Development of an immunofluorescence technique for detecting Pfiesteria piscicida

Development of an immunofluorescence technique for detecting Pfiesteria piscicida
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  Development of an immunofluorescence techniquefor detecting  Prorocentrum donghaiense  Lu Da-Zhi Wang  &  Xu-Guang HuangLeo Lai Chan  &  Hua-Sheng Hong Received: 9 May 2006 /Revised and accepted: 18 October 2006 / Published online: 19 January 2007 # Springer Science + Business Media B.V. 2007 Abstract  Prorocentrum donghaiense  Lu is a keyharmful algal bloom (HAB) species which is wide-spread along the China Sea coast. In this study,  P.donghaiense -specific antiserum was developed, and thedetection method, based on immunofluorescence (IF),was optimized. The antiserum was raised using 0.5% paraformaldehyde-fixed whole cells (WC) as antigen.The titer and the specificity were examined usingwhole-cell IF. Results showed that ethanol was aneffective decolorization reagent, and 80% ethanol wasable to minimize autofluorescence of cells. Samples preserved by freezing at   − 20°C or   − 80°C remainedabove 85% detection efficiency after 1-month storage.The antiserum against WC had a high titer (1:10000),and exhibited high specificity at species level. Theantiserum showed a weak cross reaction with the closelyrelated species  P. dentatum  HK,  P. dentatum CCMP1517 and  P. minimum  HK only at very lowdilution (1:5). However, it did not cross-react with thespecies from the same genus or other phytoplanktonspecies when the dilution reached or exceeded 1:100.Results from different   P. donghaiense  samples collectedat different growth phases or grown under different nutrient conditions showed no significant difference inIF intensity. In addition, the antiserum exhibited highspecific binding to  P. donghaiense  in both mixed phytoplankton samples and field samples. The resultsindicate that the technique is a potentially useful tool for the rapid identification of   P. donghaiense  and canfacilitate the analysis of various natural samples. Key words  Prorocentrum donghaiense  Lu.Antiserum.Immunofluorescence Introduction  Prorocentrum donghaiense  Lu, a key harmful algal bloom (HAB) species in China Sea coastal waters,was first reported and identified by Lu and Goebel(2001). In the past few years, this species has caused extensive blooms in the East China Sea, and has become of economic and public concern due to itsimpact on the marine ecosystem, aquaculture and public health (Zhou et al., 2003). Therefore, it has become essential to monitor   P. donghaiense , to provide important data for assessment and earlywarning of the hazards of   P. donghaiense  to fisheriesand aquaculture.Generally, the use of light-microscopy is sufficient toclassify and enumerate  P. donghaiense  from fieldsamples. However, this becomes difficult when  P. donghaiense  is only a minor component of planktonic J Appl Phycol (2007) 19:325  –  332DOI 10.1007/s10811-006-9139-2D.-Z. Wang ( * ) :  X.-G. Huang : H.-S. HongState Key Laboratory of Marine Environmental Science/ Environmental Science Research Center,Xiamen University, Xiamen 361005, Chinae-mail: L. ChanDepartment of Ecology and Biodiversity,The University of Hong Kong,Hong Kong, HKSAR, China  assemblages, or when trying to distinguish betweenmorphologically similar species or strains, such as  P.minimum, P. dentatum  and  P. micans . Moreover, thismethod involves training and experience and is time-consuming and labor-intensive, especially when largenumbers of samples are to be analyzed. This presentsserious problems for routine monitoring programs, anddiagnostic tools are needed urgently to identify,enumerate and separate cells of this species frommixed phytoplankton samples. One alternative is theuse of molecular or cellular probes to reveal target species.Antibody probes have shown great potential toadvance the pace of harmful algal research andmonitoring (Anderson 1995; Vrieling and Anderson1996), and indirect immunofluorescence assays using polyclonal antibodies have become the most commontechnique used to identify harmful algae, and todistinguish toxic and non-toxic harmful algal species.Thus, Anderson et al. (1990) used