Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.10.

EPIZOOTIC ULCERATIVE SYNDROME

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SUMMARY

Epizootic ulcerative syndrome (EUS) is a seasonal epizootic condition of great importance in wild and farmed freshwater and estuarine fish. It was first reported in farmed ayu (Plecoglossus altivelis) in Japan in 1971 (6). It was later reported in estuarine fish, particularly grey mullet (Mugil cephalus) in eastern Australia in 1972 (13). The outbreak has extended its range through Papua New Guinea into South-East and south Asia, and recently into west Asia, where it has now reached Pakistan (8, 14). Outbreaks of ulcerative disease in menhaden (Brevoortia tyrannus) in the United States of America (USA) have been shown to be very similar to EUS in Asia (1).
 
EUS is also known as red spot disease (RSD), and mycotic granulomatoses (MG). The fungus involved in EUS is also known variously as Aphanomyces invadans, A. piscicida, A. invaderis and ERA (EUS-related Aphanomyces). Rhabdoviruses have also been associated with particular outbreaks, and secondary Gram-negative bacteria invariably infect EUS lesions.
 
Region-wide, over 50 species of fish have been confirmed by histological diagnosis to be affected by EUS (8), but some important culture species, including tilapia, milk fish and Chinese carp, have been shown to be resistant.
 
EUS occurs mostly during periods of low temperatures and after periods of heavy rainfall (2). These conditions favour sporulation of Aphanomyces invadans (12), and low temperatures have been shown to delay the inflammatory response of fish to fungal infection (3, 5).
 
Control of EUS in natural waters is probably impossible. In outbreaks occurring in small, closed water-bodies, liming water and improving water quality, together with removal of infected fish, is often effective in reducing mortalities.
 

DIAGNOSTIC PROCEDURES

Diagnosis of epizootic ulcerative syndrome (EUS) is based on clinical signs and confirmed by histopathology.
 

1.	Clinical Signs
	EUS outbreaks have been associated with mass mortality of various species of freshwater fish in the wild (including rice-fields, estuaries, lakes and rivers) and in farms.
	The early signs of the disease include loss of appetite, and fish become darker. Infected fish may float below the surface of the water, and become hyperactive with a very jerky pattern of movement. Red spots may be observed on the body surface, head, operculum or caudal peduncle. Large red or grey shallow ulcers, often with a brown necrosis, are observed in the later stages. Large superficial lesions occur on the flank or dorsum. Most species other than striped snakeheads and mullet will die at this stage.
	In highly susceptible species, such as snakehead, the lesions are more extensive and can lead to complete erosion of the posterior part of the body, or to necrosis of both soft and hard tissues of the cranium, so that the brain is exposed in the living animal.
2.	Clinical Pathology
	Nonseptate hyphae of Aphanomyces invadans (12-25 µm in diameter) can be observed in muscle squash preparations of the infected area around the lesion.
	Lesion scrapes generally show secondary fungal, bacterial and/or parasitic infections.
3.	Histopathology
	Histology is required for a confirmatory diagnosis.
	3.1.	Sampling procedure
		i)	Sample only live or moribund specimens of fish with clinical lesions.
		ii)	Take samples of skin/muscle (<1 cm3), including the side of the lesion and the surrounding tissue.
		iii)	Fix the tissues immediately in 10% formalin. The amount of formalin should be 15-20 times the volume of the tissue to be fixed.
		iv)	Gently agitate the fixative 2-3 times during the first hour after adding the tissue.
	3.2.	Histological procedure
		Processing of the fixed tissue involves dehydration through ascending alcohol grades, clearing in a wax-miscible agent and impregnation with wax (4). The blocks of fish tissue are cut at about 5 µm and mounted on a glass slide. Before staining, the section must be completely de-waxed and stained in haematoxylin and eosin (H& E) (4).
	3.3.	Confirmatory diagnosis
		H& E and general fungus stains (e.g. Grocott's) will demonstrate typical granulomas and invasive hyphae.
		Early EUS lesions are erythematous dermatitis with no obvious fungal involvement. Aphanomyces invadans hyphae are observed growing in skeletal muscle as the lesion progresses from a mild chronic active dermatitis to a severe locally extensive necrotising granulomatous dematitis with severe floccular degeneration of the muscle. The fungus elicits a strong inflammatory response and granulomas are formed around the penetrating hyphae.

