Fish 17

599
CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
17
Saprolegnia and other Oomycetes
D.W. Bruno and B.P. Wood
FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen AB11 9DB,
UK.
INTRODUCTION
Fungal infections of fish by Oomycetes, commonly known as water moulds, are
widespread in fresh water and represent the most important fungal group
affecting wild and cultured fish. The Saprolegniaceae, in particular members of
the genus Saprolegnia, are responsible for significant infections, involving both
living and dead fish and eggs, particularly in aquaculture facilities. Oomycetes
are classical saprophytic opportunists, multiplying on fish that are physically
injured, stressed or infected (Pickering and Willoughby, 1982a). Members of
this group are generally considered agents of secondary infection arising from
conditions such as bacterial infections, poor husbandry, infestation by parasites
and social interaction. However, there are several reports of Oomycetes as
primary infectious agents of fish (Willoughby, 1978; Pickering and Christie,
1980) and their eggs (Walser and Phelps, 1993). They can readily become
pathogens, resulting in epizootics among salmonids and other teleosts (Hatai and
Hoshiai, 1992, 1993; Bly et al., 1994; Diguez-Uribeondo et al., 1996).
Saprolegnia may occur anywhere on the body of fish, but normally appears as a
conspicuous, circular or crescent-shaped, white, cotton-like mycelium,
particularly around the head and the caudal and anal fins (Neish and Hughes,
1980; Willoughby, 1989; Noga, 1993a). It may spread over the body by radial
extension until adjacent lesions merge.
The Oomycetes are an economically important group of mycotic agents that
affect salmonids and other teleosts. Four orders are recognized in this class and
the most important are the Saprolegniales. Although eight genera have been
reported in naturally or artificially induced infections, Saprolegnia, Achlya,
Aphanomyces, Calyptratheca, Thraustotheca, Leptolegnia, Pythiopsis and
Leptomitus, only Saprolegnia, Achlya and Aphanomyces are significant in
aquaculture (Aller et al. 1987; Hatai and Hoshiai, 1993; Bly et al., 1994). Some
species are consistently isolated from fish and generally these are assigned to a
600
single major cluster, which form a coherent, separate taxon, Saprolegnia
parasitica Coker (synonym Saprolegnia diclina Humphrey type 1) (Willoughby,
1978). Type 1 material is distinguished by long-hooked hairs or bifurcate
appendages on primary and secondary zoospore cysts and indirect spore
germination (Willoughby et al., 1983). A limited number of fungal species from
fish living in brackish waters are reported (Schperclaus et al., 1992). In the
marine environment, Oomycetes are significant pathogens of lobsters and
crayfish. The reader is referred to Taugboel et al. (1993) and Diguez-Uribeondo
et al. (1994) for recent reviews.
The ultrastructure, biochemistry and molecular sequences of the Oomycetes
show their phylogenetic roots with the Chromista, the chromophyte algae, and
other Protista rather than the true fungi (Beakes, 1989; Dick, 1990;
Kwon-Chung and Bennett, 1992). Several characteristics, including cell-wall
composition and ribosomal deoxyribonucleic acid (DNA) sequences,
differentiate the water moulds from the true fungi. Polymerase chain reaction
(PCR) techniques are improving our understanding of taxonomic relationships
within the Oomycetes, and developments such as random amplified polymorphic
DNA (RAPD)-PCR (Diguez-Uribeondo et al., 1996; Whisler, 1996) are likely
to help resolve taxonomic problems and enable additional comparative studies
within the Oomycetes.
The Oomycetes have a complex life cycle, involving sexual and asexual
stages (Kanouse, 1932; Noga, 1993a). Sexual reproduction involves the
production of the antheridium and the oogonium gametangia, which fuse to
allow fertilization. Asexual reproduction may occur by the production of
chlamydospores or gemmae, but generally zoospores are produced. Motile
primary zoospores, produced by the zoosporangium, encyst and may germinate
producing a vegetative growth or a secondary motile zoospore. Germination
produces a hypha, which may develop into a mycelium.
Fungal outbreaks among farmed fish stocks are frequently associated with
poor water quality, injuries associated with handling and grading, temperature
shock and spawning. For many years infections have been largely controlled
with malachite green (Bailey, 1983a; Willoughby and Roberts, 1992a).
Unfortunately, the potential teratogenic or mutagenic properties of malachite
green (Meyer and Jorgenson, 1983; Clemmensen et al., 1984; Fernandes et al.,
1991) have resulted in its limited or curtailed use in many countries. The search
for alternative chemical treatments or other means of controlling fungal
infections has resulted in many investigations being conducted. However, to
date, only a limited number of chemical compounds show any potential as
fungicides and none are considered as effective as malachite green. Currently,
the most successful strategy for the control of Saprolegnia in farmed fish
represents a combination of farm management, husbandry practices and
chemical bath treatments. Generally, a combination of these practices is
important in preventing or limiting fungal outbreaks.
Histological changes resulting from oomycete infection include loss of
integrity of the integument, oedema and degenerative changes in the muscle
mass. More severe lesions show deeper myofibrillar and focal cellular necrosis,
spongiosis or intracellular oedema and sloughing of the epidermis (Copland
601 Saprolegnia and Other Oomycetes
and Willoughby, 1982), allowing hyphae to penetrate the basement membrane
(Bootsma, 1973).
Most of the literature on oomycete infections of teleost fish concentrates on
salmonids (Coker, 1923; Willoughby, 1985; Noga and Dykstra, 1986) and forms
the predominant part of this review. The genus Saprolegnia with reference to
Achlya and Aphanomyces is included and the term fungal is used to represent
fungal-like Protoctista or flagellated water moulds.
ECONOMIC SIGNIFICANCE
Fish mycoses are considered difficult to prevent and treat, particularly in
intensive fresh-water systems, and are reported to be second only to bacterial
disease in economic importance to aquaculture (Meyer, 1991). Water mould
epizootics are generally rare in wild fish populations, however, during the 1960s,
thousands of wild Atlantic salmon, Salmo salar, died during their migration into
rivers in the British Isles and southern Ireland from ulcerative dermal necrosis
(UDN) (see review by Roberts, 1993). Such outbreaks increased public
awareness of the disease in wild salmon stocks and consequently promoted an
increase in research. Saprolegnia parasitica has been identified from many
fishes and is considered by several investigators as a significant pathogen
(Willoughby, 1969). Despite a great deal of work, the cause of UDN was
controversial (Stuart and Fuller, 1968) and was not established (Carberry, 1968).
In the eastern USA, up to 80% of the commercial catches of Atlantic
menhaden, Brevoortia tyrannus, were affected with the disease ulcerative
mycosis (synonyms: epizootic ulcerative syndrome (EUS), red-spot disease)
(Callinan, 1985; Callinan et al., 1995; Lilley and Roberts, 1997). The ulcerative
mycosis was attributed to an Aphanomyces infection, which occurred during the
unusually wet spring and summer of 1984, which created conditions of low
salinity. These losses, with sporadic outbreaks in earlier years, represented a
severe threat to this industry, which, at the time, was worth $27 million per
annum (NCDMF, 1983). In economic terms, Aphanomyces, particularly within
the Australo-Pacific and southern Asia, is considered the most significant
infection affecting cultured fish (Frerichs et al., 1986; Hatai et al., 1994).
Outbreaks of Saprolegnia spp. in farmed fish are usually restricted to
chronic but steady losses. However, egg mortality can increase rapidly, causing
significant numbers to die, which may have a major economic impact. During
severe winters, some farmers in the USA have reported losses of up to 50% in
farmed catfish, with an annual economic cost of about $40 million (Bly et al.,
1994). Saprolegnia infection is believed to contribute significantly to overall
losses in elver, Anguilla anguilla, culture (Copland and Willoughby, 1982). In
Japan, significant losses, exceeding 50% per annum, attributed almost
exclusively to S. parasitica, have been reported in farmed coho salmon,
Oncorhynchus kisutch (Hatai and Hoshiai, 1992, 1993). Significant mortality
from fungal outbreaks has also occurred in wild and farmed brown trout, Salmo
trutta, in Spain (Aller et al., 1987; Diguez-Uribeondo et al., 1996).
602
THE DISEASE AND AGENT
Historical
Descriptions of Oomycetes infecting fish date from the mid-eighteenth century
(Humphrey, 1893). In 1877, Oomycetes were first reported in association with an
epizootic infection known as salmon disease in rivers between England and
Scotland. A report of the Royal Commission on the Salmon Disease in England
and Scotland (18771879), published by Buckland et al. (1880), failed to reach
any conclusions regarding the cause of the disease. However, some investigators
believed it was due to a single species of fungus, namely Saprolegnia ferax
(Stirling, 1878, 1880, 1881; Huxley, 1882a,b). Others considered that the fungus
was a secondary parasitic infection and that the primary cause of the disease was
bacterial in nature (Rutherford, 1881; Hume Patterson, 1903). Retrospectively,
the salmon disease epizootic was clearly the first recognized case of UDN of
salmonids (Roberts, 1972; Murphy, 1973), the primary aetiology of which is
thought to be viral (Roberts, 1972; OBrien, 1974).
Ulcerative dermal necrosis was first noted in Atlantic salmon in the
Republic of Ireland, with epizootics subsequently occurring in Great Britain
(Munro, 1970) and Europe (de Kinkelin and Le Turdu, 1971). The condition is
unlike other infections involving Saprolegnia, in that lesions occur on unscaled
areas of the body, primarily the head. These lesions develop from small oval
patches to become ulcerated, haemorrhagic and infected with Saprolegnia.
Some evidence for the role played by Saprolegnia in UDN comes from studies
by Dunne (1970) and Roberts et al. (1971), who found that these early lesions
would heal if treated with malachite green.
Species of fish affected and geographical distribution
Infections of fish involving members of the Oomycetes are reported extensively
in both wild and farmed fish (Willoughby and Pickering, 1977; Neish and
Hughes, 1980) and are considered ubiquitous in freshwater ecosystems (Neish
and Hughes, 1980; Waterstrat, 1997). Many researchers reported infections on
salmonids and, in the UK, Saprolegnia sp. has been isolated from Atlantic
salmon (Willoughby, 1986), rainbow trout, Oncorhynchus mykiss, brown trout
and Arctic char, Salvelinus salvelinus (Pickering and Christie, 1980; Wood and
Willoughby, 1986). Hatchery-reared and wild brown trout succumbed to
infection by S. diclina at spawning time (Pickering and Christie, 1980), while
increased losses of cultured rainbow trout eggs have been associated with
infection by S. ferax. Bruno and Stamps (1987) reported losses of farmed
Atlantic salmon first-feeding fry due to S. diclina. Saprolegniasis is a serious
problem in Atlantic salmon hatcheries in Ireland (Smith, 1994) and in Norway
(Langvad, 1994), where infections of Saprolegnia sp. occur in wild Atlantic
salmon parr. In Japan, epizootics due to S. parasitica have occurred in farmed
coho salmon (Hatai and Hoshiai, 1992, 1993). Saprolegnia parasitica and S.
diclina have also been implicated in mortality of cultured rainbow trout, coho
salmon and ayu, Plecoglossus altivelis, in Japan (Yuasa and Hatai, 1995a). The
infection of salmonids with S. parasitica has occurred in Australia (Puckridge et
al., 1989) and the USA (Mueller and Whisler, 1994). Furthermore, Chien (1980)
discovered S. diclina and Aphanomyces laevis in spawning rainbow trout in
Taiwan, and found Pythium debaryanum saprophytic on dead eggs. Reports of
oomycete infections on live fish are summarized in Table 17.1.
There have also been records of oomycete infections on non-salmonid
fishes. In the UK, isolations of members of genera including Achlya,
Aphanomyces, Leptolegnia, Leptomitus, Pythiopsis and Saprolegnia have been
made from perch, Perca fluviatilis (Willoughby, 1970; Pickering and
Willoughby, 1977; Bucke et al., 1979). In France, mortality was caused by
Saprolegnia australis in cultured roach, Rutilus rutilus (Papatheodorou, 1981).
Other coarse fish can be hosts for S. diclina and they include orfe, Leuciscus
idus, carp, Cyprinus carpio, tench, Tinca tinca, and pike, Esox lucius (Pickering
and Willoughby, 1982b). These authors also isolated the fungus from eels and
the river lamprey, Lampetra fluviatilis. Elvers of A. anguilla intensively cultured
in warm-water effluent suffered high mortality due to Saprolegnia sp. (Copland
and Willoughby, 1982). Gajd sek and Rubcov (1985) reported damage to wild
carp eggs in the former USSR caused by Saprolegnia sp. and, in the USA, Xu
and Rogers (1991) isolated it from channel catfish, Ictalurus punctatus, in which
it caused a massive mortality during the winter months (Bly et al., 1992).
Saprolegniasis also affects cultured Atlantic sturgeon, Acipenser oxyrhynchus, at
spawning (Smith et al., 1980) and cultured juvenile barramundi, Lates
calcarifer, in Australia (Glazebrook and Campbell, 1987).
