245

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

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
6
Rickettsial and Chlamydial Infections
C.N. Lannan, J.L. Bartholomew and J.L. Fryer
Department of Microbiology and Center for Salmon Disease Research, 220
Nash Hall, Oregon State University, Corvallis, Oregon 97331-3804, USA.
INTRODUCTION
Bacteria in the orders Rickettsiales and Chlamydiales share a number of
characteristics, including obligate intracellular growth, replication by binary
fission and a typical Gram-negative cell wall. However, the chlamydiae are
separated from the rickettsiae by their unique developmental cycle, involving
transition from a rigid-walled infectious cell to a flexible replicative form. The
rickettsiae are a more diverse group than the chlamydiae and, with the exception
of Coxiella burnetti, all are transmitted only by means of an arthropod vector.
A number of species of rickettsiae and chlamydiae are pathogens of humans
and other animals. Those best described from fish are the rickettsia that causes
piscirickettsiosis (PRS; Fryer et al., 1990) and the chlamydia-like organism
(CLO) that causes epitheliocystis (EP; Hoffman et al., 1969). Although the agent
that causes EP has never been isolated, the disease has been reported in fish from
both warm- and cold-water habitats and from a variety of hosts, including
marine, anadromous and freshwater species.
The number of rickettsiae recognized in association with disease in fish has
increased since 1989, when the first of these agents was isolated from coho
salmon (Oncorhynchus kisutch) in Chile (Fryer et al., 1990) and subsequently
classified (Fryer et al., 1992). Since then, rickettsia-like organisms (RLO) have
been observed and/or isolated from other species of fish at a variety of
geographical locations.
246
C.N. Lannan et al.
RICKETTSIAL INFECTIONS OF FISH
Piscirickettsiosis
Aetiological agent
Piscirickettsia salmonis (Fryer et al., 1992) is a Gram-negative, non-motile,
obligately intracellular bacterium. The type strain, LF-89
T
(ATCC VR 1361) was
isolated from a diseased coho salmon collected during an epizootic at a sea-
water net-pen facility in Region X, Chile (Fryer et al., 1990).
The bacterium, P. salmonis, is non-motile, pleomorphic but predominantly
coccoid and most often occurs as pairs of curved rods which may also appear as
rings. The diameter of individual cells is 0.51.5 m (Fig. 6.1). Piscirickettsia
salmonis replicates within membrane-bound cytoplasmic vacuoles in cells of
infected fish and in certain cultured fish cell lines (Fig. 6.2). Observed in thin
section, the cytoplasm is enclosed by three layers: a rippled outer membrane, a
typical Gram-negative cell wall and an inner membrane.
The organism replicates and produces a cytopathic effect (CPE) in many
salmonid cell lines and in certain cell lines from warm-water fish, but does not
grow in cell-free media. Optimal temperatures for in vitro growth are 1518C
and titres of 10
6
10
7
tissue culture infective dose at 50% end-point (TCID
50
)
ml
1
are obtained. Replication is retarded below 10C and above 20C and does
Fig. 6.1. Scanning electron micrograph of Piscirickettsia salmonis, LF-89
T
, released from
cultured CHSE-214 cells. Rickettsiae of varied sizes are each enclosed in a highly rippled outer
membrane. Bar =1 m.
247 Rickettsial and Chlamydial Infections
not occur above 25C (Fryer et al., 1990). Piscirickettsia salmonis is sensitive to
freezing and titres may be decreased by 99% with a single cycle of freezethaw
at 70C. However, the addition of 10% dimethylsulphoxide to the freezing
medium as a cryoprotectant improves survival tenfold over cultures frozen
without additives. Addition of antibiotics to the cell-culture media inhibits or
retards in vitro replication of P. salmonis (Fryer et al., 1990).
Rickettsial isolates from salmonids in Chile, Norway, Ireland and British
Columbia (BC), Canada (Table 6.1) are morphologically similar and have been
identified as P. salmonis using serological techniques (Lannan et al., 1991;
Alday-Sanz et al., 1994). The 16S ribosomal ribonucleic acid (rRNA) gene
sequences of five isolates from Chile, BC Canada and Norway were compared
(Mauel, 1996) to assess the genetic variability of this group. The isolate
(SLGO-94) from rainbow trout (Oncorhynchus mykiss) collected in Chile, the
Atlantic salmon (Salmo salar) isolate (ATL-4-91) from BC Canada and the
Atlantic salmon isolate (NOR-92) from Norway were > 99.4% similar to the
type strain LF-89
T
from coho salmon. The fifth isolate (EM-90) from Atlantic
salmon in Chile was less similar (98.598.9%) but differences were not
Fig. 6.2. Transmission electron micrograph of Piscirickettsia salmonis, LF-89
T
, replicating within
cytoplasmic vacuoles in a CHSE-214 cell. Bar =1 m.
248
C.N. Lannan et al.
sufficient to warrant a new species (Mauel, 1996). While isolates of P. salmonis
from the northern and southern hemispheres are similar, it is apparent that those
from Chile are more virulent for coho salmon than those from BC Canada and
Norway.
Disease
In Chile, mortality from P. salmonis usually occurs in the autumn, approximately
