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Microb Ecol (1990) 19:21-41

MICROBIAL ECOLOGY
@Springer-VerlagNew York Inc. 1990

Bacterial Flora of Fishes: A Review

Marian M. Cahill
Department of Microbiology,University of Queensland, St Lucia, Queensland 4067, Australia

Abstract. Bacterial floras isolated from eggs, skin, gills, and intestines
have been described for a limited number of fish species. Generally, the
range of bacterial genera isolated is related to the aquatic habitat of the
fish and varies with factors such as the salinity o f the habitat and the
bacterial load in the water. In many investigations, identification of isolates
to the genus level only makes it difficult to determine the precise relation-
ships o f aquatic and fish microfloras. Bacteria recovered from the skin and
gills may be transient rather than resident on the fish surfaces. Microfloras
o f fish intestines appear to vary with the complexity of the fish digestive
system. The genera present in the gut generally seem to be those from the
environment or diet which can survive and multiply in the intestinal tract,
although there is evidence for a distinct intestinal microflora in some species.
While obligate anaerobes have been recovered from carp and tilapia in-
testines, low ambient temperatures may prevent colonization by anaerobes
in species such as rainbow trout.

Introduction

Various aspects o f the normal microbial flora associated with fish have been
studied. These include the changes in flora during storage [36], the effects of
catching methods or handling on microflora which might lead to deterioration
[ 12, 15], the relationship between environmental and fish microfloras [ 17, 41,
47-50], and the establishment of bases for monitoring changes in fish farms
[1-3, 25].
In attempting to define the bacterial groups normally associated with various
fish species, many researchers have come across problems with taxonomy.
Shewan [37] stated that when he began to study the bacteriology of fresh and
spoiling fish he found that it was difficult to identify m a n y isolates even to the
genus level. Most papers written before 1980 only refer to the genera of bacteria
isolated, but recently, with the use of numerical taxonomy and determination
o f % G + C, identification to the species level and the recognition of new phena
is occurring. For this review, I have used the identifications given by the authors
of the articles quoted, although some of these identifications are now out of
date. The genus Achromobacter, for example, is now Moraxella-Acinetobacter.
Differentiation o f the Flavobacterium-Cytophaga group has been rationalized,
and there has been much revision o f the species within the genus Vibrio. Most
22 M. M. Cahill

Table 1. Bacterial flora of fish eggs

Fish species Location Bacteria isolated Reference


Chum salmon (On- Vancouver Pseudomonas spp., Cytophaga sp., Bell et al. [9]
chorhynchus keta) I., Canada Sarcina sp., Chromobacterium
and pink salmon (O. lividum, Aeromonas liquefaciens,
gorbuscha) Enterobacter, Klebsiella
Masu salmon (O. ma- Japan Flavobacterium/Cytophagaceae, Yoshimizu et
sou) and chum Pseudomonas al. [50]
salmon (O. keta)
Goldfish (Carassius Japan Aeromonas hydrophila, A. puncta- Sugita et al.
auratus) ta, Pseudomonas, Flavobacteri- [42]
um, Micrococcus; other spp. iso-
lated less frequently

m a r i n e c o r y n e f o r m bacteria h a v e been s h o w n to be very similar to the arthro-


bacters.

The Microflora of Fish Eggs

T h r e e reports [9, 42, 50] suggest similar m i c r o b i a l floras on the surfaces o f


freshwater fish eggs (Table 1). R e p o r t s o f total viable counts on fish eggs were
103 to 106 g-i [42, 50].
Bell et al. [9] c o m p a r e d the bacterial floras o f b o t h healthy a n d d e a d stream-
incubated s a l m o n eggs with those o f s i m u l a t e d eggs (polyethylene spheres) and
a m b i e n t stream water to d e t e r m i n e any relationship between egg microflora
and egg survival. C y t o p h a g a a n d P s e u d o m o n a s species were isolated m o s t
frequently f r o m healthy e m b r y o n a t e d eggs, while d e a d eggs yielded m a i n l y
fluorescent P s e u d o m o n a s species. Simulated eggs had a m o r e diverse flora,
c o m p a r a b l e to that o f the stream. A e r o m o n a s liquefaciens (=A. hydrophila) was
isolated f r o m live and s i m u l a t e d eggs a n d f r o m s t r e a m water. T h e P s e u d o m o n a s
species isolated f r o m the d e a d eggs were p r o b a b l y stimulated b y nutrients
leaching f r o m the dead egg and were unlikely to be the cause o f death. T h e
physical a n d chemical conditions o f the e n v i r o n m e n t were considered to be
the m o s t i m p o r t a n t factors in ensuring the survival o f eggs, as no specific
bacterial pathogen responsible for egg m o r t a l i t y was isolated. T h e s e results
were based on the study o f 67 strains o f bacteria isolated on t r y p t o n e glucose
extract agar. This m e d i u m would support the growth o f a limited range o f
freshwater bacteria, and the use o f additional m e d i a would p r o b a b l y d e m o n -
strate a m o r e diverse flora.

Skin Microflora

E x a m p l e s o f the range o f bacterial genera isolated f r o m fish skin are given in


Table 2.
Colwell's survey was u n d e r t a k e n to d e t e r m i n e the microbial flora o f Puget
Sound fish caught by different fishing m e t h o d s [ 12]. T h e p u r p o s e o f the survey
Bacterial Flora of Fishes 23

