isolation and identification of perkinsus olseni from feces

Journal of Invertebrate Pathology xxx (2010) xxx–xxx

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Journal of Invertebrate Pathology

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Isolation and identification of Perkinsus olseni from feces and marine sedimentusing immunological and molecular techniques

Kyung-Il Park a, Hyun-Sung Yang b, Hyun-Sil Kang b, Moonjae Cho c, Kwang-Jae Park d, Kwang-Sik Choi b,⇑a Department of Aquatic Life Medicine, Kunsan National University, Gunsan 573-701, Republic of Koreab Faculty of Marine Biomedical Science (POST BK21) and Marine and Environmental Research Institute of Jeju (Cheju) National University, 66 Jejudaehakno,Jeju 690-756, Republic of Koreac Department of Biochemistry, College of Medicine, Jeju (Cheju) National University, 66 Jejudaehakno, Jeju 690-756, Republic of Koread Tidal Flat Research Institute of National Fisheries Research and Development Institute (NFRDI), Kunsan, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 March 2010Accepted 28 July 2010Available online xxxx

Keywords:Perkinsus olseniRuditapes philippinarumTransmissionPolyclonal antibodyFecal dischargeKorea

0022-2011/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jip.2010.07.006

⇑ Corresponding author. Fax: +82 64 756 3493.E-mail address: [email protected] (K.-S. Choi).

Please cite this article in press as: Park, K.-I., et amolecular techniques. J. Invertebr. Pathol. (2010

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a pro-tozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decompos-ing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseniantibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa.Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, includ-ing the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells weresuccessfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, whereP. olseni–associated clam mortality has recurred for the past decade. Purified cells were positively stainedwith the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time thepresence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia wereobserved in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from thefeces and pseudofeces of infected clams and confirmed by PCR. The clams released 1–2 prezoosporangiaper day through feces. The data suggested that the fecal discharge and decomposition of the infected clamtissue could be the two major P. olseni transmission routes.

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1. Introduction

Perkinsosis is an epidemic disease that occurs in commerciallyimportant marine mollusks, including oysters, clams, and abalones.Perkinsus marinus and P. olseni are the two pathogens primarilyresponsible for perkinsosis (Perkins, 1996; Bondad-Reantasoet al., 2001; Office International des Epizooties, 2004; Villalbaet al., 2004). Perkinsosis of Manila clam Ruditapes philippinarumis caused by P. olseni and has been reported from tidal flats andsand beaches along the coastal Yellow Sea of Korea and Chinaand in the Ariake Sound in Japan (Choi and Park, 1997; Hamaguchiet al., 1998; Park and Choi, 2001; Park, 2005; Liang and Liang,2007; Park et al., 2008; Uddin et al., 2010). High levels of P. olseniinfection often cause severe host tissue inflammation and interferewith the gametogenesis in clams (Villalba et al., 2005; Park, 2005;Park et al., 2006). According to Park and Choi (2001), P. olseni infec-tion among Manila clam populations in Korean waters varies spa-tiotemporally, and the infection intensity and prevalence are oftenhigher in commercial clam beds, which have a much higher clam

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density than natural habitats. Mass mortalities of Manila clamsin commercial clam beds in late summer or early spring have beenreported in Korea, and extremely high levels of P. olseni in clamswere partly responsible (Park et al., 2006). Recently, a new Perkin-sus species was isolated and propagated from Manila clam in Japanand reported as P. honshuensis n. sp. (Dungan and Reece, 2006).

Auzoux-Bordenave et al. (1995) demonstrated the P. olseni lifecycle as a trophozoite in infected host tissue, a prezoosporangiumand a motile bi-flagellated zoospore (see also Villalba et al., 2004).As observed in P. marinus, the P. olseni prezoosporangium stage canbe induced when infected host tissue containing trophozoites areplaced in an anaerobic medium such as fluid thioglycollate med-ium fortified with antibiotics and salt (RFTM, see Ray, 1952 andRay, 1966). Numerous zoospores are subsequently produced insidethe prezoosporangia and released through a discharge tube whenthe prezoosporangia are placed in aerated seawater (Auzoux-Bordenave et al., 1995; Ahn and Kim, 2001; Park et al., 2005). How-ever, the P. olseni prezoosporangium and zoospore stages have onlybeen confirmed under laboratory conditions and have yet to be ob-served in the field.

