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Research in Microbiology 153 (2002) 1318www.elsevier.com/locate/resmic

Mini-review

Bacteriophage therapy of infectious diseases in aquacultureToshihiro Nakai, Se Chang Park

Laboratory of Fish Pathology, Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima 739-8528, Japan

Received 27 June 2001; accepted 11 September 2001

Abstract

Bacteriophages may be candidates as therapeutic agents in bacterial infections. Here we describe the protective effects of phages agaiexperimentally induced bacterial infections of cultured sh and discuss the potential for phage therapy in aquaculture.2002 ditionsscientiques et mdicales Elsevier SAS. All rights reserved.

Keywords: Bacteriophage; Phage therapy; Fish disease;Lactococcus garvieae ; Pseudomonas plecoglossicida

1. Introduction

Two decades have elapsed since bacteriophages (phages)were reassessed scientically as a therapeutic and prophy-lactic agent for bacterial infections. In the 1980s, followingearly enthusiastic but uncontrolled studies on the applica-tion of phages to prevention and treatment of human bac-terial infections [13,12], epoch-making studies were car-ried out by Smith and colleagues [2528]. They indicated,usingEscherichia coli models with mice and farm animals,that phagescould be used for both treatmentandprophylaxisagainst bacterial infections. Independently of these studies,a series of successful clinical usages of phages for drug-resistant suppurative infections in humans were described byPolish and Soviet groups [1,24]. Thereafter, many success-ful results on phage therapies have been reported using var-ious animal models [2,4,13,23,2931]. Potential advantagesof phage treatment over chemotherapy are: 1) the narrowhost range of phages, indicating that the phages do not harmthe normal intestinal microora; and 2) the self-perpetuatingnature of phages in the presence of susceptible bacteria, in-dicating the superuousness of multiple administrations [3,25]. The latter lead to autonomous transfer of the adminis-tered phages between animals in a yard [4,28].

Cultured sh and shellsh, like other animals and hu-mans, are constantly threatened by microbial attacks. Al-thoughchemotherapy is a rapid and effectivemethod to treator prevent bacterial infections, frequent use of chemother-

* Correspondence and reprints. E-mail address: [email protected] (T. Nakai).

apeutic agents has allowed drug-resistant strains of bacte-ria to develop. In particular, this problem in chemotherapymay be serious in Japan where 25 drugs are now licensedfor sheries use [10]. Needless to say, vaccination is an idealmethod for preventinginfectious diseases, but commerciallyavailable vaccines are still very limited in the aquacultureeld. This is partly due to the fact that many different kindsof infectious diseases occur locally in a variety of sh andshellsh species. Studies on biological control such as pro-biotics have been sporadically reported in the eld of shpathology [6,18,34]; however, they involve substantial dif-culties in scientic demonstration of the causal sequence, asmentioned in human use of probiotics [33]. In view of a sci-entic demonstration of phage treatment, the causal effectof phages in successful phage therapy can be denitivelyproven by conrming an increase in phage particles in thenumber or the presence of phages in the survivors, whichis the result of the death of host bacterial cells. The feasi-bility of this demonstration distinguishes phage treatmentfrom other biological controls, which fail to utilize scien-tic methodologyin demonstratingcausal relationships. Un-der thesecircumstances, phages, as specic pathogenkillers,could be attractive agents for controlling sh bacterial in-fections. Phages of some sh pathogenic bacteria, such as Aeromonas salmonicida , A. hydrophila , Edwardsiella tardaandYersinia ruckeri, have been reported. However, no stud-ies on phages have been made with a view towardpreventingbacterial infections in sh until our recent works [15,21].

In this paper, we briey review our studies on phageeffects against experimentally induced bacterial infectionsof cultured sh, focusing onLactococcus garvieae infec-

0923-2508/02/$ see front matter2002 ditions scientiques et mdicales Elsevier SAS. All rights reserved.PII: S0923-2508(01)01280-3


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14 T. Nakai, S.C. Park / Research in Microbiology 153 (2002) 1318

tion of yellowtailSeliora quinqueradiata andPseudomonas plecoglossicida infection of ayuPlecoglossus altivelis , andwe discuss the potential for controlling bacterial infectionsin aquaculture by means of phages.