Table 2. Bacterial flora of fish skin

Fish species Location Bacteria isolated Reference


Various ~ Puget Pseudomonas 43.9%, Vibrio 3.4%, Colwell [12]
Sound, Achromobacter 18.1%, Flavo-
USA bacterium 8.6%, Corynebacte-
rium 7.8%, Micrococcus 6.9%,
enterobacteria 5.2%, Bacillus
0.9%, others 5.2%
Atlantic salmon (Sal- Dee R. and Moraxella, Acinetobacter, Pseudo- Horsley [17]
mo salar) Cove monas, Flavobacterium/Cytoph-
Bay, aga, Enterobacteriaceae, coryne-
Scotland forms, Micrococcaceae,
Bacillus, Aeromonas
Sea mullet (Mugil Moreton Micrococcus49%, Pseudomonas Gillespie and
cephalus), whiting Bay, 18%, coryneforms 12%, Morax- MacRae [15]
(Sillago ciliata), Queens- ella 8%, Flavobacterium/Cy-
bream (Mylio aus- land, tophaga 8%, plus Staphylococ-
tralis), and flathead Australia cus, Bacillus, Acinetobacter,
(Platycephalus fus- Aeromonas, and Vibrio
cus)
Plaice (Pleuronectes Scotland Pseudomonas Groups I, II, and Gilmour et al.
platessa L.) IV/Achromobacter/Alcaligenes/ [16l
Agrobaeterium, Vibrio/anaero-
genic Aeromonas, Enterobacteri-
aceae, Moraxella
Turbot (Scophthalmus Lowest&t, Acinetobacter calcoaceticus, Alcali- Austin [3]
maximus L.) Suffolk, genes spp., Bacillus spp., Caulo-
England batter spp., coryneforms, Cy-
tophaga-Flavobacterium-
Flexibaeter spp., Enterobacteri-
aceae, Hyphomierobium-Hypho-
monas spp., Lucibacterium
harveyi, Photobacterium spp.,
Prosthecomicrobium spp., Pseu-
domonas spp., Vibrio spp., un-
identified spp.
" Dogfish (Squalus acanthius L.), cod (Gadus macrocephalus Tilesius), English sole (Parophrys
vetulus Girard), Pacific hake (Merluccius productus Ayres), black rockfish (Sebastodes melanops
Girard), ocean perch (Cymogaster aggregata Gibbons), lingcod (Ophiodon elongatus Girard), starry
flounder (Platichthys stellatus Pallas), sea perch (Embiotoca lateralis Agassiz), sculpin (Scorpaen-
ichthys marmoratus Ayres), king salmon (Onchorhynchus tschawytscha Walbaum), silver salmon
(0. kisutch Walbaum), chum salmon (O. keta Walbaum), steelhead (Salmo gairdneri Richardson),
and Pacific herring (Clupea pallasi Valenciennes)

was to a t t e m p t to relate v a r i a t i o n s in the flora to possible c o n t a m i n a t i o n d u e


to the k i n d o f h a n d l i n g the fish r e c e i v e d d u r i n g purse a n d b e a c h seining, otter
trawling, a n d c a t c h i n g by h a n d line. T h i s c o n t a m i n a t i o n c o u l d alter the n o r m a l
flora adversely, resulting in m o r e r a p i d d e t e r i o r a t i o n a n d thus a r e d u c t i o n in
the k e e p i n g q u a l i t y o f the fish. Results suggested t h a t the m e t h o d s o f h a n d l i n g
fish, a n d their p r e c a p t u r e e n v i r o n m e n t , influence the c o m p o s i t i o n o f the skin
flora. N o t all c o m p a r i s o n s o f skin flora f r o m different e n v i r o n m e n t s h a v e u s e d
Standardized h a n d l i n g m e t h o d s (e.g., see H o r s l e y [17]).
24 M.M. Cahill

Horsley examined bacteria from the skin of Atlantic salmon in marine,


estuarine, and fresh waters. The frequency of the genera isolated varied at the
different sampling sites, and the major components of the skin flora were similar
to those present in the water, again indicating that the external flora of fish are
a reflection o f their environment. There were 10 2 to 10 3 viable heterotrophic
bacteria cm -2 on the skin and similar numbers ml -L in the water.
The above studies and those of Gillespie and MacRae [15] and Gilmour et
al. [16] show similar ranges of genera on fish skin, although the proportions o f
the different types varied with geographical location.
Austin studied a coastal marine fish-rearing unit using numerical taxonomy
techniques [2, 3]. About 600 isolates were tested for 130 unit characters, and
72 reference strains were included in the study. Isolates from sea water, slime
and tank effluent, as well as from the surface of healthy turbot Scophthalmus
rnaximus, and from lesions of moribund turbot, were examined. Many of the
bacteria were identified to the species level, and it was found that the greatest
diversity o f bacterial taxa, totaling 25, was isolated from the skin of healthy
turbot. Of the surface microflora, Photobacterium angustum, 'Photobacterium
logei,' Alcaligenes faecalis, Pseudomonas fluorescens, and Bacillus firmus, were
isolated exclusively from the surface o f healthy fish. In spite o f this abundant
growth from skin samples, scanning electron micrographs of the skin o f healthy
turbot did not show bacterial colonization, only adherent debris. The fixation
procedure used in the preparation of the skin for electron microscopy may
have removed any loosely associated bacteria, or bacteria may have been masked
by debris. Whether the organisms isolated exclusively from the surface of
healthy fish were obligate or opportunistic inhabitants was not really considered
in this study. These bacteria may also have been present in the water as a result
o f being shed from fish, but in numbers too low to be detected by the sampling
methods used.
The surface microflora isolated from turbot by Austin [2, 3] did not closely
reflect the types o f bacteria found either in the sea water which supplied the
tanks, or in the tankwater in which the fish lived. This contrasts with studies
such as those of Horsley [17], and Gilmour et al. [16]. However, in the latter,
isolates were identified to the genus level only on the basis of comparatively
few characters. This approach may have simplified the actual situation, where
the genera may be similar but the species or strains within the genera vary
between the water and the fish surface.
The mucus o f the gills, gut, and skin of fish contains lysozyme and immu-
noglobulins which presumably act as defense mechanisms against bacteria [18,
27]. Therefore, true commensal bacteria on these surfaces are likely to resist
such mechanisms and may have interesting properties associated with such
resistance. However, whether bacteria isolated from fish skin are a true resident
microflora, or are merely loosely associated with the surface, is debatable.
Thoroughly flushing the eyes and skin of rainbow trout, Salmo gairdneri, with
sterile river water before microbiological examination can remove nonresident
or transient organisms, leaving the eye and skin surfaces apparently free of
bacteria, although bacterial cells remain on the tail, fin, and gills after flushing.
Mucus, eye washings, and eye extracts of rainbow trout have been shown to
have inhibitory activity against various bacteria isolated from unflushed fish
Bacterial Flora of Fishes 25

skin, including Aerornonas hydrophila, Cytophaga, Flavobacterium, Micrococ-


cus roseus, Pseudomonas fluorescens, and Staphylococcus epidermidis. The na-
ture of the inhibitory substance has not yet been determined, but preliminary
work suggests that it may be a glycoprotein [8]. Murray and Fletcher [23]
suggested that lysozyme released from leucocytes might bind to proteoglycans
or glycoproteins in the scale pockets o f fish skin, and the resulting complex
could be ejected to the surface to combine with mucus produced when the fish
is stressed. The inhibitory substance recovered from rainbow trout, however,
had properties distinct from those of Iysozyme, and so could be a new anti-
bacterial compound.
Bacteria present on the skin of fast-swimming fish may change the surface
properties offish skin to reduce drag and thus assist in locomotion [33]. Bacteria
resident on the skin of such fish might have (a) the ability to attach strongly
to the skin, (b) high surface hydrophobicity, (c) the ability to release drag-
reducing polymers to dampen local turbulent flow, and (d) mechanisms for
overcoming antimicrobial agents in fish skin or mucus. An initial study of
bacteria adherent to the skins of fast-swimming marine fish showed that two
of 13 isolates had very high colonial hydrophobicity (measured as the ability
of the edge of a lawn of bacteria to repel water droplets). There was evidence
that some isolates could produce a hydrophobic slime which might reduce drag.
The isolates were not identified, and further investigations into the surface
properties of these bacteria and their densities on fish skin would be needed
to support claims for their ability to help fish swim faster.