612 weeks after fish are transferred from freshwater to salt-water net-pens, and
again the following spring (Bravo and Campos, 1989; Fryer et al., 1990;
Cvitanich et al., 1991). Although PRS is usually associated with fish in estuaries
or marine environments, there have been reports of the disease in juvenile
salmonids held in fresh water (Gaggero et al., 1995).
External signs of disease are variable but include lethargy, anorexia, pale
gills and darkened body coloration. Infected fish often have skin lesions that
range from small areas of raised scales to shallow haemorrhagic ulcers (Branson
and Nieto Diaz-Munoz, 1991). However, in acute infections, mortality may
occur without gross signs of disease. Internally, the rickettsiae spread
systemically, resulting in swollen kidneys and spleens. Although not common in
Table 6.1. Piscirickettsia salmonis LF-89
T
observed and/or isolated from fish.
Geographical Salt/fresh Observed/
Fish host species location water isolated Reference
Coho salmon Chile S I Fryer et al. (1990)
(Oncorhynchus
kisutch)
Rainbow trout Chile S O/I Garces et al.
(Oncorhynchus (1991)
mykiss)
Chinook salmon
(Oncorhynchus
tshawytscha)
Atlantic salmon
(Salmo salar)
Chinook salmon British S O/I Evelyn (1992)
Atlantic salmon Columbia
Coho salmon Canada
Pink salmon
(Oncorhynchus
gorbuscha)
Atlantic salmon Ireland S O Rodger and
Drinan (1993)
Atlantic salmon Norway S O/I Olsen et al. (1993)
Coho salmon Chile F I Gaggero et al.
(1995)
Rainbow trout
S, host collected from salt water; F, host collected from fresh water; I, isolated
in cell culture; O/I, observed and subsequently isolated in cell culture; O,
observed only.
249 Rickettsial and Chlamydial Infections
infected fish (510%), the most diagnostic lesions occur in the liver as grey to
yellow mottled areas or as ring-shaped foci (Cvitanich et al., 1991; Lannan and
Fryer, 1993; Fig. 6.3). Petechial haemorrhages also occur in visceral organs and
adipose tissue. Microscopically, these changes are accompanied by extensive
necrosis of haematopoietic tissues. Haematocrits generally fall below 25% and
large numbers of macrophages containing P. salmonis cells and cellular debris
can be detected in peripheral blood smears. The rickettsiae are seen within
cytoplasmic vacuoles of cells from the kidney and other organs (Fig. 6.4).
Macrophages filled with rickettsial cells are common.
The histopathology associated with natural and experimental infections of P.
salmonis has been described (Branson and Nieto Diaz-Munoz, 1991; Cvitanich
et al., 1991; Garcs et al., 1991). Pathological changes occur throughout internal
organs, with the most severe manifestations of the disease in the intestine,
kidney, liver and spleen. In the kidney and spleen, the normal haematopoietic
and lymphoid tissues are replaced with chronic inflammatory cells and host-cell
debris. Liver lesions are severe and rickettsiae are often seen in the cytoplasm of
degenerating hepatocytes. Inflammation of the lamina propria of the large
intestine is common and often results in necrosis and sloughing of the mucosal
epithelium. Hyperplasia of gill epithelia results in fusion of the lamellae.
Pathological changes in the heart and other organs are variable but disseminated
intravascular coagulation is common.
Fig. 6.3. Gross pathology associated with piscirickettsiosis in the coho salmon (Oncorhynchus
kisutch). Note the enlarged spleen, petechiae on internal organs and mottled lesions on the liver.
Photograph courtesy of Sandra Bravo, Puerto Montt, Chile.
250
C.N. Lannan et al.
The humoral response of rainbow trout experimentally infected with P.
salmonis was investigated by Kuzyk et al. (1996). The infected fish did not
produce a strong antibody response. The antibody response was most probably
directed against a lipo-oligosaccharide component of the lipopolysaccharide
located in the outer membranes of rickettsiae. Absence of a significant
antibody-mediated response is typical of infections caused by intracellular
pathogens, and the role of cell-mediated immunity against P. salmonis should be
investigated.
Geographical distribution
Observation and/or isolation of P. salmonis is currently confined to salmonids
from Chile, BC Canada, Norway and Ireland (Table 6.1). It is probable that the
condition was first recognized in Canada during the early 1970s and referred to
as parenthesis disease (Evelyn, 1992). Numerous descriptive names for the
disease include: coho salmon syndrome, piscirickettsiosis, salmonid rickettsial
septicaemia and UA (unknown agent).
By the mid-1980s, the disease was recognized among cultured salmonids in
Region X of Chile (Bravo and Campos, 1989). Here, recurring outbreaks
resulted in losses of coho salmon exceeding 90% at numerous production fish
farms. Piscirickettsia salmonis (ATL-4-91) was isolated in 1992 from diseased
Atlantic salmon held in salt water in BC Canada (Brocklebank et al., 1992).
A bacterium similar to P. salmonis is associated with low-level mortality in
Fig. 6.4. Kidney impression from moribund coho salmon (Oncorhynchus kisutch). Note the
