Aquaculture 292 (2009) 166171

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Enzyme-liked immunosorbent assay for the detection of pathogenic spiroplasma in commercially exploited crustaceans from China
Junhai Wang a, Hua Huang a, Qi Feng a, Tingming Liang a, Keran Bi a, Wei Gu a, Wen Wang a,, Jeffrey D. Shields b
a b

Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, China Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia 23062, USA

a r t i c l e

i n f o

a b s t r a c t
Spiroplasmas are emerging pathogens in commercially exploited freshwater crustaceans from China. The bacteria infect Chinese mitten crab (Eriocheir sinensis), craysh (Procambarus clarkii) and pacic white shrimp (Litopenaeus vannamei) in Jiangsu and Zhejiang provinces of China. A rabbit antiserum was prepared against the spiroplasma isolated from a Chinese mitten crab that had obvious signs of tremor disease. An indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of pathogen was developed. The titer of antibody against the pathogen was 1:31,250. The lowest detectable antigen protein was 0.573 g/ mL (3 105 CCUml 1 pathogens) in pure culture. No color reaction was observed with seven other bacteria (Acinetobacter spp, Bacillus subtilis, Streptomyces sp., Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, Aeromonas hydrophila). Very weak color reaction was observed in tests with spiroplasma species from plants and honeybees. The coefcient of variability of the assay was less than 10%, with an excellent reliability in moderate and heavy infections. The whole test procedure was rapid, being completed within 3 h. The results showed that the assay is a simple and applied method for diagnosis of spiroplasma diseases in crustaceans. 2009 Elsevier B.V. All rights reserved.

Article history: Received 27 August 2008 Received in revised form 13 February 2009 Accepted 19 April 2009 Keywords: Spiroplasma Pathogenic bacteria ELISA Detection Eriocheir sinensis Procambarus clarkii Litopenaeus vannamei

1. Introduction Eriocheir sinensis, the Chinese mitten crab, supports a valuable aquaculture industry in China valued at over US$ 1 billion (Li et al., 2007). The industry is centered in Jiangsu province with other provinces (Zhejiang, Anhui) also contributing signicantly to the production of and market for the crab. In recent years, increasing economic losses have occurred in aquaculture facilities of the crab due to a serious epidemic disease known as tremor disease (TD) (Wei, 1999). A novel pathogen, a spiroplasma, was found to be the causative agent (Wang et al., 2004a). Spiroplasmas are prokaryotes belonging to the class Mollicutes. Their traditional hosts are insects and plants (Gasparich, 2002). However the report of a spiroplasma in a freshwater crustacean, Chinese mitten crab E. sinensis (Wang et al., 2004b), is beginning to change our understanding of the host range of these organisms (Regassa and Gasparich, 2006). The species isolated from E. sinensis is undergoing taxonomic review, with specimens deposited at the China Center for Type Culture Collection (CCTCC, Wuhan, China) as CCTCC M 207170 and Deutsche Sammlung von Mikroorganismen

Abbreviation: CCU, colour change unit. Corresponding author. Tel.: +86 25 85891955; fax: +86 25 85891526. E-mail address: njnuwang@263.net (W. Wang). 0044-8486/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.04.022

und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) as DSM 21848. Hereafter we refer to the species from E. sinensis and other freshwater crustaceans as Spiroplasma ex Eriocheir. Outbreaks of Spiroplasma ex Eriocheir, or a similar agent, have also occurred in the American craysh (Procambarus clarkii) and freshwater-cultured Pacic white shrimp (Litopenaeus vannamei) in Jiangsu and Zhejiang Provinces in China (Wang et al., 2005). At the same time, a spiroplasma was associated with mortalities in Pacic white shrimp (L. vannamei) from South America (Nunan et al., 2004, 2005). These outbreaks indicate that spiroplasmas have become serious, novel pathogens that can potentially threaten global aquaculture of crustaceans. Because of the need to effect efcient disease control and to address regulatory issues, fast detection of these agents is wanted badly. Some methods, such as light microscopy, electron microscopy (Wang et al., 2004b) and PCR-based molecular diagnostics (Ding et al., 2007), have been used for detection of the spiroplasmas from crustaceans and other aquatic animals. However, it is difcult to implement these methods in aquaculture farms due to the requirement of special equipment and expensive reagents. Therefore, a rapid, as well as handy method would facilitate detection of spiroplasma infections in cultured animals. Such equipment should be inexpensive, and developed for direct use by culturists. The enzyme-linked immunosorbent assay (ELISA) method has been used for many years as a eld diagnostics (Chen et al., 2007). The ELISA methodology