Gill Microflora
Both marine and freshwater fishes have been shown to support quite high
populations of a range of bacterial genera on their gills. Studies reviewed by
Shewan [36] recorded Pseudomonas, Achromobacter, Flavobacter, and Vibrio
species, in descending order of frequency, on gills of marine fish from the North
Sea and Norwegian waters, whereas only Bacillus and Micrococcus were isolated
from gills of fish from warmer waters off India. These differences probably
reflect differences in environmental temperatures, with more psychrophiles and
fewer mesophiles in the cold North Sea waters. Results of more recent studies
are shown in Table 3.
Horsley's study [17] of bacterial genera on the gills of Atlantic salmon mi-
grating up the Dee River in Aberdeenshire, Scotland showed that the relative
numbers o f the different genera changed with changes in the environment of
the fish, from marine to freshwater. Moraxella comprised 32% of the flora of
fish from marine sampling sites but only 8% of that from upland sites. Also,
Vibrio spp. were isolated only from fish caught at marine sites, whereas Aero-
rnonas spp. were present in highest numbers on gills offish taken from upstream
Waters. From all of the sampling stations the major components of the flora
of the fish were similar to those present in the water, which supports the
hypothesis that the external flora of fish is a reflection of their environment.
The Atlantic salmon can be caught in marine, estuarine, and freshwater en-
vironments and so allows comparison of the flora of one fish species with that
of different aquatic environments.
26 M. M. Cahill

Table 3. Bacterial flora of fish gills

Fish species Location Bacteria isolated Reference


Various" Puget Pseudomonas 44.7%, Vibrio 9.5%, Colwell [ 12]
Sound, Achromobacter 16.9%, FIavo-
USA bacterium 7.4%, Corynebacte-
rium 4.2%, Micrococcus 8.4%,
enterobacteria 1.5%, others
7.4%
Flatfish (Kareius bico- Fish mar- Vibrio, Aeromonas, Pseudomonas, Simidu et al.
loratus) ket, Ja- Achromobacter, Flavobacterium, [38]
pan Corynebacterium, Micrococcus,
yeast
Atlantic salmon (Sal- Dee R. and Moraxella, coryneforms, Flavo- Horsley [17]
mo salar) Cove bacterium-Cytophaga, Pseudo-
Bay, monas, Micrococcaceae, Acine-
Scotland tobacter,Bacillus spp.,
Enterobacteriaceae, Aeromonas,
Vibrio spp.
Salmonids British Co- Pseudomonas, Cytophaga, Flavo- Trust [44]
lumbia, bacterium, Aeromonas, coryne-
Canada forms, Bacillus, Brevibacterium,
Micrococcus, Acinetobacter, Vib-
rio, and others less frequently
Rainbow trout (Salmo Hatchery, Pseudomonas/Xanthomonas, Nieto et al.
gairdnert) N.W. Aeromonas, Vibrio, Flavobacteri- [26]
Spain um-Cytophaga, enterobacteria,
corynebacteria, gram-positive
cocci
Turbot (Scophthalmus Fish farm, Janthinobacterium lividum 32%, Mudarris and
maximus) UK Vibrio spp. 24%, Hyphomicro- Austin [21]
bium 18%, Pseudomonas fluo-
rescens 12%, Asticacaulis spp.
10%, Prosthecomicrobium sp.
4%
"See Table 2 for explanation

Trust [44] sampled gills of free-living and hatchery-reared salmonids from


British Columbia and also demonstrated a difference between the gill micro-
floras of freshwater and marine fish. The mean values of numbers of bacteria
ranged from 6 x 102 to 2.2 • 106 organisms/g wet weight of gills. Although
this study probably underestimates the bacterial population of the gills as the
media used would be unlikely to support the growth of all bacteria present, it
was calculated that the area of gills covered by bacteria would be only 0.02%
of the gill surface. How fish are able to maintain such a low number of bacteria
on this large surface area exposed to continual water flow, and with potential
nutrient availability, is uncertain. One possible mechanism has been shown in
an experimental study where extensive colonization of gill surfaces by Flavo-
bacterium species was followed by host gill epithelial hyperplasia and subse-
quent shedding of most of the bacteria from the gill surfaces [22]. Cytophaga
species are apparently members of the microflora of normal gills, but are able
Bacterial Flora of Fishes 27

to cause disease under certain circumstances, as myxobacterial gill infections


are widespread, especially in fish hatcheries [44].
After testing media formulations of various compositions for their ability to
recover gill microflora, Mudarris and Austin [21] found that the highest count
of aerobic heterotrophic bacteria on the gills of healthy turbot, Scophthalmus
maximus L., was 7.0 • 103 g-1 wet weight of gill tissue. Identification of these
bacteria showed that all of the isolates were gram-negative rods, many of which
possessed prosthecae (Table 3). Scanning electron microscopy of gill prepara-
tions indicated that only protected sites on the gills, such as clefts between the
secondary lamellae, were colonized by bacteria. These bacteria covered only
0.1 to 1.0% of the gill surface in these areas, although higher coverage (5%)
occurred where gills were damaged. The gill microflora was shown to be quite
distinct from that of the surrounding water, and from that of fish skin. These
observations differ from those of previous authors who found that the gill
microflora resembled that of the surrounding water (see [7], p. 38). The use of
more appropriate methods for isolating these bacteria probably accounts for
these differences.

