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Pathological disorders caused due to metazoan parasitic infestation were studied in the hill stream fishes of northwest Himalayan region. Host specificity was found to be one of the fundamental characteristics features of metazoan parasites which belonged to the class cestoda, nematoda, trematoda and phylum acanthocephala. By this study it is assumed that successful co-evolution of the host and its parasite has caused the adaptation of the later by developing evading mechanisms in order to avoid extinction. Besides this it was also observed that some parasites have even understood to benefit from the well developed antiparasitic armament in fish intestinal epithelia. Thus parasites are exploiting the antiparasitic response mechanism of the host to optimize host finding, invasion and survival in the host. Such interaction between host and parasites are considered phylogenetically old. Some monogeneans, cestodes, digeneans and acanthocephalans were found to resist pronounced cellular and host reactions which even improved the attachment of parasite into the host predilection site. SEM study, hitstopathological examinations were conducted on in-situ parasites to understand the interaction between fish and its parasite and damage inflicted by parasite on the fish.

WSSV and AHPND are two of the most important shrimp diseases in the Philippines. Shrimp samples were gathered from 14 out of 17 regions of the Philippines during a disease surveillance study in 2014-2015. The samples were analyzed by PCR for detection of WSSV (2014-2015) and AHPND (starting mid 2015). Out of 5 regions visited in 2014, Region 6 (Negros Occidental), and Region 9 (Zamboanga) were positive for WSSV. In 2015, samples were gathered from 13 regions of the country. WSSV-positive regions include Regions 5, 6, 8, and 10. Highest occurrence was in Region 5 with 53.13% of the samples. WSSV-positive provinces include Camarines Norte, Camarines Sur, Masbate, and Catanduanes. In Region 6, WSSV was detected in Iloilo, Aklan and Negros Occidental. Shrimp samples from the wild were bought from a public market in Samar (Region 8), and one sample was found to be WSSV-positive. In Region 10, one sample from Lanao del Norte was WSSV-positive. For AHPND detection, samples were gathered from 9 regions. Only Regions 3, 4B, 7, 8, and 11 had AHPND-positive samples from the provinces of Pampanga, Oriental Mindoro, Marinduque, Cebu, Bohol, Leyte, and Davao del Sur. The study showed that WSSV is an established disease with numerous occurrences every year in major shrimp farming areas in the country, whereas AHPND is a newly emerging disease that occurred much earlier in other SE Asian countries but has only recently (2015) been reported in the Philippines. An online shrimp pathogen information resource is being developed incorporating elements of biobanking, remote data entry, and early warning system.

Nowadays, shrimp culture industry face up with the high risk cause by diseased. White spot syndrome and early mortality syndrome” (EMS) are considered to be serious diseases for shrimp culture. Vaccine was considered to be alternative for shrimp diseases control. This study was aimed to evaluate oral vaccine for controlling of WSSV and EMS infection in shrimp.
Oral vaccination could prevention of WSSV and V. parahaemolyticus infection. Vaccine against EMS and WSSV have been developed; fed 5 g of vaccine per 1 kg of diet for 1 day could increase the relative percent survival up to 80.76 % and 66.7% Vibrio parahaemolyticus and WSSV infection, respectively. Moreover, this vaccine induced the expression of shrimp immune gene.