In Nigeria, Ogbonna (1989) showed that a wide range of Oomycetes,
including members of the genus Achlya, Aphanomyces and Saprolegnia, have
been found in various freshwater hosts. Also in Nigeria, Okaeme et al. (1988)
found the eyes of hatchery-reared tilapia, Oreochromis niloticus, infected with
Myxosoma sarigi and with Saprolegnia sp. Fingerlings of another tilapia
species, Oreochromis mossambicus, cultured in South Africa, were reported as
common hosts for Saprolegnia sp. (Oldewage and van As, 1987). In Brazil,
silver mullet, Mugil curema, reacclimatized in fresh water were susceptible to
Saprolegnia sp. (Conroy et al., 1986). Another species of mullet, Liza abu, and
carp cultured in Iraq were infected with S. ferax, Saprolegnia terrestris and
Achlya polyandra (Butty et al., 1989).
Saprolegniasis has also occurred in cultured teleosts in Indonesia (Sirikan,
1981) and India (Srivastava, 1980; Sati, 1991). Das and Das (1993) described an
outbreak known as EUS, in which Saprolegnia was implicated with Aeromonas
hydrophila as affecting a range of teleosts. High mortality, attributed to S.
parasitica, was recorded by Krishna et al. (1990) in various species of carp
cultured in ponds and in silver carp, Hypophthalmichthys molitrix, cultured in
cages (Jha et al., 1984). Furthermore, infection with this oomycete species was
the cause of losses in carp and Indian major carp, Labeo rohita, during the winter
when the water had a high organic loading (Toor et al., 1983). Singhal et al.
(1990) detected a significant suppression of growth in cultured carp, in which
Saprolegnia sp. was associated with an Argulus indicus infection. Air-breathing
teleosts, Anabas testudineus, from a river in India were also found to carry S.
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608
parasitica infections (Mohanta and Patra, 1992). Freshwater tropical aquarium
species commonly living in waters of 23C have also shown clinical signs of
saprolegniasis. These include Plecostomus spp. (Leibovitz and Pinello, 1980)
and the Mexican platyfish, Xiphophorus maculatus (Vishniac and Nigrelli,
1957).
Oomycete infections have also been recorded in the marine environment.
Both Aphanomyces sp. and Saprolegnia sp. have been associated with an
ulcerative mycosis in the USA (Dykstra et al., 1986). The disease was recorded
from a range of estuarine species, primarily the Atlantic menhaden in the north-
western Atlantic Ocean. Deep skin lesions were characteristic of the infection,
which often involved the internal organs and induced an intense inflammatory
reaction (Dykstra et al., 1986; Noga et al., 1988). This is in contrast to the
superficial lesions and mild inflammatory response normally observed in
oomycete infections of freshwater fish (Bly et al., 1992; lvarez et al., 1995). In
many regions of Asia, EUS is a serious disease of estuarine fishes and has been
attributed to Aphanomyces invadens (Lilley et al., 1997a,b). This infection
appears identical to isolates recovered from fish with red-spot disease in
Australia and New Guinea and a mycotic granulomatosis in Japan (Callinan,
1985; Callinan et al., 1995; Lilley and Roberts, 1997). Large but shallow ulcers
resulted from this type of infection involving mullet, Mugil cephalus (Roberts et
al., 1986); although in this latter case the fungus was not cultured, its
morphology was characteristic of an oomycete.
General signs
Saprolegniasis is frequently observed as a superficial and chronic infection, with
the appearance of cotton-wool-like tufts on the integument and gills of host fish
or eggs (Neish and Hughes, 1980), which may spread over the entire body
surface (Richards and Pickering, 1979a). In severe cases, 80% of the body may
be covered (Fig. 17.1). In early infections, skin lesions are grey or white in
colour, with a characteristic circular or crescent shape (Willoughby, 1989),
which can develop rapidly, causing destruction of the epidermis. Lethargy and
loss of equilibrium follow as the infection proceeds, making the fish more
susceptible to predation.
Pathogenic members of the Saprolegniaceae may penetrate major organs
(Bootsma, 1973; Nolard-Tintigner, 1973, 1974; Dukes, 1975; Wolke, 1975;
Hatai and Egusa, 1977), and the terms progressive dermatomycosis or mycotic
dermatomycosis have been proposed (Hatai, 1980b; Wada et al., 1993). The
actual cause of death is likely to be associated with impaired osmoregulation
(Gardner, 1974; Hargens and Perez, 1975). Respiratory difficulties may also
feature when infection is associated with the gills (Bruno and Stamps, 1987).
Lesions do not normally appear at random, but are initially localized in
specific areas associated with physical insult, concurrent infection with another
pathogen (Neish and Hughes, 1980) or sexual differences of the host (White,
1975; Richards and Pickering, 1978). The latter seem attributable to differences
in the number of goblet cells in the skin of male and female fish (McKay, 1967;
Neish, 1977). However, the initial site of infection may not to be confined to the
skin or gills. Staffs disease in carp involves a saprolegniasis of the olfactory pits
(Bauer et al., 1973), infecting parts of the body such as the lateral line and cornea
(Leibovitz and Pinello, 1980). Outbreaks of S. ferax involving the gut epithelium
have been reported in brook trout, Salvelinus fontinalis (Agersborg, 1933), and
rainbow trout fingerlings (Davis and Lazar, 1941). More recently, the alimentary
tracts of rainbow trout and amago salmon, Oncorhynchus rhodurus, fry were
sites of infection for S. diclina (Hatai and Egusa, 1977; Miyazaki et al., 1977).
Members of the genus Saprolegnia can be considered opportunistic
facultative parasites, which are both saprotrophic and necrotrophic (Cooke,
1977). Peduzzi and Bizzozero (1977) suggested isolates of the S. parasiticaS.
diclina complex and S. ferax were capable of progressing from saprotrophs to
necrotrophs, due to their possessing a proteolytic enzyme, which resembles
chymotrypsin. However, there is evidence that some Saprolegniaceae act as
primary pathogens (Neish, 1977; Willoughby and Pickering, 1977; Willoughby,
1978; Noga et al., 1988). Many workers have successfully infected fish with
Saprolegnia under experimental conditions (Tiffney, 1939a; Vishniac and
Nigrelli, 1957; Hoshina et al., 1960; Scott and Warren, 1964; Nolard-Tintigner,
1970, 1971, 1973, 1974; Srivastava and Srivastava, 1977a,b,c), but it is
questionable whether these parasites can cause primary infection.
Fig. 17.1. Saprolegnia growth on the dorsal areas of an Atlantic salmon, Salmo salar, brood
fish.
610
Predisposing factors
It is generally assumed that fish are continually exposed to potentially
pathogenic fungi. It therefore follows that a change in some predisposing factor
or factors is necessary for infection to occur. Salmonids are susceptible to
saprolegniasis throughout the freshwater stage of their life cycle, particularly
leading up to and during smoltification (Pickering, 1994). Although S. parasitica
can survive low salinity (Langvad, 1994), it cannot withstand full salinity sea
water and therefore the infection is absent from the marine phase in anadromous
salmonid hosts. Saprolegniasis also shows a distinct seasonality, and this varies
with the species of Saprolegnia. For example, S. diclina infections are more
common in winter months (Hughes, 1962), whereas S. ferax occurs
predominantly in the spring and autumn (Coker, 1923; Hughes, 1962; Klich and
Tiffney, 1985).
Role of sexual maturation
The association of Saprolegnia infection with sexual maturation and a similar
increase in susceptibility to some common skin parasites, e.g. Ichthyophthirius
and Trichodina are well documented (Pickering and Christie, 1980). Sexual
maturation in the brown trout is accompanied by dramatic changes in epidermal
structure and a decrease in mucus cells at the end of the spawning period
(Pickering, 1977; Pickering and Richards, 1980), which is considered to
exacerbate their susceptibility (Noga, 1993a). Although precocious mature male
Atlantic salmon parr are susceptible to saprolegniasis, they have an increased
number of mucus cells (Murphy, 1981). However, no decrease in mucus cells
during the prespawning period occurs, even with marked differences in
susceptibility to fungal infections (Pickering and Christie, 1980). The retention
of zoospores of S. diclina on the epithelium of rainbow trout was also enhanced
if experimentally challenged fish were previously implanted with the androgen
11-ketotestosterone (Cross and Willoughby, 1989). Interestingly, the gross
crescentic patterns of fungal growth reflected those previously seen only on wild
salmonids. Cross and Willoughby (1989) postulated that viable hyphae persisted
only at the circumference of the advancing colony and this form of growth
created the characteristic pattern.
Richards and Pickering (1978) noted that fungal lesions were common on
the dorsum in mature males and on the caudal fin in mature females. The
activation of the pituitaryinterrenal axis in teleosts is recognized as an almost
ubiquitous component of the response to many different factors, most of which
are considered stress related (Pickering, 1981). An increase in circulating
corticosteroids has been used to assess the importance of the stress response in
fish with Saprolegnia infection (Pickering and Duston, 1983). Prolonged oral
administration of cortisol or natural increases in this hormone resulted in a
marked increase in the susceptibility of the fish to fungal infection. However,
their plasma cortisol levels were within the levels capable of being produced by
fish under natural stress (Pickering and Pottinger, 1985). Several authors have
reported the immunosuppressive role of raised cortisol in salmonids (Pickering
and Pottinger, 1985; Bennett and Wolke, 1987). A chronic increase in
corticosteroid levels in brown trout from 14 ng ml
1
to 910 ng ml
1
increased
the susceptibility of the fish to Saprolegnia infection (Pickering and Pottinger,
1985). Observed changes may be due to the fungal infection in salmonids being
associated with an increase in cortisol and certain sex hormones and therefore an
increase in susceptibility to saprolegniasis (Pickering, 1977). When brown trout
parr were treated with the chemosterilant methallibure, during the later stages of
spermatogenesis, hyperplastic changes in the epidermis were prevented and the
prevalence of saprolegniasis reduced (Pottinger and Pickering, 1985). A similar
finding was noted by Murphy (1981) with precocious male 1+ Atlantic salmon
parr.
Integument integrity
Increased susceptibility to Saprolegniaceae from damage to the epidermis has
been shown in fish under experimental conditions (Tiffney and Wolf, 1937;
Tiffney, 1939b; Hoshina and Ookubo, 1956; Vishniac and Nigrelli, 1957;
OBier, 1960; Scott and Warren, 1964; Srivastava and Srivastava, 1977a,b; Hatai
and Hoshiai, 1994). Mechanical damage from high stocking densities of farmed
brown trout was thought to be responsible for an increased incidence of
Saprolegnia infection (Richards and Pickering, 1978). The association of sexual
maturity with the elevated occurrence of infection may, in part, be attributable to
tegument damage sustained during spawning (Richards and Pickering, 1978).
Concurrent infection
Many species of Saprolegnia act as secondary invaders, and prior infection with
a primary pathogen renders the host more susceptible to the opportunistic
fungus. The condition UDN is a classical example where the disease was
characterized by secondary Saprolegnia infection following an initial viral
infection (Stuart and Fuller, 1968; Willoughby, 1968; OBrien, 1974).
Primary bacterial infection associated with Saprolegnia sp. has been
recorded in the Japanese eel, Anguilla japonica (Hoshina and Ookubo, 1956;
Egusa, 1965; Egusa and Nishikawa, 1965). Concurrent infestations with
Saprolegnia sp. were observed in wild Atlantic salmon infected with
Gyrodactylus salaris (Johnsen, 1978) and Gyrodactylus sp. (Heggberget and
Johnsen, 1982), which damaged the skin of the host.
Environmental stress
Environmental stress factors, including poor water quality, adverse water
temperatures and, in aquaculture, handling or overcrowding, can all result in
increased occurrences of fungal infections (Bailey, 1984). Annual outbreaks of
saprolegniasis in wild brown trout were partially the result of an increase in
organic debris in the water and a decreased flow rate (White, 1975). High
organic loadings were also identified as a cause of increased infection by S.
parasitica (Toor et al., 1983). Furthermore, rainbow trout exposed to sublethal
levels of ammonia and nitrite increased their susceptibility to experimental
infection with S. parasitica (Carballo and Muoz, 1991). Social aggression in
rainbow trout can increase susceptibility to this fungus (Cross and Willoughby,
1989).
612
THE MICROORGANISM
Taxonomic position
The identification and classification of Saprolegnia and other Oomycetes have
historically been a problem, and the taxonomic position of this group remains
uncertain. This is largely because oomycete taxonomy is essentially based upon
morphology of reproductive structures and certain biochemical and
physiological characteristics (Dick, 1973; Dick et al., 1984; Beakes et al.,
1994a). Delimiting genera on the basis of morphological features of
reproductive structures is unsatisfactory (Hughes, 1994), as these structures are
variable under different environmental conditions. For example, Salvin (1941)
and Scott (1956) showed variations in zoosporangia structure, resulting in
Saprolegnia isolates behaving like Achlya species. Furthermore,
zoosporogenesis of Achlya can be affected by calcium ion deficiency (Griffin,
1966) and sexual structures of Saprolegnia are often not produced when
cultivated in vitro (Seymour, 1970; Pickering and Willoughby, 1982b). None the
less, the distinctiveness and importance of the group are also acknowledged
(Beakes et al., 1994a). Consequently, identification of asexual pathogenic
Saprolegnia isolates is impractical using classical taxonomic criteria. Sexually
sterile Saprolegnia from living fish are usually referred to as Saprolegnia spp.