Intestinal Microflora

The microbiology of the intestinal tract of marine and freshwater fish has been
investigated by many researchers. There is evidence that dense microbial pop-
ulations occur within the intestinal contents, with numbers of bacteria much
higher than those in the surrounding water, indicating that the intestines provide
favorable ecological niches for these organisms (see [7], p. 26). Table 4 sum-
marizes the results of a number of these investigations.
The method of sampling of intestinal bacteria varied in these surveys. The
use of anal swabs by Colwell [ 12] has been criticized (see [7], p. 37) as it probably
does not give a representative sample of gut flora. In some cases [16, 24, 30,
41-43, 46] only the intestinal contents were sampled, while in others the in-
testinal tract plus contents were homogenized and used for culturing [19, 26,
38, 45, 46]. Austin and A1-Zahrani [6] distinguished between the flora of the
gut contents and that intimately associated with the wall of the gastrointestinal
tract, and noted that scanning electron microscopy showed only sparse micro-
bial colonization of the wall. In this study, there was a progressive decline in
the numbers of aerobic heterotrophic bacteria along the digestive tract. An-
aerobes were detected only in the upper intestine and in the intestinal contents.
However, Trust and Sparrow [46] found that numbers of bacteria in fresh-
water salmonids increased between the stomach and the posterior portion of
the intestine. They suggested that these numbers must represent active mul-
tiplication in the tract as they could not be accounted for by ingestion. The
numbers detected in this survey are probably an artificially low estimate since
the methods used did not allow for the isolation and growth of strict anaerobes,
species sensitive to oxygen, nutritionally fastidious species, or those requiring
low growth temperatures (below 20~ Also, the counts obtained were based
on the total tissue weight in each sample, while the bacteria actually populate
only the epithelial surface of the tract, and the rest of the tissue of the tract is
Table 4. Intestinal microflora o f fish"
to
Achro- Flavo- Coryne- oo
Pseudo- mobac- bacte- bacte- Aero- Micro-
Fish species Location monas Vibrio ter rium rium Bacillus monas coccus
Various (see Table 2) Puget Sound, U S A 48.1 13.9 17.7 2.5 1.3 5.1 5.1
Flatfish (Kareius bicoloratus) Fish market, J a p a n + + + +
Bluefish (Pomatomus saltatrix) Long Island Sound, 22.4 28.4 18.2 1.6 0.9 1.4
USA
Striped bass (Morone saxatilis) H u d s o n River, U S A 23 10 5 2 0.5 33 1
(estuarine)
Long Island Sound, 11 27 18 1 0 21 3
U S A (marine)
Plaice (Pleuronectus platessa) Scotland + + + + + +
Salmonids Freshwater lakes, + + + + + + +
Canada
R a i n b o w T r o u t (Salmo gairdnerO Fish farm, Scotland 12 4 12 8 16 12

Dace, field gudgeon, c o m m o n m i n n o w , T a m a River, J a p a n + + + + + + +


stone m o r o k o , carp, crucian carp,
a n d brackish goby
Goldfish (Carassius auratus), Pond, Japan + + +
1-year-old
R a i n b o w trout (Salmo gairdnert) Hatchery, Spain + + + +
Carp (Cyprinus carpio) Rearing tank, J a p a n + + + +
Salmonids Lakes, British + + + + + + +
Columbia, C a n a d a
G r a s s carp (Ctenopharyngodon Fish tanks, British + + + + + +
idella) Columbia, C a n a d a
Tilapia zillii Freshwater tank, + + +
Japan .~
Tilapia zillii Seawater tank, J a p a n + + +
Tilapia (Sarotherodon niloticus) Culture ponds, J a p a n + + =:
Figures give the percentage incidence o f different groups o f bacteria; + indicates that representatives o f the group were isolated
Table 4. Continued
9.
Acineto-
Entero- bacter- Bacte- 0
bacte- Alcalig- Mora- Staphyl- Clos- roi- P,
Fish species riaceae enes xella OCOCCItS tridium daceae Yeasts Others Reference o

Various (see Table 2) 2.5 3.8 Colwell [12]


Flatfish (Kareius bicoloratus) Simidu et al. [38]
Bluefish (Pomatomus saltatrix) 21.4 2.5 3.0 N e w m a n et al. [24]

Striped bass (Morone saxatilis) 15 MacFarlane et al. [19]

14
Plaice (Pleuronectus platessa) + Gilmour et al. [ 16]
Salmonids + + Trust and Sparrow
[46]
R a i n b o w Trout (Salmo gairdneri) 2O Austin and Al-Zahrani
[6]
Dace, field gudgeon, c o m m o n minnow, + + Sugita et al. [41 ]
stone moroko, carp, crucian carp,
and brackish goby
Goldfish (Carassius auratus), + Sugita et al. [42]
1-year-old
R a i n b o w trout (Salrno gairdnerO + + Nieto et al. [26]
Carp (Cyprinus carpio) Sugita et al. [43]
Salmonids + Trust and Sparrow
[46]
Grass carp (Ctenopharyngodon + Trust et al. [45]
idella)
Tilapia zillii Sakata et al. [30]
Tilapia zillii Sakata et al. [30]
Tilapia (Sarotherodon niloticus) Sakata and Koreeda
[29]
bo
30 M.M. Cahill

sterile. There was no significant difference in the bacterial flora offish o f different
species, sex, breeding status, weight, or geographical source.
The predominant species were Enterobacter, Aeromonas, and Acinetobacter
in these freshwater fishes. This contrasts with those in digestive tracts o f marine
fishes, where Vibrio, Pseudomonas, Achromobacter, Corynebacterium, Flavo-
bacterium, and Micrococcus predominated [12, 24].

Relationship of Intestinal Microflora to That of Aquatic Habitat

MacFarlane et al. [19] noted differences in the composition o f the intestinal


floras o f marine and estuarine striped bass (Morone saxatilis). Bacterial num-
bers were consistently higher in the intestines of the latter fish, perhaps reflecting
a higher organic content of estuary water (Hudson River) as compared with
marine water (Long Island Sound). It was also found that the numbers o f the
various genera isolated varied from month to month in both environments.
Results such as this give evidence that the bacteria present in the aquatic
environment influence the composition of the gut flora.
The intestinal microflora o f salmonids reared in both fresh and sea water,
and of salmonids collected from rivers and lakes, was determined and compared
with the microflora of the waters from which the fish were taken [47, 48]. In
the farmed fish, the number of viable bacteria ranged from 104 to 107 g-~
intestinal contents. The intestinal microfloras were simpler than those of the
surrounding waters, consisting of Aeromonas and Enterobacteriaceae in flesh
water-reared fish, and Vibrio in fish from sea water. The waters contained about
103 bacteria m1-1, with no seasonal variation, most of which were Flavobac-
terium-Cytophaga, Achromobacter, and Pseudomonas. Fish taken from rivers
and lakes also had Aeromonas and Enterobacteriaceae as the predominant
intestinal microflora, and again this microflora was simpler than that o f the
water. In this case there was seasonal variation, with counts being higher from
spring to summer and lower in winter. In anadromous salmon, the intestinal
slime o f nonfeeding fish was sampled and cultured, and its microflora was
found to consist o f Vibrio, Pseudomonas, and Aeromonas. In mature masu
salmon cultured in fresh water, Aeromonas was the predominant genus isolated
from intestinal slime [49].
A similar study was undertaken [41 ] in which aerobic and anaerobic bacteria
were isolated from the guts of seven types o f fish from different sites along a
river in Japan and were compared with the bacterial flora of water, sediment,
and aquatic insects. Seasonal variations were also measured. The total viable
counts o f the gut contents were 105 to 108 g-l, and Enterobacteriaceae and the
Vibrio-Aeromonas group were predominant in all samples. Similar ranges of
genera were isolated from intestines, water, sediment, and aquatic plants and
insects, with some variations according to sampling site, time of year, and
origin of sample. These results were considered to suggest that the flora o f the
gastrointestinal tract o f the fish originated from their environment.
Again, the intestinal micro flora of fingerling rainbow trout from hatcheries
in northwest Spain was mainly enterobacteria and Aeromonas. Numbers o f
Pseudomonas, Aeromonas, and enterobacteria isolated from fish at monthly
intervals paralleled numbers isolated from water, although the numbers in water
Bacterial Flora of Fishes 31