IDENTIFICATION

1.	Fungal Isolation
	The following are two methods of isolation of A. invadans fungus adapted from refs 7 and 15.
	1.1.	Moderate, pale, raised, dermal lesions are most suitable for fungal isolation attempts. Remove the scales around the periphery of the lesion and sear the underlying skin with a red-hot spatula so as to sterilise the surface. Using a sterile scalpel blade and sterile fine-pointed forceps, cut through stratum compactum underlying the seared area and, by cutting horizontally and reflecting superficial tissues, expose the underlying muscle. Ensure the instruments do not make contact with the contaminated external surface and thereby contaminate the underlying muscle. Using aseptic technique, carefully excise pieces of affected muscle, approximately 2 mm3, and place on a Petri dish containing Czapek Dox agar with penicillin G (100 units/ml) and oxolinic acid (100 µg/ml). Seal plates, incubate at room temperature and examine daily. Repeatedly transfer emerging hyphal tips on to fresh plates of Czapek Dox agar until cultures are free of contamination.
	1.2.	Lesions located on the flank or tail of fish <20 cm in length can be sampled by cutting the fish in two using a sterile scalpel, and slicing a cross-section through the fish at the edge of the lesion. Flame the scalpel until red-hot and use this to sterilise the exposed surface of the muscle. Use a small-bladed sterile scalpel to cut out a circular block of muscle (2-4 mm3) from beneath the lesion and place it in a Petri dish of GP (glucose/peptone) medium (see Table 1) with 100 units/ml penicillin-K and 10 µg/ml oxolinic acid. Instruments should not contact the contaminated external surface of the fish. Incubate inoculated media at approximately 25°C and examine under a microscope (preferably an inverted microscope) within 12 hours. Repeatedly transfer emerging hyphal tips to plates of GP medium with 12 g/litre technical agar, 100 units/ml penicillin-K and 10 µg/ml streptomycin sulphate until axenic cultures are obtained. They may then be maintained at 10°C on GP agar and subcultured at intervals of no greater than 7 days.
2.	Fungal Identification
	Aphanomyces invadans does not produce any sexual structures and should thus not be diagnosed by morphological criteria alone. However the fungus can be identified to the genus level by inducing sporogenesis and demonstrating typical asexual characteristics of Aphanomyces as described in ref. 8. Aphanomyces invadans is characteristically slow growing in culture and fails to grow at 37°C on GPY agar (Table 1). Detailed temperature - growth profiles are given in ref. 11. Two procedures that can use to confirm the A. invadans are experimental fish infection and molecular technique. The experimental infection can be made by injecting intramuscularly a 0.1 ml suspension of 100+ motile zoospores in EUS-susceptible fish (preferably Channa striata) at 20°C, and demonstrating histologically growth of aseptate hyphae 12-25 µm in diameter in muscle of fish sampled after 7 days, and of typical mycotic granulomas in muscle of fish sampled after 14 days. For the molecular technique, this pathogen is readily identified by RAPD-PCR or sequencing of the internal spacer region (ITS) region of nuclear rDNA. RAPD-PCR is rapid and does not require DNA sequencing, but it is necessary to isolate the pathogen in pure culture for reliable results. Details regarding DNA preparation, PCR primers for amplification can be found in refs 9 and 10.
	All Aphanomyces invadans isolates found so far belong to a single genotype, which facilitates identification. Alternatively, sequencing of the internal spacer region can be performed and the result compared with the sequence deposited in public gene data banks. A. invadans is different to A. astaci etiological agent of Crayfish plague (Chapter 4.1.7). As A. invadans does not have a sexual reproductive stage. Both pathogenic fungi can also be differentiated using RAPD-PCR.
3.	Inducing sporulation in Aphanomyces invadans cultures
	The induction of asexual reproductive structures is necessary in order to identify fungal cultures as members of the genus Aphanomyces. To induce sporulation, place an agar plug (3-4 mm in diameter) of actively growing mycelium in a Petri dish containing GPY broth and incubate for 4 days at approximately 20°C. Wash the nutrient agar out of the resulting mat by sequential transfer through five Petri dishes containing autoclaved pond water, and leave overnight at 20°C in autoclaved pond water. After about 12 hours, the formation of achlyoid clusters of primary cysts and the release of motile secondary zoospores should be apparent under the microscope. Media for inducing sporulation are shown in Table 1.

Table 1. Media for isolation, growth and sporulation of Aphanomyces invadans cultures

GP (glucose/peptone) broth

	3 g/litre glucose
	1 g/litre peptone
	0.128 g/litre MgSO4.7H2O
	0.014 g/litre KH2PO4
	0.029 g/litre CaCl2.2H2O
	2.4 mg/litre FeCl3.6H2O
	1.8 mg/litre MnCl2.4H2O
	3.9 mg/litre CuSO4.5H2O
	0.4 mg/litre ZnSO4.7H2O

GPY (glucose/peptone/yeast) broth

GP broth + 0.5 g/litre yeast extract

GPY agar

GPY broth + 12 g/litre technical agar

Autoclaved pond water
 

Sample pond/lake water known to support fungal growth. Filter through Whatman 541 filter paper. Combine one part pond water with two parts distilled water and autoclave. pH to 6-7.

