APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1986. p. 1299-1304 Vol. 52, No. 60099-2240/86/121299-06$02.00/0Copyright ©) 1986. American Society for Microbiology

Rapid Serological Identification of Vibrio vulnificus byAnti-H Coagglutination

JANET SIMONSON AND R. J. SIEBELING*

Departtnent )f lMirobiologx,, Louisiainai Staite Univ.ersity, Baton Roige, Louisiatnaz 70803

Received 11 June 1986/Accepted 23 September 1986

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbitsimmunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed bycoagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen isexpressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identifiedbacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V.vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V.fluvialis isolates recovered from seafood and the marine environment. The serological resolution of thecoagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0. 1 mMEDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased thesensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinizedphosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, andinexpensive procedure for identifying V. vulnificus one step beyond primary isolation.

Because of the rapid progression and high mortality ratesof Vibrio vulnificuts infections in humans, especially thosevictims with underlying chronic disease, seawater and sea-food are being scrutinized for the presence and level of thisorganism. The recovery, identification, and enumeration ofthe Vibrio pathogens in the marine environment and in theshell fish which inhabit it can be overwhelming in logistics,material, and time, while the expeditious and specific iden-tification of V. vulnificuts in the laboratory setting is desir-able. One approach to condense the time required presentlyto confirm the presence of V. vulnificits in the marineenvironment, seafood, or clinical specimens is through theuse of a rapid serological test which detects the species-specific flagellar (H) antigens. The premise for serologicalidentification is predicated upon the observation of Gardnerand Venkatraman (5), who reported that Vibrio choleraeisolates, both 01 and non-O1 serological varieties, expressan H antigen unique to the species. This observation wasconfirmed independently by Sakazaki et al. (13) andBhattacharyya and Mukerjee (1, 2); the latter reported thatboth Vibrio mnetschnikovii and Vibrio anguiillarumin eachexpress species-specific H antigens. As additional species ofVibrio were examined serologically, it was reported that V.paraihaeemolvticus (22), V. alginolyticius (8, 16), V. fluliialis(20, 21), and V. lviulnijicus (21) each express species-specificH antigens.Tassin et al. (21) described a tube flocculation test in

which anti-H serum raised against seven Vihrio speciescorrectly identified within 2 h 99% of the V. cliolerae, 100%of the V. inetschnikovii, 98% of the V.flui'ialis, 97% of the V.viulnuficus, and 93% of the V. pairalhaenolyticus isolates fromamong 467 environmental isolates. The tube test, thoughreliable, is labor intensive and uses anti-H serum producedin rabbits immunized with Formalin-killed whole-cell vac-cines, which have to be absorbed repeatedly to removeanti-O and anti-K antibody activity.

Corresponding author.

For a number of reasons, it is difficult to visualize theanti-H reaction within the time constraints of a direct im-mune slide test (9). The anti-H antibody is restricted tobridging antigens located on a single polar flagellum, whichdelays the buildup of a visible immune aggregate. If theflagella become detached from the bacterial cells and arereacted upon by anti-H antibody, there is insufficient mass toproduce a discernible aggregate within the time frame of theslide test. In addition, the sheath which shrouds the polarflagellar core may mask the species-specific H determinants,located on the core, making them inaccessible to the Hantibody. The sheath which is contiguous with the outermembrane envelope of the cell wall may express 0 determi-nants, which make it imperative that anti-H serum possessno anti-O activity.

Coagglutination is a serological event which permits thedetection of microgram quantities of soluble antigen inclinical specimens and has been used to detect withinminutes cell wall and capsular antigens on the bacterialpathogen or in fluids collected from the infected host (6). Theserological reagent, Staphylococcus{aSiruueis cells armed withantibody through its cell wall Fc receptors, permits thedevelopment of a discernible agglutination reaction withsmall quantities of soluble or particulate antigen, reactionsthat may not evolve into discernible complexes if antiserumalone were used. It was the objective of this investigation toassess the utility of Staphyviococcus cells armed with anti-Hantibody to detect in a rapid and perceptible manner thespecies-specific H determinants on V. vulnificuts. The coag-glutination reagent was used to screen 1,168 environmentalVibrio isolates serologically by a slide test for V. vlnuifihcus;the results were correlated by bacteriological identification.

