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7 Infectious Diseases of Warmwater Fish in Fresh Water

Gilda D. Lio-Po1 and L.H. Susan Lim2 1Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, 5021 Iloilo, Philippines; 2Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

Introduction (Hypophthalmichthys molitrix) together with Puntius gonionotus and Leptobarbus Cage culture of freshwater fish, which hoevenii dominate the cyprinids (Table 7.1). began in Cambodia in the late 1800s, is now Due to the variety of common names avail- commonly practised in Southeast Asia and able for a particular fish species in Southeast gaining popularity in India (Chapter 1). In Asia, the scientific names will be used as developing tropical countries, this type of much as possible. fish culture is still either at the subsistence Publications and reports are available or semi-intensive level or is at the experi- on diseases of feral and cultured fish in mental stage, as for Chrysichthys spp. in warm fresh water (Lio-Po, 1984; Kabata, Africa (Aqua Farm News, 1993). 1985; ADB/NACA, 1991; Lim 1991d, 1992; Fish cultured in cages in Southeast Asia Paperna, 1991, 1996; Arthur, 1992; Thune include tilapia, carp, catfish, snakeheads et al. 1993; Arthur and Lumalan-Mayo, and eleotridids (Table 7.1). The tilapias, one 1997; Fijan, 1999). However, there is a of the common species in freshwater cages, paucity of information on diseases of fish are also cultured in cages in warm marine in freshwater cage culture, even though waters (Chapter 6) (Aqua Farm News, 1993). cage culture began in Southeast Asia The catfish cultured include the Ictaluridae (Chapter 1) (Christensen, 1989; Aqua Farm (Ictalurus spp.), Claridae (Clarias spp.), News, 1993). Diseases are normally either Pangasiidae (Pangasius spp.), Siluridae mentioned in passing or are not included, (Silurus glanis) and Bagridae (Hemibagrus particularly in those publications dealing spp.) (Aqua Farm News, 1993). Most catfish with cage culture (Christensen, 1989; are of Southeast Asian origin, the exception ADB/NACA, 1991; Dharma et al., 1992; being channel catfish cultured in the Nasution et al., 1992; Alawi and Rusliadi, USA, which have been introduced into 1993; Aqua Farm News, 1993). In addition, cages in Indonesia (Rabegnatar et al., 1990). publications on diseases in fish culture The most common catfish species cultured do not distinguish between diseases in cages in Southeast Asia is Pangasius found in cage culture and pond culture hypophthalmus. Exotic Chinese carp, (Davy and Chouinard, 1982; Arthur, common carp (Cyprinus carpio), grass 1987; ADB/NACA, 1991; Aqua Farm News, carp (Ctenopharyngodon idellus), bighead 1993). This is further exacerbated by the carp (Aristichthys nobilis), silver carp lack of comprehensive investigation into

©CAB International 2002. Diseases and Disorders of Finfish in Cage Culture (eds P.T.K. Woo, D.W. Bruno and L.H.S. Lim) 231

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232 G.D. Lio-Po and L.H.S. Lim

Table 7.1. Freshwater fish species cultured in cages in some tropical countries. Country Fish species References

Bangladesh Catla catla Karim and Harun-al-Rashid Khan (1982) Cirrhina mrigala Cyprinus carpio Hypophthalmichthys molitrix Oreochromis niloticus Cambodia Channa micropeltes Thana (2000) Cirrhinus microlepis Labeo sp. Clarias sp. Guerrero (1979) Leptobarbus hoevenii Oxyeleotris sp. Pangasius sp. India C. catla Natarajan et al. (1983) C. mrigala Labeo bata Labeo rohita Channa striata Sukumaran and Sanjeeviraj (1983) Oreochromis mossambicus Jameson (1983) Indonesia C. striata Indra (1982) Oxyeleotris marmoratus Tilapia C. carpio Jangkaru and Rustami (1979) Malaysia Aristichthys nobilis Annual Fisheries Statistics (1998) C. striata Ctenopharyngodon idellus C. carpio Hemibagrus nemurus (also known as Mystus nemurus) H. molitrix L. hoevenii O. marmoratus Puntius gonionotus Tilapia Philippines A. nobilis Palisoc (1988) Chanos chanos C. carpio H. molitrix O. niloticus Sri Lanka O. niloticus Siriwardena (1982) Thailand Clarias spp. Tugsin (1982) C. carpio Goby sp. O. niloticus Vietnam Barbus spp. Pantulu (1979) Leptobarbus sp. C. striata T.T. Dung (personal communication) O. marmoratus Pangasius bocourti Pangasius conchophilus

diseases encountered in cage culture sys- Disease outbreaks in cage culture have a tems in warm waters. greater impact because of high stocking

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Infectious Diseases of Warmwater Fish in Fresh Water 233

densities and close proximity of cultured affected. Most viral infections occur in fish with each other as well as with feral fish. fish at low water temperatures. This may For example, there were 64 reported disease explain the paucity of viral infections outbreaks in cage-cultured channel catfish recorded in warm freshwater fish. Stress in the USA during 1990 with mortality from handling, poor water quality, water in 91% of these cases (Masser et al., temperature, age of fish, high stocking 1991). Also, diseases appear to occur more density and poor nutrition are factors frequently in cages than in ponds (Collins, that facilitate the development of viral 1988). Cage culture exposes fish to diseases. pathogens of feral fish and perhaps to a Among viral infections in fish, the greater number of intermediate hosts in channel catfish virus disease has the most parasitic diseases. Fish reared in cages may impact on cage culture while the grass carp also present a potential health threat to haemorrhagic virus and the spinning tilapia man, especially when they are reared in syndrome are also potential viral problems. unsanitary waters in areas where fish-borne In addition, other viral epizootics have been zoonotic diseases are prevalent (see reported in common carp and tilapia that Ko, 1995) or when located in polluted may have implications in fresh warmwater areas. Diseases afflicting pond-reared and cage culture systems (Sano et al., 1993; cage-cultured fish are in most cases similar, Oyamatsu et al., 1997; Fijan, 1999). hence those that are important in pond The epizootic ulcerative syndrome (EUS), aquaculture will be treated as potential a disease associated with a rhabdovirus, problems for the cage culture. For example, bacteria and the pseudofungi, Aphanomyces Piscinoodinium pillulare, the causative invadans, is discussed in the section on agent of velvet disease of cyprinids, was first Diseases of Complex Infectious Aetiology. reported on pond-reared fish but is now An insufficient number of susceptible found on cage-cultured fish (F. Shaharom, fish cell lines hampers isolation and diagno- personal communication). The paucity of sis of viral pathogens. Cell lines currently information on diseases in cage-cultured used for isolation of warm freshwater fish is partly due to the lack of studies on virus are from: bluegill fry (BF-2) (Wolf identification of pathogens/disease mecha- and Quimby, 1966), brown bullhead (BB), nisms and/or the absence of mandatory channel catfish ovary (CCO) (Bowser and reports on disease outbreaks in many Plumb, 1980), Epithelioma papulosum countries. Hence, we expect diseases to cyprini (EPC) (Fijan et al., 1983), grass carp become more prevalent in the future as we kidney (GCK-84), grass carp gonad (GCG), move into more intensive fish culture, grass carp fin (GCF) (Wolf, 1988), rainbow find out more about infectious agents, and trout gonad (RTG-2), snakehead fry (SSN-1) adopt a system where it is mandatory to (Frerichs et al., 1993), catfish spleen (CFS) report disease outbreaks. In the current and snakehead spleen (SHS) (Lio-Po et al., review, we have also included unpublished 1999). information from colleagues as well as Electron microscopy for the diagnosis from personal observations, and wherever of viral infections is not commonly used possible we have provided the correct iden- due to inaccessibility to this equipment in tification of pathogens and supplementary most tropical countries. As an alternative, information on them. serological tests are applied such as neutral- ization index determination, Western blot, ELISA, fluorescent antibody technique Viral Infections (FAT) and indirect fluorescent antibody test (IFAT). Recent molecular biology Viral infections can cause mass mortality, techniques such as PCR, RT–PCR and especially in fry or fingerlings, while older gene probes are becoming popular for the fish may develop resistance or are hardly diagnosis of fish viral infections.

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234 G.D. Lio-Po and L.H.S. Lim

Channel catfish virus disease (CCVD) and in the spleen. Thereafter, the virus is transported via the blood to the intestine, Channel catfish (lctalurus punctatus) is the liver, heart and brain (Plumb and Gaines, principal host of channel catfish virus 1975). Thus, hyperaemia of the visceral (CCV). Outbreaks occur in most southern cavity, enlarged spleen, and empty stomach states in the USA, while low-grade mortal- and intestine have been observed (Plumb, ity can be induced in blue catfish (Ictalurus 1994). Necrosis of the renal haematopoietic furcatus) and channel catfish × blue catfish tissue and tubules, oedema, necrosis and hybrids by experimental injection (Plumb congestion of the liver, intestinal oedema et al., 1975). and congestion and haemorrhage in the spleen are characteristic histopathological Pathology. CCV causes acute infection in findings. Skeletal muscle haemorrhage is cultured channel catfish fry and fingerlings seen in experimentally infected fish. The less than 10 cm in length. It can also infect virus can be isolated from the kidney, intes- channel catfish juveniles and adults follow- tine, liver, spleen, brain and muscle tissues ing waterborne exposure to CCV (Plumb, (Plumb, 1971; Plumb and Gaines, 1975). 1971; Hedrick et al., 1987). Clinical signs The portal of entry for CCV from water is are abdominal distension, exophthalmia, through the gills and the gut (Nusbaum and pale or haemorrhagic gills and petechial Grizzle, 1987). Channel catfish surviving haemorrhage at the base of the fins and a CCV infection grow slowly; e.g. experi- throughout the skin (Fig. 7.1). Infected fish mentally induced CCVD survivors ranged swim erratically at the surface in head-high from 11 to 15 g compared with 73–93 g in or hanging position. Mortality approaching unexposed channel catfish 6 months after a 100% in channel catfish younger than standardized feeding regime (McGlamery 4 months old occurs at water temperatures and Gratzek, 1974). above 25°C within 7–10 days. The virus does The virus remains viable in dead − ° not induce mortality below 15°C. Secondary fish kept on ice for 14 days and at 20 C external lesions caused by bacteria, e.g. for 100 days (Plumb et al., 1973). It Flavobacterium columnare or Aeromonas remains infective for 2 days in pond water ° hydrophila, or by aquatic stramenopiles may at 25 C and for 11 days in dechlorinated develop. tap water. However, it is rapidly inacti- CCVD develops into a haemorrhagic vated in pond mud and by drying (Plumb, viraemia after replicating in the kidney 1994).

Fig. 7.1. Channel catfish (Ictalurus punctatus) infected with the channel catfish virus (CCV) (courtesy of Dr John Plumb).

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Infectious Diseases of Warmwater Fish in Fresh Water 235

Transmission of CCV occurs both Grass carp haemorrhagic disease (GCHD) horizontally and vertically. The virus is readily transmitted from fish to fish. The The disease was first observed in China exact mode of transmission is unknown but in the 1980s. It commonly affects grass is most likely through the branchial and carp but can also infect black carp intestinal epithelium. Upon intraperitoneal (Mylopharyngodon piceus), topmouth injection, the virus is detected in the kidney gudgeon (Pseudorasbora parva) and rare after 24–48 h, in the intestine and liver after minnow (Gobiocypris rarus). It can also 72–96 h and in the brain after 96–120 h replicate in silver carp and in Chinese post-injection (Plumb, 1971). In experi- minnow (Hemiculter bleekeri) without any mental infections, fry die within 3 days clinical signs. Outbreaks occur in Southern of exposure (Wolf, 1988). The virus also China during the summer when water occasionally persists in apparently healthy temperatures range from 24 to 30°C (Nie adult channel catfish broodfish but in most and Pan, 1985; Wolf, 1988; Jiang, 1995; cases CCV cannot be isolated from adult Fijan, 1999). fish, and has been isolated from fingerlings in only two of seven farms with positive Pathology. Acute infections cause sig- broodfish (Bowser et al., 1985). nificant mortality of more that 80% in fingerlings and up to 70% in yearlings. Diagnosis. CCV, designated as Herpesvirus Clinical signs include exophthalmia and ictaluri, is a herpesvirus of the family severe haemorrhage of the gills and fin Herpesviridae (Wolf and Darlington, 1971). bases. Internally, haemorrhage occurs in the It is enveloped, with icosahedral symmetry musculature, oral cavity, intestinal tract, and measures 90–100 nm in diameter. liver, spleen and kidney. Naturally and The virus can be isolated from the kidney experimentally infected fish have reduced of fish with active infections using CCO erythrocytes, plasma protein, calcium and or BB cells. Inoculated cells develop urea nitrogen but serum potassium is cytopathic effects (CPE) 24–48 h post- elevated. Experimental infection by bath exposure, with optimal viral replication at and by injection induced typical signs ° 25–30 C. Identification is confirmed using of the infection. Disease and mortality are electron microscopy, serum neutralization observed within 1–2 weeks exposure of fish tests, IFAT, ELISA using monoclonal anti- in water at temperatures of 25°C or higher bodies, CCV DNA probes and PCR (Wise (Wolf, 1988; Fijan, 1999). et al., 1985; Office International des Epizootie (OIE), 1995; Baek and Boyle, Diagnosis. The grass carp haemorrhagic 1996). virus (GCHV) is a non-enveloped, doubly encapsidated icosahedron with 5:3:2 sym- Prevention and control. Detection of CCV metry, 92 capsomeres, with an overall in catfish broodstock will help prevent diameter of 60–80 nm and a 40 nm inner its spread to young catfish. The use of capsid (Wolf, 1988). It is resistant to ether virus-free stock is the best preventive and chloroform. It is presently classified method. Alternatively, the use of resistant under the genus Aquareovirus (Family fish stocks or hybrids of channel catfish Reoviridae) (Li et al., 1997). The virus can is recommended. Quarantine and killing of be propagated in GCK-84, GCG and GCF 8 9 −1 CCV-infected stock including surveillance cells yielding titres of 10 –10 TCID50 ml . for feral fish carriers should be practised. In vitro viral replication is optimum at This should be of utmost consideration 28–30°C inducing CPE in 3–4 days before introduction of channel catfish into post-inoculation (Wolf, 1988). The virus in tropical countries. Vaccination is still at fish with clinical signs and in carrier fish the experimental stage and there is no can be confirmed using RT–PCR (Li et al., chemotherapy. 1997).