Despite the difficulties, S. parasitica can be distinguished by the fine structure of
the secondary zoospore cyst cases (Pickering et al., 1979; Sderhall et al., 1991)
and a presumptive identification can be made using phase-contrast microscopy
(Willoughby, 1985). Molecular studies may clarify the taxonomic status of the
Oomycetes.
The Oomycetes are no longer considered true fungi and have been placed in
the kingdom Protoctista (Table 17.2) (Dick, 1990; Kwon-Chung and Bennett,
1992). The class Oomycotea forms part of the phylum Heterokonta (Dick, 1990),
which comprises around 800 species in four orders, namely Saprolegniales,
Leptomitales, Lagenidiales and Peronosporales (Sleigh, 1989). Attention has
also been drawn to the similarity between the oomycete fungi and chromophyte
algae (Beakes, 1989), as the Saprolegniales and xanthophyte algae share many
structural and physiological features. Several workers have suggested a possible
phylogenetic relationship and shared line of evolution between heterokont fungi
and chromophyte algae (Gunderson et al., 1987; Beakes, 1989; Frster et al.,
1990; Barr, 1992).
Beakes (1989) proposed that zoospore and cyst characteristics could be
employed as taxonomic and phylogenetic markers. On this basis, Beakes (1989)
identified two major groupings of genera and species within the Oomycetes,
which were divided according to their structural characteristics instead of their
accepted taxonomic position. In an attempt to introduce biochemical rather than
morphological characteristics to oomycete taxonomy, Nakamura et al. (1995)
investigated the ubiquinone (coenzyme Q) system of Oomycetes. However, they
concluded that this system, which has proved a useful taxonomic criterion for
other fungi, could not be used to separate genera from the Saprolegniales or
Lagenidiales. Monoclonal antibodies raised against secondary cyst-coat matrix
components of S. parasitica were not specific, reacting with all Saprolegnia
(Burr and Beakes, 1994). Similarly, monoclonal antibodies to a wide range of
determinants of Phytophthora cinnamomi were unreactive with Saprolegnia
(Hardham, 1989; Hardham et al., 1991). Murine polyclonal (Bullis et al., 1990)
and monoclonal (Bullis et al., 1996) antibodies produced against various
determinants of S. parasitica reacted strongly against other isolates of the same
species in an immunofluorescence assay. In the same study, reactions with S.
diclina were also intense, whereas titres against S. australis and Aphanomyces
sp. were notably lower.
The name S. parasitica has clearly been used in two different senses: firstly,
as a repository for any isolate from fish or fish eggs which is incapable of
producing oogonia and, secondly, to describe those isolates which fit the
descriptions of Kanouse (1932) and Coker and Matthews (1937). It is these
different uses to which this name has been put that have led to much confusion
and inaccuracy. In an attempt to solve these problems, Neish (1976) proposed
that the name S. parasitica should be rejected, due to its ambiguity. The author
argued that the criteria of oospore size and type used to differentiate between S.
parasitica and other Saprolegnia isolates were of little diagnostic value and
members of this species would be better assigned to S. diclina Humphrey.
Willoughby (1978) considered the taxonomy of S. parasitica and S. diclina
in detail, and noted that there was sufficient similarity for all the isolates to be
considered in the S. parasiticadiclina complex, and included Saprolegnia
kauffmaniana Pieters, Saprolegnia shikotsuensis and S. australis. Furthermore,
Willoughby (1978) subdivided the S. diclina into three subspecific groups, based
upon oogonia morphology, with S. diclina type 1 infecting salmonid fish species
and being synonymous with S. parasitica (Kanouse, 1932) and Saprolegnia sp.
type 1 (Willoughby, 1969, 1971, 1972; Pickering and Willoughby, 1977), type 2
occurring as a parasite of coarse fish and type 3 being entirely saprophytic, with
the zoospores showing direct germination (Hatai and Hoshiai, 1992). Any
Table 17. 2. Taxonomic relationship of Oomycetes recorded in aquaculture.
Kingdom: Protoctista
|
Subkingdom: Mastigobionta
|
Division: Oomycota
|
Phylum: Heterokonta
|
Class: Oomycotea
|
Order: Saprolegniales Leptomitales Lagenidiales Peronosporales
| | | |
Family: Saprolegniaceae Leptomitaceae Lagenidiaceae Pythiaceae
| | | |
Genera: Achlya Leptomitus Lagenidium Pythium
Aphanomyces
Saprolegnia
614
sexually sterile isolates were termed Saprolegnia sp. In support of these
findings, Beakes and Ford (1983) could distinguish between type 1 and 3
isolates on the basis of their esterase isoenzyme profiles. Many subsequent
studies have adopted this approach (Beakes, 1983; Willoughby et al., 1984;
Wood and Willoughby, 1986; Wood et al., 1988; Cross and Willoughby, 1989).
However, the confusion was perpetuated when Hatai and Willoughby (1988)
classified a non-fruiting Saprolegnia isolated as S. parasitica. Research has
moved away from the S. parasiticadiclina complex, referring simply to S.
parasitica Coker (syn. S. diclina Humphrey type 1) (Hatai et al., 1990), and
isolates continue to be classified in this taxon without sexual characteristics
being observed (Willoughby and Roberts, 1992a). This taxonomic description
and terminology are widely accepted (Singhal et al., 1987; Willoughby, 1989;
Willoughby and Roberts, 1992a; Beakes et al., 1994a).
Most of the Oomycotea reported as parasites of live fish are in the
Saprolegniales, with one in each of the orders Leptomitales, Lagenidiales and
Peronosporales (see Table 17.1). The table includes isolations made from living
fish from either natural infections or experimental challenges. The most
prominent of these genera, with respect to the number of species and the
frequency of their isolation, are Aphanomyces and Saprolegnia. Consequently,
most of the following discussion concentrates upon species from these two
genera and Saprolegnia in particular.
THE LIFE CYCLE
The Saprolegniaceae are filamentous, coenocytic organisms, producing a
profusely branching aseptate vegetative mycelium. The life cycle of S.
parasitica (Fig. 17.2) was first described by Kanouse (1932).
Primary zoospores
Unicellular, biflagellate zoospores are produced in sporangia, which are usually
terminal and separated from the hyphal filaments by basal septa. The zoospores
are diplanetic and dimorphic, comprising a pyriform primary and reniform
secondary form, each with a different point of flagellum insertion. Primary
zoospores (Fig. 17.3) are normally covered in single unbranched hairs of some
1.5 mm in length (Pickering et al., 1979). They are poor swimmers, serving
merely to achieve a minimal level of dispersal from the parent colony upon
release from the zoosporangia. Upon settlement, zoospores encyst to form thin-
walled cysts, often called encysted zoospores or cytospores.
Secondary zoospores
Primary cysts germinate and release the secondary zoospore (Fig. 17.4), the
main motile stage, which remains active for extended periods until encystment
occurs on a suitable substrate or host. It is assumed that the secondary zoospore
is an important dispersive phase of the life cycle and represents the main
infective spore. Pickering and Willoughby (1982b) suggested that these spores
may be chemotrophic, thereby enhancing the probability of pathogenic strains
locating a fish host, although Smith et al. (1985) suggested that zoospores of
pathogenic strains of Saprolegnia spp. were not attracted to live fish eggs. In
contrast, Rand and Munden (1993b) demonstrated that extracts prepared from
the chorionic membrane of live brook trout eggs induced a strong chemotactic
response in zoospores of S. diclina.
Secondary zoospores are capable of polyplanetism, by which repeated
cycles of encystment and release are undergone if an appropriate substrate is not
initially found. This represents an important adaptive feature of the life cycle,
Fig. 17.2. Life cycle of Saprolegnia and other water moulds (based on Neish and Hughes, 1980).
616
which enables propagules (Fig. 17.5) to make several attempts at locating
appropriate substrates before they finally germinate to form hyphal elements
terminated with zoosporangia (Beakes et al., 1994a). The actual stimuli for
encystment and germination remain uncertain, but the phenomenon of
polyplanetism suggests they are different. None the less, Willoughby et al.
(1983) demonstrated differences between species of Saprolegnia in their ability
to encyst under different environmental conditions. Willoughby et al. (1983)
discovered that pathogenic isolates encysted more readily in sterilized lake
water, which led Pickering and Willoughby (1982b) to propose that such isolates
require a lower level of nutrient to stimulate encystment. Pickering and
Willoughby (1982b) demonstrated that fish mucus stimulates encystment of
zoospores from pathogenic isolates. Beakes et al. (1994b) reported other factors
that induced encystment of S. diclina zoospores, including vortexing and
incubation in dilutions of bovine serum albumin, haemoglobin and hemp-seed
extract.
The surface of primary and secondary zoospores contains a glucomannan
glycocalyx, which binds concanavalin A (ConA) (Lehnen and Powell, 1993;
Burr and Beakes, 1994). In thin-sectioned material, binding is associated with
Fig. 17.3. Scanning electron micrograph (SEM) of primary zoospore of Saprolegnia parasitica
isolate (TP41). Spore width = 5 mm. (A.W. Burr and G.W. Beakes, University of Newcastle,
unpublished material.)
Fig. 17.4. Scanning electron micrograph (SEM) of secondary zoospore of isolate TP41. The
mastigoneme hairs on the anterior flagellum appear as bobbles. (A.W. Burr and G.W. Beakes,
unpublished material.)
Fig. 17.5. Cryofixed scanning electron micrograph (SEM) of a germinating encysted zoospore
of Saprolegnia parasitica (A.W. Beakes, unpublished material).
618
the outermost layer of the cyst wall and particularly the short primary cyst-coat
spines (Burr and Beakes, 1994). Encystment vesicles, formerly known as
bar-bodies due to their characteristic shape in S. ferax (Heath and Greenwood,
1970), are found in the Saprolegniaceae (Beakes, 1983; Randolph and Powell,
1992). Upon encystment, these form circular plaques on localized areas of the
cyst surface in S. ferax (Heath and Greenwood, 1970), which, in S. parasitica
and S. diclina, appear as a thin electron-dense layer (Beakes, 1983; Burr and
Beakes, 1994). At the time of encystment, kinetosome-derived vesicles (K
2
bodies) have been observed to release a proteinaceous adhesive substance
(Lehnen and Powell, 1991; Durso et al., 1993; Burr and Beakes, 1994), which
contains catalase (Powell et al., 1985) and is thought to play a role in attachment
of the zoospore to a specific substrate (Lehnen and Powell, 1989).
Ornamentation of the cyst surface in Saprolegnia species isolated from
salmonid lesions was established on whole-mount preparations by electron
microscopy. Bundles of four and 16 elongate (2.514.0 mm), bifurcate, hooked
spines or hairs (Fig. 17.6), often called boat-hooks (Meier and Webster, 1957),
have been observed (Pickering et al., 1979; Beakes, 1983; Willoughby et al.,
1984; Puckridge et al., 1989; Hatai et al., 1990, Beaks et al., 1994b; Mueller and
Whisler, 1994). Similar structures are also present in cysts from saprophytic
species, including S. diclina (sensa stricto) and S. ferax. However, they are
shorter (0.51.0 mm) and occur singly or in small bundles of around four spines.
The hooked spines in Saprolegnia hypogyna also occur singly, but are thicker
Fig. 17.6. Scanning electron micrograph (SEM) of germinating cyst, showing the appearance of
long boat-hook bundles. The individual spines cannot be resolved but bundles (hoops) are clearly
resolved. (A.W. Burr and G.W. Beakes, unpublished material.)
and longer than in other saprophytic species (Pickering and Willoughby, 1982b).
Variation in the number and arrangement of these hooks and their
microarchitecture is reported (Beaks et al., 1994b), with some isolates
demonstrating a high degree of variation in cyst ornamentation (Hatai et al.,
1990). However, Beakes et al. (1994b) and Mueller and Whisler (1994) were
unable to relate any of these differences in Saprolegnia to variation in
morphological type within a geographical area. Interestingly, several workers
have reported a direct relationship between the number of hooks in a bundle and
their length (Pickering et al., 1979; Puckridge et al., 1989; Hatai et al., 1990),
but the significance of this uncertain.
Germination and growth
Germination and growth of pathogenic species of Saprolegnia are rapid when
compared with saprophytic members of the genus and may be determined by the
nutrient levels of the supporting medium (Willoughby et al., 1983). Saprophytic
species, including S. diclina and S. ferax, were normally unable to germinate in
sterilized lake water, whereas isolates from salmonid lesions, including S.
diclina type 1 and Saprolegnia sp., germinated readily in the same media
(Willoughby et al., 1983). The rate of germination of these strains was enhanced
two to fivefold in water taken from the effluent of a salmonid fish farm, whereas
saprophytic isolates failed to germinate.
