vibrio diseases of marine fish populationsvibrios often occur in association with invertebrate and...

HELGOL~NDER MEERESUNTERSUCHUNGEN Helgolander Meeresunters. 37, 265-287 (1984)

Vibr io diseases of marine fish populat ions

R. R. C o l w e l l & D. J. G r i m e s

Depar tment o f Microbiology, Univers i ty o f Maryland; Col lege Park, M D 20742 USA

ABSTRACT: Several Vibrio spp. cause disease in marine fish populations, both wild and cultured. The most common disease, vibriosis, is caused by V. anguillarum. However, increase in the intensity of mariculture, combined with continuing improvements in bacterial systematics, expands the list of Vibrio spp. that cause fish disease. The bacterial pathogens, species of fish affected, virulence mechanisms, and disease treatment and prevention are included as topics of emphasis in this review,

INTRODUCTION

It is most appropr ia te that the introductory pape r at this Sympos ium on Diseases of

Marine Organisms should be about vibrios. The d isease syndrome t e rmed vibriosis was

one of the first d iseases of mar ine fish to be descr ibed (Sindermann, 1970; Home , 1982). Cal led "red sore", " red pest" , " red spot" and " red d i sease" because of character is t ic

hemorrhagic skin lesions, this d isease was r ecogn ized and desc r ibed as ear ly as 1718 in

Italy, with numerous epizootics be ing documen ted throughout the 19th century (Crosa et al., 1977; S indermann, 1970). Today, wh i l e " red sore" is, wi thout doubt, the best

unders tood of the mar ine bacter ia l fish diseases, new diseases have b e e n added to the

list of those caused by Vibrio spp. The more recent ly publ i shed work descr ib ing Vibrio disease, pr imar i ly that appear -

ing in the l i terature since 1977, wi l l be the focus of this paper. Bacter ial pa thogens , the

species of fish affected, v i ru lence mechanisms, and d isease t rea tment and p reven t ion are inc luded as topics of emphasis . For discussions of ear l ie r work, the reader is referred to

S indermann (1970) and Roberts (1982).

VIBRIOS AS BACTERIA P A T H O G E N I C FOR FISH

All members of the genus Vibrio are Gram-n e g a t i v e rods r ang ing in size and

morphology from coccobaci l l i to def ini te rod-shape cells that may exhibi t some d e g r e e of

curvature, i.e., vibrioid. Under condi t ions of nutr ient dep le t ion or in the natura l envi ron-

ment, inc luding estuaries and the ocean, where ol igotrophic condit ions occur, vibrios

will occur as small coccoid forms, r ep resen t ing a s t rategy for survival (Single ton et al., 1982; Xu et al., 1982; Novi tsky & Morita, 1976). Vibrio spp. are mot i le by means of a

sheathed, polar f lagel lum, one or more at one pole of the cell. Some species also form non-shea thed lateral f lagella, w h e n grown on solid media . Lateral f lage l la play a role in

a t tachment to surfaces and also appea r to be invo lved in swa rming (Belas & Colwel l ,

1982a; 1982b).

© Biologische Anstalt Helgoland, Hamburg

2 6 6 R . R . C o l w e l l & D. J . G r i m e s

B i o c h e m i c a l f e a t u r e s of t h e g e n u s i n c l u d e f e r m e n t a t i o n of g l u c o s e , u s u a l l y w i t h o u t

t h e p r o d u c t i o n of g a s ; s e n s i t i v i t y to 150 btg v i b r i o s t a t 0 / 1 2 9 ( 2 , 4 - d i a m i n o - 6 , 7 - d i i s o p r o -

p y l p t e r i d i n e ) p e r m l of m e d i u m ; p o s i t i v e o x i d a s e r e a c t i o n , e x c e p t fo r V. m e t s c h n i k o v i i

a n d V. g a z o g e n e s ; a n d a n a b s o l u t e r e q u i r e m e n t fo r N a C I ( W e s t & C o l w e l l , 1984) . T h e

o v e r - a l l D N A b a s e c o m p o s i t i o n , i .e . , p e r c e n t g u a n i n e p l u s c y t o s i n e , for t h e g e n u s r a n g e s

f r o m 3 8 to 5 1 % , w i t h t h e t y p e s p e c i e s , V. cholerae, h a v i n g a n a v e r a g e % G + C of 47.

T a b l e 1 l i s t s s a l i e n t f e a t u r e s of t h e g e n u s , u s e f u l i n i d e n t i f y i n g t h e f i s h p a t h o g e n s .

T a b l e 1. C h a r a c t e r i s t i c s u se fu l in i d e n t i f y i n g those m e m b e r s of the g e n u s Vibrio tha t h a v e b e e n s h o w n to be p a t h o g e n i c for fish*

Vibrio

C h a r a c t e r i s t i c algino- anguil- carcha- cho- dam- orda- para- vulni- lyticus larum riae lerae sela 1ii haemoly- ficus

ticus 1 2

0/129 10 ~tg R S R S S S R S S

150 ~tg S S S S S S S S S

S w a r m i n g + -- + -- -- - V A m i n o a c i d s * *

a r g i n i n e -- + -- -- + -- -- l y s i n e + -- + + -- -- + + + o r n i t h i n e + - + + - - + + -

F e r m e n t a t i o n g l u c o s e A A A A AG A A A A l ac to se . . . . . . . A A m a n n o s e A A A A AG -- A A A s a l i c i n -- - A . . . . A A suc rose A A A A -- A -

U r e a s e -- -- + - + - V V -

I n d o l e + + + + - -- + + - V o g e s - P r o s k a u e r + + - + + -- -- G r o w t h a t % NaC1

0 -- V - V -- - -

3 + + + + + + + + + 6 + + + V V + + + -

8 + V + - - - - - - + 10 + . . . . . .

G r o w t h a t 42 °C + -- - + - - + + -

* + = 9 0 % of a l l s t r a i n s g i v e a p o s i t i v e reac t ion , ( + ) = w e a k a n d / o r d e l a y e d pos i t ive , - = 90 % of a l l s t r a ins a r e n e g a t i v e , V = v a r i a b l e r eac t ions , A = acid, AG = ac id + gas, R = res i s t an t , S = s ens i t i ve ; t e s t p r o c e d u r e s a re g i v e n b y W e s t & C o l w e l l (1984}.

* * a r g i n i n e d i h y d r o l a s e , l y s i n e a n d o r n i t h i n e d e c a r b o x y l a s e

T h e n a t u r a l h a b i t a t o f Vibr io s p p . i s e s t u a r i n e a n d m a r i n e w a t e r a n d s e d i m e n t .

V i b r i o s o f t e n o c c u r i n a s s o c i a t i o n w i t h i n v e r t e b r a t e a n d v e r t e b r a t e a n i m a l s u r f a c e s a n d

i n t e r n a l o r g a n s ( H u q e t a l . , 1 9 8 3 ; G r i m e s e t a l , 1984b} , S t r e s s c a n c o m p r o m i s e t h e

a n i m a l h o s t , r e s u l t i n g i n a c o m m e n s a l s p e c i e s s w i t c h i n g to p a t h o g e n i c i t y a s t h e m o d e of

i n t e r a c t i o n ( G r i m e s e t a l . , 1984c) .

Vibrio diseases 267

Vibr io a l g i n o l y t i c u s

Formerly classif ied as V. parahaemolyt icus biotype 2, V. alginolyticus often exhibi ts

p ronounced swarming on complex media , p roduces a posi t ive Voges-Proskauer reaction, tolerates 10 % NaC1, and grows at t empera tu res up to 42 °C. It fe rments sucrose and

decarboxyla tes lysine and o m i t h i n e (Table 1). V. alginolyticus is f requent ly isolated from fin fish, shellfish, seawater , and sed imen t

(Gjerde & Boe, 1981; Joseph et al., 1982). It is not w ide ly r ecogn ized as a fish pa thogen,

even though it has b e e n associated with u lcer d isease (Akazawa, 1968) and mortal i ty of sea-bream (Colorni et al., 1981; Iwata et al., 1978) (see Tab le 2). Recently, Burke &

Table 2. Vibrio species known to cause disease in marine fish

Organism Disease

V. alginolyticus ulcer disease V. anguillarum vibriosis V. carchariae vasculitis V. cholerae non-01 petechial hemorrhage V. damsela skin lesions V, ordalii * vibriosis V. parahaemolyticus ulcer disease V. vulnificus 2 skin lesions

* Formerly 1/. anguillarum biovar II

Rodgers (1981) found V. alginolyticus to be a secondary invade r of " red spot", a d isease

caused by V. "anguillarum. Gauth ie r & C l e m e n t (1979) obse rved a s t reptomycin-res is tant

strain of V. alginolyticus to be present not only in sed imen t but also water , w h e n the water t empera ture e x c e e d e d 16 °C. However , food chain t ransmiss ion was not obse rved

w h e n reconstructed mar ine food chains w e r e s tudied in the laboratory (Gauthier &

Clement , 1979).

It is wel l es tabl i shed that V. alginolyticus can colonize the h u m a n gut (Sakazaki et

al., 1963). In fact, Hira tsuka et al. (1980) observed the cause of acute enterocol i t is in a pat ient who consumed trout roe con tamina ted with the same bacter ia to be V. alginolyti-

cus. More frequently, V. alginolyticus is associa ted with skin infect ions (Blake et al., 1980), such as conjunct ivi t is and s tump ulcer cases descr ibed by Schmidt et al. (1979). Even though Blake et al. (1980) repor ted that l i t t le work has b e e n done on v i ru lence

mechanisms, it is probable , based on ava i lab le ev idence , that the co l l agenase and

several extracel lular proteases p roduced by V, alginolyticus (Long et al., 1981) partici- pate in the invas ion of fish and h u m a n skin.

Vibr io a n g u i l l a r u m

This vibrio, the first r ecogn ized to be a fish pa thogen , inc ludes strains capable of

ut i l iz ing arg in ine and tolerant of 6 % NaC1; some strains tolerate 8 % salt and others are

capable of growth in the p resence of t race amounts of salt, i.e., they are repor ted to grow

268 R. R. Colwell & D. J. Grimes

in 0 % salt, but in fact, require the concent ra t ion of NaC1 present in peptone used to prepare the m e d i u m (Singleton et al., 1982). 1/. anguillarum is sensi t ive to 10 ~g 0/129, gives a posit ive Voges-Proskauer react ion and ferments sucrose (Table 1). Recently, strains referred to as V. anguillarum biovar II were descr ibed as a ne w species, V. ordalii sp. nov. (Schiewe et al., 1981) vide infra.

Vibriosis of V. anguillarum etiology has b e e n descr ibed in over 42 species of fish, i nc lud ing ayu, eel, cod, pike, b rown trout, f lounder, striped bass and salmon. Vibriosis has b e e n especia l ly devas ta t ing to sa lmon reared in Pacific coastal waters {Crosa et al., 1977). Vibriosis is a typical Gram-nega t ive septicemia, exhib i t ing extensive hemor- rhag ing and necrosis in the in terna l organs and muscula ture . It is a rapidly fu lminat ing disease, affecting both cul tured and wild mar ine fish. Gross cl inical signs of the disease inc lude hemor rhag ing of the fins, eyes, and ventra l surface.

It is in teres t ing to note the progression of discovery of V. anguillanlm throughout the world. It was first descr ibed in Europe, as the agen t of "red spot" in eels (Bergman, 1909; Horne, 1982; S indermann , 1970). Its recovery from diseased fish in North America did not occur unt i l 1953 (Crosa et al., 1977), and Muroga et al. {1976) suggest that the first isolat ion of V. anguillarum in J apan in 1975 may have resul ted from contamina ted eels imported from Prance. Whether or not there was a true geographic spread (natural or anthropogenic) of V. anguillarum, or merely a c i rcumstant ia l sequence of discovery is open to speculat ion.

Two of the more recent deve lopments with regard to 1/. anguillarum are very exci t ing and should lead to effective control of this fish pathogen. Virulence factors, and their gene t ic control, are rece iv ing much a t tent ion in several laboratories. Also promi- s ing are several approaches to vacc ina t ion be ing invest igated.

An in teres t ing p h e n o m e n o n was recent ly reported for V.. anguillarum isolates from ayu cul tured in Japan. Muroga et al. (1979a) found that 27 of 52 isolates were not sensi t ive to vibriostatic agen t 0/129, previously thought to be a s ignif icant at tr ibute of Vibrio spp. These isolates were ob ta ined from ayu that had b e e n raised in ponds that con ta ined prophylact ic t r imethopr im-sul fadoxine (TS). Similar f indings were reported by Aoki et al. (1981), after examina t ion of 132 strains of V. anguillarum for resistance to 0/129 and tr imethoprim. Transferable R plasmids were suspected, bu t could not be demonstrated.