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236 G.D. Lio-Po and L.H.S. Lim

Prevention and control. Experimental vac- and restriction of transfer of stocks cination using inactivated virus induced from endemic to non-endemic areas is 80% protection by day 4 at temperatures recommended. above 20°C, by day 20 at 15°C, and by day 30 at 10°C, and this protection lasts for up to 14 months (Wolf, 1988). Zhu et al. (1993) Bacterial Diseases as cited by Fijan (1999) reported that the ‘Kelieao–Yufukang’, a combination of two High stocking density of fish leads to drugs, has in vitro and in vivo anti-GCHV increased feed rations and waste. This activity. also results in bacterial problems with con- comitant increases in ammonia and nitrite toxicity (Mitchell, 1997). Stress and trauma Spinning tilapia (ST) syndrome from handling are also predisposing factors. Most bacterial pathogens produce enzymes This virus was recently detected in Moz- that facilitate their entry/invasion into the ambique tilapia, blue tilapia (Oreochromis fish host tissues. Although they may cause aureus), Nile tilapia (Oreochromis nilo- primary infection, they may also act as ticus), and mango tilapia (Sarotherodon secondary disease agents to a primary virus galilaeus) in Australia. The disease is or parasite. The major bacterial infections caused by an iridovirus (Ariel and Owens, among warm freshwater fish are motile 1997). Aeromonas septicaemia, Pseudomonas sep- ticaemia, edwardsiellosis, enteric septicae- Pathology. Affected tilapia fry swim in a mia, columnaris disease and streptococcal spiral pattern, sink to the bottom then rise septicaemia/meningoencephalitis. and hang at a 45° angle just under the water surface, gasping for air. They do not feed, are darker in colour and exhibit ‘fin clamping’. Motile Aeromonas septicaemia (MAS) Tilapia fry manifesting the spinning syn- drome die within 24 h and a 100% mortality This disease was formerly known as haem- often occurs within 60 days. Naive tilapia orrhagic septicaemia, infectious dropsy, fry experimentally exposed to diseased fry infectious abdominal dropsy, red pest, red via cannibalism developed similar signs disease, red sore or rubella. The syndrome after 12 days. Histopathologically, the renal is caused by the motile A. hydrophila tubules are shrunken, haemorrhaging and (previously named Aeromonas punctata infiltrated with eosinophilic granular cells. or Aeromonas liquefaciens). Aeromonas In addition, focal myolysis occurs in sobria and Aeromonas caviae are rarely muscles. These histopathological lesions associated with fish epizooties. and the size range (110–140 nm) of the virus MAS affects freshwater and occasion- are similar to those caused by the Bohle ally brackishwater and marine warmwater iridovirus (BIV) in tilapia fingerlings (Ariel fish worldwide. It is the most frequently and Owens, 1997). The Bohle iridovirus also diagnosed bacterial fish disease and was the infects amphibians (Cullen et al., 1995). most severe disease problem among cage- cultured channel catfish in the USA between Diagnosis. So far, the virus has not been 1972 and 1980 (Plumb, 1994). Subsequently, isolated in cell culture from diseased tilapia it became the third most common bacterial but the disease is usually diagnosed based infection (1987–1991) among cage-cultured on clinical signs and is confirmed by channel catfish in the USA, accounting for electron microscopy. 13–22% of disease outbreaks (Duarte et al., 1993). The infection occurs mostly from Feb- Prevention and control. No treatment is ruary to July, with some outbreaks occurring available but prevention through quarantine in September and November. In the tropics,

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Infectious Diseases of Warmwater Fish in Fresh Water 237

MAS infections are often reported in pond- natural infection in tilapia caused by and pen-cultured milkfish (Chanos chanos), A. hydrophila was associated with epider- common carp, grass carp, Nile tilapia and mal lesions so severe that the vertebrae giant gourami (Osphronemus goramy) (Kou, were exposed (Lightner et al., 1988). This 1972; Ruangpan et al., 1985; Karunasagar severe condition is not uncommon among et al., 1986; Lio-Po et al., 1986; Saitanu et al., EUS-affected fish (Roberts et al., 1994b). 1986; Supriyadi, 1986; Areerat, 1987; Angka Hence, it is not surprising that the bacterium et al., 1988; Okaeme et al., 1989; Yambot, has been consistently isolated from EUS- 1997). Moreover, A. hydrophila has been affected fish (Llobrera and Gacutan, associated with epizootic ulcerative syn- 1987; Boonyaratpalin, 1989; Costa and drome (EUS)-affected striped snakeheads Wejeyaratne, 1989; Subasinghe et al., 1990; (Channa striata, also known as Torres, 1990; Lio-Po et al., 1992; Pathiratne Ophicephalus striatus) and walking catfish et al., 1994; Angka et al., 1995; Karunasagar (Clarias batrachus) in the wild, as well as et al., 1995; Thanpuran et al., 1995; Rahman in ponds and cages (Llobrera and Gacutan, et al., 1999). 1987; Boonyaratpalin, 1989; Subasinghe In channel catfish, A. hydrophila et al., 1990; Torres, 1990; Lio-Po et al., 1992; infection has three categories: (i) motile Pathiratne et al., 1994; Angka et al., 1995; aeromonad septicaemia with external signs; Karunasagar et al., 1995; Thanpuran et al., (ii) cutaneous, manifesting lesions that are 1995; Rahman et al., 1999). limited to the skin and underlying muscle; and (iii) latent septicaemia with no external Pathology. A. hydrophila is a free-living, signs (Grizzle and Kiryu, 1993). Internal mesophilic bacterium found in soil, fresh- clinical signs include oedema, haemorrhage water lakes, ponds, streams, bottom mud, and necrosis. The disease is acute in very domestic tap water and sewage. It is often young fish while adults generally develop associated with the normal flora of fish. chronic infections (Plumb, 1994). Thus, the bacterium has been isolated from Motile aeromonad infections are predis- both healthy and diseased fish (Lio-Po and posed by stress from temperature shock, low Duremdez-Fernandez, 1986; Lio-Po et al., dissolved oxygen, high ammonia, handling 1986, 1992; Torres, 1990). It causes infec- or hauling, and an ongoing primary infection tions not only in aquatic animals but also in (Plumb et al., 1978; Lio-Po et al., 1986). avian hosts, cows and humans. These predisposing conditions are possibly Infected fish lose their appetite, become immunodepressive, and the virulence of lethargic and swim near the surface. Exter- the Aeromonas strain is an important nal signs may vary according to fish species factor in the development of MAS epizootics but are generally similar to clinical signs (Thune et al., 1993). Moreover, A. of other bacterial septicaemia infections hydrophila is often reported in mixed in fish, i.e. exophthalmia and distended infections with Edwardsiella tarda, E. abdomen. However, septicaemia in acute ictaluri, Flavobacterium columnare, Strep- MAS can be fatal with no clinical signs. tococcus spp. or with parasites (Kanai et al., Among milkfish reared in pens in a fresh- 1977; Liu et al., 1990; Duarte et al., 1993). In water lake in the Philippines, acute signs of carp dropsy, A. hydrophila was a compo- petechial haemorrhage of the skin and fin nent in the pathology of the disease, which is bases including dermal and caudal fin rot attributed to a virus as prime aetiological were observed (Lio-Po et al., 1986). Yambot agent (Roberts, 1993). (1997) also isolated A. hydrophila from Experimental A. hydrophila infections cage-cultured tilapia in the Philippines with may be induced in milkfish with up to haemorrhagic skin, ulceration, loss of scales, 100% mortality in 2 days after immersion mouth sores, eye abnormalities, fungal exposure of scarified fingerlings, but not in growth and/or tail and fin rot. Fingerlings fish with intact skin (Lio-Po and Duremdez- to adult Nile tilapia can be infected by Fernandez, 1986). In addition, intraperi- A. hydrophila (Yambot, 1997). A case of toneal injection with the bacterium causes

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238 G.D. Lio-Po and L.H.S. Lim

mortalities within 12 h of injection of the intraperitoneally with A. hydrophila dev- pathogen (Lio-Po and Duremdez-Fernandez, eloped focal necrosis in the liver, kidney, 1986). In walking catfish and snakeheads, intestine and dorsal musculature (Angka, A. hydrophila induced dermal lesions after 1990). The infection elicits an intense intramuscular injection of at least 105 cells inflammatory response, with massive per fish, which eventually ulcerated (Fig. infiltration of monocytic and granulocytic 7.2) (Lio-Po et al., 1992). Compared with cells into infected tissues (Huizinga et al., other bacteria associated with EUS lesions, 1979; Ventura and Grizzle, 1988). Infected such as Pseudomonas sp., Aquaspirillum sp. goldfish are anaemic, e.g. low red blood and Streptococcus sp., A. hydrophila cell, haematocrit and haemoglobin counts induced the most severe lesions upon intra- (Brenden and Huizinga, 1986). In addition, muscular injection of snakeheads (Lio-Po there is a shift in the differential counts et al., 1998). Experimental infection of Nile of lymphocytes to a predominance of tilapia fingerlings by immersion yielded an neutrophils. × 6 LD50 of 1.5 10 colony-forming units (cfu) Motile aeromonads secrete extracellular − − ml 1 with 100% mortality at 108 cfu ml 1 and products (ECPs) and these include toxins, − no mortality at 103 cfu ml 1 (Yambot, 1997). protease, cytotoxin, haemolysin, leuco- Attempts to induce external gross lesions in cidin, gelatinase, elastase, staphylolysin, walking catfish by dermal cut, dermal scrap- caseinase, enterotoxin and a dermonecrotic ing, fish bite, oral feeding, gastric lavage factor (Hsu et al., 1981; Olivier et al., 1981; and cohabitation with a golden snail carrier Kanai and Wakabayashi, 1984; Lallier et al., (Ampullarius sp.) were unsuccessful (Lio-Po 1984; Krovacek, 1989; Yadav et al., 1992). et al., 1996). In contrast, in channel Moreover, cytotoxin-producing strains were catfish with mechanically abraded skin, associated with EUS-affected fish (Yadav A. hydrophila experimentally induced sys- et al., 1992). Dermonecrotic strains of this temic infection in 80% of exposed fish while bacterium secrete haemolysin at 10 and 30°C cutaneous lesions developed in the remain- (Olivier et al., 1981). However, correlation ing fish (Matsche and Grizzle, 1999). between virulence and ECP production was Histopathologically, marked necrosis of not consistent (Leaño et al., 1996). Recently, the muscle fibrils occurred in snakeheads Cascon et al. (2000) described the molecular intramuscularly injected with A. hydrophila characteristic of an elastase secreted by (Lio-Po, 1998). Walking catfish injected A. hydrophila that is important in its

Fig. 7.2. Catfish (Clarias batrachus) showing ulcerative lesions 4 days post-intramuscular injection with Aeromonas hydrophila.

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Infectious Diseases of Warmwater Fish in Fresh Water 239

pathogenicity. This protease has a high and Carnahan (1994) have further classified amino acid sequence similarity to proteases these into seven species (Table 7.2). Mol- secreted by Pseudomonas aeruginosa, ecular identification can be applied by Helicobacter pylori and Vibrio spp. An ribotyping of restriction genomic DNAs of investigation of 12 strains of A. hydrophila aeromonads using different fragments of the isolated from fish showed that protease pro- 16S rDNA gene of Escherichia coli as a probe duction varied among strains, with peak pro- (Lucchini and Altwegg, 1992). Also, ampli- tease production optimum at 27.6 ± 4.9°C. fied fragment length polymorphism (AFLP) (Uddin et al., 1997). The protease levels as a high-resolution genotype tool for classi- increased during the late log phase to early fication of Aeromonas spp. and pulse-field stationary phase. gel electrophoresis as a rapid technique for Siderophore production is also descri- typing of A. hydrophila have been devel- bed but this is not related to virulence of the oped (Huys et al., 1996; Talon et al., 1996). bacterium (Santos et al., 1988; Leaño et al., Igbal et al. (1998) furthur recommend the 1995). Virulence of A. hydrophila varies, application of genetic identification using even among isolates from the same epizootic DNA–DNA hybridization. (Lio-Po et al., 1992). Also, Rahman et al. Both virulent and non-virulent strains (1997) reported that A. hydrophila stored in have been isolated from diseased fish 0.60 and 0.85% NaCl solutions had higher (Torres, 1990; Lio-Po et al., 1992; Leaño virulence than the cultured bacterium when et al., 1996). Definitive identification of injected intraperitoneally into carp and either strain is a major difficulty and has goldfish. Subsequent studies showed that been the subject of a number of research a significantly higher number of starved A. efforts. Cartwright et al. (1994) developed hydrophila adhered to the skin of crucian monoclonal antibodies for detection of viru- carp (Carassius carassius) than the cultured lent strains using either ELISA or fluorescein bacterium (Rahman and Kawai, 1999). isothiocyanate (FITC) immunofluorescence. Attachment of A. hydrophila to carp epithe- Virulent strains of A. hydrophila require lial cells is attributed to a 43 kDa outer about 30 min to induce CPE in EPC cells, membrane protein adhesin because of an while avirulent strains do not induce this abundance of this particular receptor on the pathological effect (Leung et al., 1996). A cell surface (Lee et al., 1997). Dooley et al. PCR method that is reported to be rapid, (1986) earlier described a crystalline surface sensitive and specific for the detection of layer, or S-layer, of 52 kDa protein, and virulence factors of Aeromonas spp. has further correlated this to strain virulence been developed (Bin Kingombe et al., 1999). (Murray et al., 1988; Ford and Thune, 1991). Recently, identification of the genetic differ- ences and virulence genes among different Diagnosis. Motile Aeromonas spp. are flag- strains of A. hydrophila using a suppression ellated, Gram-negative, short rods. They subtractive hybridization (SSH) technique do not produce pigments and are resistant was reportedly successful (Zhang et al., to vibriostat 0/129 (2,4-diamino-6,7- 2000). diisopropylpteridine phosphate). The bacte- ria grow at a temperature range of 18–39°C Prevention and control. MAS outbreaks are (Uddin et al., 1997). In tryptic soy agar (TSA) common in eutrophic lakes and ponds. or in brain heart infusion agar (BHIA) at Outbreaks of A. hydrophila infections in 25–30°C incubation for 24–48 h, Aeromonas fish-pen-reared milkfish are usually related spp. produce white to creamy, convex, moist to transport and handling stress, adverse colonies. In Rimler–Shotts medium, the environmental conditions of low oxygen bacteria form orange–yellow colonies at concentration, low pH, and increased levels 35°C (Shotts and Rimler, 1973). The three of ammonia and carbon dioxide (Walters important Aeromonas spp. in fish can and Plumb, 1980; Lio-Po, 1984; Lio-Po et al., be differentiated using biochemical tests 1986). Moreover, tilapia fingerlings during (Lio-Po et al., 1992; Plumb, 1994). Joseph seining can get caught between nets and

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240 G.D. Lio-Po and L.H.S. Lim

Table 7.2. Comparison of distinguishing profiles of mesophilic clinical Aeromonas species (reprinted from Annual Review of Fish Diseases, Vol. 4, Joseph and Carnahan, 1994, with permission from Elsevier Science). Resulta for:

A. veronii A. veronii A. hydrophila bv. sobria bv. veronii A. caviae A. schubertii A. jandaei A. trota Characteristic (n = 46) (n = 26) (n = 8) (n = 33) (n = 6) (n = 9) (n = 13)

Esculin hydrolysis + – + + – – – Voges–Proskauer + + + – V + – reaction Pyrazinamidase + – – + – – – activity cAMP-like factor + + + – – V – (aerobic only) Fermentation Arabinose V – – + – – – Mannitol + + + + – + + Sucrose + + + + – – – Susceptibility Ampicillin R R R R R R S Carbenicillin R R R R R R S Cephalothin R S S R S R R Colistinb V S S S S R S Decarboxylase Lysine + + + – + + + Ornithine – – + – – – – Arbutin hydrolysis + – + + – – V Indole + + + + – + + c H2S + + + – – + + Glucose (gas) + + + – – + + Haemolysis (TSA + + + V + + V with 5% sheep erythrocytes)

a+, positive for > 70% of isolates; –, negative, i.e. positive for < 30% of isolates; V, variable; R, resistant; S, susceptible. bMIC (single dilution), 4 µg ml−1. c H2S from GCF medium.

subsequently develop MAS causing more (Clarias macrocephalus) is more effective than 25% mortality after stocking in cages than immersion or oral administration (J.A. Plumb, personal communication). (Areechon et al., 1992). Biofilm vaccine − Therefore, prevention of these stressful at 1013 cfu g 1 of A. hydrophila in catla conditions will minimize MAS outbreaks. (Catla catla), rohu (Labeo rohita) and com- Limited success has been achieved mon carp for 15–20 days elicited high serum with vaccination against MAS in milkfish antibody titre and protective response (G.D. Lio-Po, unpublished data) but vaccina- for 60 days (Azad et al., 1999). Blue tion is protective in tilapia (Ruangpan et al., gourami (Trichogaster trichopterus), when 1985). Indian major carp vaccinated with A. intraperitoneally immunized with major hydrophila yielded increased agglutinating adhesin (43 kDa) in Freund’s complete antibody titre (Karunasagar et al., 1991). adjuvant, developed protective immunity to Vaccination by intraperitoneal injection challenge by homologous and heterologous of formalin-killed A. hydrophila to catfish strains of A. hydrophila and one virulent