Germination has been described as direct or indirect. Typically, germination
is direct where cytoplasm-filled germ tubes are wide and coenocytic
(Willoughby, 1977). In contrast, Willoughby et al. (1983) and Willoughby and
Roberts (1992a) have shown that germination of parasitic Saprolegnia isolates,
grown in either fish mucus or sterilized lake water, was indirect and
characterized by a partially evacuated germination, whereby the germ tube is
uniform, narrow and septate, with the cytoplasm being confined to the apical tip
alone. This pattern of growth may enable the rapid growth of pathogenic isolates
(Willoughby et al., 1983). However, such a growth pattern varies with the
nutrient loadings of the medium and only occurred when nutrient levels were
either very low or high (Willoughby et al., 1983). Pickering and Willoughby
(1982b) proposed that the evacuated germling type may represent the infective
agent, with the increased length facilitating attachment to the host. Pickering and
Willoughby (1982b) speculated that the extended morphology of these
germlings might enable increased nutrient uptake.
Once germination has occurred, the normal life cycle is sometimes
abbreviated, with the production of an additional zoospore stage, in situations
where nutrient availability is low (Willoughby, 1977). A single sporangium is
formed, releasing one or two secondary zoospores that subsequently encyst in
the normal way. This phenomenon may represent a mechanism that ensures
dispersal during periods when conditions are suitable for encystment and
germination, but not growth.
620
Sexual reproduction
Most Oomycetes species are homothallic, with both male and female sexual
reproductive structures appearing on a single mycelium. In the genus Achlya,
sexual reproduction is heterothallic, occurring only when opposite mating
strains are brought together. In some species, notably S. ferax, the oospores
develop parthenogenetically, without antheridia. Sexual reproduction in
Saprolegnia occurs by gametangia contact, resulting in the fusion of the haploid
oosphere with sperm nuclei (Beakes and Gay, 1977). The oosphere is produced
in lateral or terminal oogonia, reached by sperm via antheridia branches. Both
antheridia and oogonia are separated from the hyphae by septa. Resulting
oospores have a central ooplast (Howard, 1971), which is granular in appearance
and contains large quantities of a lipid, thought to represent feed reserves. In
subgeneric oospores, the ooplast is displaced on to one side. Germination
normally occurs following a resting period, although Saprolegnia oospores are
typically reluctant to do so (Dick, 1972; Dick and Win-Tin, 1973).
DIAGNOSTIC METHODS
Isolation techniques
One primary difficulty encountered when investigating fungal diseases of fish is
isolating the fungus. Many Saprolegnia infections occur in the dermis and
therefore more than one species of fungus may easily occur in lesions at the same
time (Pickering and Willoughby, 1982a,b). Many saprophytic species may be
present and their growth in culture may be rapid, thereby masking the primary
species. This is particularly true of older infections, where the number of
secondary saprophytic organisms is likely to be higher (Alderman, 1982).
Consequently, sampling of dead animals is to be avoided wherever possible
(Willoughby, 1971). A squash preparation of a skin scrape from the fungal lesion
can be usefully employed as a preliminary screening method for fungi and other
ectoparasites (Pickering and Christie, 1980).
Initial isolation is usually made from small pieces of the affected tissue
(approximately 5 mm
3
). Hatai (1989) advised that deep-seated tissues should be
used wherever possible, to limit contamination with bacteria or other fungi.
All tissues should receive some form of pretreatment to remove any contaminant
species. Several regimes have been employed to achieve this, including surface
sterilization by immersion in ethanol, washing with sterile water and irradiation
(Tiffney, 1939b). Glass rings were included by Raper (1937) to reduce bacterial
contamination. Willoughby (1978) described a procedure whereby infected
salmonid tissues were cut into small pieces and incubated in sterile lake water
for up to 5 days at 7C. These were observed regularly for different forms and
a decision was then made whether to make isolations from zoospores or
hyphae. This method, although extremely thorough, is impractical as a routine
diagnostic procedure. Instead, viable fungi can be extracted from the tissues,
following washing, and cultured using an appropriate artificial medium.
To ensure unifungal growth, isolations should be made from single spores or
hyphal tips of the initial culture wherever possible (Neish and Hughes, 1980;
Alderman, 1982). Furthermore, these isolates should be subjected to infection
experiments using the same host species to determine pathogenicity. In practice,
however, this is rarely carried out, and, for salmonids, it would appear that small
numbers of isolations from affected fish are sufficient to determine the
pathogenic role of the fungal isolate (Neish, 1977; Willoughby, 1978). This was
supported by Neish and Hughes (1980), who concluded that Saprolegnia lesions
in salmonids are frequently unifungal and that this species is an aggressive
saprotroph.
Impression smears (Krishna et al., 1990) or squashes (Pickering and
Christie, 1980) are used to provide a presumptive diagnosis. These techniques
are useful in providing an indication of the organism prior to cultivation. They
may also be used for identification to genera. Imprints may be stained with a
lactophenol blue (Krishna et al., 1990) or methylene blue (Bruno and Stamps,
1987) and viewed using light microscopy. Squashes are usually observed using
dark-field or phase-contrast microscopy.
Culture methods
A wide range of media have been used for the isolation and culture of fungi from
fish. In recent years, these have had antibiotics incorporated to help reduce
contamination. However, in some circumstances, the fungi may be too sensitive
to permit the use of antibiotics, as with Aphanomyces astaci, the causative agent
of crayfish plague (Unestam, 1965; Lilley and Inglis, 1997). Penicillin and
streptomycin are the most commonly used antibiotics and are incorporated into
appropriate media at 1501000 iu ml
1
and 2501000 mg ml
1
, respectively.
Other antimicrobial agents, such as chloramphenicol, gentamycin, neomycin,
oxolinic acid, potassium tellurite and carbenicillin, are used to a lesser extent.
Agar plates are inoculated with pieces of infected tissue, using aseptic
technique. The plates are incubated and observed at frequent intervals for
emerging hyphal tips (Noga and Dykstra, 1986) and then transferred to fresh
media to obtain a unifungal culture. Isolations have also been made by
inoculating mycelium directly from fish or eggs on to an agar medium (Neish,
1975; Willoughby and Pickering, 1977). Alderman (1982) suggested that,
whenever bacterial contamination can be kept to a minimum by using
antibiotics, flooding agar plates with a thin layer of fresh or sea water, as
appropriate, is advantageous.
A broad range of media have been used for the culture of fungi from fish
(Table 17.3). Often, the media are supplemented with antibiotics and a low-
nutrient medium is preferred, to reduce growth of saprophytic species and
bacteria (Alderman, 1982; Seymour and Fuller, 1987). For marine or estuarine
species, media can be supplemented with salt, usually at a concentration up to
3% (Hatai, 1989), or prepared using sterile sea water (Fuller et al., 1964; Bian et
al., 1979; Lightner, 1988). Stevens (1974) described a range of media devised
specifically for fungal isolation from the marine environment. Incubation
622
temperatures range between 5 and 37C; however, temperatures of 10, 15 and
20C are most common.
An alternative method to using agar plates is the more traditional system of
baiting. Using techniques described by Johnson (1956), Seymour (1970) and
Stevens (1974), infected fish tissues, soil or water samples are incubated with
hemp seeds. The resulting fungal colonies are transferred either to fresh hemp-
seed media or to sterile water to obtain unifungal cultures. Currently, most
workers appear to use an agar medium. However, Abdul-Karim et al. (1989)
used hemp seeds for the initial isolation of S. ferax, S. terrestris and A. polyandra
from carp and mullet. Chien (1981) isolated S. diclina and A. laevis from
rainbow trout, using the same technique as Ogbonna (1989) in his investigations
of saprolegniaceous fungi in freshwater fish in Nigeria. Furthermore,
Willoughby (1985) advocated the use of culturing on hemp seeds in water as a
satisfactory and rapid method for obtaining germination of Saprolegnia
zoospores, essential if the species of an isolate is to be determined. Following
culture for 13 days, the water was filtered to produce a filtrate containing high
numbers of viable zoospores. This application of hemp-seed culture has
subsequently been employed by many investigators, including Rand and
Munden (1993b), who studied zoospore attachment of S. diclina in brook trout,
and by Wood and Willoughby (1986) in their survey of the Saprolegniaceae
present in Lake Windermere, England. The presence of zoospores and oogonia
Table 17.3. Isolation media employed for infectious Oomycetes.
Media Fungal species Reference
Glucose yeast extract Various Saprolegnia Willoughby and Pickering
agar (GYA) species (1977)
S. dicilinia and S. ferax Hatai and Egusa (1979)
S. parasitica Smith et al. (1985)
Hatai and Hoshiai (1992)
S. diclina, S. parasitica Hatai et al. (1990)
and S. hypogyna
Glucose peptone agar Various Saprolegnia Willoughby and Pickering
(GPA) species (1977)
Willoughby et al. (1984)
Wood and Willoughby (1986)
Sabourauds dextrose S. diclina Chien (1981)
agar (SDA) Bruno and Stamps (1987)
S. parasitica Krishna et al. (1990)
Corn-meal agar (CMA) Various Saprolegnia Willoughby and Pickering
species (1977)
Bullis et al. (1990)
Bly et al. (1992)
Dykstra et al. (1986)
Aphanomyces sp.
Peptone glucose yeast- Lagenidium callinectes Lightner (1988)
extract agar (PYGEA) S. diclina Rand and Munden (1993a)
Peptone yeast glucose Lagenidium sp. Fuller et al. (1964)
sea-water agar (PYGS) Bian et al. (1979)
and the mode of cyst germination of S. parasitica from coho salmon were also
determined following hemp-seed culture (Hatai and Hoshiai, 1992).
Wallpaper paste (Polycell) has been used as an alternative to traditional agar
(Willoughby et al., 1984), particularly for the culture of Saprolegnia from water
samples (Willoughby, 1986; Wood and Willoughby, 1986) and fish (Singhal et
al., 1987). The paste seems to act as a good physical support for the fungus,
allowing separation of the growing colonies. Samples are mixed with nutrients
and the paste in a Petri dish and incubated at 20C for 2472 h, after which time
single colonies may be transferred to sterile water for identification of sporangia,
zoospores and oogonia. More recently, Celio and Padgett (1989) reported
improved recovery of Oomycetes using hydroxyethyl cellulose (Natrosol 250) as
a replacement for wallpaper paste.
Preservation of cultures may be carried out by frequent subculturing
(Bullock, 1989) or by conserving in mineral oil (Buell and Weston, 1947).
Identification
Identification of oomycete fungi has relied largely upon micromorphology and
sporulation characteristics (Coker, 1923; Sparrow, 1960; Seymour, 1970;
Willoughby, 1978). Sexual reproductive stages are usually required to enable
accurate identification of a species (Wood and Willoughby, 1986). However, one
problem facing the diagnostic mycologist is the lack of sexual structures
produced under culture conditions, making accurate identification to species
very difficult (Pickering and Willoughby, 1982b). In spite of such difficulties,
secondary cyst ornamentation, including features such as size, shape and nature
of the oogonia surface and wall, has been successfully described in S. parasitica
(Pickering et al., 1979; Hallett and Dick, 1986; Puckridge et al., 1989; Hatai et
al., 1990; Sderhll et al., 1991). The origin of the antheridial branch is another
important diagnostic feature. Antheridial cells may be hypogynous, i.e. borne
immediately beneath the oogonium on the oogonial stalk, or androgynous,
whereby the antheridial branch originates from the oogonial stalk. A
monoclinous antheridium does not come from the oogonial stalk, but originates
from the same hypha as the oogonium, whereas a diclinous antheridium arises
from a different hypha.
Many investigators have adopted the approach of Willoughby (1985), who
examined the mode of cyst germination in water alone, along with secondary
zoospore cyst ornamentation, using electron microscopy or phase-contrast light
microscopy to identify cultures of Saprolegnia from fish. Using these methods,
Willoughby (1985) could distinguish between parasitic and saprophytic species
of Saprolegnia, based upon the presence of long hooked hairs on the zoospore
cyst in the former group.
Due to the variation in gross morphological features, efforts have been made
taxonomically to identify species at the ultrastructural and molecular level.
Shadow-cast preparations, displaying the presence of hooked hairs on secondary
zoospore cysts, have been used to differentiate species of Saprolegnia (Pickering
et al., 1979; Hallett and Dick, 1986; Hatai et al., 1990). Furthermore, Yuasa and
624
Hatai (1994) attempted to identify Saprolegnia, Achlya and Aphanomyces from
their biological characteristics. They investigated optimal growth temperature
and sensitivity to a range of chemicals and found differences between genera and
their sensitivity to malachite green, sorbose and Polyphenon
1
00. They were also
able to differentiate between a saprophytic and a pathogenic strain of Achlya
piscicidia.
Initial indications suggest that developing an immunological assay to
identify oomycete infections in clinical outbreaks may be feasible (Bullis et al.,
1990). Murine polyclonal antibodies to S. parasitica were observed to cross-
react with other Saprolegnia species, but with further investigation these authors
were confident a more specific assay could be developed. Unfortunately, cross-
reaction with all Saprolegnia genera was also observed with monoclonal
antibodies raised against the same species (Burr and Beakes, 1994). These
authors also found that the monoclonal antibody cross reacted with other genera
within the Saprolegniales, including A. astaci and Achlya sp.
The DNA base composition analysis has also been examined and is thought
to be a promising potential taxonomic tool for infrageneric separation of
Oomycetes (Green and Dick, 1972). However, Neish and Green (1976)
subsequently suggested that Saprolegnia may be a homogeneous taxon and
showed that DNA base analyses cannot be employed in the separation of species
of this genus. None the less, these authors discovered that the guaninecytosine
(GC) ratios of all members of the genus fell within a very narrow range and they
were therefore able to exclude species on this basis. In contrast, Chaplitski et al.
