Appendix 1
FACILITIES REQUIRED FOR FLUORESCENT DIAGNOSTIC PROCEDURE

Big refrigerator +4°C;
Deep freezer -27°C;
Cold room;
100 g balance (Mettler);
Electrophoretic instrument for immune-electrophoresis;
High-speed centrifuge;
Micropipettes (5-10-25-50-100 μ1);
Sonicator;
Cages for rabbits and room with water and heating;
pH meter with drop-electrodes, combined electrode;
Stabilized power supply, with 500–1 000 volts;
Shaker for producing large amount of bacteria;
Chemicals.

Fig. 1 - The rapid identification procedure for Aeromonas spp.

Aeromonas spp. (Carp - red spot disease, Eel - red fin disease)

Fig. 1

Fig. 2 - The rapid identification for Pseudomonas sp.

Pseudomonas sp. (Carp - pseudomonas infection)

Fig. 2

Fig. 3 - The rapid identification for Edwardsiella tarda Edwardsiella tarda (Eel - edwardsiella infection)

Fig. 3

Fig. 4 - The rapid identification for Pseudomonas anguilliseptica Pseudomonas anguilliseptica (Eel - red spot disease)

Fig. 4

Fig. 5 - The rapid identification for Flexibacter columnalis Flexibacter columnalis (Carp, eel, salmonid, ayu - columnalis disease)

Fig. 5

Fig. 6 - The rapid identification for Vibrio anguillarum Vibrio anguillarum (Vibrio spp.) (Eel - vibrio infection)

Fig. 6

Fig. 7 - The rapid identification for Aeromonas salmonicida Aeromonas salmonicida (salmonids - furunculosis)

Fig. 7

Fig. 8 - The rapid identification for Vibrio anguillarum Vibrio anguillarum (salmonids, ayu - vibrio infection)

Fig. 8

Fig. 9 - The rapid identification for Corynebacterium sp. Corynebacterium sp. (salmonids - bacterial kidney disease)

Fig. 9

Fig. 10 - The rapid identification for Hemophilus piscium Hemophilus piscium (salmonids - ulcer disease)

Fig. 10

Fig. 11 - The rapid identification for Yersinia ruckeri Yersinia ruckeri (salmonids - red mouth disease)

Fig. 11

1*. Rimler-Shotts medium
	L-lysine hydrochloride	5.0g
	L-ornithine hydrochloride	6.5g
	Maltose	3.5g
	Sodium thiosulfate	0.3g
	Bromthymol blue	0.03g
	Iron ammonium sulfate	0.8g
	Sodium deoxycholate	1.0g
	Novobiocine	0.05g
	Yeast extract	3.0g
	Sodium chloride	5.0g
	Agar	13.5g
	Distilled water	1000 ml
	pH	7.0

2*. Broth with 7.5% NaCl
	Peptone	10.0g
	Sodium chloride	75.0g
	Distilled water	1000 ml

3*. Production of green fluorescent pigment
	Cultivation on King's medium*
	25°C, 1 week
	↓
	Production of yellow-green fluorescence
	* King's medium
	Peptone		20.0g
	Potassium phosphate monobasic		1.5g
	Magnesium sulfate		1.5g
	Glycerin		10.0g
	Agar		15.0g
	Distilled water		1000 ml
	pH		7.2

4*. Cytophaga agar
	Tryptone	0.5g
	Yeast extract	0.5g
	Beef extract	0.2g
	Sodium acetate	0.2g
	Agar	9.0g
	Distilled water	1000 ml
	pH	7.2–7.4

5*. Congo red reaction
	Colony on cytophaga agar
	↓← 0.01% Congo red solusion
	Edge of the colony
	↓
	Turning red (+)

6*. Arginine test

	25°C, 3–7 days	* Arginine agar
	↓
	Turning red (+)	Peptone	1.0g
		Sodium chloride	5.0g
		Potassium phosphate dibasic	0.3g
		Phenol red	0.01g
		L-arginine hydrochloride	10.0g
		Agar	3.0g
		Distilled water	1000 ml
		pH	7.4

7*. Furunculosis agar
	Tryptone	10.0g
	Yeast extract	5.0g
	L-tyrosine	1.0g
	Sodium chloride	2.5g
	Agar	15.0g
	Distilled water	1000 ml
	pH	7.5
8*. Ordal agar
	Tryptose	10.0g
	Beef extract	3.0g
	Yeast extract	0.5g
	Sodium chloride	5.0g
	Agar	15.0g		Distilled water	800 ml
	L-cystein hydrochloride	1.0g		Distilled water	160 ml
	pH	6.6
	Human or horse serum	40 ml

Fig. 12 - The manufacturing and immunization method of vaccines

Oral vaccine preparation (ayu)

Fig. 12

Oral vaccine administration ( ayu)

Adsorption with vaccine on and into dryed pellet

Administration

0.1g packed cell / kg fish / day
for 10 – 14 days

Fig. 13 - The manufacturing and immunization method of vaccines
Immersion vaccine (bath immunization) preparation and administration (ayu)

Preparation

Fig. 13

Administration

* Hyperosmotic infiltration

Fig. 13

* Immersion

Fig. 13

Fig. 14 - The manufacturing and immunization method of vaccines Spray vaccine preparation (ayu)

Fig. 14

Spray administration ( ayu )

Fig. 14

Vaccine was sprayed with spray gun for 5–7 sec at a distance of 20–25 cm from fish

Fig. 15 - The manufacturing and immunization method of vaccines

Challenging procedure 1 (ayu)

Fig. 15

Challenging procedure 2 ( ayu )

Fig. 15

Fig. 16 - The manufacturing and immunization method of vaccines
Preparation and administration of pseudotuberculosis vaccine (yellowtail)

Fig. 16

Challenging procedure

Fig. 16