In the early description of P. marinus, the first Perkinsus sp. de-scribed, studies have reported enlarged trophozoites in moribund

Perkinsus olseni from feces and marine sediment using immunological and6

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2 K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx

oysters (Ray and Mackin, 1954; Mackin, 1962). Subsequently,Perkins (1968) and Valiulis and Mackin (1969) observed Perkinsussp. zoosporulation when prezoosporangia isolated from moribundclams were placed in aerated seawater. These studies suggestedthat dispersal of P. marinus from gaping infected oysters in natureis an important means of pathogen transmission. In addition to therelease of P. marinus from cadavers, fecal discharge is an importantroute for P. marinus transmission (Andrews and Hewatt, 1957;Scanlon et al., 1997). For example, Bushek et al. (1997, 2002b) ob-served a massive release of P. marinus in the feces of live C. virginicaartificially infected with P. marinus. Unlike efforts to identify andisolate various P. marinus life stages, no studies have reportedobservations of various P. olseni life stages in the field.

Immunological techniques are excellent tools for investigatingparasitic organisms in marine environment. Once an antibody israised from a molecule of interest such as protein extracts of sin-gle-celled or metazoan pathogen, the target molecule can be visu-alized or quantified using various immunological techniques.Using P. marinus hypnospore protein extract as immunogen,Dungan and Roberson (1993) first developed P. marinus-specificmonoclonal as well as polyclonal antibodies to detect the parasitein C. virginica. The murine and rabbit polyclonal antibodies raisedagainst P. marinus hypnospore did show a strong positive immuno-logical reaction to hypnospore as well as to trophozoites and zoo-spores (Dungan and Roberson, 1993). Accordingly, P. marinustrophozoites distributed in digestive gland and intestine of an in-fected oyster was successfully visualized using fluorescence immu-nostaining technique in their study. Monoclonal antibody againstP. marinus was also developed for quantification of P. marinus tro-phozoite and Romestand et al. (2001) estimated the parasite bur-dens from infected oysters using the monoclonal antibody in acompetitive ELISA.

In an effort to detect various P. olseni life stages under labora-tory and field conditions, we developed a polyclonal antibodyagainst P. olseni. In the present study, we report, for the first time,the presence of P. olseni cells in commercial clam bed sediment andin infected clam feces and pseudofeces as confirmed by immuno-logical and molecular biological probes.

2. Materials and methods

2.1. Development of a P. olseni-specific antibody

Perkinsus olseni infected clams were supplied from Hwangdo(Fig. 1), where P. olseni prevalence was known to be 90–100% yearround (unpublished data) and incubated in RFTM for 1 week in thedark (Ray, 1952, 1966). To harvest P. olseni prezoosporangia, theRFTM cultivated clam gill tissues were placed in a petri-dish andminced using a blade. The prezoosporangia and minced gill tissuemixtures were filtered through 100 and 63 lm mesh screens to re-move the large clam tissue debris. The prezoosporangia retainedon the 63 lm mesh screen were harvested and washed severaltimes with phosphate buffered saline (0.15 M NaCl, pH 7.4). Thewashed prezoosporangia were further treated with 2 M NaOH at60 �C for 10 min to remove fine clam tissue particles attached onthe cell surface. The purified prezoosporangia were re-suspendedin PBS and homogenized using an ultrasonifier to extract proteinfrom the cells. Level of P. olseni protein in the homogenate wasdetermined using BCA protein assay kit (Pierce) and the proteinconcentration was adjusted to 100 lg/ml to be served as anantigen.

A New Zealand white rabbit was immunized with the prezoosp-orangia protein extract over an 8-week period according to Parkand Choi (2004). The rabbit initially received subcutaneous injec-tion of 0.5 ml of 100 lg/ml P. olseni prezoosporangia protein ex-

