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Microb Ecol (1990) 19:21-41 MICROBIAL ECOLOGY @Springer-Verlag New 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 of 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 of aquatic and fish microfloras. Bacteria recovered from the skin and gills may be transient rather than resident on the fish surfaces. Microfloras of 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 of 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 many 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 of% 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used the identifications given by the authors of the articles quoted, although some of these identifications are now out of date.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 of the Flavobacterium-Cytophaga group has been rationalized, and there has been much revision of the species within the genus Vibrio. Most

Bacterial Flora of Fishes

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Microb Ecol (1990) 19:21-41 MICROBIAL ECOLOGY @ Springer-Verlag New 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 of 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 of aquatic and fish microfloras. Bacteria recovered from the skin and gills may be transient rather than resident on the fish surfaces. Microfloras of 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 of 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 many 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 of% 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 of the Flavobacterium-Cytophaga group has been rationalized, and there has been much revision of the species within the genus Vibrio. Most

22

Table 1. Bacterial flora of fish eggs

M. M. Cahill

Fish species Location Bacteria isolated Reference

Chum salmon (On- Vancouver chorhynchus keta) I., Canada and pink salmon (O. gorbuscha)

Masu salmon (O. ma- Japan sou) and chum salmon (O. keta)

Goldfish (Carassius Japan auratus)

Pseudomonas spp., Cytophaga sp., Sarcina sp., Chromobacterium lividum, Aeromonas liquefaciens, Enterobacter, Klebsiella

Flavobacterium/Cytophagaceae, Pseudomonas

Aeromonas hydrophila, A. puncta- ta, Pseudomonas, Flavobacteri- um, Micrococcus; other spp. iso- lated less frequently

Bell et al. [9]

Yoshimizu et al. [50]

Sugita et al. [42]

mar ine coryneform bacter ia have been shown to be very s imilar to the ar thro- bacters.

The Microflora of Fish Eggs

Three reports [9, 42, 50] suggest s imilar microbia l floras on the surfaces o f freshwater fish eggs (Table 1). Repor 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 bo th heal thy and dead s t ream- incubated sa lmon eggs with those o f s imula ted eggs (polyethylene spheres) and ambien t s t ream water to de te rmine any rela t ionship between egg microf lora and egg survival. Cytophaga and P s e u d o m o n a s species were isolated mos t frequently f rom healthy e m b r y o n a t e d eggs, while dead eggs yielded main ly fluorescent P s e u d o m o n a s species. Simulated eggs had a more diverse flora, comparab le to that o f the s t ream. A e r o m o n a s l iquefaciens (=A. hydrophi la) was isolated f rom live and s imula ted eggs and f rom s t ream water. The P s e u d o m o n a s species isolated f rom the dead eggs were p robab ly s t imulated by nutr ients leaching f rom the dead egg and were unlikely to be the cause o f death. The physical and chemical condi t ions o f the e n v i r o n m e n t were considered to be the mos t impor tan t factors in ensuring the survival o f eggs, as no specific bacterial pa thogen responsible for egg mor ta l i ty was isolated. These results were based on the s tudy o f 67 strains o f bacter ia isolated on t ryptone glucose extract agar. This m e d i u m would suppor t the growth o f a l imited range o f freshwater bacteria, and the use o f addi t ional med ia would p robab ly d e m o n - strate a more diverse flora.

Skin Microflora

Examples of the range o f bacterial genera isolated f rom fish skin are given in Table 2.

Colwell 's survey was under taken to de te rmine the microbia l flora o f Puget Sound fish caught by different fishing me thods [ 12]. The purpose o f the survey

Bacterial Flora of Fishes

Table 2. Bacterial flora of fish skin

23

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 Moreton Micrococcus 49%, Pseudomonas

Bay, 18%, coryneforms 12%, Morax- Queens- ella 8%, Flavobacterium/Cy- land, tophaga 8%, plus Staphylococ- Australia cus, Bacillus, Acinetobacter,

Aeromonas, and Vibrio

Sea mullet (Mugil cephalus), whiting (Sillago ciliata), bream (Mylio aus- tralis), and flathead (Platycephalus fus- cus)

Plaice (Pleuronectes platessa L.)