2. Phage therapy of Lactococcus garvieae infection

2.1. L. garvieaeinfection

The disease caused byL. garvieae , formerlyEnterococ-cus seriolicida [11], has been responsible for the most seri-ous economic damage to the yellowtail aquaculture indus-try in Japan since its rst outbreak in 1974, mainly due tofrequent occurrences in marketable-sized sh [14]. It is be-lieved thatL. garvieae is a typical opportunistic pathogenbecause the bacterium is ubiquitous in sh and their cul-ture environments. Therefore, reducing stress factors suchas poor water quality, overcrowding, overfeeding, and insuf-cient nutrition is generally important in controlling the dis-ease. However, the difculty in putting these methods intopractice still results in heavy dependenceon chemotherapeu-tics.

2.2. L. garvieaephages

Phages specic toL. garvieae , designated as PLgY andPLgW, were isolated from diseased sh and sea waterin sh culture cages, and the phage was identied as amember of the family Siphoviridae based on morphologicaland genomic features [19,20]. One-hundred-eleven clinicaland environmental strains of L. garvieae were divided into14 phage types (A to N), with the major phage type A,which contains 66% of strains examined; however, 90%or more strains of L. garvieae were sensitive to phageisolates such as PLgW-1 and PLgW-3. This uniformity of L. garvieae in phage sensitivity will be advantageous inphage treatment. The phages appeared extracellularly frominfectedL. garvieae cells after a latent period of 1 h, andthen progeny increased until reaching the maximum numberof 1010 PFUmL 1 after 5 h.L. garvieae grows well at 17 to41 C, but lytic activity of the phage is observed at 29 C or

lower.

Anti- L. garvieae phages survived in unsterilized naturalseawater for at least 3 days and persisted well at variousphysicochemical (temperature: 5 to 37 C; salinity: distilledwater to double-strength seawater; pH: 3.5 to 11.0) and

biological conditions (feed, serum and alimentary tractextracts of yellowtail), except for acidity lower than pH 3.0[15,19]. It seems that resistance to such low acidity is not arequisite for in vivo survival of phage, since the pH levelsof digestive tracts of cultured yellowtails were higher thanpH 3.4 even after feeding. This stability of phages withrespect to environmental factors is of practical value forphage treatment. In vivo, the phage (PLgY-16) was detectedin the spleens of yellowtails up to 24 h after intraperitoneal(i.p.) injection, and the phage was recovered from theintestine of yellowtails 3 h after the oral administration of phage-impregnated feed, but was undetectable 10 h later.

Simultaneous administration of liveL. garvieae and phageenhanced the survival time of the phage; the phage wasrecovered from the spleen 5 days after i.p. injection andfrom the intestine 24 h after oral administration [15]. Therelatively long-term in vivo survival of phage is enough forthe phage to encounter the host bacterium in infected sh.

2.3. Phage therapy

Protective effects of anti- L. garvieae phage were exam-ined by i.p. or oral administration of phage against exper-imentally infected young yellowtails [15]. After i.p. chal-lenge withL. garvieae , the survival rate (100%, n= 20) of sh receiving i.p. injection of the phage was much higherthan that (10%, n= 20) of the control sh without phage injection. When sh were i.p.-injected with phages at differenthours afterL. garvieae challenge, a signicantly higher pro-tective effect (p < 0.01 or< 0.001 in a chi-square test) wasdemonstrated even in sh that received phage treatment 24 hlater (Table 1). In other sh groups, to facilitate phage intro-duction into the sh organs, phage-infected bacterial cells asa source of phage were injected into sh after bacterial chal-lenge. Interestingly, this use of bacterial cells as a protectoror vehicle did not inuence the curative effect of phage (Ta-

ble 1).Table 1Phage treatment of yellowtails infected withL. garvieae a

Administration of: Time afterL. garvieae infection when phage given No. of sh: died/examined Mortality (%) 2

Phage onlyb 0 h 0/ 20 0 p < 0.0011 h 4/ 20 20 p < 0.00124 h 10/ 20 50 p < 0.01None 18/ 20 90

Phage-infected 1 h 1/ 20 5 p < 0.001 L. garvieae c 24 h 11/ 20 55 p < 0.001

None 20/ 20 100a Reproduced from [15].b Fish were i.p. injected with the phage (PlgY-16), immediately (0 h) or 1 h or 24 h after theL. garvieae challenge.c Fish were i.p. injected with previously phage (PlgY-16)-infectedL. garvieae as the source of phage, 1 h or 24 h after theL. garvieae challenge.


