1991, 57(12):3470. Appl. Environ. Microbiol. 

Henry N. WilliamsAndrew J. Schoeffield, William A. Falkler Jr., Darshana Desai and Immunodiffusion and ImmunoelectrophoresisChesapeake Bay and Environs by Serogrouping of Halophilic Bdellovibrios from

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Vol. 57, No. 12

Serogrouping of Halophilic Bdellovibrios from Chesapeake Bay andEnvirons by Immunodiffusion and Immunoelectrophoresis

ANDREW J. SCHOEFFIELD,l WILLIAM A. FALKLER, JR. 2* DARSHANA DESAI 2AND HENRY N. WILLIAMS2

Biology Department, Loyola College, Baltimore, Maryland 21210,1 and Department of Microbiology,University of Maryland Dental School, 666 West Baltimore Street, Baltimore, Maryland 212012

Received 14 March 1991/Accepted 25 September 1991

Little has been reported on the serological relationship of halophilic bdellovibrios (Bd). Immunodiffusionanalysis performed with rabbit or mouse Bd antisera developed against eight halophilic Bd isolates and oneterrestrial Bd isolate, when reacted with soluble antigen preparations of 45 isolates of halophilic Bd, allowedseparation into seven serogroups, which were distinct from the terrestrial isolate. Soluble antigen preparationsof prey bacteria, Vibrio parahaemolyticus P-5 (P-5) and Escherichia coli ML 35 (ML 35), exhibited no reactivitywith the antisera by immunodiffusion. Immunoelectrophoresis revealed the presence of three distinct antigensin homologous reactions and one shared antigen in heterologous Bd reactions. Shared antigens were notedbetween halophilic and terrestrial Bd, in addition to between halophilic Bd strains, indicating the possibleexistence of an antigen(s) which may be shared among all Bd. Again, no shared antigen was noted when P-5or ML 35 was allowed by immunoelectrophoresis to react with the antisera. Prey susceptibility testing of theseven distinct groups of halophilic Bd, using 20 test prey, produced essentially identical spectra for each group,indicating that this was not a useful technique in delineating the Bd. While immunoelectrophoresis was able todemonstrate an antigen common to all Bd tested, immunodiffusion was able to delineate strains on the basis ofa "serogroup specific" antigen. This suggests that immunological tools may serve as important means to studythe taxonomy of halophilic Bd, as well as in the formation of a clearer taxonomic picture of the genusBdellovibrio.

The genus Bdellovibrio consists of a group of diversegram-negative, bacteriolytic organisms which prey on vari-ous gram-negative bacteria (18). The use of predation and acommon life cycle as primary criteria to classify unknownorganisms as Bdellovibrio has resulted in a very heteroge-neous group of organisms.On the basis of a requirement for sodium chloride, mem-

bers of the genus Bdellovibrio have been divided into twogroups: halophilic, or salt requiring; and terrestrial (nonhalo-philic) members which have no salt requirement. Othermethods which have been used to characterize these groupsfurther include guanine-plus-cytosine content (11, 14, 15),prey susceptibility and optimum salinity or temperaturerequirements (7, 16), DNA ribosomal binding sites, enzymemigration patterns and levels of protease activity (14), andsusceptibility to bacteriophage lysis (1). Either these meth-ods are not suitable for routine use in most laboratories,have not been standardized, or are not adaptable to stan-dardization, or they do not allow a routine convenientmethod for distinguishing between subgroups. Biochemicaltests are not reliable since it may be difficult to distinguishbetween reactions caused by contaminating prey metabolicenzymes and reactions of bdellovibrios (Bd). Little is knownof the nutritional and metabolic characteristics of the Bd,and these characteristics are not useful taxonomically (10).The result has been little or no progress in defining sub-groups of Bd and little upon which to base taxonomic statusand appropriate classification.

