Journal of Invertebrate Pathology xxx (2010) xxx–xxx



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K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx                                                  ...
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Isolation and identification of Perkinsus olseni from feces ...

  1. 1. Journal of Invertebrate Pathology xxx (2010) xxx–xxx Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip Isolation and identification of Perkinsus olseni from feces and marine sediment using 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 Korea b 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 Korea c Department of Biochemistry, College of Medicine, Jeju (Cheju) National University, 66 Jejudaehakno, Jeju 690-756, Republic of Korea d 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: Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a pro- Received 13 March 2010 tozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decompos- Accepted 28 July 2010 ing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni Available online xxxx antibody 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- Keywords: ing the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were Perkinsus olseni successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where Ruditapes philippinarum Transmission P. olseni–associated clam mortality has recurred for the past decade. Purified cells were positively stained Polyclonal antibody with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the Fecal discharge presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were Korea observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1–2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction density than natural habitats. Mass mortalities of Manila clams in commercial clam beds in late summer or early spring have been Perkinsosis is an epidemic disease that occurs in commercially reported in Korea, and extremely high levels of P. olseni in clams important marine mollusks, including oysters, clams, and abalones. were partly responsible (Park et al., 2006). Recently, a new Perkin- Perkinsus marinus and P. olseni are the two pathogens primarily sus species was isolated and propagated from Manila clam in Japan responsible for perkinsosis (Perkins, 1996; Bondad-Reantaso and reported as P. honshuensis n. sp. (Dungan and Reece, 2006). et al., 2001; Office International des Epizooties, 2004; Villalba Auzoux-Bordenave et al. (1995) demonstrated the P. olseni life et al., 2004). Perkinsosis of Manila clam Ruditapes philippinarum cycle as a trophozoite in infected host tissue, a prezoosporangium is caused by P. olseni and has been reported from tidal flats and and a motile bi-flagellated zoospore (see also Villalba et al., 2004). sand beaches along the coastal Yellow Sea of Korea and China As observed in P. marinus, the P. olseni prezoosporangium stage can and in the Ariake Sound in Japan (Choi and Park, 1997; Hamaguchi be induced when infected host tissue containing trophozoites are et al., 1998; Park and Choi, 2001; Park, 2005; Liang and Liang, placed in an anaerobic medium such as fluid thioglycollate med- 2007; Park et al., 2008; Uddin et al., 2010). High levels of P. olseni ium fortified with antibiotics and salt (RFTM, see Ray, 1952 and infection often cause severe host tissue inflammation and interfere Ray, 1966). Numerous zoospores are subsequently produced inside with the gametogenesis in clams (Villalba et al., 2005; Park, 2005; the prezoosporangia and released through a discharge tube when Park et al., 2006). According to Park and Choi (2001), P. olseni infec- the prezoosporangia are placed in aerated seawater (Auzoux- tion among Manila clam populations in Korean waters varies spa- Bordenave et al., 1995; Ahn and Kim, 2001; Park et al., 2005). How- tiotemporally, and the infection intensity and prevalence are often ever, the P. olseni prezoosporangium and zoospore stages have only higher in commercial clam beds, which have a much higher clam been confirmed under laboratory conditions and have yet to be ob- served in the field. ⇑ Corresponding author. Fax: +82 64 756 3493. In the early description of P. marinus, the first Perkinsus sp. de- E-mail address: skchoi@jejunu.ac.kr (K.-S. Choi). scribed, studies have reported enlarged trophozoites in moribund 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.07.006 Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006
