epizootic ulcerative syndrome

Pictures

Picture	Title	Caption	Copyright
Title Distribution map Caption Map showing the spread of EUS across the Asia-Pacific region. Dates indicate the time of the first serious outbreak. There is some doubt about outbreaks marked with asterisks. Copyright J.H. Lilley	Distribution map	Map showing the spread of EUS across the Asia-Pacific region. Dates indicate the time of the first serious outbreak. There is some doubt about outbreaks marked with asterisks.	J.H. Lilley
Title Symptoms Caption Sand whiting, Sillago ciliata, caught in Richmond River, Eastern Australia, infected with EUS. Copyright R. V. L. Wollongbar	Symptoms	Sand whiting, Sillago ciliata, caught in Richmond River, Eastern Australia, infected with EUS.	R. V. L. Wollongbar
Title Symptoms Caption Striped snakehead, Channa striata, bath challenge, Thailand, infected with EUS. Copyright Supranee Chinabut	Symptoms	Striped snakehead, Channa striata, bath challenge, Thailand, infected with EUS.	Supranee Chinabut
Title Symptoms Caption Silver perch, Bidyanus bidyanus, from Eastern Australia, infected with EUS. Copyright R. V. L. Wollongbar	Symptoms	Silver perch, Bidyanus bidyanus, from Eastern Australia, infected with EUS.	R. V. L. Wollongbar
Title Symptoms Caption Grey mullet, Mugil cephalus, caught in the Richmond River, Eastern Australia, infected with EUS. Copyright R. V. L. Wollongbar	Symptoms	Grey mullet, Mugil cephalus, caught in the Richmond River, Eastern Australia, infected with EUS.	R. V. L. Wollongbar
Title Histology Caption Histological pictures of EUS muscle samples with various stains: (a) Haematoxylin and Eosin; (b) Uvitex; (c) Grocott's; (d) Perodic Acid Schiff (PAS). Copyright Supranee Chinabut & Varinee Panyawachira	Histology	Histological pictures of EUS muscle samples with various stains: (a) Haematoxylin and Eosin; (b) Uvitex; (c) Grocott's; (d) Perodic Acid Schiff (PAS).	Supranee Chinabut & Varinee Panyawachira
Title Hyphae and sporangium Caption Hyphae and sporangium of Aphanomyces invadans from EUS-infected fish. Copyright J.H. Lilley	Hyphae and sporangium	Hyphae and sporangium of Aphanomyces invadans from EUS-infected fish.	J.H. Lilley

Identity

Preferred Scientific Name

• epizootic ulcerative syndrome

Other Scientific Names

• mycotic granulamotoses
• red spot disease

International Common Names

• English: Aphanomyces invadans infection

English acronym

• EUS
• MG
• RSD

Overview

Epizootic ulcerative syndrome (EUS) is a disease of freshwater and brackishwater fish that has other synonyms such as mycotic granulomatosis (MG), red spot disease (RSD) and ulcerative mycosis (UM). More than 100 species of fish species with a wide geographic distribution have been reported to be affected by EUS. EUS outbreaks occur only when a number of causal factors combine.

The causative agent of this disease is the fungus, Aphanomyces invadans. There are a number of sufficient causes that lead to exposure of the dermis to fungal spore germination. In the early stages, red spots or small haemorrhagic lesions are found on the body surface. Intermediate stage lesions become small ulcers. Advanced stage lesions expand into large necrotic open ulcers resulting in death.

Positive diagnosis of EUS is made by the presence of mycotic granulomas in histological section and isolation of Aphanomyces invadans from infected fish. Quarantine and health certification practice for the movement of live fish between countries or regions are effective means of preventing the spread of EUS to new areas. For areas where EUS is endemic, prevention programmes should include eradication, exclusion, management, surveillance and treatment.

Outbreaks of EUS have continued to occur periodically in all target countries. The immunity of fish surviving EUS infections should be the subject of interest because records from many countries showed that the severity of EUS outbreak after the first few years tends to decrease (Mohan and Shankar, 1994; Bondad-Reantaso et al., 1992). The serious implications for the development of vaccines against EUS should be deliberated because there was a report on the strong reaction between the sera from immunized fish with the spores and mycelium of pathogenic strains of Aphanomyces and the extracts of pathogenic strains of Aphanomyces (Thompson et al., 1997).