Immunological characterization has been widely acceptedas useful in determining relationships among groups ofbacteria and may be an important tool in helping to better

* Corresponding author.

define the subgroups of the Bd. Immunological studiesinvolving the terrestrial Bd have resulted in several serolog-ically distinct subgroups which may share a common antigen(2, 5, 10). Because little is known regarding the number ofdifferent halophilic subgroups of Bd, and no immunologiccharacterization of the halophilic Bd has ever been reportedin the literature, a study of the serological relationshipsamong various halophilic strains of Bd was undertaken.Subgroups based on the results of this immunologic studyand the methods developed herein may begin to contributeto the taxonomy, classification, ability to study and monitorBd in the environment, and, ultimately, the formation of amore unified concept of the genus Bdellovibrio.

MATERIALS AND METHODS

Sources of Bd. Halophilic Bd were isolated from differentareas of the Chesapeake Bay and some of its subestuaries, aChesapeake Bay tidal pond, tank 20 at the National Aquar-ium in Baltimore, Md., and the Atlantic Ocean. Water andsediment were sampled as described previously (19) andcultured for halophilic Bd, using Pp2O medium and thedouble-agar overlay technique, with Vibrio parahaemolyti-cus as prey (12, 19-21). The fouled biomass of oyster shellswas cultured for halophilic Bd. Oysters were collected bydredging at various sites. The oyster shell epifauna wasscraped from viable oysters with an alcohol-flamed spatulaand removed into 50-ml centrifuge tubes containing 20 ml ofsterile seawater until the biomass displaced 5 ml. Thecontents of the tubes were then vortexed at high speed for 1

min and centrifuged at approximately 600 x g for 10 min.The resulting supernatant fluids were then cultured for Bd. Aterrestrial strain W was obtained from John Tudor, SaintJoseph's University, Philadelphia, Pa. A list of the isolates

3470

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1991, p. 3470-34750099-2240/91/123470-06$02.00/0Copyright C 1991, American Society for Microbiology

SEROGROUPING OF BDELLOVIBRIOS 3471

TABLE 1. Bdellovibrio isolates used throughout this study

Strain . . Source ofdesignation Site of isolation isolation

SJ St. John's, U.S. Virgin Islands Water845 Chesapeake Bay, station 845 F WaterPD1 Horn Point Tidal Pond no. 2, Water

Cambridge, Md.AQ National Aquarium in Balti- Water

more, tank 20, Baltimore,Md.

W John Tudor, St.Joseph's Uni-versity, Phil-adelphia, Pa.

KS1-KS10 National Aquarium in Balti- Watermore, tank 20, Baltimore,Md.

OCi-OC1O Atlantic Ocean Water

VH1-VH10 Horn Point Tidal Pond no. 2, WaterCambridge, Md.

JS1 Patuxent River, Helen's Bar WaterJS2 Choptank River WaterJS3 Magothy River WaterJS4 Choptank River SedimentJS5 Patuxent River Crab gillJS6 Patuxent River, Helen's Bar Oyster shellJS7 Little Annemessey River WaterJS8 Severn River, Tolley Point Oyster shellJS9 Patuxent River, Hog Island Oyster shellJS10 Mid-Chesapeake Bay, station Water

856-D

of Bd, as well as the sites and sources from which they were

recovered, are given in Table 1.Antigen preparations of Bd. Isolated plaques of halophilic

Bd recovered from the various sources were transferred into5 ml of sterile 70% seawater, vortexed for 30 s, and filteredthrough a 0.2-,um membrane filter (Millipore Corp., Bedford,Ma.). The filtrate was then added to 20 ml of sterile seawatercontaining 2.4 x 108 cells of V. parahaemolyticus (PSB) perml (8). After clearing of the broth (approximately 3 to 4 days,25°C), the culture was added to 1,000 ml of PSB andincubated until clearing. The culture was then centrifuged at1,500 x g for 20 min to partially remove unlysed prey cells.The supernatant fluid was then filtered through a 0.3-,umMillipore filter. In trials conducted in this laboratory, 0.3-,umMillipore filters allowed for maximum passage of halophilicBd with minimum prey cell contamination. For terrestrial Bdstrain W, 0.4-,um Nuclepore filters (Nuclepore Corp., Pleas-anton, Calif.) gave optimal results. The filtrate was thencentrifuged at 27,000 x g for 60 min to pellet the Bd. Thepellet was then resuspended in sterile 0.01 M phosphate-buffered 0.15 M NaCl (PBS), and the organisms werewashed twice in PBS.The terrestrial strain W was grown in dual culture with

Escherichia coli ML 35 as the prey. Dual cultures wereincubated at 30°C with shaking for 3 to 4 days. Followingincubation, the cultures were purified as described for thehalophilic Bd; however, the terrestrial strains were filteredthrough a 0.4-l,m Nuclepore filter to separate the Bd fromtheir prey. The filtrate was centrifuged, resuspended, andwashed as for the halophilic isolates.