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T. Nakai, S.C. Park / Research in Microbiology 153 (2002) 1318 15

Protection was also obtained in yellowtails receivingphage-impregnated feed, and sh were challenged with ananal intubation withL. garvieae. Anal-intubatedL. garvieaewere detected constantly in the spleens of the control sh

for 72 h or longer, while they were detected sporadicallyand disappeared from the phage-treated sh 48 h later. Onthe other hand, orally administered phages were detected inthe intestines and spleens of the phage-treated sh 3 to 48h later, with a maximum of 106 PFUg 1. Phage-resistantmutants are fairly common in in vitroL. garvieae cultures,but allL. garvieae isolates from dead sh obtained duringthe in vivo experiments were still susceptible to the phageused. No neutralizing antibodies were detected in the seraof yellowtails that repeatedly received phage-impregnatedfeed.

3. Phage therapy of Pseudomonas plecoglossicidainfection

3.1. P. plecoglossicida infection

Ayu is the most popular freshwater sh for culture andsports shing in Japan. Bacterial hemorrhagic ascites causedby P. plecoglossicida [17] has been one of the most devas-tating diseases in the ayu culture industry in Japan since theearly 1990s. The disease occurs in sh at any developmen-tal stage throughout the culture period. Some antimicrobialagents, such as orfenicol and sulsozole, are used to treat

coldwater disease caused byFlavobacterium psychrophilum[32], another serious disease for cultured ayu. After suchtreatment, particularly when it is coupled with overfeeding,P. plecoglossicida infection abruptly emerges and results inheavy mortality. This is a typical example of microorganismsubstitution in sh disease. Thus, the causativeP. plecoglos-sicida was believed to be an opportunistic pathogen, thoughan infection experiment by intramuscular injection revealedthat the bacterium is highly virulent to ayu with a LD50of 101.2 CFUsh 1. P. plecoglossicida survives and pro-liferates well in ayu-rearing freshwater, indicating that thebacterium may be ubiquitous in ayu culture environmentsand will cause rapid horizontal transmission of the disease,though the precise infection mechanisms of the disease re-main unsolved. At present, there are no licensed chemother-aputics effective against the disease, and no procedures tocontrol the disease other than reducing predisposing factorssuch as overcrowding and overfeeding.

3.2. P. plecoglossicida and phages

P. plecoglossicida strains are homogeneous with respectto biochemicalcharacteristics, and all isolates obtainedfromgeographically and chronologically different sources aremembersof a singleserotype and a singlephage type [16,21,35]. However, in two previous papers the authors describedconicting results for motility of the bacterium and the

presence of bloody ascites in affected sh; both of thesecharacteristics were positive in onestudy [35], andboth werenegative in the other study [16]. The relationship betweenthemotility of thebacteriumanddifferent clinical conditions

(bloody ascites) in affectedsh remains unclear. Both motileand nonmotile strains are equally virulent to ayu.Two types of bacteriophage specic toP. plecoglossicida

were isolated from diseased ayu and the rearing pond wa-ter. One type of phage (PPpW-3), forming small plaques,was tentatively classied as Myoviridae, and another type(PPpW-4), forminglargeplaques, was classied as Podoviri-dae. All examinedP. plecoglossicida strains, either motileor nonmotile, which were isolated from diseased ayu of geographically different areas from 1991 to 1999, exhib-ited quite similar sensitivity to phages of either type [21].In in vitro conditions, PPpW-4 inhibited the growth of P.

plecoglossicida more effectively than PPpW-3, but the mix-ture of two phages exhibited the highest inhibition. The lyticactivities of phages were observed at temperatures from 10to 30 C or less, which covers the entire range of rearing wa-ter temperature in ayu culture. Interestingly, phage-sensitivestrains of P. plecoglossicida were highly virulent to ayu,while phage-resistant variants of the strain were less virulent(LD50: higher than 104 CFUsh 1). Ultraviolet irradiationor mytomicin C induced no temperate phages from any of the strains examined.