REFERENCES

1.	Blazer V.S., Vogelbein W.K., Densmore C.L., May E.B., Lilley J.H. & Zwerner D.E. (1999). Aphanomyces as a cause of ulcerative skin lesions of menhaden from Chesapeake Bay tributaries. J. Aquat. Anim. Health, 11, 340-349.
2.	Bondad-Reantaso M.G., Lumanlan S.C., Natividad J.M. & Phillips M.J. (1992). Environmental monitoring of the epizootic ulcerative syndrome (EUS) in fish from Munoz, Nueva Ecija in the Philippines. In: Diseases in Asian Aquaculture 1, Shariff M., Subasinghe R.P. & Arthur J.R., eds. Fish Health Section, Asian Fisheries Society, Manila, The Philippines, 475-490.
3.	Catap E.S. & Munday B.L. (1998). Effects of variations of water temperature and dietary lipids on the expression of experimental epizootic ulcerative syndrome (EUS) in sand whiting, Sillago ciliata. Fish Pathol., 33, 327-335.
4.	Chinabut S. & Roberts R.J. (1999). Pathology and Histopathology of Epizootic Ulcerative Syndrome (EUS). Aquatic Animal Health Research Institute, Department of Fisheries, Royal Thai Government, Bangkok, Thailand, 33 pp. ISBN 974-7604-55-8.
5.	Chinabut S., Roberts R.J., Willoughby G.R. & Pearson M.D. (1995) Histopathology of snakehead, Channa striatus (Bloch), experimentally infected with the specific Aphanomyces fungus associated with epizootic ulcerative syndrome (EUS) at different temperatures. J. Fish Dis., 18, 41-47.
6.	Egusa S. & Masuda N. (1971). A new fungal disease of Plecoglossus altivelis. Fish Pathol., 6, 41-46.
7	Fraser G.C., Callinan R.B. & Calder L.M. (1992). Aphanomyces species associated with red spot disease: an ulcerative disease of estuarine fish from eastern Australia. J. Fish Dis., 15, 173-181.
8.	Lilley J.H., Callinan R.B., Chinabut S., Kanchanakhan S., MacRae I.H. & Phillips M.J. (1998). Epizootic ulcerative syndrome (EUS) technical handbook. Aquatic Animal Health Research Institute, Bangkok, Thailand.
9.	Lilley J.H. & Chinabut S. (2000). DNA-based studies on Aphanomyces invadans, the fungal pathogen of epizootic ulcerative syndrome (EUS). In: DNA-based Molecular Diagnostic Techniques: Research Needs for Standardization and Validation of the Detection of Aquatic Pathogens and Diseases, Walker P. & Subasinghe R., eds. Food and Agriculture Organization of the United Nations Fisheries Technical Paper No. 395, FAO, Rome, Italy, 83-87.
10.	Lilley J.H., Hart D., Richards R.H., Roberts R.J., Cerenius L. & Soderhall K. (1997). Pan-Asian spread of single fungal clone results in large scale fish kills. Vet. Rec., 140, 653-654.
11.	Lilley J.H. & Roberts R.J. (1997). Pathogenicity and culture studies comparing the Aphanomyces involved in epizootic ulcerative syndrome (EUS) with other similar fungi. J. Fish Dis., 20, 135-144.
12.	Lumanlan-Mayo S.C., Callinan R.B., Paclibare J.O., Catap E.S. & Fraser, G.C. (1997). Epizootic ulcerative syndrome (EUS) in rice-fish culture systems: an overview of field experiments 1993-1995. In: Diseases in Asian Aquaculture III, Flegel T.W. & MacRae I.H., eds. Fish Health Section, Asian Fisheries Society, Manila, The Philippines, 129-138.
13.	McKenzie R.A. & Hall W.T.K. (1976). Dermal ulceration of mullet (Mugil cephalus). Aust. Vet. J., 52, 230-231.
14.	Tonguthai K. (1985). A preliminary account of ulcerative fish diseases in the Indo-Pacific region (a comprehensive study based on Thai experiences). National Inland Fisheries Institute, Bangkok, Thailand, 39 pp.
15.	Willoughby L.G. & Roberts R.J. (1994). Improved methodology for isolation of the Aphanomyces fungal pathogen of epizootic ulcerative syndrome (EUS) in Asian fish. J. Fish Dis., 17, 541-543.

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