The washed, pelleted Bd were resuspended in sterile PBSat a concentration of 109 cells per ml as determined by theacridine orange direct count method (3). Suspensions of Bdwere then divided in half. Half of each suspension (whole-cell preparation) was frozen in 1-ml volumes at -20°C untilneeded. The other half of each suspension was placedthrough a model B Carver Laboratory Press (Fred S. Carver,Inc., Summit, N.J.) and is referred to as the soluble antigenpreparation (SAP). The protein content of the SAP wasdetermined by the method of Lowry et al. (6), and thepreparation was frozen in 1-ml volumes at -20°C untilneeded.

Preparation of rabbit antisera to Bd. Rabbit antisera wereprepared by using whole-cell preparations of halophilicstrains SJ, 845, PD1, and AQ and terrestrial strain W.Female New Zealand White rabbits, weighing 3 to 4 kg, wereimmunized. Before immunization, each of the rabbits wasbled via the marginal ear vein to obtain nonimmune serum.Each was injected subcutaneously in multiple sites (0.25 mlper site) with a 1-ml emulsion containing 0.5 ml of Freundcomplete adjuvant (Difco Laboratories, Detroit, Mich.) and0.5 ml of the whole-cell preparation. These injections werefollowed by six intravenous injections given at weeklyintervals. After 8 weeks, all rabbits were bled via the centralear artery, using an I.V. Catheter Placement Unit (Critikon,Inc., Tampa, Fla.). The blood was kept at room temperaturefor 2 h and then overnight at 4°C. The sera were separatedfrom the clot by centrifugation at 800 x g for 20 min.

Preparation of mouse antisera. Mouse antisera to Bd wereprepared from whole-cell preparations of halophilic strainsJS2, JS4, JS5, and JS6. These strains were all isolated fromdifferent areas of the Chesapeake Bay. For each strain, sixSwiss-Webster mice were immunized. Each was immunizedintraperitoneally with four 0.5-ml injections of whole-cellpreparations, given at weekly intervals. After 5 weeks, allmice were bled via decapitation. Six nonimmunized micewere also decapitated to obtain nonimmune serum. The serawere separated and collected as for the rabbit antisera.

Adsorption of antisera to Bd with prey. The rabbit antiserawere each adsorbed with a 5% (packed cell volume/totalvolume) whole-cell suspension of the corresponding prey (V.parahaemolyticus for antihalophilic strain sera, E. coli forthe antiterrestrial serum). To pelleted prey cells was added 1ml of each antiserum, and after mixing and incubation at37°C for 30 min, the suspension was centrifuged at 12,800 xg for 10 min in an Eppendorf 5412 centrifuge (Eppendorf,Hamburg, Germany) and the supernatant fluid was removed.The supernatant fluid was then similarly adsorbed five moretimes with prey cells with the exception that the last adsorp-tion procedure was at 37°C for 30 min and was followed byincubation overnight at 4°C prior to centrifugation of thesuspension. The adsorbed antisera were used in all serolog-ical studies.Immunodiffusion (ID). Double diffusion in agar was per-

formed by the method of Ouchterlony (9). Agarose gels (1%)were prepared on glass slides (4 ml per slide), and 2-mm-diameter wells were punched in the agarose with a GelmanImmunodiffusion Punch (Gelman Instrument Co., Ann Ar-bor, Mich.) to a depth of 2 mm and a center-to-centerdistance of 4 mm. The SAP of the strains of Bd, as well assoluble prey cell preparations at 240 to 260 ,ug of protein perml, were placed in the peripheral wells against homologousand heterologous antisera undiluted in the center wells. Theslides were then incubated in a humidor at 4°C for 72 h andthen at 25°C overnight. Positive reactions in the form of

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3472 SCHOEFFIELD ET AL.

precipitation lines were recorded upon observation the fol-lowing morning.