species-specific polyclonal antibodies to successfully detect the browntide Chrysophyte  Aureococcus anophagefferens;  Bateset al. (1993) developed a specific polyclonal antiserumto distinguish toxic  Pseudonitzschia multiseries  andnon-toxic  P. pungens ; and Lopez-Rodas and Costas(1999) investigated morphospecies and strains of   Prorocentrum  using polyclonal antisera. Recently, Linet al. (2003) developed an immunofluorescence tech- nique to detect   Pfiesteria piscicida . Monoclonal anti- bodies have also been applied in distinguishingharmful and toxic algal species (Sako et al. 1993;Adachi et al. 1993; Lopez-Rodas and Costas 1997). In this study, antiserum against cell-surface antigenswasproducedusing0.5%paraformaldehyde-fixedwholecellsof   P. donghaiense , and the titer and specificity wereexamined. An IF protocol based on antiserum wasoptimized for samples taken from cultures at variousgrowth phases and under various culture conditions, aswell as mixed and field samples. The goal of this studyis to establish a species-specific detection method for research into, and monitoring of,  P. donghaiense . Material and methods Algal cultures  P. donghaiense  Lu was isolated from the East ChinaSea in April, 2001 and the strain (  A. donghaiense PR0201-01) was routinely maintained in naturalseawater supplemented with f/2-Si nutrients at 20°C,60  μ  E m − 2 s − 1 light intensity and a 12 h:12 h light:dark cycle.For antigen preparation and series experiments,  P.donghaiense  was grown in 5000-ml conical flaskscontaining 4000 ml of f/2-Si medium under the sameregime as above, except that the light intensity wasadjusted to 100  μ  Em − 2 s − 1 . Cell growth was moni-tored by counting cells daily under a microscope.When the cultures reached their highest density (theearly stationary phase), cells were harvested usingcentrifugation (8000 rpm for 10 min).Production of polyclonal antiserumApproximately 10 7 cells were collected using centri-fugation for each antigen preparation, and antigenswere prepared as the whole cell antigen (WCA), byresuspending the pelleted cells in 0.5% paraformal-dehyde (PFA) prepared with phosphor buffered saline(PBS), and then storing them in 15-ml microcentri-fuge tubes at 4°C prior to immunization.Antigen materials preserved in 0.5% PFA werewashed five times with PBS and then resuspended inPBS. Four New Zealand white rabbits were immu-nized for each antigen preparation. For initial inocu-lation, 10 7 cells, in 1-ml PBS, were mixed with 1 mlof Complete Freund ’ s Adjuvant (from Sigma) andinjected at multiple sites: subcutaneously in theinguinal and axillary regions, and intramuscularly inthe hind limbs. Subsequent boosts were madeintravenously on days 14, 21, 35 and 49, using thesame amount of antigen as for the initial inoculation.Test bleeds were collected on day 40, and a final bleed was obtained 10 days after the final boost (Linet al. 2003).Immunofluorescence protocolAbout 5×10 3 cells were collected from each sampleusing centrifugation. The pellets were washed with80% ethanol to extract chlorophyll, so as to avoidauto-fluorescence. Then the pellets were rinsed fivetimes with PBS to remove the ethanol. Incubationwith the primary test antiserum was performed at 37°C for 1 h. The different dilutions were designed toobtain a constant primary antibody/cell ratio. After rinsing five times with PBS, the binding activity of  326 J Appl Phycol (2007) 19:325  –  332  the antiserum was gauged using a secondary fluores-cein isothiocyanate (FITC)-conjugated goat anti-rab- bit antibody (from Sigma) employed at 1.631 mg/mlfor 1 h at 37°C. After rinsing five times with PBS,the samples were examined for the quality andquantity of antibody binding, as described previously(Lopez-Rodas and Costas 1997). The quality of stainwas estimated using epifluorescence in a Zeiss Axio-shop microscope (Carl Zeiss Ltd.) with a filter set (450  –  495 nm excitation, 520  –  560 nm emission) usingthe following scale: (3+) bright stain, 100% of cellsstained; (2+) medium stain, 100% of cells stained; (+)low stain but obviously different from controls, andless than 100% of cells stained; ( − ) non-detectablereaction. All tests were read  ‘  blind ’ , that is, the