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SID 5

Research Project Final Report
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SID 5 (2/05)
Project identification
1.
Defra Project code
2.
Project title
FC1163
Optimisation and standardisation of PCR assay protocols
for koi herpesvirus
3.
Contractor
organisation(s)
CEFAS Weymouth Laboratory
Barrack Road
The Nothe
Weymouth, Dorset
DT4 8UB
54. Total Defra project costs
5. Project:
Page 1 of 13
£
start date ................
06 September 2004
end date .................
04 September 2005
6. It is Defra’s intention to publish this form.
Please confirm your agreement to do so. ................................................................................... YES
NO
(a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They
should be written in a clear and concise manner and represent a full account of the research project
which someone not closely associated with the project can follow.
Defra recognises that in a small minority of cases there may be information, such as intellectual property
or commercially confidential data, used in or generated by the research project, which should not be
disclosed. In these cases, such information should be detailed in a separate annex (not to be published)
so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report
without including references to any sensitive or confidential data, the information should be included and
section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No"
answer.
In all cases, reasons for withholding information must be fully in line with exemptions under the
Environmental Information Regulations or the Freedom of Information Act 2000.
(b) If you have answered NO, please explain why the Final report should not be released into public domain
An edited version should be released into the public domain. Reference is made to experiments on
fish in which the end point in many cases is the death of the fish from a laboratory induced virus
infection. This may be used by some animal rights extremists as a reason to target the scientists and
technicians involved, some of whom can be identified from the outputs section. In order to protect
those individuals, we request that reference to the animal experiments be removed from the version to
be placed in the public domain.
Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
Koi herpesvirus (KHV) disease was first reported in 1998 following mass mortalities in populations of
cultured common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi) in Israel (Perelberg et
al.2003). The disease has rapidly spread around the world through international trade in ornamental carp
and devastating losses have been seen in carp populations in Europe, the USA, South Africa, South-East
Asia and Japan (Haenen et al. 2004). The virus has been difficult to detect using traditional culture
methods and therefore a range of PCR-based methods are cited in the literature.
In February of 2004 an international workshop was sponsored by Defra to raise the general awareness
amongst regulators, scientists and the ornamental fish industry of the current status of koi herpesvirus
(KHV) with respect to management, identification and potential treatment. A working group at the KHV
workshop looking at standardisation of detection procedures highlighted the need for standardisation of
detection methods, and in particular PCR assays, for KHV. Selection and standardisation of the most
accurate PCR detection methods is an important step in providing the best tools to diagnostic laboratories
to enable them to test carp populations for KHV. This one year student project was initiated in response to
this need.
The aim of this project was to 1) compare published PCR protocols for KHV with PCR protocols based on
protein coding regions of the KHV genome for sensitivity and specificity. 2) to optimise the tissue
sampling, extraction and amplification protocols of the most effective assays, and 3) develop standardised
protocols in preparation for a validation ring-trial.
Published primer sets were compared using a standard PCR protocol. The primer set targeting the
thymidine kinase gene (Bercovier et al., 2005) was the most sensitive with a detection limit three log
greater than the Gilad primers which are commonly used in KHV diagnostic laboratories. CNGV primers
that target a short region of the genome (109bp) (Pikarsky et al., 2004) also performed well in initial
studies.
Extraction protocols were compared for their suitability in routine diagnostics of KHV and of the
commercial kits the DNAzol reagent was considered the most suitable based on the duration of the
protocol and ease of use. With the exception of DNA extracted using the Aquapure DNA extraction kit, it
was shown that the other DNA extraction protocols generated DNA of sufficient quality and could be used
as an alternative to the DNAzol protocol if this was unavailable.