showed fluctuations. G r a m - p o s i t i v e cocci from these fish did not show the
same correlation with those from the water supplying the ponds, but handling
and c o n t a m i n a t e d feed m a y have affected the n u m b e r s o f staphylococci detected
in the fish [26].
Farming practices for some fish [e.g., rainbow trout (Sahno gairdneri)] require
a constant flow o f fresh water through the fish ponds. W h e r e large n u m b e r s o f
fish are being raised, the addition o f fish feed (some o f which is not utilized)
and fish feces to the water can have a detrimental effect on the microbial quality
o f the effluent water from the fish farm. A study in Finland [25] showed that
effluents from two fish farms had elevated n u m b e r s o f total coliforms and fecal
coliforms, and on one farm, effluent had m o r e fecal streptococci than the
influent. The majority o f coliforms were identified as Enterobacter and Citro-
bacter, and Aeromonas hydrophila was quite c o m m o n . High concentrations o f
fecal streptococci and total coliforms were detected in fish feces. Fecal strep-
tococci and coliforms appeared to enter the farms via influent water and feed,
and seemed to multiply in the fish intestines and possibly in the sediment. A
quantitative study o f the intestinal microflora o f the farmed fish was not done,
although such a study could have indicated m o r e accurately the relationship
between intestinal bacteria and those in the effluent.
Austin [4] also c o m p a r e d the microbial floras o f the inflow and outflow from
four rainbow trout farms. T h e r e was no consistent increase in coliforms or in
Aeromonas hydrophila in the effluents f r o m these farms, although Enterobacter
aerogenes increased from 0 (inflow) to 5% (outflow) on two farms, and there
was a similar increase in Escherichia coil on another two farms. In these cases,
increases were considered due to increasing eutrophication and were not linked
to multiplication within the fish intestinal tract.
While the microorganisms present in water can influence the microbial flora
associated with fish, the contribution o f bacteria shed from fish to water is also
worth consideration, especially where dense populations offish are maintained,
as in aquaculture.
Total viable counts and determinations o f the varieties o f bacteria in the
water o f a tank used for rearing carp were m a d e before, during, and after the
addition o f the fish [43]. Total viable counts increased after the introduction
of carp, and the diversity and p r e d o m i n a n c e o f bacterial groups also changed.
Before the introduction o f fish, Pseudomonas and the Flavobacterium-Cytoph-
aga group were most n u m e r o u s in the water, and Enterobacteriaceae, the Vibrio-
Aeromonas group, and Bacteroidaceae were not detected. T h e Vibrio-Aero-
rnonas group b e c a m e p r e d o m i n a n t after the fish had been added. As the only
bacteria present in the pelleted diet o f the fish were Bacillus and Clostridium,
it appeared that the Vibrio-Aeromonas and Bacteroidaceae in the water had
originated from the fish feces. T h e presence o f these organisms in the water
seemed d e p e n d e n t on a continual input from fish excreta, as they disappeared
once fish were removed.

The Intestinal Microflora o f Tilapia Species

Tilapia zillii is a fish which originated in Africa where it inhabits freshwater


and river estuaries. It has been introduced into other parts o f the world and
32 M.M. Cahill

has been cultured extensively in Japan and elsewhere. It was considered a good
subject for a study o f intestinal flora because o f its ability to adapt to fresh and
sea water, and its o m n i v o r o u s diet, including phytoplankton, algae, and some-
times water plants. Its intestine is five to seven times its b o d y length and has
an a b u n d a n t c o m m e n s a l flora [30]. Counts and identification o f viable hetero-
trophic aerobic bacteria were m a d e from h o m o g e n i z e d sections o f intestines
offish kept in fresh or sea water, and the variety o f these bacteria was c o m p a r e d
with that o f the corresponding water.
T h e n u m b e r o f genera isolated from the fish intestines was m u c h m o r e
restricted than that isolated from the surrounding water. Aeromonas p r e d o m -
inated in the intestines o f freshwater Tilapia, whereas Flavobacterium and
Pseudomonas p r e d o m i n a t e d in freshwater. In marine fish, Vibrio and Aero-
monas f o r m e d the greater percentage o f the intestinal flora, but Pseudomonas
was most frequently isolated from the sea water. H o w e v e r , where fish had been
kept for a long time (181 days) in sea water, there were m o r e Aeromonas than
Pseudomonas in the water, indicating that fish feces m a y be a source of Aero-
monas in water.
In a further study o f the microbial flora of Tilapia intestines, separate groups
o f fish were reared in freshwater and in 25, 50, 75, or 100% sea water [40].
T h e bacteria o f the stomach, fore-intestine, and postintestine (sic) were isolated.
Bacteroides types A and B, aerobic gram-negative rods, coryneforms, and strep-
tococci were isolated from all three regions. Bacilli, aerobic gram-positive cocci,
and yeasts were found only in the stomach and fore-intestine regions. Where
the fish were reared in 75 and 100% sea water, n u m b e r s o f obligate anaerobes
decreased a n d / o r disappeared, and aerobic and facultatively anaerobic gram-
negative rods predominated. Bacteroides type A was found to grow in NaCl
concentrations o f 0 to 3%, Bacteroides type B in 0 to 2%, and Vibrio-Aeromonas
in 0 to 4%. Therefore, sensitivity to increased salinity is a possible reason why
the bacterial flora in the gastrointestinal tract o f Tilapia nilotica changes during
the process o f the fish's adaptation to sea water.