Acid-fast bacterial diseases, Nocardiosis and Mycobacteriosis, give serious economic damages to the aquaculture industry in Japanese. This is caused by an intracellular pathogen called Nocardia seriolae and Mycobacteriumsp., respectively. In general, induction of cell-mediated immunity (CMI) is effective in infection defense against intracellular-pathogen. However, the conventional formalin-killed cell (FKC) vaccine mainly induces humoral immunity. Therefore, the prevention of the acid-fast disease infection with the vaccine deriving CMI is strongly expected. Glycolipids of the cell wall of acid-fast bacteria are known to activate the innate and adaptive immunity of the host in mammals. Thus, glycolipids were expected as a potencial CMI-inducible vaccine adjuvant in fish. In the present study, we extracted total lipid from N. seriolae and Mycobacterium sp. using chloroform and methanol and confirmed the existence of glycolipids by Thin-layer chromatography (TLC). First, to investigate the immune response against the glycolipids in fish, lipids from Mycobacterium sp. were injected to ginbuna crucian carp Carassius auratus langsdorfii. As a result of gene expression analysis after injection, inflammatory cytokine (marker of innate immunity); IL-1β and TNF-α, and Th1-related cytokine (marker of CMI induction); IFN-γ and IL-12 were induced whereas Th2-related gene; IL-10 and antibody titer (marker of humoral immunity) were suppressed in spleen leukocytes. Next, we performed challenge test to clarify the CMI-inducible adjuvant effect of the glycolipids in amberjack. The survival rate in challenge test showed that 54% of FKC + lipid vaccinated fish survived at 10 days after N. seriolae injection, whereas FKC only 20% survived. These results suggested that i) lipids delivered from acid-fast bacterium induces CMI, and ii) lipid from N. seriolae is viable CMI inducible adjuvant.

Streptococcus agalactiae is the major infectious bacterial of streptococcosis disease in tilapia and other fresh water fish species nowadays. Several trials on inactivated fish streptococcal vaccine development have been established but provide limited protection due to several serotypes of bacteria. Therefore, the improvement of the efficacy and effectiveness of streptococcoal vaccine is required due to the emerging of different serotypes of S. agalactiae. Previous study demonstrated that the surface immunogenic proteins (Sip protein) showed highly immunogenic properties in mammalian system and conserved between different serotypes of S. agalactiae. Hence, in this study, recombinant Sip protein was produced and purified from bacterial expression system. The molecular mass of recombinant Sip protein is 68.9 kDa which consists of His-tag and TRX- tag for purification. Large scale of recombinant Sip protein was purified using HisTrap HP column attached to FPLC instrument. The amount of purified protein 0.2 mg/ml was used as vaccination. This purified protein was mixed with inactivated streptococcal vaccine before immunized to the fish. It demonstrated that the combined vaccines can improve the efficacy of infected fish with approximately 75% relative percent survival (RPS). Moreover, the combination of this vaccine and adjuvant showed greater in the protection of streptococcosis disease. The immunity of vaccinated fish demonstrated that the antibody level in all vaccinated group was significantly increased in the first week of vaccination and slightly decreased thereafter. The non-specific immunity such as the activity of lysozyme, it found that the purified Sip protein group was highest; superoxide anion activity and alternative complement activity were found highest values in the combination of inactivated vaccine, Sip and adjuvant. From this study, Sip protein is the promising subunit vaccine which increasing conventional vaccine’s efficacy.

Brown-marbled grouper (Epinphelus fuscoguttatus) is an important marine fish species in many Asian counties including Taiwan, Indonesia, Malaysia, Singapore, and Thailand. These fish offering provided highly economic impact due to their fast growth, efficient feed conversion and high market value. Recently, brown-marbled grouper facing with a problem from vibriosis disease which caused by Vibrio spp. It is known that causative agents of vibriosis disease in grouper could be V. parahaemolyticus, V. alginolyticus, V. vulnificus and V. carchariae. In this study, molecular tools were used to identify Vibrio spp. isolated from diseased grouper. By 16s rDNA and phylogenetic tree analysis, it is demonstrated that 39 samples from 66 samples were identified as V. vulnificus biotype 1. Moreover, virulence genes identification can categorize V. vulnificus in this study to be 3 subgroups (G1, G2 and G3) in accordance to different number of virulence genes. Multi-locus sequence typing (MLST) revealed 2 new sequence type (ST) and one of these subgroup was classified as ST209. Interestingly, among 3 subgroups, growth rate of G2 which lacks of CM-like nab1 and CM-like nab2 allele showed slower growth rate than others and provided the lowest virulence to brown-marbled grouper. The inactivated vaccine produced from mix-subgroup V. vulnificus showed highest protection from V. vulnificus infection in grouper. Moreover, the identification of antigenic proteins was succeeding by secretome analysis which demonstrated 108 up-regulated proteins and 22 down-regulated proteins in the comparative proteome of G1 and G2, whereas 167 up-regulated proteins and 16 down-regulated proteins were found in the comparative proteome of G1 and G3. Thus, as a part of system immunology, those of secreted proteins will serve as a component of effective subunit vaccines. In the future, these proteins will be selected for formulate subunit vaccine against V. vulnificus.