MATERIALS AND METHODSProduction of anti-core H serum. New Zealand White

rabbits were immunized with the H core protein preparedfrom motile strains of V.viulnificuts ATCC 27526, V. choleraeOgawa ATCC 14035, Vibrio mimnicus ATCC 33653, and V.parahacmnolyticius ATCC 10136. H cores were isolated from

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each of the four Vibrio strains by the method of Yang et al.(23). Each vaccine strain was propagated on Trypticase soyagar (BBL Microbiology Systems) supplemented with 2%NaCl, and the bacterial lawns from 40 Roux bottles wereharvested in 0.15 M NaCl (saline), sedimented, and washedonce in saline. The pellets were suspended in 9 volumes of0.1 M Tris buffer (pH 7.8) containing 0.1 mM EDTA, 1.0%Triton X-100, and 0.001% thimerosol (TET buffer) andhomogenized for 90 s at 4°C at the high-speed setting in aSorvall Omni-Mixer. The sheared flagella were separatedfrom the bacterial cell bodies by four cycles of differentialcentrifugation at 10,000 x g for 10 min each, and the flagellawere sedimented at 30,000 x g for 2 h. The H cores wereisolated from the residual cellular debris by cesium chloridedensity ultracentrifugation at 64,000 x g for 18 h in a Vti 50rotor (Beckman Instruments, Inc.). The H cores were re-trieved by aspiration from the gradient after the piercing ofthe tube wall with a syringe. Each core fraction was dialyzedovernight at 4°C against Tris-EDTA buffer and then sedi-mented at 82,000 x g for 4 h. The core protein pellets wereresuspended in a small volume of Tris-EDTA buffer, and aportion was examined by electron microscopy to verify thatnaked cores, free of sheath material, were present. Thepurified cores, approximately 4 to 6 mg of protein, werestored at 4°C. Total protein determinations were made by themethod of Lowry et al. (7).Immunization protocol. Rabbits were immunized by a

subcutaneous injection of 10 ,ug of core protein suspended in0.5 ml of 0.067 M phosphate-buffered saline (PBS), and thenat 3-day intervals, each rabbit was immunized by intrave-nous injection with graded doses of core protein, 15 to 100,ug, over a 30-day period. The animals were exsanguinated35 to 40 days after the subcutaneous injection, and the serumwas harvested and processed.

Preparation of the anti-H coagglutination reagent. Theanti-core (i.e., anti-H) serum, was fixed to Formalin-killed S.aureus Cowan I ATCC 12598 cells by methods reportedpreviously (4). Staphylococcus cells pelleted from an 18-hbroth culture were washed three times in PBS, suspended in0.3% formalinized PBS, and incubated at room temperaturefor 3 h. The Formalin-killed cell suspension was sedimented,and the cell pellet was washed three times with PBS. Thefinal pellet was resuspended in PBS to give a 10% suspension(vol/vol) of cells, which were heated to 80°C for 1 h. S.aureus cells were armed with anti-H antibody by mixing 0.1ml of undiluted or diluted antiserum with 1 ml of the 10% S.aureus cell suspension. The mixture was incubated over-night at 4°C and washed once in PBS, and the armed cellswere resuspended in 10 ml of PBS.

Serology. The anti-V. vulnificus H serum was evaluatedserologically by the tube test for anti-H activity against thevaccine strain and 20 heterologous Vibrio spp. The protocolsfor antigen preparation and standardization and antiserumdilution schemes were reported previously (21).Each Vibrio isolate to be tested by coagglutination was

propagated on alkaline-peptone agar slants (1% peptone, 2%NaCl, 0.2% yeast extract, 1.5% agar [pH 8.0]) for 18 to 24 hat 30°C. In group 1, 528 isolates examined were harvestedfrom the slants in 2 ml of formalinized PBS and testedserologically after 1 h. In group 2, 640 isolates were har-vested in TET buffer and tested 1 and 24 h later. Each Vibrioisolate, suspended in either formalinized PBS or TET buffer,was tested in each of the four coagglutination reagents. Onedrop of the Vibrio cell suspension was placed on a glasssurface, and one drop of the coagglutination reagent wasplaced next to it. The drops were mixed with a wooden

applicator stick, and the glass plate was tilted slowly fromside to side. The reactants were observed over a 3-minperiod for evidence of agglutination, with indirect lighting asa background.