Gillespie and MacRae [15]

Scotland Gilmour et al. [16l

Pseudomonas Groups I, II, and IV/Achromobacter/Alcaligenes/ 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 va r ia t ions in the flora to possible c o n t a m i n a t i o n due to the k ind o f hand l ing the fish rece ived dur ing purse a n d beach seining, o t ter trawling, and ca tch ing by h a n d line. Th i s c o n t a m i n a t i o n cou ld al ter the n o r m a l flora adversely , resul t ing in m o r e rap id de te r io ra t ion and thus a r educ t i on in the keeping qual i ty o f the fish. Resul ts suggested tha t the m e t h o d s o f hand l ing fish, and their p recap ture 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 have used Standardized hand l ing m e t h o d s (e.g., see Hor 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 of 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 of 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 of 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 of healthy fish. In spite of this abundant growth from skin samples, scanning electron micrographs of the skin of 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 of 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 of 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 of 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 of 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 of 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

Table 3. Bacterial flora of fish gills

M. M. Cahill

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) U K 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

Tab

le 4

. In

test

inal

mic

rofl

ora

of

fish

" to

A

chro

- F

lavo

- C

oryn

e-

oo

Pse

udo-

m

obac

- ba

cte-

ba

cte-

A

ero-

M

icro

- F

ish

spec

ies

Loc

atio

n m

onas

V

ibri

o te

r ri

um

rium

B

acil

lus

mon

as

cocc

us

Var

ious

(se

e T

able

2)

Pug

et S

ound

, U

SA

48

.1

13.9

17

.7

2.5

1.3

5.1

5.1

Fla

tfis

h (K

arei

us b

icol

orat

us)

Fis

h m

arke

t, J

apan

+

+ +

+

Blu

efis

h (P

omat

omus

sal

tatr

ix)

Lon

g Is

land

Sou

nd,

22.4

28

.4

18.2

1.

6 0.

9 1.

4 U

SA

S

trip

ed b

ass

(Mor

one

saxa

tili

s)

Hu

dso

n R

iver

, U

SA

23

10

5

2 0.

5 33

1

(est

uari

ne)

Lo

ng

Isl

and

Sou

nd,

11

27

18

1 0

21

3 U

SA

(m

arin

e)

Pla

ice

(Ple

uron

ectu

s pl

ates

sa)

Sco

tlan

d +

+ +

+ +

+ S

alm

on

ids

Fre

shw

ater

lak

es,

+ +

+ +

+ +

+ C

anad

a

Rai

nb

ow

Tro

ut

(Sal

mo

gair

dner

O

Fis

h fa

rm,

Sco

tlan

d 12

4

12

8 16

12

Dac

e, f

ield

gud

geon

, co

mm

on

min

no

w,

Tam

a R

iver

, Ja

pan

+

+ +

+ +

+ +

ston

e m

oro

ko

, ca

rp,

cruc

ian

carp

, an

d b

rack

ish

goby

Gol

dfis

h (C

aras

sius

aur

atus

), P

ond,

Jap

an

+ +

+ 1 -

year

-old

R

ain

bo

w t

rout

(Sa

lmo

gair

dner

t)

Hat

cher

y, S

pain

+

+ +

+

Car

p (

Cyp

rinu

s ca

rpio

) R

eari

ng t

ank,

Jap

an

+ +

+ +

Sal

mo

nid

s L

akes

, B

riti

sh

+ +

+ +

+ +

+ C

olum

bia,

Can

ada

Gra

ss c

arp

(Cte

noph

aryn

godo

n F

ish

tank

s, B

riti

sh

+ +

+ +

+ +

idel

la)

