Pengembangan Kultur sel dan Vaksin Inaktif untuk Pencegahan Infeksi Megalocytivirus pada Ikan Gurami (Osphronemus goramy).
Date
2021Author
Gardenia, Lila
Sukenda
Zairin Jr, Muhammad
Alimuddin
Lusiastuti, Angela Mariana
Metadata
Show full item recordAbstract
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.
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- DT - Fisheries [725]