Please cite this article in press as: Park, K.-I., et al. Isolation and identification ofmolecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.00

tract mixed with an equal volume of Freund’s complete adjuvant.Two weeks after the initial injection, the rabbit received 0.5 ml of100 lg/ml P. olseni prezoosporangia protein extract mixed withFreund’s incomplete adjuvant. The booster injection was continuedon a biweekly basis over 6 weeks. The specificity of the rabbit anti-P. olseni serum obtained from the rabbit after completing theimmunization was tested using an enzyme-linked immunosorbentassay (ELISA). The rabbit antiserum initially showed weak but rec-ognizable cross-reactivity to proteins extracted from P. olseni-freeclam tissue. The cross-reacting antibodies were removed fromthe antiserum using an immunoadsorbent prepared from unin-fected Manila clam tissue according to Fuchs and Sela (1979). Afterremoving the cross-reacting antibodies, we tested the specificityagain using the ELISA, and no cross-reaction to P. olseni-free clamtissue was demonstrated. The rabbit anti-P. olseni IgG (PK-IgG)was subsequently isolated from the antiserum by precipitatingthe PK-IgG with saturated ammonium sulfate solution.

Western blotting was performed to characterize the P. olseniprotein antigens. The proteins were extracted from trophozoitescultured in vitro and prezoosporangia produced in RFTM cultureusing lysis buffer, and the proteins were precipitated with trichlo-roacetic acid. Then 10 lg of P. olseni protein was loaded onto a 10%SDS–polyacrylamide gel for electrophoresis (SDS–PAGE). The pep-tides separated on SDS–PAGE under reducing conditions weretransferred onto a polyvinyl difluoride membrane. The membranewas blocked with 5% bovine serum albumin (BSA), and 4 lg/ml PK-IgG was added to the membrane as the primary antibody. Afterthree washes with Tris-buffered saline containing 1% Tween-20(TBST), the membrane was incubated in horseradish peroxidase-conjugated goat anti-rabbit IgG (0.2 lg/ml) as the secondary anti-body. After several washes with TBST, the immunoreactive peptidebands in the membrane were visualized using enhanced chemilu-minescence (ECL) detection reagent.

2.2. Testing the immunological specificity of the PK-IgG

The specificity of PK-IgG for various P. olseni life stages as wellas other organisms was tested using indirect immunofluorescenceassay. The prezoosporangia were produced using Ray’s FTM assay(Ray, 1954). The trophozoites were prepared from in vitro cultureaccording to the method of Ordas and Figueras (1998). Zoosporeswere induced by incubating the prezoosporangia in aerated seawa-ter for 2 days (30 ppt, 25 �C). The different types of P. olseni cellswere fixed in 4% paraformaldehyde and 0.2 M cacodylate bufferfor 2 h at 4 �C. Following washes with PBS containing 1% Tween-20 (PBST), the P. olseni cells were incubated for 30 min in 5% (w/v) BSA and PBST as a blocking agent. P. olseni cells were reactedwith PK-IgG (100 lg/ml) at room temperature for 1 h and thenincubated with fluorescein isothiocyanate (FITC)-conjugated goatanti-rabbit IgG (1:400 dilution; Sigma, St. Louis, MO, USA) for1 h. Fluorescent images of prezoosporangia, trophozoites, and zoo-spores labeled with FITC were acquired using a confocal scanningsystem (Olympus FV 300). As negative controls, the pre-immunerabbit IgG and two species of microalgae Tetraselmis sp. andSkeletonema sp. were used in the immunofluorescence assay.Tetraselmis and Skeletonema spp. have been frequently reportedfrom tidal flats on the west coast of Korea where clams in the pres-ent study were collected and might have been confused with Perk-insus sp. in sediment samples (NFRDI, 2001; Moon and Choi, 2003).

An immunohistochemistry assay was also performed to localizeP. olseni trophozoites in the clam tissue. A cross-section of theManila clam was embedded in paraffin, sliced at 6-lm thickness,deparaffinized, and rehydrated. The sections were incubated for30 min in 5% (w/v) BSA and PBS Triton X-100 as a blocking agent.After blocking, the tissue was reacted with PK-IgG (100 lg/ml) atroom temperature for 1 h. The tissue was washed three times with



China

Korea

N

W E

S

120° 125°115° 130°

40°

35°

30°

45°

Yellow Sea

East Sea

10 km

Hwangdo

Fig. 1. Map of study area: Hwangdo (36�35050.8700E, 126�22053.8100N), on the west coast of Korea.

K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx 3

PBST, and FITC-conjugated goat anti-rabbit IgG (1:400 dilution,Sigma) was added. The FITC-stained P. olseni trophozoites werewashed again with PBST, mounted in glycerol-PBS solution (1:1),and observed under a fluorescence microscope.