In further studies, KHV DNA was readily detected in spleen, kidney and gut tissue but most consistently in
SID 5 (2/05)
Page 2 of 13
gill tissue. Also, KHV DNA was most readily detected in fresh (refrigerated) or frozen tissues and least
readily from tissues fixed in 70% alcohol. In studies examining the ability of selected primer sets to detect
DNA in samples of decomposed tissues, primer sets targeting smaller sequences of KHV genome were
more reliable in detecting the degraded DNA found in such samples. Both the CNGV and modified SPH
primer sets readily detected DNA in decomposed tissues but the CNGV primers produced extra bands
that could be mistaken for a KHV product.
A standardised protocol has been developed and the Bercovier-TK and modified SPH primer sets selected
as the most robust for detection of KHV DNA in a range of tissue samples. The protocol has been adopted
as the standard at the CEFAS Weymouth laboratory and other laboratories will now be contacted to
request their participation in a ring-trial to complete the validation of the protocol.
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
SID 5 (2/05)
Page 3 of 13
Scientific objectives :
1
To compare published PCR protocols for KHV with PCR protocols based on protein coding regions of
the KHV genome for sensitivity and specificity.
2
To optimise the tissue sampling, extraction and amplification protocols of the most effective assays.
3
To develop standardised protocols in preparation for a validation ring-trial.
Note : Some studies relating to objective 2 were required to be completed before commencing studies relating to
objective 1.
Methods and Results
Koi herpesvirus used for spiking of carp tissues was grown in koi fin (KF, Hedrick et al. 2000) and common carp
brain (CCB, Neukirch & Haenen 2004) cells. Preparations of other DNA viruses used in specificity studies are
listed in table 1 and were obtained as frozen aliquots from the Virology laboratory stocks.
Table 1 : Viruses used in experimental studies with growth requirements
Virus
Koi herpesvirus
Carp pox herpesvirus
Goldfish
haematopoietic
necrosis herpesvirus
Channel Catfish herpesvirus
Catfish iridovirus
Common carp iridovirus
Abbreviation
KHV
CyHV-1
CyHV-2
Cell line & growth temp.
KF or CCB cells at 20°C
KF at 20°C
KF at 20°C (limited growth)
CCV
CIV
CCIV
CCO cells at 25°C
EPC cells at 20°C
KF at 20°C
Tissues were sampled from infected and healthy carp using the routine diagnostic methods employed at CEFAS
and following recommended OIE procedures as detailed in the OIE Diagnostic manual for other virus diseases of
cyprinid fish species. Apart from those studies where tests were carried out on stored tissue samples, all tissue
sampling for comparative tests was carried out on fresh or 24-hour chilled material.
Tissue homogenates were sub-sampled for DNA purification, which was carried out using standard protocols and,
where kits were used, following manufacturers instructions. Where re-testing of infected tissue samples was
carried out as new primer sets became available the re-test was done on the –20°C stored DNA extract.
Polymerase chain reaction (PCR) amplification was performed following a standardised CEFAS KHV protocol. A
master mix was prepared with GoTaq polymerase (1.25 units) (Promega Cat. No. M8305); 10µl of buffer (1x final
conc.); 5µl Magnesium Chloride (final conc. 2.5mM) supplied; 0.5µl of dNTPs (final conc. 0.25) (Promega cat.
No. U1240); 0.5µl of each primer (final conc. 1μM) and then made up to 47.5µl using molecular grade water. 2.5µl
of DNA extract was added to this master mix and overlaid with mineral oil. The tube was then placed in a MJ
Research DNA engine Tetrad 2 thermocycler, on a 40 cycle program of 95˚C for 10 minutes followed by 40 cycles
of 55˚C for 1 minute, 72˚C for 1 minute and 95˚C for 1 minute. This was followed by a 10 minute 72˚C extension
period after which the samples were kept at 4˚C until required. PCR products were separated by electrophoresis
in agarose gels and incorporating ethidium bromide using a standard procedure. Gels were then visualised under
UV light and photographed using a UVP transilluminator system.
Details of the primer sets used in the various studies undertaken are given in Table 2. Products were cloned and
sequenced according to published protocols and analysed using a 3100-Avant Genetic Analyzer and software
supplied.