Piscirickettsia salmonis, LF-89
T
, organisms within and scattered among the host cells. Giemsa
stain. Bar =10 m. Photograph courtesy of Sandra Bravo, Puerto Montt, Chile.
251 Rickettsial and Chlamydial Infections
Atlantic salmon held in salt-water net-pens in Norway (NOR-92; Olsen et al.,
1993) and Ireland (Rodger and Drinan, 1993). These pathogenic micro-
organisms were subsequently all identified as P. salmonis using the fluorescent
antibody test (Fryer and Lannan, 1996). Analysis of the 16S rRNA gene
sequence of the isolates from Chile, Norway and BC Canada has confirmed their
identity (Mauel, 1996).
Host range
Piscirickettsia salmonis and the disease it causes has, thus far, only been
observed in members of the family Salmonidae (Table 6.1). Although coho
salmon seem to be the most susceptible species, the disease has been reported in
cultured Atlantic, chinook (Oncorhynchus tshawytscha) and masu salmon
(Oncorhynchus masou; Bravo, 1994) and rainbow trout. Juvenile pink salmon
(Oncorhynchus gorbuscha) are also reported to be susceptible to infection
(Evelyn, 1992).
Diagnostic methods
Isolation of P. salmonis in cell culture and microscopic observations of stained
tissue imprints or smears are effective methods to detect rickettsiae in infected
fish tissue. However, confirmation of P. salmonis must be made using
serological methods (Lannan and Fryer, 1991).
Kidney tissue is recommended for isolating the rickettsia in cell culture. The
tissue is aseptically removed and kept at 4C, as the bacterium is sensitive to
both elevated and freezing temperatures. Piscirickettsia salmonis replicates in a
number of fish cell lines, including chinook salmon embryo (CHSE-214) (ATTC
CRL 1681), chum salmon heart (CHH-1) (ATCC CRL 1680), rainbow trout
gonad (RTG-2) (ATCC CCL 55), epithelioma papulosum cyprini (EPC) (Fijan et
al., 1983) and fathead minnow (FHM) (ATCC CCL 71) (Wolf and Quimby,
1962; Fryer et al., 1990). Because the organism is sensitive to most of the
antibiotics routinely used in cell culture, tissue is inoculated on to cells
maintained in antibiotic-free medium. Inoculated cultures are incubated at the
optimal growth temperature of 1518C and observed up to 28 days for
appearance of CPE. Characteristic CPE appears as clusters of rounded cells
with large vacuoles and, at primary isolation, may require 21 or more days to
appear. In subsequent passages, CPE becomes apparent 47 days after
inoculation.
Although isolation in cell culture provides a sensitive method for detection,
there are disadvantages. Conditions in the field are rarely ideal for a septic
necropsy and the incubation period further delays results. The necessary absence
of antibiotics in the culture medium requires substantial efforts to prevent
introduction of contaminating organisms.
A less sensitive but more rapid method for diagnosis of a rickettsial infection
in fish is by microscopic examination of smears or imprints of kidney, liver or
spleen tissue stained with Giemsa or acridine orange stain (Lauer et al., 1981;
Lannan and Fryer, 1991). However, following initial detection in stained tissue
smears or cell cultures, the identity of P. salmonis must be confirmed by means
of serological methods, e.g. immunofluorescence (Lannan et al., 1991) and
252
C.N. Lannan et al.
immunohistochemistry (Alday-Sanz et al., 1994), or molecular techniques
(Mauel et al., 1996).
Disease transmission
The source and reservoir of P. salmonis in the natural environment and its mode
of transmission, directly from fish to fish and/or indirectly through a vector, are
unknown. In terrestrial environments, an intermediate arthropod host or vector is
required for transmission of rickettsiae (Weiss and Moulder, 1984). There are
many parasitic crustaceans in the marine environment that might serve as
vectors of P. salmonis. No sporogenic stage has been observed in P. salmonis;
however, examination of cells for such structures has been limited. One
important role of a vector in the life cycles of rickettsiae is prevention of
desiccation of these fragile bacteria during transmission from host to host.
Because the bacterial cell is protected from desiccation in an aquatic
environment, it is possible that no vector is required for P. salmonis and that fish-
to-fish transmission may occur. It has been demonstrated that P. salmonis can
survive 14 days in sea water (Lannan and Fryer, 1994), which might allow
horizontal transmission of the bacterium without the involvement of a vector.
Survival experiments in fresh water demonstrate an almost immediate
deactivation of P. salmonis, indicating that horizontal transmission in fresh
water is unlikely unless the bacterium is protected by host cell membranes or
other biological material.
Piscirickettsiosis has been induced experimentally by injection of
supernatant from an infected cell culture (Cvitanich et al., 1991; Garcs
et al., 1991). However, published experimental data concerning horizontal
transmission of P. salmonis are contradictory and differences in experimental
conditions make results impossible to compare. The organism has reportedly