Cross-agglutination and absorption. Each of the four anti-H coagglutination reagents was tested serologically againstTET buffer suspensions of 21 Vibrio species. Staphylococ-cus cells were armed with various dilutions of each of thefour anti-H sera. In addition to the four vaccine strainsidentified above, the following Vibrio species were individ-ually titrated with each coagglutination reagent armed withvarious dilutions of each of the four anti-H preparations: V.anguillarum ATCC 19264, V. alginolyticus ATCC 33787, V.carchariae ATCC 35084, V. campbellii ATCC 25920, V.costicola NCMB 701, V. damsela ATCC 35083, V.diazotrophicus ATCC 33466, V. fischeri NCMB 1281, V.fluvialis ATCC 33810, V. harveyi NCMB 1280, V.nigrapulchritudo [sic] ATCC 27043, V. pelagius ATCC25916, V. metschnikovii P206, V. natriegens ATCC 14048,V. nereis ATCC 25917, V. ordalii ATCC 33509, and V.proteolyticus NCMB 1326.Each anti-H serum was absorbed with Formalin-killed

Vibrio cell suspensions to remove anti-H activity directedagainst common H determinants shared among heterologousVibrio species, in addition to the species-specific H antigens.The bacterial growth was harvested from five slant culturesin formalinized PBS, pooled, sedimented, and washed threetimes. A 4-ml amount of the anti-H serum to be absorbedwas diluted 1:10 in PBS, mixed with the heterologous Vibriocell pellet (absorbing heterologous H antigen), and incubatedat 45°C for 2 h and then overnight at 4°C. The absorbedserum was recovered by centrifugation, and the absorptionprocess was repeated two more times. The absorbed anti-Hserum was fixed to S. aureus cells and retested by coagglu-tination for serological specificity.

Environmental Vibrio isolates tested. Over a 9-month pe-riod, 969 Vibrio isolates were sent to us from the CentralLaboratory, Division of Laboratories, Department of Healthand Human Resources, New Orleans, La., and the LakeCharles Regional Laboratory, Lake Charles La. Plankton,shellfish, sediment, and water were collected from 10 oysterfishing areas in south Louisiana and processed bacteriolog-ically for Vibrio spp. Each specimen was enriched in bothalkaline peptone broth and Horie broth, streaked to thiosul-fate-citrate bile salts agar from which both sucrose-positiveand -negative colonies were selected, and identified bacteri-ologically by methods reported previously (21). Each isolatewas number coded and sent in groups of 50 to 100 to theDepartment of Microbiology at Louisiana State Universityfor serological testing.

Altogether, 199 V. cholerae non-O1 isolates were receivedfrom the National Institute of Cholera and Enteric Diseases,Calcutta, India, and were also tested in each of the anti-Hcoagglutination reagents.

RESULTS AND DISCUSSION

The serological specificity of anti-V. vulnificus H serumproduced in rabbits immunized with H cores was assessedby both tube flocculation and slide coagglutination of Hantigens. Formalin-killed cell suspensions of 20 heterologousVibrio spp. and the homologous vaccine strain were added toserial dilutions of the antiserum, starting at a dilution of1:500. A titer of 40,000 was obtained against V. vulnificuscells, whereas only one heterologous species, V. pelagius,flocculated with a titer of 1:1,000. Bhattacharyya and

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TABLE 1. Coagglutination of Vibrio isolates suspended in PBSaIsolate by No. of isolates agglutinated with anti-H prepared to:

bacteriological No. testedidentification V. cholerae V. mimicus V. vulnificus V. parahaemolyicus

V. cholerae 71 71 71 0 0V. mimicus 6 6 6 0 0V. vulnificus 232 1 0 230 0V. parahaemolyticus 203 0 0 0 196V. fluvialis 16 16 16 0 0

a For 1 h in 0.3% formalinized PBS (pH 7.3). Isolates were tested with S. aureus cells armed with anti-H (core) antibody.