2.3. Isolation of P. olseni from marine sediment and immunologicalidentification

To locate P. olseni cells in the marine environment, we took a10 � 10 � 2 cm (ca. 200 g) section of bottom sediment from a sandymud tidal flat in Hwangdo off the west coast of Korea in late August2005 (Fig. 1). A high level of P. olseni infection has been reported inHwangdo, and mass mortalities of clams had occurred in the tidal flatduring the springs of 2004 and 2005 (NFRDI, 2007). The sedimentwas mixed with 200 ml PBS, stirred, and sieved through a 100-lmfilter to remove sand particles. Then 30-ml aliquots of each of the fil-trates were placed into 50 ml conical tubes, and 9 ml of 100% Percollfluid was added to remove fine sediment particles by gravity. Thesuspension containing low density particles was separated fromthe tube, washed several times with PBS (1200g for 10 min) and fil-tered through a 1.2-lm GF/C filter (Whatman).

An immunofluorescence assay was performed to identify P.olseni cells retained on the filter. The filter was blocked with 2%BSA in PBS, and 100 lg/ml PK-IgG was added as the primary anti-body. Pre-immune rabbit antiserum was applied to the filter as anegative control. After 1 h incubation at room temperature, the fil-ter was washed three times with PBST, and FITC-conjugated goatanti-rabbit IgG (1:500 dilution) was added as the secondary anti-body. The filter was incubated for 1 h at room temperature andwashed three times with PBST. Finally, the immunologicallystained filter was examined under a fluorescence microscope to lo-cate any P. olseni cells retained on the filter.

2.4. Microscopic examination of clam cadavers

Numerous gaping clams were collected from Hwangdo in Au-gust 2005 to examine P. olseni in dead or dying clams. Gill tissuewas excised from the cadavers, stained with Lugol’s iodine, andexamined under a light microscope.


2.5. Isolation of P. olseni from fecal discharge

Sixteen clams collected from Hwangdo (Fig. 1) in August 2005were individually placed on top of 50-ml conical tubes and placedin a 50-L tank containing filtered and aerated seawater (Fig. 2). After24 h of depuration, feces and pseudofeces of each clam accumulatedon the bottoms of the conical tubes were collected using a pasturepipette and filtered individually through GF/C filter. To identify P.olseni cells present in the fecal discharge, feces and pseudofeces offive individual clams retained on the filters were pooled and the totalDNA in the discharge was extracted using DNA extraction kit (Qia-gen). The extracted DNA was amplified using a P. olseni–specific pri-mer pair (F 50-CATTATCGAGGTCTGTGGTGACG-30, R 50-ACGATAGGTCTGCTGAGCAAGC-30, Park et al., 2002). The PCR product was elec-trophoresed in 1% agarose, and the size of the amplicon was deter-mined. The other GF/C filters of 11 clams containing their fecesand pseudofeces were incubated in RFTM for 2 weeks and stainedwith Lugol’s iodine. Meanwhile, the 11 clams were opened and incu-bated in RFTM for the same period as above. Finally, the numbers ofP. olseni prezoosporangia retained on each filter and in whole bodywere counted under a microscope.

3. Results

3.1. Specificity of P. olseni-specific antibody

SDS–PAGE revealed that the proteins extracted from P. olsenitrophozoites included peptides of 55.9, 24.0, 19.2, 10.6, 14.9,10.6, and >9 kDa (Fig. 3, lane 1). For Western blotting, 55.9-,24.0-, and 19.2-kDa peptides were coupled with the antibodydeveloped in this study (Fig. 3, lane 2). The trophozoite (T) cellwalls and membranes were stained with PK-IgG in the immunoflu-orescence assay, but the cytoplasm and the connective tissueswere not, indicating that the antibody specifically recognizedP. olseni cells in the host tissue (Fig. 4). In the immunofluorescenceassay, the PK-IgG positively reacted with all P. olseni life stages; thePK-IgG strongly reacted with proteins on the surface of prezoosp-orangia, entire trophozoites, and the zoospore heads (Fig. 5). Incontrast, the PK-IgG did not react to the negative controls, such



Fig. 2. Experimental design for collecting feces and pseudofeces of R. philippinarum in 50-ml conical tubes.