Hosts/Species Affected

The disease called mycotic granulomatosis was first found in ponds raising goldfish (Carassius auratus) (Miyazaki and Egusa, 1972), and ayu (Plecoglossus altivelis) in various regions in Japan in 1971 (Egusa and Masuda, 1971). Wild species such as the formosan snakehead (Channa maculata), Japanese trident goby (Tridentiger obscurus) and black mullet (Mugil cephalus) were also affected by the disease at that time (Miyazaki and Egusa, 1973a,b,c). Later, the causative agent of mycotic granulomtosis was identified as Aphanomyces piscicida by Hatai (1980). In the following year, 1972, red spot disease (RSD) was first reported in estuarine fish from the Burnett River, Queensland, Australia (McKenzie and Hall, 1976; Rodgers and Burke, 1981). Lately, outbreaks of RSD have been reported in many species of estuarine and freshwater fish in New South Wales (Callinan et al., 1989), North Territory (Humphrey and Langdon, 1986; Pearce, 1990) and Western Australia (D. Pass, personal communication). Sea mullet (Mugil cephalus), sand whiting (Sillago ciliata), yellow fin bream (Acanthopagrus australis) and barramundi (Lates calcarifer) were some of the economically important estuarine species affected. Outbreaks of RSD have occurred in farmed silver perch (Bidyanus bidyanus) which is a freshwater species of Southern Australia (Rodgers and Burke, 1981).

Distribution

Since 1971-1972 the disease gradually spread to many countries in the Asia-Pacific region. Catfish cultured in the Mekong delta, Vietnam were reported to be infected by EUS in 1973 (Roberts et al., 1994) but this information was not supported by histopathological confirmation. In 1975, there was a report on an EUS outbreak in some freshwater fish in Papua New Guinea. By the year 1980 it had spread to Malaysia and Thailand. In 1982-1983, a severe EUS outbreak in Thailand affecting snakehead (Channa striata), Puntius sp. and many species of rice field fish was recorded (Tonguthai, 1985). The outbreak was also reported in East Kalimantan, Indonesia, in 1982 and 1984, causing serious mortality on wild species such as snakehead, catfish, sand goby, Puntius sp. and kissing gouramy in natural water resources (Roberts et al., 1994). Cambodia, Laos and Myanmar also suffered from EUS in 1984. The first confirmed EUS outbreak of some freshwater fish in Laguna de Bay, Philippines, was recorded in 1985-1986. As early as 1987, Puntius sp. and snakehead in a western province of Sri Lanka were infected by EUS. After the severe flooding in 1988, the first EUS outbreak was reported in Bangladesh. Indian major carps seem to be the main species affected. In the same year, EUS occurred in northeast India and spread throughout the whole country, then to Nepal in the following year (Roberts et al., 1994). By 1996 it was confirmed that EUS had spread to the upper part of the Indus River in Pakistan (Kanchanakhan, 1996).

Distribution Table

The distribution in this summary table is based on all the information available. When several references are cited, they may give conflicting information on the status. Further details may be available for individual references in the Distribution Table Details section which can be selected by going to Generate Report.

Pathology

Pathogenic Agents

Based on the investigation of the pathogenic organisms found from EUS-infected samples, a wide range of aetiologies have been attributed to EUS, such as bacteria, fungi, parasites and viruses.

Several species of parasitic protozoans (Chilodonella sp., Costia sp., Epistylis sp., Glossatella sp., Ichthyophthirius sp., Scyphidia sp., Trichodina spp.), myxosporeans (Henneguya sp. and Thelohania sp.), monogeneans and crustaceans (Lernaea sp.) have been recorded from diseased fish (Callinan et al., 1997; Reungprach et al., 1983). Nevertheless, there is no evidence to confirm that these parasites are the causative agent of the disease. Parasitic infections possibly induce stress in fish, thus predisposing them to infection (Subasinghe, 1993).

Aeromonas hydrophila, A. sobria, Pseudomonas spp. and Vibrio spp. are the species of bacteria isolated from the internal organs of EUS-infected fish (Burke and Rodgers, 1981; Llobrera and Gacutan, 1987; Tonguthai, 1985). Among these bacteria, A. hydrophila is the most commonly isolated species from the advanced stages of infected fish. It is usually not isolated from the early stage of the disease. On the other hand, A. hydrophila is known to be part of the normal microflora of fish and aquatic environments and is recognised as an opportunistic pathogen.

A number of birnaviruses, reoviruses and rhabdoviruses have been isolated from diseased snakehead and other susceptible species. These viruses most probably represent adventitious infections unrelated to EUS because of their heterogeneous nature, together with a low and non-consistent level of recovery from epizootics of disease. Therefore, the role of viruses in the aetiology of this disease remains unproven (Kanchanakarn, 1996; Roberts et al., 1994).