Relationship o f Intestinal Microflora to That o f Food

The bacterial flora o f the gut o f two marine fish has been investigated in an
attempt to clarify the relationship between these bacteria and the bacterial flora
of their diets, and to d e t e r m i n e the effect o f the degree o f specialization o f the
digestive tracts on their floras [34]. Bacteria were isolated from the gastric and
intestinal contents o f two fish species, red sea bream and file-fish, at various
time intervals after the fish were fed in the laboratory. Bacteria were also isolated
from the fishes' e n v i r o n m e n t and from various diets (fishmeat, fishmeat sup-
plemented with chitin or starch, and shellfish). The file-fish has a relatively
undeveloped stomach in comparison with that o f the red sea bream. T h e
stomach and intestinal contents o f the file-fish closely reflected the bacterial
flora of their diet, whereas in red sea bream, the d o m i n a n t flora always consisted
o f one or both o f two groups o f vibrios (distinct from those present in the
diets). These two groups o f vibrios were both resistant to 2% bile and low p H
(5.5) and were considered to be m e m b e r s o f an indigenous flora o f the digestive
Bacterial Flora of Fishes 33

tract o f sea bream. This work implied that the bacterial flora of fish with
relatively undeveloped digestive tracts reflected that o f the fishes' food, whereas
fish with more specialized tracts have a distinctive gut microflora. In the red
sea bream, the composition of the bacterial floras of the stomach and intestine
changed with time after feeding. Half an hour after ingestion most o f the bacteria
isolated from the stomachs resembled those of the fishmeat diet, but after 6
hours, vibrios resistant to bile and low pH predominated.
Further work comparing representative strains of these indigenous vibrios
from the red sea bream with other isolates from the stomach and intestine
(peritrichous rods, Achromobacter species, and cocci) showed that the vibrios
were able to survive in the presence of gastric juice at pH 4, and were able to
grow, although at a reduced rate, at pH 5, while most o f the other isolates were
inhibited by these conditions [35].
N e w m a n et al. [24] sampled the microbial flora o f the bluefish (Pomatomus
saltatrix) intestine to see whether the microorganisms present were dependent
on the type o f food most recently ingested. The bacteria isolated were similar
to the flora o f some other marine fish (e.g., skate, sole, and cod) which may be
prey to bluefish. Whether these fish actually were prey was not determined,
and no observations o f the flora of prey fish from the same geographic location
was available, so this conclusion was rather conjectural. The gut flora merely
seems typical o f that o f most marine fish.

The Effect of Antibiotics on Intestinal Microflora

Medicated feeds o f the types used in the treatment o f fish diseases contain
antimicrobial agents, such as oxolinic acid, oxytetracycline, sulphafurazole,
erythromycin, and penicillin G. Administration o f these to rainbow trout caused
changes in the population composition of the gastrointestinal flora [6]. Organ-
isms sensitive to these compounds appeared to be inhibited and were succeeded
by a resistant microflora. This suggests that the presence of the normal micro-
flora o f the gut may prevent its colonization by other bacteria. Antimicrobial
treatment also resulted in higher populations of bacteria in the lower intestine,
but the reason for this was not clear.

Obligate Anaerobes in Intestinal Microflora

Most investigators offish intestinal microflora have used methods which would
isolate only aerobic or facultatively anaerobic bacteria. Trust et al. [45] isolated
obligate anaerobes from intestines of grass carp and goldfish, showing that fish
can have a resident nonpathogenic anaerobic microflora. The number of an-
aerobes in the gastrointestinal tract plus contents ranged from 6.6 x 1 0 4 to 1.6
• 109 g-l, depending on the medium used and the region of the tract sampled.
The highest count was from the hindgut of grass carp. Strict anaerobes isolated
from the different fish are shown in Table 5 [45]. Other strict anaerobes, mostly
gram-negative rods, but also some gram-positive cocci, which were isolated
had not previously been described.
34 M . M . Cahill

Table 5. Strictly anaerobic bacteria isolated from the


gastrointestinal tract of grass carp, goldfish, and rain-
bow trout

Fish Bacteria
Grass carp Actinomyces
Bacteroides
Clostridium
Eubacterium
Fusobacterium
Pepgostreptococcus
Goldfish Bacteroides melaninogenicus
Bacteroides thetaiotaomicron
Bacteroides sp.
Rainbow trout Bacteroides melaninogenicus
Clostridium
Fusobacterium
From Trust et al. [45]

As well as obligate anaerobes, aerobes and facultative anaerobes were iso-


lated. Genera resembled those cultured from freshwater fish in other studies
[46], but with the addition of Pasteurella, Proteus, Salmonella arizonae, Strep-
tococcus, and Yersinia enterocolitica. The last species was found in four fifths
o f the grass carp examined. When present, these represented the major isolate,
and they grew without previous cold enrichment.
Attempts to culture anaerobes from rainbow trout intestines produced very
few isolates. Cultivation temperatures of the fish may have been the cause o f
this difference between rainbow trout (maintained at 11 ~ and the other species
(maintained at 18 to 22~ At 1 I~ the time of passage for intestinal contents
in trout is about 12 hours. This is less than the generation time for anaerobes
at this temperature, so these bacteria are passed out before there is time for
them to reach the population level required to establish a gut microflora.
Sakata et al. [31, 32] found that a selective medium known as NBGT-1/3S
agar was useful for the quantitative isolation of obligate anaerobes from the
intestine o f freshwater fish. The medium included neomycin, sodium tauro-
cholate, and brilliant green. Anaerobic colony counts of 105 to 109 g-I wet
weight were obtained from the intestinal contents of Tilapia, carp, ayu, and
goldfish by this method. Use o f the medium showed that in Tilapia and ayu
intestines there were more obligate than facultative anaerobes. Characteristics
of the anaerobic bacteria identified them as members o f the Bacteroidaceae. It
was also found that N B G T agar with blood incubated anaerobically yielded
about 1,000-fold higher counts than when incubated aerobically.