  2. 2. 2 K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx oysters (Ray and Mackin, 1954; Mackin, 1962). Subsequently, tract mixed with an equal volume of Freund’s complete adjuvant. Perkins (1968) and Valiulis and Mackin (1969) observed Perkinsus Two weeks after the initial injection, the rabbit received 0.5 ml of sp. zoosporulation when prezoosporangia isolated from moribund 100 lg/ml P. olseni prezoosporangia protein extract mixed with clams were placed in aerated seawater. These studies suggested Freund’s incomplete adjuvant. The booster injection was continued that dispersal of P. marinus from gaping infected oysters in nature on a biweekly basis over 6 weeks. The specificity of the rabbit anti- is an important means of pathogen transmission. In addition to the P. olseni serum obtained from the rabbit after completing the release of P. marinus from cadavers, fecal discharge is an important immunization was tested using an enzyme-linked immunosorbent route for P. marinus transmission (Andrews and Hewatt, 1957; assay (ELISA). The rabbit antiserum initially showed weak but rec- Scanlon et al., 1997). For example, Bushek et al. (1997, 2002b) ob- ognizable cross-reactivity to proteins extracted from P. olseni-free served a massive release of P. marinus in the feces of live C. virginica clam tissue. The cross-reacting antibodies were removed from artificially infected with P. marinus. Unlike efforts to identify and the antiserum using an immunoadsorbent prepared from unin- isolate various P. marinus life stages, no studies have reported fected Manila clam tissue according to Fuchs and Sela (1979). After observations of various P. olseni life stages in the field. removing the cross-reacting antibodies, we tested the specificity Immunological techniques are excellent tools for investigating again using the ELISA, and no cross-reaction to P. olseni-free clam parasitic organisms in marine environment. Once an antibody is tissue was demonstrated. The rabbit anti-P. olseni IgG (PK-IgG) raised from a molecule of interest such as protein extracts of sin- was subsequently isolated from the antiserum by precipitating gle-celled or metazoan pathogen, the target molecule can be visu- the PK-IgG with saturated ammonium sulfate solution. alized or quantified using various immunological techniques. Western blotting was performed to characterize the P. olseni Using P. marinus hypnospore protein extract as immunogen, protein antigens. The proteins were extracted from trophozoites Dungan and Roberson (1993) first developed P. marinus-specific cultured in vitro and prezoosporangia produced in RFTM culture monoclonal as well as polyclonal antibodies to detect the parasite using lysis buffer, and the proteins were precipitated with trichlo- in C. virginica. The murine and rabbit polyclonal antibodies raised roacetic acid. Then 10 lg of P. olseni protein was loaded onto a 10% against P. marinus hypnospore did show a strong positive immuno- SDS–polyacrylamide gel for electrophoresis (SDS–PAGE). The pep- logical reaction to hypnospore as well as to trophozoites and zoo- tides separated on SDS–PAGE under reducing conditions were spores (Dungan and Roberson, 1993). Accordingly, P. marinus transferred onto a polyvinyl difluoride membrane. The membrane trophozoites distributed in digestive gland and intestine of an in- was blocked with 5% bovine serum albumin (BSA), and 4 lg/ml PK- fected oyster was successfully visualized using fluorescence immu- IgG was added to the membrane as the primary antibody. After nostaining technique in their study. Monoclonal antibody against three washes with Tris-buffered saline containing 1% Tween-20 P. marinus was also developed for quantification of P. marinus tro- (TBST), the membrane was incubated in horseradish peroxidase- phozoite and Romestand et al. (2001) estimated the parasite bur- conjugated goat anti-rabbit IgG (0.2 lg/ml) as the secondary anti- dens from infected oysters using the monoclonal antibody in a body. After several washes with TBST, the immunoreactive peptide competitive ELISA. bands in the membrane were visualized using enhanced chemilu- In an effort to detect various P. olseni life stages under labora- minescence (ECL) detection reagent. tory and field conditions, we developed a polyclonal antibody against P. olseni. In the present study, we report, for the first time, 2.2. Testing the immunological specificity of the PK-IgG the presence of P. olseni cells in commercial clam bed sediment and in infected clam feces and pseudofeces as confirmed by immuno- The specificity of PK-IgG for various P. olseni life stages as well logical and molecular biological probes. as other organisms was tested using indirect immunofluorescence assay. The prezoosporangia were produced using Ray’s FTM assay (Ray, 1954). The trophozoites were prepared from in vitro culture 2. Materials and methods according to the method of Ordas and Figueras (1998). Zoospores were induced by incubating the prezoosporangia in aerated seawa- 2.1. Development of a P. olseni-specific antibody ter for 2 days (30 ppt, 25 °C). The different types of P. olseni cells were fixed in 4% paraformaldehyde and 0.2 M cacodylate buffer Perkinsus olseni infected clams were supplied from Hwangdo for 2 h at 4 °C. Following washes with PBS containing 1% Tween- (Fig. 1), where P. olseni prevalence was known to be 90–100% year 20 (PBST), the P. olseni cells were incubated for 30 min in 5% (w/ round (unpublished data) and incubated in RFTM for 1 week in the v) BSA and PBST as a