been passed from experimentally infected coho salmon to uninfected coho in
both static freshwater and salt-water aquaria (Cvitanich et al., 1991). However,
in another study, P. salmonis transmission did not occur when uninfected coho
were caged in a tank with flowing fresh water and infected coho salmon (Garcs
et al., 1991).
Vertical transmission has not been demonstrated for P. salmonis. Rickettsiae
are found in the reproductive tissues of infected fish; however, the limited
number of PRS cases reported in juvenile fish in fresh water indicate that vertical
transmission, if it occurs, is a rare event.
Treatment and control
Although members of the rickettsiae are known to affect mammals (Weiss and
Moulder, 1984), P. salmonis presents no known health risk to humans. Like
many other pathogens of poikilotherms, the inability of the rickettsiae to survive
at temperatures of 25C and above precludes replication at human body
temperatures. Little is known of the host range or pathogenicity among aquatic
poikilotherms other than salmonid fish hosts. It is possible that other
cold-blooded species are susceptible to infection and, until more information on
host range and mode of transmission is available, efforts to control PRS and its
spread are impeded.
253 Rickettsial and Chlamydial Infections
Although P. salmonis is sensitive under in vitro conditions to certain
antibiotics (e.g. tetracycline, erythromycin, oxolinic acid) commonly used
against other infectious diseases, the efficacy of antibiotics in the treatment of P.
salmonis has not been clearly demonstrated. At present, oxolinic acid appears to
be the drug of choice. The intracellular location of the pathogen may make it
possible for substantial numbers of these organisms to be sequestered away from
contact with lethal concentrations of antibiotics and hence maintain the
infection.
The lack of effective chemotherapeutants available to fish culturists for
treatment of PRS has focused attention on vaccine development; however, no
efficacious preparations are currently available. The only strategy for control of
PRS is adherence to good fish culture practices and culture of the more resistant
species of salmonids in areas where P. salmonis occurs.
Other rickettsia-like organisms
Although P. salmonis is the only rickettsial pathogen of fish that has been
characterized, other RLO have been noted. The earliest report of an RLO in fish
was by Mohamed (1939), who observed the organism within cells of diseased
Tetrodon fahaka taken from the River Nile, Egypt. Diagnosis was made on the
basis of staining and intracellular location. The organism observed was not
cultured on media and no fish cell lines were available. The second observation
of an RLO was from cultured fish cells inoculated with tissue homogenates from
rainbow trout in Europe (Ozel and Schwanz-Pfitzner, 1975). These fish were
also infected with viral haemorrhagic septicaemia virus, so the relationship of
the RLO to the mortality that occurred in the trout population is unknown.
Studies were not conducted to verify the identity of the microorganism or to
determine its pathogenicity. The bacterium was not maintained and its
relationship to P. salmonis is unknown (I. Schwanz-Pfitzner, Berlin, Germany,
1990, personal communication).
Rickettsia-like organisms have since been reported from a variety of fish
species (Table 6.2). In 1986, Davies observed an RLO in the tissues of dragonets
(Callionymus lyra), collected in waters off the coast of Wales. An RLO was
observed in moribund specimens of the blue-eyed plecostomus (Panaque
suttoni), imported into the USA from Colombia, South America (Khoo et al.,
1995). In France, an RLO was observed in the brain of juvenile sea bass
(Dicentrarchus labrax) exhibiting abnormal swimming behaviour (Comps et al.,
1996). In Chile, an RLO similar to P. salmonis but smaller in size was isolated in
fish cell cultures from Atlantic salmon reared in fresh water (Cvitanich et al.,
1995). The organism was not recognized by anti-P. salmonis antibodies in a
direct fluorescent antibody test.
In the preceding observations of RLO, the relationship of the organism to
fish mortality was not clear. However, in the early 1990s, a disease outbreak
among tilapia (Oreochromis sp.) in Taiwan was directly linked to an RLO
(Chen et al., 1994; Chern and Chao, 1994). The disease eventually spread to
approximately 37 rearing facilities in both fresh and salt water; six species of
254
C.N. Lannan et al.
tilapia were affected and mortality reached 95% at certain sites. Behavioural
signs and pathologies exhibited were similar to those in salmonids infected with
P. salmonis. Morphology and culture characteristics indicated that the organism
was a rickettsia, but smaller than P. salmonis. The researchers were able to infect
nave fish with the isolated agent and recover the organism from moribund fish
thus completing Kochs postulates. Cohabitation studies demonstrated hori-
zontal transmission of the infectious agent from infected to uninfected fish
(Chern and Chao, 1994).
Table 6.2. Unidentified rickettsia-like organisms observed and/or isolated