(1986), using a different technique, obtained lower GC ratios in S. terrestris and
Saprolegnia mixta.
A PCR to examine ribosomal DNA from Saprolegnia isolates obtained from
many geographical locations was developed by Molina et al. (1995). They
discovered that the use of endonuclease BstUI produced identical fingerprints
from all strains of S. parasitica examined, and suggested that this could form the
basis of a diagnostic test to be applied in the absence of antheridia and oogonia.
Diguez-Uribeondo et al. (1996) have also developed a method employing
RAPD, using PCR with DNA from S. diclinaparasitica complex isolates. They
showed that Spanish isolates of this fungus had a genetic similarity of 85100%,
compared with only a 2045% similarity with other strains of the S. diclina
parasitica complex. Whisler (1996) noted that S. parasitica isolates from the
Columbia River basin, USA, recognized by RAPD analysis, were representative
of the vast majority of the isolates cultured from fungal lesions. The use of single
and paired primers with PCR amplification permitted identification of
pathogenic groups and their distinction from other species of the genus
considered more saprophytic in character (Whisler, 1996).
Rapid methods
Preliminary investigations using polyclonal (Bullis et al., 1990) and monoclonal
antibodies (Beakes et al., 1994b; Burr and Beakes, 1994, Bullis et al., 1996)
raised against various epitopes of Saprolegnia indicate that developing a rapid
antibody-based assay for the detection of oomycete infections of fish may be
possible. However, no such techniques have been developed to date. The
successful development of a PCR technique for the identification of the S.
diclinaparasitica complex (Diguez-Uribeondo et al., 1996) suggests that
employing this method as a rapid detection test should also be feasible.
TRANSMISSION
Fish pathogens, including members of the class Oomycetes, are transmitted via
several sources, including wild and farmed fish, their eggs, the water-supply,
transport vehicles, movement of staff between aquaculture facilities and farm
equipment, such as nets. Transmission within the Oomycetes occurs directly
between fish and/or eggs, with no intermediate host being involved (Singhal et
al., 1987). Susceptibility to infection changes with prevailing circumstances,
and several key factors are known to affect both the sensitivity of the fish and the
growth of the fungus. These include pollution, low water levels, overcrowding,
mechanical trauma, including handling, the failure to remove moribund and
dead fish or ova, changes in hormonal status and the result of infection by other
organisms (Piper et al., 1982; Plumb, 1984). In addition, there may also be some
seasonal variation in inoculum potential (Hunter, 1975). Among wild fish, redd
digging and spawning also contribute to physical damage and therefore the
possibility of increased fungal infection (Richards and Pickering, 1978).
There are at least three lines of defence against Saprolegnia infection
following the challenge of fish with zoospores (Wood et al., 1988). The skin is
the first point of contact for infection, and increased secretion of fish mucus
following contact with secondary zoospores may serve to reduce the number of
parasites on the fish surface. Pickering (1994) concluded that there was
sufficient circumstantial evidence linking a decrease in mucification from fish
with increased susceptibility to fungal infection. Willoughby (1989) also
suggested this process represented an important defence against infection.
Secondly, a morphogen from the external mucus could inhibit mycelia growing
from spores (Wood et al., 1988), and finally a cellular response was detected in
the external mucus. Thus, the mucosal layer acts primarily as a physical barrier
to colonization by fungi or other infectious agents. Components in the cyst-coat
matrix aid physical entanglement with the fish surface (Beakes et al., 1994b),
and, in the presence of a sufficiently high inoculum, surface mucus readily
accumulates propagules at 2060 times the concentration of spores in the
surrounding water (Wood et al., 1988). However, Willoughby and Pickering
(1977) noted that the number of spores on the skin of brown trout was greatly
reduced during the first 24 h following exposure of the fish to Saprolegnia.
Mucus removed from the surface of fish triggered encystment of zoospores from
pathogenic strains of Saprolegnia, with mycelial growth following rapidly
(Pickering and Willoughby, 1982b). As a result of damage to the epithelium and
loss of goblet cells, mucus production ceases and it is at this stage that fungal
spores are more likely to attach.
Adhesion of Saprolegnia to the egg membrane is believed to involve the
626
production of a non-fibrous material by the fungus (Beakes, 1983). The
formation of discrete points of attachment to the egg appear raised, and this
suggests that the associated thalli are firmly attached to the surface (Rand and
Munden, 1993a). Shortly after exposure, encysted spores and young thalli can be
recorded on the egg. The development of thalli and their spread and penetration
into the egg are considered important factors for the establishment of infection in
hatcheries. Within 124 h postexposure, the egg surface becomes covered by
branching thalli, spreading over or sometimes invading the chorionic membrane.
Dead eggs are particularly susceptible and infection may spread rapidly to viable
eggs (Rand and Munden, 1993a). In this context, it is not surprising that
chorionic membrane extracts have been identified as factors stimulating positive
chemotaxis of zoospores towards live eggs (Rand and Munden, 1993b). A
positive chemotactic response towards arginine and alanine was also detected,
but no such response was observed to other amino acids or sugars tested. These
results indicate that chemotaxis may have an important role in attracting
zoospores of S. diclina towards live salmonid eggs. Rand and Munden (1993b)
concluded that chemoattractants might be useful in the development of
strategies to control or eliminate Saprolegnia among salmonid eggs raised in
hatcheries.
Infectious disease outbreaks always pose a risk to farmed stock, despite the
quality of husbandry practices employed. The main factor determining whether
Saprolegnia infection occurs is considered to be the physiological state of the
fish (Pickering and Christie, 1980; Cross and Willoughby, 1989). Sexually
mature, stressed or damaged fish are the most susceptible to infection. Fungal
outbreaks in hatcheries can result in substantial egg mortality (Smith et al.,
1985), as hyphal growth on the chorionic membrane spreads rapidly, especially
if dead eggs are not removed.
Trade in aquatic animals, especially the movement of fish to countries where
they are not indigenous, represents a risk to fisheries and aquaculture (Lilley et
al., 1997b). The use of RAPD-PCR enabled Lilley et al. (1997b) to show that
isolates of A. invadens, obtained from fish reared in several different countries,
had identical nucleotide sequences and could not be differentiated by restriction
fragment length polymorphism. Lilley et al. (1997b) concluded that the lack of
genetic diversity might explain many cross-border outbreaks of EUS, ulcerative
mycosis or red-spot disease recorded over the last 25 years.
CONTROL AND TREATMENT
Treatment and protection
Fungal overgrowth on developing salmonid eggs and fry is a widespread
problem. Overcrowding, handling, temperature changes, parasitism, increased
organic loading and sexual maturation increase the likelihood of Saprolegnia
and other diseases (Toor et al., 1983; Pickering, 1994). Where fungal infections
are slight, lesions may be treated with some success. Fish may also recover if
they migrate naturally or are moved to estuarine or sea water (Pickering and
Willoughby, 1982b; Noga et al., 1988). However, salmonids with significant
saprolegniasis do not normally recover, since, once the fungus penetrates its
host, it is largely protected from chemical treatments.
Many potential control methods for the Oomycetes are known, but direct
comparison between such reports is difficult. For example, the range of factors
influencing exposure of fish and particular chemicals have not always been
standardized. This is particularly relevant where the relationship between an
increase in water temperature and the toxicity of malachite green has been
examined (Alderman and Polglase, 1984; Alderman, 1985).
The efficacy of antifungal compounds in the presence of excretory products
was noted by Willoughby and Roberts (1992a). They found that S. parasitica
zoospores in pure water were sensitive to penicillinstreptomycin, but to a lesser
extent in waters containing high levels of nutrients resulting from fish excretory
products. Interestingly, 0.1 mg l
1
malachite green oxalate was effective at
inhibiting germination of zoospore cysts in pure and nutrient-enriched water, but
not in the presence of dense plankton concentrations.
The main methods of fungal control on eggs involve removal of dead or
infected eggs at regular intervals, and chemical bath treatments. However, both
approaches are generally time-consuming and therefore costly in terms of staff
time. Other husbandry practices employed to reduce the likelihood of an
outbreak include the use of an elevated water flow to roll fish eggs. Rach et al.
(1995) held uninfected and fungus-infected rainbow trout eggs at various flow
rates. Eggs maintained at a flow rate of 300 and 600 ml min
1
showed no rolling,
a higher rate of infection and reduced hatching success. At 1200 ml min
1
, the
eggs were lifted into the water column and rolled moderately and hatching was
significantly increased, without fungal growth. The success of this physical
method is dependent upon maintaining flow rates at levels that induce a
moderate rolling of the eggs. Other areas considered, which achieved variable
degrees of success, include the exploitation of biological control, using bacteria,
protozoa or crustaceans that feed on or parasitize fungal hyphae (Oseid, 1977;
Willoughby and Roberts, 1992b), and the use of ultraviolet irradiation
(Kokhanskaya, 1973; Sako and Sorimachi, 1985). Overall, the most successful
strategy for the control of Saprolegnia and similar infections in farmed fish is a
combination of management techniques and chemical bath treatments.
Chemical treatment methods
In recent years, the search for alternative methods and compounds for the control
of oomycete outbreaks has increased and the efficacy of many potential
fungicides have been tested, using in vitro screening methods. Bailey (1983a,b)
outlined a method for screening aquatic fungicides using agar plugs containing
fungal hyphae removed from the edge of actively growing colonies. Plugs were
placed on to the depressions of spot plates containing the test compound. This
approach enables many compounds to be screened in a reasonable time, and
helps identify those compounds with fungicidal activity. Furthermore, it was
found that this in vitro testing correlated well with in vivo testing of surface
628
infections of fish. Bailey (1983a) recommended that the mean activity of each
test should be carried out in triplicate for each compound and be compared with
the activity obtained for malachite green. Theoretically, selected fungicides
should control aquatic fungal growth for at least 48 h (Bailey, 1983a). For in
vitro testing, he advocated using corn-meal agar (CMA) as a suitable media for
testing potential fungicides, as it is readily available, maintains a stable pH and
promotes fungal growth. In preliminary screening of Saprolegnia and Achlya, a
culture temperature of 20 2C, with a pH of 6 0.2, was proposed. Other
recommendations for standardization were also outlined, and the use of this
method, or slight variations of it has been adopted. The acceptance of
compounds with fungicidal potential depends on the adoption of these specific
criteria, and a summary of the criteria for acceptance or rejection is presented in
Table 17.4.
Bailey and Jeffrey (1989) established minimum inhibitory concentrations
for each compound they examined. Fungal growth (colony diameter) was
categorized into three levels of activity, high ( 10 mg l
1
), moderate (> 10
100 mg l
1
) or low (> 100 mg l
1
). More than 50% of compounds tested were
unsuitable as aquatic fungicides. After further testing, the activities of the
remaining compounds were tested against fungal-infected rainbow trout eggs.
The number of comprehensive studies carried out shows the range of chemicals
tested in aquaculture and the importance of finding reliable fungicidal or
fungistatic agents. Bailey and Jeffrey (1989) examined more than 200
compounds to determine their antifungal activity against pathogenic fungi, such
as S. hypogyna and Achlya flagellata.
Further work, including the testing of candidate compounds, is undoubtedly
required, as new regulations are introduced that limit the compounds used in
aquaculture. This is particularly relevant within the European Union (EU),
where all compounds used as medicines must be given official approval through
a licence or registration before their use is permitted.
Since the selection of aquatic fungicides is frequently based upon
comparative studies, using malachite green as a reference compound (Bailey and
Jeffrey, 1989), it is appropriate that a brief review on the status of this compound
is included.
Table 17.4. Criteria for acceptance or rejection of candidate aquatic
fungicide compounds (based on Bailey, 1983a; Marking et al., 1994b).
1. The activity of the candidate fungicide must be less than 100 mg l
1
2. A candidate fungicide must show the desired level of activity in 1 h
exposures, except candidates for pond treatments
3. The efficacy of the fungicide must be reproducible in repetitive tests and
be at least 50% that of malachite green after 48 h of incubation
4. The fungicide must be soluble in suitable carriers or capable of remaining
in homogeneous suspension to provide an effective contact time
5. The fungicide must kill (fungicidal action) or totally inhibit (fungistatic
action) the growth of mycelia for at least 48 h after exposure
6. There must be a satisfactory margin of safety between therapeutic and
toxic concentrations
Malachite green
Malachite green has been used in fish aquaculture facilities worldwide to control
or prevent freshwater fungal outbreaks for more than 40 years (Foster and
Woodbury, 1936) and is typically administered as a bath or flush treatment for
fish and eggs. However, it has never been registered for use on feed fish (Allen et
al., 1994). The compound is a diaminotriphenylmethane or rosaniline dye and is
related to two common laboratory compounds, basic fuscin and crystal violet.
Malachite green is a commercial dye, commonly used in the ceramic industry
and as an additive and commercial dye in the paper industry (Alderman, 1985).
It is not well defined chemically (Clemmensen et al., 1984) and is of variable
purity (Alderman, 1994). For aquaculture purposes, two main forms of
malachite green are available in the UK, the dry-powder oxalate salt and a
nominal 50% solution of a zinc-free malachite green hydrochloride. Although
the latter is the most frequently sold formulation (Alderman, 1985, 1994),
published data would clearly establish a difference between these two
formulations in terms of chronic toxicity to fish (Lanzing, 1965; Alderman,
1985).