blocking agent. P. olseni cells were reacted dark (Ray, 1952, 1966). To harvest P. olseni prezoosporangia, the with PK-IgG (100 lg/ml) at room temperature for 1 h and then RFTM cultivated clam gill tissues were placed in a petri-dish and incubated with fluorescein isothiocyanate (FITC)-conjugated goat minced using a blade. The prezoosporangia and minced gill tissue anti-rabbit IgG (1:400 dilution; Sigma, St. Louis, MO, USA) for mixtures were filtered through 100 and 63 lm mesh screens to re- 1 h. Fluorescent images of prezoosporangia, trophozoites, and zoo- move the large clam tissue debris. The prezoosporangia retained spores labeled with FITC were acquired using a confocal scanning on the 63 lm mesh screen were harvested and washed several system (Olympus FV 300). As negative controls, the pre-immune times with phosphate buffered saline (0.15 M NaCl, pH 7.4). The rabbit IgG and two species of microalgae Tetraselmis sp. and washed prezoosporangia were further treated with 2 M NaOH at Skeletonema sp. were used in the immunofluorescence assay. 60 °C for 10 min to remove fine clam tissue particles attached on Tetraselmis and Skeletonema spp. have been frequently reported the cell surface. The purified prezoosporangia were re-suspended from tidal flats on the west coast of Korea where clams in the pres- in PBS and homogenized using an ultrasonifier to extract protein ent study were collected and might have been confused with Perk- from the cells. Level of P. olseni protein in the homogenate was insus sp. in sediment samples (NFRDI, 2001; Moon and Choi, 2003). determined using BCA protein assay kit (Pierce) and the protein An immunohistochemistry assay was also performed to localize concentration was adjusted to 100 lg/ml to be served as an P. olseni trophozoites in the clam tissue. A cross-section of the antigen. Manila clam was embedded in paraffin, sliced at 6-lm thickness, A New Zealand white rabbit was immunized with the prezoosp- deparaffinized, and rehydrated. The sections were incubated for orangia protein extract over an 8-week period according to Park 30 min in 5% (w/v) BSA and PBS Triton X-100 as a blocking agent. and Choi (2004). The rabbit initially received subcutaneous injec- After blocking, the tissue was reacted with PK-IgG (100 lg/ml) at tion of 0.5 ml of 100 lg/ml P. olseni prezoosporangia protein ex- room temperature for 1 h. The tissue was washed three times with Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006
  3. 3. K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx 3 N 45° W E S 40° Korea Hwangdo East Sea China 35° Yellow Sea 10 km 30° 115° 120° 125° 130° Fig. 1. Map of study area: Hwangdo (36°350 50.8700 E, 126°220 53.8100 N), on the west coast of Korea. PBST, and FITC-conjugated goat anti-rabbit IgG (1:400 dilution, 2.5. Isolation of P. olseni from fecal discharge Sigma) was added. The FITC-stained P. olseni trophozoites were washed again with PBST, mounted in glycerol-PBS solution (1:1), Sixteen clams collected from Hwangdo (Fig. 1) in August 2005 and observed under a fluorescence microscope. were individually placed on top of 50-ml conical tubes and placed in a 50-L tank containing filtered and aerated seawater (Fig. 2). After 24 h of depuration, feces and pseudofeces of each clam accumulated 2.3. Isolation of P. olseni from marine sediment and immunological on the bottoms of the conical tubes were collected using a pasture identification pipette and filtered individually through GF/C filter. To identify P. olseni cells present in the fecal discharge, feces and pseudofeces of To locate P. olseni cells in the marine environment, we took a five individual clams retained on the filters were pooled and the total 10 Â 10 Â 2 cm (ca. 200 g) section of bottom sediment from a sandy DNA in the discharge was extracted using DNA extraction kit (Qia- mud tidal flat in Hwangdo off the west coast of Korea in late August gen). The extracted DNA was amplified using a P. olseni–specific pri- 2005 (Fig. 1). A high level of P. olseni infection has been reported in mer pair (F 50 -CATTATCGAGGTCTGTGGTGACG-30 , R 50 -ACGATAGG Hwangdo, and mass mortalities of clams had occurred in the tidal flat TCTGCTGAGCAAGC-30 , Park et al., 2002). The PCR product was elec- during the springs of 2004 and 2005 (NFRDI, 2007). The sediment trophoresed in 1% agarose, and the size of the amplicon was deter- was mixed with 200 ml PBS, stirred, and sieved through a 100-lm mined. The other GF/C filters of 11 clams containing their feces filter to remove sand particles. Then 30-ml aliquots of each of the fil- and pseudofeces were incubated in RFTM for 2 weeks and stained trates were placed into 50 ml conical tubes, and 9 ml of 100% Percoll with Lugol’s iodine. Meanwhile, the 11 clams were opened and incu- fluid was added to remove fine sediment particles by gravity. The bated in RFTM for the same period as above. Finally, the numbers of suspension containing low density particles was separated from P. olseni prezoosporangia retained on each filter and in whole body the tube, washed several times with PBS (1200g for 10 min) and fil- were counted under a microscope. 