Vibrio carchanae

The or ig inal isolate of V. carchariae was ob ta ined from a dead sandbar (brown) shark (Carcharhinusplumbeus) (Grimes et al., 1984a). Subsequent ly , it has been isolated from lemon sharks (Negaprion brevirostris) and from trematodes infest ing the skin of l emon sharks (Grimes et al., 1984c). V. carchariae is a swarming vibrio that exhibits mixed flagellat ion. Morphological ly, and in terms of several b iochemical attributes, V. carchariae is pract ical ly ind i s t ingu i shab le from V. alginolyticus and the sucrose-positive, urease-posi t ive strains of Vibrio parahaemolyticus. However, un l ike these two vibrios, V. carchariae can not grow at 42°C; other differences inc lude growth in 10 % NaC1 and Voges-Proskauer (Table 1), as wel l as DNA homology.

V. carchariae is descr ibed in detai l e l sewhere in this Sympos ium volume (GAmes et al., 1984b). The host range of V. carchariae has not yet b e e n thoroughly investigated, s ince the type strain was isolated only in 1982. With regard to sharks, 1/. carchariae

Vibrio diseases 269

causes a vasculitis, espec ia l ly in organs of the re t i cu loendothe l ia l system. It also causes,

or is f requent ly isolated from, dermal lesions and cysts in sharks. Recently, Gr imes et al.

{1984c} hypothes ized that shark t rematodes (Dermophthirius nigrellii) may serve as a vector for V. carchariae.

Vibrio cholerae

Serovars of V. cholerae, other than serovar 01, are occas ional ly i sola ted from fish. These V. cholerae serovars have b e e n referred to as non -agg lu t i nab l e (NAG) vibrios and

as non-cholera vibrios (NCV), a pract ice that has crea ted much confusion and is not acceptable nomenc la tu re [West & Colwel l , 1984). The non-01 V. cholerae strains do not

react with the 0 an t i se rum or iginal ly des igna t ed as 01. In a study of the Danube River, St rusiewicz et aL (1980) isolated V. cholerae non-01

from water, fish, and s e w a g e outfalls. The h ighes t i nc idence of posi t ive samples occurred during Sep tember (the warmes t month) and 81.9 % of the isolates were enterotoxi-

genic, measu red by rabbit i leal loop assay. Ente ro tox igenic strains w e r e r ecove red more

f requent ly from fish than from wate r samples (Strusiewicz & Serban, 1980). In a s imilar

study, Szeness et al. (1979) isolated V. cholerae non-01 from fish, frogs, and birds. Nei ther of these studies, however , c o m m e n t e d on the hea l th status of the fish from which

the V. cholerae isolates were obtained.

Muroga et al. {1979b) isolated a Vibrio sp. from diseased ayu and demons t ra ted that the isolate was the causat ive agen t of an ayu epizootic. Briefly, the d i sease was character ized by pe t ech iae on the body surface and by in ternal organ congest ion. The

isolates were morphologica l ly and b iochemica l ly very similar to V. cholerae, but did not

agglut ina te in e i ther O g a w a or Inaba antisera, i.e., they were non-01 serovar. Muroga et al. (1979b) also de te rmined the % G + C base ratio for their isolates (47 to 47.7 %), and demonst ra ted 86 % DNA homology b e t w e e n their isolates and If. cholerae NIH35A3.

There is little doubt that the cause of the ayu epizoot ic was the human pa thogen, V. cholerae.

In a later study, Yamanoi et al. (1980) demons t ra ted that one of the ayu V. cholerae isolates (strain PS-7701) surv ived for 320 days at 25°C in freshwater , 0.85 % NaC1

solution, Ringer solution, seawater , and in d i lu ted seawater . They further noted that survival in water was great ly r educed at 2 °C, a f ind ing similar to that of S ing le ton et al. {1982). Rela ted to this observat ion was the fact that hea l thy ayu could not be re infec ted

with V. cholerae, w h e n the ayu were he ld at 16°C. However , w h e n infec ted by the

immers ion method (ca. 1.26 × 104 cells/ml) and he ld at 21 °C and 26°C, most of the ayu died wi th in 4 days post infection. The V. cholerae strain was also p a th o g e n i c for eels (Anguilla japonica] and for mice.

Vibrio damse la

First isolated from damsel f i sh off the coast of southern Cal i fornia [Love et al., 1981),

V. damsela is now known to infect humans (Love et a l , 1981) and sharks (Grimes et al., 1984a). Dis t inguishing at t r ibutes of V. damsela inc lude its abi l i ty to me tabo l i ze a rg in ine

and to produce gas dur ing fe rmenta t ion of g lucose and other se lec ted sugars (Table 1).

V. damsela causes character is t ic skin ulcers in the blacksmith , a t e m p e ra t e -w a t e r

damselfish (Chromis punctipinnis), and in gar iba ld i (Hypsypops rubicunda), and was

270 R.R. Cotwell & D. J. Grimes

thought by Love et al. (1981) to have a nar row host range. However, we isolated V. damsela, along with V. carchariae, from a dead sandbar shark (C. plumbeus}, and subsequen t ly demonst ra ted V. damsela to be v i ru lent for the spiny dogfish shark (Squalus acanthias) (Grimes et al., 1984a). Al though we have not been successful in p roduc ing disease in lemon sharks (N. brevirostris) exper imenta l ly infected with V. damsela, V. damsela was cul tured from the stomach of a wild lemon shark (Grimes et al., unpubl , data).

Vibrio ordali i

V. ordalii was recent ly proposed by Schiewe et al. (1981) to inc lude all strains previously des igna ted as V. anguillarum biovar II. It is differentiated from V. anguillarum by g iv ing nega t ive react ions for: Voges-Proskauer; a rg in ine dihydrolase; citrate; starch; ONPG; lipase; growth at 37 °C; and acid from cellobiose, glycerol, sorbitol, and trehalose (Table 1). Detai led discussion of V. ordalil is provided in the recent review by Schiewe (1983).

V. ordalii causes vibriosis in sa lmonid fish in the Pacific Northwest and in Japan. This species appears to have a more hos t -dependen t mode of existence than does V. anguillarum, because it is rarely isolated from water, sediment , or other abiotic marine samples; V. ordalii is recovered only from mor ibund fish (Schiewe, 1983). V. ordalii preferent ia l ly colonizes skeletal muscle, cardiac muscle, gills, and the gastrointest inal tract of salmonids, in contrast to V. anguillarum, which presents d i s semina ted infections, with highest number s in blood, loose connect ive tissue, kidney, spleen, gills, and in the posterior gas t rointes t inal tract. Several invest igators have observed a marked leuko- pen ia in mor ibund fish, sugges t ing the presence of a powerful leukocytolytic factor in V. ordalii (Schiewe, 1983).

Vibrio p a r a h a e m o l y t i c u s

First recognized as the etiologic agen t of h u m a n seafood-borne disease in J apan in the 1950s, V. parahaemolyticus is the most thoroughly s tudied Vibrio sp., next to V. cholerae. Notable features of V. parahaemolyticus inc lude swarming, growth at 42 °C, nega t ive a rg in ine reaction, and resis tance to 10 t~g of 0/129. V. parahaemol~icus is considered to be non-sucrose fermenting. However, sucrose positive variants do exist, and are usua l ly overlooked dur in 9 rout ine cl inical isolat ion (West & Colwell, 1984).

V. parahaemolyticus causes gastroenteri t is in h u m a n s and has also b e e n implicated in ext ra intes t inal infections (Blake et al., 1980; Joseph et al., 1982). An enterotoxin appears to be involved, bu t has yet to be conclus ively demonst ra ted (Joseph et at., 1982). V. parahaemolyticus was so n a m e d because of its abi l i ty to produce beta-hemolysis on high-sal t blood agar, a react ion that has been des igna ted the Kanagawa phenomenon . For reasons as yet unknown , only h u m a n cl inical isolates seem to give a positive Kanagawa reaction; env i ronmen ta l isolates of V. parahaemolyticus, i nc lud ing fish isolates, are rarely Kanagawa posit ive (Joseph et al., 1982).

The role of V. parahaemolyticus in fish disease is more obscure, and fish may simply funct ion as a passive carrier. Like V. alginolyticus, V. parahaemolyticus is f requent ly isolated from both hea l thy and diseased fish throughout the world (Lall et al., 1979; Lhuillier, 1977; Franca et al., 1980; Qadri & Zuberi , 1977; Nair et al., 1980). Important

Vibrio diseases 271

habitats for V. parahaemolyticus inc lude sed iment and shellfish (both oysters and crustaceans), with movemen t into the water co lumn occurr ing dur ing warmer months (Kaneko & Colwell, 1978; Joseph et al., 1982; Gjerde & Boe, 1981; Natara jan et al., 1980). V. parahaemolyticus is occasional ly isolated from skin ulcers of fish, bu t usual ly in association with other vibrios (Yasunaga & Yamamoto, 1977). Kusuda et al. (1979) has isolated 1I. parahaemolyticus from vibriosis of red sea-bream cul tured in Japan, and Gilmour {1977) recovered the species from fish farm tank water. Ortiz & Decker (1976) detected anti-V, parahaemolyticus ant ibody in the sera of 13 of 26 f lounder examined; Robohm et al. (1979), on the other hand, did not detect s ignif icant titers of anti-V. parahaemolyticus ant ibody in the fish they examined. The status of V. parahaemolyticus as a fish pa thogen needs to be more completely documented.

Vibrio vuln i f icus

V. vulnificus is a lac tose-ferment ing vibrio that, in many respects resembles V. alginolyticus and V. parahaemolyticus (Table 1). Or ig ina l ly referred to as a lactose- positive vibrio, V. vulnificus is recognized as a h ighly virulent , opportunis t ic h u m a n pathogen with a low inc idence of disease (Oliver et al., 1983; Joseph et al., 1982; Blake et al., 1980). Oliver et al. (1983) s tudied the dis t r ibut ion of sucrose-negat ive vibrios in seawater, sediment , p lankton, and an imal samples collected at 80 sites from Miami, Florida to Portland, Maine. They conc luded that V. vulnificus was ubiqui tous , both geographical ly and in terms of presence in a variety of env i ronmen ta l samples. How- ever, absolute numbers of V. vulnificus were always found to be low, s ince Oliver et al. (1983) demonstra ted that only 33 isolates (0.83 %) of 3,887 sucrose-negat ive vibrios examined fermented lactose.

Tison et al. (1982) reported on a new subgroup, b iogroup 2, of V. vulnificus that is pathogenic for eels. Previously, the names V. anguillarum type B and V. anguillicida had been used to describe these vibrios (Nishibuchi et al., 1979). Briefly, the eel disease caused by V. vulnificus biogroup 2 is a vibriosis character ized by a red patch or swol len lesion on the t runk or tail. In early stages of the disease, the skin and lateral muscula ture is primari ly involved; advanced stages involve his topathological changes in the spleen, liver, kidney, heart, gills, and in tes t ine (Miyazaki et al., 1977).

V. vulnificus biogroup 2 differs from V. vulnificus biogroup I in several characteristics (Table 1), and the two biogroups do not share common surface an t igens (Tison et al., 1982). However, because of the strong reassociat ion (> 90 %), unde r s t r ingent condi- tions, of DNA from clinical V. vulnificus strains (i.e., b iogroup 1) with DNA from eel isolates, Tison et al. (1982) proposed that the eel isolates, and strains phenotypica l ly resembl ing the eel isolates, be classified as V. vulnificus biogroup 2.

L u m i n o u s Vibrio spp.

Although not known to be pa thogenic for fish, the luminous vibrios deserve brief mention. These in teres t ing species are symbionts of fish, l iv in 9 in the luminous organ (Ruby et al., 1980; Yetinson & Shilo, 1979; Has t ings & Nealson, 1977). The pr inc ipa l species are V. fischeri and 1,I. harvey1, al though occasional strains of V. cholerae are also luminous. Light organs, located variously d e p e n d i n g on the species of fish, usual ly

272 R.R. Colwel l & D. J. Gr imes

conta in approx imate ly l0 g cel ls /ml, and the organ emits constant l ight (Haygood &

Nealson, 1984}. Because of this h igh cell density, Haygood & Nea lson {1984} be l i eve that f ree- l iv ing luminous vibrios recovered from seawate r are "over - f low" from l ight organs

of fish. Presently, the prec ise reason for this unusua l species interact ion is unknown.

FISH POPULATIONS AFFECTED BY VIBRIO

Vibrio spp. infect, and often cause d isease in, a var ie ty of es tuar ine and mar ine fish, some of wh ich are l isted in Tab le 3. Gene ra l ly speaking , d isease is a t t r ibutable to some

form of stress that renders the fish more susceptible. The d isease that follows is typically

a systemic infect ion known as vibriosis or some form of skin u lcer (Table 3).

Table 3. Selected marine fish known to be susceptible to Vibrio infection and disease

Fish Disease

Ayu vibriosis, hemorrhagic disease Akame skin ulcers Sharks skin ulcers, vasculitis Cod skin ulcers, fin rot Eels vibriosis Flounder vibriosis Gray mullet fin rot Salmon vibriosis Sea-bream acute septicemia, vibriosis Sole red spot Striped bass vibriosis Turbot acute edema

A y u . Ayu (Plecoglossus altivelis), a fish cul tured in Japan, is suscept ible to a

var ie ty of infections, inc lud ing s t reptococcal and vibrio disease. Vibrio disease is usual ly a classic vibriosis caused by V. anguillarum (Jo & Muroga, 1977; Kusuda et al., 1981;

Aoki & Kitao, 1978). However , V. cholerae serovar non-01 has also been impl ica ted in

ayu d isease (Muroga et al., 1979b; Yamanoi et al., 1980}.

