Pengembangan Kultur sel dan Vaksin Inaktif untuk Pencegahan Infeksi Megalocytivirus pada Ikan Gurami (Osphronemus goramy).

Abstract

Megalocytivirus of family Iridoviridae is a known pathogen that caused fatal systemic infection lead to massive death and significant economic losses of numerous fish species in marine, fresh water and ornamental fish industries. In Indonesia, since 2011 megalocytivirus had been found as main pathogen in some cases of giant gourami outbreak especially in West Java, Central Java and Bali. Rapid expansion and intensification in aquaculture has led to many cases of disease outbreaks. Mortality rates of fish due to viral infections is high and up to now there’s no effective treatment methods to overcome the problem. Prevention is a better choice than treatment although efforts to prevent and control diseases caused by viral infections are still difficult to do. The aim of this study was to develop a primary cell culture and an inactivated vaccine for megalocytivirus infection in giant gourami. The first stage of this study was conducted to identify and characterized megalocytivirus in cultured giant gourami. The virus was identified using universal and spesific primers for megalocytivirus and infectious spleen and kidney necrosis virus (ISKNV). Sequencing and BLAST analysis were used to develop phylogenetic tree. The result showed that phylogenetic analysis of major capsid protein (MCP) gene unveiled a new megalocytivirus strain, designated as giant gourami iridovirus (GGIV). GGIV formed cluster belonged to ISKNV and has 100% homology to ISKNV complete genome. Artificial infection by intraperitoneally injection with supernatant homogenate from spleen and kidney of naturally infected fish were showed 93% mortality in 12 days. Fish showed clinical sign of infection as lethargic, loss of appetite, pale or darken body color and haemorrhages. Internal organ on death fish showed swollen spleen and kidney and also pale liver. Quantitative PCR analysis on internal organs showed spleen had the highest viral DNA copy number followed by kidney, gill and liver. Histopathological analysis showed hemorrage in these organs and many abnormally hypertrophied cells which is typical histopathological characteristic of megalocytivirus infection. On the second stage of this study was to develop primary cell cultures from its spleen as target organ for propagating megalocytivirus in vitro. We developed primary cell from spleen by enzimatic dissociation. The result showed that primary cell (GPs cell) can grow well at 27 oC and 10% serum in L-15 medium was sufficient for cells growth. GPs cells were infected with giant gourami iridovirus (GGIV) showed enlargement and rounding cells. Virus propagated in GPs cells was highly virulent when injected to giant gourami in artificial infection. Intraperitoneal injection experiments of diluted virus supernatant showed 100% mortality within 7-11 dpi and 97% mortality in 21 days by cohabitation with abnormalities were observed in spleen and kidney. GPs cell was successfully subcultured for more than 30 passages and was found to be susceptible to GGIV as a pathogen of ISKNV infection in cultured-giant gourami. GGIV was propagated in GPs cells for megalocytivirus vaccine development for giant gourami. On the third stage of this study was to develop an effective vaccine and measure its efficacy using relative percent survival (RPS), serological parameters (antibody titers), and molecular (relative expression of gouramy immune genes). The virus was propagated in GPs cell cell and produced a virus titer of 105.9TCID50/ml. Virus inactivation was carried out using two methods (heatkilled and formalin-killed). Vaccination in giant gourami by intraperitoneal injection for three weeks of vaccine induction period and challenge test was done by cohabitation method. The experimental results showed RPS value of 17.7- 73.3% wherein formalin-killed vaccine results in a higher value. Viral accumulation in fish spleen in formalin-killed vaccine trials was lower than in the heat-killed and control vaccine groups which has higher chances of survival due to viral infection. Differential leukocyte after challenge test showed a decrease in percentage of lymphocytes, increased in percentage of monocytes and neutrophils, but no significant differences were observed between treatments. Formalin-killed vaccine showed an increased of antibody titer started on the 7th day post-vaccination and decreasing after 14th day. Increased antibody titer was observed 7th day postchallenge test. Immune gene expression has increased in formalin-killed vaccine group which showed that administration of the formalin-killed vaccine induced expression of pro-inflammatory cytokines (TNF-α and IL-1β) and increased expression of MyD88 gene that plays a role in activation of signal transduction of Toll-Like Receptor (TLR) pathway. In conclusion, formalin-killed vaccine showed better efficacy than heat-killed vaccine which showed higher protection from viral infection after challenge test, higher RPS value and increased antibody titer and expression of immune genes after challenge test. The final conclusion of this study was that giant gourami iridovirus (GGIV), virus isolate from infected giant gourami, was a strain of ISKNV from megalocytivirus genus. Primary cell culture (GPs cells) which developed from spleen of giant gourami found sussceptible to GGIV infection and it can propagated on suscessfully on thoses primary cells. Vaccine was made by formalin-killed and heat-killed methods and it were safe for application in fish. Formalin-killed vaccine can be used for megalocytivirus infection prevention and disease control in giant gourami.