8/11/2019 Uptake and Clearance of Edwardsiella Ictaluri in The

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JOURNAL OF THE

WORLD AQUACULTURE SOCIETY

Vol.

28, No.

1

March, 1997

Uptake and Clearance of Edwardsiella ictaluri in the

Peripheral Blood

of

Channel Catfish

Zctalurus punctatus

Fingerlings during Immersion Challenge

DAVID . WISE,

THOMAS

.

SCHWEDLER

ND JEFFERYS.

TERHUNE

Department of Aquaculture Fisheries and Wildlife Clemson University Clemson,

South Carolina

29634-0362

USA

Abstract

Uptake and clearance of Edwardsiella ictaluri in the peripheral blood of channel catfish

Ictalurus punctatus

fingerlings were monitored for 216 h after exposure to

E. ictaluri

for 4 h

and 8 h under static conditions. Most fish exposed to

E. ictaluri

developed bacteriemia 24 h

post-exposure, and by 72 h post-exposure

E. ictaluri

was recovered from all the blood of all

exposed fish. The number of E. ictaluri colony forming units

CFU)

in the blood of moribund

fish ranged between 1.7

X

lo3 to 1.6 X

lo5

CFU/SO p,L whole blood. Clearance of bacteria

from the blood was observed by 216 h post-exposure and all fish surviving bacterial expo-

sure developed agglutinating antibody against

E. ictaluri.

The pathogenesis of the infection

was accompanied by the shedding of viable

E. ictaluri

into the water which may serve as a

mechanism by which fish to fish transmission occurs.

Enteric septicemia of catfish is caused by

a gram negative bacterium identified as E d -

wardsielfa ictafuri

(Hawke 1979). This dis-

ease most commonly affects fingerling

channel catfish and outbreaks of the disease

are common when fish are exposed to the

bacterium in water temperatures between 18

and 28 C (Francis-Floyd et al. 1987). In fin-

gerlings the disease may progress rapidly in

apparently healthy fish populations, and

mortalities may approach 1 per day

(Areechon and Plumb 1983). Fish that sur-

vive infection develop anti-E.

i c fa lur i

anti-

bodies and are less susceptible

to

subse-

quent

E.

i c fa lur i infection (Vinitnantharat

and Plumb 1993). Disease transmission

likely occurs through the cannibalism of in-

fected fish and by direct exposure of sus-

ceptible fish

to E.

icfaluri in the water (Kle-

sius 1992). The shedding of bacteria from

infected fish and the kinetics of the infec-

tion, however, have not been documented.

Laboratory challenge experiments have

been used to study the pathogenesis of ESC

t

Corresponding authors present address: Delta Re-

search and Extension Center,

Post

Office

B o x 197,

Stoneville, Mississippi 38776

USA

and evaluate potential disease treatments

using a variety of challenge methodologies.

Direct methods of exposure such as intra-

peritoneal injection (Areechon and Plumb

1983) and intragastric intubation (Baldwin

and Newton 1993) have yielded similar re-

sults with respect to the bacterial coloniza-

tion of internal organs. Route of infection

has been shown to influence infection rates

which raises concern of how well laboratory

challenges reflect natural disease outbreaks.

Immersion of channel catfish in solutions

containing

E.

ictaluri

resulted in low infec-

tivity rates in comparison to fish exposed by

IP injection (Ciembor et al. 1995). Although

IP infection ensures more uniform exposure

rates, this exposure route may increase dis-

ease susceptibility by circumventing natural

defense mechanisms of the fish. The pur-

pose of this study was

to

quantitatively de-

scribe the development of bacteriemia in

channel catfish fingerlings after immersion

exposure to

E.

i c f a f u r i .

Uptake and clear-

ance of E. ictaluri from the blood of fish

and the shedding of E. icfaluri into the wa-

ter by infected fish were evaluated. In ad-

dition, the production of anti-E. ictaluri an-

tibody was measured in fish surviving im-

8

Copyright

by

[he

World

Aquaculture Society

1997

45

8/11/2019 Uptake and Clearance of Edwardsiella Ictaluri in The

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46 WISE

ET AL.

mersion challenge to verify the occurrence

of a systemic infection.

Methods and Materials

Channel catfish fingerlings used in this

study were produced at the Clemson Uni-

versity Fish Health Complex, Clemson,

South Carolina. Sibling f r y were raised to

approximately 20 g/fish under laboratory

conditions in flow-through rearing troughs

supplied by UV-sterilized, filtered lake wa-

ter. Fingerlings had no prior history of ex-

posure to E. ictuluri and agglutination anti-

body titers of examined fish were below de-

tectable levels prior to experimentation.