from fish.
Geographical Salt/fresh Observed/
Fish host species location water isolated Reference
Fokaka Egypt F O Mohamed (1939)
(Tetrodon fahaka)
Rainbow trout Europe F I Ozel and Schwanz-
(Oncorhynchus Pfitzner (1975)
mykiss)
Dragonet Wales S O Davies (1986)
(Callionymus lyra)
Masu salmon Chile S O Bravo (1994)
(Oncorhynchus
masou)
Mozambique
tilapia Taiwan S, F I Chern and Chao
(Oreochromis (1994)
mossambicus)
Nile tilapia
(Oreochromis
niloticus)
Blue tilapia
(Oreochromis
aureus)
Redbelly tilapia
(Tilapia zillii)
Wami tilapia
(Tilapia hornorum)
Blue-eyed
plecostomus Colombia F O Khoo et al. (1995)
(Panaque suttoni)
Atlantic salmon Chile F I Cvitanich et al.
(Salmo salar) (1995)
Sea bass France S O Comps et al. (1996)
(Dicentrarchus
labrax)
F, host collected from fresh water; S, host collected from salt water; O,
observed only; I, isolated in cell culture.
255 Rickettsial and Chlamydial Infections
Topics for further study
Priority research areas should include development of therapeutic methods for
control of P. salmonis in cultured salmonids. These methods may include
antibiotic treatments, immunostimulatory compounds or specific vaccines. The
development of vaccines will require further investigations of the host immune
response to P. salmonis infection. Creative approaches may be needed, as tradi-
tional vaccine production methods are physically and financially impractical for
manufacture of such a bacterin for fish.
Determining the means of natural transmission and identifying the source of
infection in marine and freshwater environments are important in preventing
dissemination of P. salmonis to new geographical regions and to previously
uninfected stocks of fish. Use of molecular techniques for diagnosis will provide
a rapid, sensitive alternative to cell culture and enable researchers to establish
the host(s), geographical ranges and reservoirs of infection. These methods can
also provide information useful in determining the epizootiology of the
pathogen and means of preventing spread of the disease.
Biochemical comparison of the virulent isolates from Chile with the
apparently less virulent ones from Norway, Ireland and BC Canada may provide
valuable clues to the origins of P. salmonis. There is also a need to develop a
centrally located culture collection of rickettsiae isolated from fish so that
molecular, biological and antigenic properties can be compared.
CHLAMYDIAL INFECTIONS OF FISH
Epitheliocystis
Aetiological agent
The causative agent(s) of the disease referred to as EP are morphologically
diverse and may represent a group of related organisms that produce similar
pathology in varied hosts. The taxonomic placement of these diverse
intracellular microorganisms is undefined but is considered to fit most
appropriately in the order Chlamydiales (Moulder, 1984). Most exhibit the
pleomorphic developmental cycle typical of this group of intracellular bacteria
(Paperna et al., 1981). Studies of the ultrastructure of the CLO of EP show them
to range from about 0.2 to 1.2 m in diameter, with a typical Gram-negative
bacterial cell wall. Chlamydia species share a genus-specific, lipopoly-
saccharide antigen but this has not been demonstrated in the CLO observed in
fish.
Due to the inability to isolate and culture the CLO of EP, characterization is
based on observations of the ultrastructure. Development has been described as
progressing from a small, rigid infectious form to large pleomorphic stages,
which divide by fission. Four morphological stages have been commonly
described: the irregularly shaped initial or reticulate bodies; elongated cells;
round cells, which appear to be an intermediate developmental stage; and the
small cell presumed to be the infective stage (Paperna et al., 1978; Turnbull,
256
C.N. Lannan et al.
1993; Groff et al., 1996). Chained (Desser et al., 1988), branched (Paperna et al.,
1978) and head-and-tail cell forms (Bradley et al., 1988) which exhibit common
CLO features have also been described.
Disease
Epitheliocystis occurs as a benign or proliferative disease, characterized by cysts
in the branchial epithelia of the host. Clinical signs may include lethargy, flared
opercula and rapid respiration. Cysts may appear as transparent white to yellow
capsules on the gill filaments. Generally the host response is limited, and there is
little or no mortality associated with infection.
Characteristic cysts are hypertrophic host cells filled with the causative
bacterium (Fig. 6.5). The enlarged host cells range from 10 to 400 m in
diameter and are frequently surrounded by squamous or cuboidal epithelial cells.
The nature and origin of the target host cell is unclear; cysts may originate from
a single cell (Zachary and Paperna, 1977) or be a coalescence of several (Wolke
et al., 1970). The target cell may vary, as infection has been variously described
in mucus, epithelial lining and chloride cells of an unidentified species of carp
(Paperna and Alves de Matos, 1984); in capillary-forming cells of red sea bream
(Pagrus major) and tiger puffer (Takifugu rubripes; Miyazaki et al., 1986); and