Comparison of DNA extraction methods (Objective 2)
Commercially available DNA extraction kits were tested on gill tissue samples from experimentally infected carp.
Based on results of previous tests, the PCR was performed using Gray Sph primers. Four kits were tested and
compared to the standard proteinase K extraction method. These were Aqua Pure, EasyDNA, DNEasy and
DNAzol. In all, nine samples were tested in duplicate and an example of the results is presented in Figure 1. The
quality of DNA generated by the different methods, as indicated by the intensity of the PCR products, was
comparable for 4 of the 5 extraction protocols tested. The Aquapure DNA purification system consistently failed to
produce PCR amplicons of the quality generated using DNA extracted from the other four methods (Figure 1).
SID 5 (2/05)
Page 4 of 13
1 2
3
4
5 6
7 8
9 10 11
12 13 14 15 16 17 18 19 20 21 22
23 24 25 26 27 28
29 30 31 32
Figure 1. A comparison of DNA extraction protocols. Amplification of KHV DNA from clinically infected tissues
using Gray Sph primers and following DNA extraction by 5 different methods: Aquapure (Lanes 1, 2, 12, 13, 23,
24), Easy DNA (lanes 3, 4, 14, 15, 25, 26), DNEasy (Lanes 5, 6, 16, 17, 27, 28), DNAzol (Lanes 7, 8, 18, 19, 29,
30), and Proteinase K (Lanes 9, 10, 20, 21, 31, 32). 100base pair ladder (lanes 11, 22).
Comparison of tissues for detection and tissue storage methods (Objective 2)
KHV-infected tissue samples from an on-going laboratory challenge experiment were collected for use in the
sample storage comparison tests. The tissues were stored frozen and in 70%IMS and compared in tests with
fresh chilled tissue samples. Gill, gut, kidney and spleen tissues were dissected from moribund or freshly dead
carp and pooled by tissue type. The tissue pool was then divided into three and two portions stored frozen at –
20°C and in 70%IMS and the third portion immediately processed. Portions of tissue were stored for a minimum
of one month before processing.
All extractions were completed using a standard DNAzol protocol, and stored material was also processed with
an Easy DNA protocol. These extraction methods were chosen because they are easy-to-use kits and had
performed consistently well in the initial extraction tests. The quality of the DNA (ability to generate amplicons in
PCR) was seen to vary between storage conditions but not with extraction method. Consistently higher intensity
banding of KHV DNA products was seen from fresh and frozen samples than was seen from alcohol-fixed
tissues. KHV DNA was detected in all tissue types but good quality DNA was detected most consistently from gill
tissue.
Comparison of primer sets for sensitivity and specificity (Objective 1)
Twelve different primer sets were compared. Seven were published primer sets based on coding (gene) regions
and non-coding regions of the KHV genome. The other five were CEFAS-designed primers targeting different
regions of the KHV genome sequenced during previous student projects. These primers all targeted coding
regions of the genome. The twelve primer sets and target genes (or restriction enzyme sites) are listed in Table 2.
The sensitivity of the PCR assay with each of the 12 different primer sets, under standard cycling conditions, was
compared using DNA extracted from fish tissues spiked with KHV. A stock sample of KHV was diluted from 10 -1
to 10-6 and 50μl added to 50μl of tissue homogenate before DNA extraction by the DNAzol method. Although
some non specific products were observed with some primer sets, a product of the expected size was generated
for both the 10-1 and 10-2 dilutions of virus with 11 of the 12 primer sets. The exception was the primer set
targeting the helicase gene, which did not produce an amplification product. At the low dilutions, products of
greatest intensity were generated by the Gray Sph and Gilad primer sets. The Gray Bam H1, KHV helicase,
tricaspid and ORF 5 internal & external primer sets were only able to detect virus to levels of 10 -2 or less whereas
all other primers sets were able to detect to 10-3 or more. It was therefore decided that the Bam H1, Herpes &
KHV helicase, tricapsid and the two sets of ORF 5 primers would be excluded from further studies.