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% 3. Results 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 a 3.1. Specificity of P. olseni-specific antibody negative control. After 1 h incubation at room temperature, the fil- ter was washed three times with PBST, and FITC-conjugated goat SDS–PAGE revealed that the proteins extracted from P. olseni anti-rabbit IgG (1:500 dilution) was added as the secondary anti- trophozoites included peptides of 55.9, 24.0, 19.2, 10.6, 14.9, body. The filter was incubated for 1 h at room temperature and 10.6, and >9 kDa (Fig. 3, lane 1). For Western blotting, 55.9-, washed three times with PBST. Finally, the immunologically 24.0-, and 19.2-kDa peptides were coupled with the antibody stained filter was examined under a fluorescence microscope to lo- developed in this study (Fig. 3, lane 2). The trophozoite (T) cell cate any P. olseni cells retained on the filter. walls and membranes were stained with PK-IgG in the immunoflu- orescence assay, but the cytoplasm and the connective tissues 2.4. Microscopic examination of clam cadavers were not, indicating that the antibody specifically recognized P. olseni cells in the host tissue (Fig. 4). In the immunofluorescence Numerous gaping clams were collected from Hwangdo in Au- assay, the PK-IgG positively reacted with all P. olseni life stages; the gust 2005 to examine P. olseni in dead or dying clams. Gill tissue PK-IgG strongly reacted with proteins on the surface of prezoosp- was excised from the cadavers, stained with Lugol’s iodine, and orangia, entire trophozoites, and the zoospore heads (Fig. 5). In examined under a light microscope. contrast, the PK-IgG did not react to the negative controls, such Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006
  4. 4. 4 K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx Fig. 2. Experimental design for collecting feces and pseudofeces of R. philippinarum in 50-ml conical tubes. T C Fig. 4. Immunofluorescence assay performed on P. olseni trophozoites from Manila clam tissue. T, trophozoites; C, connective tissue. Scale bar = 10 lm. without nuclei, and strong fluorescence was seen along the cell surface, 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. The Fig. 3. SDS–PAGE and Western blot analyses of P. olseni proteins extracted from P. olseni cells were approximately 20 lm in diameter and were in vitro cultured trophozoites. Lane 1, SDS–PAGE analysis; lane 2, Western blot stained in Lugol’s iodine. As shown in the photograph, the cells analysis; M, marker. Marker and lane 1 were stained with Coomassie brilliant blue. were at the 2-cell (A), 4-cell (B), and 32-cell (C) stages of zoospor- ulation and contained numerous motile zoospores. as Tetraselmis sp. and Skeletonema sp. The pre-immune IgG used as a negative control also failed to show any positive reaction to the 3.4. Fecal and pseudofecal discharge of P. olseni various P. olseni life stages (Fig. 5). The presence of P. olseni in the clam fecal discharge retained on the GF/C filters was confirmed using PCR and RFTM. The filters 3.2. Isolation and identification of P. olseni from marine sediment were incubated in RFTM for 2 weeks, and up to two spherical cells (20 lm in diameter) were stained dark blue1 in Lugol’s iodine Several prezoosporangia-like cells isolated from the Hwangdo (Fig. 8A). Fig. 8B shows the PCR results for the positive control (i.e., sediment were retained on the GF/C filter and were positively stained with fluorescence in the immunofluorescence assay. The 1 For interpretation of color in Fig. 8, the reader is referred to the web version of prezoosporangia-like cells were approximately 30 lm in diameter this article. Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006
  5. 5. K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx 5 Prezoosporangia Trophozoite Zoospore A B C PK-IgG D E F Pre-immune IgG G H I J K L Fig. 5. Confocal microscopic images depicting P. olseni antibody binding to prezoosprangium, trophozoite and zoospore stages of P. olseni isolated from Manila clams and cultured 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. Scale bars = 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 the of 55.9, 24.0, and 19.0 kDa. Results of the immunofluorescence and fecal discharges of clams retained on the filters. The positive control Western blotting assay suggested that the antigens provoking anti- and the fecal discharges of three clams exhibited a 661-bp amplicon, body production were cell membrane constituents of prezoospo- indicating the presence of P. olseni. PCR confirmed that the spherical rangia and trophozoites, possibly lipoproteins forming the cell cells stained dark blue in the RFTM assay were P. olseni prezoosporangia. membrane. Fig. 5 also demonstrated that the antibody–antigen A positive relationship was found between the P. olseni total reaction