in cells described as possible transformed macrophages in brown bullhead
(Ictalurus nebulosus; Desser et al., 1988).
Fig. 6.5. Epitheliocystis cyst in the gill of a juvenile white sturgeon (Acipenser transmontanus).
Haematoxylin and eosin stain. Bar =100 mm.
257 Rickettsial and Chlamydial Infections
Molnar and Boros (1981) described the development of EP cysts in the gills
of the common carp (Cyprinus carpio). In the early stages of the disease,
infected cells are 1015 m in diameter and contain a central inclusion body,
foamy cytoplasm and excentric nucleus. As infection progresses, infected cells
increase in size to 7080 m in diameter and the granular inclusion fills the
entire host cell, compressing or replacing both the nucleus and the cytoplasm.
In benign infections, cysts may be surrounded by a layer of squamous or
cuboidal epithelial cells. Typically, no host response is apparent, even in the
presence of large numbers of cysts. However, in certain cases, the organism
induces an extensive host response, with unrestricted proliferation of gill
epithelia and extensive mucus production, a condition referred to as hyper-
infection (Paperna, 1977; Rourke et al., 1984; Bradley et al., 1988). The
proliferative form of the disease has been described in Sparus aurata (Paperna,
1977). Hyperplastic epithelial cells form concentric layers around the cysts and
proliferate throughout the gill filaments. Infiltrating macrophages and eosino-
phils combine with the hyperplastic tissue to surround and obstruct the capillary
network of the gill filament. These circumstances impair both gas transfer and
osmoregulatory processes, which may result in the death of infected fish.
The causes for variation in the severity of EP infections are not clear, but
proliferative infections with accompanying mortality often occur in cultured fish
(Paperna, 1977; Miyazaki et al., 1986; Bradley et al., 1988; Crespo et al., 1990).
Benign infections are more frequently found in free ranging fish (Grau and
Crespo, 1991). Progression from the chronic to the progressive form may occur
when host defence mechanisms are compromised by genetics or environmental
factors (Paperna and Sabnai, 1980). Because the causative organism has not
been isolated, the possibility that these observations can be explained by
differences in virulence of the pathogen cannot be excluded.
Host range and geographical distribution
Epitheliocystis was first described in the bluegill sunfish (Lepomis macro-
chirus), by Hoffman et al. (1969), who identified the aetiological agent as a
bedsonia (now termed chlamydia). Molnar and Boros (1981) verified the
chlamydia-like nature of the causative agent and determined that EP was
identical to the mucophilosis disease of common carp described by Plehn (1920)
and believed to be caused by a unicellular alga or fungus. These descriptions
were followed by reports of the disease in freshwater, marine and anadromous
fish from both warm- and cold-water environments. The documented host range
includes species from more than 20 families of fishes (Table 6.3). Probably
additional hosts have gone unreported because of the self-limiting nature of the
disease.
Diagnostic methods
Preliminary diagnosis of EP is made by observation of the white to yellow cysts
on the gills or skin of affected fish (Wolf, 1988). The thick capsule and granular
contents, which are characteristic of EP cysts, are easily seen in wet mounts. The
gill is the most frequently affected organ, but it has been suggested that the
pseudobranch also be examined for cysts (Crespo et al., 1990).
258
C.N. Lannan et al.
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1
9
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(
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9
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(
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(
1
9
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1
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(
1
9
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(
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(
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9
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(
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(
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(
1
9
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(
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(
1
9
2
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(
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a
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(
1
9
8
1
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9
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P
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(
1
9
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A
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(
1
9
9
2
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(
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l
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(
1
9
8
8
)
(
B
r
o
w
n