Fig. 3. SDS–PAGE and Western blot analyses of P. olseni proteins extracted fromin vitro cultured trophozoites. Lane 1, SDS–PAGE analysis; lane 2, Western blotanalysis; M, marker. Marker and lane 1 were stained with Coomassie brilliant blue.

C

T

Fig. 4. Immunofluorescence assay performed on P. olseni trophozoites from Manilaclam tissue. T, trophozoites; C, connective tissue. Scale bar = 10 lm.


as Tetraselmis sp. and Skeletonema sp. The pre-immune IgG used asa negative control also failed to show any positive reaction to thevarious P. olseni life stages (Fig. 5).

1 For interpretation of color in Fig. 8, the reader is referred to the web version ofthis article.

3.2. Isolation and identification of P. olseni from marine sediment

Several prezoosporangia-like cells isolated from the Hwangdosediment were retained on the GF/C filter and were positivelystained with fluorescence in the immunofluorescence assay. Theprezoosporangia-like cells were approximately 30 lm in diameter


without nuclei, and strong fluorescence was seen along the cellsurface, indicating that they were P. olseni prezoosporangia (Fig. 6).

3.3. Identification of P. olseni in clam cadavers

Fig. 7 shows P. olseni in the gills of dead Manila clams. TheP. olseni cells were approximately 20 lm in diameter and werestained in Lugol’s iodine. As shown in the photograph, the cellswere at the 2-cell (A), 4-cell (B), and 32-cell (C) stages of zoospor-ulation and contained numerous motile zoospores.

3.4. Fecal and pseudofecal discharge of P. olseni

The presence of P. olseni in the clam fecal discharge retained onthe GF/C filters was confirmed using PCR and RFTM. The filterswere incubated in RFTM for 2 weeks, and up to two spherical cells(20 lm in diameter) were stained dark blue1 in Lugol’s iodine(Fig. 8A). Fig. 8B shows the PCR results for the positive control (i.e.,



PK-IgG

Pre-immuneIgG

ZoosporePrezoosporangia Trophozoite

D

G

J K L

H I

E F

B CA

Fig. 5. Confocal microscopic images depicting P. olseni antibody binding to prezoosprangium, trophozoite and zoospore stages of P. olseni isolated from Manila clams andcultured in vitro. A, B, C, G, H, and I are fluorescent confocal images. D, E, F, J, K, and L are differential interference contrast images. The label H = head and F = flagellum. Scalebars = 50 lm in images A, D, G, and J, 10 lm in images B, C, E, F, H, and K, and 5 lm in images I and L.


P. olseni cultured in vitro) and those for the DNA extracts from thefecal discharges of clams retained on the filters. The positive controland the fecal discharges of three clams exhibited a 661-bp amplicon,indicating the presence of P. olseni. PCR confirmed that the sphericalcells stained dark blue in the RFTM assay were P. olseni prezoosporangia.

A positive relationship was found between the P. olseni totalbody burden and the number of P. olseni cells discharged throughfeces and pseudofeces. The total body burden of P. olseni in clamsused in the fecal discharge experiment varied from 0 to1,726,645 cells/g tissue wet weight (TWT). Notably, no P. olsenicells were found in the feces or pseudofeces of clams whose totalP. olseni body burden was less than 500,000 cells/g TWT.

4. Discussion

The rabbit anti-P. olseni antibody developed in this study suc-cessfully recognized the trophozoite, prezoosporangia, and zoo-spore head but did not react to the negative controls (i.e.,diatoms) in the immunofluorescence assay. As shown in Fig. 5,the antibody–antigen reaction as fluorescence was limited to theprezoosporangia and cytoplasm of trophozoites (Figs. 4 and 5).Western blotting (Fig. 3) indicated that the antigens were peptides


of 55.9, 24.0, and 19.0 kDa. Results of the immunofluorescence andWestern blotting assay suggested that the antigens provoking anti-body production were cell membrane constituents of prezoospo-rangia and trophozoites, possibly lipoproteins forming the cellmembrane. Fig. 5 also demonstrated that the antibody–antigenreaction in the zoospores was localized only in the head, and noimmunological reaction was observed for the flagella, suggestingthat the antigens were distributed only in the zoospore head.