IE. Immunoelectrophoresis (IE) was performed with glassslides containing 4 ml of 1% agarose in 0.05 M Tris-boratebuffer, pH 8.6. A template of a center trough and twosurrounding wells was punched with an electrophoresistemplate cutting device (Shandon Scientific Co., London,England). After removal of the agarose plugs, the SAP of Bdand prey were placed in the wells, which were electro-phoresed at 120 V for 5 h in a Gelman Deluxe Electropho-resis Chamber (Gelman Instrument Co.) containing approx-imately 1 liter of Tris-borate buffer. After electrophoresis,the gel in the center trough was removed and the trough wasfilled with the appropriate rabbit antisera. The slides werethen incubated as for the ID slides. All slides were stainedwith amido black 10B.

Prey range of halophilic Bd. To determine whether theprey range of halophilic Bd may also be an important tool indelineating strains of the predator, the following 20 bacteriawere tested for their susceptibility to eight halophilic isolateswhich had previously been separated (by ID) into sevendistinct serogroups: Vibrio cholerae, Vibrio sp., V. camp-bellii, V. mimicus, V. damsela, V. diazotrophicus, V. aes-turianus, V. vulnificus, Alteromonas colwelliana D, A. col-welliana W, Alcaligenes sp., Pseudomonas fluorescens, V.fluvialis, Achromobacter xylosoxydans, Salmonella arizo-niae, P. marina, V. ordalii, E. coli 5, E. coli 45, Pseudomo-nas sp.Twenty-four-hour PSB cultures of isolates SJ, 845, PD1,

AQ, JS2, JS4, JS5, and JS6 were prepared and individuallyfiltered through a 0.45-,um filter unit (Nalgene). Counts of Bdwere then adjusted to 107 per ml by acridine orange directcount. Each test bacterium was inoculated into a Pp2O topagar tube, overlaid onto bottom agar in petri dishes (150 by15 mm), and allowed to harden. Pp20 plates were dividedinto eight quadrants, to which 10 ,ul of each of the isolates ofBd was spotted. Plates were incubated at 25°C uninvertedfor 24 h to allow the Bd spot to absorb into the top agar.Plates were then inverted and incubation was continued for5 days.

RESULTS

ID. Forty-five soluble antigen preparations of Bd werescreened, using five rabbit antisera to Bd. This was per-formed to determine the existence of serologically distinctserogroups of Bd.

Lines of precipitation in proximity to the antigen wellswere only observed when antisera to Bd were reacted withtheir homologous SAP (Fig. 1). No band of precipitation wasobserved when nonimmune sera were reacted with homolo-gous or heterologous SAP. These results revealed that atleast four serogroups of halophilic Bd were present, whichwere distinct, by ID, from each other and from terrestrialisolate W.

It was then of interest to know whether these four sero-groups constituted a majority or a minority of those actuallyin existence or whether serogroups were geographicallydistributed. To examine this, 10 isolates of Bd were obtainedfrom each of the ecosystems from which the four serogroupswere isolated: the Atlantic Ocean, the Chesapeake Bay, abay tidal pond, and tank 20 at the National Aquarium inBaltimore, Md. SAP of these 40 isolates were then screenedby ID, using the five rabbit antisera. None of the isolatesobtained from the Atlantic Ocean, the Chesapeake Bay, orthe tidal pond reacted with any of the five rabbit halophilic

FIG. 1. Unstained ID gel of anti-SJ serum (center well) whenallowed to react with soluble antigen preparations of strains (1) SJ,(2) 845, (3) PD1, (4) AQ, (5) W, and (6) P-S.

