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Largemouth B
ass Virus R
esearchat A
uburn University:
Highlights for 2002
Andrew
D. N
oyes, M. J. M
aceina andJohn M
. Grizzle
Southeastern C
ooperative Fish D
isease Project
Dept. of F
isheries and Allied A
quaculturesA
uburn University, A
uburn, Alabam
a
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Funding
•S
port Fish R
estoration Funds
administered through the A
labama
Cooperative F
ish and Wildlife R
esearchU
nit
•A
labama D
ivision of Wildlife and
Freshw
ater Fisheries
•S
outheastern Cooperative F
ish Disease
Project
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Outline
•P
CR
Detection M
ethod–
Grizzle, J.M
., I. Altinok, and A
.D. N
oyes. (inpress) A
PC
R m
ethod for detection of largemouth
bass virus. Diseases of A
quatic Organism
s
•LM
BV
prevalence in LMB
populations
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Cell C
ulture
•Virus isolated in cell culture
•Isolated virus identified by polymerase
chain reaction (PC
R)
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Problem
s with C
ell Culture-P
CR
Method for D
etection of LMB
V
•T
he amount of LM
BV
in apparently healthy fishis often near the detection lim
it with cell culture
assay method
•T
herefore, some fish that test negative for
LMB
V probably have virus levels below
thedetection lim
it•
PC
R m
ethod used before 2001 was
–not specific for LM
BV
–w
as less sensitive than cell culture assay
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New
PC
R M
ethod•
New
primers designed to be specific for LM
BV
•C
an be used to identify virus isolated in cell culture•
Can be used to detect the D
NA
of LMB
V in fish
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Features of N
ew P
CR
Method
•dU
TP
and uracil-DN
A glycosylase (U
NG
) used inP
CR
mixture to reduce the possibility of false-
positives•
Prim
ers for LMB
V are m
ore specific and sensitivethan other prim
ers used for LMB
V•
Second set of prim
ers for ß-actin used as internal
control to detect false negatives•
Sensitivity im
proved by partial removal of cell
nuclei, which increases the ratio betw
een viraland cellular D
NA
•N
egative fish used as controls at each step,starting w
ith necropsy
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Non-Lethal T
esting
0 10
20
30
40
50
60
70
Viscera
FinM
ucus
Positive (%)
Culture
PC
R
•Largem
outh bass•
Wheeler R
eservoir,A
labama
•C
ollected byelectroshockingduring July-A
ugust•
Viscera w
as pool ofspleen, trunk kidney,and sw
im bladder
N=61
N=61
N=
55
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PC
R vs. C
ell Culture of Largem
outh Bass:
Locations Positive by C
ell Cultures
639405
234T
otal
580405
175N
egative
590
59P
ositive
Total
Negative
Positive P
CR
Cell
culture
(37%)
(9%)
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PC
R vs. C
ell Culture of Largem
outh Bass:
Locations where all fish w
ere cell culture negative
8181
0T
otal
8181
0N
egative
00
0P
ositive
Total
Negative
Positive
Cell
culture
PC
R
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Population Level E
ffectsof LM
BV
W.F
. George
Weiss
Wheeler
Guntersville
Dem
opolis
•5 different lakes•547 largem
outh bass•E
lectrofished samples
•July 2001 to Aug 2002
•Cell culture for detection
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Age
Frequency of LMBv+ (%)
0 2 4 6 8 10 12 14 16 18LR
- χ2 =
10.29P
= 0.07
N =
545
Length group (cm)
0 2 4 6 810 12 14 16 18
LR - χ
2 = 17.79
P <
0.01N
= 547
> 5
< 20
20-3030-38
38-51
01
23
4
> 51
N =
6
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Relative w
eight
Low (<
85)M
ed (85 - 95)H
igh (>95)
Frequency of LMBv+
0 5 10 15 20
LR - χ
2 = 7.77
P <
0.05N
= 483
LMB
> 150 m
m T
L
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logit (LMB
v 0 or 1) = - T
L + T
L2
r for concondance = 0.63
Total length (m
m)
150200
250300
350400
450500
550
Probability of LMBv+
0.26
0.28
0.30
0.32W
r < 85
Wr 85-95
Wr >
95
r of concordance = 0.67
N =
483
Logistic regression to predict the probabilityof LM
Bv+
from length and relative w
eight
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•G
rizzle, J.M., I. A
ltinok, W.A
. Fraser, R
. Francis-F
loyd. 2002. First
isolation of largemouth bass virus. D
iseases of Aquatic O
rganisms
50:233-235.
•W
oodland, J.E., C
.J. Brunner, A
.D. N
oyes, and J.M. G
rizzle. 2002.E
xperimental oral transm
ission of largemouth bass virus. Journal of
Fish D
iseases 25:669-672.
•W
oodland, J.E., A
.D. N
oyes, and J.M. G
rizzle. 2002. A survey to
detect largemouth bass virus am
ong fish from hatcheries in the
southeastern US
A. T
ransactions of the Am
erican Fisheries S
ociety131:308-311.
Recent P
ublications about LMB
V