personreading the test did not know the identity of the testedmaterial.Decolorization of cultured samplesThree organic solvents were tested for decolorization of the cultured samples fixed with 0.5% paraformalde-hyde: (1) 80% ethanol, (2) 80% acetone, and (3) 80%methanol. After incubation with the above solvents for 5, 10, 20, 60 and 120 min at ambient temperature, eachsamplewas analyzedfor decolorizationefficiencyusingan IF protocol as outlined below. A sample incubatedwith water served as the comparison.Protocol for mixed and field collected samplesIn this experiment,  P. donghaiense  cells were mixedwith various phytoplankton species, such as  Skeleto-nema costatum, Alexandrium catanella, Thalassiosiraweissflogii, P. dentatum CCMP1517   and  P. micans, and the detection efficiency was tested using theestablished IF protocol as the following:  Detection efficiency ¼ C   IF  = C   LM  C IF  = Cell number under the fluoroscope micros-copy, C LM  = Cell number under the light microscopy.Meanwhile, the detection efficiency of field sam- ples collected from the East China Sea and XiamenHarbor were also investigated. Briefly, an appropriatevolume of seawater was filtered through a 1- μ  m polycarbonate membrane, and the cells were rinsedand resuspended in 5 ml seawater. Next the cells werefixed in 0.5% buffered paraformaldehyde, thenshaken in 5 ml 80% ethanol for 2 h, collected usingcentrifugation, and rinsed five times with PBS toremove PFA. After decolorization, the cells wereincubated with primary and secondary antibodiessequentially, and the detection efficiency was tested. Results High titer and specificity antiserum producedwith whole-cell antigenThe titer and specificity of the antiserum raised withWC antigen were examined using IF (Fig. 1). Whenthe dilution was below 1:500, there was an intenseimmunofluorescence reaction (3+) (Fig. 1a). Whenthe dilution increased from 1:1000 to 1:2500, therewas a moderately intense immunofluorescence reac- Fig. 1  Labeling intensity of   P. donghaiense  cells at different dilutions of antiserum.  a  <1:500.  b  1:1000  –  1:2500.  c  1:5000  –  1:10000.  d >1:10000J Appl Phycol (2007) 19:325  –  332 327  tion (2+) (Fig. 1 b). From 1:5000 to 1:10000 dilution,the samples showed a weak immunofluorescencereaction (+) (Fig. 1c) and, with further dilution, IFexhibited a non-detectable reaction ( − ) (Fig. 1d). Anantiserum dilution of 1:10000 was the lowest concen-tration that produced positive labeling.Results of the test for specificity and cross reactivityagainst 19 phytoplankton species or strains representing10 genera are shown in Table 1. The antiserum against   P. donghaiense  showed specificity at species level. It did not cross-react with cells from relatively closelyrelated species nor cells from the Bacillariophyceae,Chlorophyceae or Cryptophyceae, although it exhibiteda weak cross reaction (1+) with  P. dentatum  HK,  P. dentatum  CCMP1517 and  P. minimum  HK at verylow dilution (1:5). When the dilution reached or exceeded 1:100, the cross-reactions disappeared.Decrease of autofluorescenceThe cells of   P  .  donghaiense  produce intense redautofluorescence due to chlorophyll under epifluo-rescence microscopy (Fig. 3a), and this masks thegreen fluorescence produced by FITC. Completeextraction of chlorophyll was thus necessary beforeanalysis under an epifluorescence microscope. In thisstudy, the decolorization efficiencies of three organicsolvents  —  80% ethanol, 80% acetone and 80% meth-anol  —  were examined (Fig. 2). Ethanol and acetonewere found to decrease the autofluorescence morerapidly than methanol and, in both, about 70  –  80% of chlorophyll was extracted within 60 min, so that almost no red autofluorescence was observed under epifluorescence microscopy (Fig. 3c and d). However,intense green fluorescence was detected only in thetargeted cells extracted with 80% ethanol. Theefficiency of cell extraction with methanol was verylow; only 60% of chlorophyll was extracted within60 min, and red fluorescence was still observed insome cells (Fig. 3 b).Preservation methodTo examine the stability of preserved samples, four  preservation methods were examined. One samplewithout fixation was stored at 4°C; three other