SID 5 (2/05)
Page 5 of 13
Table 2: Primer sets used in comparative tests with the target genes or restriction sites.
[Note : For primer sequences refer to published paper or, for sequences developed
at Cefas, contact the report author. ]
Primer
Gilad
Target
KHV genome Kpn1/Sac1
Reference
Gilad et al., 2002
Gray Sph
KHV genome Sph1
Gray et al., 2002
Gray Bam H1
KHV genome Bam H1
Gray et al., 2002
Bercovier TK
KHV Thymidine Kinase
Bercovier et al., 2005
CNGV
KHV Genome
Pikarsky et al., 2004
KHV Helicase
Waltzek et al., 2005
Herpes Helicase
General Herpesviruses
Including CyHV and IcHV
CyHV specific
ORF 5 internal
KHV genome
CEFAS
ORF 5 external
KHV Genome
CEFAS
Reductase
KHV genome
CEFAS
Tricapsid
General Herpesviruses
Including CyHV and IcHV
KHV Thymidine Kinase KHV Thymidine Kinase
CEFAS
Waltzek et al., 2005
CEFAS
Of the other 6 primer sets (CNGV, TK, Reductase, Gray Sph, Gilads, Bercovier TK) the most sensitive was
Bercovier TK (Figure 2), which detected KHV DNA at the 10-5 dilution.
The specificity of the primer sets selected was tested against 5 other DNA viruses listed in table 1. These viruses
were first tested with primer sets broadly specific for the virus sub-families (Cyprinid herpesviruses and Fish
iridoviruses), where they were all detected by the expected primer set apart from CCIV, which was not detected
by the iridovirus primer set. Of the 6 KHV-specific primer sets, none of those tested produced amplification
products from DNA extracted from the other viruses, all were shown to be specific for KHV.
Figure 2. Result of the senstivity test on the Bercovier-TK primers
Amplifications with Bercovier TK primers of serial dilutions of KHV. Dilutions from neat to 10 -5 in 10x steps (Lanes
3 to 8) DNA ladder (Lane 1) Negatives (Lane 2, 9) Positive (Lane 10). Products were size separated through 2%
agarose gel.
SID 5 (2/05)
Page 6 of 13
Comparison of the ability of selected primer sets to amplify KHV DNA in clinically infected carp tissues (Objective
1)
The six primer sets (Sph, Gilad, Reductase, TK, CNGV and Bercovier-TK) were tested on gill tissue or pooled
visceral organ tissues sampled from carp mortalities from an experimental KHV challenge trial. For the trial, carp
were injected with a high dilution of KHV (~10 virus particles/fish) and co-habited with naïve (non-injected carp)
and mortalities collected and frozen as the disease progressed. Tissues were dissected from carp carcasses
frozen whole at –70°C during the trial and DNA extraction achieved using the DNAzol protocol. The results from
21 fish tested are detailed in table 3. In the first run of tests 5 of the 6 primer sets (not TK-Bercovier) were
compared and in the second run the DNA extracts were tested again with CNGV primers and compared with the
TK-Bercovier set. The CNGV and TK-Bercovier were the most sensitive and reliable of the 6 primer sets tested.
Table 3 : Comparison of six primer sets for amplification of KHV DNA in clinically infected tissue samples.
