IDENTIFICATION OF EDWARDSIELLA ICTALURI AND AEROMONAS HYDROPHILA ISOLATED FROM PANGASIANODON HYPOPHTHALMUS SIMULTANEOUSLY USING MULTIPLEX PCR (M-PCR)

N Amira-Syahidah¹, M Nur-Nazifah^1*, R Rimatulhana², AA Sabuti¹

1Department of Marine Science, Kulliyyah of Science, International Islamic University Malaysia, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia

2National Fish Health Research Centre, Department of Fisheries Malaysia, 11960 Batu Maung, Penang, Malaysia

*Corresponding author: nurnazifah@iium.edu.my

Striped catfish, Pangasianodon hypophthalmus is one of the major species cultured in Malaysia, especially in Pahang River. However, the challenge faced by farmers culturing this species is bacterial diseases The two main pathogens identified as the cause of disease were Edwardsiella ictaluri and Aeromonas hydrophila. Thus, this study was conducted to identify both pathogens simultaneously using multiplex PCR (M-PCR). Firstly, sampling of Pangasianodon hypophthalmus was conducted at two local farms located at Pekan and Temerloh, Pahang. 30 samples were collected from each farm and four organ tissues were taken which were liver, spleen, kidney and brain. DNA were extracted from all tissues and pursued for M-PCR to detect presence of Aeromonas hydrophila and Edwardsiella ictaluri . The primer used for detection of Edwardsiella ictaluri are IVS and IRS, while for Aeromonas hydrophila are AER-16-R and AER-16-F. The size of amplicon for Edwardsiella ictaluri is 2000bp and Aeromonas hydrophila is 1328bp. Both bands will appear on agarose gel if the fish was infected with bacteria Aeromonas hydrophila and Edwardsiella ictaluri. Only one band will appear if the fish was infected by either one of the bacteria, while no band appear if the fish was not infected by any of the bacterium. Our result suggest that this method can be used to detect both bacteria in Pangasianodon hypophthalmus in Pahang River.

Keywords: Pangasianodon hypophthalmus, Edwardsiella ictaluri, Aeromonas hydrophila, multiplex polymerase chain reaction (M-PCR)

RAPID AND ACCURATE SNP GENOTYPING OF CLONAL BACTERIAL FISH PATHOGENS, NOCARDIA SERIOLAE WITH MISMATCH AMPLIFICATION MUTATION ASSAY

Cuong T. Le^1,2*, Erin P. Price^2,3, Derek S. Sarovich^2,3, Thu Thi Anh Nguyen⁴, Hung Vu-Khac⁵, Ipek Kurtböke², Wayne Knibb², Shih-Chu Chen⁶, Mohammad Katouli²

 ¹Institute of Aquaculture, Nha Trang University, Nha Trang, Vietnam

²Centre for Bioinnovation and School of Science, Technology and Engineering University of the Sunshine Coast, Maroochydore 4558, Australia

³Sunshine Coast Health Institute, Birtinya, Queensland, Australia

⁴Institute for Biotechnology and Environment, Nha Trang University, Nha Trang, Vietnam

⁵Central Vietnam Veterinary Institute, Nha Trang, Vietnam

⁶Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

 ^*Corresponding author: cuonglt@ntu.edu.vn

Nocardia seriolae, a Gram-positive, filamentous bacterium that causes granulomatous diseases with high losses for both marine and freshwater fish aquaculture worldwide. Our previous genomic analysis of this pathogen revealed recent importation and subsequent widespread dissemination in mariculture farms in Vietnam. Since they are genetically clonal and traditional molecular techniques have limited discriminatory power, surveillance for their spread and source tracing currently remains a challenge. To overcome this, we targeted two phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. We then designed and optimized two methods that genotype strains based on melt analysis of mismatch amplification mutation assay (Melt-MAMA) and agarose gel-based mismatch amplification mutation assay (Agarose-MAMA). All assays were validated across DNA from 60 strains and fish tissues. Our MAMA assays accurately genotyped strains both from culture and fish tissues using either real-time or conventional PCR instrumentation. These assays provide rapid, discriminative, simple, reproducible typing tools for epidemiological investigations of N. seriolae infections and disease source tracking.

Keywords: Nocardia seriolae, Disease, Genotyping, Genomic, MAMA assays, Aquaculture

DEVELOPMENT OF ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) FOR VNN DETECTION IN OREOCHROMIS SPP.

