Part A | 4.7
44 Part A Marine Flora and Fauna
Serological Methods
Several serology-based diagnostic methods have been
applied for shrimp virus disease diagnosis. Polyclonal
antibodies (pAbs) and monoclonal antibodies (mAbs)
have also been virus diagnosis. A fluorescent pAb test
for BMNV, the ELISA-based pAb test for BP, and pAb
for rhabdovirus of penaeid shrimp (RPS) have been reported, and many attempts have been made to develop
mAbs against many other viruses, such as HPV, YHV,
WSSV, BP, and IHHNV. However, the development
of mAbs involves problems related to specificity. Although mAbs may be specific for a particular virus, under some conditions, they may react nonspecifically to
shrimp tissue due to the nature of IgM (immunoglobulin
M) antibodies [4.62]. Specific mAb-based diagnostic
tools are likely to be used because of their good sensitivity, versatility, low cost, speed, and simplicity.
Western blotting is a specific method for detecting
antigens using mAb or pAb developed against viral capsid protein. This method requires the separation and
transfer of the antigens prior to antibody treatment.
Therefore, the component of the pathogen at a particular molecular weight can be identified with a high
degree of accuracy. However, this is a time-consuming
process that requires specialized equipment for protein
separation and transformation. Therefore, this method
is not commonly used for pathogen diagnosis [4.63, 64].
Recently, diagnostic lateral flow strip-based kits
have been developed for WSSV and YHV using mAbs
to VP28 and structural protein p20 [4.65, 66]. An mAb
was raised to PmDNV recombinant VP protein, which
was applied to detect HPV infection in Thailand. However, the detection sensitivity of this method is not comparable to sensitive molecular diagnostic tools [4.67]. In
this method, a pre-coated mouse antiviral protein antibody is used as a capture antibody at the test line (T),
an anti-mouse IgG (immunoglobulin G) is used as the
capture antibody at the control line (C), and a colloidal
gold-conjugated monoclonal antibody pad is located
adjacent to the sample pad. The sample is applied to the
sample pad located at one end of the strip and allowed
to flow by chromatography through the nitrocellulose
membrane from one end to the other. Virus particles
in test samples bind to the colloidal gold-conjugated
monoclonal antibody and the resulting complex is captured by the antiviral antibody at the test line, yielding
a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moves across the
test line and is captured by the IgG to form a band
at the control line (C). However, this method is about
500 times less sensitive than a one-step real-time polymerase chain reaction, but is slightly more sensitive
than dot blotting.
Molecular Diagnostic Methods
Molecular diagnostic methods include radioactive and
nonradioactive labeled genomic probes and DNA amplification methods based on polymerase chain reaction
(PCR), and additional PCR-based methods are being
applied for the development of efficient diagnostic techniques. The first gene probe introduced into shrimp
viral detection involved using a radioactive tag for
IHHNV. However, it could only be applied in wellequipped laboratories due to the use of radioactive tags.
Therefore, an alternative nonradioactive gene probe
was developed using digoxigenin 11-dUTP (DIG). This
technology allowed the development of many DIGlabeled probes for viral pathogens, such as IHHNV,
HPV, TSV, WSSV, MBV, BP, and the ssRNA virus
YHV [4.68]. A commercial kit for making DIG-labeled
probes is available for IHHNV [4.68].
Dot blot hybridization is a simplification of the
Northern, Southern, and Western blotting methods. In
dot blot hybridization, the biomolecules to be detected
are not first separated by chromatography. Instead,
a mixture containing the molecule to be detected or
a homogenized tissue sample is applied directly onto
a negatively charged nitrocellulose membrane or a positively charged nylon membrane as a dot.
Dot blotting is a quick, convenient, and relatively
cheap method to detect pathogens in hemolymph or tissue homogenate. The nitrocellulose membrane is the
most common solid matrix used for sample adsorption.
However, the major disadvantage of this method is that
the sensitivity is limited to 1000 times less than that of
PCR, and in most cases, the sensitivity is insufficient
for detecting asymptomatic infection and it is usually
associated with high background staining due to nonspecific binding of antibodies to the high concentration
of shrimp proteins [4.63]. The background staining resulting from the nonspecific binding of antibodies to
various shrimp components usually causes difficulty of
interpretation in the case of light infection.
In situ hybridization (ISH) is a unique and powerful
hybridization technique that uses a labeled complementary DNA or RNA probe to localize a specific DNA or
RNA sequence in a portion or section of tissue. This
method has been developed to detect shrimp pathogens
using nonradioactive DIG-labeled gene probes [4.61].
