DETECTION OF VIRAL NERVOUS NECROSIS (VNN) DISEASE IN ASIAN SEABASS (LATES CALCARIFER) IN AT EAST COAST AREA OF MALAYSIA

Safwan, M. K. A², Azila, A¹, Firdaus-Nawi, M^2*, Shaharah, M., I³ and, Nur-Nazifah, M²

1National Fish Health Research Centre, 11960 Batu Maung, P.Pinang.

2Aquatic Animal Health Lab, Institute of Oceanography and Maritime Studies, International

Islamic University Malaysia, Cherok Paloh, 26060, Kuantan, Pahang, Malaysia. 3Marine Fish Aquaculture Research Division, FRI Tanjong Demong, 22200, Besut, Terengganu, Malaysia.

*Corresponding author: firdausn@iium.edu.my

Asian seabass (Lates calcarifer) is one of the important marine fish cultured in Malaysia which accounted for 36% of the total marine production in 2019. Mass mortality among Asian seabass larvae and fry were regularly reported at several farms in Malaysia located at the east coast area. This area is among the main producer for marine fish like seabass and groupers. Clinical signs observed in the affected fish were suspected to be due to betanodavirus infection. Betanodavirus infection causes a fast spreading disease known as viral nervous necrosis (VNN) disease in fish. The disease could be transmitted by horizontal or vertical, and caused mass mortaöity up to 100% in larvae and juvenile. Hence, this study was conducted to determine the status of VNN in cultured Asian seabass in a district located at the east coast of Malaysia. Five farms were randomly selected based on the farmer’s agreement to join the sampling. Five samples were collected from each live stages of Asian seabass; egg, larvae, fry, juvenile, adult and brood stock available at each farm. The collected samples were then subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to detect betanodavirus infection. Results from this study showed that VNN-positive samples were detected in 80% of the selected farms. Fry is the most susceptible to VNN whereby samples from this stage recorded 100% positive detection. As a conclusion, the status of VNN in this district alone is considered endemic due to the high occurrence as detected by RT-PCR. Thus, to be able to maintain the fry production and also fast spreading of VNN, control and prevention of VNN at this area should be further explore since they are among the biggest marine fish fry producers that supply the seeds all over Malaysia.

Keywords: VNN, Betanodavirus, Asian seabass, RT-PCR

IDENTIFICATION OF B-CELL AND T-CELL EPITOPES OF THE BETANODAVIRUS CAPSID PROTEIN THROUGH IMMUNOINFORMATICS TOOLS

Hazreen-Nita M. K.*^1,2, Azzmer-Azzar A.A², Nur-Nazifah M.² and Azila A.³

1Department of Agricultural Sciences, Faculty of Agro-Based Industry, University Malaysia Kelantan, Jeli Campus, 17600 Jeli, Kelantan, Malaysia

2Kulliyyah of Science, International Islamic University Malaysia, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia.

3National Fish Health Research Centre, 19600 Jalan Batu Maung, Pulau Pinang, Malaysia.

*Corresponding author: hazreen.mk@umk.edu.my

Betanodavirus is an important pathogen that causes viral nervous necrosis (VNN) disease and has significant impacts through mass mortalities at larval stage in many marine fish species. In Malaysia, the re-occurrence of VNN disease was noticed at the same culture facilities, indicating the virus is endemic in the local environment. To date, there is no local VNN vaccine available in Malaysia and those commercial vaccines, namely ALPHA JECT micro®1 Noda, an injectable-inactivated vaccine for farmed Mediterranean seabass, and CIBA-Nodavac-R, an injectable-recombinant RGNNV vaccine, are not suitable to be used locally due to lack of information regarding host-specificity and impractical vaccination routes for the smaller size of fish such as larvae. Therefore, this study was conducted to identify B-cell and T-cell epitopes that are potential for formulating a vaccine against local betanodavirus as a preventive strategy for VNN disease. Characterization of two betanodavirus local isolates were done through molecular identification of capsid protein. The amplicons of 1026-bp were sequenced and showed 99% nucleotide sequence identity with 10 other betanodavirus capsid protein sequences in GenBank which revealed the genotype of local isolates as Redspotted grouper nervous necrosis virus (RGNNV). The B-cell epitope prediction analysis predicted 12 epitopes, and these antigenic sequences were further analyzed for T-cell epitopes of MHC class I and class II. Results revealed 4 T-cell epitopes were found on MHC class I while 15 T-cell epitopes were detected on MHC class II. The capsid protein of betanodavirus is mainly conserved across genotypes and is known as the main determinant of betanodavirus immunoreactivity during infection. Therefore, the portrayed epitopes shall be promising for the practical designing of a peptide-based vaccine against betanodavirus through recognition by B-cell and T-cell to elicit an effective humoral and cell-mediated immune response.