 Irfan-Hakimi, R¹, Azila Abdullah^2*, Nur-Nazifah, M¹, Firdaus-Nawi, M¹, Hazreen-Nita, M³

 ¹Kulliyah of Science, International Islamic University Malaysia, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia

²National Fish Health Research Centre, Batu Maung, 11960 Penang, Malaysia

³Faculty of Agro-Based Industry, University Malaysia Kelantan, Jeli Campus, 17600 Jeli, Kelantan, Malaysia

^*Corresponding author: azila@dof.gov.my

Viral Nervous Necrosis (VNN), also known as viral encephalopathy and retinopathy is a serious viral disease affecting more than 120 species including marine and freshwater farmed fishes. Adult fishes infected with VNN are usually asymptomatic and eventually become carrier that transmit the virus to the offspring through eggs. In responds to the needs of practical and validated tests for the study of Nervous Necrosis Virus (NNV) immunity, Enyzme Linked Immunosorbent Assay (ELISA) protocols were developed, improved and compared for the detection of serum antibodies against the virus in vaccinated fishes. Hence, in this study, ELISA method for VNN detection in Oreochromis spp. was developed. Indirect ELISA using non-purified NNV from cell culture supernatant as coating antigen, Tilapia anti VNN (TaVNN) as primary antibody, Rabbit anti Tilapia (RaT) as secondary antibody and Goat anti Rabbit (GaR) HRP as tertiary antibody were performed in this study. Optimum dilution for antibodies that involved for the detection process were determined. This assay was considered efficient as it does not depend on purified virus or recombinant capsid NNV protein as coating antigen. Moreover, the improved protocol was flexible as it could be applied to wider range of species.

Keywords: Viral Nervous Necrosis, Nervous Necrosis Virus, ELISA, Viral disease

COMPARISON OF OUTER MEMBRANE PROTEIN PROFILING OF STREPTOCOCCUS AGALACTIAE AND STREPTOCOCCUS INIAE

M Faizal¹, Syafiq Ridzuan², M Nur-Nazifah^1*

 ¹Department of Marine Science, Kulliyah of Science, International Islamic University Malaysia, Bandar Indera Mahkota, Jalan Sultan Ahmad Shah, 25200 Kuantan, Pahang, Malaysia

²National Fish Health Research Centre, Jalan Batu Maung, 11960 Batu Maung, Penang, Malaysia

^*Corresponding author: nurnazifah@iium.edu.my

Tilapia is the third important finfish species group cultured worldwide in terms of production. However, a major setback in tilapia aquaculture is the outbreak of Streptococcosis. Streptococcosis, mainly caused by Streptococcus agalactiae and Streptococcus iniae has caused enormous economic losses in tilapia aquaculture worldwide. The infection can cause up to 100% mortality of tilapia culture. To address this issue, the study on bacteria structure is very essential for control and prevention measures of this disease. Thus, the objective of this study is to profile the outer surface protein of Streptococcus agalactiae and Streptococcus iniae isolated from tilapia. First, Streptococcus agalactiae and Streptococcus iniae were isolated from tilapia case in Kuala Lipis and Tasik Banding. The bacteria were then cultured and inoculated into solid and liquid medium before centrifuged at 5000rpm at 4°C for 10 minutes. Pellet collected were washed and stored in PBS. The pellet was sonicated 20 times, one minute each to break the protein cell. Extraction of Outer Surface Protein (OSP) then was conducted using Ultracentrifuge at 28500rpm, 4°C for 4 hours. Sodium Dodecyl Sulfate Polyacrylamide Gel-Electrophoresis (SDS PAGE) was used to determine the protein profiling. As a result, the major protein found in Streptococcus agalactiae was ranging from 85 kDa until 31 kDa. Meanwhile, the major protein found in Streptococcus iniae was ranging from 75 kDa until 31 kDa. The major Outer Surface Protein of Streptococcus agalactiae was 72 kDa, 67 kDa, 54 kDa and 44 kDa. Meanwhile, the major Outer Surface Protein of Streptococcus iniae was 48 kDa and 41 kDa. Our results suggest that the outer surface protein might be suitable as the bivalent vaccine candidate for tilapia in the future.