Fish were transferred from rearing

troughs into 48, 15-L aquaria at 10 fish/

aquaria. UV-sterilized, filtered lake water

(26-28 C) was supplied to each aquaria at

0.25 L/min. Fish were fed a maintenance ra-

tion of feed (1 body weighdday) and al-

lowed to acclimate to culture conditions for

five days. Infections were then established

by

stopping the flow of water to each

aquaria and exposing fish to approximately

2.6 X

lo6

E.

ictuluri

colony forming units

(CFU)/mL of water. Water flow was re-

sumed to half of the aquaria after 4 h (4-h

immersion trial) and water flow was re-

sumed to the remaining aquaria after 8 h

(8-h immersion trial). Four aquaria of unin-

fected fish (10 fish/aquaria) served as con-

trols. Dead fish were recorded and removed

from aquaria daily. Water quality was mea-

sured at the end of the immersion period

and every 48 h after the water flow was re-

sumed. All water quality parameters re-

mained within acceptable limits for the cul-

ture of channel catfish fingerlings (Tucker

and Robinson 1990).

The development of bacteremia was

monitored by bleeding fish at 24, 72, and

216 h post-exposure to

E . ictaluri.

All fish

from eight randomly selected aquaria from

each sampling period and exposure group

were anesthetized in MS-222 and bled from

the caudal artery into sterile lithium hepa-

rinized evacuated tubes. In addition, serum

was collected from fish surviving infection

216

h

post-exposure using sterile non-

heparinized evacuated tubes. After bleed-

ing, fish were necropsied and the posterior

kidney and brain were cultured for

E.

ictu-

luri. The number

of E.

ictuluri

CFU

in the

water of each aquaria was determined im-

mediately after bacteria was added to

aquaria, at the end of the static exposure pe-

riod, and every 24 h thereafter. Bacterial

isolates were identified from biochemical

characteristics.

Standard plate counts were performed

in duplicate on peripheral blood and

water samples using

E.

ictuluri

culture me-

dia (Shotts and Waltman 1990). Samples

were serially diluted (1 to 10) in sterile

phosphate-buffered saline pH 7.2). Col-

lected serum was serially diluted in twofold

steps using

PBS

as the diluent. Agglutinat-

ing antibody titers were determined by in-

cubating equal volumes (25 pL of serially

diluted serum and antigen (formalin-killed

E. ictaluri) at 24 C for 24 h (Conrath and

Coupe 1978). The titer was determined to

be the reciprocal of the highest dilution that

showed a visible agglutination.

An additional exposure trial was per-

formed to ensure that the increase in bacte-

rial numbers observed in aquarium water

during the 4-h and 8-h exposure trials was

not related to the growth of bacteria in the

water. Eight of 12, 15-L aquaria were

stocked with 10 fiswaquaria and the re-

maining four aquaria were not stocked with

fish. After fish were acclimated to experi-

mental conditions for five days, challenges

were performed as described except that the

static exposure period was increased to 12

h. The number of

E.

ictaluri CFU in the wa-

ter of each aquaria was determined imme-

diately after bacteria was added to aquaria,

at the end of the static exposure period, and

every 24 h thereafter.

The median number of CFU/SO

IJ.L

periphrial blood was determined at each

sampling period for both challenge trials.

Nonparametric median comparisons were

made among sampling periods for each

challenge trial (Zar 1984). The number of

8/11/2019 Uptake and Clearance of Edwardsiella Ictaluri in The

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E D W A R D S I E U A

ICTALURI

IN CATFISH

BLOOD

47

TABLE

Numbers of Edwardsiella ictaluri colony forming units ( C F U ) of peripheral blood and percent offish

infected afrer exposure

of

channel carfish to

E.

ictaluri by immersion fo r either

4

or 8 hours. Data represent

the maximum minimum and median number of

CFUl5Op.L

whole blood. Median values followed by different

letters are significantly different with each exposure treatment P 0.05).

ND represents water samples which did not have detectable

l eve l s

of E.

ictaluri in the

water

8/11/2019 Uptake and Clearance of Edwardsiella Ictaluri in The

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EDWARDSIEL L A ICTAL U RI

IN

CATFISH

BLOOD

8

49

t

L

CI

I

A

3

T ime hou rs , pos t expo sure )

FIGURE

.

Numbers of

Edwardsiella ictaluri

colony forming units

C F U )

isolated from the water of aquaria

after inoculation with

E .

ictaluri. Open and closed symbols represent the log

of

the number of E . ictaluri

CFlJIml of water in aquaria with and without fi sh . respectively. Water samples were taken at the beginning

0

h) and end 1 2 h ) of the static immersion treatment and every 24 h after the waterflow was resumed.

Vertical lines represent the standard errors. Values represented by similar letters aquaria with fi sh ) or similar

numbers aquaria without fi sh ) are not statistically different

P