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has been shown to detect pathogens with good sensitivity and give rapid detection based on observable color changes. Moreover, it does not require specialized, expensive equipment in the eld (Keisuke et al., 2000.). There are a few reports of ELISA detection for spiroplasmas from plants and insects tissues. A serological method has been used for the detection of Spiroplasma citri in plant host tissues (Clark et al., 1978; Saillard et al., 1978). Raju and Nyland (1981) produced antiserum against an isolate of corn stunt spiroplasma (CSS; 1-747) and established an ELISA testing method for that agent. However, there are currently no serological methods available for detection of spiroplasmas from aquatic animals. This study reports the use of an indirect ELISA method for the detection of a spiroplasma in commercially grown crustaceans from China. 2. Materials and method 2.1. Reagents and apparatus

by ELISA using a checkerboard titration. The goat anti-rabbit HRP-IgG was diluted by 1:3000. 2.5. Indirect ELISA method All of the ELISA operations were carried out at room temperature (25 C). Aliquots of 100 l per well were used in each step. The sample was diluted 1:20 with the coating solution and incubated for 30 min at 37 C followed by three washes with PBS. Positive and negative controls were treated in the same way. The polyclonal antiserum was added at the optimized working concentration and incubated for 30 min at 37 C. After removing the unbound polyclonal antibody, the goat anti-rabbit IgG (1:3000 dilution) was added and incubated for 30 min at 37 C, followed by three washes with PBS. The substrate solution was added and the color reaction was terminated after 10 min by addition of 2 M H2SO4. The color reaction was read on a BIO-RAD microplate reader. 2.6. Determination of sensitivity, specicity and reproducibility

2.1.1. Buffers and solutions Phosphate buffered saline (PBS, pH7.4): 0.135 M NaCl, 1.5 Mm KH2PO4, 8 Mm Na2HPO412H2O and 2.7 mM KCl. Coating buffer (pH = 9.6): a 15 mM Na2CO3 and a 35 mM NaHCO3 solution. Wash solution (PBS-Tween20, PBST): PBS with 0.05%(v/v) Tween20. Dilution buffer: 10%(v/v) calf serum. Substrate buffer (pH = 5.2): a 2.0 mg ml 1 TMB (3,3,5,5-tetramethy1 benzidine) and 0.02%(v/v) H2O2 in 40 mM citric acid and 35 Mm Na2HPO412H2O solution. Stop buffer: 2 M H2SO4. Enzyme labelled antibody: HRP-goat anti-rabbit IgG (Beijing Biosynthesis Biotechnology Co. Ltd.). 2.1.2. Apparatus Polystyrene 96-well microtitre plates (JET FSP11012), Microtiter plate washer (BIO-RAD Model 1575), ELISA microplate reader (BIORAD Model 680), Ultraviolet spectrophotometer (UNIC UV-2100), Mettler Toledo GmbH (LE 438), Transmission Electron Microscope (TEM, HITACHI-600). 2.2. Strains for experiments Spiroplasma ex Eriocheir was isolated from E. sinensis that were exhibiting the tremor disease using a previously described method (Wang et al., 2004a). Experimental inoculation trials conrmed that the spiroplasma was pathogenic, causing TD in E. sinensis (Wang et al., 2004b) and craysh (Procambarus clarkii) (Wang et al., 2005). Other aquaculture-associated bacterial isolates (Acinetobacter spp, Bacillus subtilis, Streptomyces sp., Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, Aeromonas hydrophila) were obtained from courtesy of the Nanjing Agricultural University, Freshwater Fisheries Research Institute of Jiangsu Province, and the Jiangsu Center for Control and Prevention of Aquatic Animal Infections Disease. 2.3. Antigen and immune serum preparation Three different preparation methods (viable organism, formaldehydekilled bacteria, and ultrasonic fragmentation of the agent) were used to obtain antigens. In addition, three different immunization techniques (ear vein injection, hypodermic injection with Freund's complete adjuvant and without adjuvant) were compared for optimization. 2.4. The titer of antisera and optimization of working concentration The antiserum titer was determined via ve-fold dilutions of the antisera from 1:50 to 1:156,250. Coating antigen for the indirect ELISA was 106 CCUml 1 pathogens in the previously mentioned coating solution. The best working concentration of antisera was monitored