At least two species of fungi have been isolated from EUS-infected fish, namely, Achlya sp. which was isolated from the superficial area of the lesions (Pittchayangkula and Bodhalamik, 1983; Subasinghe et al., 1990) and Aphanomyces sp. which is normally isolated from the muscle area near the lesions (Egusa, 1992, Fraser et al., 1992; Hatai et al., 1994; Roberts et al., 1993). Recently, it has been shown that all Aphanomyces identified from EUS samples not only are the same species but also a single clonal lineage (Lilley et al., 1997). Therefore, it is confirmed that the fundamental aetiological agent of EUS is an oomycete fungus, Aphanomyces invaderis (Willoughby et al., 1995). However, the valid taxonomic name according to the International Code of Botanical Nomenclature (ICBN) of this species is A. invadans (Lilley et al., 1998). This specific pathogenic fungus showed the ability to invade the skin of EUS-infected fish (Kiryu et al., 2003). Isolation of this particular fungus is difficult as it is slow growing and most of the hyphae in the muscle are dead except for the tips that are penetrating deeper into the muscle tissue (Roberts et al., 1993)

Pathogenicity of the Fungus

After success in isolating the slow growing fungus, Aphanomyces invadans, from EUS-infected fish in various countries (Egusa, 1992; Fraser et al., 1992; Willoughby et al., 1995), pathogenicity studies were conducted. Intramuscular injection of spore suspensions into some susceptible fish species (barbs and snakehead) demonstrated a severe necrotising myogranulomatous condition at the injected site similar to the pathological feature of fish naturally infected with EUS. At the later stages, the fungus rapidly invaded the other internal organs, causing death to the host. The diameter of the hyphae of the fungus in lesions was always larger and the walls thicker than in cultured specimens (Roberts et al., 1993). Co-habitation transmission experiments with EUS-infected fish were successful (Balasuriya et al., 1990; Cruz-Lacierda and Shariff, 1995).The exposure of healthy Atlantic menhaden to suspension of zoospores of A. invadans demonstrated the germination and penetration of the germ tube through the epithelium layer of the experimental fish (Kiryu et al., 2003).

Histopathology

Lesions in all EUS-infected fish are similar, except that in snakehead chronic lesions can develop. This is because snakehead survive longer and the lesions are able to develop to a very advanced stage; other species die before this stage is reached.

Grossly the lesions are generally small red erosions on the body, head and fins. In the advanced stage, deep haemorrhaged ulcers spread through the whole body. Fungal mycelium is commonly found on top of the ulcerative lesions. The internal organs in the early stage of infection show only minimal inflammatory response. It is important to note that diseased fish with not too advanced lesions, kept in better water quality conditions, often recover and undergo a healing process leaving dark scars at the sites of infection.

Histopathological observation of early stage lesions reveal acute spongiosis with loss of epithelial cells. Degenerative changes are observed in the dermis, with hyperaemia, haemorrhages and inflammatory cells infiltrating between the fibres of the dermis. Inflammatory exudate and haemorrhages are also found in the hypodermis. Sarcolysis with haemorrhages and inflammatory exudate are obvious in the more advanced stage. The fungal hyphae are enclosed by a well developed epithelioid cell layer. Mycotic granulation of these non-septate fungal hyphae spreads through the infected muscle and other internal organs in the very late stages. In the advanced stage, there are no muscle fibres in the granuloma tissues. They are replaced by fibrosis, inflammatory cells and newly developing capillaries (Chinabut et al., 1995; Chinabut and Roberts, 1999). This distinct feature of the typical mycotic granulomas in the lesions of EUS-infected fish provides important histopathological information which can be used to distinguish EUS from other fish ulcerative diseases.

Diagnosis

Clinical signs

EUS is associated with mass mortality of susceptible fish in areas that are initially affected. However, evidence from EUS-endemic areas shows that it can also occur at low intensity, with fish often recovering towards the end of the cool season. Gross pathology of EUS infected fish is similar to other cutaneous ulcerative syndromes of freshwater and estuarine fishes and it is therefore important not to rely on clinical signs as a means of diagnosis. Outbreaks in estuarine fish have been recorded only in water with salinity less than 2 ppt. The gross appearance of lesions varies between species, habitat and stage of lesion development (Callinan et al., 1989, Viswanath et al., 1997, Chinabut and Roberts, 1999). The most distinctive EUS lesion is the open dermal ulcer. This is often most conspicuous in snakehead which is often used as an "indicator species" of EUS in an area.