Establishment of Intestinal Microflora

Masu salmon Oncorhynchus masou and chum salmon O. keta were cultured
from the egg to the fry stage to determine when the normal intestinal microflora
Bacterial Flora of Fishes 35

was established [50]. In sac fry and advanced fry the bacterial counts from the
whole digestive tract ranged from nil to 104 g-J, whereas in fingerlings similar
counts yielded 103 to 107 bacteria g-~. In sac fry, coryneforms and Pseudomonas
were isolated from the digestive tract, and these organisms appeared to be
derived from bacterial flora of the water and the fishes' diet. In later stages
(i.e., in advanced fry and fingerlings), the intestinal flora consisted mainly of
Aerornonas. Therefore, it seems that the normal intestinal microflora of sal-
monids is established when the yolk is absorbed and the digestive tract is
activated at the advanced fry stage.
An investigation of the aerobic and anaerobic heterotrophic intestinal flora
of goldfish Carassius auratus demonstrated that the intestinal microflora be-
came relatively stable at about 67 days after hatching, and consisted of Aero-
rnonas hydrophila, Pseudomonas, Clostridium, and Bacteroides type A. Other
transient bacteria appeared in intestinal samples, including Plesiomonas shi-
gelloides, Enterobacteriaceae, and Moraxella. These transients were also de-
tected in fish diets and fish eggs, and in water or sediment, but did not become
established in the intestines. Bacteroides type A was first detected at about 44
days and became the predominant organism in the intestinal contents of the
adult fish. Thus, the permanent intestinal microflora consisted of bacteria which
were also present in the surroundings but which were able to persist and grow
in the environment provided by the intestinal tract [42].

The Possible Role of Intestinal Microflora in Fish Nutrition

Another aspect o f the intestinal microflora o f fish is their role in fish nutrition.
Microbial breakdown of substances, such as cellulose and chitin, in the gut
could make nutrients available for absorption. Cellulase activity was found to
occur in the stomachs of 17 of 62 fish species examined and was apparently
due to the production o f this enzyme by gut microflora [39].
Chitin-decomposing bacteria were isolated from the digestive tracts of ayu
(Plecoglossus altivelis), carp (Cyprinus carpio), and rainbow trout (Salmo gaird-
neri), in relatively high numbers and frequencies (up to 4.4 x 107 g-~ wet weight
of whole digestive tract, and 3 to 91% of the total count). Most of these chitin-
decomposers were identified as Aeromonas species from freshwater fish, and
mainly Vibrio from marine fish [20]. Direct evidence o f a nutritional role for
these bacteria was not given.
Commercial fish feeds contain food materials held together by various bind-
ers to form pellets or particles suitable for fish of different sizes to eat. In a
study o f farmed plaice [16], bacteria isolated from the intestines of fish which
had eaten feeds bound with different binders were tested for their ability to use
each binder as a sole carbon source. Samples of intestinal contents were in-
cubated in modified ZoBell's medium for 4 days before being plated out, and
the bacteria which grew were identified. Because of the method used, relative
numbers o f the genera from the intestines could not be determined. The iden-
tification methods used did not distinguish between the Pseudomonas and
Achromobacter/Alcaligenes/Agrobacterium species which were isolated. Other
bacterial groups were Vibrio/anaerogenic Aeromonas, Enterobacteriaceae, Mor-
36 M.M. Cahill

axella, Aeromonas, Acinetobacter, gram-positive cocci, coryneforms, and a


group o f unidentified gram-negative rods. These intestinal isolates could use
two o f the fish-feed binders, alginate and guar-gum, as sole sources o f carbon,
but few could use h y d r o x y p r o p y l methyl cellulose in this manner. N o rela-
tionship between particular genera and the ability to use a binder as the sole
carbon source was detected.
Trust et al. [45] tested the ability o f bacteria from the intestine o f grass carp
to break down cellulose. There was no evidence that anaerobic isolates were
i n v o l v e d in cellulose or cellobiose degradation, but Aeromonas hydrophila, the
p r e d o m i n a n t facultative anaerobe from this source, was capable o f breaking
down cellobiose but not cellulose or earboxymethyl-cellulose. T h e role o f the
extracellular enzymes ofAeromonas hydrophila in degradative processes in the
intestinal tract o f fish is not clear, but its high n u m b e r s suggest that it must be
metabolically active.

Bacterial Flora of Fish Grown in W a s t e Water

Intensive aquaculture systems which depend on the addition o f agricultural


wastes to provide feed for the fish a n d / o r prawns being raised are in operation
in various countries (e.g., China, Taiwan, Malaysia, and Israel). Where animal
wastes, such as pig, poultry, or cow manure, are a d d e d to the p o n d water, there
is a possibility that potential h u m a n pathogens might remain on or in the
product harvested from the ponds. In Malaysia, ponds receiving pig m a n u r e
were m o n i t o r e d to determine types and n u m b e r s o f parasites and pathogens
on and in the tissues o f the fish and prawns raised in the ponds [28] (Table 6).
Samples were taken by swabbing external tissues, dissecting out portions o f
muscle tissue, and excising foreguts and hindguts. Samples were h o m o g e n i z e d
and serially diluted, then plated out on nutrient agar to detect total heterotrophs,
and on selective media to culture pathogenic Enterobacteriaceae and Vibriona-
ceae. T h e bacterial populations on the external tissues o f fish from m a n u r e d
and u n m a n u r e d ponds were c o m p a r a b l e in n u m b e r s and types. N o Sahnonella
or Shigella were found in or on any fish. It was concluded that it was unlikely
that h u m a n infection would result from the c o n s u m p t i o n o f fish or prawns
from these ponds.
In another fish p o n d system, waste from a cattle feedlot was treated in a high
rate algal p o n d then passed into a fish pond, where three species o f carp were
raised [l 1]. The microbial flora o f these fish was c o m p a r e d with that o f fish
from a natural population in a freshwater dam. Gill surfaces, skin, blood, tissue,
and intestinal contents were sampled. It was found that 'salmonellae were
present in all fish, including those derived from the natural population.' Sub-
sequent investigations showed that o f the 20 isolates o f p r e s u m p t i v e salmo-
nellae from the waste-pond fish, only three actually were Salmonella. N o patho-
genic bacteria were detected in the blood or tissues o f the waste-pond fish,
although the sensitivity o f the isolation m e t h o d used would only detect n u m b e r s
above 102 per sample. T h e r e were high n u m b e r s o f bacteria associated with
the skin ( < 5 0 to 0.35 x 105 cm-2), gills ( < 102 to 1.4 x 105/gill), and intestine
(7.5 • 106 to 8.5 X 108/intestine) whether the fish were grown in the waste
Bacterial Flora of Fishes 37