Mukerjee (2) reported that at low dilutions (1:500 or less),anti-H serum showed cross-reactions among heterologousVibrio species. It is possible that the flocculation of V.pelagius in anti-V. vulnificus H serum reflects such a situa-tion in view of the low titer. Previously, we reported (21) thatthe anti-V. vulnificus H serum that was produced in rabbitsimmunized with the Formalin-killed whole-cell vaccinefailed to flocculate V. pelagius cells. Since the core proteinused in the present investigation was prepared from the V.vulnificus vaccine strain used in the previous study (21), it ispossible that the physical state of the flagellum influencedthe character of the immune response. The naked coreproteins may present a greater spectrum of more accessibleH determinants to the immune facilities of the rabbit than dothe sheath-shrouded core proteins in the whole-cell vaccine.The advantage H core protein offers as an immunogen

over the whole-cell vaccine resides with the fact that theresultant antiserum does not possess detectable anti-cell wallor anti-capsular serological activity.

Cognizant of the low anti-V. pelagius H activity in theanti-V. vulnificus serum, we used it to prepare the coagglu-tination serological reagent. S. aureus cells armed withanti-V. vulnificus H immunoglobulin were tested for serolog-ical activity and species specificity. V. vulnificus cells wereagglutinated within 1 min, V. pelagius cells were aggluti-nated after 3 min, and none of the remaining 19 heterologousVibrio spp. was coagglutinated.Examination of environmental Vibrio isolates by anti-H

coagglutination. The 528 isolates of group 1 were inoculateddirectly onto alkaline-peptone agar slants without benefit ofmotility enhancement. Each isolate was harvested in 0.3%formalinazed PBS and tested serogically after a 1-h incuba-tion at room temperature. The primary intent was to assessthe efficiency of the anti-V. vulnificus H coagglutinationreagent to detect in a rapid, discernible, and specific mannerthose isolates which had been identified bacteriologically asV. vulnificus. Of the 232 isolates identified as V. vulnificus,230 (99.1%) were coagglutinated within 2 min after theaddition of the homologous coagglutination reagent (Table1). None of the 296 isolates identified as V. cholerae, V.mimicus, V. fluvialis, or V. parahaemolyticus was aggluti-nated with this reagent. The two isolates, identified as V.vulnificus, which failed to coagglutinate in the anti-V.vulnificus reagent were retested bacteriologically. One fit allthe physiological tests for V. vulnificus yet failed to coag-glutinate even though the isolate was motile, whereas theother isolate was not reidentified as V. vulnificus and agglu-tinated in the anti-V. cholerae H reagent.Each of the 528 Vibrio isolates was also tested by coag-

glutination with S. aureus cells armed with anti-V. cholerae,anti-V. mimicus, or anti-V. parahaemolyticus H serum pro-duced in rabbits immunized with H core protein. The pur-pose of this portion of the survey was twofold; first, to

ascertain if the anti-H coagglutination reagents with impliedspecificity for these Vibrio pathogens possessed serologicalactivity for V. vulnificus, and second, to assess each of thesereagents for its capacity to coagglutinate precisely the rele-vant species from among the 528 environmental isolates.The anti-V. cholerae H coagglutination reagent aggluti-

nated all 71 isolates identified as V. cholerae (100%), all 6isolates identified as V. mimicus, and all 16 isolates identifiedbacteriologically as V. fluvialis (Table 1). We reported pre-viously (21) that V. cholerae and V. mimicus express similiarif not identical H determinants. Bhattacharyya and Mukerjee(2) examined the H antigen relationships among V. cholerae,V. metschnikovii, and V. anguillarum. They reported thatthese species express H antigens unique to each, at least oneH determinant common to all three, and another two Hantigens shared by two of the three species in differentcombinations. Tassin et al. (20, 21) reported that V. fluvialisexpresses a species-specific H antigen and also possessesserologically detectable H determinants in common with thethree species studied by Bhattacharyya and Mukerjee (2, 9).It is most probable that the anti-V. cholerae coagglutinationreagent (Table 1) detected these common or public H anti-gens on V. mimicus and V. fluvialis.The anti-V. parahaemolyticus H coagglutination reagent

agglutinated 196 of the 203 isolates (96.5%) identified bacte-riologically and did not agglutinate any of the heterologousVibrio isolates. The seven isolates which did not agglutinatewere reexamined bacteriologically, and each was reidenti-fied as V. parahaemolyticus. One was found to benonmotile.