Keywords: betanodavirus, VNN, antigenic determinant, capsid protein, immunoinformatics tool

MOLECULAR EPIDEMIOLOGY OF MEGALOCYTIVIRUS IN FRESHWATER ANGELFISH (PTEROPHYLLUM SCALARE) FROM MALAYSIA

Che Azarulzaman Che Johan¹, Muhd Danish Daniel Abdullah^1,2, Sharifah Noor Emilia Syed Jamil Fadaak¹, Sandra Catherine Zainathan^1,2,*

¹Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

²Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

*Corresponding author: sandra@umt.edu.my

Malaysia has more than 630 culturists that are involved in the ornamental fish industry for culturing 250 species including local and exotic species. However, megalocytiviruses have been associated globally with severe systemic disease and economic loss in ornamental fish. Thus, this study aimed to detect the occurrence of Megalocytivirus while discovering its associated risk factors and the genotypes of its causative agent in an ornamental fish farm in Malaysia. A total of seven pairs of freshwater angelfish broodstock were used in this study to follow the fish’s life stages from eggs to market size. The water samples and other samples such as mucus swabs, gill swabs, freshwater angelfish eggs, fries, juvenile, snail, snail eggs, live feed (tubifex worms and moina), sediment samples and wild fish were collected periodically for preliminary sampling and from day 0 to day 60. Nested PCR amplifications were carried out on megalocytiviruses-related sequences. The phylogenetic tree was inferred from the major capsid protein (MCP) genes from all known Iridoviridae species using Molecular Evolutionary Genetics Analysis (MEGA). Pearson’s correlation coefficient values used to determine the strength of the correlation between the presence of megalocytiviruses in P. scalare samples and its associated risk factors. A total of 312 samples out of 935 pooled and individual samples, demonstrated positive results in tests for the presence of megalocytiviruses-related sequences except snail eggs and wild fish (Poecilia reticulata). Detection of Megalocytivirus-associated viruses in water samples demonstrated evidence of horizontal transmission of the disease. All the nucleotide sequences found in this study had high nucleotide identities of 88% to 99 % to each other and were closely related with Megalocytivirus genotype I infectious spleen and kidney necrosis (ISKNV). Overall, this study was the first to confirm the existence of different possible routes of megalocytiviruses distribution in the ornamental fish farm in Malaysia.

Keywords: Epidemiology, ISKNV, Megalocytivirus, Ornamental fish, Risk factors

ISOLATION AND IDENTIFICATION OF BACTERIA ISOLATED FROM NILE TILAPIA (OREOCHROMIS NILOTICUS) AND TROPICAL CATFISH (HEMIBAGRUS NEMURUS) IN TERENGGANU, MALAYSIA

Mei Rifqi Mursyidah¹, Mohd Naqib Azfar Mohd Roslan¹, Hasrul Hizat Hassin¹, Norraidah Abd Azif¹, Nasrenim Suhaimin¹, Mulham Alfatama², Hassan Sheikh Ibrahim¹, Laith A. Abdulrazzak¹,Nurul Aqilah Iberahim¹, Nadirah Musa¹, Lee Kok Leong¹, Najiah Musa¹

¹Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu (UMT), 21030 Kuala Nerus, Terengganu, Malaysia

²Faculty of Pharmacy, Universiti Sultan Zainal Abidin (UniSZA), Besut Campus, Besut 222000, Terengganu, Malaysia

Corresponding author: najiah@umt.edu.my

Bacterial infection is one of the major problems in freshwater aquaculture that may cause high mortalities and incur heavy losses. The aims of this study are to isolate and identify bacterial pathogens from diseased Nile tilapia (Oreochromis niloticus) and tropical catfish (Hemibagrus nemurus) in Terengganu, Malaysia. Diseased fishes were collected from commercial farm and UMT Hatchery. The clinical signs shown included lethargy, ulcers, haemorrhage, exophthalmos and erratic swimming. Bacterial isolation was conducted on kidney, spleen, brain and liver using trypticase soy agar (TSA) with 24 to 36 hours incubation at 30°C. Bacterial colonies were subjected to morphological characterisation, and biochemical identification using BD PHOENIX^TM ID/AST System. The results showed Aeromonas veronii vs sobria (liver, H. nemurus), Plesiomonas shigelloides (brain, O. niloticus), Streptococcus agalactiae (brains, H. nemurus and O. niloticus), Streptococcus porcinus (kidney and skin, O. niloticus), Dermacoccus nishinomiyaensis (brain, O. niloticus), Staphylococcus pettenkoferi (liver and skin, O. niloticus), Staphylococcus pasteuri (liver, O. niloticus) and Bacillus coagulans (kidney, H. nemurus). Blood agar test showed absence of β-haemolytic activity.

