other alternatives method such as probiotic,
vaccines and plant-derive bioactive compounds are being sought after. Nonetheless, unlike
vaccines and probiotics, plant-derive bioactive compounds are cheaper and possess a broader
spectrum of host range which make them an idea substitute to antibiotics.
1. Characterization of Aeromonas hydrophila infections in Heterotilapia buttikoferi
Heterotilapia buttikoferi is a freshwater fish species, which belongs to the family Cichlidae.
The fish is native to West Africa and mostly found endemic to coastal rivers and streams in
Guinea-Bissau and Liberia. Nevertheless, due to their high aesthetic value and rapid growthefficacy, they are mostly imported and bred as fishes for ornamental purposes and/or food forhuman consumption. In Japan, they are only bred for ornamental purposes.

Nonetheless in recent times, disease outbreak caused by bacterial pathogen Aeromonas
hydrophila swept through the ornamental fish industry, hence resulting in large fish kills andmorbidity. Nonetheless information on the pathogenicity of A. hydrophila to H. buttikoferi wasnot reported. So, in this study using the infection challenge trial, we described for the fish time
the clinical signs and pathogenicity of A. hydrophila to H. buttikoferi. It was observed from thestudy that fish challenged with specific lethal doses (LD50) of the bacterium elicited strong
clinical signs ranging from exophthalmia, corneal opacity, ascites, liquefaction of internalorgans and fin rot. The median lethal dose of the bacterium at which it is able to cause 50%
mortality in the fishes was calculated at 2.0×106cfu/ml.

2. Antimicrobial effect of Mallotus japonicus against fish pathogenic bacteria
In this study the antimicrobial activity of the medicinal plant Mallotus Japonicus Müeller
Argoviensis was evaluated against the fish pathogenic bacteria −Aeromonas hydrophila, A.
salmonicida, Vibrio anguillarum and Edwardsiella tarda. It was observed from the study thatthe diethyl ether- , ethanol- and methanol extract of the plant, when challenged using the disk
diffusion assay, minimum inhibitory concentration (MIC) assay and minimum bactericidal
concentration (MBC) assay elicited strong antibacterial activities against the challenged
bacteria species. It was however observed that, an extract of a diethyl ether group elicited thestrongest bactericidal activity against the studied bacteria species at concentrations of 0.625,
2.5 and 5 mg ml-1, respectively. Also, some minor active compounds present within the plant
extracts and detected by LC-MS such as citrinin, corilagin, fraxetin and furfuryl acetate
demonstrated a corresponding strong antibacterial activity against the challenged bacterial
species; hence confirming the potential of the plant as an alternative antimicrobial to antibiotics.
3. Immunomodulatory effect of M. japonicus on fish innate-immune response
Additionally, upon evaluating their role in improving the immune response of fish after dietaryintake, it was observed that the diethyl ether extract of the plant, when supplemented in the dietof fish significantly improve the innate-immune response against pathogenic disease infectionsof A. hydrophila. The lysozyme, serum bactericidal activity, globulin, total protein, albumin andphagocytic activity of the fish’s blood was significantly improved after feeding on dietsupplemented with the plant extract− M. japonicus.
3. Effect of M. japonicus supplemented diet on fish intestinal microbiota
The role of the extract on the microbial richness and diversity of the fish microbiota was alsoevaluated. It was observed from the study that feed supplemented with M. japonicus− diethylether extracts significantly enrich certain microbial taxa whereas diminishing others. For
instance, the phylum Firmicutes, which was the second most predominantly identified OTUs;
was significantly enriched after consumption of the supplemented plant diet contrarily to thatof the phylum Proteobacteria which was the most dominant. It was observed that as theconcentration of the plant additive increases, microbial richness and diversity skewed toward
the phylum firmicutes contrary to the phylum proteobacteria. This was in correspondence toseveral other studies in which plant base diet was used as supplement in animal feed. Weobserved that the most commonly detected bacterial genus−Bacillus, often increases as fishes
feed supplemented diet increases contrary to that of opportunistic pathogenic bacteria.
Suggesting that the antimicrobial peptides in the plant additives might have counteract theproliferation of these taxa.
However, a fairly diverse microbial community was observed after feeding of plantsupplemented diet. For instance, 5 phyla were identified (Proteobacteria, Fusobacteria,Firmicutes, Bacteroidetes and Actinobacteria), with 7 class (Actinobacteria, Flavobacteria,Bacilli, Fusobacteriia, α−proteobacteria, β−proteobacteria and γ−proteobacteria), 14 families(Nocardiaceae,Flavobacteriaceae,Bacillaceae,

Staphylococcaceae,Fusobacteria,Caulobacteraceae, Rhizobiaceae, Comamonadacea, Aeromonadaceae, Enterobacteriaceae,
Pseudomonadaceae, Shewanellaceae, Xanthomonadaceae) and 21 genera (Rhodococcus,

Nocardia,Flavobacterium,

Lysinibacillus,

Chryseobacterium,Staphylococcus,

Cetobacterium,

Haloanella,Bacillus,Brevundimonas,

Fictibacillus,

Shinella,

Delftia,

Diaphorobacter, Pelomonas, Aeromonas, Plesiomonas, Citrobacter, Pseudomonas, Shewanella,Pseudoxanthomonas). This effect of enrichment of specific taxa were observed not only inplant-supplemented diets but also in synthetic antimicrobial supplemented diet (Kokou et al.,2020).

3. Conclusion
In conclusion, the present study demonstrated that the M. japonicus has a significant
antimicrobial effect against some important pathogenic bacteria of fishes . Therefore, as analternative to antibiotics, the plant extracts, particularly the diethyl ether-extract, can beconsidered as a prophylactic to disease control in fish. Also, the diethyl ether extract, can beused as a dietary supplement to improve the innate-immune response of the host. Since, thehematological parameters of the fish blood serum further confirms that the type of extract,
dosage and duration of feeding significantly enhances the immune response of the fish against
the challenged bacterial species. M. japonicus supplemented plant diet selected for beneficial
microbial communities such as Bacillus, Rhodococcus, Cetobacterium and Lysinibacillus,
whereas decreasing the abundance of pathogenic groups such as Aeromonas, Pseudomonas,
Staphylococcus and Nocardia (Table 5.1). Bacillus, Rhodococcus, Cetobacterium andLysinibacillus enriched within the fish microflora are involved in biodegradation, bioconversion
and fermentation which helps in nutrient recycling whereas Aeromonas, Pseudomonas,
Staphylococcus and Nocardia constitute important disease-causing pathogens of fish.

Keywords: microbiota, innate-immune response, pathogenecity, M. japonicus andHeterotilapia buttikoferi, Aeromonas hydrophila, A. salmonicida, Vibrio
anguillarum and Edwardsiella tarda