(G = gill tissue, V = visceral organ tissues)
Challenge
route
Injected/cohab
Injected
Cohab 1
Cohab 1
Cohab 1
Injected
Cohab 1
Injected
Cohab 1
Injected
Injected
Injected
Injected
Injected
Injected
Cohab 2
Cohab 2
Cohab 2
Cohab 2
Cohab 2
Cohab 2
Cohab 2
Total detected
PCR protocol
1st testing
2nd Testing
Reductase Grays Gilads CNGV Bercovier
SPH
TK
CNGV
TK
V
G+V
G+V
G+V
G+V
G+V
V
G+V
G+V
G
G
G+V
G
G+V
G+V
V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G
G+V
G+V
V
G+V
G+V
G+V
G+V
G
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G
G
G
G+V
G
G
G
G
G+V
13G
16V
G+V
7G
5V
G+V
9G
7V
G+V
9G
7V
4G
0V
G+V
19G
20V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
16G
15V
16/21
(76%)
7/21
(33%)
9/21
(43%)
10/21
(48%)
4/21
(19%)
20/21
(95%)
16/21
(76%)
Modification of primer sets to improve sensitivity (Objective 1 & 3)
The CNGV primer set targets a small sequence of only 109bps and it is suggested that the sensitivity of the
CNGV primers is because of their ability to detect viral DNA degraded in the freeze/thaw cycle step in the
processing of the tissue sample. To test this, other primer sets were modified and new primer sets designed to
target a smaller sequence. Gilad and Gray Sph primers were modified to only target 110bps and a new set of
primers targeting the Thymidine kinase gene were designed. Primer sets were initially tested on a KHV dilution
series with original Gray Sph and CNGV primer sets as controls. The 110bp TK primers produced non specific
banding and no obvious virus specific product and the modified-Gilad primers produced non-specific banding and
smeared results. The remaining primer sets were equivalent in sensitivity detecting virus at 10-4 with both the
modified-Gray Sph and CNGV primer sets giving clean results comparable to original Gray Sph primers.
Modified-Gray Sph and CNGV primers were further compared with Bercovier TK by re-testing the DNA extracts
SID 5 (2/05)
Page 7 of 13
from the clinical samples detailed in table 3. The results for tests on 17 of the samples are given in table 4.
Table 4 : Comparison of CNGV, Modified-Gray Sph and Bercovier TK primer sets for
amplification of KHV DNA in clinically infected tissue samples.
(G = gill tissue, V = visceral organ tissues)
Challenge route
Injected/cohab
Injected
Cohab 1
Cohab 1
Cohab 1
Injected
Cohab 1
Cohab 1
Injected
Injected
Injected
Cohab 2
Cohab 2
Cohab 2
Cohab 2
Cohab 2
Cohab 2
Cohab 2
No. of Gill and visceral
samples positive for
KHV
Total number KHV
positives detected
Primer set
Modified Sph CNGV Bercovier TK
V
G+V
G+V
G
G+V
G+V
G+V
G
G+V
G
G+V
G+V
G+V
G+V
G+V
14G
12V
15 / 17
(88%)
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
V
G+V
15G
16V
16 / 17
(94%)
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
G+V
13G
13V
13 / 17
(76.5%)
The three primer sets detected virus in, between 76.5 and 94% of samples. There were two samples where only
CNGV primers detected KHV DNA and one where the Bercovier-TK and Modified-Sph primers detected KHV
DNA and CNGV primers did not.
However, although the CNGV primers appeared to be the most sensitive of the 3 sets, it became apparent that
CNGV primers were creating false positive banding when PCR products were visualised in agarose gels. Seen as
spurious product bands in negative control lanes (Fig 3). So, as Bercovier-TK and modified-Gray Sph primers had
given constantly accurate and clean results they were chosen for further studies on degraded tissue
homogenates. Gill and visceral organ tissue homogenates were obtained from carp mortalities from a KHVchallenge trial and stored for 3 weeks at 4°C. Tissues from six carp mortalities were shown positive for KHV DNA
with Bercovier-TK and modified-Sph primers before storage at 4°C. After 3 weeks storage, tissues from 5 of the 6
carp showed positive with the modified-Sph primers but none were positive in the PCR test using Bercovier-TK
primers.