Table 6. Bacteria isolated from fish grown in ponds


fertilized with pig manure

Source Taxa isolated


Gut walls and Enterobacteriaceae, E. coli,
contents Citrobacter, Providencia
rettgeri, Vihrionaceae,
Aeromonas hydrophila.
Pseudomonas spp.,
Micrococcaceae,
Acinetobacter spp.
Scales Vibrionaceae, A. hydrophila,
Mierococcaceae
Muscle Vibrionaceae, A. hydrophila
From Rice et al. [28]

ponds or in a natural freshwater environment. The bacterial load o f the water


containing waste from the feedlot was reduced by 99.6% by the time it reached
the outflow o f the fish pond. Thus, health risks of the feedlot waste were greatly
reduced by this water treatment system, although further work on the risks to
human health associated with waste-grown fish is indicated.
Buras et al. [ 10] also studied public health implications of fish grown in waste
water. Fish were kept in clean tap water for 2 weeks before the experiment,
then suspensions of various concentrations o f bacteria (Escherichia coli, Ci-
trobacter freundii, Streptococcus faecalis, and Salmonella montevideo) and vi-
ruses were inoculated into the esophagus. The fish were killed 30 min, 2 hours,
and 24 hours after inoculation, and tissues and organs were cultured to recover
these organisms. There were threshold concentrations for inoculated bacteria,
above which the bacteria appeared in the muscles after 2 hours, indicating that
they had passed the normal barriers o f the immune system which prevent
foreign organisms from invading fish tissues. These barriers include phagocytic
cells localized in fish intestines, and the macrophages and lymphoid cells in
the spleen and kidneys. The threshold values for 0.1 ml aliquots of inoculated
bacteria in carp weighing 60 to 100 g were: E. colL 2.5 x 106; C. freundii, 9.3
x 103; S.faecalis, 1.9 x 104; and S. montevideo, 1.8 x 104.
In another experiment, carp were exposed for 9 days to water containing S.
montevideo at concentrations of 2.5 x 103 ml -~ or 5 x 105 ml -~. After this
time, the bacteria were recovered from muscle (2 cfu g-l) and from kidney,
liver, and spleen (20 to 31 cfu g-l, and the concentration of S. montevideo in
the intestinal contents was 6.0 x 105 g-l, equal to or greater than the concen-
tration in the water. Depuration was effective in removing bacteria from the
internal organs. After exposure to S. montevideo, fish were transferred to clean-
water tanks and kept there for 1 week with daily water changes. After this
period no S. montevideo cells were recovered from the internal organs, although
they were still at a concentration of 3 x 102 ml-~ in the contents o f the intestine.
These results imply that a high bacterial load in the water stresses the fish
immune system and can result in the penetration of internal organs by envi-
ronmental bacteria. Thus, it is important that waste water used for growing
38 M.M. Cahill

fish should not contain populations of potential human pathogens which are
larger than the threshold values for these organisms. Threshold values for
viruses (as demonstrated by the exposure of fish to phage T2) were quite low
(102 per fish).

Isolation of Bacteria from Internal Tissues

The spleen, liver, and kidneys should be sterile in healthy fish [26], and the
presence of bacteria in these organs could indicate breakdown of immunological
defense mechanisms as the result of stress. Stress factors include poor water
quality, temperature changes, nutritional deficiencies, overcrowding, trauma,
parasitism, or primary viral infections [14]. There are reports of isolations of
bacteria from internal organs of apparently healthy fish [18, 26, 46]. Such
organisms may have been particularly resistant to the host's defense mecha-
nisms. Presumably, they would eventually either be destroyed, or survive to
initiate an infection.
Some breakdown of the fish's natural defenses could also explain the occur-
rence of bacteria in organs such as the liver and kidney. The immune response
in fish is significantly affected by temperature [ 13]. When the immune response
is suppressed by lower temperatures, fish have no immune protection against
the pathogenic mechanisms of bacteria in their environment, whether these
organisms are obligate pathogens or environmental organisms, such as Aero-
m o n a s hydrophila.

Conclusions

It is apparent from the foregoing that fish have considerable populations of


bacteria on their skin, gills, eggs, and in their gastrointestinal tracts. The mi-
croflora of skin and gill surfaces is diverse, and varies in marine, estuarine,
and freshwater species. Since the bacteria associated with fish surfaces seem to
reflect the diversity ofbacteria in the surrounding water, it is debatable whether
there is a specific microflora adapted to colonizing these surfaces, or whether
these organisms are merely transients. The method of sampling surfaces, usually
by swabbing an area, has not resolved this problem. A more rigorous means
of separating environmental organisms temporarily trapped in surface mucus
from indigenous floras (if there actually are such) is required.
Most identification of isolates has been only to the genus level, which does
not allow recognition of particular species which may form a resident microflora
distinct from that in the water. Numerical taxonomic techniques applied to
bacteria from various sites at a marine fish farm have shown some phena which
were isolated exclusively from the surface of healthy fish [2]. Colonists of fish
surfaces must be resistant to the antibacterial components and the flushing
activity of mucus, but the extent of such adaptations in the normal fish mi-
croflora is not evident in the studies reviewed.
If the microflora of fish reflects that of their habitat, fish could harbor patho-
Bacterial Flora of Fishes 39

gens if grown in contaminated waste water. More work is required to determine


safety levels for bacterial loads in fish ponds receiving animal wastes.
The relationship between normal microflora and opportunistic pathogens is
unclear. Certainly the genera Pseudomonas, Aeromonas, Vibrio, and Cytophaga
include such pathogens and are commonly isolated from normal healthy fish,
but only certain strains of these bacteria possess the virulence factors necessary
to induce disease. The incidence of virulence factors in strains associated with
Populations of healthy fish could be determined for epidemiological purposes.
It is interesting to speculate on the significance of prosthecate organisms on
gills. Are such bacteria more readily trapped on gill filaments as water passes
Over the gills? Again, are they indigenous or transient?
The intestinal microflora shows evidence of some selection of certain genera
which can multiply in the conditions of this environment to form large pop-
ulations of facultative and obligate anaerobes. To survive passage through the
digestive tract, bacteria must be able to resist low pH, digestive enzymes, the
effects oflysozyme and immunoglobulins in gut mucus, and possibly anaerobic
Conditions in some regions. The structure of the digestive tract has some in-
fluence on whether gut microflora differs significantly from that of the fish's
diet and environment. Sea bream, with a complex digestive tract, had a more
distinctive gut microflora than file-fish with its simple system. The role of gut
rnicroflora in fish nutrition is yet to be clearly determined, and probably varies
with the fish species. The existence ofcommensal bacteria intimately associated
with gut epithelia, as in mammals, has not been demonstrated in fish.
"Aspects of the taxonomy and ecology of many bacterial fish pathogens have
been neglected making identification of fresh isolates difficult" [5]. This applies
equally to the normal flora of fish, which may include many opportunistic
pathogens.
As yet the bacterial floras of relatively few species, mainly commercially
important wild-caught or cultured fish, have been examined. The identification,
ecological adaptations, and pathogenic potential of bacteria which live in close
association with healthy fish, are worthy of further study.

Acknowledgment. This work was supported by funding from the Mayne bequest fund of the
University of Queensland.

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