in the zoospores was localized only in the head, and no body burden and the number of P. olseni cells discharged through immunological reaction was observed for the flagella, suggesting feces and pseudofeces. The total body burden of P. olseni in clams that the antigens were distributed only in the zoospore head. used in the fecal discharge experiment varied from 0 to Antibodies developed using P. marinus or P. olseni prezoospo- 1,726,645 cells/g tissue wet weight (TWT). Notably, no P. olseni rangia protein extracts as antigens often fail to show an immuno- cells were found in the feces or pseudofeces of clams whose total logical reaction to other Perkinsus spp. life stages (Choi et al., 1991; P. olseni body burden was less than 500,000 cells/g TWT. Montes et al., 1995). In contrast, P. marinus or P. olseni-specific anti- bodies raised from the trophozoite protein extracts have shown a strong positive immunological reaction not only for trophozoites 4. Discussion but also for prezoosporangia and zoospores (Dungan and Roberson, 1993; Romestand et al., 2001; Montes et al., 2005). These results The rabbit anti-P. olseni antibody developed in this study suc- indicate that Perkinsus spp. protein expression varies depending cessfully recognized the trophozoite, prezoosporangia, and zoo- on the developmental stage, as suggested by Choi et al. (1991) spore head but did not react to the negative controls (i.e., and Montes et al. (2005), and that fewer proteins are expressed diatoms) in the immunofluorescence assay. As shown in Fig. 5, in prezoosporangia than in trophozoites. It is interesting that, un- the antibody–antigen reaction as fluorescence was limited to the like in Choi et al. (1991) and Montes et al. (1995), the antibody prezoosporangia and cytoplasm of trophozoites (Figs. 4 and 5). developed in the present study recognized all P. olseni life forms, Western blotting (Fig. 3) indicated that the antigens were peptides even though it was raised against the prezoosporangium, which Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006
  6. 6. 6 K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx Fig. 6. Detection of P. olseni prezoosporangia from fecal and speudofecal samples using a polyclonal antibody (RbaPKIgG). FITC-labeled RbaPKIgG recognized the P. olseni prezoosporangium 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- anti-P. marinus antibody bound to P. olseni and P. atlanticus tropho- terization at each developmental stage helped explain the varia- zoites, suggesting that P. marinus and P. olseni may share common tion in antibody specificity. It is believed that at least one of the immunogens. Romestand et al. (2001) also examined cross-reactiv- 55.9-, 24.0-, and 19.2-kDa P. olseni proteins observed in the present ity of anti-P. marinus monoclonal antibody to P. olseni infecting study are commonly present in P. olseni regardless of the develop- R. decussatus. The monoclonal antibody also bound to P. olseni pre- mental stage. served in histological preparations of R. decussatus in an immunof- Dungan and Roberson (1993) tested the immunological cross- lourescence assay. In the present study, the specificity of the rabbit reactivity of rabbit anti-P. marinus polyclonal antibody to P. atlan- anti-P. olseni IgG was not tested with other species of Perkinsus or ticus (=P. olseni) infecting the clam, R. decussatus, and P. olseni in with P. olseni occurring in other host organisms; therefore the abalone, Haliotis laevigata. In an immunofluorescence assay the antibody developed in this study may cross react with other Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006
  7. 7. K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx 7 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 stained with 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 cultured P. olseni; lane 7, no P. olseni DNA was added. Scale bar = 50 lm. Perkinsus species, as was reported by Dungan and Roberson (1993) such small numbers of voiding cells in Manila clams suggest that fe- and Romestand et al. (2001). cal discharge from P. olseni is an insignificant method for pathogen A new Perkinsus species co-infecting R. philippinarum with transmission in a natural clam bed. Bushek et al. (2002b) reported P. olseni has been reported in Japan. Dungan and Reece (2006) iso- that P. marinus fecal discharge was highly correlated with P. marinus lated Perkinsus spp. cells from apparently healthy and moribund infection intensity in whole tissue, and it was suggested that clams collected from Mie Prefecture Japan and propagated them P. marinus cells in feces could be useful for determining P. marinus in vitro. A PCR assay and subsequent DNA sequence analysis per- total body burden without sacrificing oysters. Although we also formed on the in vitro cultured isolates indicated that there were found a relationship between P. olseni cells in the pseudofeces and at least two Perkinsus spp. infecting Manila clams at this site. feces of clams and total