A k a m e . Wild a k a m e (Liza akame) were obse rved by Muroga {1979} to have skin ulcers caused by a parasi t ic copepod, Caligus orlentalls. The copepod was de tec ted on

35 of 36 a k a m e examined , with an infestat ion rate of 27 copepods per fish. Several

species of bac ter ia w e r e i so la ted from the lesions, inc lud ing Vibrlo spp.

C o d . Atlant ic cod (Gadus morhua) are suscept ib le to vibriosis, especia l ly w h e n exposed to po l lu ted waters. Larsen et al. (1978) observed that cod l iv ing in Danish

coastal waters suffered from a specif ic ulcer syndrome, but only w h e n exposed to water

con tamina ted wi th mun ic ipa l s e w a g e or eff luent from sugar and ce l lu lose plants. Larsen

et al. (1978} were not ab le to e luc ida te e t io logy of the pr imary lesion, but could demons t ra te that V. anguillarum in fec ted the u lcer the reby es tabl i sh ing a genera l i zed

vibriosis. In a later study, it was shown that V. angufllarum could not el ici t the disease,

e v e n w h e n the o rgan ism was inocu la ted into the an imal by a var ie ty of routes (Jenson & Larsen, 1982). Interest ingly, an icosahedra l virus isolated from the d iseased cod was

Vibrio diseases 273

shown to produce the ulcer syndrome. Thus, it was hypothes ized that the icosahedral virus was necessary to ini t iate pathogenesis , with V. anguillarum p lay ing a role in progression of the disease. Most in teres t ing was the re la t ionship b e t w e e n the inc idence of V. anguillarum and wastewater discharge. The f requency of occurrence of V. anguillarum was highest in areas of known contaminat ion, and the number s of V. anguillarum in water and sed iment immedia te ly increased in direct response to eff luent discharge from a sugar p lan t (Larsen et al., 1978). Robohm et al. (1979) also s tudied the effect of pol lut ion on fish disease, and were able to demonst ra te s ignif icant ly h igher an t ibody titers to V. anguillarum in f lounder collected from the New York Bight apex than in f lounder from unpol lu ted coastal waters.

In a study of captive Atlant ic cod, it was observed that 320 of 621 cod died from fin rot, over an 8-year period. Most deaths occurred wi th in 2 months after capture, and death was probably due to excessive blood loss associated with fin erosion. Three gene ra of bacteria were isolated from the infected tissues, ma in ly Pseudomonas spp., but also Aeromonas and Vibrio spp. (Khan et al., 1981).

E e 1 s . Cul t ivated eels are suscept ib le to a variety of bacter ia l infections, p roduc ing several clinical symptoms. Correct d iagnosis is, therefore, d e p e n d e n t on isolat ion and identification of the pa thogen (Nishibuchi et al., 1980). In addi t ion to diseases caused by Aeromonas and Pseudomonas spp., discussed above, eels are suscept ible to infect ion with V. anguillarum and 11. vulnificus biogroup 2 (Nishibuchi et al., 1980; Tison et al., 1982). Rodsaether et al. (1977) conc luded that V. anguillarum is part of the normal flora of eels (A. anguilla}, and that copper con tamina t ion changes the commensa l association be tween fish and the bac te r ium to pathogenici ty . In contrast, is the f ind ing of Kanai et al. (1977) who reported they could not recover Vibrio spp. from heal thy eels (A. japonica}, except dur ing the month of October. However the results of Kanai et al. (1977) must be seriously quest ioned, with regard to Vibrio recovery, s ince the recovery m e d i u m used was nut r ient agar without salt added. Vibrios, in general , require NaC1 for growth (Singleton et al., 1982).

F 1 o u n d e r. Both winter f lounder (Pseudopleuronectes americanus} and smooth flounder (Liopsetta putnami} are suscept ible to infect ion and disease caused by V. anguillarum (Hoornbeek et al., 1982; Watkins et al., 19811 Robohm et al., 1979). Myxobacter and protozoan parasi tes have also b e e n shown to be associated with f lounder mortali ty observed by H o o m b e e k et al. (1982), bu t it was not stated whe the r there were mutualist ic, mixed infection, or vector re la t ionships with V. anguillarum. Watkins et al. (1981) s tudied laboratory infections of win te r f lounder, and found that the LDs0 was lower (by 2-3 logs) for cha l l enge by in jec t ion of V. anguillarum, compared with oral chal lenge. In addition, the m e a n LDs0 for fish collected dur ing the win te r was about 3 logs less than that for spec imens collected in the summer.

G r a y m u 11 e t. Heal thy gray mul le t (Mugil cephalus} were shown by Hamid et al. (1978) to carry Enterobacter, Bacillus, and Micrococcus spp. as the pr inc ipa l in tes t inal bacteria. When transferred to seawater, the dominan t gene ra recovered from the fish included Vibrio, Pseudomonas, and Aeromonas. In subsequen t studies, it was shown that the intest inal Vibrio and Enterobacter spp. were strongly proteolytic and also capable of producing amylase, chitinase, and leci thinase (Hamid et al., 1979). In a later study, Kakimoto & Mowlah (1980) showed these gray mul le t isolates to be V. anguillarum and E. aerogenes.

274 R.R. Colwel l & D, J. Gr imes

M i n c h e w & Yarbrough (1977) demons t ra ted that expe r imen ta l crude oil spills s ignif icant ly inc reased the inc idence of fin rot in mullet , from 6 % in clean, control ponds

to 96 % in ponds con tamina ted wi th 4 -5 ~l/1 (ppm) crude oil. A Vibrio sp. was considered to be the pr imary pa thogen invo lved in the oi l -associa ted fin erosion. Interestingly, Hada

& S izemore (1981) showed that the inc idence of Vibrio spp. in the wate r co lumn near a Gulf of Mexico oil f ield product ion platform was much grea ter than inc idence in a non-

con tamina ted control area. Fur thermore , these vibrios were more l ike ly to contain one or more plasmids.

S a I m o n i d s. Vibriosis of sa lmonid fish, both wi ld and cultured, is a serious disease of wor ldwide distr ibut ion and importance. As discussed above, both V. anffuillarum and V. ordafii can cause vibriosis in sa lmon (Schiewe, 1983; Crosa et al., 1977;

Harre l l et al., 1976) a l though in specif ic cases other Vibrio sl0p. have been isolated {Tajima et al., 1981).

Yoshimizu et al. (1976a, 1976b, 1976c) pub l i shed a series of s tudies on the intest inal

microflora of salmonids, Masu sa lmon reared in f reshwater were found to contain Aeromonas as the p redominan t bacter ia l genus in the intestine, When the fish were m o v e d to seawater , a success ion in the bacter ia l flora composi t ion occurred, go ing from Aeromonas to Pseudomonas to Vibrio dominance (Yoshimizu et al., 1976a). Yoshimizu et

al. (1976b) also s tudied the intes t inal microflora of sa lmonids l iv ing in the Bering Sea, as we l l as bac te r ia associa ted wi th seawate r and zooplankton samples col lec ted from the

Ber ing Sea. Vibrio spp. were found to be dominan t in the intest ine of salmonids. The

microbia l flora of the an imals dif fered from that isola ted from seawate r and zooplankton. Finally, Yoshimizu et al. (1976c) compared the intest inal microflora of adult p ink salmon

(Oncorhynchus gorbuscha) with that of anadromous chum salmon (0. keta) and cul tured

masu sa lmon (0. masu). Predominan t bacter ia l gene ra in the anadromous p ink and

chum salmon were Vibrio, Pseudomonas, and Aeromonas. In the masu salmon, Aeromo- has dominated . It would appear , therefore, that Vibrio spp. comprise the normal intesti-

nal flora of salmonids, with exposure to seawater serving as a se lec t ive pressure.

Sawyer et al. (1979) s tud ied the compara t ive suscept ibi l i ty of a t lant ic sa lmon (Salmo salar) and coho sa lmon (Oncorhynchus kisutch) to V. anguillarum adminis te red via water. Exposure to ~ 105 ce l l s /ml for 1 h at 10 °C and at 15 °C resul ted in the dea th of 80

to 100 % of both sa lmonid species. It was conc luded that newly re leased smolts could

encoun te r le tha l concent ra t ions of V. anguiltarum if the an imals were re leased in Maine (USA) estuaries.

Finally, wi th regard to salmon, MacMi l l an et al. (1980) s tudied infect ion of chum

salmon (0. keta) suffering from erythrocytic necrosis virus (ENV). Fish infected with

ENV were less tolerant of d i sso lved oxygen deple t ion , exh ib i t ed character is t ic cytopa- thogen ic effects in erythroid cells, and were at least 3 t imes more suscept ible to vibriosis.

S e a - b r e a m. Acute sep t i cemia was obse rved by Colorni et al. (1981) to be a

f requent cause of mortal i ty among g i l thead sea -b ream (Sparus aurata) raised in a

laboratory at Elat, Israel. The most f requent ly i sola ted bac te r ium was 1/. alginolyticus, a l though 6 dist inct strains were ident if ied. Yasunaga & Yamamoto (1977) s tudied a s imilar ou tbreak a m o n g cul tured red s ea -b ream (Pagrus major) and isolated several

cul tures of Vibrio that were able to be classif ied as 3 dist inct groups. Iwata et al. (1978),

in a study of the v i ru lence of 1/. alginolyticus for red sea-bream, d iscovered that red sea-

b ream mortal i ty increased if rotifers w e r e present in the seawate r a long with the V.

Vibrio diseases 275

alginolyticus, If Chlorella was mixed with V. alginolyticus, mortal i ty was less than if V. alginolyticus was present alone. W h e n all three microorganisms, V. alginotyticus, Chlorella, and rotifers, were mixed in an aquar ium, the n u m b e r of V. alginolyticus increased as the rotifers grazed and consumed the Chlorella.

S h a r k s. Diseases of sharks have b e e n descr ibed recent ly (Grimes et al., 1984a, b, c), with Vibrio spp. def ini te ly involved in certain of the shark infections reported. Gene ra other than Vibrio have b e e n isolated from shark spec imens examined in our laboratory, and these bacteria are now be ing characterized. C h e u n g et al. (1982) isolated and described a vibrio associated a long with a t rematode (Dermophthirius nigrellii) (Cheung & Ruggieri, 1983) which induced ulcers in captive lemon sharks. Unfortunately, the few biochemical characteristics reported by C h e u n g et al. (1982) are insuff ic ient to classify their isolate as a Vibrio sp.; the characteristics reported also fit the descr ipt ion for Aeromonas hydrophila and A. salmonicida. Vibrio spp. have b e e n isolated from the great white shark (Carcharodon carcharias), but not in associat ion with specific disease (J. Buck, pets. comm).

S o 1 e. Vibrio spp. can cause disease in sole. Wil l iams & Caldwel l (1978) reported that laboratory-reared Engl ish sole [Parophyrs vetulus] exper ienced vibrio mortality, and Fluechter (1979) descr ibed a red spot disease of the common sole (Solea solea] caused by a Vibrio sp.

S t r i p e d b a s s. Substant ia l losses of str iped bass (Morone saxatilis] have occurred in the Chesapeake Bay dur ing the last decade. Eight distinct b iochemica l groups of Vibrio spp. have been isolated from mor ibund striped bass (Toranzo et al., 1983a), two groups of which were ident i f ied as V. anguillarum (Toranzo et al., 1983b). Hetrick et al. (1984) found that the v i ru lence of V. anguillarum for striped bass was directly in f luenced by age of the fish and water temperature . Exposure of the str iped bass to sub- le tha l chlorine levels (0.05-0,20 rag/l, total res idual chlorine) did not affect suscept ibi l i ty to infection. Virulence (LDs0), cytotoxicity, hemolyt ic activity, and adhes iveness of V. anguillarum have b e e n s tudied (Toranzo et al., 1983b), as wel l as p lasmid content and envelope protein profiles (Toranzo et al., 1983a). Details of this work are discussed in the section on mechanisms of v i ru lence (vide infra).

T u r b o t . A vibriosis descr ibed by H o m e et al. (1977) as an acute edematous disease and characterized by orbital and abdomina l swel l ing and high mortali ty (20 %), occurred in turbot (Scophthalmus maximus] in Scotland. V. anguillarum was isolated, in pure culture, from all organs of all of the fish examined . Ant ibiot ic therapy was not successful, but reduct ion of the water tempera ture to less than 10 °C reduced mortal i ty from 30 % to 5 %. Aust in (1982) s tudied the surface bacter ial flora of heal thy turbot, as well as specimens collected from turbot suspected of hav ing vibriosis, and failed to isolate V. anguillarum from any of the samples.

VIRULENCE MECHANISMS

The most recent work on Vibrio diseases of mar ine fish has deal t wi th e luc ida t ion of virulence mechanisms. Much of this exci t ing work involves molecular genet ics and the bulk of the studies have b e e n directed toward V. anguillarum and V. ordalii because of the extensive invo lvement of these pa thogens in fish diseases throughout the world.