Rapid muscle squash

A provisional diagnosis of EUS can be made by demonstrating aseptate fungal hyphae (12-30 µm in diameter) in a squash preparation of skeletal muscle taken from beneath the surface of an ulcer.

Histology

Confirmatory diagnosis requires histopathological demonstration of typical for necrotizing mycotic granulomas using haematoxylin and eosin stain (H&E) and a general fungus stain such as Periodic Acid Schiff (PAS), Grocott's or Uvitex.

Fungus culture and characterization

Isolation and identification of Aphanomyces invadans from infected fish are reported by Fraser et al. (1992) and Willoughby and Roberts (1994). Moderate, pale, raised, dermal lesions are most suitable for fungal isolation. Remove the scales around the lesion and sear the underlying skin with a red-hot spatula to sterilize the surface. Using a sterile scalpel blade and sterile, fine pointed forceps, cut through skin underlying the seared area and then by cutting horizontally and reflecting superficial tissues, expose underlying muscle. Ensure the instruments do not contact the contaminated external surface and otherwise contaminate the underlying muscle. Using aseptic techniques, carefully excise pieces of affected muscle, approximately 2 mm³, and place them in a Petri dish containing Czapex Dox agar with penicillin G (100 units mL^-1) and oxolinic acid (100 mg mL^-1). Seal plates and incubate at room temperature, examining daily. Repeatedly transfer emerging hyphal tips on to fresh plates of Czapex Dox agar until cultures are free of contamination.

Lesions located on the body or tail of fish less than 20 cm in length can be sampled by cutting the fish in two using a sterile scalpel, slicing a cross-section through the fish at the edge of the lesion. Flame the scalpel until red-hot and use this to sterilize 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 medium (3 g L^-1 glucose, 1g L^-1 peptone, 0.128 g L^-1 MgSO₄.7H₂O, 0.014 g L^-1 KH₂PO₄, 0.029 g L^-1 CaCl₂.2H₂O, 2.4 mg L^-1 FeCl₃.6H₂O, 1.8 mg L^-1 MnCl₂.4H₂O, 3.9 mg L^-1 CuSO₄.5H₂O, 0.4 mg L^-1 ZnSO₄.7H₂O) with 100 units mL^-1 penicillin-K and 10 mg mL^-1 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 L^-1 technical agar, 100 units mL^-1 penicillin-K and 10 mg mL^-1 streptomycin sulfate until pure cultures are obtained. They may be maintained at 10°C on GP agar and subcultured at intervals of no greater than 7 days.

The fungus can be identified to genus by inducing sporogenesis and demonstrating typical asexual characteristics of Aphanomyces as described in Lilley et al. (1998). A. invadans is characteristically slow growing in culture and fails to grow at 37°C on GPY agar (GP broth with 0.5 g L^-1 yeast extract and 12 g L^-1 technical agar). Confirmation that the isolate is A. invadans can be made by injecting a 0.1 ml suspension of 100+ motile spores intramuscularly in EUS susceptible fish at 20°C, and demonstrating histologically growth of aseptate hyphae 12-30 µm in diameter in muscle of fish sampled after 7 days, and typical mycotic granulomas in muscle of fish sample after 14 days.

Epidemiology

Cutaneous ulcerative disease, named epizootic ulcerative syndrome (EUS), has been a seasonal disease of freshwater and brackishwater fish in Asia, Australia and the United States of America since 1971. Earlier outbreaks of mycotic granulomatosis (MG) in Japan, red spot disease (RSD) in Australia and ulcerative mycosis (UM) in the United States of America are now known to be the same as EUS (Kiryu et al., 2003). EUS commonly occurs during periods of low temperature and after periods of heavy rainfall (Bondad-Reantaso et al., 1992). These conditions favour sporulation of the Aphanomyces pathogen (Lumanlan-Mayo et al., 1997) and low temperature has been shown to delay the inflammatory response of fish to fungal infection (Chinabut et al., 1995; Catap and Munday, 1998). It has been defined as "a seasonal epizootic condition of freshwater and estuarine warm water fish of complex infectious aetiology characterized by the presence of invasive Aphanomyces infection and necrotising ulcerative lesions typically leading to a granulomatous response" (Roberts et al., 1994). Recent studies suggest that a complex infectious aetiology is not necessarily involved in all cases of EUS (Callinan, 1997, Kiryu et al., 2003); and one species of invasive fungus, Aphanomyces invadans, is involved in the outbreaks. Region wide, over 100 species have been confirmed affected by histological diagnosis in Asia (Lilley et al., 1998), but some important cultured species, including Chinese carps, milkfish and tilapia, have been shown to be resistant.

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Distribution Maps