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Infectious Diseases of Warmwater Fish in Fresh Water 241

strain of Vibrio anguillarum (Fang et al., associated with skin lesions. Histopatho- 2000). Vaccination studies in carp immu- logical findings in Nile tilapia include focal nized with crude lipopolysaccharide necrosis, abscess and granulomas in the showed that the mechanism of immunity eyes, gills, liver, swim-bladder, kidney is attributed to a sensitized thymocyte– and spleen (Miyashita et al., 1984). The bac- macrophage system (Baba et al., 1988a,b). terium also causes mortalities in 2-week- Recently, immersion vaccination of old Nile tilapia fry (Lio-Po and Sanvictores, carp with A. hydrophila bacterins showed a 1987). distinct increase of lysozyme level in fish P. fluorescens is part of the normal flora mucus, with stronger bacteriolytic proper- of tilapia gut (Sugita and Kadota, 1980). ties 7 and 28 days after immunization It remains viable in fresh water for up to (Kozinska, 2000). Moreover, a single intra- 150 days (Duremdez and Lio-Po, 1984) and peritoneal injection of 20 mg β(1,3)-D-glucan secretes an extracellular proteinase (Li and − kg 1 into blue gourami enhanced the Fleming, 1967). immune response against A. hydrophila for up to 29 days (Samuel et al., 1996). Subse- Diagnosis. As clinical signs of Pseudo- quent studies showed that the use of four monas septicaemia resemble those of MAS, glycans, namely Bar (glycan extracted from isolation and identification of the bacterial barley), krestin, scleroglucan and zymosan, pathogen is required. P. fluorescens is a significantly increased survival rates of Gram-negative rod with one to three polar tilapia and grass carp after infection with flagella. It grows on nutrient agar, Pseudo- A. hydrophila (Wang and Wang, 1997). monas F agar and blood agar (Austin and Prophylactic bath treatments with Austin, 1987). For strains pathogenic to 1–3% NaCl will help reduce post-handling fish, the optimum growth temperature is infections. Likewise, bath treatments with ° − 20–25 C. These secrete oxidase, catalase and 2–4 mg potassium permanganate l 1 are also gelatinase but not amylase, galactosidase, effective for external lesions. Medicated − urease or hydrogen sulphide. It is citrate- feed with 2–4 g oxytetracycline kg 1 feed − positive, oxidative for glucose and produces (50–100 mg kg 1 fish) for 14 days is a fluorescent pigment (Plumb, 1994). recommended (Plumb, 1994). However, drug-resistant strains of A. hydrophila may Prevention and control. Stress from low evolve (Aoki, 1999). dissolved oxygen concentrations, high stocking density, physical trauma and poor nutrition are predisposing factors in the Pseudomonas septicaemia development of Pseudomonas septicaemia (Post, 1983). Therefore, avoidance of these Pseudomonas spp. are ubiquitous in water conditions is necessary in the prevention and are opportunistic pathogens. In fresh- of its outbreak. Suggested bath treatments water culture systems, Pseudomonas during the early stage of the disease include − fluorescens has been implicated in 1–2 mg benzalkonium chloride l 1 for 1 h, − epizootic outbreaks in Nile tilapia, grass 0.5–1 mg furanace l 1 for 5–10 min or − carp, silver carp and bighead carp (A. 1–5 mg malachite green l 1 for 1 h (Austin nobilis) (Miyashita, 1984; Lio-Po and and Austin, 1987). Sanvictores, 1987; Thune et al., 1993).

Pathology. The clinical signs in fish Edwardsiellosis and enteric septicaemia affected with Pseudomonas septicaemia are very similar to those with MAS. Gross signs E. tarda is synonymous to Paracolobactrum include ascites, exophthalmia, septicaemia anguillimortiferum and to the E. anguilli- and ulcers. The infection may be acute mortiferum described by Wakabayashi or chronic, with the latter commonly and Egusa (1973) and Kuo et al. (1987),

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242 G.D. Lio-Po and L.H.S. Lim

respectively. Edwardsiellosis has been occurs in Asia, the USA and Europe, reported in 25 countries worldwide (Austin affecting warmwater fish like channel and Austin, 1987). The disease affects eels catfish and other ictalurids, cultured eels, (Wakabayashi and Egusa, 1973), channel common carp and tilapia (Plumb, 1994). catfish (Meyer and Bullock, 1973), mullet The pathogen is Flavobacterium colum- (Kusuda et al., 1976), tilapia (Lio-Po et al., nare, formerly called Flexibacter colum- 1982), carp (Sae-Oui et al., 1984) and naris, Cytophaga columnaris, Chondrococ- striped bass (Herman and Bullock, 1986). cus columnaris and Bacillus columnaris. Although, there are no reports of cage- cultured fish affected by edwardsiellosis, it Pathology. Infection primarily begins at the poses a health threat. E. tarda can also cause mouth, fins and gills. Clinical signs include serious infections in humans. frayed fins with greyish to white margins, Enteric septicaemia is attributed to depigmented, necrotic skin lesions with E. ictaluri in freshwater fish. This is a major yellowish or pale margins, which can pathogen of cage-reared channel catfish develop into shallow ulcers, yellowish and accounts for about 30% losses in the mucoid material at the mouth and light to southeastern USA. The estimated annual dark brown gill discoloration. Gill lesions loss attributed to this pathogen is US$20–30 initiate at the distal end of the filaments, million (Plumb and Vinitnantharat, 1993). which extend to the base. Epithelial The majority of cases occur in May and June vacuolation, necrosis, congestion, oedema, and again in September and October when fusion and degeneration of the secondary water temperatures are between 22 and 28°C lamellae subsequently follow. Acute mortal- (Plumb and Schwedler, 1982). A morbidity ity is usually associated with gill lesions. rate as high as 68% was observed in May Internal pathology or host inflammatory 1987, and an estimated 10–32% yearly response may occur, and the pathogen morbidity rate among the primary diagnostic may be isolated from internal tissues cases of cage-cultured and pond-cultured (Thune et al., 1993; Plumb, 1994; Shotts and channel catfish in 1987–1991 (Duarte et al., Starliper, 1999). 1993). Farkas and Olah (1986) described the E. ictaluri has been reported in three stages of gill necrosis. The first stage is Thailand, the USA and Australia. It is initiated and maintained by environmental pathogenic to channel catfish but only stress (probably ammonia, pH, temperature very slightly pathogenic to blue catfish. or any toxins in the rearing water) but White catfish (Ictalurus melas) and brown F. columnare is seldom detectable on gills bullhead (Ictalurus nebulosus) are occasion- that are pale or dark purple. The second ally infected, while natural infections of stage consists of bacterial invasion of the walking catfish have been reported in damaged gill at water temperatures above Thailand (Plumb, 1994). Information on 20°C, causing gill necrosis, resulting in the pathology, epizootiology, diagnosis, a grey-white coating of the gills. In the prevention and control of Edwardsiella is third stage, the white coating of the gills detailed in Chapter 4 and in Plumb (1999). disappears and the infected gills become distorted. Different stages of gill necrosis may be observed in the same fish Columnaris disease population. Transmission of the bacterium is Columnaris disease is an acute to chronic via water. The disease is most commonly infection of freshwater fish and a common associated with stress from high tempera- bacterial infection in the southeastern tures, elevated organic loads, high stocking USA (Duarte et al., 1993; Mitchell, 1997). density, low dissolved oxygen and trauma Outbreaks are from March to September from excessive handling. In channel catfish, with peaks in June, and usually follow it occurs more often at temperatures between outbreaks of other diseases. The disease 25 and 32°C with significant mortality.

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Infectious Diseases of Warmwater Fish in Fresh Water 243

Young fish are more susceptible than older Diagnosis. F. columnare is a slender, fish. It may occur as a primary infection or as Gram-negative, non-flagellated rod (about a mixed infection with another bacterium, 0.5 × 4–12 µm) with gliding motility and E. ictaluri or A. hydrophila, or in association forms ‘hay stacks’ or columns. Primary with a parasite, e.g. Henneguya sp. or Ichthy- isolation of the pathogen can be achieved obodo sp. (Hawke and Thune, 1992; Duarte on selective Cytophaga agar supplemented − et al., 1993; Plumb, 1994). Columnaris dis- with 5 µg neomycin ml 1 and 200 IU − ease appears to follow outbreaks of other polymyxin B ml 1 (Hawke and Thune, diseases (Duarte et al., 1993). 1992). F. columnare colonies are yellow to Survivors of columnaris disease release orange and rhizoid. This aerobic organism the pathogen into the water at rates of up cannot tolerate more than 0.5% NaCl and − − to 5 × 103 cells ml 1 h 1 (Fujihara and it grows between 4 and 36°C, producing Nakatani, 1971), and surviving fish may gelatinase, caseinase, catalase, oxidase release the bacterium for up to 140 days and chondroitin sulphatase (Song et al., post-infection. The severity of lesion 1988). depends on the virulence of the strain Diagnosis of the disease is dependent on and the ability of the pathogen to produce the appearance of typical lesions on the skin, proteolytic enzymes. F. columnare produces fins and gills, including the detection of the an extracellular chondroitin AC lyase that filamentous bacterial cells in wet mounts degrades chondroitin and hyaluronic acid made from lesions. Based on the gene in fish connective tissue (Griffin, 1991). sequence of the 16S rRNA of the bacteria, Bertolini and Rohovec (1992) also reported Bader and Shotts (1998) designed primers four extracellular proteases with molecular for its detection using PCR. weights of 32, 34, 40 and 47 kDa. Newton et al. (1997) further observed that more Prevention and control. Disease prevention protease is secreted into a medium with is by maintenance of fish under optimal low nutrients and salt (Ordal’s medium) environment conditions, proper handling than into media with high concentrations of fish, prophylactic treatment and good of nutrients or salt (TYES, Hsu–Shotts, health management practices (Plumb, modified Shieh’s media). 1994). Daily oral vaccination with heat- Gills or dermal/muscular capillaries killed F. columnare for 4 weeks reportedly of infected fish become congested and reduced mortality of rainbow trout from degenerate (Plumb, 1994). Kuo et al. (1981) 48 to 8%, with protection correlated with showed that survival of fish given − antibody levels (Fujihara and Nakatani, 0.35–1.4 mg iron 100 g 1 fish prior to 1971). Moore et al. (1990), however, showed challenge with the pathogen was reduced that immunization of channel catfish from 3 days to 1 day. Furthermore, highly using formalin-inactivated F. columnare virulent strains of F. columnare adhered bacterin by immersion yielded inconsistent more readily to the gills than low virulence results. − strains, and were enhanced in ion-rich Potassium permanganate at 5 mg l 1 water, in the presence of nitrite or organic (depending on the organic load of the rearing ° matter and at 28 C temperature (Decostere water) in combination with oxytetracycline − − et al., 1999). added to feed at 50 mg kg 1 fish day 1 for The bacterium can survive up to 16 days 10 days is effective in controlling outbreaks ° at 25 C in hard, alkaline water with a high in cages. Potassium permanganate (based on organic load, but survival decreases at pH 7 the cage volume) mixed with a few litres of or less and in waters with less than 50 mg water and then poured through a 7.5 cm −1 CaCO3 l and with low organic matter diameter polyvinyl chloride (PVC) pipe into ° (Fijan, 1968). In sterile mud at 25 C, the the cage and allowed to dissipate into the organism survives for 16 days (Becker and pond by diffusion is also effective (Duarte Fujihara, 1978). et al., 1993).

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244 G.D. Lio-Po and L.H.S. Lim

Streptococcal stomach, small intestine, brain, eyes septicaemia/meningoencephalitis and musculature. Multiple necroses with granuloma occur in the hepatic parenchyma. In freshwater cage-cultured Mozambique The spleen develops hyperplasia of the tilapia (Oreochromis mossambicus), epizo- reticuloendothelial cells with necrotic foci. otics attributed to streptococcal septicaemia Degenerative changes in the renal tubules, were reported in Taiwan (Tung et al., catarrhal enteritis in the small intestine and 1985). Other outbreaks have included the stomach, bacterial meningitis and abscess disease in Nile tilapia, hybrid tilapia formation in the muscles have been noted. (O. niloticus × O. aureus), rainbow trout The disease was experimentally repro- (Oncorhynchus mykiss), striped bass duced in trout and tilapia using 107 and 108 (Morone saxatilis) and hybrid striped bass cfu of S. shiloi and S. difficile, respectively, (Morone chrysops × M. saxatilis) in Israel, with virulence increased to 102 and 105 cfu Japan and the USA (Kitao et al., 1981; Kitao, after in vivo passage (Eldar et al., 1995a). 1993; Eldar et al., 1994; Perera et al., 1994; Streptococcus is also more pathogenic to Baya et al., 1996; Stoffregen et al., 1996). Nile tilapia than to channel catfish (Chang Pathogenic species are Streptococcus iniae and Plumb, 1996). In a mixed infection (phenotypically identical to Streptococcus experiment with Streptococcus sp. and A. shiloi) (Eldar et al., 1995b), Streptococcus hydrophila as inocula, mortality was higher difficile and other Streptococcus spp. among experimental fish inoculated with Most reports on streptococcal infections both bacterial pathogens compared with have occurred among wild and cultured those inoculated with either Streptococcus marine chinook salmon (Oncorhynchus or A. hydrophila (Liu et al., 1990). Infection tshawytscha), rabbitfish (Siganus canali- via the nares is a potential route in Nile culatus) and barramundi (Lates calcarifer) tilapia and hybrid striped bass (Evans in the USA, Singapore and Japan (Moring, et al., 2000). Experimental transmission 1982; Foo et al., 1985; Bromage et al., 1999). occurs by immersion, injection, orally or by S. iniae specifically causes infections in cohabitation and is enhanced by injury to marine finfishes, as discussed in Chapter 5. the skin or stressful environment. Sources of infection are water, mud, contaminated feed Pathology. Among tilapia (15–20 cm in or carrier fish (Plumb, 1994). length) cage-cultured in a dam, this Environmental factors influenced the bacterium caused cumulative mortality development of streptococcal disease in of 50–60% within 1 month (Tung et al., Nile tilapia. Shoemaker et al. (2000) showed 1985). Clinical signs include unilateral that significantly higher mortality (about − and bilateral exophthalmia with or without 28.4%) developed in medium (11.2 g l 1), − conjunctival haemorrhage and corneal opac- compared with 4.8% in low (5.6 g l 1) fish ity. Petechiae occur on the underside of density treatments exposed to 2.5 × 107 − the operculum, around the anus, caudal cfu ml 1 S. iniae by immersion. Moreover, and pectoral fins and mouth, with darkening the infection could be transmitted by of the body and discoloration of the dorsal cohabitation with S. iniae-infected Nile and lateral trunk and peduncle with nodular tilapia for 48 h. In another study, Bunch or abscess formation. Abdominal swelling and Bejerano (1997) demonstrated that low with ascites is common. Affected fish are oxygen and high nitrite levels increased anorexic, swim sluggishly in a circle, turn- mortality in hybrid tilapia exposed to ing laterally, and eventually die. Streptococcus sp. However, these factors Internal signs include petechiae and had no additive effect. Furthermore, haemorrhage of the intestinal tract, liver streptococcal infection in Nile tilapia and pyloric caeca. Systemic infection has fingerlings may occur in association with been observed with evidence of bacterial Trichodina infestation (J.A. Plumb, personal dissemination in the heart, liver, kidney, communication).