Co

lum

bia

, C

anad

a Ti

lapi

a zi

llii

F

resh

wat

er t

ank,

+

+ +

Jap

an

.~

Tila

pia

zill

ii

Sea

wat

er t

ank,

Jap

an

+ +

+ T

ilap

ia (

Saro

ther

odon

nilo

ticus

) C

ultu

re p

onds

, Ja

pan

+

+ =:

Fig

ures

giv

e th

e pe

rcen

tage

inc

iden

ce o

f di

ffer

ent

grou

ps o

f ba

cter

ia;

+ in

dica

tes

that

rep

rese

ntat

ives

of

the

grou

p w

ere

isol

ated

Tab

le 4

. C

onti

nued

9.

Fis

h sp

ecie

s

Ent

ero-

ba

cte-

ri

acea

e A

lcal

ig-

enes

Aci

neto

- ba

cter

- M

ora-

xe

lla

Stap

hyl-

OC

OC

CIt

S

Clo

s-

trid

ium

Bac

te-

roi-

da

ceae

Y

east

s O

ther

s R

efer

ence

0 P,

o

Var

ious

(se

e T

able

2)

Fla

tfis

h (K

arei

us b

icol

orat

us)

Blu

efis

h (P

omat

omus

sal

tatr

ix)

Str

iped

bas

s (M

oron

e sa

xatil

is)

Pla

ice

(Ple

uron

ectu

s pl

ates

sa)

Sal

mon

ids

Rai

nb

ow

Tro

ut (

Salm

o ga

irdn

eri)

Dac

e, f

ield

gud

geon

, co

mm

on

min

now

, st

one

mor

oko,

car

p, c

ruci

an c

arp,

an

d br

acki

sh g

oby

Gol

dfis

h (C

aras

sius

aur

atus

), 1 -

year

-old

R

ain

bo

w t

rout

(Sa

lrno

gai

rdne

rO

Car

p (C

ypri

nus

carp

io)

Sal

mon

ids

Gra

ss c

arp

(Cte

noph

aryn

godo

n id

ella

) Ti

lapi

a zi

llii

Tila

pia

zilli

i T

ilap

ia (

Saro

ther

odon

nilo

ticus

)

2.5

21.4

15

14 + 2O +

+ + + +

+

2.5

3.8

3.0 + + +

Col

wel

l [1

2]

Sim

idu

et a

l. [

38]

New

man

et

al.

[24]

Mac

Far

lane

et

al.

[19]

Gil

mou

r et

al.

[ 16]

T

rust

and

Spa

rrow

[4

6]

Aus

tin

and

Al-

Zah

rani

[6

] S

ugit

a et

al.

[41

]

Sug

ita

et a

l. [4

2]

Nie

to e

t al

. [2

6]

Sug

ita

et a

l. [4

3]

Tru

st a

nd S

parr

ow

[46]

T

rust

et

al.

[45]

Sak

ata

et a

l. [3

0]

Sak

ata

et a

l. [3

0]

Sak

ata

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eeda

[2

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bo

30 M.M. Cahill

sterile. There was no significant difference in the bacterial flora offish of 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 of marine fishes, where Vibrio, Pseudomonas, Achromobacter, Corynebacterium, Flavo- bacterium, and Micrococcus predominated [12, 24].

Relationship o f Intestinal Microflora to That o f Aquatic Habitat

MacFarlane et al. [19] noted differences in the composition of the intestinal floras of 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 of 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 of 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 of 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 of nonfeeding fish was sampled and cultured, and its microflora was found to consist of 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 of 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 of 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 of the gastrointestinal tract of 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 of 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. Gram-pos i t ive cocci f rom these fish did not show the same correlat ion with those from the water supplying the ponds, but handling and contaminated feed may have affected the numbers o f staphylococci detected in the fish [26].