Antibodies developed using P. marinus or P. olseni prezoospo-rangia protein extracts as antigens often fail to show an immuno-logical reaction to other Perkinsus spp. life stages (Choi et al., 1991;Montes et al., 1995). In contrast, P. marinus or P. olseni-specific anti-bodies raised from the trophozoite protein extracts have shown astrong positive immunological reaction not only for trophozoitesbut also for prezoosporangia and zoospores (Dungan and Roberson,1993; Romestand et al., 2001; Montes et al., 2005). These resultsindicate that Perkinsus spp. protein expression varies dependingon the developmental stage, as suggested by Choi et al. (1991)and Montes et al. (2005), and that fewer proteins are expressedin prezoosporangia than in trophozoites. It is interesting that, un-like in Choi et al. (1991) and Montes et al. (1995), the antibodydeveloped in the present study recognized all P. olseni life forms,even though it was raised against the prezoosporangium, which



Fig. 6. Detection of P. olseni prezoosporangia from fecal and speudofecal samples using a polyclonal antibody (RbaPKIgG). FITC-labeled RbaPKIgG recognized the P. olseniprezoosporangium surface (A) but pre-immune IgG did not (B). (C and D) show light microscopic images of A and B, respectively. Scale bar = 30 lm.

Fig. 7. Various developmental stages of P. olseni zoosporulation in moribund clam tissue collected from a commercial clam bed. From the 2-cell stage to motile zoospores.Scale bar = 30 lm (A–C), 20 lm (D).


had been washed with strong NaOH. Accordingly, protein charac-terization at each developmental stage helped explain the varia-tion in antibody specificity. It is believed that at least one of the55.9-, 24.0-, and 19.2-kDa P. olseni proteins observed in the presentstudy are commonly present in P. olseni regardless of the develop-mental stage.

Dungan and Roberson (1993) tested the immunological cross-reactivity of rabbit anti-P. marinus polyclonal antibody to P. atlan-ticus (=P. olseni) infecting the clam, R. decussatus, and P. olseni inabalone, Haliotis laevigata. In an immunofluorescence assay the


anti-P. marinus antibody bound to P. olseni and P. atlanticus tropho-zoites, suggesting that P. marinus and P. olseni may share commonimmunogens. Romestand et al. (2001) also examined cross-reactiv-ity of anti-P. marinus monoclonal antibody to P. olseni infectingR. decussatus. The monoclonal antibody also bound to P. olseni pre-served in histological preparations of R. decussatus in an immunof-lourescence assay. In the present study, the specificity of the rabbitanti-P. olseni IgG was not tested with other species of Perkinsus orwith P. olseni occurring in other host organisms; therefore theantibody developed in this study may cross react with other



Fig. 8. Detection of P. olseni prezooporangia using the Percoll gradient-GF/C filter method and PCR analysis. After a 2-week incubation in RFTM, prezoosporangia were stainedwith Lugol’s iodine. (A) An expected amplicon size (661 bp) was found in three of five samples. Lanes 1–5, DNA from feces and pseudofeces; lane 6, DNA from in vitro culturedP. olseni; lane 7, no P. olseni DNA was added. Scale bar = 50 lm.


Perkinsus species, as was reported by Dungan and Roberson (1993)and Romestand et al. (2001).

A new Perkinsus species co-infecting R. philippinarum withP. olseni has been reported in Japan. Dungan and Reece (2006) iso-lated Perkinsus spp. cells from apparently healthy and moribundclams collected from Mie Prefecture Japan and propagated themin vitro. A PCR assay and subsequent DNA sequence analysis per-formed on the in vitro cultured isolates indicated that there wereat least two Perkinsus spp. infecting Manila clams at this site.Accordingly, Dungan and Reece (2006) reported one of the isolateas P. honshuensis, a new Perkinsus species. Recently, co-infection ofP. honshuensis and P. olseni in Manila clams was confirmed in Hiro-shima, Japan by Takahashi et al. (2009). In the present study, cross-reactivity of the rabbit anti-P. olseni IgG to P. honshuensis was nottested, although a certain level of serological affinity is expectedbetween P. olseni and P. honshuensis.