Malachite green is a successful therapeutant in fish culture (Olah and
Farkas, 1978; Bailey, 1984; Willoughby and Roberts, 1992a) and is also
effective against some protozoa, including the PKX organism (Leteux and
Meyer, 1972; Clifton-Hadley and Alderman, 1987). The chemical controls
Saprolegnia infections on eggs with minimal mortality if used at concentrations
between 3 and 5 mg l
1
for a 60 min exposure (Bailey, 1983a; Marking et al.,
1994a,b). Cline and Post (1972) reported that 2 mg l
1
malachite green was
fungicidal. During continuous exposure, malachite green is effective at killing
zoospores and inhibiting hyphal growth at 0.25 mg l
1
(Willoughby and Roberts,
1992a). Furthermore, the inhibition of zoospore germination (0.060.50 mg ml
1
)
and hyphal growth (0.52.0 mg ml
1
) is reported for Saprolegnia, Aphanomyces
and Achlya (Srivastava and Srivastava, 1978; Hatai et al., 1994; Yuasa and
Hatai, 1995b). Other studies report similar values (Cline and Post, 1972;
Srivastava and Srivastava, 1978; Hatai et al., 1994; Yuasa and Hatai, 1995b).
Willoughby and Roberts (1992a) considered that a contact time of 15 min at
10C would be sufficient to kill mycelia established on fish within the previous
24 h, and therefore control could be achieved through a single flush treatment of
malachite green. In the same study, the sensitivity of fine, delicate and wider
mycelia to malachite green was reported as a function of mycelium diameter.
The production of fine mycelia is assumed to occur in the fish mucus within 24 h
of infection, supporting the theory that pulse treatments of malachite green
would be effective in controlling outbreaks. Wider fungal mycelia were believed
to develop in areas of higher nutrient levels such as those occurring when the
epidermis was breached.
Several studies associated with malachite green have identified
toxicological (Bills et al., 1977; Meyer and Jorgenson, 1983) and potentially
mutagenic properties (Clemmensen et al., 1984; Fernandes et al., 1991). In
addition, there are implications for this compound as a teratogen (Steffens et al.,
1961; Meyer and Jorgenson, 1983) and a tumour promoter in animals
(Fernandes et al., 1991). In some studies, however, results relating to toxicity
630
have been carried out using a product of undefined or varying quality. Minor
impurities may contribute to reported toxicity and their function requires further
study (Alderman, 1985). Furthermore, malachite green is reported as irritating to
mucous membranes and mutagenic in the Salmonella microsome mutagenicity
test (Clemmensen et al., 1984). Fish subjected to excessive exposure to
malachite green may also show respiratory distress (Alderman, 1994). Meyer
and Jorgenson (1983) reported spinal, head, fin and tail abnormalities in trout fry
hatched from eggs exposed to standard repetitive malachite green treatments,
although the dose was far higher than that generally used in aquaculture. An
assessment of genotoxic impact with malachite green was carried out on
fertilized eggs of the convict cichlid, Cichlasoma nigrofasciatum, by Schwaiger
et al. (1995). Analysis of the chromosomes revealed significant aberrations,
including chromosome breaks and bridges, after exposure to 0.25 mg l
1
malachite green oxalate for 48 h (Schwaiger et al., 1995; J. Schwaiger, Germany,
1997, personal communication). Liver obtained from trout exposed to
therapeutic levels of malachite green showed focal necrosis and congestion,
which was considered to be a generalized response to toxic cell damage
(Gerundo et al., 1991).
The use of malachite green has been severely curtailed or prohibited in
many countries (Schnick and Meyer, 1978; Hatai and Willoughby, 1988). The
US Center for Veterinary Medicine (CVM) recommended that the USA Fish and
Wildlife Service (FWS) submit a request for a very restricted permit for
malachite green. In May 1981, CVM granted a restricted investigational new
animal drug (INAD) exemption to selected FWS facilities and later permitted
several States to use malachite green throughout the 1980s. However, the
Agency cancelled the INAD exemption for malachite green in August 1991 at all
facilities, the only exception being its use under an INAD at an FWS facility on
one endangered species. In Norway, malachite green can be used as a
prophylactic to reduce fungal infections in aquaculture (Langvad, 1994).
However, fish cannot be marketed for a year following treatment and therefore,
in practice, its use is restricted to eggs and fry (K.E. Thorud, Norway, 1996,
personal communication). Since 1993 in Germany, malachite green has been
restricted to the treatment of fish eggs (Schwaiger et al., 1995). It is not listed
in Annex IV of the European Community (EC) regulation 2377/90/EEC,
which defines substances that may not be present in feed-animal species. As
Alderman (1997) points out, malachite green residues in market-ready fish are
unacceptable. Currently, therefore, in the UK the compound is restricted to
eggs and fish under 5 g, and growing fish must be free of malachite green
residues.
Formalin
Formalin, an aqueous solution containing approximately 37% formaldehyde by
weight, has been widely used to control fungal infections in aquaculture (Cline
and Post, 1972; Walser and Phelps, 1993). Effective control of fungal outbreaks
has been recorded for rainbow trout following a 60 min exposure at 250 mg l
1
(Bailey and Jeffrey, 1989). Similar concentrations of 150300 mg l
1
gave
effective control of Saprolegnia in rainbow and brown trout (Cline and Post,
1972). Marking et al. (1994a) showed that 250 mg l
1
formalin prevented fungal
infections on eggs. At 1000 mg l
1
,

a decrease in existing infection and an
increase in hatching rates were also recorded. Recently, Schreier et al. (1996)
concluded that S. parasitica was controlled on rainbow trout eggs which
received prophylactic formalin treatments on alternate days. In laboratory
challenge tests, Bly et al. (1996) found that formalin could be used
prophylactically to control winter saprolegniasis in channel catfish, inhibiting
Saprolegnia zoospore production at 12.5 mg l
1
and being suppressive at 7.5 mg
l
1
. Walser and Phelps (1993) reported an improvement in the hatching of catfish
eggs relative to untreated eggs, following a twice-daily flush treatment of
formalin at 400 mg l
1
. However, other studies showed that lower concentrations
of around 250 mg l
1
were also effective as flush treatments for this species
(Rogers, 1985; Meyer and Schnick, 1989). Currently, formalin is the only
registered fungicide available for use in fish culture in the USA, but it is limited
to use on eggs of salmon, trout and escoids (Marking et al., 1994a). Despite
increased efforts in the USA, through an INAD permit, to extend the registration
to include other species of fish and eggs, concerns regarding its safety and the
effect of the effluent on the environment remain (Meyer and Schnick, 1989).
Such concerns make it unlikely that formalin will be considered for widespread
use in the short term.
Hydrogen peroxide
Hydrogen peroxide is effective against a variety of organisms, such as bacteria,
yeasts, viruses and fungal spores, and is potentially an important fungicide for
fish culture, exhibiting little environmental impact (Schreier et al., 1996). It has
also recently been used as a bath treatment for sea lice (Johnson et al., 1993;
Bruno and Raynard, 1994). Several groups, including Dawson et al. (1994),
Marking et al. (1994a), Waterstrat and Marking (1995) and Schreier et al.
(1996), have reported on the effectiveness of hydrogen peroxide for controlling
Saprolegnia on developing eggs of rainbow trout and chinook salmon,
Oncorhynchus tshawytscha. Preliminary tests by Dawson et al. (1994) noted that
exposure of eggs to a prophylactic treatment of 250500 ml l
1
hydrogen
peroxide (based on 100% active ingredient) for 15 min, on alternate days,
inhibited fungal infections on healthy eggs. However, treatments of 1000 ml l
1
were necessary to control infection where 10% of the eggs were infected.
Dawson et al. (1994), Marking et al. (1994a) and Schreier et al. (1996) reported
an increase in the hatch rate of eggs that had received a treatment of hydrogen
peroxide at a concentration of 1000 ml l
1
. Hydrogen peroxide has also been
shown to control fungal infections on adult chinook salmon, using a
flow-through dose of 25 mg l
1
. In the USA, the Food and Drug Administration
(FDA) has classified hydrogen peroxide as a low regulatory priority (LRP) for
the control of fungi on all species and life stages of fish (Schreier et al., 1996).
This has enabled its use at concentrations of up to 500 ml l
1
as a fungicide
without an INAD permit or a new animal drug application (NADA).
A commercial sanitizing agent containing 20% hydrogen peroxide and 5%
peracetic acid has also been effective in decreasing fungal infection on rainbow
trout eggs and subsequent hatch rate, following a treatment at 100 mg l
1
for 60
632
min (Marking et al., 1994a). However, toxicity to trout eggs was recorded in this
study at a concentration of 250 mg l
1
.
Sodium chloride
The use of sodium chloride (NaCl) as a fish therapeutant was recommended
during the 1940s by Cranfield (1947), who advised using a 0.5% salt dip for
fungus control on fish and eggs. Phelps and Walser (1993) reported a significant
improvement in the hatching success of channel catfish eggs using a continuous
sea salt bath treatment at 12.5% until hatching. Two- to three-day-old eggs
were held in systems with recirculating water at salinities of 05% until
hatching. Mean hatching success rates were 79.4% for eggs treated at salinities
of 0.52.5% and 67.5% for those treated at a salinity of 0.5%, while the hatch
rate of untreated eggs averaged 57.6%. Phelps and Walser (1993) concluded that
this treatment reduced the unionized ammonia in the water as the salinity
increased, thus reducing ammonia stress. This is relevant, since the
accumulation of ammonia and nitrite by the biological degradation of
nitrogen-rich products is a prerequisite for outbreaks of saprolegniasis (Toor et
al., 1983). As the salt treatment reduced fungal outbreaks, usage of other, harsher
chemicals could be decreased. In contrast, Waterstrat and Marking (1995)
reported that 1.5% NaCl failed effectively to control fungal infections of
chinook salmon eggs. As Saprolegnia isolates tolerated high salt concentrations
without any appreciable reduction in growth (Langvad, 1994), it is not surprising
that growth was not adequately controlled at this concentration. With 1.75%
NaCl in the growth medium, these authors reported that growth was around 65%
of the salt-free control, and, with 3.5% salt, growth was completely checked.
Sodium chloride has similarly been classified as an LRP for the control of fungal
outbreaks in the USA. However, Marking et al. (1994a) and Waterstrat and
Marking (1995) considered that the large quantities of salt required for treatment
presented logistical difficulties, and this was likely to limit its usefulness.
Sodium/calcium chloride
A mixture of sodium and calcium chloride (26:1), applied at 20 g l
1
for 1 h on
three occasions in 1 week, yielded an egg mortality between 6.2 and 9.7%
(Edgell et al., 1993). These results compare favourably with 5.28.8% for 1 mg
l
1
malachite green tested in an identical manner. Marking et al. (1994a) and
Schreier et al. (1996) showed a decrease in Saprolegnia infection of trout eggs
and an improved hatching rate using an NaCl bath at 30 g l
1
. At 20 g l
1
, the salt
mixture was as effective for the control of Saprolegnia on uneyed eggs as
malachite green. Edgell et al. (1993) found an increase in the observed
abnormalities among alevins exposed to malachite green (16%) when compared
with the salt solution (9.3%). However, they speculated that the increase in
coagulated yolk observed within the yolk sac after treatment was unlikely to be
serious in terms of alevin development. These authors also suggested that
rearing alevins in soft water or with other stress-associated factors might induce
or contribute to a higher mortality in the treated group. Fungus control seems
effective with these combined salt solutions, without causing any increase in
yolk-sac abnormality. However, at 25 mg l
1
or above, the salt solutions cause the
death of chinook salmon eggs. The pink salmon, Oncorhynchus gorbuscha, and
chum salmon, Oncorhynchus keta, may have a higher salt tolerance. Future
research into the use of salt solutions for fungus control should consider the
efficacy, logistics and costs associated with the treatment of large groups of eggs
(Edgell et al., 1993).
Sea-water flushes
A sea-water flush into aquaculture facilities offers a cheap means of limiting
fungal growth and has been successfully employed (Taylor and Bailey, 1979). A
23 h flush of sea water for 5 out of 7 days over 10 weeks effectively controlled
S. diclina on eggs of pink salmon. In gravel incubators, there was also a higher
survival of treated eggs to the fry stage, compared with tray incubators, where no
difference in survival levels was recorded. When compared with malachite
green, no difference in the survival of eggs was reported over several years of
testing and Taylor and Bailey (1979) suggested that, where sea water is
available, this method of control would be worthy of investigation.
Copper
Compounds containing copper are usually effective as fungistatic agents when
used at high concentrations (Somers, 1967; Richmond, 1977). Rainbow trout
continuously exposed to sublethal concentrations of copper and challenged with
S. parasitica spores showed changes in total immunoglobulin levels and the
mitogenic responses of pronephric lymphocytes (Carballo et al., 1992).