Nervous Necrosis Virus (NNV) is an aquatic virus that can infect more than 30 species including the grouper, which is a valuable fish species in Taiwan. NNV causes up to 90-100% mortality in the aquaculture industry and that results in great economic loss in Taiwan grouper culture industry.
In present, the anti-NNV vaccines such as inactivated vaccines, subunit vaccines and recombinant vaccines which rely on adaptive immunity can only be applied to 30-days-post-hatching grouper larvae. However, the NNV acute infection is before adaptive immunity development. By using small interfering RNA (siRNA), specific degradation of viral mRNA could suppress the replication of virus effectively. The plasmid contained NNV RNA2 and EGFP reporter gene was co-transfected with siRNA plasmid into cells to examine the efficiency of siRNA. In previous study, we constructed the vector containing siRNA sequence against NNV and verified that it has effective suppression to the replication of NNV either in vitro or in vivo.
Moreover, the successful breeding rate of grouper larvae was increased to 27-40% after feeding with RNAi vaccine in our field trial compared to 2-5% in traditional grouper aquaculture. By large scale experiment, we also tried to feed our siRNA to the grouper in outbreak farm. The survival rate also significantly increased from 10% to 50-75%. These techniques were already developed and suitable for grouper industry in the future.

Screening of Bacteria from Salina tilapia (Oreochromis niloticus) Cultured in Floating Net Cages in Indonesian Marine Waters
Sutanti, Sugiyo Hadi Pranoto, and Ratu Siti Aliah
The Center of Agricultural Production Technology, Agency for the Assessment and Application of Technology (BPPT), Indonesia
*E-mail: sutanti@bppt.go.id
Salina (Saline Indonesian Tilapia) is a saline tolerant tilapia strain that has been developed by BPPT. Recently Salina tilapia has been cultured in floating net cage at Pramuka island, Kepulauan Seribu, DKI Jakarta and coastal area of Bantaeng, South of Sulawesi. In marine cultured fishes, streptococcal and vibriosis infections often develop into lethal condition. The aims of this study were to screen and identify the bacteria isolated from Salina tilapia that grew-up in marine environment by using 16S rRNA gene. The bacteria were isolated from infected Salina tilapia both from Pramuka Island and Bantaeng coastal area using selective medium thiosulfate citrate bile salt sucrose agar (TCBS) and Kenner-Fecal (KF) Streptococcus agar. Identification of bacteria was accomplished by PCR, using 16S rRNA, which revealed the 1500 bp PCR product. The PCR product was direct sequenced and analized using BLAST homology. The 16S rRNA sequence code VO2 analysis confirmed that have 100% similarity with Protobacterium leognathi. The bacteria were non pathogenic, symbiotic bacteria, and have a luminescence. The 16S rRNA sequence code StL311 analysis confirmed that have 91% similarity with Streptococcosis iniae Dan 1. The bacteria were a pathogen and listed among the emerging problem in aquaculture as streptococcosis. The 16S rRNA sequence code BT08 analysis confirmed that have 98% similarity with Salinococcus sesuvii. The bacteria were an endophytic and halophytic. The sequence described in this paper has been deposited in GenBank under accession number of KJ408270.1 for Streptococcus iniae and KU320975.1 for Protobacterium leognathi.
Keywords: Salina Tilapia, 16S rRNA, Protobacterium leognathi, Streptococcus iniae, Salinococcus sesuvii