Often V. parahaemolyticus isolates, especially those re-covered recently from the environment, produce mucoidcolonies which, when suspended in 0.3% formalinized PBSfor testing by coagglutination, give rise to a viscous puddleon the glass surface which is not miscible with the anti-Hcoagglutination reagent. The subsequent coagglutinationevent was obscured and difficult to discern and interpret. Anoccasional V. vulnificus isolate presented the same obstacleto serological resolution. Presented with a related problem,Sil and Bhattacharyya (19) exposed V. cholerae cells to 1.5%phenol in saline for 1 min to enhance the serological resolu-tion of the H flocculation event in a direct immune slide test.Phenol treatment, they proposed, damages or removes thesheath which encases the H core and uncovers the species-specific H determinants. An extensive serological survey byCastellani-Pastoris et al. (3), in which 1,081 V. choleraeisolates were examined by the phenol slide agglutinationtest, correctly identified 98.9% as V. cholerae. One draw-back to the phenol slide agglutination test is that it may haveto be performed as many as three times on each isolate tochange phenol concentrations in the suspending buffer or tochange exposure time to the phenol before an isolate may bedeclared negative. In the coagglutination test (Table 1), V.

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TABLE 2. Coagglutination of Vibrio isolates suspended in TETa

Isolate by No. of isolates agglutinated with anti-H prepared to:bacteriological No. testedidentification V. cholerae V. mimicus V. vulnificus V. parahaemolyticus

V. chholerae 219 216 216 0 0V. mimicus 16 16 16 0 0V. vulnificus 203 0 0 202 1V. parahaemolyticus 192 0 0 1 184

a For 24 h in 0.1 M Tris buffer containing 0.1 mM EDTA, 1% Triton X-100, and 0.001% thimerosol. Isolates were tested with S. aureus cells armed with anti-H(core) antibody.

cholerae and V. mimicus cells suspended in PBS wereagglutinated within 1 min, with no interference by slime orother impediments.To produce a clean and uniform coagglutination reaction,

free of artifacts, the H determinants on the flagella must beaccessible to the anti-H reagent, and the viscous characterthat some Vibrio cell suspensions exhibit must be eliminatedwithout diminishing the sensitivity of the serological test.During the preparation of the H core protein immunogen, theH sheath was stripped from the core by a suspension of thesheared intact flagella in TET buffer (23). Six mucoid V.vulnificus isolates, which produced viscous cell suspensionsin PBS, were propagated and harvested from the slants in 2ml of TET buffer and tested after 1 h by coagglutination.Each of the six isolates suspended in TET buffer aggluti-nated within 30 s with the anti-V. vulnificus H coagglutina-tion reagent producing a clean reaction with no backgroundturbidity. There was no evidence of the viscous mucoidstrands which had obscured the coagglutination reactionwhen these cells had been suspended in formalinized PBS.The TET buffer served to liquefy the troublesome slimewithout obvious loss in serological activity or specificity.

Examination by anti-H coagglutination of environmentalvibrio isolates suspended in TET buffer. Each of the 640vibrios in group 2 of the environmental isolates tested was

suspended in 2 ml of TET buffer and then tested by coag-glutination at 1 and 24 h after suspension (Table 2). Alto-gether, 203 isolates in this group were identified bacteriolog-ically as V. vulnificus, and 202 of these were agglutinatedwithin 60 s by the homologous coagglutination reagent whentested 24 h after suspension in TET buffer. The single isolatewhich failed to agglutinate in the anti-V. vulnificus H reagentwas agglutinated in the anti-V. parahaemolyticus reagent.The agglutination reactions were easy to visualize and,importantly, there was no obvious loss in serological speci-ficity in that none of the 202 isolates was coagglutinated withthe other three coagglutination reagents. The V. vulnificusTET buffer cell suspensions, if capped, retained serologicalactivity and specificity for at least 1 month. Often, theturbidity of the cell suspension disappeared after 24 h and

gave rise to a clear fluid, which enhanced the resolution ofthe coagglutination reaction.