Although this compound induced notable stress, manifested in enhanced plasma
cortisol levels, there was no alteration to the mitogenic response of lymphocytes
from the pronephros to phytohaemagglutinin (PHA), ConA or
lipopolysaccharide (LPS) measured at 1, 3 and 8 h and 1, 3, 7, 14 and 21 days
postexposure. After 21 days, fish were challenged with S. parasitica and the
same immunological parameters were analysed at 3 h and 7, 14 and 21 days after
challenge. No changes in the immune parameters were observed, except an
increase in the immunoglobulin levels. Saprolegniasis was not present in fish
exposed to copper and a low number of infected fish were observed in the
control group. The apparent lack of correlation between stress and the immune
response during copper exposure may suggest a direct effect of this toxicant,
rather than via a stress-mediated mechanism. Copper is reported as immunotoxic
and is considered to act by decreasing both humoral and cellular immune
responses, reducing circulating lymphocytes and the phagocytic response.
Further work by Carballo et al. (1995) into the sublethal effects of copper and
other compounds on juvenile rainbow trout and their subsequent challenge
with S. parasitica was carried out using sublethal concentrations of copper
(0.25 mg l
1
), cyanide (0.07 mg l
1
), ammonia (0.50 mg l
1
) and nitrite (0.24 mg l
1
).
After 24 h, no enhancement of infection by S. parasitica was recorded. Similar
increases in raised cortisol levels were found for the four chemicals tested and
were used as an indicator of the stress response. Fish that had cortisol levels
<370 ng ml
1
showed an increase in susceptibility to saprolegniasis, while only
24% of the fish with cortisol levels >370 ng ml
1
were infected (Carballo et al.,
1995).
634
Iodophores
Buffered bicarbonate iodophores are often used to disinfect eyed ova; they are
generally applied within 1 h following fertilization. The aim is to destroy
potentially infectious agents, including fungal hyphae on the egg surface. Eggs
are agitated gently in the disinfectant bath for 515 min and then rinsed
thoroughly. A gentle, even flow of water over the eggs ensures sufficient
oxygenation. Alderman and Polglase (1984) confirmed the effectiveness of
iodophores commonly used in fish farming (e.g. Wescodyne and Buffodyne) at
their recommended concentrations of 100 mg l
1
available iodine, although in
their study exposure was 30 min as opposed to the normal 15 min. The potential
of iodine as a fungicide for eggs of rainbow trout (Marking et al., 1994a) and
channel catfish (Walser and Phelps, 1993) has been examined. Iodine
administered twice daily as a flush at concentrations of 50, 100 or 200 mg l
1
increased the hatching rate of channel catfish eggs. Their potential for further
development was considered limited due to the high concentrations required.
Although iodophor baths are sometimes reused until the strong yellow colour
has faded, the iodophor becomes ineffective at 25 mg l
1
(Ross and Smith, 1972)
and must be changed regularly to maintain efficacy. When treating a large
number of fish, a bath or flush method is the most economic, but, for small
numbers of fish, a dip treatment of around 30 s is advised. Treatment with
buffered iodine following water-hardening of eggs is permitted as an LRP by the
FDA in the USA and other countries when used at a level of 100 mg l
1
for 10 min
as a disinfectant (Marking et al., 1994b).
Bacterial antagonists
Several studies have identified bacterial antagonists to S. parasitica and a
variety of Pythium and Rhizophthora species (Hatai and Willoughby, 1988;
Petersen et al., 1994). Hatai and Willoughby (1988) reported that the bacterium
Pseudomonas fluorescens could strongly inhibit the radial growth of S.
parasitica in vitro, and natural control might therefore be feasible. Fungal
inhibition attributed to this bacterial antagonist has been suggested to result from
the production of an antibiotic by the bacteria (Gurusiddaiah et al., 1986) or
from an efficient iron-capturing siderophore that deprives the fungus of this
essential ion (Weller and Cook, 1983). However, Bly et al. (1997) reported that
inhibition was not associated with culture supernatants, but might depend on the
secretion of a chemical or nutrient acting between the bacteria and the
Saprolegnia. It is possible that the fungus may remove a nutrient or secrete a
chemical that the bacteria recognize, and then react by secreting an inhibitor. The
commercial use of bacterial antagonists seems unlikely, as antibiotics and other
chemicals are used in commercial fish farming, and Pseudomonas sp. and P.
fluorescens are now recognized causal agents of bacterial haemorrhagic
septicaemia in fish (Roberts and Horne, 1978; Nakatsugawa and Iida, 1996).
Biological control
A few reports highlight the potential of naturally grazing organisms, such as
amphipods, to control fungal outbreaks. Oseid (1977) noted Gammarus
pseudolimnaeus and the isopod Asellus militaris feeding on fungal growth on
dead eggs. In laboratory studies, G. pseudolimnaeus prevented fungal growth on
developing eggs from walleye, Stizostedion vitreum, at a ratio of one
invertebrate to ten fish eggs. Although some live eggs were consumed, this
approach demonstrated an improvement over batches where dead eggs were
removed by hand. Asellus militaris also controlled fungal growth on eggs, but
here no difference was noted between these eggs and the hand-picked controls.
Fungal parasitism was examined by Willoughby and Roberts (1992b), who
reported that an obligate pathogen of Saprolegnia, Woronia polycystis, infected
zoospore germlings and established mycelia of S. parasitica in vitro. After 6
days incubation, the hyphae were grossly swollen and contained parasitic
plasmodia. Further development of the potential use of biological controls such
as these has not been reported.
Chitosan
The Oomycetes have a high calcium requirement for the stabilization of their cell
membranes, particularly during differentiation (Griffin and Coley-Smith, 1975).
Min et al. (1994) exposed oospores and vegetative hyphae from S. parasitica to
chitosan (a deacetylated form of chitin), reporting complete inhibition by 0.06%
and 0.05% chitosan, respectively. Yuasa and Hatai (1995b) also reported on the
effectiveness of chitosan for S. parasitica and A. piscicida, finding that it acted
as a competitor for calcium-binding sites on the cell surface. Min et al. (1994)
and Yuasa and Hatai (1995b) considered that these findings warranted further
investigation into the application of chitosan for preventing saprolegniasis.
Ultraviolet irradiation
Ultraviolet irradiation has been used successfully to kill viruses and bacteria in
freshwater systems (Sako and Sorimachi, 1985; Liltved and Landfald, 1996), but
this treatment seems ineffective towards Saprolegnia hyphae in vitro (Sako and
Sorimachi, 1985).
Vaccine development
In teleosts, adaptive immune responses are well developed, and specifically
acquired antibodies have been demonstrated towards a variety of organisms
following artificial immunization (Leong, 1993). However, there are currently
no vaccines available for saprolegniasis or other water-mould diseases of fish.
Naturally occurring serum proteins have been demonstrated to react with
636
mycelial extracts and culture filtrates in several fish species, and Seelinger
(1960) showed that such reactions between fungal antigens and antisera are not
specific. Any vaccine development for saprolegniasis might best concentrate on
stimulating the immune system at the mucosal surface. However, the majority of
fish are likely to have been exposed to Saprolegnia naturally, due to the
widespread nature of the freshwater fungi (Pickering and Willoughby, 1982b).
Beakes et al. (1994b) postulated that the specific surface glycoprotein on the
cyst-coat spines may represent epitopes to which antibodies might be directed,
thereby reducing the stickiness of the spores and their subsequent attachment
to the fish surface. However, Burr and Beakes (1994) demonstrated that
monoclonal antibodies raised against secondary cyst coat matrix components of
S. parasitica were not specific and reacted with all Saprolegnia genera plus A.
astaci and Achlya sp., but not members of the Peronosporoycetidae.
The potential efficacy of any fungal vaccine must be questioned in light of
the evidence produced by Bly et al. (1996) that, during periods of low water
temperature, when susceptibility to Saprolegnia infection occurs, channel
catfish were immunosuppressed (Bly and Clem, 1991). Understanding the
relationship between the environmental temperature, fungal agent and nature of
the immune response will be important for future development in this area.
PATHOGENESIS AND IMMUNITY
Fungal disease attributed to the genus Saprolegnia results from opportunistic
and primary infection (Willoughby and Pickering, 1977; Noga et al., 1988) and
is generally assigned to a single major cluster and separate taxon, S. parasitica
Coker (syn. S. diclina Humphrey type 1), in salmonids. The secondary zoospore
cysts of S. parasitica (S. diclina type 1) and Saprolegnia sp. were shown by
Pickering et al. (1979) to bear bundles of long (510 mm) hooked spines,
whereas S. diclina types 2 and 3, S. ferax and S. australis produced secondary
cysts with short (<1.0 mm), single, hooked hairs. This, and subsequent studies
suggest that the long hairs associated with the former species could facilitate
infection by enabling the spores to attach to fish more efficiently by remaining at
the waterair interface (Hallett and Dick, 1986). Willoughby and Roberts
(1992a) considered that the elongate hairs present on the zoospore cysts of S.
parasitica might allow them to remain suspended in the water column for longer
periods, thereby increasing the probability of them encountering a fish host.
These observations have promoted the suggestion that the length of hooked hairs
is related to pathogenicity (Beakes, 1983; Hatai and Hoshiai, 1993).
Manton et al. (1951) and Meier and Webster (1954) considered that the long
bifurcate hooks of the secondary cyst of Saprolegnia play a role in attachment to
the fish host, and Roberts (1989) proposed that these ornamentations are
important in the pathogenicity of saprolegniasis. In support of this, a series of
challenge experiments, using brown trout and Arctic char, showed that S.
parasitica remained localized on the fish surface for longer periods than the
saprophytic S. diclina. However, Hatai et al. (1990) found that the number and
size of hooks are not necessarily related to pathogenicity, with some highly
pathogenic isolates possessing small bundles of relatively short hairs.
Furthermore, Rand and Munden (1993a) found no evidence that attachment of S.
diclina zoospores to brook trout eggs was mediated by the cyst-wall appendages.
Instead, they proposed that attachment was facilitated by the release of an
adhesive substance, supporting earlier findings regarding the adhesion of this
species to non living surfaces (Willoughby, 1977; Beakes, 1983).
Histopathology
Early circular or crescent-shaped skin lesions associated with Saprolegnia
infection in salmonids are often characterized by growths of thin, white or grey
threads (Willoughby, 1989). Microscopic examination of hyphal growth reveals
the characteristic, branched, coenocytic mycelium, with many zoosporangia.
The histopathology associated with early, superficial infection in salmonids
shows rapid degenerative changes in the epidermis and dermis. More aggressive
lesions, with deeper myofibrillar and focal cellular necrosis, spongiosis or
intracellular oedema and ultimate sloughing of the epidermis, may follow
(Neish, 1977; Pickering and Richards, 1980). Associated inflammatory reactions
are absent (Hatai and Hoshiai, 1992). As the fungus radiates from the focus of
infection, more of the epidermis is destroyed, and consequently hyphae can
penetrate the basement membrane, with growth sometimes continuing into the
hypodermis and musculature (Neish, 1977). Thrombi are frequently observed in
the blood-vessels as a result of the penetrating hyphae. Primary infectious
lesions with many hyphae have been reported in the pyloric region of the
stomach of amago salmon, and these hyphae may also invade other abdominal
tissues (Miyazaki et al., 1977). Furthermore, the gill lamellae and pharynx have
been the sites for primary infection of farmed Atlantic salmon fry (Bruno and
Stamps, 1987).
Saprolegniasis has been correlated with an extensive haemopoietic
pathology in brown trout (lvarez et al., 1988). In addition to a marked
lymphocytopenia (Pickering and Pottinger, 1988), significant impairment of the
haemopoietic organs is reported. Lymphoid cell degeneration, cell depletion,
vascular alterations within blood-vessels and enlargement and hypertrophy of
sinusoidal endothelial cells also occur (lvarez et al., 1988). lvarez et al.
(1995) recorded histological changes in the thymus and, although the hyphae
had not directly invaded this organ, the tissue was oedematous, with epithelial
hypertrophy, increased pyknosis and phagocytic activity, involving
macrophages and epithelial cells. Considerable changes occurred in the structure
of the parenchyma, with large areas showing a marked decrease in cellular
density. Most thymocytes were pyknotic, and both epithelial cells and
macrophages contained engulfed dead cells. Darkly staining epithelial cells
showed cytoplasmic vesicles and clear signs of degeneration, including a
vacuolar cytoplasm. No inflammatory response to the fungal invasion was
observed.
Copland and Willoughby (1982) reported a loss of integrity of the
integument, an oedema of the hypodermis, with degenerative changes in the
638
muscle mass, accompanied by marked myofibrillar degenerative changes in
farmed elvers infected with Saprolegnia. Severe infection causes swelling in the
intermyotomal connective tissue, which has a fenestrated appearance, due to
loss of nuclei. In farmed pike, muscle lesions attributed to fungal infection are
uncommon, but hyphae occasionally invade deeper areas, including nervous
tissue (Bootsma, 1973). The location of the hyphae suggests that pathogenic
Saprolegnia are not generally tissue-specific.
Saprolegnia lesions in channel catfish initially occur at the site of injury,
containing a central zone of either necrotic skin, with fungal mycelia throughout
the lesion, or, in more severe lesions, a necrotic core of sloughed tissue, which
leaves a crater-shaped cavity (Xu and Rogers, 1991). In some lesions, the
epidermis has been reported as completely sloughed, leaving the dermis exposed
(Xu and Rogers, 1991; Bly et al., 1992). Adjacent tissue becomes infected
following the spread of hyphae on the skin surface, and mucus cells present in
normal skin are absent in the infected skin.