SID 5 (2/05)
Page 8 of 13
1 2
3 4
5
6 7
8 9 10 11 12 13
14
Figure 3. Comparison of Modified Sph and CNGV primers
Amplifications of various KHV-infected carp tissue samples using Sph Modified (upper panel) and CNGV (lower
panel) primers in the standard PCR protocol. Test samples (Lanes 2 to 11) Negative controls (Lanes 1 and 12)
Positive control (Lane 13) and 100bp ladder (Lane 14). Products were size separated through 4% agarose gel.
The proposed standardised PCR protocol for detection of KHV DNA (Objective 3)
The following has been adopted as standard protocol at the Cefas Weymouth laboratory for detection of KHV
DNA in fish tissues. The amplification reaction incorporates Bercovier-TK or modified-Gray Sph primer sets
depending on the state of decomposition of the tissue sample.
Sample processing and storage
1.
Remove gill and visceral (kidney, spleen, gut) tissue place into viral transport medium and chill at 4°C.
Store at 4°C for max 24 hours before homogenising tissue and sub-sampling into DNAzol. If more than 24 hours
will elapse before processing then freeze tissues at –20˚C.
Extraction of total DNA from fish tissue homogenates using the DNAzol® method (Invitrogen Cat No. 10503-027)
1.
2.
3.
4.
5.
Add 100µl of Tissue homogenate (5% w/v in transport medium) to 1 ml of DNAzol® in a 1.5ml
microfuge tube and incubate for at least 5min. (Samples can be stored in DNA for 18hours at 1530°C and 72 hours at 2-8°C).
Centrifuge at 10,00rpm for 10 minutes and take 1 ml of the clarified solution (leaving behind the
protein pellet) into a fresh tube containing 500µl of ethanol.
Vortex well, and then centrifuge at 13,000rpm for 30 minutes to pellet the DNA.
Wash the DNA pellet with 250µl 70% ethanol and centrifuge for a further 5min.
Remove all traces of the ethanol and re-suspend the DNA in 50µl of molecular biology grade (Dnase
and RNase free) water.
PCR amplification
Polymerase chain reaction (PCR) amplification is performed following a standardised CEFAS KHV protocol.
1.
2.
SID 5 (2/05)
Prepare a master mix containing the following for each sample: 0.25µl GoTaq polymerase (1.25
units) (Promega Cat. No. M8305); 10µl of buffer (1x final conc.); 5µl Magnesium Chloride (final conc.
2.5mM) supplied; 0.5µl of dNTPs (final conc. 0.25) (Promega cat. No. U1240); 0.5µl of each primer
(final conc. 1μM) and then made up to 47.5µl using molecular grade water.
Dispense 47.5µl into a 0.5ml thin wall thermal cycler tube and add 2.5µl of DNA extract and overlay
Page 9 of 13
3.
4.
with 20µl mineral oil.
Place the tube in a MJ Research DNA engine Tetrad 2 thermocycler, on a 40 cycle program of 95˚C
for 10 minutes followed by 40 cycles of 55˚C for 1 minute, 72˚C for 1 minute and 95˚C for 1 minute.
Followed by a 10 minute 72˚C extension period after which the samples are kept at 4˚C until
required.
Electrophorese 20µl of the 50µl reaction on a 2% agarose containing ethidium bromide (4% when
separating smaller products of <300bp) at 120V for 20min and visualise under UV light.
Discussion & Conclusions
In objective 2, five DNA extraction methods were compared for efficiency in performance (DNA recovered and
amplification by PCR) and ease in completion of the protocol. All extraction methods tested gave similar
successful results with the exception of the Aquapure protocol.
The EasyDNA and DNAzol extraction protocols were chosen for the testing of storage methods as they were the
more user friendly of the four comparable methods. The methods compared were short-term chilled (4°C)
storage, freezing at -20˚C and fixation in 70% IMS. Frozen or chilled storage gave consistently better results than
alcohol fixation, possibly because of degradation or masking of the KHV DNA in the fixed tissues. Further trials
need to be conducted to test the reliability of detection in tissues frozen at temperatures below -20˚C and to
ascertain the reliability of the alcohol storage method. If the DNA in alcohol stored tissues has degraded because
of the slow rate of fixation then the method needs to be re-assessed to identify if tissues could be better prepared
before alcohol storage and also if the later modified primers are more efficient at detecting the shorter DNA
fragments in the samples.