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Infectious Diseases of Warmwater Fish in Fresh Water 245

Diagnosis. Streptococcal organisms can be Pseudofungal Diseases isolated in culture from the brain, kidney, heart, spleen and exophthalmia in Todd– Stramenopiles are pseudofungal organisms Hewitt (TH) broth (DIFCO), nutrient agar previously classified as mycotic microbes supplemented with sheep or goat’s blood, (Alexopoulos et al., 1996). Infections brain heart infusion agar or TSA for 24–48 h induced by the stramenopiles (Family ° at 20–30 C (Kitao et al., 1981). Modified Saprolegniaceae, Class Oomycetes) are Hucker’s Gram-staining showing small, Gram- commonly called ‘water mould infections’, µ positive cocci, approximately 0.3–0.5 min cotton tuft disease or saprolegniasis. Bran- diameter, most often occurring in chains, is a chiomycosis and mycotic granulomatosis presumptive diagnosis. These organisms are also occur in cultured fish in fresh waters. non-motile and encapsulated. Plumb (1994) The EUS is associated with a rhabdovirus, divided Streptococci associated with fish the bacterium Aeromonas hydrophila epizootics into four major groups: (i) group and/or the stramenopile, Aphanomyces B, which is non-haemolytic; (ii) group D invadans (see section on Diseases of alpha- and group D beta-haemolytic; (iii) Complex Infectious Aetiology, p. 246). alpha-haemolytic strains that do not react with Lancefield antisera; and (iv) other Streptococci from freshwater and marine fish. The pathogen does not grow in 40% Saprolegniasis bile, 6.5% saline, 0.1% methylene blue milk or at 10 or 45°C (Kusuda and Salati, 1999). The Oomycetes are distributed worldwide Details on the classification of Streptococci and affect warmwater fish in ponds, lakes, spp. based on biochemical and serological dams and rivers. In India, Achlya spp., tests are in Kitao (1993) and Plumb (1994). Aphanomyces, Dictyuchus, Saprolegnia All isolates from freshwater fish are beta- and Pythium were isolated from rohu, grass haemolytic (Kitao et al., 1981; Tung et al., carp, common carp, catla, banded gourami 1985). (Colisa fasciatus), Labeo bata, climbing perch (Anabas testudineus) and giant Prevention and control. Avoidance of stress snakehead (Channa micropeltes); Aphano- due to adverse or poor water quality, myces spp. from rohu and Puntius ticto; rough handling, high stocking density, and Saprolegnia spp. from dwarf gourami non-removal of infected or dead fish (Colisa lalia), banded gourami, Nandus and overfeeding should be followed. nandus, Heteropneustis fossilis and Formalin-killed S. difficile vaccine injected Notopterus notopterus (Srivastava, 1980; intraperitoneally protects tilapia (Eldar Bisht et al., 1996). Saprolegniasis was also et al., 1995c). Recently, Klesius et al. (2000) reported in Nile tilapia, mango tilapia and showed that intramuscular injection of a common carp in Taiwan, Egypt, Nigeria combined vaccine prepared from two strains and Hungary (Chien, 1981; Okaeme et al., of S. iniae obtained from Nile tilapia 1989; ElSharouny and Badran, 1995; Jeney provided relative percentage survivals of and Jeney, 1995). 63.1 and 87.3% when challenged with its homologous pathogens. Medicated feed Pathology. Aphanomyces piscicida causes − with enteroflaxin at 5 mg kg 1 body weight mycotic granulomatosis in ayu (Plecoglos- for 10 days (Stoffregen et al., 1996) or sus altivelis) and dwarf gourami. External with erythromycin–doxycycline mixture at clinical signs include red spots on the body − 100 mg and 70 mg kg 1 body weight for surface due to fungal growth, swelling, ero- 6 days are also effective (Tung et al., 1985). sion and ulcers. Histologically, fungal-like Formalin treatment was used for the hyphae and granulomas are seen in the inter- associated Trichodina (J.A. Plumb, personal nal organs and musculature. It is also highly communication). pathogenic to goldfish (Carassius auratus),

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246 G.D. Lio-Po and L.H.S. Lim

Rhodeus ocellatus, bluegill (Lepomis is commonly named ‘gill rot’ (Post, 1983). macrochirus) and crucian carp (Hatai Secondary bacterial invasion of the filament and Egusa, 1977; Hatai et al., 1994). edges follows. In experimentally infected ayu, typical The presence of organic matter, algal mycotic granulomatosis occurred, while in blooms, dissolved fertilizer, low dissolved common carp no inflammatory response oxygen, pH between 5.8 and 6.5, high was observed (Wada et al., 1996). stocking density and temperatures between Fungal-like Aphanomyces spp., 25 and 32°C are predisposing factors. Under Achlya, Allomyces and Saprolegnia are favourable conditions, the disease may also associated with EUS in snakeheads develop in 2–4 days although in vitro culture (Roberts et al., 1993; Paclibare et al., of the pathogen produced spores on day 1994; Willoughby et al., 1995). However, 14 of culture (Post, 1983). only Aphanomyces has been experimen- tally shown to induce lesions in naive Diagnosis. Two species have been snakeheads (Chinabut et al., 1995; Lilley described: Branchiomyces sanguinis and and Roberts, 1997). Bruno and Wood B. demigrans. Squash preparations of the (1999) provided a recent review on gills examined using light microscopy can saprolegniasis, which is also discussed in be used to differentiate the two species. detail in Chapter 4. B. sanguinis has a thin hyphal wall (0.2 µm), spores of 5–9 µm diameter and affects the gill filaments and gill lamellar capillaries. Branchiomycosis B. demigrans has a thicker hyphal wall (0.5–0.7 µm), spores of 12–17 µm diameter Another fungal-like pathogen, Branchio- and infects the parenchyma of the gills (Post, myces, has also been implicated as a cause 1983). of loss of 85% of juvenile red tilapia hybrid (O. niloticus × O. mossambicus) and green Prevention and control. Affected fish should tilapia hybrid (O. niloticus × O. aureus)in be burned and/or buried. Survivors of the Israel (Paperna and Smirnova, 1997). Carp epizootic are carriers of the pathogen and are also susceptible (Post, 1983). should not be cultured with naive fish or transported into Branchiomyces-free Pathology. Affected fish are lethargic with geographical areas. ragged or corroded gills, which are either bright red or white to brown depending on Diseases of Complex Infectious the degree of necrosis. Histological examina- tion of the gill filaments of infected fish Aetiology demonstrates the proliferation of hyphae of up to 11 µm in diameter. At the onset Epizootic ulcerative syndrome (EUS) of sporulation, the hyphae contain multi- nucleated plasmodia, which develop into EUS affects wild and cultured snakeheads, daughter plasmodia. The final stage of cell catfish (Clarias spp.), Mastacembelus division yields a sporont filled with spores. armatus, Puntius spp., giant snakehead, Spores are released from the necrotic gills Oxyeleotris marmoratus, Glossogobius and remain suspended in the water or fall to giurus, blue gourami, snakeskin gourami the bottom. (Trichogaster pectoralis), Trichopsis vittata, In severe infection, some filaments Siamese fighting fish (Betta splendens), undergo complete degeneration with necro- swamp eels (Monopterus albus) and several tic residues of the pseudofungus. As a result, wild fish species (Lilley et al., 1998). Major the pseudofungi reduce the blood supply to outbreaks occurred in Malaysia in 1979, the gills, causing necrosis and sloughing in Indonesia in late 1980, in Thailand in away of the gill tissue. Hence, the disease 1981, in Kampuchea, Myanmar and Lao

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Infectious Diseases of Warmwater Fish in Fresh Water 247

PDR in 1984, in the Philippines in 1985, in In less severe infections, there is scale loss Sri Lanka in 1987, in Bangladesh and India with erosion of the skin surface with or in 1988, and in Bhutan and Nepal in 1989 without haemorrhagic signs. To date, EUS is (Tonguthai, 1985; Lilley et al., 1998; Lio-Po, defined as a seasonal epizootic condition of 1998). In addition, EUS was observed in freshwater and estuarine warmwater fish of Vietnam, Singapore and Pakistan. EUS-like complex infectious aetiology characterized lesions on fish were also reported in 1972 in by the presence of invasive Aphanomyces Australia (Rodgers and Burke, 1977) where and necrotizing ulcerative lesions typically infected fish included mullet (Liza spp., leading to a granulomatous response Mugil sp.), sand whiting (Sillago ciliata), (Roberts et al., 1994a). Acanthopagrus australis and Arrhamphus In general, EUS outbreaks show a sclerolepis. The disease was then called red seasonal pattern (Phillips and Keddie, spot disease (RSD). Similarly, in Papua New 1990). In Laguna de Bay, the Philippines, the Guinea, Toxotes chatareus, Kurtus gulliveri, EUS morbidity rate among snakeheads was Bunaka spp., goby, freshwater anchovy estimated to be 59% in January, 1986 (Mines and spotted scat (Scatophagus argus) were and Baluyot, 1986). Outbreaks are more severely affected in 1975 (Haines, 1983). common from September to March, which Fish with EUS were found in all types of correlates with the period when the water freshwater systems, including lakes, rivers, temperature in the region is at its lowest streams, culture ponds, rice paddies, irriga- range of below 25°C. Such low temperatures tion canals and reservoirs. Cage-cultured reduce the immune response of fish (Catap snakeheads in the Philippines are very and Munday, 1998). susceptible to the disease (Lio-Po et al., The spreading pattern of outbreaks of 1992). Similarly, it was reported among EUS in Southeast and East Asia strongly cage-cultured P. gonionotus and L. hoevenii indicates the infectious nature of the (Christensen, 1989). aetiological agent. The actual pathogen of this disease has been in dispute for years. Pathology. Lesions associated with EUS are A rhabdovirus, Aeromonas hydrophila characterized by severe, ulcerative, dermal and Aphanomyces invadans have been necrosis with extensive erosion/sloughing associated with EUS-affected fish of the underlying musculature (Fig. 7.3). The (Frerichs et al., 1986; Llobrera and Gacutan, necrotic muscular tissue emits a foul odour. 1987; Boonyaratpalin, 1989; Costa and Fish have frank ulcers that consist of eroded Wejeyaratne, 1989; Lio-Po et al., 1992, 2000; dermal layer, exposing the underlying Pathiratne et al., 1994; Chinabut et al., 1995; musculature, which may be haemorrhagic. Karunasagar et al., 1995; Thanpuran et al.,

Fig. 7.3. Snakehead (Channa striata) affected with epizootic ulcerative syndrome.

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248 G.D. Lio-Po and L.H.S. Lim

1995; Kanchanakhan, 1996; Lilley and temperature range of 18–39°C and secretes Roberts, 1997; Lilley et al., 1998). Saitanu a dermonecrotic factor at temperatures of et al. (1986) also detected a virus associated 10 and 30°C (Olivier et al., 1981; Uddin with EUS. et al., 1997). Moreover, cytotoxin-producing The association of a rhabdovirus with strains were associated with EUS-affected EUS in Thailand and in the Philippines fish and hypothesized to play an important was first reported by Frerichs et al. (1986) role in the pathogenesis of the disease and by Lio-Po et al. (2000). The virus is bul- (Yadav et al., 1992). let-shaped, typical of the rhabdovirus genus The pseudofungi Aphanomyces spp., (Family: Rhabdoviridae) and induces a CPE Achlya, Allomyces and Saprolegnia have in BF-2, SSN-1, CFS, CCO and SHS cells, also been reported in EUS-affected snake- producing virus titres in the latter cells of 106 heads (Roberts et al., 1993; Paclibare et al., −1 ° TCID50 ml at 25 C in 2–3 days (Lilley and 1994; Willoughby et al., 1995). Isolates of Frerichs, 1994; Lio-Po et al., 2000). Opti- A. invadans were experimentally shown to mum replication in SHS cells is at 15–25°C. induce lesions in most test snakeheads or Characterization and serological comparison sand whiting (Roberts et al., 1993; Chinabut of the virus with other fish rhabdoviruses et al., 1995; Catap and Munday, 1998). Other associated with EUS-affected fish in Thai- studies have reported that the pseudofungi land showed that the Philippine virus iso- grow invasively through the fish muscle late is morphologically similar and slightly causing severe myonecrosis (Callinan et al., antigenically related to the ulcerative 1995; Chinabut et al., 1995; Lilley and dermal rhabdovirus (UDRV) (Lio-Po et al., Roberts, 1997). Granuloma development 2000). Earlier experiments on the pathoge- was observed at 26°C or above, while fish at nicity of rhabdovirus from EUS fish were not lower temperatures showed acute inflam- demonstrated (Frerichs et al., 1993). How- mation (Chinabut et al., 1995). In addition, ever, subsequent studies experimentally Catap and Munday (1998) observed that induced lesion development and mortality sand whiting injected with zoospores of in virus-injected snakeheads reared at Aphanomyces sp. at 26°C developed highly 20–22.5°C but not at 28–32°C (Lio-Po et al., inflamed, haemorrhagic external lesions, 2001). Similarly, Kanchanakhan (1996) while similarly treated fish held at 17°C had reported that rhabdoviruses can experimen- slightly inflamed injection sites. The tem- tally cause skin damage in juvenile snake- perature-related growth rate of this pathogen heads at ~20°C. This lower temperature appears to correlate with the findings that range corresponds to the water temperature Aphanomyces isolates from EUS-affected during the cooler months of December fish generally thrive better at 26–30°C than through to February when outbreaks of EUS at lower temperatures (Lilley and Roberts, among freshwater fish occur in the Philip- 1997). pines and in other EUS-affected countries. A. hydrophila has been consistently Diagnosis. The virus is typical of the bullet- isolated from lesions of EUS-affected fish shaped rhabdoviruses with an estimated (Llobrera and Gacutan, 1987; Boonyarat- size of 65 × 175 nm (Lio-Po et al., 2000). palin, 1989; Costa and Wejeyaratne, 1989; Filtrates derived from the visceral organs Subasinghe et al., 1990; Torres, 1990; Lio-Po of EUS-affected fish can induce a CPE when et al., 1992; Pathiratne et al., 1994; Angka inoculated into susceptible cells. The bacte- et al., 1995; Karunasagar et al., 1995; ria, A. hydrophila, and the pseudofungus, Thanpuran et al., 1995; Rahman et al., 1999). Aphanomyces sp., can be isolated from Pure cultures of the bacterium inoculated ulcers and muscles of EUS-affected fish by intramuscularly induced dermonecrotic methods described in the section on motile lesions in healthy catfish and snakeheads Aeromonas septicaemia (Chapter 4) and in (Lio-Po et al., 1992, 1996, 1998; Pathiratne Lilley et al. (1998). Histopathology of et al., 1994; Angka et al., 1995; Karunasagar muscular lesions of affected fish shows the et al., 1995). This bacterium grows at a development of a necrotic granulomatous

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Infectious Diseases of Warmwater Fish in Fresh Water 249

mycosis, which may eventually invade culture of Oxyeleotris marmorata in cages the abdominal viscera (Lilley et al., 1998). in Thailand (ADB/NACA, 1991). Thus, Bacterial colonies are also histologically subsequent details of the diseases encoun- demonstrated in EUS-affected snakeheads tered in cage culture systems are discussed (Lacierda, 1995). under generic and other taxonomic group- ings, rather under the specific pathogens in Prevention and control. Quarantine and question. restricted movement of EUS-susceptible fish Generally, wild/feral fish have greater from endemic areas to non-endemic sites parasite species diversity but lower popula- should be practised. Prophylactic treatment tion abundance and the converse is true with 5 ppm Coptrol (a chelated copper com- for cultured fish but further studies are pound) was reported to prevent induction required (L.H.S. Lim, personal observation; of EUS lesions while a proprietary mixture, Lerssutthichawal, 1999). Personal observa- CIFAX, may be curative (Lilley et al., 1998). tions and discussions with tropical fishery Moreover, recent studies showed that fish scientists and the current literature indicate fed with the immunostimulant Salar-bec that not all parasites known from other forms survived better when challenged with A. of culture systems have a similar impact on invadans (Miles et al., 2001). cage-cultured fish.