Vibrio isolates identified as V. parahaemolyticus, whensuspended in TET buffer, produced agglutination reactionsin the homologous coagglutination reagent that were easierto discern and developed more rapidly than when cells weresuspended in PBS. In the present format, each isolate shouldbe exposed to the TET buffer for 24 h before testing tocompletely eliminate the viscosity of the cell suspension. Ofthe 192 V. parahaemolyticus isolates tested, 184 (95.8%)were coagglutinated in the anti-V. parahaemolyticus reagentonly. One of the eight isolates that failed to agglutinate in thehomologous H reagent was agglutinated with the anti-V.vulnificus H reagent, four auto-agglutinated in the unarmedS. aureus cell reagent, and three isolates failed to agglutinatein any of the four H reagents.The serological sensitivity of the anti-H coagglutination

test was significantly enhanced when the Vibrio isolatesunder test were suspended in TET buffer for as short a timeas 1 h. S. aureus cells armed with anti-V. vulnificus H serumdiluted to 1:500 agglutinated V. vulnificus cells suspended inTET buffer within 60 s, whereas V. vulnificus cells sus-

pended in formalinized PBS were agglutinated in the sametime interval only by S. aureus cells armed with undiluted Hantiserum (Table 3). These findings reinforce the suppositionthat TET buffer may expose H determinants which aremasked by sheath protein when the cells are suspended inPBS. It is tempting to suggest that TET buffer treatment mayliberate outer membrane proteins, which Richardson andParker (12) detected in motile strains of V. cholerae. Theseproteins exhibited serological similarity to H- core proteins.The serological sensitivity of the coagglutination reactionwas similarly enhanced for the anti-V. cholerae and V.parahaemolyticus H reagents when isolates under test weresuspended in TET buffer for 1 and 24 h after suspension.

In light of the expression of public, non-species-specificantigens by some vibrios, the question of serologicalspecficity in the coagglutination test was examined. The 21Vibrio species identified in Table 4 were propagated andsuspended in TET buffer for 24 h. Each isolate was tested by

TABLE 3. Titration of anti-V. vulnificus H serum by coagglutination with V. vulnificus cells suspended in formalinized PBSand TET buffer

V. vulnificus Suspension Agglutination time for S. aureus cells armed with dilutions of anti-Ha

solution time (h) undiluted 1:10 1:100 1:500 1:1,000 1:2,500 1:5,000 1:10,000 unarmed

F-PBSb 1 + + + + 0 0 0 0 0 0 0TET'c 1 + + + + ++ + + + + + + +++ + + + + 0 0F-PBS 24 + + + + + 0 0 0 0 0 0 0TET 24 + + + + + + +++ + + + +++ + + + 0 0

a Agglutination reactions were examined and scored to indicate agglutination at 1 min or less (+ + +), 1 to 2 min (+ +), or 2 to 3 min (+).bF-PBS, 0.3% Formalinized PBS (pH 7.3).' TET, 0.1 M Tris buffer containing 0.1 mM EDTA, 1.0% Triton X-100, and 0.001% thimerosol.

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TABLE 4. H antigen relationships among 21 Vibrio spp. examined by coagglutination with S. aureus cells armed with variousdilutions of anti-H serum

Coagglutination titer in anti-H raised against:Suspensiona

V. cholerae V. mimicus V. vulnificus V. parahaemolyticus

V. cholerae 5,000 2,500 0 0V. mimicus 5,000 2,500 0 0V. fluvialis 500 100 0 0V. metschnikovii 100 100 0 0V. anguillarum 500 100 0 0V. ordalii 100 100 0 0V. proteolyticus 50 10 0 0V. vulnificus 0 0 5,000 0V. pelagius 0 0 50 0V. parahaemolyticus 0 0 0 1,000V. alginolyticus 0 0 0 50V. natriegens 0 0 0 50V. campbellii 0 0 0 50V. harveyi 0 0 0 100V. carchariae 0 0 0 100

a Organisms were suspended for 24 h in TET (0.1 M Tris buffer containing 0.1 mM EDTA, 1.0% Triton X-100, 0.001% thimerosol). Each of the following Vibriospp. was also tested and did not agglutinate: V. costicola, V. damsela, V. diazotrophicus, V. fischeri, V. nereis, and V. nigripulchritudo.