b
u
l
l
h
e
a
d
)
259 Rickettsial and Chlamydial Infections
I
c
t
a
l
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r
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s

p
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n
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t
a
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s
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(
1
9
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4
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(
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c
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a
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(
1
9
7
0
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(
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p
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(
1
9
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0
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(
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(
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9
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(
1
9
7
9
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(
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(
1
9
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1
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(
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9
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(
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(
1
9
8
1
)
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a

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a
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M
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d
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d

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a
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a
p
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(
1
9
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7
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(
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(
1
9
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1
9
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1
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R
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(
1
9
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(
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(
1
9
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0
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(
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(
1
9
8
1
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d
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b
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(
1
9
8
0
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(
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o
a
t
f
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O
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n
a
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c
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a
p
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n
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a

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t

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.

(
1
9
8
7
)
(
R
o
c
k

s
e
a

b
r
e
a
m
)
P
A
R
A
L
I
C
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T
H
Y
I
D
A
E
P
a
r
a
l
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c
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s

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t
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s
N
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h
-
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t

A
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l
a
n
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O
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s

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t

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.

(
1
9
9
2
)
(
S
u
m
m
e
r

f
l
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u
n
d
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r
)
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a
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a
l
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t
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s

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b
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s
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h
-
w
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t

A
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l
a
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c

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a
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L
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s

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t

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(
1
9
9
2
)
(
F
o
u
r
s
p
o
t

f
l
o
u
n
d
e
r
)
P
E
R
C
I
D
A
E
L
a
t
e
s

c
a
l
c
a
r
i
f
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r
A
u
s
t
r
a
l
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a
A
n
d
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r
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n

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d

P
r
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o
r

(
1
9
9
2
)
(
B
a
r
r
a
m
u
n
d
i
)
C
o
n
t
i
n
u
e
d

o
v
e
r
260
C.N. Lannan et al.
P
H
Y
C
I
D
A
E
U
r
o
p
h
y
c
i
s

r
e
g
i
a
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h
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A
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a
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s

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l
.