strain antisera. By contrast, all 10 of the isolates obtainedfrom the aquarium tank formed lines of precipitation withrabbit anti-AQ serum. In addition, the results of theseinteractions demonstrated lines of identity (Fig. 2) among theaquarium isolates.Mouse antisera to Bd were then raised against four of the

bay isolates which had tested negative against rabbit anti-sera: JS2, JS4, JS5, and JS6. This was done to further clarifythe question of the possible number of halophilic serogroupsof Bd which may exist within a particular ecosystem andwhether this information can be of value in the classificationof the predators. Precipitation lines similar to those alreadyobserved were noted when homologous antigen and antiserawere reacted by ID. No line of precipitation was observedwith nonimmune sera. Reaction of the four mouse antiserawith heterologous antigen preparations of bay isolates dem-onstrated lines of identity when anti-JS2 serum was reactedwith JS6 and, conversely, when anti-JS6 serum was reactedwith JS2. No other heterologous antisera reacted with bayisolates (Table 2). No lines of precipitation formed when thefour mouse antisera were allowed to react with ocean, pond,and aquarium isolates. ID utilizing rabbit and mouse antiserahas, therefore, demonstrated at least seven serotypes ofhalophilic Bd.

IE. IE was performed on the five strains of Bd which hadpreviously been used to immunize rabbits. This was done inan effort to separate antigens of the Bd and to observeantigens common to various strains of the predator. Allcombinations of antisera and SAP were tested.Homologous reactions revealed the presence of two to

three electrophoretically distinct antigen bands (Fig. 3 and4). Heterologous reactions showed at least one antigencommon to all strains tested. No common antigens wereobserved in P-5 or ML 35. Cross-reactivity was demon-strated between halophilic isolates (Fig. 3), as well asbetween halophilic isolates and terrestrial isolate W (Fig. 4).

Prey range of serologically distinct isolates of Bd. The preyrange spectra of seven serologically distinct groups of Bd,using 20 test bacteria, were obtained to determine whether

APPL. ENVIRON. MICROBIOL.

SEROGROUPING OF BDELLOVIBRIOS 3473

TABLE 2. ID results of mouse antisera to Bd when allowed toreact with Chesapeake Bay halophilic strain antigen preparations

Antigen Result with antiserum:prepn JS2 JS4 JS5 JS6

JS2 + _ _ +JS4 _ + - _JS5 - - + _JS6 + - - +845 - - - -

JS1 - - - -

JS3 - - - -JS7 - - - -

JS8JS9 - - - -

Js10 - - - -

FIG. 2. Unstained ID gel of anti-AQ serum (center wells) whenallowed to react with soluble antigen preparations of aquariumisolates (1) AQ, (2-6, 8-12) KS1-10, and (7) P-5.

this might be a useful technique in delineating strains of thepredator. Prey range spectra of the eight isolates of Bd wereessentially identical. Of the 20 bacteria tested, only Alcali-genes sp. did not serve as prey for the eight halophilic Bdtested. JS5 was the only isolate able to prey on S. arizoniae.

DISCUSSION

The present classification scheme utilized for Bd does nottake into consideration immunological similarities and/ordifferences which may exist in these organisms and hasresulted in a very heterogeneous assemblage of organisms.Immunological characterization has been widely accepted asuseful in determining relationships among groups of bacte-ria. Immunological parameters alone, or in combination withother techniques, may be useful tools in the taxonomy ofhalophilic Bd. Nonhalophilic Bd have been separated intoseveral serologically distinct subgroups which may share a

common antigen (2, 5, 10). Little is known regarding thenumber of different halophilic subgroups of Bd.

Forty-five antigen preparations of Bd were screened,using rabbit antisera prepared to five halophilic isolates. Allfive of the antisera represented individual serogroups of Bd.None of the antisera reacted with any antigen preparationsof Bd: 10 from the Atlantic Ocean, 10 from the ChesapeakeBay, and 10 from a bay tidal pond. However, all 10 antigenpreparations of Bd from tank 20 at the National Aquarium inBaltimore cross-reacted with anti-AQ, developed from anisolate of that tank. Because of the lower than expectedamount of cross-reactivity, mouse antisera were developedagainst four additional Chesapeake Bay isolates of Bd.Cross-reactivity was noted between just two isolates.