3.3. Phage therapy

Oral administration of phage-impregnated feed to ayu in-creased resistance to experimental infection withP. pleco-glossicida [21]. In the rst trial, sh were orally challengedwith liveP. plecoglossicida -loaded feed and immediately re-ceived phage (PPpW-3/PPpW-4 mixture)-impregnated feed.Mortality in the control sh groups receiving feed withoutphage was initiated at 7 d after the bacterial challenge, andthe cumulative mortality in 2 weeks was 65.0%, while shreceiving phage-impregnated feed immediately after bacte-rial challenge survived to live longer, and there was only22.5% cumulative mortality (Table 2). Such protective ef-fects of phage treatment, signicantly (p < 0.001) decreas-ing mortalities, were demonstrated in sh receiving phage 1h or 24 h after bacterial challenge. InoculatedP. plecoglossi-cida was isolated from all the kidneys of dead sh irrespec-tive of phage treatment and from some survivors in controlgroups, but not from any of the sh that had received phagesand survived. In addition, the bacteria isolated from shthat had received phage treatment and died were still sus-ceptible to both phages used. Although PPpW-4 producedhigher protection than did PPpW-3 in the second trial withsingle use of each phage, the mixture of both phages ex-hibited the highest protective effect (Table 2). When phagetherapy was evaluated under cohabitation conditions withsh which had been previously infected withP. plecoglos-sicida , phage (PPpW-3/PPpW-4)-receiving sh showed sig-


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Table 2Phage treatment of ayu infected withP. plecoglossicida a

Experiment no. Phage used Time afterP. plecoglossicida No. of sh: Mortality (%) 2infection when phage given died/examined

1b

PPpW-3+ PPpW-4 0 h 8/ 40 22.5 p < 0.001None 26/ 40 65.0PPpW-3+ PPpW-4 1 h 0/ 50 0.0 p < 0.001

None 39/ 50 78.0PPpW-3+ PPpW-4 24 h 5/ 40 12.5 p < 0.001

None 32/ 40 80.0

2b PPpW-3 0 h 16/ 30 53.3 p < 0.001PPpW-4 0 h 12/ 30 40.4 p < 0.001

PPpW-3+ PPpW-4 0 h 6/ 30 20.2 p < 0.001None 28/ 30 93.3

3c PPpW-3+ PPpW-4 24 h & 72 h 8/ 30 26.7 p < 0.001None 30/ 30 100

4c PPpW-3+ PPpW-4 24 h & 72 h 8/ 30 26.7 p < 0.001None 27/ 30 90.0

a Reproduced in part from [21].b Fish were challenged by oral administration of P. plecoglossicida -impregnated feed and immediately (0 h) or 1 h or 24 h later received phage-impregnated

feed.c Fish were challenged by cohabitation with previously infected sh with intramuscular-injection of P. plecoglossicida , and 24 h and 72 h later sh received

phage-impregnated feed or phage-free feed (control).

Table 3Growth dynamics of P. plecoglossicida and phage administered to ayua

Time after inoculation (h) P. plecoglossicida (log10 CFUg 1) in sh fed: Phage count (log10 PFUg 1) in sh fed:

bacteria alone bacteria and phage phage alone bacteria and phage0 < 2 < 2 < 2 < 23 3.5 4.8 5.2 4.0

12 4.7 < 2 3.5 3.624 5.1 < 2 < 2 3.548 4.6 < 2 < 2 < 272 4.2 < 2 < 2 < 2

a Modied from [21].Fish were fedP. plecoglossicida -impregnated feed, feed impregnated with phages (PPpW-3+ PPpW-4), or phage-impregnated feed after bacterial challenge.

nicantly lower (p < 0.001)mortality than untreatedcontrolsh (Table 2).