body burden, estimating P. olseni total body Accordingly, Dungan and Reece (2006) reported one of the isolate burden in Manila clams using this method is inappropriate, as only a as P. honshuensis, a new Perkinsus species. Recently, co-infection of few P. olseni cells are discharged through feces or pseudofeces. P. honshuensis and P. olseni in Manila clams was confirmed in Hiro- Enlarged P. marinus trophozoites have been observed in decom- shima, Japan by Takahashi et al. (2009). In the present study, cross- posed oyster tissue (see review by Villalba et al. (2004)). In addi- reactivity of the rabbit anti-P. olseni IgG to P. honshuensis was not tion, zoospores were released when prezoosporangia were tested, although a certain level of serological affinity is expected isolated from decomposing tissue and placed in aerated seawater between P. olseni and P. honshuensis. (Perkins, 1968). According to Mackin (1962), P. marinus can be In the summer of 2009, a survey for P. honshuensis infections in transmitted directly from **P. marinus–infected decaying oysters Manila clams was conducted in Korea. Using P. honshuensis-specific (i.e., gaping oysters) to other uninfected oysters, as all P. marinus (Dungan and Reece, 2006; Takahashi et al., 2009) and P. olseni-spe- life stages appear to be infective. These studies suggest the exis- cific (Park et al., 2005) primers in a PCR assay, Kang et al. (unpub- tence of prezoosporangia and zoospores in nature. Evidence that lished data) investigated P. olseni and P. honshuensis co-infection in these Perkinsus spp. cells occur in nature was also observed in clams collected from 23 localities including the present study site, the present study: P. olseni prezoosporangia and zoospores were Hwangdo. Of the 250 clams analyzed in the survey, all exhibited formed in the same decomposing clams collected from a commer- P. olseni-positive nucleotide bands, while none showed P. honshuen- cial clam bed. This result strongly suggests that prezoosporangia sis-positive bands, suggesting that clams in Korean waters are free and zoospores occur naturally and develop simultaneously in the from P. honsheunsis infection and P. olseni is the only Perkinsus spe- same moribund host organism. cies responsible for Perkinsosis at the Korean sites. The fact that Bushek et al. (2002b) suggested that P. marinus discharged from the PCR assay showed that clams collected from Hwangdo were dead oysters was a more significant mode of infection than feces not infected with P. honshuensis is a strong indication that the Perk- and pseudofeces, because only 5% of P. marinus in the whole body insus cell isolated from the marine sediment in this study (Fig. 6) is P. of the host was shed through feces. Ragone Calvo et al. (2003) ob- olseni. served a high occurrence of P. marinus cells in the water column Although little information is currently available on the trans- during a mass mortality of oysters, strongly supporting the idea mission routes of Perkinsus spp. infection in marine mollusks in of Bushek et al. (2002b). The present study also observed a great natural habitats, it is hypothesized that live host organisms such number of prezoosporangia and zoospores in the carcasses of gap- as oysters and clams void Perkinsus spp. cells through feces and ing clams, suggesting that release from decomposing clam tissue pseudofeces in hosts, and Perkinsus spp. cells are released from may be a major means of P. olseni transmission in Manila clams. decaying host tissue in dead samples. Vector species transmission Park et al. (2006) also reported that the highest infection intensity (e.g., ectoparasitic snail, Boonea impressa, White et al., 1987; of P. olseni observed in clams in Gomso Bay, Korea, coincided with Wilson et al., 1988) is another possible way that Perkinsus spp. is mass mortality of the Manila clam observed in the habitat. spread. However, as no vector species for P. olseni has been identi- Although it is unclear whether such high infection intensity was fied in Asian waters, we focused on the first two transmission caused by P. olseni proliferation in the host or transmitted by filtra- methods in the present study. tion of pathogen-contaminated seawater, clams could be exposed We observed that P. olseni in Manila clams shed only one to two to high levels of P. olseni cells released from dead clams during a cells through feces and pseudofeces within 24 h. This result stands mortality event. Andrews (1988) also found that P. marinus trans- in stark contrast to the number of cells shed by P. marinus from oys- mission occurred during a period of high oyster mortality in sum- ters: 10–100,000 P. marinus cells were discharged in the feces of mer and early fall, as infective P. marinus cells were disseminated their hosts within 24 h (Bushek et al., 2002b). However this may upon dead and decomposing infected oysters. Mackin (1962) not be an appropriate comparison because the oysters were chal- emphasized that a large dose of P. marinus is required to develop lenged by injection of 1,000,000 P. marinus cells/g oyster. Even so, a rapid infection in the field. Accordingly, it is recommended that Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006