coagglutination with S. aureus cells armed with variousdilutions of anti-V. vulnificus, -V. cholerae, -V. mimicus, or-V. parahaemolyticus H serum. The anti-V. cholerae Hcoagglutination reagent agglutinated six heterologous Vibriospecies, each previously reported (2, 15, 20, 21) to express Hdeterminants in common with V. cholerae. With the excep-tion of V. mimicus, the other five heterologous Vibriospecies were not agglutinated in optimal dilutions of theanti-H serum (Table 4). A 1:10 dilution of anti-V. cholerae Hserum was absorbed with Formalin-killed V. fluvialis cellsfixed to S. aureus cells and was then retested by coaggluti-nation with TET suspensions of those Vibrio spp. whichagglutinated previously. The absorbed serological reagentagglutinated V. cholerae and V. mimicus cells only. In asimiliar fashion, the anti-V. vulnificus coagglutination Hreagent could be rendered species specific if the antiserumwas absorbed with motile V. pelagius cells before the armingof the S. aureus cells. Likewise, the anti-V. parahae-molyticus H reagent would no longer have agglutinated thefive heterologous Vibrio species identified in Table 4 if theantiserum had been absorbed with V. campbellii cells.Shinoda et al. (14, 15, 17, 18) reported in a series of

investigations that the lateral, nonsheathed flagella (HL),which are produced by a limited number of Vibrio spp.,express H antigens which are not related serologically to thepolar H antigens of those species. They also reported (18)that anti-HL serum raised in rabbits immunized with lateralflagella that were separated from V. parahaemolyticus ag-glutinates this Vibrio by a direct immune slide test in 1 or 2min. Their studies revealed that at least three serologicallyunique HL serotypes exist for V. parahaemolyticus, andtheir surveys showed excellent agreement between serolog-ical and bacteriological identification when they examined192 marine and clinical V. parahaemolyticus isolates. Itn rareinstances, marine isolates positive for O-nitrophenyl-p-D-galactopyranoside were agglutinated in anti-V. parahaemo-lyticus HL serum, whereas an occasional isolate identifiedbacteriologically as V. parahaemolyticus failed to aggluti-nate. With respect to the latter finding, Shinoda et al. (18)concluded that these isolates were not V. parahaemolyticus,since they failed to agglutinate in anti-V. parahaemolyticus0-typing serum. Alternatively, it could be reasoned thatthese nonagglutinating isolates may constitute a fourth HL

serotype, a proposition that was not addressed in theirreport. Relevant to the serological findings of Shinoda et al.(18), a small number of Vibrio isolates encountered in ourserological survey which were identified bacteriologically asV. parahaemolyticus failed to agglutinate (Table 1 and 2) inthe relevant H coagglutination reagent, whereas isolatepositive for o-nitrophenyl-p-D-galactopyranoside and identi-fied as V. vulnificus (Table 2) agglutinated in the anti-V.parahaemolyticus H reagent. It is interesting that the twoo-nitrophenyl-p-D-galactopyranoside- and lactose-positiveisolates which Shinoda (18) reported to be agglutinated inanti-V. parahaemolyticus HL serum were identified bacteri-ologically as V. vulnificus and were shown to produce lateralflagella, structures that this Vibrio species has not beenreported to express.

In our investigation, the method of Yang et al. (23) wasused to isolate the H cores from V. parahaemolyticus, and itis quite possible that these preparations contained bothlateral and polar H cores. As a result, the antiserum used inour surveys possessed both antipolar and -lateral H activity,a situation which remains to be clarified in our serologicalsystem. We chose to use anti-polar H serum for a number ofreasons. First, all Vibrio species that we have examinedserologically thus far, with the exception of V. mimicus,express species-specific H antigens on the polar flagellum,whereas only six or seven Vibrio species, which include thehuman pathogens V. alginolyticus, V. fluvialis, and V.parahaemolyticus, produce lateral flagella. Second, vibriosproduce the polar flagellum under most cultural conditions,whereas lateral flagella are produced only when the organ-isms are propagated on an agar substrate. Shinoda et al. (18)recommended that those isolates to be tested serologicallywith anti-HL serum should be grown on modified marineoxidative fermehtative agar, since many isolates they exam-ined failed to produce lateral flagella when propagated onnutrient agar or brain heart infusion agar supplemented with3% NaCl. In addition, they recommended that the isolateunder test be incubated at 25°C for no longer than 20 h toensure optimal lateral flagella production. Third, the lateralflagella are readily separated from the cell body by handshaking, once the organisms are suspended in buffer, anddetached flagella lack sufficient mass to lead to the develop-ment of a discernible immune aggregate in the presence of