P. plecoglossicida in sh receiving bacteria-loaded feedrst appeared in the kidney 3 h after feeding, and thenwere detected at levels of 106.3 and 103.9 CFUg 1 fromall kidneys examined 72 h or later. In sh receiving phage-loaded feed, inoculated phage emerged at concentrationsof 105.2 and 103.5 PFUg 1 in kidneys 3 and 12 h laterrespectively, but disappeared 24 h later. On the other hand,when sh received phage-impregnated feed after bacterialchallenge,P. plecoglossicida were not detected in kidneysof sh at 12 h or later, after a slight appearance at 3 hpostchallenge, and phage was detected from the kidneysat 3, 12 and 24 h postchallenge (Table 3). These growthdynamics of administeredbacteria andphages in sh explainthat phage killing of bacteria in the internal organ causedsurvival of sh.

4. Potential for phage control in aquaculture

As stated above, rediscoveryof phage therapy beganwithSmith and his colleagues works using various animal mod-els of cattle and human diseases. Our phage therapy studiesfor aquatic animals are fairly well under way but its efcacyhas been demonstrated against experimentally induced in-fections [15,21]. In our very recent eld trials of phage ther-apy, we succeeded in markedly reducing the mortality of ayudue toP. plecoglossus infection. When phage-impregnatedfeed was administered to ayu in a diseased pond (200 m3,sh no.= 120,000), daily mortality decreased at a constantlevel (5% a day) and phage therapy lowered mortality to one-third during the two-week period. Results obtained so far in-dicate the potential for phage control of bacterial diseasesin aquaculture. In particular, successful phage treatments byoral administration, as demonstrated in both sh models, are


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T. Nakai, S.C. Park / Research in Microbiology 153 (2002) 1318 17

of practical value as a route for therapeutic administration of phages toa large numberof sh. Acidsensitivity of phages isnot a determinant for phage treatment due to relatively highpH levels in sh digestive tracts as shown in yellowtail. Such

a high pH of the stomach and abomasum contents was alsonoticed in very young animals, and the abomasum at this pHlevel has little harmful effect on orally administered phages[28]. This route of phage administration will be directly ad-vantageous for cases in which the oral route is the majorroute for pathogen transmission, as areL. garvieae infectionof yellowtail andP. plecoglossicida infection of ayu. Inter-estingly, orally administered phages quickly appeared in thekidneys of sh, without host bacterial cells as a transport ve-hicle. Easy movement of phage from the alimentary tract tothe blood circulation system was also observed in humansand rats [8,24]. It has already been noted that phages arenot as rapidly inactivated in animal tissue uids and bloodas previously thought [2,25]. A technical problem which re-mains is how to select or nd the most aggressive strain toenhance the therapeutic effect. Phages with high activity invitro are more active in vivo [25], as was ourP. plecoglos-sicida phage. Successive in vivo passages will be an alter-nate method to obtain long-term in vivo survival phage mu-tants [13].

There is also an indication that phages can invade thesh body through skin and/or gills, since phages are easilydemonstrable in the kidney after dipping sh in phagesolution. Bath administration of phages will be effective forthose in which infection is initiated by bacterial colonization

on the skin and gills. Cold water disease caused byF. psychrophilum is the typical example of this infectiontype. Yet there are no reports on phages of this world-important pathogen. In addition to these diseases, otherbacterial infections of sh will be suitable for phage therapy.Furthermore, our preliminary studies suggest that phagetreatment is useful for controllingVibrio infection of PacicoysterCrassostrea gigas larvae. Thus, phage therapy mayhave many applications in the aquaculture eld.

Although many previous review papers have pointed outand addressed a number of intrinsic obstacles to phagetreatment and prevention for terrestrial animals [1,3,12],there still remain some issues to be addressed for phageapplication in aquaculture. The supposed obstacles are asfollows:

First, the narrow host specicity of phages is a disad-vantage for phage therapy. It is strain-specic rather thanspecies-specic, which leads to difculty when preparingphages of highly diverse bacterial variants. Our aquatic ani-mal models, however, do not indicate that this is always anessential weak pointof phage therapy.Theremay exist a major phage type inL. garvieae. Some phage isolates are sobroad in their infectivity that they can be lytic for 90% ormore strains of the organism [19]. For other bacteria such asP. plecoglossicida, only a single phage type is known [21].A specic cell surface substance as a virulence factor, suchas the capsule of E. coli orof L. garvieae , may determine this

low diversity of the organism [2]. Such a surface substance,however, has not been identied inP. plecoglossicida .

