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

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

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

Cell C

ulture

•Virus isolated in cell culture

•Isolated virus identified by polymerase

chain reaction (PC

R)

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

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

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

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

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%)

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

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

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

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

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

•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