  8. 8. 8 K.-I. Park et al. / Journal of Invertebrate Pathology xxx (2010) xxx–xxx gaping clams be removed from clam beds as quickly as possible eries Technology Development Program funded by Ministry for during periods of high mortality to diminish the spread of P. olseni Food, Agriculture, Forestry and Fisheries of Republic of Korea and cells by tide and wave action. we appreciate for the support. To determine the transmission mechanisms of P. olseni in the natural environment, it is essential to develop a sensitive diagnos- References tic technique to detect P. olseni in seawater or sediment. Although RFTM and 2 M-NaOH tissue lysis methods (Choi et al., 1989) have Ahn, K.-J., Kim, K.H., 2001. Effect of temperature and salinity on the in vitro been successfully used to diagnose Perkinsus spp. infections, sev- zoosporulation of Perkinsus sp. in Manila clams Ruditapes philippinarum. Dis. eral molecular biology techniques have been developed to identify Aquat. Org. 48, 43–46. Andrews, J.D., 1988. Epizootiology of the disease caused by the oyster pathogen different Perkinsus species (see review by Villalba et al. (2004)). Perkinsus marinus and its effects on the oyster industry. Am. Fish. Soc. Spec. Audemard et al. (2006) developed a real-time PCR method and Publ. 18, 47–63. used it to diagnose and quantify P. marinus in the Chesapeake Andrews, J.D., Hewatt, W.G., 1957. Oyster mortality studies in Virginia. II. The fungus disease caused by Dermocystidium marinum in oysters of Chesapeake Bay, Maryland, USA. Using real-time PCR, they estimated 1200 P. Bay. Ecol. Monogr. 27, 1–26. marinus cells/L in August 2003. Ragone Calvo et al. (2003) esti- Audemard, C.L., Calvo, M.R., Paynter, K.T., Reece, K.S., Burreson, E.M., 2006. Real- mated the number of P. marinus cells in the water column using time PCR investigation of parasite ecology: in situ determination of oyster a flow cytometric immunodetection method and reported 11,900 parasite Perkinsus marinas transmission dynamics in lower Chesapeake Bay. Parasitology 132, 827–842. P. marinus cells/L in the lower York River, Virginia, USA, in August Auzoux-Bordenave, S., Vigario, A.M., Ruano, F., Domart-Coulon, I., Doumenc, D., 1995. These studies strongly suggest that seawater is an important 1995. In vitro sporulation of the clam pathogen Perkinsus atlanticus medium through which P. marinus can be actively transmitted as (Apicomplexa, Perkinsea) under various environmental conditions. J. Shellfish Res. 14, 469–475. oysters in the infected area filter the pathogen-contaminated sea- Bondad-Reantaso, M.G., McGladdery, S.E., Subasinghe, R.P., 2001. Asia Diagnostic water for feeding. Recently, real-time PCR technique was also suc- Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper No, 402, cessfully applied in detection of QPX (hard clam Mercenaria Supplement 2. Rome, FAO. 240 pp. Bushek, D., Allen, S.K., Alcox, K.A., Gustason, R., Ford, S.E., 1997. Response of mercenaria pathogen) in marine sediments (Liu et al., 2009). In Crassostrea virginica to in vitro-cultured Perkinsus marinus: preliminary addition to molecular technique, immunological techniques have comparison of three inoculation methods. J. Shellfish Res. 16, 479–485. been developed to diagnose and localize pathogens in host tissue. Bushek, D., Dungan, C.F., Lewitus, A.J., 2002a. Serological affinities of the oyster pathogen Perkinsus marinus (Apicomplexa) with some dinoflagellates Polyclonal and monoclonal antibodies against P. marinus and their (Dinophyceae). J. Eurkaryot. Microbiol. 49, 11–16. extracellular proteins were also developed in several studies to Bushek, D., Ford, S.E., Chintala, M.M., 2002b. Comparison of in vitro-cultured and investigate Perkinsosis (Dungan and Roberson, 1993; Ottinger wild type Perkinsus marinus. III. Fecal elimination and its role in transmission. Dis. Aquat. Org. 51, 217–225. et al., 2001; Romestand et al., 2001; Bushek et al., 2002a). Choi, K.-S., Park, K.-I., 1997. Report on the occurrence of Perkinsus sp. in the Manila We applied the rabbit anti-P. olseni antibody for the first time to Clam, Ruditapes philippinarum in Korea. Korean J. Aquacult. 10, 227–237. locate P. olseni cells in the bottom substrate, which is inhabited by Choi, K.-S., Wilson, E.A., Lewis, D.H., Powell, E.N., Ray, S.M., 1989. The energetic cost infected clams. It is likely that P. olseni is released from infected of Perkinsus marinus parasitism in oysters: quantification of the thioglycollate method. J. Shellfish Res. 8, 125–131. clams via feces and pseudofeces as well as decomposing clam tis- Choi, K.