2.6.1. Sensitivity test The concentration of the coating antigen was examined using serial dilutions to determine the lowest concentration of antigen present (Fan et al., 2006), based on the working concentration of antiserum and enzyme-labelled antibody. 2.6.2. Specicity test Seven common bacteria found in aquaculture were tested against the ELISA: Acinetobacter spp, B. subtilis, Streptomyces sp., P. aeruginosa, E. coli, V. cholerae, A. hydrophila. Two spiroplasma from plants and honeybees were also tested (CH from honey bees (Apis mellifera): CH-1, CR from cole owers(Rassica capestris): CR-1). The concentration of antigen was 183 g/mL protein from the pure culture of the spiroplasma. An experimental infection, or inoculation, trial was conducted with 4 groups of 6 crabs each to establish a doseresponse test. Crabs were obtained from culturists from Baoying county in Jiangsu province. Pure cultures of Spiroplasma ex Eriocheir in serial densities from 105 to 108 CCU/mL were administered to crabs via injection directly into the hemolymph at the joint between the pereopod and the thorax (the juncture of the base and the ischium) (Wang et al., 2004b). Ethanol was used to sterilize the injection site. Approximately 100 L of crab hemolymph was sampled from each crab 3 days after inoculation, and every 2 days thereafter. Hemolymph samples were diluted with sterile PBS 1:1 and the mixture was tested directly via ELISA. 2.6.3. Variability of the ELISA All of the test samples from the inoculation trial were divided into three groups randomly categorized as their infection status: heavy infection (color intensity of 0.30.8), moderate infection (color intensity of 0.10.3), light or no infection (color intensity of 0 0.1), based on the OD (optical density) value of all the samples tested. Three samples of each group were selected without bias and each of these samples was retested ve times to examine variability of the ELISA procedure. Infections were conrmed as light, moderate or heavy based on the presence of spiroplasma in the hemolymph as in Wang et al. (2005). 2.7. Diagnosis and detection of spiroplasma from cultured crustaceans Crabs, craysh and shrimps from an aquaculture farm in Jiangsu Province were brought to the lab for further diagnoses. Crustaceans were held in small aquaria and were fed during the experiments. Prior to sampling, the body of each crustacean was rinsed well with water and disinfected with 75% ethanol. Hemolymph samples were then taken from the joint between the pereopod and the thorax (the juncture of the base and the ischium) (Wang et al., 2004b). A small

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Fig. 1. Determination of the titer value of antisera. The highest readable titer of antiserum was 1:31,250 by the serial dilution method. 3rd antisera: the third immunization of antisera; 4th antisera: the fourth immunization antisera; OD450: optical density.

Fig. 3. Analytical sensitivity of the indirect ELISA. The abscissa was the ratio of antigen dilution and longitudinal coordinates was ELISA OD value. The original antigen protein content was 183 g/mL. The lowest concentration that antigen could be detected by indirect ELISA was 1/320, 0.573 g/mL.

aliquot of hemolymph (about 0.4 mL) was obtained with 1-mL sterilized syringe and then the hemolymph samples were diluted 1:1 with PBS in 1.5-mL centrifuge tube. In addition, approximately 1 cm3 (about 0.22 g) of muscle from each crustaceans was collected and cut into smaller pieces in an 1.5 mL test tube with 500 L PBS. All of the collected samples were stored at 4 C and tested for spiroplasma using the indirect ELISA method. To determine the infection status, a negative staining technique using transmission electron microscopy (Wang et al., 2004a) was the standard against which ELISA tests were compared. The negative staining method used with TEM was considered the most powerful method for diagnosing infections in of the spiroplasma in crustaceans. The sensitivity and specicity of the ELISA test was also estimated in terms dened by Pestal et al. (2003). The crab samples were examined by both diagnostic methods, and the results were summarized in a cross-classied 2 2 table (Table 3). From this, the epidemiological sensitivity, Se, and specicity, Sp, of the ELISA method could be estimated as: Se Sp infected determined by both methods/infected determined by electron microscopy not infected determined by both methods/not infected determined by electron microscopy

2.8. Testing the ELISA in serial dilutions of cultured spiroplasma We further tested the analytical sensitivity of the ELISA test using a serially diluted culture of the spiroplasma. An aliquot of 100 mL of cultured Spiroplasma ex Eriocheir from the logarithmic phase (about 108/mL or 1010 total bacteria) was concentrated by 100 fold using centrifugation. The nal protein concentration was 0.9 mg/mL or an approximate protein content per bacteria of 10 7 g. According to the OD value and protein content from the analytical sensitivity test, the relationship between the ELISA test and the serial dilutions of cultured spiroplasma was highly correlated (Fig. 4). 3. Results 3.1. Antigen preparation Three different methods were used to produce antibodies for testing with ELISA and included injection of viable bacteria, injection of formaldehyde-killed bacteria, and injection of ultrasonically-produced bacterial fragments. We found that the antigen that consistently produced the highest titer of antibody for antiserum production was from the bacteria that had been inactivated by formaldehyde. The titer of antibody prepared by either viable bacteria or bacteria fragments was lower than

Table 1 Analytical specicity of the antiserum against Spiroplasma ex Eriocheir at OD450. Treatment or species Positive control Negative control Blank control Escherichia coli W22 VC BS AY SDD CR XcZ CH Absorbance 0.779, 0.765 0.041, 0.04 0.003, 0.004 0.021, 0.029 0.05, 0.05 0.005, 0.008 0.035, 0.049 0.011, 0.015 0.039, 0.039 0.197, 0.202 0.036, 0.037 0.055, 0.057

Fig. 2. Results of the titration of spiroplasma and antisera. OD value of the optimized concentration of antisera was read at 450 nm. The antigen to distilled water ratio was 1:20, the working concentration of antiserum was 1:400, and for enzyme labelled antibody, 1:3000.