Farming practices for some fish [e.g., ra inbow trout (Sahno gairdneri)] require a constant flow o f fresh water through the fish ponds. Where large numbers o f fish are being raised, the addit ion o f fish feed (some o f which is not utilized) and fish feces to the water can have a detr imental effect on the microbial quali ty of the effluent water f rom the fish farm. A study in Finland [25] showed that effluents from two fish farms had elevated numbers of total coliforms and fecal coliforms, and on one farm, effluent had more fecal streptococci than the influent. The majori ty o f coliforms were identified as Enterobacter and Citro- bacter, and Aeromonas hydrophila was quite comm o n . High concentrat ions 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 mult iply in the fish intestines and possibly in the sediment. A quanti tat ive study o f the intestinal microflora o f the farmed fish was not done, although such a study could have indicated more accurately the relat ionship between intestinal bacteria and those in the effluent.

Austin [4] also compared the microbial floras of the inflow and outflow from four rainbow trout farms. There was no consistent increase in coliforms or in Aeromonas hydrophila in the effluents f rom these farms, al though 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 eutrophicat ion and were not linked to mult ipl icat ion within the fish intestinal tract.

While the microorganisms present in water can influence the microbial flora associated with fish, the contr ibut ion o f bacteria shed f rom fish to water is also worth consideration, especially where dense populat ions off ish are maintained, as in aquaculture.

Total viable counts and determinat ions o f the varieties o f bacteria in the water of a tank used for rearing carp were made before, during, and after the addit ion o f the fish [43]. Total viable counts increased after the in t roduct ion of carp, and the diversi ty and predominance o f bacterial groups also changed. Before the int roduct ion of fish, Pseudomonas and the Flavobacterium-Cytoph- aga group were most numerous in the water, and Enterobacteriaceae, the Vibrio- Aeromonas group, and Bacteroidaceae were not detected. The Vibrio-Aero- rnonas group became predominant 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. The presence o f these organisms in the water seemed dependent 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 in t roduced 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 of intestinal flora because of its ability to adapt to fresh and sea water, and its omnivorous diet, including phytoplankton, algae, and some- t imes water plants. Its intestine is five to seven t imes its body length and has an abundant commensa l flora [30]. Counts and identification o f viable hetero- trophic aerobic bacteria were made from homogenized sections o f intestines off ish kept in fresh or sea water, and the variety of these bacteria was compared with that o f the corresponding water.

The number o f genera isolated f rom the fish intestines was much more restricted than that isolated from the surrounding water. Aeromonas predom- inated in the intestines o f freshwater Tilapia, whereas Flavobacterium and Pseudomonas predomina ted in freshwater. In marine fish, Vibrio and Aero- monas formed the greater percentage o f the intestinal flora, but Pseudomonas was most frequently isolated f rom the sea water. However , where fish had been kept for a long t ime (181 days) in sea water, there were more Aeromonas than Pseudomonas in the water, indicating that fish feces may be a source of Aero- monas in water.

In a further study of 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]. The bacteria of the s tomach, fore-intestine, and postintestine (sic) were isolated. Bacteroides types A and B, aerobic gram-negative rods, coryneforms, and strep- tococci were isolated f rom all three regions. Bacilli, aerobic gram-posi t ive cocci, and yeasts were found only in the s tomach and fore-intestine regions. Where the fish were reared in 75 and 100% sea water, numbers o f obligate anaerobes decreased and /or disappeared, and aerobic and facultatively anaerobic gram- negative rods predominated. Bacteroides type A was found to grow in NaCl concentrat ions of 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 of the fish's adaptat ion to sea water.