Keyword: Bacterial infection, freshwater aquaculture, Gram-negative, Gram-positive bacteria

DISINFECTION OF ROTIFER, BRACHIONUS PLICATILIS LEADING TO BACTERIA-FREE POPULATIONS USING SIREHMAX^TM SOLUTION

Nur Fatin Afifah Osman Manah^1*, Shaharah Mohd Idris¹, Azila Abdullah², Nur Nazifah Mansor³

¹Fisheries Research Institute (FRI), Tanjung Demong, Terengganu, Malaysia

²National Fish Health Research Division (NaFish), Batu Maung, Penang, Malaysia

³International Islamic University Malaysia (IIUM), Kuantan, Pahang, Malaysia

*Corresponding author: fatinafifah22@gmail.com

Rotifer is one of an essential food sources for marine fish larvae. Organic loads and microbial accumulation in rotifer cultures especially in batch culture are very high. Some of these bacteria can be pathogenic to rotifers. As rotifers are the first feed of fish larvae, their bacterial load can have a major impact on the survival. A reliable method was developed to disinfect rotifer, Brachionus plicatilis by using SirehMAX™.  SirehMAX^TM is a herbal product containing betle leaves extract that has potential as an alternative to commercial antibiotic in aquaculture. The research was focused on determining the optimum exposure time and concentration dosage of SirehMAX™ for rotifer disinfection. The results indicate that the use of 10 ppm of SirehMAX™ for one-hour time is a very effective protocol for rotifer disinfection with negligible lethal effects on rotifers. The study has successfully tested disinfection protocols that were highly effective with regard to survival of rotifers.

Keywords: Rotifer, Brachionus Plicatilis, disinfection, SirehMAX^TM

THE FIRST DESCRIPTION OF HISTOPATHOLOGY OF LATES CALCARIFER HERPESVIRUS (LCHV) INFECTION IN BARRAMUNDI (LATES CALCARIFER)

 Mai Dang^{1, a, *}, Tran Duc Dien^{2, a, *}, Ut Phan Van³, Vo Thi Ha², Vu Viet Dung², Nguyen Trinh Duc Hieu⁴, Viet Cuong Hua¹, Nguyen Thi Kim Hue¹, Nguyen Thi Thu Giang¹, Vu Huu Trương¹, Ha Thanh Dong⁵

¹ Institute of Veterinary Research and Development of Central Vietnam, Km 4, 2/4 Street, Vinh Hoa, Nha Trang, Khanh Hoa 57000, Vietnam

² Coastal Branch of the Joint Vietnam - Russia Tropical Science and Technology Research Center, 30 Nguyen Thien Thuat, Nha Trang, Khanh Hoa 57000, Vietnam

³ Institute of Aquaculture, Nha Trang University, No 2 Nguyen Dinh Chieu Street, Nha Trang, Khanh Hoa 57000, Vietnam

⁴ Institute of Oceanography, Vietnam Academy of Science and Technology (VAST), 01 Cau Da, Nha Trang, Khanh Hoa, 57000, Vietnam

⁵ AARM/FAB, School of Environment, Resources and Development, Asian Institute of Technology, Pathum Thani, Thailand

 ^a Joint First author, ^* Corresponding authors:

Mai Dang, Institute of Veterinary Research and Development of Central Vietnam, Km 4, 2/4 Street, Vinh Hoa, Nha Trang, Khanh Hoa 57000, Vietnam. Phone number: +84 766486859. Email: morning46@gmail.com

Tran Duc Dien, Coastal Branch of the Joint Vietnam - Russia Tropical Science and Technology Research Center, 30 Nguyen Thien Thuat, Nha Trang, Khanh Hoa 57000, Vietnam. Phone number: +84  986019997. Email: mrtran_cnvb@yahoo.com

Barramundi (Lates calcarifer), an economically important species for mariculture in the Asia-Pacific region, has been threatened by various infectious diseases. Lates calcarifer herpes virus (LCHV) is a newly emerging virus which was first reported in farmed barramundi in 2015. Up to now, the pathognomonic histopathological lesion of this disease has not yet been described for the purpose of disease diagnosis. This study investigated the histology of five natural LCHV outbreaks throughout a year and consistently detected the presence of a typical type of intra-nuclear inclusion bodies (IIBs) in various organs of infected fish. Experimental infection was conducted by injecting the healthy fish with filtered supernatant of crude extract from the LCHV-positive barramundi from the field. Histological examination of experimentally infected barramundi clearly indicated the presence of the same type of IIBs in multiple organs, including the liver, pancreas, kidney, eyes, gills, and adipose tissue. The presence of IIBs in barramundi infected with LCHV was a pathognomonic feature that can be used for the histological diagnosis and particularly for surveillance of this emerging viral disease in barramundi farming countries.

Keywords: pathognomonic, intra-nuclear inclusion bodies (IIBs), diagnosis, coinfection, herpes virus