In 1977, Crosa, Schiewe, and Falkow (Crosa et al., 1977) discovered that h igh

276 R.R. Co lwe l l & D. J. Gr imes

v i ru l ence s t ra ins of V. anguitlarum, i.e., s t ra ins that cause dea th of infec ted salmonids, possess a l a rge p l a s m i d wi th a mass of ca 47 m e g a d a l t o n s (Mdal). This p lasmid , pJM1, was found in aH high virulence strains examined, judged by plasmid homology experi-

ments (Crosa et al., 1977). Fur thermore , cured s t ra ins lost the i r v i ru lence (Crosa et al., 1977, 1980). Crosa (1979, 1980) s u b s e q u e n t l y demons t r a t ed that the p l a smid pJM1 codes for p roduc t ion of a h igh ly eff icient i ron s eques t e r ing sys tem which a l lows V. angufllarum to ob ta in i ron necessa ry for me tabo l i sm, even in the p re sence of host defense factors that have h igh aff ini ty for u n b o u n d iron (i.e., t ransfer r in and lactoferrin, iron che l a t i ng pro te ins p re sen t in body f luids a n d secre t ions of ver tebra tes) . Briefly, the i ron s eques t e r ing sys tem consists of a low m o l e c u l a r we igh t s ide rophore and two outer m e m b r a n e prote ins , OM2 and OM3, wh ich have m o l e c u l a r we igh t s of 86,000 and 79,000 dal tons, r e spec t ive ly (Crosa et al., 1983). Both the s ide rophore and OM2 are coded by pJM1; OM3 is ch romosome produc t of u n k n o w n invo lvemen t in the system. The s ide rophore diffuses into the env i ronment and complexes wi th iron, even i ron that has b e e n b o u n d by ve r t eb ra t e t ransferr in (Crosa et al., 1983). The s ide rophore - i ron complex is a t t ached to outer m e m b r a n e p ro te in OM2, w h e r e i ron is then p r e s u m a b l y t ranspor ted into the cel l (Crosa et al., 1983).

Trust e t al. (1981) s tud i ed the ab i l i ty of mar ine vibrios, i nc lud ing V. anguillarum, to a g g l u t i n a t e e ry throcy tes and to resis t the bac t e r i c ida l effect of normal (nonimmune) serum. H igh v i ru l ence s t ra ins of V. anguillarum e x h i b i t e d res i s tance to normal trout (Salmo gairdneri) se rum and a g g l u t i n a t e d trout ery throcytes , even in the p resence of mannose . However , ne i the r se rum res i s tance nor ma nnose - r e s i s t a n t h e m a g g l u t i n a t i o n was coded by v i ru l ence p l a s m i d pJM1.

Recently, Sch iewe & Crosa (1981) demons t r a t ed the p r e s e n c e of a 20 Mda l p lasmid , pMJ101, in V. ordalii. H o m o l o g y was not obse rved b e t w e e n pJM1 and pMJ101, and the funct ion of pMJ101 was conc luded to be cryptic. In teres t ingly , the p re sence of this 20-Mdal p l a s m i d is un ive rsa l in al l s t rains of V. ordalii e x a m i n e d thus far, and it is the only p l a s m i d present .

Toranzo et al. (1983a, b, c) e x t e n d e d the molecu la r gene t i c a n d v i ru lence s tudies of V. anguillarum to s t ra ins i so la ted from mor ibund s t r iped bass (M. saxatilis) cap tu red in C h e s a p e a k e Bay. In a s tudy of mo lecu l a r factors a s soc ia t ed wi th v i ru lence , Toranzo et ak (1983b) found that, of 8 dis t inct g roups of Vibrio spp. i so la ted from s t r iped bass and C h e s a p e a k e Bay wa te r samples , two vibr io g roups we re p a t h o g e n i c for s t r iped bass. These two groups were iden t i f i ed as V. anguillarum, b a s e d on b iochemica l tests and DNA homology wi th re fe rence strains. None of the V. anguillanzm strains conta ined de t e c t ab l e p lasmids , a l t hough al l s trains were c a p a b l e of g rowing unde r condi t ions of i ron l imi ta t ion and in normal fish serum. W h e n g rown u n d e r i ron l imitat ion, new outer m e m b r a n e p ro te in b a n d s a p p e a r e d in SDS-po lyac ry l a mide gels. Fur thermore , the stri- p e d bass V. anguillarum strains were shown to syn thes ize a s ideophore , and p re l imina ry e x p e r i m e n t s r e v e a l e d the p r e s e n c e of ch romosomal DNA sequences that hybr id ized wi th pJM1 (Toranzo et al., 1983b). In a r e l a t ed study, Toranzo et al. (1983c) showed that the s t r iped bass 1I. anguitlarum s t ra ins we re not cytotoxic for a ch inook sa lmon embryo cel l cul ture, a l though al l s t rains were hemoly t i c for s t r iped bass, trout, and sheep erythrocytes . M u n n (1978) has also i nves t i ga t ed h e m o l y s i n p roduc t ion by V. anguillarum, and he found the toxin to be a t he rmo lab i l e p ro te in p r o d u c e d in s ta t ionary growth phase . In addi t ion , the V. anguillarum s t ra ins s tud ied by Toranzo et al. (1983c) also

Vibr io d i seases 277

p roduced strong h e m a g g l u t i n i n s for trout, s t r iped bass, and other erythrocytes . Adhes ion of V. anguillarum to r a i nbow trout in tes t ina l t issue sect ions was recen t ly e x a m i n e d by H o m e & Baxenda le (1983). They found that m a x i m u m a t t achmen t occur red wi th in 100 min, and that al l reg ions of the gut were subjec t to h e a v y (ca. 103 cel ls p e r cm 2) colonizat ion.

Kreger (1984) s tud ied the v i ru lence of V. damsela, and de t ec t ed the p roduc t ion of a cytolyt ic toxin. The toxin has a mo lecu l a r w e i g h t of 57,000 da l tons and is a n t i ge n i c a l l y dis t inct from toxins p r o d u c e d b y V. cholerae, V. parahaemolyticus, and V. vulnificus. Using mouse ery throcytes as the cytolyt ic a s say system, Kreger found that pa r t i a l l y pur i f ied cytolys in from an u n n a m e d fish i so la te con ta ined ca. 3 x 105 hemoly t i c uni ts pe r mg of protein. Mice in j ec t ed subcu t aneous ly wi th 1 LDs0 (1,000 hemoly t i c units) of the cytotysin b e c a m e le tharg ic , h a d ruff led fur, d e v e l o p e d encrus ta t ions a round the eyel ids , and exh ib i t ed severe local e d e m a at the si te of inocula t ion; mice so in jec ted usua l ly d i ed wi th in 24 h. To date, the re have b e e n no r epor t ed v i ru l ence s tudies conducted on fish.

Both V. vulnificus (Smith & Merke l , 1982) and V. anginolyticus (Long et al., 1981) produce co l l agenase which p r e s u m a b l y aids in the cu taneous infect ions caused by these two vibrios. Other Vibrio spp. have also b e e n shown to be c a p a b l e of e l a b o r a t i n g co l lagenase , but the role of the pro teo ly t ic enzyme in p a t h o g e n e s i s has not b e e n repor ted (Merkel & Dreisbach, 1978}.

TREATMENT AND PREVENTION

A n t i m i c r o b i a l a g e n t s

At tempts to control fish d i sease have b e e n r epor t ed a lmost s ince d e v e l o p m e n t of the first c l in ica l ly successful an t imicrob ics in the la te 1940's (Snieszko et al., 1952). Treat - ment of f reshwater fish species , w i ld or cul tured, is r e l a t ive ly easy, s ince one is u sua l ly dea l ing with a smal l body of water . Sa l twa te r spec ies on the o ther hand, un less they are cul tured or confined, p resen t obvious diff icul t ies b e c a u s e of the i m m e n s e size of the habi tat . Gene ra l ly speak ing , the most w i d e l y used an t imic rob ia l s have b e e n the sulfona- mides, the nitrofurans, the te t racycl ines , and, more recent ly , subs t i tu ted quinol ines . Regardless of wha t an t imicrob ic is used, bac t e r i a can deve lop res i s tance by aquis i t ion of R p lasmids (Aoki et al., 1980; Kimura et al., 1983; Toranzo et al., 1983a).

One of the more useful an t imic rob ia l s has b e e n the combina t ion of two bac te r io- stat ic chemicals , su l fonamides and t r imethopr im. In concert , these two chemica l s have a synergis t ic effect, and can be used ef fec t ive ly to control res i s tan t o rgan i sms (Kimura et al., 1983; Sako & Kusuda, 1978; Sako et al., 1979). Recent ly , Keck et al. (1980) r epo r t ed that inc reased oxygen tens ion (ca 2.2 atm.) m a r k e d l y p o t e n t i a t e d a bac te r ios ta t i c effect (i.e., l owe red the m i n i m u m inh ib i to ry concent ra t ion , MIC) of fol ic ac id inhibi tors , such as su l fonamides and t r ime thopr im-su l fonamides .

Some of the n e w e r agen t s inc lude p i romid ic ac id (Tashiro et al., 1979), fu ranace (Egidius & Andersen , 1979), and subs t i tu ted quinol ines , such as ha lqu ino l (Austin et al., 1981, 1982). Also, some d e g r e e of success in p r ev e n t i ng t ransmiss ion of severa l Gram- nega t ive fish pa thogens , i nc lud ing V. anguillarum, has b e e n a c h i e v e d by u l t rav io le t

278 R. R. C o l w e l l & D. J. G r i m e s

Table 4. Recommended rates of appl icat ion of fish ant imicrobics

Antimicrobic Dosage b Route Target Reference

Piromidic acid 10-40 mg /kg oral salmonids & eels Tashiro et al. (1979) Sulfadoxine-TMI ~ 30 mg /kg oral ayu Sako et al. (1979) Sulfamethoxazole-TMP 25 mg/l bath turbot Aust in et al, (1981~ Furanace 2 mg/I bath ra inbow trout Egidius & Andersen (1979) Halquinol 25 mg/1 ba th turbot Aust in et al. (1982)

75 mg /kg oral turbot Aust in et al. (1982)

a TMP-tr imethopr im b wt /kg indicates amount per kg fish body wt.; wt/1 indicate amount per 1 of immersion water

i r r a d i a t i o n of t h e f i sh c u l t u r e w a t e r ( B u l l o c k & S t u c k e y , 1977). In T a b l e 4 a r e l i s t e d s o m e

of t h e m o r e e f f e c t i v e a n t i m i c r o b i a l s .

I m m u n i z a t i o n

As w i t h a n t i m i c r o b i a l t h e r a p y , i m m u n i z a t i o n h a s a l s o b e e n p r a c t i c e d for m a n y y e a r s

( S n i e s z k o & F r i d d l e , 1949; K r a n t z e t al., 1963; H a y a s h i e t al., 1964). E a r l y a t t e m p t s at

i m m u n i z a t i o n i n v o l v e d c r u d e a n t i g e n p r e p a r a t i o n s , if no t s i m p l y w h o l e cel ls , a n d w a s

l a b o r i n t e n s i v e . R e c e n t a p p r o a c h e s to i m m u n i z a t i o n a re n o v e l a n d m u c h m o r e ef f ica-

c ious t h a n t h e e a r l y p r o c e d u r e s . A w i d e v a r i e t y of a n t i g e n p r e p a r a t i o n s , a d m i n i s t e r e d in

d i f f e r e n t ways , h a v e b e e n t r i e d a n d a r e p r e s e n t e d in T a b l e 5.

T h e s i m p l e s t d e l i v e r y s y s t e m a p p e a r s to b e t h e a d m i n i s t r a t i o n of t h e v a c c i n e b y t h e

ora l rou te , u s u a l l y w i t h food. K u s u d a e t al . (1978) s t u d i e d four d i f f e r e n t o ra l v a c c i n e s

Table 5. Summary of approaches to immuniza t ion against Vibrio anffuillarum

Delivery route

Protection

Average Site Ant ibody Duration Reference dose (titer)

Oral 0.4 g /kg

Hyper- 0.2 g /kg osmotic

Direct 5 x 105 immers ion cells/ml

Spray 100 mg/g

Intraperi- 150 mg /kg toneal inj.