Parasitic Diseases Diseases caused by protistans

Although there is information on parasitic The protozoan or protistan parasites that diseases of fish in tropical aquaculture cause disease in fish belong to several phyla (Kabata, 1985; Lim, 1991d, 1992; Paperna, and these include the Ciliophora, Myxozoa, 1991, 1996; Arthur, 1992; Arthur and Microspora, Sarcomastigophora and Api- Lumalan-Mayo, 1997), there is little or complexa (Dickerson and Dawe, 1995; no information dealing specifically with Dykova, 1995; Lom, 1995; Lom and Dykova, parasitic diseases in cage culture systems. 1995; Molnar, 1995; Noga and Levy, 1995; This paucity of information on disease Woo and Poynton, 1995). The commonly pathogens and control measures and the reported pathogenic protistans in or on fish lack of regulations concerning movement reared in cages in warm waters include the of diseased fish and mandatory reporting myxosporeans, trichodinids and the dino- of diseases and mortalities in developing flagellates (Christensen, 1989; T.T. Dung, countries, coupled with the diverse species personal communication; F. Shaharom, cultured, have made management of para- personal communication). Leptobarbus sitic diseases in warmwater cage culture a hoevenii cultured in cages in Indonesia are difficult task. The diseases and specific infected with myxosporeans (Christensen, identity of the parasites infecting warm 1989). In Vietnam, fish in cage culture are freshwater cultured fish (in particular cage plagued by Trichodina, Balantidium (in cultured fish) are seldom known and at the intestines of catfish) and Glossatella best only the genera are recorded (Paperna, (T.T. Dung, personal communication). 1991). Overall, there is also a lack of knowl- The oodinid dinoflagellate Piscinoodinum edge about the actual disease patterns, the sp. infects grass carp, bighead carp and pathology and prevailing factors predispos- P. gonionotus in pond culture, as well ing fish to the disease (Christensen, 1989; as catfish and tilapia in cage culture Dharma et al., 1992; Nasution et al., 1992; (Shaharom-Harrison et al., 1991; F. Alawi and Rusliadi, 1993). The lack of Shaharom, personal communication). comprehensive investigations into the Various other protistan parasites have also diseases encountered in cage culture been recorded but their prevalence is not systems has resulted in the abandonment known. For example, Ichthyobodo (Costia) of some lucrative projects such as the and Oodinium are known to affect hybrids

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250 G.D. Lio-Po and L.H.S. Lim

of Clarias in tropical warm freshwaters (Christensen, 1989). Myxobolus koi has (Paperna, 1991), resulting in pale gills and been found on the gills of common carp and excessive mucus secretions, causing the goldfish in Japan (Egusa, 1992) and on fish fish to gasp for air. The lack of reports on farms in Israel, Indonesia and the Indian protistan diseases in warmwater cage cul- continent, causing high mortality among ture systems could be due to lack of exper- the younger fish (ADB/NACA, 1991; tise in diagnosing the disease and/or the Paperna, 1991), while Myxobolus artus absence of reporting procedures, rather than is found on common carp in East and the absence of the disease agents. Southeast Asian countries (Lom and Movement of fish for culture has Dykova, 1995). contributed to the worldwide distribution of many of their parasites, especially parasitic Pathology. M. koi infections on the gills of protistans. For example, Eimeria cheni and common carp and goldfish result in many Eimeria sinensis, originally found in farmed small white to large pinkish to red cysts in carp in China, are now found in Europe the gill tissue (Paperna, 1991; Egusa, 1992). (Molnar, 1976). Nile tilapia imported into Large cysts are enclosed in the host connec- Thailand from Egypt were also infected tive tissues, which turn dark red due to with Eimeria vanasi (Paperna, 1991), while haemorrhaging, leading to congestion and cichlid fish farmed in Israel (Landsberg and degeneration of the gill capillaries. The Paperna, 1985) were infected by E. vanasi movement of the opercula and respiratory and Gousia cichlidarum. A few protozoan processes are further affected by increased diseases found in cold waters could be mucus production and epithelial prolifera- regarded as emerging disease problems in tion. Spores of M. koi were also observed cage culture in warm waters since these in the heart, liver, kidney and intestine could be carried with their host species. (Hoshina, 1952). According to Lom and Dykova (1995), Thelohanellus pyriformis Myxosporean diseases forms large plasmodia in the subcutaneous Myxosporeans are observed as cysts, infect- tissue and muscle of cyprinids causing fatal ing the skin and subcutaneous layer, epizootics in Indonesia. Little is known muscle, gills, central nervous system as about the pathology caused by the other well as visceral organs. These cause myxosporeans. extensive lesions as cysts break, and mortal- ity occurs in cultured as well as feral fish Diagnosis. One characteristic sign of myxo- (Lom and Dykova, 1995). In most cases in sporean infections is small white and/or Southeast Asia, the specific myxosporidean large cysts on the gills. For example, M. koi pathogens are not known and at best are observed as small white cysts and large the identification is at generic level. pinkish to reddish cysts in the gill tissues Thelohanellus (Myxobolidae), Myxobolus of common carp and goldfish (Paperna, (Myxobolidae) and Myxosoma (Myxidiidae) 1991; Egusa, 1992). Opercular movements of have been reported from exotic carp infected fish are hampered and respiration is and indigenous cyprinids in the Indian affected by the increased mucus secretion continent, Southeast Asia and China and epithelial proliferation (Hoshina, 1952). (ADB/NACA, 1991; Paperna, 1991). Some myxosporeans are confined to the Thelohanellus has been reported on P. body and these occur as white cysts gonionotus, common carp and Clarias spp. under the scales, often near the tail or fins, in Peninsular Malaysia (Paperna, 1991; resulting in sores or ulcers on the skin ADB/NACA, 1991). Myxosporeans are a (Christensen, 1989). Identification of the major problem in Central Java (Indonesia), myxosporeans is based on the morpho- infecting L. hoevenii and P. gonionotus logical characteristics of the spores. Cysts reared in ponds. However, in cages, the on the skin or gills are removed and gently parasite was only found on L. hoevenii broken to release the spores (preferably) on

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Infectious Diseases of Warmwater Fish in Fresh Water 251

glass slides. The multicellular spores are Diseases caused by ciliates unique in possessing nematocyst-like polar capsules (Lom and Dykova, 1995). They are The ciliates (Phylum Ciliophora) are usually oval–pear to round shaped, anterior common ectoparasites of fish, especially in end pointed, posterior end rounded, 1–2 hatcheries and on young fish in grow-out polar capsules with polar filaments, sporo- ponds. Ichthyophthirius multifiliis is the plasm with or without iodophilic vacuole most well known pathogenic ciliate and is and with or without posterior processes related to the marine pathogen, Crypto- (Shulman and Shtein, 1962; Lom and caryon irritans. Others include the tricho- Dykova, 1995). dinids and Chilodonella. However, in the The spores of Myxobolus are oval to majority of reported cases in tropical pear-shaped with two polar capsules at aquaculture, the specific identities of these their pointed anterior; the posterior end is ciliates are not known. Besides the known rounded and lacks processes. The spores of obligatory parasitic (pathogenic) ciliates, Henneguya are round, oval or fusiform with there are also facultative parasites (Tetra- two anterior polar capsules and valves with hymena, for example), which are opportu- two caudal processes from the posterior end. nistic organisms. The oval to round spores of Myxosoma are different in having two polar capsules at Trichodinid diseases one end and lack processes and iodophilic vacuoles, while Thelohanellus has oval to Pathogenic trichodinids include Chilodo- round spores with smooth valves without nella, Trichodina, Tripartiella and Tricho- processes and one medially displaced polar denella. A large number of trichodinids are capsule. associated with the goldfish, common carp, grass carp, silver carp and bighead carp and Prevention and control. There is no effec- these were introduced into Israel and tive treatment and the best method is to Southeast Asia from China (Chen, 1955; remove and destroy heavily infected fish Paperna, 1991). The trichodinids (Tricho- from cages (Christensen, 1989). In light dina acuta, Trichodina centrostrigeata and (early) infections, the cysts should be Trichodina heterodentata) from African carefully removed and destroyed. Treatment cichlids have also been introduced into with saline (0.23–5.0%), copper sulphate Southeast Asia (Albaladejo and Arthur, (0.025–0.05%), potassium permanganate, 1989; Bondad-Reantaso and Arthur, 1989). formalin, methylene blue, glacial acetic acid Trichodinids can be found on snake- or phenol is not effective (Hoshina, 1952) as heads and Pangasius conchophilus cultured myxosporean spores are highly resistant to in cages in Vietnam (T.T. Dung, personal chemicals. The inclusion of certain drugs communication). Although chilodonellosis (such as Proguanil and furazolidone) in the occurs mainly in cold waters, Chilodonella fish feed has been shown to reduce spore hexasticha has also been found on the production and alleviate lesions (Lom and bighead carp in Malaysia (Shariff, 1984). Dykova, 1995). Although the life cycles of Trichodinids commonly cause mortal- some species of myxosporeans are known ity in hatcheries and these may continue to to involve intermediate hosts such as be a problem after fish are transferred to cage oligochaetes (Lom and Dykova, 1995), for culture systems. Trichodinids are prevalent the majority of cases, the life cycles have not on young clariid hybrids of African catfish been elucidated and the actual intermediate (Clarias gariepinus) and Clarias sp. in cages. hosts not identified. Hence, control of myxo- These are also found on silver carp, bighead sporeans via eradication of intermediate carp and grass carp in hatcheries in China hosts (oligochaetes) is not a viable option at and Vietnam, and are also on pangasiids the present time. Eradication of heavily and Catla sp. in cage culture. In Nepal, infected hosts appears to be the most viable trichodinids cause mortality among the fry option for the moment. during spring and autumn. Although there

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252 G.D. Lio-Po and L.H.S. Lim

are many species of trichodinids, only a few components (Lom, 1995). Trichodinids are are known to be pathogenic (Lom, 1995). essentially flat discs, with somatic ciliature consisting of 3–4 ciliary wreaths around Pathology. Pathological effects are depend- the aboral surface of the body, which is ent on the host’s response, the intensity transformed into an adhesive disc. The disc of infections and environmental conditions, is a proteinaceous skeleton, composed of since stressful conditions can compromise a ring of hollow conical denticles. The the host’s ability to counteract infections denticles consist of blades (centrifugal flat (Paperna, 1996). Some trichodinids live projections) and horns (rod-like centripetal specifically on the body surface or on the projections), connected to each other by gills, while others are found both on the radial pins (Fig. 7.4). skin and the gills (Paperna, 1996). In skin There are five genera of fish tricho- infections, the preferred sites are the bases dinids (Lom, 1995). In warm freshwater cage of fins. These parasites damage epithelial culture systems, only Trichodina spp. has tissue through adhesion and crawling been identified. Trichodina is characterized actions (Paperna, 1996). They feed on by denticles with massive central conical the epithelial cells causing abrasion and parts, flat semi-circular blades, straight µ some trichodinids may suck out cellular thorns and a diameter of 50–100 m. For contents, damaging cells, which degenerate general identification, skin and gill smears and disintegrate resulting in erosion and containing trichodinids should be air-dried, desquamation of the epidermis (Paperna, fixed in Bouins for 20 min, washed in 70% 1996). The host responds to the infection ethanol, rehydrated and stained in a haema- by increased mucus secretion and epi- toxylin stain, dehydrated and mounted. For thelial hyperplasia, cellular destruction specific identification of the trichodinids, and inflammation. The damaged gills and the adhesive disc is studied using a silver epidermal tissues are targets for bacterial impregnation method (Welborn, 1967; invasion. The infected epidermis thickens, Paperna, 1996). Air-dried smears should be becomes turbid with mucus and sloughed fixed in 2% silver nitrate for 7–9 min in the epithelial cells, and the fish becomes dark, washed in distilled water and exposed emaciated. When the gills are infected, to sunlight or UV light for 5–10 min. excessive mucus is produced, with massive destruction of the gills, and proliferation Prevention and control. In most cases, an of epithelial cells causing difficulty in outbreak of trichodinid infections is the respiration. Trichodinids are usually found result of adverse environmental conditions, in association with monogenean and other which are common in intensive culture protozoan infections. Massive infections systems. The best preventative measure is causing damage in the epidermis as to ensure that good quality environmental described above result in mortality due to conditions are maintained. To eliminate disruption in the respiratory functions of trichodinids from aquaculture systems, the gills (Paperna, 1996). Young fish in over- several chemicals have been recommended crowded and confined stressful habitats are (Lom, 1995): saline solution (0.1–0.2% as a usually heavily infected with trichodinids, dip for 1–2 days), formalin (150–250 ppm as while older fish have fewer but more host- a dip for 30–60 min), acriflavine (indefi- specific species (Paperna, 1996). nitely in water at 10–20 ppm) and potassium permanganate (0.1% as a dip for 30–45 min). Diagnosis. Trichodinids are easily obser- Formalin has been used effectively to con- ved microscopically from skin and gill trol trichodinids in warm waters. The effi- scrapings (Paperna, 1996). Taxonomy of the cacy of formalin in controlling trichodinids trichodinids is based on the structure of depends on water quality (pH, salinity and the buccal ciliature, the morphology of the ambient temperature) and species of fish adhesive disc and the number and size of its treated. Van As et al. (1984) showed that

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Infectious Diseases of Warmwater Fish in Fresh Water 253

Fig. 7.4. Trichodina acuta from the skin of Ctenopharyngodon idellus (Klein’s silver impregnation) (courtesy of Dr Richard Arthur, Canada).

25 ppm for 24 h was effective in cleaning as a low subclinical (enzootic) infection and infected carp, while 45 ppm for 24 h was as encysted tomonts. It persists in the envi- needed to clean tilapia. ronment, becoming epizootic clinical infec- tions when fish are stressed as a result of Ich or white spot disease poor management practices (e.g. poor feed, overcrowding and poor sanitation). The Ichthyophthirius multifiliis is a pathogenic pathogen is not host-specific and recovery ciliate infecting freshwater fish causing ich- from the disease confers resistance to rein- thyophthiriosis (also known as ich or white fection (Paperna, 1996). spot). This pathogen was first reported from China (Dickerson and Dawe, 1995), but is Pathology. The feeding or trophont stage is now a cosmopolitan pathogen in temperate located within the epidermis (gills or skin) and tropical warmwater fish (ADB/NACA, of the fish (feeding on the basal layer of 1991). It is predicted to spread with the the epidermis). The matured tomonts leave increase in aquaculture activities and also the fish and damage the epidermis causing via the aquarium trade (Paperna, 1996). The detachment from its basal membrane; they outbreaks of ich are dependent on water secrete a gelatinous cyst wall and divide temperature and, as temperature increases, asexually to form tomites, which differenti- the life cycle of this parasite is completed in ate into infective theronts and are released a shorter time (Dickerson and Dawe, 1995), into the water. The tomites develop into making them a potential danger to cage infective theronts, which penetrate the culture systems in tropical warmwaters. epidermis of the fish becoming established This parasite is maintained within the fish in the basal layer of the epithelium just

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254 G.D. Lio-Po and L.H.S. Lim

above the basal membrane, and feed on consideration when chemicals are used epithelial cells. The rate of development since some species, especially catfish, do not of these stages is dependent on water tem- respond well to malachite green (Paperna, perature (see above). Intense and prolonged 1996). Potassium permanganate has been infections cause epithelial proliferation, used successfully in ponds to control ich but haemorrhagic inflammation and subsequent its effectiveness is affected by the amount disintegration of the integument. of organic matter in the water (Dickerson and Dawe, 1995). Malachite green in a non- Diagnosis. Clinical signs include anorexia water-soluble formulation in feed has been and lethargy, and the disease is character- reported to be effective against trophonts ized by white spots on the skin and gills (Schmahl et al., 1992). Immersion of fish (Dickerson and Dawe, 1995). Skin and gill infected with ich in Toltrazuril or triazinone µ −1 scrapings examined under the microscope (10 gml ) for 4 h (repeated daily for 3 reveal the ciliate (1 mm in diameter) with days) has been shown to be effective a small cytostome. Ichthyophthirius fixed against trophonts (Dickerson and Dawe, and stained with Giemsa or haematoxylin 1995). However, malachite green has been reveals a large crescent-shaped macro- reported to be carcinogenic and its use nucleus and small micronucleus. is limited to aquarium fish, and should not be used in fish cultured for human Prevention and control. This pathogen is consumption (Dickerson and Dawe, 1995). particularly difficult to control. An inte- Studies have shown that fish that have grated approach incorporating appropriate recovered from ich infections develop culture practices (locating cages in areas immunity against the parasite (Dickerson where water movement is continuous and and Dawe, 1995). Immunization and vac- stocking of clean and healthy fish), immuni- cination offer another way to protect fish zation and chemotherapy in cases of heavy against ich. Experimental immunization infestations are probably the most effective using killed vaccines, intraperitoneal inoc- means of disease control (Dickerson and ulation with live theronts and controlled Dawe, 1995; Paperna, 1996). exposure to infective tomites have been The chemicals recommended for treat- used (Paperna, 1996; Sin et al., 1996). An ment include sodium chloride, malachite experimental recombinant vaccine (from a green, formalin and potassium permanga- 316 bp gene fragment of the immobilizing nate (Dickerson and Dawe, 1995; Paperna, antigens, or i-antigens, of I. multifiliis and 1996). The efficacy of these chemicals is expressed in Escherichia coli) has been dev- dependent on a number of factors such as eloped for ichthyophthiriosis (Woo, 1998). environmental conditions, the fish species Goldfish inoculated with the recombinant in question and the different developmental protein vaccine in Freund’s adjuvant sur- stages of the parasites (see below). For vived a parasite challenge (He et al., 1997). example, encysted tomonts in the environ- ment are resistant to antiparasitic chemicals (Paperna, 1996). The stages of the parasite Diseases caused by dinoflagellates that can be destroyed are the dividing tomonts and the newly released tomites. There are five genera of parasitic oodinid Several chemicals have been listed for dinoflagellates: Amyloodinium, Piscino- use against this pathogen, and the cost- odinium, Crepidoodinium, Ochthyodinium effective chemicals suitable for large- and Oodinioides on fish (Noga and Levy, scale farming systems are malachite 1995). The ichthyotoxins produced by dino- green (0.05–0.15 ppm used continuously for flagellates cause massive mortality in cul- 3–4 days) and a mixture of formalin and tured and feral fish (Steindinger and Baden, malachite green (50 and 0.05 ppm) (Paperna, 1984). The important freshwater pathogenic 1996). The fish species has to be taken into dinoflagellate in fish is Piscinoodinium,