Results of ID indicated that at least seven serologicallydistinct groups of halophilic Bd can be delineated. Becausethe majority of antigen preparations of the 45 collected Bdisolates failed to react with any of the eight antihalophilic orone antiterrestrial sera, we can only conclude that there mayexist many more serogroups which await characterization.These results indicated, however, that ID may be a quickand reliable method to group these predators serologically.The serologic heterogeneity of the halophilic Bd is manifestby these findings. This is consistent with findings that theremay exist several serologic groups of nonhalopililic Bd (4,10) and that one nonhalophilic species alone (B. bacterio-vorus) may contain as many as nine distinct serogroups. Ofspecial interest was the finding that all 10 aquarium antigen

FIG. 3. Stained IE gel of anti-SJ serum (trough) when allowed toreact with soluble antigen preparation of strains (1) 845 and (2) SJ.Arrowheads indicate bands of precipitation.

VOL. 57, 1991

3474 SCHOEFFIELD ET AL.

FIG. 4. Stained IE gel of anti-845 serum (trough) when allowedto react with soluble antigen preparations of strains (1) 845 and (2)W. Arrows indicate bands of precipitation.

preparations of Bd cross-reacted with antisera producedagainst an aquarium isolate. Possibly, the closed system ofthe aquarium tank selected for a single serologic type. Itshould be noted that the tank received 25% water changesand additions to the tank community at periodic intervals,yet these factors did not seem to affect the indigenous(homogeneous) population of Bd. The isolate to which theantiserum was raised was collected approximately 3 yearsprior to the isolation of the 10 additional strains. Thisrepresents an extended period of time in which the serotypeof the tank remained, essentially, unchanged. The factorsselecting for one particular strain of the predator over othersare unknown at this time. This was in sharp contrast to allother data collected. Halophilic Bd collected from variousnatural ecosystems may consist of several to many serolog-ically distinct groups.

In a previous study (4), halophilic and nonhalophilicisolates were observed to be distinct by ID. However, in thisstudy, using a more sensitive technique (IE), halophilic andnonhalophilic Bd revealed in homologous systems the pres-ence of two to three electrophoretically distinct antigenbands, while heterologous reactions demonstrated at leastone antigen common to all five strains tested. These commonantigens were shared between halophilic and nonhalophilicstrains, as well as between halophilic strains. This indicatesthe possible existence of an antigen(s) which may be sharedamong all Bd.The prey range of halophilic Bd was determined to see

whether this may be a useful technique in separating strainsof the predator. Each of the seven distinct serogroupsresulted in identical spectra of prey, with the exception ofone isolate which deviated from the others solely by itsability to prey on one additional prey. Seidler and Mandel(13) noted that B. starrii could not lyse enterobacteria insolid culture; however, Torrella et al. (17) demonstrated nosuch distinctive prey-range spectrum for 11 other nonhalo-philic isolates. On the basis of prey range, Marbach et al. (7)were able to separate halophilic Bd into 10 groups andTaylor et al. (16) were able to separate 13 isolates into threegroups. These data are in sharp contrast to those presentedin this report, which indicates that prey range may not be a

useful technique in delineating species. Prey range was notable to further divide determined serogroups of the predator.In addition, it has already been shown that culture mediumcomposition may drastically affect the ability of an isolate todevelop pla .ue on a given prey (7, 16); therefore, prey rangestudies may not be amenable to standardization.

Although halophilic Bd appear to be a serologically heter-ogeneous assemblage of microorganisms, capable of utilizingmany different species as prey, IE was able to identify anantigen of Bd common to the halophilic and nonhalophilicisolates, but not found in prey. ID was able to delineatestrains on the basis of a "serogroup specific" antigen. Theseresults suggest that such immunological tools may well serveas important tools in the taxonomy of halophilic Bd. Futureserologic studies may provide both insight into the diversityof the halophilic Bd and evidence of the evolutionary patternof the predator.

ACKNOWLEDGMENTS

We thank Judy Pennington for preparing the manuscript.This material is based on work supported by grant OCE-8746188

from the National Science Foundation.

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