In the summer of 2009, a survey for P. honshuensis infections inManila clams was conducted in Korea. Using P. honshuensis-specific(Dungan and Reece, 2006; Takahashi et al., 2009) and P. olseni-spe-cific (Park et al., 2005) primers in a PCR assay, Kang et al. (unpub-lished data) investigated P. olseni and P. honshuensis co-infection inclams collected from 23 localities including the present study site,Hwangdo. Of the 250 clams analyzed in the survey, all exhibitedP. olseni-positive nucleotide bands, while none showed P. honshuen-sis-positive bands, suggesting that clams in Korean waters are freefrom P. honsheunsis infection and P. olseni is the only Perkinsus spe-cies responsible for Perkinsosis at the Korean sites. The fact thatthe PCR assay showed that clams collected from Hwangdo werenot infected with P. honshuensis is a strong indication that the Perk-insus cell isolated from the marine sediment in this study (Fig. 6) is P.olseni.

Although little information is currently available on the trans-mission routes of Perkinsus spp. infection in marine mollusks innatural habitats, it is hypothesized that live host organisms suchas oysters and clams void Perkinsus spp. cells through feces andpseudofeces in hosts, and Perkinsus spp. cells are released fromdecaying host tissue in dead samples. Vector species transmission(e.g., ectoparasitic snail, Boonea impressa, White et al., 1987;Wilson et al., 1988) is another possible way that Perkinsus spp. isspread. However, as no vector species for P. olseni has been identi-fied in Asian waters, we focused on the first two transmissionmethods in the present study.

We observed that P. olseni in Manila clams shed only one to twocells through feces and pseudofeces within 24 h. This result standsin stark contrast to the number of cells shed by P. marinus from oys-ters: 10–100,000 P. marinus cells were discharged in the feces oftheir hosts within 24 h (Bushek et al., 2002b). However this maynot be an appropriate comparison because the oysters were chal-lenged by injection of 1,000,000 P. marinus cells/g oyster. Even so,


such small numbers of voiding cells in Manila clams suggest that fe-cal discharge from P. olseni is an insignificant method for pathogentransmission in a natural clam bed. Bushek et al. (2002b) reportedthat P. marinus fecal discharge was highly correlated with P. marinusinfection intensity in whole tissue, and it was suggested thatP. marinus cells in feces could be useful for determining P. marinustotal body burden without sacrificing oysters. Although we alsofound a relationship between P. olseni cells in the pseudofeces andfeces of clams and total body burden, estimating P. olseni total bodyburden in Manila clams using this method is inappropriate, as only afew P. olseni cells are discharged through feces or pseudofeces.

Enlarged P. marinus trophozoites have been observed in decom-posed oyster tissue (see review by Villalba et al. (2004)). In addi-tion, zoospores were released when prezoosporangia wereisolated from decomposing tissue and placed in aerated seawater(Perkins, 1968). According to Mackin (1962), P. marinus can betransmitted directly from **P. marinus–infected decaying oysters(i.e., gaping oysters) to other uninfected oysters, as all P. marinuslife stages appear to be infective. These studies suggest the exis-tence of prezoosporangia and zoospores in nature. Evidence thatthese Perkinsus spp. cells occur in nature was also observed inthe present study: P. olseni prezoosporangia and zoospores wereformed in the same decomposing clams collected from a commer-cial clam bed. This result strongly suggests that prezoosporangiaand zoospores occur naturally and develop simultaneously in thesame moribund host organism.

Bushek et al. (2002b) suggested that P. marinus discharged fromdead oysters was a more significant mode of infection than fecesand pseudofeces, because only 5% of P. marinus in the whole bodyof the host was shed through feces. Ragone Calvo et al. (2003) ob-served a high occurrence of P. marinus cells in the water columnduring a mass mortality of oysters, strongly supporting the ideaof Bushek et al. (2002b). The present study also observed a greatnumber of prezoosporangia and zoospores in the carcasses of gap-ing clams, suggesting that release from decomposing clam tissuemay be a major means of P. olseni transmission in Manila clams.Park et al. (2006) also reported that the highest infection intensityof P. olseni observed in clams in Gomso Bay, Korea, coincided withmass mortality of the Manila clam observed in the habitat.Although it is unclear whether such high infection intensity wascaused by P. olseni proliferation in the host or transmitted by filtra-tion of pathogen-contaminated seawater, clams could be exposedto high levels of P. olseni cells released from dead clams during amortality event. Andrews (1988) also found that P. marinus trans-mission occurred during a period of high oyster mortality in sum-mer and early fall, as infective P. marinus cells were disseminatedupon dead and decomposing infected oysters. Mackin (1962)emphasized that a large dose of P. marinus is required to developa rapid infection in the field. Accordingly, it is recommended that




gaping clams be removed from clam beds as quickly as possibleduring periods of high mortality to diminish the spread of P. olsenicells by tide and wave action.