Several DNA amplification methods based on PCR
have been developed, and PCR has been applied in numerous methodologies for pathogen detection. Small,
44 Part A Marine Flora and Fauna
Serological Methods
Several serology-based diagnostic methods have been
applied for shrimp virus disease diagnosis. Polyclonal
antibodies (pAbs) and monoclonal antibodies (mAbs)
have also been virus diagnosis. A fluorescent pAb test
for BMNV, the ELISA-based pAb test for BP, and pAb
for rhabdovirus of penaeid shrimp (RPS) have been reported, and many attempts have been made to develop
mAbs against many other viruses, such as HPV, YHV,
WSSV, BP, and IHHNV. However, the development
of mAbs involves problems related to specificity. Although mAbs may be specific for a particular virus, under some conditions, they may react nonspecifically to
shrimp tissue due to the nature of IgM (immunoglobulin
M) antibodies [4.62]. Specific mAb-based diagnostic
tools are likely to be used because of their good sensitivity, versatility, low cost, speed, and simplicity.
Western blotting is a specific method for detecting
antigens using mAb or pAb developed against viral capsid protein. This method requires the separation and
transfer of the antigens prior to antibody treatment.
Therefore, the component of the pathogen at a particular molecular weight can be identified with a high
degree of accuracy. However, this is a time-consuming
process that requires specialized equipment for protein
separation and transformation. Therefore, this method
is not commonly used for pathogen diagnosis [4.63, 64].
Recently, diagnostic lateral flow strip-based kits
have been developed for WSSV and YHV using mAbs
to VP28 and structural protein p20 [4.65, 66]. An mAb
was raised to PmDNV recombinant VP protein, which
was applied to detect HPV infection in Thailand. However, the detection sensitivity of this method is not comparable to sensitive molecular diagnostic tools [4.67]. In
this method, a pre-coated mouse antiviral protein antibody is used as a capture antibody at the test line (T),
an anti-mouse IgG (immunoglobulin G) is used as the
capture antibody at the control line (C), and a colloidal
gold-conjugated monoclonal antibody pad is located
adjacent to the sample pad. The sample is applied to the
sample pad located at one end of the strip and allowed
to flow by chromatography through the nitrocellulose
membrane from one end to the other. Virus particles
in test samples bind to the colloidal gold-conjugated
monoclonal antibody and the resulting complex is captured by the antiviral antibody at the test line, yielding
a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moves across the
test line and is captured by the IgG to form a band
at the control line (C). However, this method is about
500 times less sensitive than a one-step real-time polymerase chain reaction, but is slightly more sensitive
than dot blotting.
Molecular Diagnostic Methods
Molecular diagnostic methods include radioactive and
nonradioactive labeled genomic probes and DNA amplification methods based on polymerase chain reaction
(PCR), and additional PCR-based methods are being
applied for the development of efficient diagnostic techniques. The first gene probe introduced into shrimp
viral detection involved using a radioactive tag for
IHHNV. However, it could only be applied in wellequipped laboratories due to the use of radioactive tags.
Therefore, an alternative nonradioactive gene probe
was developed using digoxigenin 11-dUTP (DIG). This
technology allowed the development of many DIGlabeled probes for viral pathogens, such as IHHNV,
HPV, TSV, WSSV, MBV, BP, and the ssRNA virus
YHV [4.68]. A commercial kit for making DIG-labeled
probes is available for IHHNV [4.68].
Dot blot hybridization is a simplification of the
Northern, Southern, and Western blotting methods. In
dot blot hybridization, the biomolecules to be detected
are not first separated by chromatography. Instead,
a mixture containing the molecule to be detected or
a homogenized tissue sample is applied directly onto
a negatively charged nitrocellulose membrane or a positively charged nylon membrane as a dot.
Dot blotting is a quick, convenient, and relatively
cheap method to detect pathogens in hemolymph or tissue homogenate. The nitrocellulose membrane is the
most common solid matrix used for sample adsorption.
However, the major disadvantage of this method is that
the sensitivity is limited to 1000 times less than that of
PCR, and in most cases, the sensitivity is insufficient
for detecting asymptomatic infection and it is usually
associated with high background staining due to nonspecific binding of antibodies to the high concentration
of shrimp proteins [4.63]. The background staining resulting from the nonspecific binding of antibodies to
various shrimp components usually causes difficulty of
interpretation in the case of light infection.
In situ hybridization (ISH) is a unique and powerful
hybridization technique that uses a labeled complementary DNA or RNA probe to localize a specific DNA or
RNA sequence in a portion or section of tissue. This
method has been developed to detect shrimp pathogens
using nonradioactive DIG-labeled gene probes [4.61].
Several DNA amplification methods based on PCR
have been developed, and PCR has been applied in numerous methodologies for pathogen detection. Small,