-S., Lewis, D.H., Powell, E.N., Frelier, P.F., Ray, S.M., 1991. A polyclonal sue and then is retained in the sediment. To confirm this, we used antibody developed from Perkinsus marinus hypnospores fails to cross-react polyclonal antibodies to detect P. olseni in the sediment in Hwang- with other life stages of P. marinus present in oyster Crassostrea virginica. J. Shellfish Res. 10, 411–415. do, where the prevalence is almost 100% during any season, and Dungan, C.F., Reece, K.S., 2006. In vitro propagation of two Perkinsus spp. Parasites the infection intensity ranges seasonally from 500,000 to from Japanese Manila clams Venerupis philippinarum and description of 3,000,000 cells/g TWT (unpublished data). As shown in Fig. 6, Perkinsus honshuensis n. sp.. J. Eukaryot. Microbiol. 53, 316–326. Dungan, C.F., Roberson, B.S., 1993. Binding specificities of monoclonal and P. olseniprezoosporangia-like particles retained on filter paper polyclonal antibodies to the protozoan oyster pathogen Perkinsus marinus. were stained positive with the rabbit anti-P. olseni antibody. This Dis. Aquat. Org. 15, 9–22. result confirms that the particles are possibly prezoosporangia Fuchs, S., Sela, M., 1979. Immunoadsorbents. In: Weir, D.M. (Ed.), Handbook of Experimental Immunology, vol. 1. Immunochenistry. Blackwell Scientific and that P. olseni is present on the surface of the sediment. Conse- Publications, Philadelphia, USA, pp. 10.1–10.6. quently, it is believed that P. olseni prezoosporangia present in the Hamaguchi, M.N., Suzuki, N., Usuki, H., Ishioka, H., 1998. Perkinsus protozoan sediment are re-suspended and circulated in the environment by infection in short-necked clam Tapes (=Ruditapes) philippinarum in Japan. Fish Pathol. 33, 473–480. tide and wave action and then ingested by clams through active Liang, Y., Liang, B., 2007. Spatial distribution of the protozoan parasite Perkinsus sp. seawater filtering. It is notable that we could detect P. olseni cells found in the Manila clams Ruditapes philippinarum in China. In: World in only 200 g marine sediment. This is attributable to the extraor- Aquaculture 2007 Abstract Book. San Antonio Convention Center, San dinarily high P. olseni infection level in the study area. Our field Antonio, Texas, pp. 533. Liu, Q.Q., Allam, B., Collier, J.L., 2009. Quantitative real-time PCR assay for QPX survey of P. olseni infection in Manila clams on the west coast of (Thraustochytriidae), a parasite of the hard clam (Mercenaria mercenaria). Appl. Korea indicated that the prevalence and infection intensity in Environ. Microbiol. 75, 4913–4918. Hwangdo was the highest among 20–25 different clam habitats Mackin, J.G., 1962. Oyster disease caused by Dermocystidium marinum and other microorganisms in Louisiana. Public. Ins. Mar. Sci. Univ. Texas 7, 132–229. surveyed in 2007 and 2009 (unpublished data). Montes, F., Durfort, M., García-Valero, J., 1995. Characterization and localization of In conclusion, the rabbit anti-P. olseni antibody developed in an Mr 225kDa polypeptide specifically involved in the defense mechanisms of this study enabled us for the first time to detect P. olseni prezoosp- the clam Tapes semidecussatus. Cell Tissue Res. 280, 27–37. Montes, F., Durfort, M., García-Valero, J., 2005. Ultrastructural localization of orangia in the sediment of a clam culture ground. Our study indi- antigenic determinants conserved during Perkinsus atlanticus trophozoites to cates that P. olseni prezoosporangia and zoospores, which are prezoosporangium differentiation. Dis. Aquat. Org. 66, 33–40. discharged mainly from decomposing clam tissue, are distributed Moon, S.-G., Choi, C.-M., 2003. A list of important species and distribution of marine phytoplankton in Korea. J. Environ. Sci. 7, 725–733 (in Korean with English in the sediment and transmitted to new host clams by active feed- abstract). ing activity (i.e., filtration) when P. olseni cells are re-suspended NFRDI, 2001. Development of Optimal Technology for Sustaining Production in and circulated in the water column. Shellfish Farm. Ministry of Maritime Affairs and Fisheries, p. 930 (in Korean with English abstract). NFRDI, 2007. Studies on the Management of the Tidal Flat Aquaculture Animals. Acknowledgments 2007 Annual Report of National Fisheries Research and Development Institute (NFRDI) of Korea, p. 113 (in Korean with English abstract). We appreciate the staff of the Shellfish Research and Aquaculture Office International des Epizooties, 2004. <http://www.oie.int/eng/maladies/ en_classification2007.htm?e1d7>. Laboratory at Jeju National University for their data acquisition. This Ordas, M.C., Figueras, A., 1998. In vitro culture of Perkinsus atlanticus, a parasite of study was supported by a grant (F20814208H220000110) from Fish- the carpet shell clam Ruditapes decussates. Dis. Aquat. Org. 33, 129–136. Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006
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Fish. Bull. 86, 553–566. Please cite this article in press as: Park, K.-I., et al. Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques. J. Invertebr. Pathol. (2010), doi:10.1016/j.jip.2010.07.006

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