Second, rapid appearance of phage-resistant bacteria is aproblem for treatment, as in chemotherapy. Phage-resistant

mutants are fairly common inL. garvieae and P. plecoglos-sicida cultures. However, allL. garvieae and P. plecoglos-sicida isolates from dead sh obtained during therapy ex-periments were still susceptible to phages used for treat-ment. Furthermore, phage-resistant variants of P. plecoglos-sicida , which were induced in vitro, lacked virulence forayu [21]. In successful phage control against a systemic in-fection of mice withE. coli (O18:K1:H7 ColV+ ) or diar-rhea in calves by enteropathogenicE. coli (O9:K30,99 andO20:K101,987P),only the less virulent K types emerged asphage-resistant organisms [25,26]. A surface component as-sociated with bacterialvirulencealso seemedto be therecep-tor for phage attachment, and consequently phage-resistantvariants of a virulent organism would not be pathogenic asstated in previous papers [2,3,21].

Third, phage-neutralizing antibodies produced as the re-sult of phage administration, either by the oral or parenteralroute, will be an obstacle for phage therapy against recurrentinfections. EnteropathogenicE. coli phage-neutralizing an-tibodies were found in the sera of human beings, cattle andpigs, and in bovine colostrum. The neutralizing antibodieswere induced by orally administered phage or its parenteralinoculation at much higher levels of antibody [28]. However,no such neutralizing antibodies were detected from yellow-tail and ayu that repeatedly (successive seven days) received

phage-impregnated feed (about 107 PFUsh

1) or evenfrom ayu after receiving intramuscular injections (4 times,weekly) of phage solution at a dosage of 109 PFUsh 1.Conversely, this low immunogenicity of phage to sh mightprovide an advantage for phage therapy in sh.

Lastly, the risk that phages might mediate genetic ex-change among bacteria, i.e. transduction or phage conver-sion, may be the nal problem raised. It is well knownthat some temperatephages contributeto bacterial virulence.Temperate phages with broad infectivity over species wouldstrongly support antiphage therapy views [9]. However, thispossibility is probably unlikely for our therapeutic phagesbecause of their extremely narrow host specicity.

5. A hypothetical role of phages in nature

The high abundance of viruses in aquatic environmentsindicates that phage infection is an important factor in theecological control of planktonic microorganisms [5,22] andmany other biogeochemical processes [7]. Through phagetherapy studies using sh models, however, we postulatethat naturally occurring phages might contribute not onlyto the killing of such planktonic bacteria but also to thekilling of pathogens proliferating in the internal body of shwhere there exists a range of defense factors against invad-ing microorganisms. This idea was derived from the follow-


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ing facts: 1) lytic phages are frequently isolated from rear-ing water where a specic bacterial infection prevails amongsh; 2) phages appear in the internal organs (ex. kidney) im-mediately after oral or water-borne administration of phages

to sh, and can survive for a relatively long time in situ; and3) phage-neutralizing antibodies are not easily produced bysh under either experimental or natural conditions. In or-der to demonstrate this hypothesis, we examined live shin ayu culture ponds in whichP. plecoglossicida infectionprevailed.P. plecoglossicida phages at high concentrations(maximum: 103 PFUg 1) were detected in the kidneys of apparently healthy sh at an incidence of 2.8% (n= 534).There is no doubt that the presence of phages at high num-bers in live sh results from in vivo bacterial killing of phage. Therefore, naturally occurring phages must at leastpartly contribute to the survival of sh after otherwise fatalbacterial infection.

Acknowledgements

This work was partlysupported by a grant-in-aid fromtheMinistry of Education, Science, Sports and Culture, Japan(No. 12660171).

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17. c6. phagetherapy-r

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