Gill, kidney, spleen and gut tissues from clinically infected carp were shown by Gilad et al. (2004), to have high
titres of KHV present when tested in real-time Taqman PCR experiments. The results in this study were
consistent with these findings as virus was readily detected in gill, spleen, kidney and gut tissues and the most
consistent results were achieved when using DNA extracted from the gill. In further studies gill tissue was
sampled separately and kidney, spleen and gut were combined into a visceral tissue sample. The DNAzol method
was chosen for all future extractions as it was a rapid method that gave good, consistent results.
In objective 1, seven published KHV PCR primer sets and five developed at CEFAS were tested both for
sensitivity and specificity. Four of the published primer sets targeted non-coding regions of the KHV genome
while all of the other primer sets targeted coding regions. The primer sets also varied in the size of their amplified
DNA products.
The most sensitive were the Bercovier-TK primers, which detected KHV DNA in virus preparations diluted down
to 10-5. This agrees with Bercovier et al., (2005) who state that their PCR protocol is 10 to 1000 times more
sensitive than the PCR assays using Gray Sph and Gilad primers and is estimated to detect as little as 10
femtograms (equivalent to <10 copies) of KHV DNA. The CNGV primer set (Pikarsky et al. 2004) also performed
well in the same studies. All 6 primer sets included in specificity tests were shown to be specific for KHV and, in
particular, none of the primers amplified DNA from the carp pox herpesvirus. Outbreaks of carp pox are not
uncommon in populations of koi carp during winter months.
The same 6 primer sets were then taken forward to trials where they were tested for their ability to amplify KHV
DNA from clinically infected tissue samples. The most successful of the primer sets in these tests were the CNGV
primers, which detected KHV DNA in 95% of carp tested. Then followed Bercovier-TK (76%), Gray Sph (48%),
Reductase (43%), TK (33%) and least successful were the Gilad primers detecting KHV DNA in only 19% of the
carp tested. The CNGV primers target a non-coding region of the genome but have an amplification product of
only 109bp. It was suggested that the primer sets targeting smaller sequences of KHV genome should be more
reliable in detecting the degraded DNA that is found in such samples and this explained the greater reliability of
the CNGV primers. In further studies other primer sets were then modified to amplify smaller regions of their
target sequence and from comparative studies a modified Gray Sph primer set (110bp) was shown to be
comparable in performance to the CNGV primers and gave a cleaner, more accurate result when PCR products
were visualised in agarose gels. Gray Sph primers were then shown to be more reliable than the Bercovier-TK
primers for amplification of KHV DNA from decomposed tissue samples.
In objective 3, the Bercovier-TK and modified SPH primer sets were selected as the most robust for detection of
KHV DNA in a range of tissue samples and a protocol (detailed above) has been adopted as the standard at the
CEFAS Weymouth laboratory
Further work & actions resulting from the research
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Preliminary trials have indicated that Bercovier-TK and modified Sph primer sets can be used together in the
standard KHV PCR mastermix and detect KHV DNA in a range of tissue samples. However, further tests need to
be carried out to determine if the sensitivity of either of the two primer sets is compromised by the presence of the
other primer set in the reaction mixture. If a mixture of these two primer sets can be used it will considerably
decrease the number of PCR amplifications performed because of testing duplication.
A further objective arising from this study is to be completed under the new Defra-funded project F1167
‘Research into Koi herpesvirus and other important viral pathogens of cyprinid fish species’. As part of objective 2
it is hoped to validate standardised PCR protocols in participating laboratories in Europe and around the world
and the aim is that this will be jointly co-ordinated by CEFAS and the CRL in Aarhus.