samplesfixed with 0.5% paraformaldehyde were stored at 4°C, − 20°C and  − 80°C respectively for 1 month. Thedetection efficiency decreased significantly in bothsamples stored at 4°C for 1 month, particularly for thenon-fixed sample, which showed an efficiency of only27%. The fixed samples stored at   − 20°C and  − 80°Cremained above 85% detection efficiency (Fig. 4).Therefore, preservation by freezing at   − 20°C or   − 80°C is an ideal method for long-term sample storage.Quality of antiserum in various  P. donghaiense  samplesQuality of antiserum was also examined with cellscollected at different growth phases and under different nutrient conditions. Antiserum against   P. donghaiense collected at the exponential phase was active against cells at various growth phases. At a dilution of 1:100,no difference was observed in labeling intensity (it was 3+) among cells collected at the initial, mid-exponential, late exponential, stationary or dissipation phase. Cells from nutrient-deplete and nitrate-limitedcultures showed a moderate immunofluorescencereaction (2+), while cells from phosphate-limitedand nutrient-replete cultures showed an intenseimmunifluorescence reaction (3+) (Table 2). Table 1  Cross reaction of different phytoplankton species/ strains and genera with antiserum against   P. donghaiense Species/strains Dilution1:5 1:100  Prorocentrum dentatum  HK +  –   P. dentatum  CCMP1517 +  –   P. triestinum  HK1  – –   P. triestinum  HK2  – –   P. minimum  HK +  –   P. micans  DYW  – –   P. donghaiense  3+ 3+  Alexandrium tamarense  – –   A. catenella  – –   A. minutum  – –   Karenia brevis  – –   Dunaliella salina  – –  Chlorella  sp.  – –   Dicrateria zhangjianggensis  – –   Phaeodactylum tricornutum  – –  Chaetoceros  sp.  – –  Skeletonema costatum  – –  Thalassiosira weissflogii  – –  T. pseudonana  – –  Labeling intensity detected by immunofluorescence, 3+ =intense label; 2+ = moderate label; 1+ = low intensity;  –   =nondetectable reaction.328 J Appl Phycol (2007) 19:325  –  332  Application of the IF protocol to mixed and fieldcollected samplesThe detection efficiency of   P. donghaiense  cells inmixed samples and field collected samples is shownin Fig. 5.  P. donghaiense  cells showed a bright greencolor, which was clearly distinguishable in the mixed phytoplankton sample, and the efficiency was over 90% (Fig. 5a and b). This protocol was also applied tonatural samples collected from the East China Sea andTaiwan Strait in May 2005, and  P. donghaiense  cellswere clearly detected in the former samples (5C and5D), while no positive staining was observed insamples collected from the Taiwan Strait, where no  P. donghaiense  cells have been observed. It was notedthat there was no difference in IF between fresh andPFA fixed samples. Discussion Generally, the use of light microscopy and settlingchambers are sufficient to classify and enumerateHAB species or strains. However, it is difficult todistinguish between morphologically similar speciesor strains in samples under the light microscope.Another problem is that it is time-consuming toidentify and count a particular species using light microscopy, and it is not simple enough for routine 0204060801000 20 40 60 80 100 120 Time (min)    R   e   l   a   t   i   v   e   a   u   t   o   f   l   u   o   r   e   s   c   e   n   c   e   (   %   ) Fig. 2  Decrease of autofluorescence in cell samples after chlorophyll extraction with different solvents.  “ Decreasing rateof autofluorescence ”  was determined by comparison withautofluorescence without decolorization treatment (relativeautofluorescence at 0 min).  ♦  water; ▪ 80% methanol; × 80%acetone;  ▲  80% ethanol Fig. 3  Labeling intensity of   P. donghaiense  cells decolorized with different solvents.  a  water;  b  80% methanol;  c  80% acetone;  d  80%ethanol Fig. 4  Effects of different preservation methods on detectionefficiency of   P. donghaiense .  “ Detection efficiency ”  wasdetermined in comparison with the fresh sample.  1  = Freshsample;  2  = non-fixed sample at 4°C;  3  = fixed sample at 4°C; 4  = fixed sample at   − 20°C;  5  = fixed sample at   − 80°CJ Appl Phycol (2007) 19:325  –  332 329
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