Relationship o f Intestinal Microflora to That o f Food

The bacterial flora o f the gut of two marine fish has been investigated in an a t tempt to clarify the relationship between these bacteria and the bacterial flora of their diets, and to de termine 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 t ime intervals after the fish were fed in the laboratory. Bacteria were also isolated from the fishes' env i ronment and f rom various diets (fishmeat, f ishmeat sup- p lemented with chitin or starch, and shellfish). The file-fish has a relatively undeveloped stomach in compar ison with that of the red sea bream. The s tomach and intestinal contents o f the file-fish closely reflected the bacterial flora of their diet, whereas in red sea bream, the dominan t flora always consisted of one or both o f two groups o f vibrios (distinct f rom those present in the diets). These two groups of vibrios were both resistant to 2% bile and low pH (5.5) and were considered to be members o f an indigenous flora o f the digestive

Bacterial Flora of Fishes 33

tract of sea bream. This work implied that the bacterial flora of fish with relatively undeveloped digestive tracts reflected that of 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 of 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 of the other isolates were inhibited by these conditions [35].

Newman et al. [24] sampled the microbial flora of the bluefish (Pomatomus saltatrix) intestine to see whether the microorganisms present were dependent on the type of food most recently ingested. The bacteria isolated were similar to the flora of 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 of the flora of prey fish from the same geographic location was available, so this conclusion was rather conjectural. The gut flora merely seems typical of that of most marine fish.

The Effect of Antibiotics on Intestinal Microflora

Medicated feeds of the types used in the treatment of fish diseases contain antimicrobial agents, such as oxolinic acid, oxytetracycline, sulphafurazole, erythromycin, and penicillin G. Administration of 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 of 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 of 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 of 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 of 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 of 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 of the Bacteroidaceae. It was also found that NBGT 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 of the intestinal microflora of 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 of 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 of 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 of 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 of 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-posi t ive 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 hydroxypropyl methyl cellulose in this manner . No rela- t ionship 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 f rom the intestine o f grass carp to break down cellulose. There was no evidence that anaerobic isolates were involved in cellulose or cellobiose degradation, but Aeromonas hydrophila, the predominan t facultative anaerobe from this source, was capable o f breaking down cellobiose but not cellulose or earboxymethyl-cellulose. The role o f the extracellular enzymes ofAeromonas hydrophila in degradative processes in the intestinal tract o f fish is not clear, but its high numbers suggest that it must be metabolically active.

Bacterial Flora of Fish Grown in Was te Water

Intensive aquaculture systems which depend on the addi t ion o f agricultural wastes to provide feed for the fish and /or prawns being raised are in operat ion in various countries (e.g., China, Taiwan, Malaysia, and Israel). Where animal wastes, such as pig, poultry, or cow manure , are added to the pond water, there is a possibility that potential human pathogens might remain on or in the product harvested f rom the ponds. In Malaysia, ponds receiving pig manure were moni to red to determine types and numbers 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 port ions o f muscle tissue, and excising foreguts and hindguts. Samples were homogenized and serially diluted, then plated out on nutr ient agar to detect total heterotrophs, and on selective media to culture pathogenic Enterobacteriaceae and Vibriona- ceae. The bacterial populat ions on the external tissues o f fish from manured and unmanured ponds were comparable in numbers and types. No Sahnonella or Shigella were found in or on any fish. It was concluded that it was unlikely that human infection would result from the consumpt ion o f fish or prawns from these ponds.

In another fish pond system, waste f rom a cattle feedlot was t reated in a high rate algal pond then passed into a fish pond, where three species of carp were raised [l 1]. The microbial flora o f these fish was compared with that o f fish from a natural populat ion 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 der ived f rom the natural populat ion. ' Sub- sequent investigations showed that o f the 20 isolates o f presumpt ive salmo- nellae f rom the waste-pond fish, only three actually were Salmonella. No patho- genic bacteria were detected in the blood or tissues o f the waste-pond fish, although the sensitivity of the isolation me thod used would only detect numbers above 102 per sample. There were high numbers of 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

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]

37

ponds or in a natural freshwater environment. The bacterial load of the water containing waste from the feedlot was reduced by 99.6% by the time it reached the outflow of 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 of 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 of 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 of 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- monas 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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40 M . M . Cahill

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