* NA-data not avai lable

body agglu t in in > 4 wk Kawai et al. (1981) surface (1 : 64)

serum agglut in in > 100 wk Antipa & Amend (1977) (1:8) Naka j ima & Chikaha ta (1979)

NA* NA > 200 da Gould et al. (1979) Johnson et al, (1928b)

serum agglut in in > 16 wk Gould et al. (1978) (1 : 147) Itami & Kusuda (1980b)

serum agglu t in in > 6 mo Antipa (1976) (1:64) Antipa & Amend (1977)

Home et al. (1984) Agius et aL (1983)

Vibrio diseases 279

prepared from formal in-ki l led V. anguillarum: (1) formal in-k i l led cells, (2) formalin- killed, ul t rasonicated cells, (3) formalin-ki l led, hea ted cells, and (4) formal in-k i l led cells mixed with Freund ' s incomple te adjuvant . The four bacter ins were admin i s te red to cultured ayu (P. attivelis) at a rate of 0.4 g (wet wt ) of cells per kg body wt. per day. Formal in-ki l led and formalin-ki l led, u l t rasonica ted bacter ins were the most effective, lowering mortali ty from 90 % in controls to approximate ly 10 %. Interest ingly, circulat- ing ant ibody could not be detected. In a follow-up study, Kawai et al. (1981) compared orally immunized ayu with n o n - i m m u n i z e d controls to de te rmine the site of immuni ty . Immunized fish were highly resistant to cha l lenge with V. anguillarum by immersion, but were equal ly susceptible, as were the controls, w h e n the cha l lenge was by in t ramus- cular injection. It was de te rmined that the skin of i m m u n i z e d ayu was free from V. anguillarum, w h e n cha l l enged by immersion, and serological examina t ion of the body surface mucus revealed an agg lu t in in titer of 1 : 64 (Kawai et al., 1981). Nei ther the serum nor the in tes t inal mucus of immun ized fish con ta ined increased agg lu t in in titers. It was concluced that the mechan i sm of i m m u n e resis tance involved p reven t ion of bacterial a t tachment by agg lu t in ins secreted in the surface mucus (Kawai & Kusuda, 1981).

Vaccinat ion by direct immers ion of fish has b e e n pract iced in the commercia l sector by Antipa & A m e n d (1977). They d ipped the sa lmon (O. kisutch and O. tshawytscha) in a hyperosmotic salt solution, a step that was hypothes ized to e n h a n c e a n t i ge n uptake. The salmon were next d ipped in ki l led V. anguillarum an t igen preparat ions, and i m m u n i z e d animals were compared with n o n - i m m u n i z e d controls and in t raper i tonea l ly- in jec ted immune controls. Ant ipa & A m e n d (1977) concluded that the hyperosmotic infi l t rat ion method of vaccine del ivery afforded the best protect ion and y ie lded the most rapid rise in ant ibody titer. Croy & A m e n d (1977) s tudied the hyperosmotic infi l t rat ion method of immuniz ing sockeye sa lmon (0. nerka) agains t vibriosis, us ing ki l led V. anguillarum as ant igen. They found that p re - immers ion of the fish in Hanks ' b a l a n c e d salt solut ion with 8 % NaC1 was markedly superior to immuniza t ion wi thout hyperosmotic t reatment . In subsequent studies on i m m u n i z i n g agains t V. anguillarum, L a n n a n (1978) appl ied the hyperosmotic approach for chum sa lmon (0. keta) and Naka j ima & Chikaha ta (1979) tried it for ayu (P. altivelus). Most recently, Aoki et al. (1984) modif ied the hyperosmotic approach by removing undes i rab le toxic components of V. anguillarum, Specifically, they formalized a culture and sed imented the cells by centr i fugat ion to remove soluble toxic factors in the s u p e r n a t a n t The precipi ta ted ceils were used to immun ize ayu previously immersed in a 5.32 % NaC1 solution. Controls consisted of n o n - i m m u n i z e d and in t raper i toneal ly i m m u n i z e d ayu. Removal of the superna tan t increased the survival of immunized ayu. The conclusion was that an exotoxin was presen t in the formalized cultures. Upon in t raper i toneal cha l lenge with v iable V. anguillarum, survival was found to be greatest (near 100 %) among ayu immersed in precipi ta ted cells, next highest (60-70 %) among ayu injected intraper i toneal ly , and least (ca. 20 %) among non- immunized controls. One month after immuniza t ion , no agg lu t ina t ing titers were detected in the serum or mucus of fish immunized by immers ion or in n o n - i m m u n i z e d controls. In t raper i toneal ly immun ized ayu had e levated serum titers, but no ant i- V. anguillarum activity was detected in the mucus (Aoki et al., 1984).

Later studies revealed prior t rea tment with hyperosmotic salt immers ion was u n n e - cessary. Gould et al. (1979) ob ta ined long- las t ing immun i ty in sockeye sa lmon (0. nerka)

280 R.R. Colwell & D. J. Grimes

by immers ing the fish in formal in-ki l led V. anguillarum (ca 5 × 105 cells per ml of immers ion solution), types I and II (i.e., V. anguillamm and V. ordalii, respectively). Mortali ty was reduced to 2 %, with little cross protect ion observed by immuniza t ion with the two different Vibrio bacterins. Bivalent immuniza t ion , i.e., s imul taneous immersion in both bacterins, was as effective as type I and II bacter ins ind iv idua l ly appl ied (Gould et al., 1979). Johnson et al. (1982a, b) examined the direct immers ion method as a means of i m m u n i z i n g the fry of different salmonids. In general , the best results were obta ined with fry 1.0 to 2.5 g, total body wt, and the immers ion t ime required was ca 5 seconds. Direct immers ion as a means of i m m u n i z i n g agains t vibriosis has also been appl ied to cul tured ayu (Kusuda et al., 1980; Takej i et al., 1981) and to striped bass (Roberson et al., 1982). Roberson et al. (1982) used a 2-min immers ion in I/. anguillamm 0-ant igen (acetone-ethanol extract), and e n h a n c e d an t ibody levels were detected in serum collected from the striped bass.

Spray, or shower, vacc ina t ion is bas ical ly a modif icat ion of direct immersion, in that fish are i m m u n i z e d by a h igh pressure spray directed at ne t ted fish or at fish in shallow channels . This approach has b e e n used successfully to immunize both ayu and salmonids agains t V. anguillarum (Gould et al., 1978; Itami & Kusuda, 1978, 1980a, b; Johnson et al., 1982b), In general , immun i ty was comparable to that ob ta ined with direct immersion, and bet ter than that by oral (Gould et al., 1978).

In t raper i toneal in jec t ion is perhaps the most effective, in terms of producing a high degree of immuni ty , but is both expens ive and labor intensive. It also subjects the fish to stress associated with handl ing . S ingle inject ions of V. anguillarum bacter ins (heat- kil led, formalin-ki l led, or both) gave s ignif icant protect ion in sa lmonids for over 7 months pos t - inocula t ion (Antipa, 1976). Sawyer & Strout (1977) compared immuniza t ion wi th oxytetracycline t rea tment as methods for control l ing vibriosis in coho salmon, and found in t raper i toneal immuniza t ion s ignif icant ly superior to ant ibiot ic prophylaxis. Finally, H o m e et al. (1982) compared intraperi toneal , direct immersion, and oral administration of formalized V. anguillarum to ra inbow trout (Salmo gairdneri)using a vaccine con ta in ing 35 mg (wet wt.) of bac ter in per ml of vaccine and 2.5 % (w/v) potassium a l u m i n u m sulfate adjuvant . The in t raper i toneal route employed 100 ~1. Fish were immersed for 30 min in a 10-fold di lut ion for direct immersion. For oral route of entry, 3.5 mg of bacter ia were g iven with food over a 32-day period, After 85 days, all fish, i nc lud ing n o n - i m m u n i z e d controls, were cha l l enged by in t raper i toneal inject ion of 2 x l0 T v iable cells of V, anguillarum. Survival rates at 21 days after cha l lenge were 93 % for in t raper i toneal del ivery of the vaccine; 47 % for direct immersion, 6 % for oral, and 0 % for controls. In a fol low-up study, Agius et al. (1983) used a perchloric acid extract as the an t igen preparat ion, and compared it to formal in-ki l led V. anguillarum cells as an i m m u n i z i n g agent . The an t igen extract was conc luded to be superior when in t raper i tonea l del ivery was employed, but formalized cells were bet ter in del ivery by the oral route. A lum appears to offer little addi t iona l protection, l ead ing H o m e et al. (1984) to ques t ion the use of adjuvants .

In addi t ion to improvements in methods of immuniza t ion , improvements have also b e e n made in serological techniques . Roberson (1981) combined the methods of thin layer immunoassay and enzyme- l i nked immunoso rben t assay (ELISA) to detect anti- V. anguillarum ant ibody in striped bass i m m u n i z e d by in t raper i toneal inject ion with 0- ant igen. The sensi t ivi ty was found to be comparable to passive hemagglut ina t ion .

Vib r io d i s e a s e s 281

G o s t i n g et al. (1981) u s e d an i m m u n o c y t o a d h e r e n c e a s s a y to m o n i t o r p r o g r e s s i o n of t h e

i m m u n e r e s p o n s e in s o c k e y e s a l m o n i m m u n i z e d a g a i n s t V. angui l larum b y i m m e r s i o n

a n d i n t r a p e r i t o n e a l i n j ec t i on . Both d e l i v e r y m e t h o d s i n i t i a t e d an e l e v a t e d i m m u n e

a d h e r e n c e in l e s s t h a n 1 day , a n d t h e r e s p o n s e p e r s i s t e d 1 w e e k l o n g e r in i n j e c t e d t h a n

in i m m e r s e d fish.

C O N C L U S I O N

T h e u n d e r s t a n d i n g of Vibrio d i s e a s e s of m a r i n e f i sh p o p u l a t i o n s h a s i n c r e a s e d

s i g n i f i c a n t l y s i n c e the first d e s c r i p t i o n of 1/. angui l larum a p p e a r e d in 1893 (Canes t r i n i ,

1893). W e n o w r e c o g n i z e s e v e r a l Vibrio spp . as b e i n g c a p a b l e of c a u s i n g d i s e a s e in fish.

T h e n u m b e r of s p e c i e s of Vibrio i n v o l v e d wi l l no d o u b t i n c r e a s e as m a r i c u l t u r e ac t iv i t i e s

are e x t e n d e d . H o w e v e r , m o d e r n i m m u n i z a t i o n t e c h n i q u e s , m o r e e f f e c t i v e a n t i m i c r o -

bials , a n d b e t t e r u n d e r s t a n d i n g of t h e e c o l o g y of t h e g e n u s Vibrio offer m e a n s for

i m p r o v e d con t ro l of Vibrio d i s e a s e . In t h e n e a r fu ture , it wi l l b e p o s s i b l e to p r e v e n t

v ib r ios i s by s p e c i f i c g e n e t i c m a n i p u l a t i o n s of t h e p a t h o g e n s (Co lwe l l , 1983, 1984).

LITERATURE CITED

Agius, C., Horne, M. T. & Ward, P. D., 1983. Immunization of rainbow trout, Salmo gairdneri Richardson, against vibriosis: comparison of an extract antigen with whole cell bacterins by oral and intraperitoneal routes. - J. Fish Dis. 6, 129-134.

Akazawa, H., 1968. Bacterial disease of marine fishes. - Bull. Jap. Soc. scient. Fish. 34, 271-272. Antipa, R., 1976. Field testing of injected Vibrio anguillarum bacterins in pen reared Pacific salmon.

- J. Fish. Res. Bd Can. 33, 1291-1296. Antipa, R. & Amend, D. F., 1977. Immunization of Pacific salmon: comparison of intraperitoneal

injection and hyperosmotic infiltration of Vibrio anguillarum and Aeromonas salmonicida bacterins. - J. Fish. Res. Bd Can. 34, 203-208.

Aoki, T. & Kitao, T., 1978. Vibriosis of ayu. - Fish Pathol. 13, 19-24. Aoki, T., Jo, Y. & Egusa, S., 1980. Frequent occurrence of drug resistant bacteria in ayu (Plecoglos-

sus altivelis) culture. - Fish Pathol. 15, 1-6. Aoki, T., Kitao, T. & Kawano, K., 1981. Changes in drug resistance of Vibrio anguillarum in cultured

ayu, Plecofflossus altivelis Temminck and Schlegel, in Japan. - J. Fish Dis. 4, 223-230. Aoki, T., Kitao, T., Fukudome, M., Takahashi, S. & Egusa, S., 1984. Modification of the hyperosmatic

infiltration method of vaccination against vibriosis in cultured ayu, Plecoglossus altivelis Temminck and Schlegel. - J. Fish Dis. 7, 149-156.

Austin, B., 1982. Taxonomy of bacteria isolated from a coastal marine fish rearing unit. - J. appI. Bact. 53, 253-268.

Austin, B., Morgan, D. A. & Alderman, D. J., 1981. Comparison of antimicrobial agents for control of vibriosis in marine fish. - Aquaculture 26, 1-12.

Austin, B., Johnson, C. & Alderman, D. J., 1982. Evaluation of substituted quinolines for the control of vibriosis in turbot (Scophthalmus maximus}. - Aquaculture 29, 227-239.

Belas, M. R. & Colwell, R. R, 1982a. Scanning and electron microscope observation of the swarming phenomenon in Vibrio parahaemolyticus. - J. Bact. 150, 956-959.

Belas, M. R. & Colwell, R. R., 1982b. Adsorption kinetics of laterally flagellated Vibrio. - J. Bact. 15I, 1568-1580.