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Infectious Diseases of Warmwater Fish in Fresh Water 255

which is closely related to the marine complete lysis (Lom and Schubert, 1983; dinoflagellate pathogen, Amyloodinium. Paperna, 1991). Piscinoodinium is not host-specific and has been reported on feral, aquarium and Diagnosis. Initial diagnosis can be based on cultured food fish species from diverse clinical signs and confirmed by microscopic families in warm waters (Lom and examination of the trophont stage. Piscino- Schubert, 1983; Paperna, 1991, 1996; odinium infects skin and gills with clinical Shaharom-Harrison et al., 1991). signs similar to amyloodiniosis. Infected fish have a yellow to rust-coloured (velvety) Velvet or rust disease skin, dense covering of mucus resulting in darkening of the skin, dyspnoea, anorexia Fish with excessive mucus covering the and skin ulcers (Shaharom-Harrison et al., body together with a rust-coloured appear- 1991). ance of the skin are infected with Piscino- All oodinids have a parasitic trophont odinium pillulare, the causative agent for stage and a sessile, stalked, sac-like tropho- velvet rust diseases, gold dust disease, zoite stage, which feeds on the skin and gill pillularis disease and freshwater Oodinium epithelia. The trophont has a prominent disease (Shaharom-Harrison et al., 1991). stalk, which anchors the parasite to the host. Piscinoodinium, like its marine relative It probably uses the stalk to absorb nutrients. Amyloodinium, is found on a wide range of After feeding, the trophont detaches, with- host species and is known to cause mortal- draws the stalk and forms an encysted ity in warmwater fish (Paperna, 1996). tomont (reproductive cysts). The tomont P. pillulare has been reported from 14 divides asexually forming dinospores, the tropical ornamental fish species as well as mobile infective stages. The trophonts and cultured carp and cyprinids (Shaharom- tomonts are important for definitive diagno- Harrison et al., 1991; Noga and Levy, 1995). sis, and microscopic identification of these In Peninsular Malaysia, P. pillulare occurs stages is necessary. Trophonts are oval with on aquarium fish, cultured grass carp, smooth walls, usually visible to the naked bighead carp, P. gonionotus and L. hoevenii, eye as white spots (80–100 µm) and in causing mortality in the latter (Shaharom- Lugol’s iodine turn dark blue. Harrison et al., 1991). This pathogen also Piscinoodinium is distinguished from causes disease in cage-cultured Hemibagrus other oodinid dinoflagellates on the basis of nemurus in the Trengganu River and in the morphology of the trophont, especially Tilapia cultured in Kenyir dam, Malaysia the type of host attachment and mode (F. Shaharom, personal communication), of nutrition (Lom, 1981). Fish should be although not to the same extent as that found examined live or immediately after death, on pond cultured fish. and snips of the gills can be removed from live or recently dead fish and examined. Pathology. Histopathological changes of Trophonts are removed by brushing the fish gill structure occur with a massive prolifera- gently in a dish of water and the sediment tion of the gill epithelium, fusion of adjacent is examined under the microscope. The lamellae and separation of the gill res- trophont of Piscinoodinium is a yellow- piratory epithelium resulting in a severe green, pyriform or sac-like cell, almost hyperplasia of the entire gill filament round, 12 × 29 µm, with a rudimentary (Shaharom-Harrison et al., 1991). The sulcus and a short stalk with an attachment trophonts of P. pillulare penetrate the host disc extending from its base and thin hold- cells by nail-like extensions resulting in fasts (rhizocysts) radiating from the stalk degeneration and collapse of the cells, (Lom and Schubert, 1983). Head parts of the leading to focal erosion and proliferation of rhizocysts are inverted in separate compart- the epithelium and obliteration of the gill ments (rhizothecas) in the sole of the disc, lamellae. The inner strata of the epithelium while their shafts are firmly embedded in become spongious and may undergo the host cell cytoplasm. The theca covers

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256 G.D. Lio-Po and L.H.S. Lim

the entire cell except for the area of the vectors of bacterial and viral diseases, but attachment disc. further confirmation is needed. In intensive culture systems, where intensity of infec- Prevention and control. Outbreaks of tion can be high on the gills, monogeneans oodinid infections result from stress due can cause death directly by inhibiting respi- to poor environmental conditions. Hence, ration through physical damage to the gills. environmental manipulation is probably a Fish mortality from monogenean infections viable approach to control outbreaks of Pis- may result from damage to gill tissues and cinoodinium. Formalin detaches trophonts, skin caused by attachment organs, and by but does not inhibit division (Paperna, feeding on the integument, which stimu- 1996). A copper ion concentration of about lates cell proliferation and secretion of 0.15 ppm (mixture of 5-hydrate copper copious amounts of mucus (Paperna, 1991). sulphate with citric acid monohydrate) in Cage culture in tropical areas is usually water is effective in controlling Piscinoodin- conducive to the perpetuation of parasitic ium (Paperna, 1996). A salt dip for 1–3 min diseases with high stocking density. The dislodges the trophonts, while immersion nets trap eggs, infective larvae and food − for 3–5 days in a combination of 7 g salt l 1 debris around the cages, which attract − and 40 mg potassium permanganate l 1 carrier/reservoir feral fish. is also effective. However, freshwater fish Most monogenean genera are specific to cannot tolerate high salt concentration and a group of related host species. Dactylogyrus potassium permanganate higher than 2 mg is found on cyprinids and catfish harbour − l 1 (van Dujin, 1973; Plumb, 1979). Thaparocleidus. Although at species level most species are specific to a particular host species, some species, like Thaparocleidus caecus, are found on a number of pangasiids Diseases caused by monogeneans (Lerssutthichawal, 1999). Many of the monogenean species on Monogeneans are among the most com- warm freshwater cultured fish have not been monly reported parasitic agents of fish identified or are incorrectly classified. For (ADB/NACA, 1991). They are mainly example, Dactylogyrus spp. have also been ectoparasitic on the gills, buccal cavity, incorrectly implicated as being pathogenic body surface and fins of freshwater fish to snakeheads, tilapia and clariids cultured although some are endoparasitic (Gussev in Southeast Asia (Kabata, 1985). These fish and Fernando, 1973; Euzet and Combes, possess their own unique and specific 1998). Monogeneans are oviparous with the monogeneans (Lim and Furtado, 1983, exception of the viviparous gyrodactylids. 1986; Lim, 1986, 1991a). Trianchoratus Although they rarely cause disease in wild is found on snakeheads other than fish, apart from the benedenids (Paperna, giant snakehead, which is infected by 1975), they are important pathogens in Sundanochus spp., while Cichlidogyrus intensive fish culture (Paperna et al., 1984). spp. infect the tilapias and Quadriacanthus Their direct life cycle results in rapid and spp. and Bychowskyella spp. infect the continuous recruitment, especially in warm Southeast Asian clariids. The gyrodactylids, waters; this makes monogeneans especially on the other hand, are ubiquitous, although dangerous in intensive culture. Disease at species level they might be host-specific. caused by monogeneans is normally more The most commonly reported monogen- debilitating than fatal, and subsequent mor- eans on warm freshwater cultured fish are tality is usually attributed to viral or bacte- the Dactylogyrus spp. on carp, Cichlidogyrus rial infection. Monogeneans stress the fish spp. on cichlids, Bychowslyella spp. and hosts by destroying the epidermal integrity Quadriacanthus spp. on clariids, Trian- of the fish, thus predisposing their hosts to choratus spp. on snakehead, Pseudo- other pathogens. Cone (1995) suggested that dactylogyroides spp. on O. marmorata, and monogeneans could be the mechanical Thaparocleidus spp. on catfish other than

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Infectious Diseases of Warmwater Fish in Fresh Water 257

clariids and Gyrodactylus spp. (see below). common carp, grass carp, bighead carp, Pseudodactylogyrus spp. have been silver carp and Catla spp., as well as other recorded from eels (Anguilla spp.) in warm Southeast Asian carp such as P. gonionotus waters of Indonesia (K. Buchmann, personal and L. hoevenii (see Chapter 1). communication), and it should be noted that The four important species of Dactylo- Pseudodactylogyrus infections caused mass gyrus that cause disease in cultured common mortalities of cultured eels, especially carp in Israel are Dactylogyrus anchoratus, Anguilla japonicus, in Europe in the 1980s Dactylogyrus extensus, Dactylogyrus min- (Buchmann et al., 1987; Buchmann, 1997). utus and Dactylogyrus vastator (Paperna, In the majority of cases, the specific 1991). These have different temperature identity of the pathogenic monogeneans, preferences: for example, D. extensus flour- signs and pathology of the infection, disease ishes at low water temperatures (optimum mechanism and control and preventative temperatures of 16–17°C), while D. vastator measures have not been specifically prefers warmer waters (20–24°C). Currently, elucidated and documented. For instance, D. minutus can be found on common carp in it is known that Thaparocleidus siamensis Taiwan (Paperna, 1991). The grass carp are occurs in greater intensity than Thaparo- infested with Dactylogyrus lamellatus and cleidus caecus on cultured P. hypophthal- Dactylogyrus ctenopharyngodonis, silver mus in Peninsular Malaysia and Thailand carp with Dactylogyrus hypophthalmich- (Lim, 1990, 1996; Lerssutthichawal, 1999), thys, Dactylogyrus suchengtaii and Dacty- but it is not known which of the two species logyrus scriabini and bighead carp with is pathogenic. The translocation of mono- Dactylogyrus aristichthys and Dactylogyrus geneans, along with their hosts, has been nobilis (Paperna, 1991). In Peninsular well documented for the various Dacty- Malaysia, D. nobilis and D. aristichthys are logyrus spp. on imported Chinese carp, found on cultured bighead carp and D. lam- Cichlidogyrus spp. on tilapia and recently ellatus on grass carp (Shaharom, 1988). P. for Quadriacanthus clariadis on the C. garie- gonionotus and L. hoevenii are infested with pinus imported into Thailand (Paperna, Dactylogyrus leptobarbus and Dactylogyrus 1991; Lerssutthichawal, 1999). lampam (Mizelle and Price, 1964; Lim and There is information on the signs, Furtado, 1986; Lim, 1991b), respectively, in pathology and control measures for some Peninsular Malaysia. In Thailand, however, species of Dactylogyrus, Gyrodactylus and there are seven species of Dactylogyrus Cichlidogyrus, but not for other monogenean on feral P. gonionotus (Chinabut and Lim, pathogens. In most cases, the information 1993). Dactylogyrus has also been shown to is derived from pond culture systems and cause mass mortality of fry, small fish and not from cage culture systems. It should be broodfish (Paperna, 1991). also noted that habitat can affect parasitic infections, as indicated by the infestation Pathology. The pathology caused by Dacty- of tilapia by Neobenedenia spp. instead of logyrus spp. on exotic carp has been reported Cichlidogyrus spp. when farmed in cages in in studies done in Europe, but not for the estuarine waters (see Chapter 5). species infecting the indigenous cyprinids of Southeast Asia. Feeding on epithelial Diseases caused by Dactylogyrus species cells and anchorage (attachment) by the monogeneans cause severe destruction of Dactylogyrus species are specific to the the gills resulting in haemorrhage and meta- Cyprinidae although they are also found on plasia of the gill tissue. Secondary bacterial Hemiramphidae (L.H.S. Lim, unpublished infections usually occur and result in death data) and one species on a catfish (Gussev, of the fish. The pathologies caused by 1976). This genus is frequently listed as a D. vastator and D. lamellatus are similar disease-causing agent since cyprinids are (Molnar, 1972; Paperna, 1991). D. vastator the most cultured fish group. Cyprinids infestations cause severe hyperplasia of cultured in cages and pens include the the epithelium of gill filaments. Extensive

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258 G.D. Lio-Po and L.H.S. Lim

proliferation of the respiratory epithelium of added underneath the coverslip to clear the gills interferes with respiratory functions and fix the specimens, which are examined and may be a direct cause of death. The sites using a phase contrast microscope. Mono- of proliferation are dependent on the pre- genean species are usually identified on ferred sites of the monogenean species. D. the basis of the sclerotized reproductive vastator prefers the tips of the gill filaments and haptoral armaments on the cleared and and causes mass mortality in young fish but flattened specimens. The Dactylogyrus are seldom on fish greater than 32–35 mm since oviparous monogeneans with or without the functions of the remaining gill filaments four eye-spots, 14 marginal hooks, two are not affected. Massive infestations of anchors, one to two connective bars and two D. extensus can cause mortality in 4–7 kg needle-like structures and spindle-shaped broodfish (Paperna, 1991). dactylogyrid-type seminal vesicles. The descriptions for the various Dactylogyrus are Diagnosis. Fish infected with Dactylogyrus found in Gussev (1985) for imported carp, in spp. are lethargic and usually found swim- Lim and Furtado (1986) and Chinabut and ming on the surface of the water. Fish Lim (1993) for P. gonionotus and in Mizelle heavily infected with Dactylogyrus have and Price (1964) for L. hoevenii. Presently, pale to greyish gills, swollen at the edges, other diagnostic techniques (such as immu- and the opercula appear to open wider nological) are not known. than normal and secrete excessive amount of mucus (Christensen, 1989). Heavily infected Prevention and control. The main method fish are also anorexic and are usually found for control of monogeneans is the applica- gasping for air and exhibiting abnormal tion of chemicals. Chemotherapeutic treat- behaviour such as jumping out of the water. ments include dips or baths in salt, formalin Dactylogyrus spp. are usually found on or organophosphates (Dylox, Dipterex, the gills, although in massive infections they Neguvon, Chlorophos), Bromex-50 and can also be found on the buccal cavity. potassium permanganate (Paperna, 1996; Dactylogyrus spp. can kill directly by T.S. Thana, personal communication; T.T. damaging gill structures and affecting respi- Dung, personal communication). The rec- ration. In warm eutrophic waters with low ommended doses and concentrations vary oxygen, this becomes serious. Dactylogyrus according to host and parasite species, as infections usually result in secondary well as physico-chemical properties of the bacterial infections with subsequent mortal- waters. A 1 h bath with formalin at 1:4000 ity. At present, Dactylogyrus infections are (< 10°C), 1:5000 (10–15°C) or 1:6000 (15°C) confirmed by examination of the gills and a bath with 3–5 ppm potassium per- and infected fins for presence of the manganate for 1–2 h (Hoffman and Meyer, monogeneans. 1974) has been recommended. Trichlorfon − The signs and pathology of monogenean (Dylox) may be added in the food (50 mg kg 1 infections are neither generic- nor species- fish) four times at 3 day intervals each month specific. Hence, diagnosis of monogenean during the critical periods. Lime and other infection is based on the identification chemicals have been recommended for pond of the pathogen itself. Correct diagnosis of applications: 0.4–0.5 ppm of trichlorphon monogeneans requires proper preparation of (0.0-dimethyl-2,2,2 trichloro-1-hydroxy- the parasite specimens. Gills can either be ethyl phosphanate) has been used in Japan completely removed or gill clippings can be and 0.2 ppm dimethyl-1,2 dibromo-2,2 taken from the infected fish. Each parasite is dichloroethyl phosphate (Bromex) in Israel removed carefully from the gills under a dis- (Egusa, 1992). These chemicals will be effec- secting microscope, placed on a slide and tive if the cages are in ponds. However, they covered with a coverslip. Excess water is will not be effective for large bodies of water removed and the corners of the coverslip and rivers where cages are usually located. sealed with nail polish to prevent it from The above chemotherapeutic formulations moving (Lim, 1991c). Ammonium picrate is are for specific regions, and effective doses