Keywords: Streptococcus agalactiae, Streptococcus iniae, Outer surface protein, Protein profiling, Vaccine candidate

ANTIBACTERIAL ACTIVITY OF GOLD NANOPARTICLES SYNTHESIZED BY MELALEUCA CAJUPUTI LEAVES EXTRACT AGAINST VIBRIO PARAHAEMOLYTICUS

Nurul Ashikin Elias¹, Marina Hassan¹, Mohamad Sofi Abu Hassan¹, Noor Aniza Harun^1,2,3

 ¹Higher institution Centre of Excellence (HIcoE) Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

²Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

³Advanced Nano Materials Research Group, Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

^*Corresponding author: marina@umt.edu.my

Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimps has been a major threat to the farmers since 2009 which caused by invaded of the bacteria known as Vibrio parahaemolyticus. The conventional method to treat AHPND was using antibiotics. The overuse of antibiotics can create “antibiotic resistance genes” of the pathogens and was reported can leech to the environment and most of the residues are remain in the shrimp’s biomass and can easily be transferred through the food chain. Little known, the application of nanotechnology in the aquaculture industry is expected to be a promising method to curb the disease, yet it is still under-discovered and found to be scarce in the aquaculture industry. Thus, this study mainly focused on synthesizing the gold nanoparticles (AuNPs) by using aqueous leaves extract of Melaleuca cajuputi. The synthesized nanoparticles have been characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction. The synthesized AuNPs were revealed surface plasmon resonance (SPR) band of 530-535nm and in ranges sized of 15-30nm with a spherical shape, as confirmed by TEM. X-ray diffraction (XRD) confirmed there is no pre-existed Au metal in the leaves extract, but synthesized AuNPs was successfully reduced by leaves extract with Au precursors. In vitro, the antibacterial activity of AuNPs synthesized by M. cajuputi leaves extract was tested by using the well diffusion method, broth-dilution microtiter plate assay (minimum inhibitory concentration, MIC and minimum bactericidal concentration test, MBC). The result revealed that the zone of inhibition (ZoI) of AuNPs against V. parahaemolyticus was 17.3 ± 0.58 mm. The MIC and MBC of AuNPs against V. parahaemolyticus was 18.7 mg/ml respectively. This study indicates that AuNPs possessed a strong antimicrobial activity and can be one of the promising methods to replace antibiotics and thus contributing to the sustainability of the aquaculture industry. 

Keywords: Gold nanoparticles, Melaleuca cajuputi, Vibrio parahaemolyticus, Acute hepatopancreatic necrosis disease (AHPND)

EFFECT OF PROBIOTICS ON BACTERIAL COMMUNITY COMPOSITION IN THE CULTURE MEDIA OF AFRICAN CATFISH (CLARIAS GARIEPINUS BURCHELL, 1822)

 Indah Istiqomah^1*, Alim Isnansetyo¹, Endang Sulistyaningsih¹, Toto Sudargo¹, Andita Ratih Dewanti¹

 ¹Faculty of Agriculture, Gadjah Mada University, Sleman, D.I. Yogyakarta, Indonesia

²Faculty of Medicine, Public Health and Nursing, Gadjah Mada University, Sleman, D.I. Yogyakarta, Indonesia

^*Corresponding author: indah_ist@ugm.ac.id

Controlling the fish microbiome is a strategic step toward fish health promotion and disease prevention. Oral autochtonous probiotics have been shown to improve fish digestion, growth, and immunity. The effect of oral probiotic application on the bacterial microflora in fish culture media, on the other hand, is rarely studied. To address this issue, we used metabarcoding (16S rRNA gene) to assess the impact of oral probiotics on the microbiome profile of African catfish (Clarias gariepinus BURCHELL, 1822). Bacteria were collected from fish tank water using 0.45um filter paper. The following steps were DNA isolation, amplicon generation (16SV3-V4) using the Illumina paired-end platform, and  analysis. The bacterial microflora of catfish culture water had an average Operational Taxonomic Unit (OTU), unique taqs, taxon taqs, and total taqs of 410, 3103, 131486, and 134590, respectively. Taxonomic annotations revealed that the use of oral probiotics increased the composition of the bacterial community in the water, with Firmicutes and Proteobacteria dominating. The use of probiotics reduced the relative abundance of Deinococcus, Rhodobacter, Meganema, Dechloromonas, Hydrogenophaga, and Bacteriodes while increasing the relative abundance of Paludibacter, Christencenellaceae, Acitenobacter, Parabacterioides, Macellibacteriodes, and Vibrio. Our findings indicate that using oral probiotics in African catfish (Clarias gariepinus BURCHELL, 1822) farming may change the bacterial community composition in the culture media.

Keywords: oral, probiotics, water, catfish, mibrobiome