Bergman, A. M., 1909. Die rote Beulenkrankheit des Aals. - Ber. K. bayer, biol. Vers.-Stn 2, 10-54. Blake, P. A., Weaver, R. E. & Hollis, D. G., 1980. Diseases of humans (other than cholera) caused by

vibrios. - A. Rev. Microbiol. 34, 341-367. Bullock, G. L. & Stuckey, H. M., 1977. UV treatment of water for destruction of 5 gram-negative

bacteria pathogenic to fishes. - J. Fish. Res. Bd Can. 34, 1244-1249. Burke, J. & Rodgers, L., 1981. Identification of pathogenic bacteria associated with the occurrence of


" r e d spo t" in s e a mul le t , Mugil cephalus L., in s o u t h - e a s t e r n Q u e e n s l a n d . - J. F i sh Dis. 4, 153-159.

Canes t r i n i , G , 1893. La m a l a t t i a d o m i n a n t e de l l e a n g u i l l e . - Atti Ist. v e n e t o Sci. 7, 809-814, C h e u n g , P. J. & Rugg ie r i , G. D., 1983. Dermophthirius nigrellii n. sp. ( M o n o g e n e a : Microbothr i i -

dae) , a n e c t o p a r a s i t e f rom t h e s k i n of t h e l e m o n shark , Neffaprion brevirostris. - Trans . Am. microsc . Soc. 102, 129-134.

C h e u n g , P. J., Nigre l l i , R. F., Rugg ie r i , G. D. & Cil ia, A., 1982. T r e a t m e n t of s k i n l e s ions in cap t ive l e m o n sha rks , Neffaprion brevirostris (Poey), c a u s e d b y m o n o g e n e a n s (Dermophthirius sp.). - J . F i sh Dis. 5, 167-170.

Colorni , A., P a p e r n a , I. & Gord in , H., 1981. Bac te r ia l i n f ec t i ons in g i l t h e a d s e a - b r e a m (Sparus aurata) c u l t u r e d at Elat, Israel . - A q u a c u l t u r e 23, 257-268 .

Colwel l , R. R., 1983. B i o t e c h n o l o g y in t h e m a r i n e s c i ences . - Sc ience , N. Y. 222, 19-24. Colwel l , R. R., 1984. T h e i n d u s t r i a l po t en t i a l of m a r i n e b i o t e c h n o l o g y . - O c e a n u s 27" 3-12 . Crosa , J. H., 1979. Vibrio anguillarum: An i ron u p t a k e m e c h a n i s m as a factor of v i ru lence . - F ish

H e a l t h N e w s 8, 7-10 . Crosa , d. H., 1980. A p l a s m i d a s s o c i a t e d w i t h v i r u l e n c e in t he m a r i n e f ish p a t h o g e n Vibrio

anguillarum spec i f i e s a n i ron s e q u e s t e r i n g s y s t e m . - Na tu r e , Lond. 284, 566-568 . Crosa , d. H., S c h i e w e , M. H. & Fa lkow, S., 1977. E v i d e n c e for p l a s m i d con t r ibu t ion to t he v i ru l ence

of t h e f i sh p a t h o g e n Vibrio anguillarum. - Infect. I m m u n . 18, 509-513 . Crosa , d. H., H o d g e s , L. L. & S c h i e w e , M. H., 1980. C u r i n g of a p l a s m i d is co r re la ted wi th an

a t t e n u a t i o n of v i r u l e n c e in t h e m a r i n e f i sh p a t h o g e n Vibrio anguillarum. - Infect. I m m u n . 27, 897-902 .

Crosa , J. H., Wal te r , M. A. & Potter, S. A., 1983. T h e g e n e t i c s of p l a s m i d - m e d i a t e d v i r u l e n c e in the m a r i n e f i sh p a t h o g e n Vibrio anguitlarum. In: Bac te r i a l a n d vi ra l d i s e a s e s of f ish, m o l e c u l a r s tud ies . Ed. b y J. H. Crosa . Univ . of W a s h i n g t o n , Seat t le , 21-30 .

Croy, T. R. & A m e n d , D. F., 1977. I m m u n i z a t i o n of s o c k e y e s a l m o n (Oncorhynchus nerka) a g a i n s t v ib r ios i s u s i n g t h e h y p e r o s m o t i c in f i l t ra t ion t e c h n i q u e . - A q u a c u l t u r e 12, 317-326 .

Eg id ius , E. & A n d e r s e n , K., 1979. T h e u s e of F u r a n a c e a g a i n s t v ibr ios i s in r a i n b o w trout Salmo galrdnerl R i c h a r d s o n in sa l t wa te r . - J. F i sh Dis. 2, 79-80 .

F luech te r , J., 1979. Iden t i f i ca t ion a n d t r e a t m e n t of d i s e a s e s in t he c o m m o n sole (Solea solea). - A q u a c u l t u r e 16, 271-274 .

Franca , S. M. C., Gibbs , D. L., S a m u e l s , P. & J o h n s o n , W. D. Jr., 1980. Vibrioparahaemolyticusin Braz i l i an coas ta l wa te r s . - J. A m . m e d . Ass. 244, 587-588 .

G a u t h i e r , M. J. & C l e m e n t , R., 1979. E x p e r i m e n t a l s t u d y of t h e t r ans fe r of Vibrioparahaemolyticus b i o t y p e 2 f rom w a t e r a n d s e d i m e n t s to o r g a n i s m s in b e n t h i c m a r i n e food cha ins . - Can. J. Mic rob ioL 25, 499-507 .

G i lmore , A., 1977. C h a r a c t e r i s t i c s of m a r i n e v ibr ios i so la t ed f rom f ish f a rm t a n k s . - A q u a c u l t u r e I 1, 51-62 .

G je rde , J. & Boe, B., 1981. I so la t ion a n d c h a r a c t e r i z a t i o n of Vibrio alginolyticus a n d Vibrio parahaemolyticus f rom t h e N o r w e g i a n coas ta l e n v i r o n m e n t . - Ac t a vet . s cand . 22, 331-343.

G o s t i n g , L., M i r a n d o , D. M. & Gou ld , R. W., 1981. A n t i g e n b i n d i n g ce l l s in t he p e r i p h e r a l b lood of s o c k e y e s a l m o n (Oncorhynchus nerka) i n d u c e d b y i m m e r s i o n or i n t r a p e r i t o n e a l in jec t ion of Vibrio anguillarum bac te r ia . - J. F i sh Biol. 19, 83-86 .

Gould , R. W., O 'Lea ry , P. J., Gar r i son , R. L., Rohovec , J. S. & Fryer , J. L., 1978. Spray vacc ina t ion : A m e t h o d for t h e i m m u n i z a t i o n of fish. - F i sh Pathol . 13, 63-68 .

Gou ld , R. W., Anf ipa , R. & A m e n d , D. F., 1979. I m m e r s i o n v a c c i n a t i o n of s o c k e y e s a l m o n (Onco- rhynchus nerka) w i t h two p a t h o g e n i c s t r a ins of Vibrio anguillarum. - J. Fish. Res. Bd Can. 36, 220-225 .

Gr imes , D. J., S t e m m l e r , J., H a d a , H., May , E. B., M a n e v a l , D., Het r ick , F. M., Jones , R. T., Stoskopf, M. & Colwel l , R. R., 1984a. Vibrio s p e c i e s a s s o c i a t e d w i t h mor ta l i ty of s h a r k s h e l d in captivi ty. - Microb . Ecol. 10 (In press ) .

G r i m e s , D. J., Colwel l , R. R , S t e m m l e r , J., H a d a , H., M a n e v a l , D., Het r ick , F. M., May, E. B., Jones , R. T. & Stoskopf , M., 1984b. Vibrio s p e c i e s as a g e n t s of e l a s m o b r a n c h d i sease . - He lgoRinde r M e e r e s u n t e r s . 37, 309-315 .

Gr imes , D. J., Gruber , S. H. & May , E. B., 1984c. E x p e r i m e n t a l i n fec t ion of l e m o n sharks , Negaprion brevirostris, w i t h Vibrio spec i e s . - J. F i sh Dis. (Submi t ted) .

V i b r i o d i s e a s e s 2 8 3

Hada , H. S. & S izemore , R. K., 1981. I n c i d e n c e of p l a s m i d s in m a r i n e Vibrio spp . i so l a t ed f rom a n oil f ie ld in t h e N o r t h w e s t e r n Gul f of Mex ico . - Appl . env i ron . Microbio l . 41, 199-202.

H a m i d , A., Saka ta , T. & Kak imoto , D., 1978. Microf lo ra in t h e a l i m e n t a r y t rac t of g r a y mul l e t . Par t 2. A c o m p a r i s o n of t h e m u l l e t i n t e s t i n a l mic rof lo ra in f r e sh w a t e r a n d s e a wa te r . - Bull. J ap . Soc. sc ient . Fish. 44, 53-58.

I-Iamid, A., Saka ta , T. & Kak imoto , D., 1979. Microf lo ra in t h e a l i m e n t a r y t rac t of g r a y mul le t . Par t 4. E s t i m a t i o n of e n z y m i c ac t iv i t i es of t h e i n t e s t i n a l bac te r i a . - Bull. J ap . Soc. sc ien t . Fish. 45, 99-106.

Harrel l , L. W., Novo tny , A. J., S c h i e w e , M. H. & H o d g i n s , H. O., 1976. I so la t ion a n d d e s c r i p t i o n of two v ibr ios p a t h o g e n i c to Paci f ic s a l m o n in P u g e t Sound , W a s h i n g t o n . - Fish. Bull . U. S. 74, 447-449 .

Has t ings , J. W. & Nea l son , K. H., 1977. Bac te r ia l b i o l u m i n e s c e n c e . - A. Rev. Microbiol . 31,549-595. H a y a s h i , K., K o b a y a s h i , S., K a m a t a , T. & Ozaki , H., 1964, S t u d i e s on t h e v ib r io d i s e a s e of r a i n b o w

trout (Salmo gairdneri irideus) II. P rophy lac t i c v a c c i n a t i o n a g a i n s t t h e v ibr io d i sease . - J. Fac. Fish., pref. Univ . Mie. 6, 181-191.

Haygood , M. G. & N e a l s o n , K. H., 1984. Effects of i ron on bac t e r i a l g r o w t h a n d l u m i n e s c e n c e : Ecologica l impl i ca t ions . In: C u r r e n t p e r s p e c t i v e s in m ic rob i a l eco logy . Ed. by M. J. K l u g & A. Reddy. Amer . Soc. Microbiol . , W a s h i n g t o n , D.C., 56-61 .

Hetr ick, F. M., Hal l , L. W. Jr., Wolsk i , S., Graves , W. C., Rober son , B. S. & Bur ton, D. T., 1984. Effect of e x p o s u r e of s t r iped b a s s (h4orone saxatilis) to ch lo r ine on the i r s u s c e p t i b i l i t y to Vibrio anguillarnm. - Can . J. Fish. aqua t . Sci, (In press}.

H i ra t suka , M., Sai toh, Y. & Y a m a n e , N,, 1980. I so la t ion of Vibrio alginolyt icus f rom a p a t i e n t w i t h a c u t e enterocol i t i s . - T o h o k u J. exp. M e d . 132, 469-472 .

H o o r n b e e k , F. K., Sawyer , P. J. & Sawyer , E. S., 1982, G r o w t h of w i n t e r f l o u n d e r Pseudopleuronec- tes americanus a n d s m o o t h f l o u n d e r I~'opsetta pu tnami in h e a t e d a n d u n h e a t e d wa te r . - A q u a c u l t u r e 28, 363-374.

H o m e , M. T., 1982. T h e p a t h o g e n i c i t y of Vibrio anguil larum (Bergman}. In: Mic rob ia l d i s e a s e of fish. Ed. b y R. J. Roberts . Acad . Press , N e w York, 171-187.

Horne , M. T. & B a x e n d a l e , A., 1983. T h e a d h e s i o n of Vibrio anguil larum to hos t t i s s u e s a n d its role in p a t h o g e n e s i s . - J. F i sh Dis. 6, 461 -471 .

H o m e , M. T., Richards , R. H., Roberts , R. J. & Smi th , P. C., 1977. Per a c u t e v ib r ios i s in j u v e n i l e t u rbo t (Scophthalmus max/ runs) . - J. F i sh Biol. 11, 355-362 .

Hornme, M. T., Ta tne r , M., M c D e r m e n t , S., A g i u s , C. & Ward , P., 1982. V a c c i n a t i o n of r a i n b o w trout, Salmo gairdneri Richardson , at low t e m p e r a t u r e s a n d t he l o n g - t e r m p e r s i s t e n c e of pro tec t ion . - J. F i sh Dis. 5, 343-345 .

H o m e , M. T., Roberts , R. J., Ta tne r , M. & Ward , P., 1984. T h e ef fec ts of t he u s e of p o t a s s i u m a l u m a d j u v a n t in v a c c i n e s a g a i n s t v ib r ios i s in r a i n b o w trout , Salmo gairdneri Richa rdson . - J. F i sh Dis. 7, 91-99 .

Huq , A., Smal l , E. B., Wes t , P. A., H u q , M. I., R a h m a n , R. & Colwel l , R. R., 1983. Eco log ica l r e l a t i o n s h i p s b e t w e e n Vibrio choterae a n d p l a n k t o n i c c r u s t a c e a n c o p e p o d s . - Appl . env i ron . Microbiol . 45, 2?5-283 .