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Infectious Diseases of Warmwater Fish in Fresh Water 259

have to be tested for different waters. If to cause severe gill damage in tilapias chemicals prove ineffective, most farmers cultured in the Philippines (Kabata, 1985). will simply destroy their heavily infected Neobenedenia spp. found on tilapia in cages fish (personal communication with farm- in estuarine waters are more pathogenic than ers). Eradication of feral reservoir fish from Cichlidogyrus spp. (see Chapter 5). ponds is possible but not when the cages are in rivers or large lakes. The best alternative Diagnosis. The behaviour of the fish can management strategy includes good hus- indicate the presence of parasites and this is bandry based on knowledge of the reproduc- similar to Dactylogyrus infection. However, tive biology and ecological requirements of accurate diagnosis requires removing the the parasites such as temperature depend- gills or gill clippings; the monogeneans ency. Using healthy fish fry from reliable are collected and prepared as stated above hatcheries, limiting stocking density of fish, for Dactylogyrus. Cichlidogyrus can be providing good quality feed and sanitation distinguished from other monogeneans by of nets will help to keep infestation at a having a haptor with four anchors, with two low level. Some fish are able to acquire bars, one of which is V-shaped and the immunity against monogenean infections other made up of three parts. To identify (Paperna, 1964, 1991) and more studies the different Cichlidogyrus species, consult should be done to see if this could be used in Paperna (1980). the control of monogenean infections. Prevention and control. As for Dactylogyrus infections. Diseases caused by Cichlidogyrus species

Cichlids are cultured in warm fresh- Trianchoratus and Sundanonchus infections water cages as well as in warm estuarine waters. Tilapias cultured in freshwater Monogenean species belonging to these two are affected by Cichlidogyrus spp., while genera are found to infect the channids. in marine waters they are infected by Sundanonchus spp. are restricted to C. the marine monogenean, Neobenedenia micropeltes while Trianchoratus spp. are melleni (see Chapter 5). Tilapia (cichlids) found on the other channids. Although is cultured in cages in freshwater in these monogenean species are found on and Indonesia, Vietnam and the Philippines known to plague cultured snakeheads, there as well as Malaysia. Cichlids are hosts to is no report of mortality due to these species of Cichlidogyrus, Onchobdella and monogeneans. Enterogyrus (an endoparasitic monogenean present on Sri Lankan cichlids). Sev- Diagnosis. Methods for collecting and eral species of Cichlidogyrus and E. preparation of monogenean species for cichlidarium have been introduced with diagnosis are the same as for the Dactylo- their fish hosts into the Philippines, gyrus spp. above. Trianchoratus spp. have Indonesia and Peninsular Malaysia (L.H.S. four anchors, of which one pair is vestigial, Lim, unpublished data; Shaharom, 1985). no connective bars, 14 marginal hooks The Cichlidogyrus species on tilapia in and a dactylogyrid-type seminal vesicle Indonesia have been incorrectly identified (Lim, 1986), while Sundanonchus spp., as Dactylogyrus spp. (ADB/NACA, 1991). infecting giant snakeheads, can be differ- Cichlidogyrus spp. are also found on tilapia entiated from the other monogeneans in in cages in Vietnam (T.T. Dung, personal having four anchors, with two connective communication). bars (dorsal bar may be split into two), 16 As noted by Paperna (1980) and Paperna marginal hooks, a dactylogyrid-type seminal et al. (1984), no reports of mortality due vesicle and an X-shaped vitelline duct to Cichlidogyrus spp. have been recorded, (Lim and Furtado, 1985; Kritsky and Lim, but Cichlidogyrus sclerosus was found 1995).

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260 G.D. Lio-Po and L.H.S. Lim

Prevention and control. As for Dactylogyrus Prevention and control. As for Dactylogyrus infections. infections.

Diseases caused by Gyrodactylus species Diseases caused by Pseudodactylogyroides marmoratae The gyrodactylids are easily differentiated from the dactylogyrids since they are vivi- Pseudodactylogyroides marmoratae has parous with developing embryos in the been found on cage-cultured O. marmorata, uterus. The young gyrodactylids do not a highly priced fish in Malaysia and Viet- need to search for a host. It has been nam (Leong and Wong, 1998; T.T. Dung, postulated that gyrodactylids are able to personal communication). O. marmorata is disengage and reattach to new hosts, espe- cultured in cages in Peninsular Malaysia, cially under intensive culture where fish Indo-China and Thailand. However, this are in close proximity to each other. fish is no longer cultured in Thailand Some Gyrodactylus spp. have wide because of disease problems (ADB/NACA, host specificity and cause fish mortality. 1991). Other than the fact that this parasite Gyrodactylids are easily translocated via the causes disease, practically nothing is live fish trade, for example Gyrodactylus known about the signs, its pathology or how turnbulli is spread via the aquarium trade to to control this pathogen. England, New England States, Nova Scotia and Peru from Singapore (Cone, 1995). Diagnosis. The monogenean species are col- Although the gyrodactylids are important lected and prepared as given for the Dactylo- pathogens in warmwater culture systems, gyrus species above. Morphologically, Pseu- there is a paucity of information on this dodactylogyroides spp. (Fig. 7.5) possess four group. Studies on the pathogenicity of the anchors, of which one pair is usually under- Gyrodactylus spp. are mostly from temper- developed and small and the larger pair has a ate countries (Paperna, 1991; Cone, 1995). patch-like inner root, two connective bars, Gyrodactylus infection is common on 14 marginal hooks and a dactylogyrid-type Clarias spp. such as C. batrachus, C. macro- seminal vesicle (Lim, 1995). cephalus, C. gariepinus and the hybrid of C. macrocephalus and C. gariepinus reared Prevention and control. As for Dactylogyrus in cages in Thailand (Aqua Farm News, infections. 1993). Paperna (1991) reported Gyrodact- ylus rysavyi and Macrogyrodactylus on C. Diseases caused by Thaparocleidus species gariepinus in Africa. These parasites may have an impact on the future of cage culture Species belonging to this monogenean of C. gariepinus in the Ivory Coast as well as genus are found on cultured pangasiids and the Clarias culture in Thailand, the Philip- bagrids in Southeast Asia (Lim, 1990; pines and Indonesia. Gyrodactylus fuscus Lerssutthichawal, 1999; A. Pariselle, per- has been found on Clarias fuscus in North sonal communication). As with Pseudo- Vietnam. Unidentified Gyrodactylus spp. dactylogyroides, little is known about infect cage-cultured L. hoevenii and P. the pathology caused by this group of gonionotus in Indonesia (Christensen, monogeneans, or its level of pathogenicity. 1989). Gyrodactylus is found on tilapia cultured in freshwater and brackish waters Diagnosis. Monogenean species from (Natividad et al., 1986). pangasiids are collected and prepared as for Dactylogyrus infection above. Thaparo- Pathology. Gyrodactylus spp. are usually cleidus spp. (Fig. 7.6) have four anchors, two found on the skin and fins, although there connective bars, one of which may be whole are species that can be found on the gills. or separated into two, 14 marginal hooks and They are also found in conjunction with a sac-like seminal vesicle (Lim, 1996). protozoan and bacterial infections. Mucus

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Infectious Diseases of Warmwater Fish in Fresh Water 261

Fig. 7.5. Pseudodactylogyroides marmoratae from the gills of Oxyeleotris marmorata (reproduced with permission from Systematic Parasitology).

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262 G.D. Lio-Po and L.H.S. Lim

Gyrodactylus exhibit abnormal behaviour, i.e. rubbing against the net, anorexia, hyper- production of skin mucus, haemorrhagic ulcers on the body sides, fin rot (mainly anal and caudal fins) and thickening and opacity of the eye cornea. At this stage, it is easy to detect parasites on the eyes, skin and fins. The skin usually appears whitish. At the later stage of infections, reddish inflamed areas develop on the skin and the eyes may become opaque and blind (Christensen, 1989).

Diagnosis. Initial diagnosis can be based on clinical signs with confirmation by examina- tion of the parasites. The monogeneans are collected from skin and gill scrapings and prepared as for Dactylogyrus spp. (see above). The anterior region of the gyrodactylid is divided into two lobes with two sets of head glands. Its haptor is armed with 16 hinged, marginal hooks, two anchors and two con- nective bars. Gyrodactylus spp. are difficult to identify (Paperna, 1991). The body size, excretory systems, dimensions and morpho- logy of the sclerotized parts (reproductive spines, anchors, marginal hooks, connective bars) are important criteria for species differ- entiation (Malmberg, 1970). Gyrodactylus spp. in the tropical regions are poorly stud- ied and more investigations are required.

Prevention and control. A formalin bath using 20–25 ml of 40% formalin in 100 l of well-aerated, clean water for 30 min is used. Other formulations include formalin at 1:2000 for 10 min and ammonia solution − at 1.5 ml ammonia 1 1. The latter two methods reduce infections but do not eradicate them. Trichlorphon (0.25 ppm) is also effective (Meyer, 1968).

Diseases caused by other helminths Fig. 7.6. Thaparocleidus caecus from the gills of Pangasius hypophthalmus (reproduced with Although there are pathogenic trematodes, permission from The Raffles Bulletin of Zoology). nematodes, cestodes and acanthocephalans in tropical aquaculture (Paperna, 1996), secretion is increased during heavy infec- the pathogenic species causing disease in tions, fins become frayed, skin ulcerated and tropical cage culture systems are unknown. gills damaged by the feeding and attachment For instance, trematodes and cestodes have processes of the worm. Fish infected with been found in cage-cultured Pangasius

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Infectious Diseases of Warmwater Fish in Fresh Water 263

bocourti and in snakeheads in Vietnam but Kabata, 1985) but little is known about their their identities are unknown (T.T. Dung, effect in cage culture. personal communication). With the intro- duction of more exotic species into tropical Cestode infection waters there will probably be more reports of helminthic parasites in aquaculture in The adult Asian tapeworm, Bothriocephalus the future. The impact of helminthic infec- acheilognathii, causes mortality in heavily tions in aquaculture is not known: although infected grass carp in Europe (Paperna, the intestines of several feral giant snake- 1996). The Asian tapeworm is not confined heads infected with the acanthocephalan to cultured imported carp but has spread to species Gorgorhynchus ophicephali were native fish in warm waters of Asia (Peninsu- observed with perforations (personal obser- lar Malaysia) and Israel with the imported vation), its impact on cultured giant grass carp, silver carp and bighead carp (Sha- snakeheads is not known. Since there are harom, 1985; ADB/NACA, 1991; Paperna, no reports of massive mortalities caused 1991, 1996). Paperna (1996) has provided a by cestodes, trematodes or nematodes, the detailed account of the disease caused by following sections will briefly deal with this cestode species. Cestodes are also pres- known helminth infections in fish species ent in cultured and wild fish in warm that are cultured in cages in warm waters. waters: Lytocestus spp. are found in cul- tured and wild C. batrachus, while Senga Trematode infection spp. are found in cultured and wild snake- heads, Channa spp. (Furtado, 1963; Furtado Sanguinicola (blood fluke) has been and Lau, 1971; Furtado and Tan, 1973). recorded on exotic cultured grass carp and Cestode infections in fish and resulting bighead carp (Anderson and Shaharom- mortality are sporadic. Fish infested with Harrison, 1986). Thus far, no Sanguinicola intestinal (adult) cestodes have retarded has been reported on clariids of Southeast growth, erratic swimming behaviour and Asia although Sanguinicola dentata is distended abdomen, become emaciated, found on Clarias lazera (now known as C. cease to feed, develop a haemorrhagic enteri- gariepinus) from Africa (Paperna, 1996) and tis caused by the destruction of the intestinal this species has been imported into Thai- epithelium, and heavily infected fish have land for culture purposes. Metacercariae varying degrees of aseptic dropsy (Paperna, causing ‘black spots’ in cichlids and clariids 1996). The cyclopoid copepod is the inter- in Africa (Paperna, 1996) could spread to mediate host, and the cestodes could be an other tropical waters. Kabata (1985) noted important pathogen in cage culture systems the presence of clinostomatids and hetero- since fish are in intimate contact with the phyids in farmed fish in the warm waters, environment. but thus far none have been reported among cage-cultured fish. Diseases caused by parasitic arthropods Nematode infection Lernaea and Ergasilus spp. (Copepoda), Nematodes are common on feral as well Argulus (Branchiura) and Alitropus as food fish (L.H.S. Lim, unpublished data; (Isopoda) have been recorded on a wide Kabata, 1985). The nematode Anguillicola range of cultured fish species (Kabata, 1985; crassa could become important since its ADB/NACA, 1991). Lernaea and Argulus host, A. japonicus, is cultured in Taiwan cause the most problems in warmwater and on a smaller scale in Indonesia. The aquaculture in Southeast Asia and the other nematode of importance is Philomet- Indian continent. They were introduced roides cyprini in common carp (Paperna, into these countries via fish importation 1996). Camallanids are common on feral (ADB/NACA, 1991) and will be discussed catfish (L.H.S. Lim, unpublished data; in greater detail below. The isopod

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264 G.D. Lio-Po and L.H.S. Lim

Alitropus is another common arthropod in aquaculture systems (Fig. 7.7) and is associ- ated with poor fish growth and increased fish mortality (L.H.S. Lim, unpublished data; Lester and Roubal, 1995). It could be a potential pathogen in warm freshwater cage cultures. However, nothing much is known about its impact in aquaculture. Infestation by the copepods Ergasilus, besides causing cage-cultured fish to lose weight and appear unsightly, causes gill damage and, in heavy infestations, results in gill dysfunction (Kabata, 1985). Ergasilus was recorded to cause fish mortality in Indonesia, especially in young fish (ADB/NACA, 1991). Ergasilus is a common crustacean parasite of fish and a potential pathogen in cage culture systems; however, little is known about its ecology or pathology.