Observations in India, on pond-reared Chela laubuca, showed typical
colonization and destruction of the epidermis and hypodermis by S. diclina and a
profuse hyphal growth, associated with inflammation of the cornea and
concavity of the retina (Srivastava et al., 1994). Infection of the cornea in other
oomycete outbreaks is uncommon.
Oomycetes, other than Saprolegnia, which have been reported as pathogens
of wild and farmed fish species include Aphanomyces and Achlya (Miyazaki and
Egusa, 1973; Fraser et al., 1992; Kitancharoen et al., 1995). Hatai et al. (1994)
and Wada et al. (1994) observed multiple granulomas within the internal organs
and musculature of the dwarf gourami, Colisa lalia, infected with Aphanomyces.
These consisted of mononuclear cells, neutrophils, macrophages and fibrillar
structures and were considered to resemble the mycotic granulomatosis recorded
in farmed ayu infected with Saprolegnia (Hatai, 1980b). Multinuclear giant cells
were absent in the Aphanomyces and Achlya infections.
In the eastern USA, Noga et al. (1988) assigned an ulcerative mycosis in
Atlantic menhaden to an oomycete infection. Aphanomyces are more commonly
cultured from these lesions, although Saprolegnia is also isolated (Dykstra et al.,
1986). The lesions are classified based upon gross and histological features.
Advanced lesions consist of open ulcers containing many hyphae, interspersed
with necrotic muscle. Hyphae are surrounded by intense inflammation, with
many basophilic granulomas possessing necrotic centres. Some lesions are
haemorrhaged and infiltrated by lymphocytes and granular cells. Peduzzi and
Bizzozero (1977) speculated that the secretion of lytic enzymes ahead of the
fungal hyphae may account for tissue damage. Deeply basophilic granulomas
and other haemorrhagic lesions, infiltrated with lymphocytes and occasional
multinucleated giant cells, are also observed. However, it is considered unusual
for granuloma formation to occur in association with oomycete infection.
Hatchery mortality among juvenile ayu has been attributed to S. diclina type 1
infection, and diagnosed as a mycotic granulomatosis (Hatai, 1980b; Wada et al.,
1993). Histologically, numerous hyphae observed in the stomach penetrate into
the pyloric caeca and other visceral organs, resulting in severe necrosis.
Multinuclear giant cells are abundant in these lesions. Wada et al. (1993)
recognized that primary infectious lesions in farmed ayu were initially
established in the pyloric region, with hyphae invading from the mucous
membrane into these regions.
Ultrastructural studies
The first electron-microscopy study on the ornamentation of zoospore cysts was
carried out by Manton et al. (1951), who recorded curious double-headed hooks
on slender stalks, likened to boat-hooks. Meier and Webster (1954) extended
these early observations, and demonstrated that the secondary rather than the
primary cysts from several S. parasitica isolates bore stalked, double-headed
hooks.
Electron-microscopy studies of experimentally infected channel catfish
revealed the appearance of lesions typically between 7 and 9 days postinfection
(Xu and Rogers, 1991). Hyphae penetrated the epidermis and dermis, with
necrosis and sloughing in areas next to the hyphae. Damage to fibroblasts and
change in the collagen orientation were noted in both naturally and
experimentally infected fish. Some epithelial cells were more electron-dense
than normal cells and the nuclear membrane in other cells had ruptured, showing
an increase in cytoplasmic vacuoles. Earlier, lvarez et al. (1988) had
hypothesized that cytoplasmic vesicles observed in the endothelial cells were
derived from the fungi. Xu and Rogers (1991) also noted the destruction of
collagen, with significant debris containing melanosomes between the lamellae.
The ultrastructure of vegetative (somatic) hyphae of S. parasitica was
examined by Xu et al. (1990), who found that hyphae were aseptate and thin-
walled and appeared to comprise three zones. It was also noted that
presporangium hyphae were aseptate and thicker than the vegetative hyphae,
possessing conspicuous, dense-body vesicles and primary bars, called
encystment vesicles by Beakes (1983). These organelles were present before
sporangium delimitation and were thought to play a role in the thickening of the
wall of presporangium hyphae. These were associated with dictyosomes and
mitochondria, and may produce and transport materials, giving rise to the dense
bodies. Primary bars appear to play a role in cyst coat formation in the primary
zoospores and may originate from the rough endoplasmic reticulum (Beakes,
1983).
The invasion of brook char eggs by S. diclina thalli was shown, using
scanning electron microscopy, to occur from a combination of mechanical
pressure and extracellular enzyme digestion (Rand and Munden, 1992). Brook
trout eggs exposed to zoosporulating hyphae were colonized by encysted spores,
spore germlings and young thalli within 15 min postexposure (Rand and
Munden, 1993a). Some thalli penetrated the outer layer of the chorion and
appeared to spread in a lateral direction just beneath the membrane surface.
Between 1 and 24 h postexposure, infected eggs were covered by a few
branching thalli, either spreading over or penetrating the chorionic membrane.
By 24 h postinfection, however, a light to moderately heavy mycelial mat
covered the egg surface.
640
Osmoregulatory failure
The primary sequel of uncomplicated saprolegniasis is an osmotic imbalance,
due to loss of epithelial integrity and tissue destruction, caused by penetration of
the hyphae (Copland and Willoughby, 1982; Noga, 1993b). It is generally
accepted that death is due to severe haemodilution, caused by haemorrhage, and
the progressive destruction of the epidermis by hyphae (Hatai and Hoshiai,
1994). A significant decrease in serum ions in Saprolegnia-infected mature
brown trout has been correlated with infection (expressed as the percentage of
body-surface area covered by the fungus), serum osmotic pressure and sodium
ion concentration (Richards and Pickering, 1979b; Duran et al., 1987). The
pathology observed can be directly attributed to the tissue destroyed in the
immediate area of the hyphae (Neish, 1977), and the oedema in infected fish can
be assigned to osmoregulatory changes (Richards and Pickering, 1979b; Noga,
1993a). However, Noga et al. (1988) considered that these observations did not
fully explain the lesions in deep muscle fibres in menhaden.
Enzyme activity
Some 20 years ago, Peduzzi and Bizzozero (1977) showed that the thalli of
certain fish-pathogenic Saprolegnia exhibit chymotrypsin-like activity and
postulated that this enzymatic activity contributed to pathogenesis. The work
was extended by Rand and Munden (1992), who reported high lipase and
alkaline phosphatase activity surrounding the thalli of S. diclina infesting the
egg membrane of brook char. These enzymes may alter the integrity of the
chorionic membrane by solubilizing structural polymers, thereby easing
penetration of the thalli. Enzyme changes noted in Saprolegnia-infected brown
trout include lactate dehydrogenase, glutamateoxyalate transaminase (GOT),
glutamatepyruvate transferase (GPT), creatine phosphokinase, alkaline
phosphatase and acid phosphatase, with the presumptive liver enzymes GOT
and GPT being elevated, which suggests hepatocellular damage (Duran et al.,
1987). Fish dying from Saprolegnia infection suffer a severe haemodilution,
associated with elevated serum enzymes (Noga, 1993a; Hatai and Hoshiai,
1994) and a hypoproteinaemia in several fish species, including brown trout
(Richards and Pickering, 1979b), Atlantic salmon (Mulcathy, 1969) and coho
salmon (Hatai and Hoshiai, 1994). Severe hypoproteinaemia and a significant
reduction in the albumin:globulin ratio is reflected in the electrophoretogram of
the serum proteins from infected fish (Richards and Pickering, 1979b).
Immunology
Resistance to infectious diseases may be innate or specifically acquired. In fish,
there is increasing evidence that fungal infections are associated with
immunosuppression (lvarez et al., 1988; Hayman et al., 1992), with many
outbreaks occurring after a sharp decrease in water temperature, to levels near
the physiological minimum for a particular fish species. Fungal growth occurs
rapidly, with the formation of characteristic skin lesions, being accompanied by
significant fish mortalities (Bly et al., 1994) and, at temperatures 12C, the
culture of channel catfish is under threat from outbreaks of Saprolegnia spp.
Many farmed catfish in the southern USA are frequently killed following such
infections, known locally as winter kill or winter saprolegniasis (Bly et al.,
1992, 1994). Affected fish show fungal-associated skin lesions and
mucus-depleted skin. Experimental studies demonstrated that this condition
involved complex reactions between a rapid decrease in water temperature, in
vivo immunosuppression, lack of an inflammatory response and the Saprolegnia
(Xu and Rogers, 1991; Bly et al., 1992; lvarez et al., 1995). Hayman et al.
(1992) found that channel catfish were also complement-deficient in the winter
months. Serum CH
50
values became severely depressed, suggesting that immune
and non-immune functions generally associated with complement were not
functioning correctly. These authors proposed that the lack of leucocytic
infiltration in fungal-infected tissue was related, in part, to the inactivity of the
complement system, but it is possible that low water temperature also affected
the synthesis of complement proteins. Bly and Clem (1992) considered that low
water temperature, elevated corticosteroid and an increase in cytotoxic factors
secreted by the fungus may account for immunosuppression. Bly et al. (1994)
suggested that catfish and possibly other fish could clear fungal infection via a
cell-mediated response. When catfish were held at 22C and injected
intramuscularly with viable Saprolegnia, the hyphae were rapidly destroyed in a
classical foreign-body response (Bly et al., 1994). lvarez et al. (1995) further
postulated that the disappearance of the thymic parenchyma in infected brown
trout and a combination of some or all of the above factors were related to
Saprolegnia infection.
There are a few reports on the relationship between genetic variation and
disease resistance of farmed salmonids (reviewed by Chevassus and Dorson,
1990). In a recent study, Nilsson (1992) reported a positive relationship between
mortality of replicated families of Arctic char with Saprolegnia infection.
Heritability estimates for mortality showed that tolerance to fungal infection by
the char could be improved by selective breeding of fish.
TOPICS FOR FURTHER STUDY
Our understanding of the Saprolegniaceae has expanded rapidly in recent years.
Molecular studies show that the Oomycetes have their phylogenetic origins with
the chromophyte algae, rather than the true fungi, and PCR techniques are
enhancing our taxonomic knowledge of this group. These and other
developments, such as RAPD-PCR, are likely to help resolve some existing
taxonomic problems and enable species and subgroups within the Oomycetes to
be compared. Eventually, this may lead to the development of diagnostic kits
specific for S. parasitica and subgroups that could be applied in the absence of
antheridia and oogonia.
The importance of specific surface topography recognition at the host
642
surface during the initial interaction with the disease agent has been
demonstrated. Future studies of such interactions in Oomycetes might be
directed towards the molecular analysis of cell-surface components, using
monoclonal antibodies, and the identification of the cell types involved in host
defence. Monoclonal antibodies are likely to provide precise tools for probing
and identifying receptor functions of antigens and therefore represent an area
which would benefit from further development.
Many researchers have screened chemicals and advised on their suitability
as fungicides. Despite this, no agent has been identified which matches the
effectiveness of malachite green, although hydrogen peroxide, formalin and
NaCl show some potential, and prophylactic bath treatments using a buffered
iodophor are apparently successful for eggs. Both hydrogen peroxide and NaCl
have been granted LRP drug status by the FDA in the USA and can be used as
approved treatment regimes without an INAD permit, or a NADA. This process
has avoided the high costs associated with registration of new compounds for
aquaculture and goes some way towards allowing further study into their
potential for the management of fungal infections.
To date, only a limited number of chemical compounds show any potential
as fungicides and none are considered more effective than malachite green. The
current view that the Oomycetes are more closely related to the chromophyte
algae should enable particular chemical groups to be selected, since many
fungicides that are effective against higher fungi are ineffective against
Oomycetes and vice versa. Clearly, there is a need for safe and effective
fungicides for use in aquaculture, and development in this area remains a
priority. Supporting studies aimed at examining the permeability of the cell
membrane to particular fungicides, the existence of suitable binding sites and the
rate at which compounds accumulate and are detoxified are also required.
Measurement of uptake is also essential for a thorough understanding of the
mode of action of each fungicide.
Studies identifying mechanisms of immunostimulation in immuno-
suppressed fish may help to develop a vaccine or therapeutic approach for
controlling saprolegniasis. This will require research into the absence of
leucocytic infiltration in fungal-infected tissue seen in the winter months, its
relationship to the inactivity of the complement system and an understanding of
the connection between environmental temperature, fungal agents and the nature
of the immune response. Studies would usefully be aimed at producing
antibodies to specific surface components, thereby reducing the stickiness of
the spores and their attachment to the fish surface.
There is sufficient evidence to show that the infestation of fish eggs by
zoospores results from a chemotactic response to compounds associated with the
eggs themselves. Although further work is necessary to identify the role of
specific compounds, it may become practical to manipulate these chemical
messengers with amino acid baits, allowing the development of more targeted,
specific control measures.
ACKNOWLEDGEMENTS
We thank Dr G.W. Beakes for providing several illustrations and Dr A.S. Wood
for reviewing the manuscript.
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CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

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