I tami, T. & K u s u d a , R., 1978. Eff icacy of a v a c c i n a t i o n b y s p r a y a d m i n i s t r a t i o n a g a i n s t v ib r ios i s in c u l t u r e d ayu . - Bull. J ap . Soc. sc ien t . Fish. 44, 1413-1414 .

Itami, T. & K u s u d a , R., 1980a. S t u d i e s o n s p r a y v a c c i n a t i o n a g a i n s t v ib r ios i s in c u l t u r e d a y u (Plecogtossus altivelis). 1. Effect of b e n t o n i t e a n d p H on v a c c i n a t i o n eff icacy. - Bull . J ap . Soc. sc ient . Fish. 46, 533-536 .

I tami, T. & K u s u d a , R., 1980b, S t u d i e s on s p r a y v a c c i n a t i o n a g a i n s t v ibr ios i s in c u l t u r e d a y u (Plecoglossus altivelis). 2. D u r a t i o n of v a c c i n a t i o n e f f icacy a n d ef fec t of d i f fe ren t v a c c i n e p repa ra t ions . - Bull. Jap . Soc. sc ient . Fish. 46, 699-704 .

Iwata, K., Yanoha ra , Y. & I sh ibash i , O., 1978, S t u d i e s on fac tors r e l a t ed to mor t a l i t y of y o u n g r ed s e a - b r e a m (Pagrus major) in t h e ar t i f ical s e e d p roduc t ion . - F i sh Pa thol . 13, 97-102 .

J e n s e n , N. J. & Larsen , J. L., 1982. T h e u l c u s s y n d r o m e in cod Gadus m o r h u a . 4. T r a n s m i s s i o n e x p e r i m e n t s w i t h 2 v i r u s e s i so la t ed f rom cod a n d Vibrio anguillarum. - Nord. V e t M e d . 34, 136-142.

Jo, Y. & M u r o g a , K., 1977. S tud ie s o n v ibr ios i s in ayu . Par t 1. V i r u l e n c e of a cu l tu re of Vibrio anguillarum. - Fish Pathol . 12, 151-156,


J o h n s o n , K. A., F lynn , J. K. & A m e n d , D. F., 1982a. O n s e t of i m m u n i t y in s a l m o n i d fry v a c c i n a t e d by d i rec t i m m e r s i o n in Vibrio anguillarum a n d Yersinia ruckeri bac te r ins . - J. F i sh Dis. 5, 197-206.

J o h n s o n , K. A., F lynn , J. K. & A m e n d , D. F., 1982b. D u r a t i o n of i m m u n i t y in s a l m o n i d s v a c c i n a t e d by d i rec t i m m e r s i o n w i t h Yersinia ruckeri a n d Vibrio anguillarum bac te r ins . - J. F i sh Dis. 5, 207-214 .

J o s e p h , S. W., Colwel l , R. R. & Kaper , J. B., 1982. Vibrio parahaemolyticus a n d re la t ed ha loph i l i c vibr ios . - C R C crit. Rev. Microbiol . 10, 77-124 .

Kak imoto , D. & M o w l a h , A. H., 1980. Microf lora in t h e a l i m e n t a r y t ract of g r ay m u l l e t (h4ugil cephalus). 8. Ut i l i za t ion of a m i n o ac ids b y Vibrio a n d Enterobacter i sola tes . - M e m . Fac. Fish. K a g o s h i m a Univ . 29, 349-354 .

Kana i , K., W a k a b a y a s h i , H. & E g u s a , S., 1977. C o m p a r i s o n of i n t e s t i na l microf lo ra b e t w e e n h e a l t h y a n d d i s e a s e d p o n d c u l t u r e d eels . - F i sh Pathol . 12, 199-204.

K a n e k o , T. & Colwel l , R. R., 1978. T h e a n n u a l cyc le of Vibrioparahaemolyticusin C h e s a p e a k e Bay. - M i c r o b . Ecol. 4, 135-155.

Kawai , K. & K u s u d a , R., 1981. M e c h a n i s m s of p ro tec t ion in a y u (Plecoglossus altivelis) oral ly v a c c i n a t e d for v ibr ios is . 1. Pro tec t ion ac t iv i ty in b o d y su r f ace m u c u s . - F i sh Pathol . 16, 1-8.

Kawai , K., K u s u d a , R. & I tami, T., 1981. M e c h a n i s m s of p ro t ec t i on in a y u (Plecoglossus altivetis) oral ly v a c c i n a t e d for v ibr ios is . - F i sh Pathol . 15, 257-262 .

Keck, P. E., Got t l ieb , S. F. & Conley , J., 1980. In te rac t ion of i n c r e a s e d p r e s s u r e s of o x y g e n and s u l f o n a m i d e s o n t h e in vitro g r o w t h of p a t h o g e n i c bac te r ia . - U n d e r s e a b i o m e d . Res. 7, 95-106.

Khan , R. A., C a m p b e l l , J. & Lear, H., 1981. Mor ta l i ty in c ap t i ve A t l an t i c cod Gadus morhua a s s o c i a t e d w i t h f in rot d i sease . - J . Wildl . Dis. 17, 521-528 .

K i m u r a , T., Y o s h i m i z u , M. & W a d a , M., 1983. In vitro an t i bac t e r i a l ac t iv i ty of t he c o m b i n a t i o n of s u l p h a d i a z i n e a n d t r i m e t h o p r i m on bac t e r i a l f i sh p a t h o g e n s . - J. F i sh Dis. 6, 525-532 .

Krantz , G. E., Reddecl i f f , J, M. & Heis t , C. E., 1963, D e v e l o p m e n t of a n t i b o d i e s a g a i n s t Aeromonas salmonicida in trout. - J. I m m u n . 91, 757-760 .

Kreger , A. S., 1984. Cyto ly t ic ac t iv i ty a n d v i r u l e n c e of Vibrio damsela. - Infect. I m m u n . 44, 326-331 . K u s u d a , R., Kawai , K., Jo, Y., Ak izuk i , T., F u k u n a g a , M. & Kotake , N., 1978. Eff icacy of oral

v a c c i n a t i o n for v ibr ios i s in c u l t u r e d ayu. - Bull. Jap . Soc. sc ient . Fish. 44, 21-26 . K u s u d a , R., Sake , H. & Kawa i , K., 1979. C l a s s i f i c a t i o n of v ib r ios i so l a t ed f rom d i s e a s e d f i shes . Par t 1.

O n t h e m o r p h o l o g i c a l a n d b i o c h e m i c a l p roper t ies . - F i sh Pathol . 13, 123-138. K u s u d a , R., Kawai , K. & I tami, T., 1980. Eff icacy of b a t h i m m u n i z a t i o n a g a i n s t v ibr ios i s in cu l t u r ed

a y u (Plecoglossus altivelis). - Bull. Jap . Soc. sc ien t . Fish. 46, 1053. K u s u d a , R., S u g i y a m a , A., Kawai , K., Inada , Y. & Y o n e da , M., 1981. P a t h o g e n i c i t y of Streptococcus

sp. a n d Vibrio anguillarum in c u l t u r e d ayu. - Bull. Jap . Soc. sc ient . Fish. 47, 993-998.

LalI, R., Sen, D., Saha , M. R., Bose, A. K., De, S. P., P a l c h o w d h u r y , N. C. & Pal, S. C., 1979. P r e v a l e n c e of Vlbrio parahaemolyticus in Port-Blair , India . - I n d i a n J. reed. Res. 69, 217-221.

L a n n a n , J. E., 1978. Vibr ios i s v a c c i n a t i o n of c h u m s a l m o n by h y p e r o s m o t i c inf i l t rat ion. - P rogve F ish Cult . 40, 43-45 .

Larsen , J. L., J e n s e n , N. J. & C h r i s t e n s e n , N. D., 1978. W a t e r po l l u t i on a n d t he u l ce r s y n d r o m e in the cod Gadus morhua. - Vet. Sci. C o m m u n . 2, 207-216 .

Lhui l l ie r , M., 1977. Vibrio parahaemolyticus a n d Vibrio alginolyticus i so la t ion , d i a g n o s i s a n d e p i d e m i o l o g y : 74 s t r a in s i so la t ed in M a d a g a s c a r . - Archs Inst. P a s t e u r M a d a g a s c a r 45, 27-50.

Long, S., Moth ibe l i , M. A., Robb, F. T. & Woods , D. R., 1981. R e g u l a t i o n of ex t r ace l lu l a r a lka l ine p r o t e a s e ac t iv i ty b y h i s t i d i n e in a c o l l a g e n o l y t i c Vibrio alginolyticus s t ra in . - J. g en . Microbiol . 127, 193-199.

Love, M., T e e b k e n - F i s h e r , D., Hose , J. E., F a r m e r III, J. J,, H i c k m a n , F. W. & F a n n i n g , G. R., 1981. Vibrlo damsela, a m a r i n e b a c t e r i u m , c a u s e s s k i n u l ce r s on t he d a m s e l f i s h Chromispunctlpinnis. - Sc ience , N. Y. 214, 1139-1140.

M a c M i l l a n , J. R., M u l c h a y , D. & Landol t , M,, 1980. Vira l e ry th rocy t i c necros is . Some phys io log i ca l c o n s e q u e n c e s of in fec t ion in c h u m s a l m o n (Oncorhynchus keta). - Can . J. Fish. aqua t . Sci. 37, 799-804 .

Merke l , J. R. & D r e i s b a c h , J. H., 1978. Pur i f i ca t ion a n d c h a r a c t e r i z a t i o n of a m a r i n e bac te r ia l c o l l a g e n a s e . - B i o c h e m i s t r y 17, 2857-2863 .

M i n c h e w , C. D. & Y a r b r o u g h , J. D., 1977. T h e o c c u r r e n c e of f in rot in m u l l e t (Mugil cephalus)

V i b r i o d i s e a s e s 2 8 5

a s s o c i a t e d w i t h c r u d e oil c o n t a m i n a t i o n of a n e s t u a r i n e p o n d e c o s y s t e m . - J, F i sh Biol. 10, 319-323,

Miyazak i , T., Jo, Y., Kubota , S. S. & E g u s a , S., 1977. H i s t o p a t h o l o g i c a l s t u d i e s on v ib r ios i s of t h e J a p a n e s e eel Anffuilla japonica. Part 1. N a t u r a l in fec t ion . - F i sh Pa thol . 12, 163-170.

M u n n , C. B., 1978. H a e m o l y s i n p r o d u c t i o n b y Vibrio anguillarum. - FEMS Microb ioL Lett. 3, 265-268.

M u r o g a , K., 1979. Ulcer d i s e a s e of a k a m e (Mug i l i dae ) in t he e s t u a r y of t he r iver A s h i d a , J a p a n . - F i sh Pathol . 13, 163-168.

Muroga , K., Jo, Y. & N i s h i b u c h i , M., 1976. Vibrio anguillarum i so la t ed f rom the E u r o p e a n ee l Anguilla anguilla c u l t u r e d in J a p a n . - J. Fac. F i sh A n i m . Husb . H i r o s h i m a Univ. 15, 29-34 .

M u r o g a , K., Y o n e y a m a , N. & Jo, Y., 1979a. Vibr ios ta t ic a g e n t r e s i s t a n t Vibrio anguillarum i so l a t ed from ayu. - F ish Pathol . 13, 159-162.

Muroga , K., T a k a h a s h i , S., Y a m a n o i , H. & N i s h i b u c h i , M., 1979b. N o n c h o l e r a v ibr io Vibrio anguillarum i so la t ed f rom d i s e a s e d a y u Plecoglossus altivelis. - Bull. J ap . Soc. sc ient . Fish. 45, 829-834.

Nair , G. B., A b r a h a m , M. & N a t a r a j a n , R., 1980. D i s t r i bu t ion of Vibrioparahaemolyticusin f in f i sh h a r v e s t e d f rom Porto N o v o (S. India) e n v i r o n s : a s e a s o n a l s tudy . - Can . J. Mic rob ioL 26, 1264-1269.

N a k a j i m a , M. & C h i k a h a t a , H., 1979. Ef f ic iency of oral a n d h y p e r o s m o t i c v a c c i n a t i o n s for v ibr ios i s in a y u (Plecoglossus altivelis). - Fish Pathol . 14, 9-14 .

Na ta ra j an , R., A b r a h a m , M. & Nair , G. B., 1980. D i s t r i bu t ion of Vibrio parahaemolyticus in t he Porto-Novo, India e n v i r o n m e n t . - I n d i a n J. m e d . Res. 71, 679-687 .

N i sh ibuch i , M., M u r o g a , K., Seidler , R. J. & Fryer, J. L., 1979. P a t h o g e n i c Vibrio i so la t ed f rom cu l tu red eels . IV. D e o x y r i b o n u c l e i c ac id s tud ies , - Bull. Jap . Soc. sc ient . Fish. 45, 1469-1473.