Lernaea infections

Lernaea or anchor worm causes the most damage in warm freshwater fish and is usually associated with high mortality. Lernaea spp. seem to prefer warm waters of 26–30°C (Shields and Tidd, 1974). Although it is known that this parasite causes disease in cage-cultured fish in Fig. 7.7. Alitropus species (Isopoda) found on the skin of Channa micropeltes (picture by K.S. Liew). Southeast Asia, the extent of its impact and damage to aquaculture has not been esti- mated (Kabata, 1985). Java and North Sumatra (ADB/NACA, 1991). In Southeast Asia, Lernaea polymorpha is Pathology. Lernaea cyprinacea is distrib- found on bighead carp and silver carp uted widely with the global translocation of (Shariff and Sommerville, 1986). carp and is now recorded in 45 species of Haemorrhaging and gross lesions occur cyprinids as well as in other orders of fish, at the site of Lernaea infections and are asso- especially the siluriforms (Lester and Roubal, ciated with bacterial and other secondary 1995). Lernaea is found in India, Nepal, infections. There are relatively few studies Bangladesh, Thailand, Indonesia, Pen- on the effects of anchor worm infection on insular Malaysia, Vietnam, China and Japan the fish hosts in warm waters. Some authors (ADB/NACA, 1991). Lernaeosis occurs in suggest that the attached females feed on China on silver carp, bighead carp, grass host blood, while others suggest that they carp and black carp, in India and Bangladesh probably ingest host cells and absorb tissue on all the major carp, in Vietnam on bighead fluids (Egusa, 1992). Lester and Roubal (1995) carp, grass carp, silver carp, common carp, provided detailed information on the other crucian carp and snakehead, and in Indone- signs associated with Lernaea infections, and sia on common carp, P. gonionotus, spotted these included blindness, epidermal and gourami, mudfish and catfish. In 1976, these dermal necrosis and haemorrhage and encap- parasites reached epizootic levels, destroy- sulation of the embedded horns of Lernaea. ing about 30% of fish in over 7500 ha of Copepodids of Lernaea may cause disrup- ponds, ricefields and open waters in West tion and necrosis of the gill epithelia, and

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large numbers of larvae on the gills may Copepodid stages are killed in 24–36 h cause fish mortality. Lesions caused by pen- at 0.2 ppm and in 12–18 h at 0.5 ppm at etration of metamorphosing females are gen- 20°C, but Dipterex is not effective on adult erally associated with punctuate haemor- females. However, in cages located in rivers rhage, and muscle necrosis is evident at the or large lake systems, the use of chemicals is point of penetration of parasites (Khalifa and ineffective and dipping fish in chemicals Post, 1976). Penetrating female L. polymor- seem to be insufficient to get rid of all the pha cause punctuate haemorrhage in big- copepodid stages (Lester and Roubal, 1995). head carp resulting in mortality in heavily infected fish (Shariff and Sommerville, 1986). Argulus infections L. cyprinacea in the eyes cause blindness. The majority of the branchiurans are fresh- water parasites (about 75% of the 120 Diagnosis. There are over 40 species of species of Argulus), with few estuarine or pathogenic Lernaea (Kabata, 1983), but marine species (Kabata, 1985). Argulus or in most outbreaks, the specific identity of fish louse (Fig. 7.8) is macroscopic and eas- the parasite is unknown. Lernaea spp. are ily observable on the skin and fins and also macroscopic and easily seen with the naked in the oral cavity. Infected skin becomes eye on the surface of fish. Only females opaque with frayed fins. This ectoparasitic of Lernaea are parasitic and are highly crustacean feeds on the mucus layer, flesh modified so they do not resemble free-living and blood of the fish. The prolonged feed- copepods. Adult Lernaea females have ing and strong attachment of Argulus by their anterior end embedded into the body its suckers on to the host result in direct musculature of their host, while their long mechanical damage to the skin and dis- rod-shaped body with two egg sacs pro- ruption of epithelial structure, resulting in trudes outside the host tissue. The anterior lesions and subsequent invasions by oppor- head region is modified as a small hemi- tunistic pathogens such as pseudofungi spherical cephalothorax, which contains (Singhal et al., 1986; van der Salm et al., the mouth, with a well-developed holdfast, 2000). There are at least four species of bifurcate dorsal process and simple ventral argulids (Branchiura, Argullidae) that are process (anchor). The anterior region may economically important as parasites of fish even penetrate into the body cavity and in warm freshwater aquaculture, i.e. Arg- embed into visceral organs. Lernaea spp. are ulus japonica, Argulus foliaceus, Argulus distinguished by the shape of the anterior indicus and Argulus siamensis, and these anchors, which may be modified by bone or have been introduced along with their other structures encountered during devel- cyprinid hosts and are now reported opment in their host tissue. Ergasilus,onthe from both local indigenous cyprinids and other hand, is recognizably a copepod with a second antenna modified for attachment and a pair of multiseriate egg sacs arising from the genital segment.

Prevention and control. Several chemicals are recommended but their efficacy requires further careful testing (Kabata, 1985; Egusa, 1992; Paperna, 1996). Kasahara (1962) effectively used Dipterex (organophosphate trichlorphon) to control and eradicate the larval stages of L. cyprinacea in the water column. At temperatures of 20–27°C, con- Fig. 7.8. Argulus species (Branchiuran) found centrations of 0.5 and 0.2 ppm kill on the skin of Channa micropeltes (picture by the nauplii in 1 and 2 days, respectively. K.S. Liew).

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266 G.D. Lio-Po and L.H.S. Lim

non-cyprinid hosts in the introduced areas secondary infections. Kabata (1970), (Paperna, 1991). A. japonica is in Israel, Paperna and Zwener (1976) and Paperna while A. foliceaus is in Thailand, Penin- (1980) noted that lytic and toxic substances sular Malaysia and Sri Lanka on carp and were secreted into the dermal area, while native cyprinid species (Kabata, 1985). feeding caused acute haemorrhagic, A. indicus, an Asiatic species, is on anaban- inflamed wounds. Argulus feeding on blood tids, chaniids, tilapias and native cyprinids causes fish to become anaemic and its pierc- in Indonesia, Thailand and India. A. sia- ing proboscis stylet causes haemorrhagic mensis is reported in Thailand from ana- spots on the epidermis. The spots are formed bantids and Cirrhina spp. (Gopalakrishnan, by epidermal hyperplasia. Bacterial infec- 1968; Kabata, 1985) and in India on a tions occur around the site of infection. snakehead species (Channa gachua) (Rama- Argulus may also be a vector of viral infec- krishna, 1951). A. japonica is an important tions. Ahne (1985) showed that spring parasite of warm freshwater fish, while viraemia of carp (SVC) was transmitted by A. Argulus coregoni parasitizes cold fresh- foliaceus, and in Israel, carp pox (carp water fish. papilloma) occurred in conjunction with A. japonica infestation (Sarig, 1971). Pathology. This parasite is not host-specific and is found on a wide range of fish species Diagnosis. The parasite is oval to round, from cyprinids to siluriforms and perciforms dorso-ventrally flattened (about 4–8 mm (see above). The life cycle of the parasite in diameter), with a pair of modified is direct and the eggs hatch into free- sucker-like first maxillae. Its proboscis or swimming larvae, which must find a host feeding organ is for inserting into the epider- within 2–3 days. It is reported to cause mas- mis and the underlying tissue of the fish sive mortality of fish in Bangladesh, and in hosts to feed on blood (Fig. 7.8). the majority of cases the outbreaks were sea- sonal, usually in the colder months (Kabata, Prevention and control. Several chemicals, 1985). Argulus usually infects the young fish especially organophosphate insecticides, from spring until early summer. The parasite formalin, chlorine, sodium chloride and is also common in India, affecting the major even antimalarial drugs, are recommended Indian carp, especially Rohu spp. In Penin- (Kabata, 1985; Egusa, 1992; Lester and sular Malaysia, argulids have been found Roubal, 1995; Paperna, 1996), but their on wild fish such as C. micropeltes (L.H.S. efficacy in different types of water bodies Lim, unpublished data), the imported fry is not known. Studies carried out in warm of bighead carp and grass carp (Shaharom, waters of Israel and Africa show that some 1988). Argulus spp. are found on the sand (see below) of the insecticides are effective goby and snakeheads in cages in Vietnam in killing argulids within the safety margin (T.T. Dung, personal communication). for fish (Paperna, 1996). Lindane has been Heavily infected fish are lethargic, listless, used to clean fish of argulids prior to market- cease to feed and rub themselves on the sub- ing (Paperna, 1996). The chemicals in use strate in an attempt to dislodge the parasite. are gemmexane (this is toxic to fish and The lesion or wound made by the feed- man), Pyrethrum (not tested on a large ing Argulus may be restricted to the epider- scale yet) (Paperna, 1996), Dipterex, tricho- mis or may penetrate through to the stratum lorphon, Neguvon, malathion, formalin spongiosum of the dermis and even the and antimalarial drugs such as quinine stratum compactum turning the dermis hydrochloride (13 ppm) (Puffer and Beal, oedematous (Lester and Roubal, 1995). The 1981; Kabata, 1985; Singhal et al., 1986). area may become necrotic with secondary However, not all the chemicals are equally bacterial and fungal infections. Mortality effective for the different developmental may be associated with changes in the ionic stages of argulids. For example, Dipterex and osmotic homeostasis, anorexia and (0.2–0.3 ppm) is effective against the adults

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and larvae causing them to fall off the fish Despite the long history of aquaculture and die but has no effect on the eggs (Egusa, in the tropics and the importance of disease 1992). The water chemistry and temperature in aquaculture, there have been few con- are important factors in the use of these certed efforts to document and investigate chemicals. The occurrence of these para- the diseases of fish cultured in cages and sites, despite the amount of chemicals ponds. This may be due to lackof trained used, indicates that the eggs are still in the manpower and institutional support. The system and that the chemicals used are not diversity of fish cultured in warm waters effective in destroying the eggs. The strategy does not help to alleviate this problem. of not stocking the ponds until the larval The usual approach to disease and health stages have died could be effective. Other management is to use chemicals (usually methods of control include the use of indiscriminately) or, if this does not work, to substrates such as wooden slats to trap eggs, discard the fish species and start afresh with filtering incoming water to remove larval another species. Also, there is no mandatory stages, stocking clean fish, quarantining requirement in many of the developing and incoming fish with treatment if necessary underdeveloped countries to report fish before stocking, and stocking with argulid- death, and until recently fish were usually predatory fish (Kabata, 1985). imported and stocked without quarantine. It must be taken into consideration that the movement of fish, especially across Conclusions and Recommendations international boundaries, may transfer fish for Future Research pathogens as well (Hedrick, 1996). In this regard, the provisions of the Office Inter- Microorganisms and parasites are normal national des Epizooties (OIE) (1995) Inter- flora and fauna inhabiting the skin, fins, national Aquatic Animal Health Code gills and the gastrointestinal tract of fish. should be adhered to. Under normal conditions, many of these The lackof institutional support results organisms do not induce disease in their in reduced research on pathogens and conse- fish host. However, man-made pollutants quently an inability to control and prevent and/or intensification of fish culture diseases. There is also a lackof trained per- result in increased environmental changes, sonnel in disease management and little which may be stressful to fish. Bacterial reliable information on the specific identity multiplication, for instance, is enhanced of pathogens. A related issue is the lackof with increasing organic matter from legislation and guidelines pertaining to the uneaten feeds. The stress predisposes fish use of drugs and chemicals in aquaculture. to invasion by opportunistic pathogens, Currently, drugs and chemicals are used with subsequent morbidity and mortality. indiscriminately (usually the aetiological Stress is also associated with handling, agents are not identified), and without a stocking, grading and shipping of fish. specific withdrawal period prior to the sale Often fish mortality can be attributed to of the fish. Trained competent fish disease several factors (e.g. fish condition, patho- managers, who are able to diagnose patho- gens and environment) and it is difficult to gens and are capable of dispensing proper determine the significance of any one of prevention and control measures, are impor- these factors (Mitchell, 1997). Parasites such tant to sustain aquaculture. as the monogeneans may not have a direct effect on fish mortality but they debilitate the fish, making it more susceptible to other Acknowledgements pathogens. Their organs of attachment usu- ally create portals of entry for viral, bacterial We would like to thank and acknowledge and pseudofungal pathogens of fish. In addi- the information provided by Dr Richard tion, some parasites are reservoirs of viral Arthur (Canada), Dr F. Shaharom pathogens. (Universiti Putra Malaysia, Trengganu

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Branch, Malaysia), Dr S. Chinabut (AAHRI, batrachus. In: Suwignyo, P., Tjitrosoma, S.S. Bangkok, Thailand), Dr T.S. Thana (Depart- and Umaly, R.C. (eds) Practical Measures for ment of Fisheries, Phnom Penh, Cambodia), Preventing and Controlling Fish Diseases. Dr T.T. Dung (Department of Freshwater Proceedings of the Symposium, Bogor, Indonesia, 24–26 July 1985. Biotrop Special Aquaculture, College of Agriculture, Cantho Publication No. 28, pp. 129–137. University, Cantho, Vietnam) and Dr E.M. Angka, S.L., Lam, T.J. and Sin, Y.M. (1995) Some Leaño (SEAFDEC, Iloilo, Philippines). Dr virulence characteristics of Aeromonas J.A. Plumb (Department of Fisheries and hydrophila in walking catfish (Clarias Allied Aquacultures, College of Agricul- gariepinus). Aquaculture 130, 103–112. ture, Auburn University, Alabama, USA) is Annual Fisheries Statistics (1998) Volume 1. also gratefully acknowledged for sharing Department of Fisheries Malaysia, Ministry information and for his critical review. of Agriculture Malaysia, Kuala Lumpur, 193 pp. Aoki, T. (1999) Motile aeromonads (Aeromonas References hydrophila). In: Woo, P.T.K. and Bruno, D.W. (eds) Fish Diseases and Disorders. Vol. 3. Viral, Bacterial and Fungal Infections. CAB ADB/NACA (1991) Fish health management. In: International, Wallingford, UK, pp. 427–453. Asia-Pacific Report on a Regional Study and Aqua Farm News (1993) Catfish Culture, Vol. Workshop on Fish Diseases and Fish Health XI(6). SEAFDEC Aquaculture Department, Management. ADB Agriculture Department Iloilo, The Philippines. Report Series No. 1. Network of Aquaculture Areechon, N., Kitancharoen, N. and Tonguthai, K. Centers in Asia-Pacific, Bangkok, 627 pp. (1992) Immune response of walking catfish Ahne, W. (1985) Argulus foliaceus L. and Piscicola (Clarias macrocephalus Gunther) to vaccina- geometra L. as mechanical vectors of spring tion against Aeromonas hydrophila by injec- viraemia of carp virus (SVCV). Journal of Fish tion, immersion and oral administration. Diseases 8, 241–242. In: Langdon, J.S., Enriquez, G.L. and Alawi, H. and Rusliadi (1993) Culture of tilapia Sukimin, S. (eds) Proceedings of the Sympo- (Oreochromis niloticus) fish in cage culture sium on Tropical Fish Health Management in in Sungai Kanpar (Sumatra): growth and Aquaculture, Bogor, Indonesia, 14–16 May production of tilapia with different stocking 1991. Biotrop Special Publication No. 48, densities. Terubuk (Indonesia) 19, 12–31 (in pp. 143–151. Indonesian with English summary). Areerat, S. (1987) Clarias culture in Thailand. Albaladejo, J.D. and Arthur, J.R. (1989) Some Aquaculture 63, 355–362. trichodinids (Protozoa: Ciliophora: Peri- Ariel, E. and Owens, L. (1997) Epizootic mortali- trichida) from freshwater fishes imported ties in tilapia Oreochromis mossambicus. into the Philippines. Asian Fisheries Science Diseases of Aquatic Organisms 29, 1–6. 3, 1–25. Arthur, J.R. (ed.) (1987) Fish Quarantine and Fish Alexopoulos, C.J., Mims, C.W. and Blackwell, M. Diseases in South and Southeast Asia: 1986 (1996) Introductory Mycology, 4th edn. John Update. Asian Fisheries Society Special Pub- Wiley & Sons, New York, 869 pp. lication 1, Asian Fisheries Society, Manila, Anderson, I.G. and Shaharom-Harrison, F. (1986) 86 pp. Sanguinicola armata infection in bighead Arthur, J.R. (ed.) (1992) Asian Fish Health Biblio- carp (Aristichthys nobilis) and grass carp graphy and Abstracts 1: Southeast Asia. Fish (Ctenopharyndogodn idella) imported in Health Section Special Publication No.1, Malaysia. In: Maclean, L.B., Dizon, L.B. and Fish Health Section of Asian Fisheries Soci- Hosillos, L.V. (eds) The First Asian Fisheries ety, Philippines and International Research Forum. Asian Fisheries Society, Manila, Centre of Canada, 252 pp. pp. 247–250. Arthur, J.R. and Lumalan-Mayo, S. (1997) Check- Angka, S.L. (1990) The pathology of the walking list of the Parasites of Fishes of the Philip- catfish, Clarias batrachus (L.), infected intra- pines. Food and Agriculture Organization of peritoneally with Aeromonas hydrophila. the United Nation, Fisheries Technical Paper Asian Fisheries Science 3, 343–351. 369, 102 pp. Angka, S.L., Wongkar, G.T. and Karwani, W. Austin, B. and Austin, D.A. (1987) Bacterial (1988) Blood picture and bacteria isolated Pathogens: Disease in Farmed and Wild Fish. from ulcered and crooked-back Clarias Ellis Horwood Ltd, UK, 364 pp.

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