Also as part of objective 2 of FC1167 the effectiveness of the selected PCR protocols for detection of sub-clinical
KHV infections will be tested. It is hoped to identify the tissues most likely to harbour latent virus (sampled from
carp in long-term challenge studies) and detect KHV DNA with the existing optimised PCR assay protocols.
A summary of selected results from this study have been presented at a workshop on KHV at the 12 th
International Conference of the EAFP held in Copenhagen in September 2005. At the workshop it was stated that
it was hoped to validate the PCR methods by proficiency testing of the selected protocols through ring-trials with
other diagnostic laboratories around the world. Also, attending the workshop was a project officer (Arne Flåøyen)
for DG research of the European Commission who suggested that the commission may be keen to fund the type
of ring trials needed to validate diagnostic methods for KHV. Further to this an expression of interest entitled ‘Koi
Herpesvirus – the need for diagnostic and surveillance tools’ was submitted to Dr Flåøyen on 3 October 2005
from the community reference laboratory for fish diseases and 3 national reference laboratories for fish diseases,
including CEFAS Weymouth. However, if funding is not made available in the very near future by the EU then it is
proposed that Cefas Weymouth will push ahead with the validation of the standardised protocol that has been
developed from the FC1163 study.
Small parts of the data obtained in this project need to be ‘tidied up’ to publication standard and this will be carried
out under FC1167. When this is complete the development, comparison and standardisation of the PCR protocols
will be published in a peer-reviewed journal as soon as possible.
References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
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References
Bercovier H, Fishman Y, Nahary R, Sinai S, Zlotkin A, Eyngor M, Gilad O, Eldar A, Hedrick RP (2005).
Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive
PCR based diagnosis. BMC Microbiology 5:13 (Open access article on http://www.biomedcentral.com).
Gilad O, Yun S, Andree KB, Adkison MA, Zlotkin A, Bercovier H, Eldar A, Hedrick RP (2002). Initial
characteristics of Koi herpesvirus and development of a polymerase chain reaction assay to detect the
virus in koi, Cyprinus carpio koi. Dis.Aquat.Org. 48: 101-108
Gilad O, Yun S, Zagmutt-Vergara FJ, Leutenegger CM, Bercovier H, Hedrick RP (2004) Concentrations of
a Koi Herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time
Taqman PCR. Dis.Aquat.Org. 60: 179-187
Gray WL, Mullis L, LaPatra SE, Groff JM, Goodwin A (2002) Detection of koi herpesvirus DNA in tissues
of infected fish. J. Fish Dis. 25: 171-178
Haenan OLM, Way K, Bergmann SM, Ariel E (2004) The emergence of koi herpesvirus and its
significance to European aquaculture. Bull. Eur. Ass. Fish Pathol. 24 (6) 293-307
Hedrick RP, Gilad O, Yun S, Spangenberg JV, Marty GD, Nordhausen RW, Kebus MJ, Bercovier H, Eldar
A (2000) A Herpesvirus Associated with Mass Mortality of Juvenile and Adult Koi, a Strain of Common
Carp. Journal of Aquatic Animal Health 12: 44-57
Neukirch M, Haenen OLM (2004) Susceptibility of CCB cell line to different fish viruses. Bull. Eur. Ass.
Fish Pathol. 24 (4) 209-211
Perelberg A, Smirnov M, Hutoran M, Diamant A, Bejerano Y, Kotler M (2003) Epidemiological description
of a new viral disease afflicting cultured Cyprinus carpio in Israel. The Israeli Journal of Aquaculture –
Bamidgeh 55(1), 5-12
Pikarsky E, Ronen A, Abramowitz J, Levavi-Sivan B, Hutoran M, Shapira Y, Steinitz M, Perelberg A,
Soffer D, Kotler M (2004) Pathogenesis of Acute Viral Disease Induced in Fish by Carp Interstitial
Nephritis and Gill Necrosis Virus. J. Virol. 78 : 9544-9551
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