To determine the transmission mechanisms of P. olseni in thenatural environment, it is essential to develop a sensitive diagnos-tic technique to detect P. olseni in seawater or sediment. AlthoughRFTM and 2 M-NaOH tissue lysis methods (Choi et al., 1989) havebeen successfully used to diagnose Perkinsus spp. infections, sev-eral molecular biology techniques have been developed to identifydifferent Perkinsus species (see review by Villalba et al. (2004)).Audemard et al. (2006) developed a real-time PCR method andused it to diagnose and quantify P. marinus in the ChesapeakeBay, Maryland, USA. Using real-time PCR, they estimated 1200 P.marinus cells/L in August 2003. Ragone Calvo et al. (2003) esti-mated the number of P. marinus cells in the water column usinga flow cytometric immunodetection method and reported 11,900P. marinus cells/L in the lower York River, Virginia, USA, in August1995. These studies strongly suggest that seawater is an importantmedium through which P. marinus can be actively transmitted asoysters in the infected area filter the pathogen-contaminated sea-water for feeding. Recently, real-time PCR technique was also suc-cessfully applied in detection of QPX (hard clam Mercenariamercenaria pathogen) in marine sediments (Liu et al., 2009). Inaddition to molecular technique, immunological techniques havebeen developed to diagnose and localize pathogens in host tissue.Polyclonal and monoclonal antibodies against P. marinus and theirextracellular proteins were also developed in several studies toinvestigate Perkinsosis (Dungan and Roberson, 1993; Ottingeret al., 2001; Romestand et al., 2001; Bushek et al., 2002a).

We applied the rabbit anti-P. olseni antibody for the first time tolocate P. olseni cells in the bottom substrate, which is inhabited byinfected clams. It is likely that P. olseni is released from infectedclams via feces and pseudofeces as well as decomposing clam tis-sue and then is retained in the sediment. To confirm this, we usedpolyclonal antibodies to detect P. olseni in the sediment in Hwang-do, where the prevalence is almost 100% during any season, andthe infection intensity ranges seasonally from 500,000 to3,000,000 cells/g TWT (unpublished data). As shown in Fig. 6,P. olseniprezoosporangia-like particles retained on filter paperwere stained positive with the rabbit anti-P. olseni antibody. Thisresult confirms that the particles are possibly prezoosporangiaand that P. olseni is present on the surface of the sediment. Conse-quently, it is believed that P. olseni prezoosporangia present in thesediment are re-suspended and circulated in the environment bytide and wave action and then ingested by clams through activeseawater filtering. It is notable that we could detect P. olseni cellsin only 200 g marine sediment. This is attributable to the extraor-dinarily high P. olseni infection level in the study area. Our fieldsurvey of P. olseni infection in Manila clams on the west coast ofKorea indicated that the prevalence and infection intensity inHwangdo was the highest among 20–25 different clam habitatssurveyed in 2007 and 2009 (unpublished data).

In conclusion, the rabbit anti-P. olseni antibody developed inthis study enabled us for the first time to detect P. olseni prezoosp-orangia in the sediment of a clam culture ground. Our study indi-cates that P. olseni prezoosporangia and zoospores, which aredischarged mainly from decomposing clam tissue, are distributedin the sediment and transmitted to new host clams by active feed-ing activity (i.e., filtration) when P. olseni cells are re-suspendedand circulated in the water column.

Acknowledgments

We appreciate the staff of the Shellfish Research and AquacultureLaboratory at Jeju National University for their data acquisition. Thisstudy was supported by a grant (F20814208H220000110) from Fish-


eries Technology Development Program funded by Ministry forFood, Agriculture, Forestry and Fisheries of Republic of Korea andwe appreciate for the support.

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isolation and identification of perkinsus olseni from feces

Technology

olseni life

olseni antibody

olseni igg

olseni infec

olseni prezoosporangium

infected clam tissue

olseni transmission

higher clam