(
1
9
9
2
)
(
S
p
o
t
t
e
d

h
a
k
e
)
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a
c
h
a
r
y

a
n
d

P
a
p
e
r
n
a

(
1
9
7
7
)
P
L
E
U
R
O
N
E
C
T
I
D
A
E
H
i
p
p
o
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l
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M
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d

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h
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m

(
1
9
8
3
)
(
A
m
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c
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n

p
l
a
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e
)
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m
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A
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(
1
9
9
2
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(
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t
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r

f
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(
1
9
9
2
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(
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f
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(
1
9
9
0
)
O
n
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c
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(
1
9
8
4
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(
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t
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O
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1
9
9
7
,

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(
1
9
9
0
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(
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m
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G
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t

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(
1
9
8
8
)
(
L
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k
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t
r
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t
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262
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No serological techniques are available for identification of the organism or
for diagnosis of infection. Electron microscopy is required for definitive
diagnosis of this disease. With this technique, the intracellular forms, too small
to be clearly differentiated under the light microscope, can be observed and
differentiated from cysts having a viral aetiology (Wolf, 1988).
Disease transmission
Natural transmission of the CLO is not understood, but horizontal transmission
apparently occurs within some host species. Hoffman et al. (1969) described
experiments in the bluegill and Wolf (1988) listed experiments conducted by
D.S. Wyand in goldfish (Carassius auratus) that demonstrated direct transmis-
sion. In both studies EP developed in the experimental animals 34 weeks after
the addition of infected gill tissue to aquaria where uninfected fish were held.
Paperna (1977) suggested that transmission from infected fish may occur in
culture facilities through contaminated nets and other equipment. There is little
information on interspecies transmission and it is not known if differences in the
morphology of the agent relate to different host specificities or the CLO itself.
No information is available concerning temperature requirements for repli-
cation of the CLO or on transfer of the CLO from fish to humans or other
warm-blooded animals. However, these microorganisms have only been
reported in fish.
Treatment and control
Most studies on EP are descriptive (e.g. reports of host species, characterization
of the histopathology, ultrastructural studies of the aetiological agents) and there
is little information on therapeutic methods. Frequently, the disease produces
limited mortality, and hence treatment is not necessary. One exception is a report
by Paperna (1977), who treated hatchery-reared juvenile S. aurata, suspected of
having EP, by feeding 1% chloramphenicol mixed in ground chick livers.
Although no information on untreated fish was included, it was implied that the
infection was controlled by the antibiotic therapy.
The development of vaccines against EP is unlikely at this time. The
infection is frequently benign, the aetiological agents appear diverse and none
have been isolated or grown in culture. Mortality associated with EP occurs
principally in hatcheries, where prevention and control of the disease can best be
accomplished through careful attention to proper fish husbandry.
Topics for further study
Future studies of the CLO of EP should be directed toward isolation, in vitro
propagation of the aetiological agents, biochemical characterization, determina-
tion of taxonomic placement and clarification of the relationship among
morphologically diverse groups of these organisms. Elucidation of the enigmatic
nature of the host target cell will also aid in the identification and classification
process.
The mode(s) of transmission of the CLO of EP should be clarified, and
identification of the factors leading to development of a proliferative rather than
a benign infection is a priority. Studies on the antimicrobial sensitivity of the
263 Rickettsial and Chlamydial Infections
aetiological agent(s) and development and testing of therapeutic procedures for
control of the proliferative infection are important. Development of more rapid
and precise diagnostic procedures for identification of the disease would also be
helpful.
ACKNOWLEDGEMENTS
This chapter is dedicated to William Q. Wick (24 October 1927 to 15 December
1991), who served as director of the Oregon Sea Grant programme, Oregon State
University, from 1973 to 1990 and was a staunch advocate of fish disease
research. The authors thank R.P. Hedrick of the School of Veterinary Medicine,
University of California, for providing histological preparations of EP-infected
fish. Preparation of the manuscript was made possible by Oregon Sea Grant,
with funds from the National Oceanic and Atmospheric Administration Office of
Sea Grant, Department of Commerce, under grant NA89AA-D-SG108, project
R/FSD-17. This is Oregon Agricultural Experiment Station Technical Paper No.
9846.
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