Attempted Vaccination of Rainbow Trout ...

4 downloads 0 Views 338KB Size Report
Attempted. Vaccination of Rainbow. Trout Oncorhynchus mykiss. Against Bacterial Kidney Disease. Masahiro Sakai,* Shizuo Atsuta,* and Masanori Kobayashi*.
Nippon Suisan Gakkaishi Attempted

55(12),2105-2109(1989)

Vaccination

of Rainbow

Against

Bacterial

Trout

Oncorhynchus

Kidney

mykiss

Disease

Masahiro Sakai,* Shizuo Atsuta,* and Masanori

Kobayashi*

(ReceivedJune 30, 1989) Immune responses in rainbow trout Oncorhynchusmykiss intraperitoneally injected with formalin killed Renibacteriumsalmoninarumcells (with or without Freund's complete adjuvant), were evaluated by monitoring serum agglutinating antibody and phagocytic activity of kidney leukocytes, and the challenge test. Serum antibody titers (range 8-16) were detected in the vac cinated groups 5 weeks after vaccination. The phagocyticactivitiesin the kidneycells of vaccinated groups increasedafter 15 days and 25 days. However, experimental intraperitoneal challenge(107 or 106 cells/fish) of immunized groups showed no protective effects. Bacterial kidney disease (BKD) is a systemic and clonic infection of salmonid fishes characteriz ed by the presence of granulomatous lesions.1) The etiological agent Resnibacterium salmoninarum is a Gram-positive diplobacillus which is very fastidious in its growth requirements. The disease is considered important because of the serious losses it causes and because of the difficulty in controlling it. Some successful results in chemo therapy with erythromycin,2,3) sulfonamides,4) penicillin,5) clindamycin, kitasamycin and spira mycin6) have been reported. However, it has been proved difficult to eliminate the pathogen from the host because of its intracellular existence in macrophages.7) Vaccination against BKD has been attempted by Paterson et al.8) and McCarthy et al..9) Pater son et al.8) reported a significant increase in serum agglutinating titer and a reduced incidence of BKD in Atlantic salmon Salmo salar. However, because the potency of vaccine was demonstrated by the gross lesions of BKD and the detection of R. salmoninarum antigen with the indirect fluore scent antibody technique (IFAT), the exact efficacy of vaccine was not determined. McCarthy et al.9) also reported that rainbow trout Oncorhynchus mykiss formerly Salmo gairdneri, injected intra

mune

小 林 正 典:北

Fisheries

Sciences,

里 大 学 水 産 学 部).

Kitasato

of

salmoninarum

the

effect

of

lenging

rainbow

trout

vaccinated

bacterins

was

investigated

vaccination

with

the

was

with and

estimated

intraperitoneal

by

chal

of

living

50

g and

injection

organisms.

Materials

and

Methods

Animals Rainbow 10

g were

and

a

trout

of

Iwate

University

a mean

an test,

the

Bacterin

The

with

and

salmon

Oncorhynchus

was

used

for

was

cultured

4•Ž.

washed

adjusted The

to

equal

with

g-fish)

killed fish

by

trout

or were

0.05

ml

adjuvant

Sanriku, Iwate

at

15•Ž

by

adjuvant

were

in

sterile

with

O22-01,Japan(酒

at

same

nm.

cells

MS-222

0.1 of

ml

(for

formalin

bacterin. the

and

520

(Difco).

with

g-fish)

h

emulsifying

(FCA)

with 10

7.2)

washed

anesthetized

(for

48

cells

1.0

killed

and

for

pH

prepared

emulsified

administrated

days

(PBS,

intraperitoneally or

1985, strain

formalin

formalin

were

a dise in

centrifugation

was of

15

of O.D.

complete

injected

from

inactivated

saline

bacterin

Rainbow

50

formalin

volume

12•Ž

The

0.3%

a concentration

Freund's

and

for of

times

adjuvant

an

KDM-210)

buffered

at

kisutch

preparation.

addition

three

phosphate

isolated

bacterin

The

kept

Vaccination

coho

at

were

were Station

pellets.

ased

in

fish

Fish

fish

KU8501

the

The

commercial

Preparation

by

experiment

Experimental

salmoninarum

killed

of

response

respectively.

lwate

daily

weight

immune

Prefecture.

fed

R.

in

challenge by

•@ and

with

used

supplied

- peritoneally with lysed cell preparations of R. salmoninarum were protected against the artificial challenge of living cells, although serum agglutinat ing antibody was not investigated. Thus, the relation between immune response and protection must be investigated in fish vaccinated with R. salmoninarum. In the present study, the im * School of

response

R.

Control volume

井 正 博,厚

of

田 静 男,

2106

Sakai,

FCA.

Vaccinated

maintained 12•Ž

and

tanks

fed

Serum fish

of

each

five

fish

water

The

were

temperature

of

in

obtained

from

weeks

after

microtiter

method.

indirect used

detect

kidney

Five

vessels

of

were

The

serum fish

agglutinating

determined

Serum

titer using

the

Agglutinating

antibody

inarum

were

cinated

by

cination The

vaccinated

and

5,

kidney

phagocytosis

to

the

of

was

and

or

non-vaccinated

25

days

after

were

collected.

leukocytes

assay

method

difference

15

was

Sakai

performed

only

et

estimated

al.11)

by

statistical

by

The

Student

Method

Thirty

after

non-vaccinated itoneal

fish

injection

suspended were

were ed

were with

in

PBS. at

daily necropsied

on

of

the

to

the

higher

60

and

kidney

intraper

bacterial

cells

challenge,

and

days.

the

fish

mortalities All

were

moribund

samples

of

value

is

in days all

was

percent

method

Sixty

cinated

for

by

106

the

12•Ž

vaccine

relative

immunity

from

or

and

were

fish inoculat

after

the

A to

calculat according

RPS

of

indicate

60

Phagocytic

5,

fish

the

kidney or

smears non-vac

non-fluorescent

activities

in kidney

without

the

injected

with

days

after the

cination

were

controled

5,

15

and

and

57.6%

respectively. activities

from

8.5

of to

25

higher

the

10.8%.

leukocytes

and

significantly

cells

40.2%

of 15

the

formalin-killed

were

measured

in

Leukocytes

vaccination,

activities

fish

shown

without

48.6%

phagocytic

FCA

phagocytic

and

10.9%,

hand, with

munized

are

respectively.

were

of

leukocytes cells

28.1%

immunized

25

of

of

days

after

than

im vac

those

of

fish.

Protection

Experiments

slides.

leucocytes

pattern

controled cells died

fish are by

of rainbow

shown 25

days

trout

of

mortalities

challenged in after

of with

Fig.

1.

the

challenge.

vaccinated

with

vaccinated

2.2•~107 All

or bacterial

controled In

that

Renibacterium

Vaccine

Phagocytic (n =5) activity (%)

Singificance

FK*t FK with FCA*2 Control

14.7±3.9 10.9±1.6 9.5±2.1

N.S*3 N.S

15

FK FK with FCA Control

28.1±1.9 40.2+6.6 10.6±2.0

p