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1304 SIMONSON AND SIEBELING

antiserum alone. It was such an event which probablyprompted Shinoda et al. (17) to resort to the tube flocculationtest when they examined the utility of anti-HL serum raisedin rabbits to V. fluvialis to assess serologically the speciesspecificity of this reagent. The particulate coagglutinationreagent armed with species-specific polar (i.e., H) or lateral(i.e., HL) antibody should circumvent most of the obstaclesencountered during examination of Vibrio spp. by the directimmune agglutination slide test, since detached flagellashould be agglutinated as readily as H cells.

Nishibuchi and Seidler (10, 11) recently described amicroimmunodiffusion test which correctly identified su-crose-negative colonies such as V. vulnificus within 10 to 20h after being picked from the isolation medium. Their systemused antiserum raised against an apparent species-specificantigen present in the supernatant fluid of sonicated V.vulnificus cells. The resultant antiserum formed lines ofidentity with distilled-water lysates of 78 V. vlulnificus iso-lates that they examined. Heterologous Vibrio, Aeromonas,and Pseudomonas spp. failed to produce precipitin lines withthe anti-V. vulnificus serum. Superficial inspection of theirspecies-specific antigen suggests it is not related to the Hcore antigens, because their antiserum did not agglutinatewhole-cell suspensions of V. vulnificus.

Staphylococcus cells armed with species-specific anti-Hantibody offer several advantages for rapid identification ofV. vulnificus isolates over the use of anti-H serum alone.Either the anti-H serum fails, when mixed on a glass surfacewith the Vibrio cell suspension under test, to produce adiscernible agglutination reaction, or the event is delayed,giving rise to minute trivial patterns. To visualize H agglu-tination by the direct immune approach, the flagella mustremain attached to the bacterial cell body, the latter provid-ing the mass which contributes to perceptible clumping. Inthe coagglutination reaction, (S. aureus the carrier particlebearing the anti-H antibody) provides the mass, and theantigen in question consequently need not be particulate; asa result, the H coagglutination reagent produces visibleimmune aggregates with detached or attached flagella. Inaddition, the Vibrio sp. under test can be treated to exposethe core-associated H determinants without regard to thefate of the Vibrio cell. The H coagglutination reagents can bestored at 4°C for at least 6 months with no detectable loss inreactivity or specificity. Most importantly, the anti-V.vulnificus H reagent correctly identified 432 (99.3%) of 435isolates determined bacteriologically to be V. vulnificus.

ACKNOWLEDGMENTS

We thank N. C. Roberts, T. Donovan, G. B. Nair, and P. West forproviding bacterial cultures used in this investigation. We alsoexpress our appreciation to Tina Teague and Connie Neff for theirundaunted secretarial assistance.

This research was supported in part by grant NA85AA-D-SG141from the Louisiana Sea Grant College Program, a part of theNational Sea Grant College Program maintained by the NationalOceanic and Atmospheric Administration, U.S. Department ofCommerce. The Louisiana Program is administered by the Centerfor Wetland Resources, Louisiana State University, Baton Rouge,La.

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15. Shinoda, S., R. Kariyama, M. Ogawa, Y. Takeda, and T.Miwatani. 1976. Flagellar antigens of various species of thegenus Vibrio and related genera. Int. J. Syst. Bacteriol.26:97-101.

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20. Tassin, M. G., R. J. Siebeling, and A. D. Larson. 1984. H-antigen relationships among several Vibrio species, p. 73-82. InR. R. Colwell (ed.), Vibrios in the environment. John Wiley &Sons, Inc., New York.

21. Tassin, M. G., R. J. Siebeling, N. C. Roberts, and A. D. Larson.1983. Presumptive identification of Vibrio species with H anti-serum. J. Clin. Microbiol. 18:400-407.

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