N i sh ibuch i , M., M u r o g a , K. & Jo, Y., 1980. P a t h o g e n i c Vibrio i so la t ed f rom c u l t u r e d eels . 6. D iagnos t i c tes t s for t h e d i s e a s e d u e to p r e s e n t b a c t e r i u m . - F i sh Pathol . 14, 125-132.

Novi tsky, J. A. & Mori ta , R. Y., 1976. M o r p h o l o g i c a l c h a r a c t e r i z a t i o n of s m a l l ce l ls r e s u l t i n g f rom n u t r i e n t s t a rva t ion of a p s y c h r o p h i l i c m a r i n e Vibrio. - AppL env i ron . Microbiol . 32, 617-622 .

Oliver , J. D., W a m e r , R. A. & C l e l a n d , D. R., 1983. D i s t r i bu t ion of Vibrio vulnificus a n d o t h e r l a c t o s e - f e r m e n t i n g v ibr ios in t h e m a r i n e e n v i r o n m e n t . - Appl . env i ron . Microbio l . 45, 985-998 .

Ortiz, J. S. & Decker , D. L., 1976. D e m o n s t r a t i o n of a g g l u t i n a t i n g a n t i b o d i e s a g a i n s t Vibrio parahaemolFticus in t h e ye l lowta i l f lounder , Limanda ferruginea. - H e a l t h Lab. Sci. 13, 197-202.

Qadri , R. B. & Zuber i , R., 1977. S u r v e y on t h e o c c u r r e n c e of Vibrio parahaemolyticus a n d Vibrio alginolyticus in f ish a n d she l l f i sh f rom t h e Karachi , P a k i s t a n coas ta l wa te r s . - Pakis t . J. sc ient . ind. Res. 20, 183-187.

Roberson, B. S., 1981, De tec t i on of f i sh a n t i b o d y b y t h i n - l a y e r ELISA. - D e v s biol. S tand . 49, 113-118.

Roberson , B. S., Wolski , S. & Hetr ick , F. M., 1982. Anti-Vibrio anguillarum a n t i b o d y a m o n g s t r i ped b a s s (Morone saxatilis) fo l lowing in j ec t ion w i t h v i a b l e bac te r ia , or a m b i e n t e x p o s u r e to i so l a t ed 0 - a n t i g e n or e n v i r o n m e n t a l vibr ios . - A b s t r a c t s of t h e A n n u a l M e e t i n g of t h e A m e r i c a n Soc ie ty for Mic rob io logy 82, 33 (B91).

Roberts , R. J. (Ed.)., 1982. Mic rob ia l d i s e a s e s of f ish. Acad . Press , N e w York, 305 pp . Rohohm, R. A., Brown, C. & M u r c h e l a n o , R. A., 1979. C o m p a r i s o n of a n t i b o d i e s in m a r i n e f i sh f rom

c l e a n a n d p o l l u t e d w a t e r s of t h e N e w York Bight : Re la t ive l eve l s a g a i n s t 36 bac te r i a . - Appl . envi ron . Microbiol . 38, 248-257 .

Rodsae ther , M. C., O la f sen , J., Rea, J., M y h r e , K. & Steen , J. B., 1977. C o p p e r as a n i n i t i a t i ng factor of v ibr ios is (Vibrio anguillarum) in eel (Anguilla anguilla). - J. F i sh Biol. 10, 17-21.

Ruby, E. G., G r e e n b e r g , E. P. & H a s t i n g s , J. W., 1980. P l a n k t o n i c m a r i n e l u m i n o u s bac te r ia : s p e c i e s d i s t r ibu t ion in the w a t e r c o l u m n . - Appl . env i ron . Microbiol . 39, 302-306 .

Sakazak i , R., I w a n a m i , S. & F u k u m i , H., 1963. S t u d i e s on t h e e n t e r o p a t h o g e n i c f acu l t a t i ve ly h a l o p h i l i c bac ter ia , Vibrio parahaemoIFticus. - Jap . J. reed. Sci. Biol. 15, 161-168.

Sako, H. & K u s u d a , R., 1978. C h e m o t h e r a p e u t i c a l s t u d i e s on t r i m e t h o p r i m a g a i n s t v ibr ios i s of p o n d c u l t u r e d ayu. Par t 1. Mic rob io log ica l e v a l u a t i o n of t r i m e t h o p r i m a n d s u l f o n a m i d e s on t h e c a u s a t i v e agen t , Vibrio anguillarum. - Fish Pa thol . 13, 91-96 .

Sako, H., K u s u d a , R. & T a b a t a , T., 1979. C h e m o t h e r a p e u t i c a l s t u d i e s on t r i m e t h o p r i m a g a i n s t

286 R . R . C o l w e l l & D. J. G r i m e s

vibriosis of pond cultured ayu (Plecoglossus altivelis). 2. Tissue levels after administration of tr imethoprim-sulfadoxine mixture. - Fish Pathol. I4, 65-70.

Sawyer, E. S. & Strout, R. G., 1977. Survival and growth of vaccinated, medicated and untreated coho salmon (Oncorhynchus klsutch] exposed to Vlbrio anguillarum. - Aquaculture I0, 311-315.

Sawyer, E. S., Strout, R. G. & Coutermarsh, B. A., 1979. Comparative susceptibility of Atlantic (Salmo salar) and coho (Oncorhynchus kisutch) salmon to 3 strains of Vibrio anguillarum from the Maine-New Hampshire coast. - J. Fish. Res. Bd Can. 36, 280-282.

Schmidt0 U., Chmel, H. & Cobbs, C., 1979. Vibrio alginolyticus infections in humans. - J. clin. Microbiol. 10, 666--668.

Schiewe, M. H., 1983. Vibrio ordalii as a cause of vibriosis in salmonid fish. In: Bacterial and viral diseases of fish, molecular studies. Ed. by J. H. Crosa. Univ. of Washington, Seattle, 31-40.

Schiewe, M. H. & Crosa, J, H., 1981. Molecular characterization of Vibrio anguillarum biotype 2. - Can. J. Microbiol. 27, 1011-1018.

Schiewe, M. H., Trust, T. J. & Crosa, J. H., 1981. Vlbrlo ordalii sp. nov.: a causative agent of vibriosis in fish. - Curt. Microbiol. 6, 343-348.

Sindermann, C. J., 1970. Principal diseases of marine fish and shellfish. Acad. Press, New York, 369 pp.

Singleton, F. L., Attwell, R., Jangi, S. & Colwell, R. R., 1982. Effects of temperature and salinity on Vlbrio cholerae growth. - Appl. environ. Microbiol. 44, 1047-1058.

Smith, G. C. & Merkel, J. R., 1982. Collagenolytic activity of Vibrio vulnificus: Potential contribu- tion to its invasiveness. - Infect. Immun. 35, 1155-1156.

Snieszko, S. F. & Friddle, S. B,, 1949. Prophylaxis of furunculosis in brook trout (Salvelinus fontinalis) by oral immunization and sulfamerazine. - Progve Fish Cult. I1, 161-168.

Snieszko, S. F., Griffin, P. J. & Friddle, S. B., 1952. Antibiotic treatment of ulcer disease and furunculosis in trout. - T r a n s . N. Am. Wildl. Conf. 17, 197-213.

Strusiewicz, B. & Serban, D., 1980. The enterotoxicity of nonagglut inat ing vibrios. - Rev. Ig. Bacteriol. VirusoL Parazitol. Epidemiol. Pneumoftiziol. (Ser, Bacteriol. Virusol. Parazitol. Epi- dermiol.) 28, 215-218.

Strusiewicz, B., Cvasniuc, D., Florian, V., Cvasniuc, T. & Busca, M., 1980. Nonagglutinat ing vibrios, etiologic agents of diarrheic disease: Their presence and dissemination in the external environment. - Rev. Ig. Bacteriol. Virusol. Parazitol. Epidemiol. PneumoftizioL (Ser. Ig.) 29, 135-140.

Szeness, L, Sey, L. & Szeness, A., 1979. Bacteriological studies of the intestinal content of aquatic birds, fishes, and frogs with special reference to the presence of noncholera vibrios. - Zbl. Bakt. ParasitKde Infektionskr. Hyg. (Abt. 1, Orig., R. A.: Med. Mikrobiol. Parasitol.) 245, 89-95.

Tajima, K., Yoshimizu, M., Ezura, Y. & Kimura, T., 1981. Causative organisms of vibriosis among pen cultured coho salmon (Oncorhynchus kisutch) in Japan. - Bull. Jap. Soc. scient. Fish. 47, 35-42.

Takeji, S., Susumu, I., Shiteru, M. & Akira, G., 1981. Immersion and oral bacterins used in combination to immunize ayu (Plecoglossus altivelis) against vibriosis. - Devs biol. Stand. 49, 461-462.

Tashiro, F., Morikawa, S., Motonishi, A., Sanjo, L, Kimura, N., Inoue, K., Nomura, T., Ushiyama, M. & Jo, Y., 1979. Chemotherapy of fish diseases with piromidic acid. 2. Its clinical studies against bacterial infections in cultured salmonids and eels (Anguilla japonica). - Fish PathoL 14, 93-101.

Tison, D. L., Nishibuchi, M., Greenwood, J. D. & Seidler, R. J., 1982. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. - Appt. environ. Microbiol. 44, 640--646.

Toranzo, A. E., Barja, J. L, Colwell, R. R. & Hetrick, F. M., 1983a. Characterization of plasmids in bacterial fish pathogens. - Infect. Immun. 39, 184-192.

Toranzo, A. E., Barja, J. L, Potter, S. A., Colwell, R. R., Hetrick, F. M. & Crosa, J. H., 1983b. Molecular factors associated with virulence of marine vibrios isolated from striped bass in Chesapeake Bay. - Infect. Immun. 39, 1220--1227.

Toranzo, A. E., Barja, J. L., Colwell, R. R., Hetrick, F. M. & Crosa, J. H., 1983c. Hemagglutinating, haemolytic and cytotoxic activities of Vibrio anguiflarum and related vibrios isolated from striped bass on the Atlantic Coast. - FEMS Microbiol. Lett. 18, 257-262.

Trust, T. J., Courtice, I. D., Khouri, A. G., Crosa, J. H. & Schiewe, M. H., 1981. Serum resistence and hemagglut inat ion ability of marine vibrios pathogenic for fish. - Infect. Imrnun. 34, Y02-707.

V i b r i o d i s e a s e s 287

Watkins, W. D., Wolke, R. E. & Cabelli , V. J., 1981. Pa thogenic i ty of Vibrio anguillarum for juveni le winter f lounder, Pseudopleuronectes amencanus. - Can. J. Fish. aquat . Sci. 38, 1045-1051.

West, P. A. & Colwell, R. R., 1984. Identif icat ion and classif icat ion of Vibr ionaceae - an overview. In: Vibrios in the env i ronment . Ed. by R. R. Colwell . Wiley, N e w York, 285-363.

Williams, S. F. & Caldwell , R. S., 1978. Growth , food convers ion, and survival of 0 g roup Engl i sh sole {Parophrys vetulus] at 5 t e m p e r a t u r e s and 5 rat ions. - Aquacu l tu r e 15, 129--140.

Xu, H.-S., Roberts, N., Singleton, F. L., Attwell, R. W., Grimes, D. J. & Colwell , R. R., 1982. Survival and viabili ty of noncu l tu rab le Escherichia coli and Vibrio cholerae in the es tua r ine and mar ine environment . - Microb. Ecol. 8, 313-323.

Yamanoi , H., Muroga, K. & Takahash i , S., 1980. Physiological character is t ics and pa thogen ic i ty of NAG vibrio isolated from d i seased ayu. - Fish Pathol. 15, 69-73.

Yasunaga , N. & Yamamoto, N., 1977. Character is t ics of bacter ia l s t ra ins isola ted f rom so-cal led vibriosis of cul tured red s e a - b r e a m in the win te r of 1977. - Fish Pathol. 12, 209-214.

Yetinson, T. & Shilo, M., 1979. Seasona l and g e o g r a p h i c d is t r ibut ion of l u m i n o u s bac te r ia in the eas te rn Med i t e r r anean Sea and the Gulf of E l a t - Appl. environ. Microbiol. 37, 1230-1238.

Yoshimizu, M., Kimura, T. & Sakai, M., 1976a. Studies on the intest inal microflora of sa lmonids . Part 2. Effects of artificial t r ansp l an t ing from fresh wa te r into sea wa te r on the intes t inal microflora of feed ing and non feed ing fish. - Bull. Jap. Soc. s c i e n t Fish. 42, 863--873,

Yoshimizu, M., Kimura, T. & Sakai, M., 1976b. Studies on the intest inal microflora of sa lmonids . Part 3. The intest inal microflora of s a l m o n l iving in the o p e n sea. - Bull. Jap. Soc. scient. Fish. 42, 875-884.

Yoshimizu, M., Kimura, T. & Sakai, M., 1976c. S tudies on the intes t inal microflora of sa lmonids . Part 5. The intest inal microflora of the a n a d r o m o u s sa lmon. - Bull. Jap. Soc. s c i e n t Fish. 42, 1291-1298.

vibrio diseases of marine fish populationsvibrios often occur in association with invertebrate and...

Documents