N: negative control, P: positive control, B: blank control, each sample was duplicated. Antigens in cross reaction: W22: Acinetobacter spp, BS: Bacillus subtilis, SDD: Streptomyces sp. XCZ: Pseudomonsa aeruginosa, E.coli: Escherichia coli, HL: Vibrio cholerae. AY: Aeromonas hydrophila, CH: Spiroplasma sp.CH-1, CR: Spiroplasma sp.CR-1, P: Spiroplasma.

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that produced by inactive bacteria injection. Therefore, the antigen chosen for further testing was that of the whole bacteria inactivated formaldehyde. This antigen was used in all subsequent procedures. The density of the bacteria in PBS solution was estimated at 183.4 g/mL using an ultraviolet (260/280 nm) spectrophotometer, based on the protein density measurement of Bradford (Zhao et al., 2007). The highest antibody titer in antiserum was obtained via the immunization method comprising two injections of killed bacteria with Freund's complete adjuvant (1:1, volume ratio) and two rabbit ear-vein injections. 3.2. The titer of antisera and best working concentration determination Two immunization methods gave excellent results for ELISA: the injection of antigen into the peritoneal cavity and injection directly into an ear vein four times. Both methods provided a higher titer of

antisera against spiroplasma than did the hypodermic injection method without adjuvant. The twice hypodermic injection with adjuvant and then twice injection directly into an ear vein produced the highest antisera titer. The best antiserum titer produced was 1:31,250 by the serial dilution method (Fig. 1); and the OD value of the best working concentration of antisera was read at 450 nm (Fig. 2): the antigen to distilled water ratio was 1:20; the working concentration of antiserum was 1:400, and for enzyme labelled antibody, 1:3000. 3.3. Determination of sensitivity, specicity and variability in the ELISA The detectable antigen concentration varied from 0.573 g/mL to 36.6 g/mL. The optical density (OD) value was shown in the gure (Fig. 3). For determination of analytical sensitivity and specicity, the working concentration of antiserum and enzyme labeled antibody was

Table 2 Intra- and inter-assay variability of the indirect ELISA for Spiroplasma ex Eriocheir. Series Heavy infection 0.30.8 Sample OD OD Within series Mean 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 I 0.633 0.655 0.623 0.683 0.612 0.56 0.497 0.601 0.539 0.538 0.409 0.419 0.416 0.389 0.386 0.238 0.279 0.256 0.262 0.21 0.238 0.233 0.304 0.232 0.243 0.175 0.145 0.166 0.148 0.178 0.042 0.035 0.041 0.046 0.04 0.049 0.06 0.052 0.056 0.058 0.056 0.059 0.059 0.068 0.053 0.021 0.023 0.031 0.02 0.026 0.673 0.684 0.648 0.701 0.638 0.51 0.533 0.577 0.508 0.542 0.384 0.401 0.457 0.39 0.384 0.251 0.249 0.287 0.24 0.233 0.218 0.253 0.282 0.266 0.241 0.179 0.146 0.162 0.145 0.173 0.043 0.036 0.04 0.048 0.044 0.048 0.062 0.05 0.052 0.057 0.059 0.055 0.055 0.069 0.054 0.024 0.025 0.027 0.022 0.026 0.653 0.6695 0.6355 0.692 0.625 0.535 0.515 0.589 0.5235 0.54 0.3965 0.41 0.4365 0.3895 0.385 0.2445 0.264 0.2715 0.251 0.2215 0.228 0.243 0.293 0.249 0.242 0.177 0.1455 0.164 0.1465 0.1755 0.0425 0.0355 0.0405 0.047 0.042 0.0485 0.061 0.051 0.054 0.0575 0.0575 0.057 0.057 0.0685 0.0535 0.0225 0.024 0.029 0.021 0.026 0.028284 0.020506 0.017678 0.012728 0.018385 0.035355 0.025456 0.016971 0.02192 0.002828 0.017678 0.012728 0.028991 0.000707 0.001414 0.009192 0.021213 0.02192 0.015556 0.016263 0.014142 0.014142 0.015556 0.024042 0.001414 0.002828 0.000707 0.002828 0.002121 0.003536 0.000707 0.000707 0.000707 0.001414 0.002828 0.000707 0.001414 0.001414 0.002828 0.000707 0.002121 0.002828 0.002828 0.000707 0.000707 0.002121 0.001414 0.002828 0.001414 0 0.043314 0.030629 0.027817 0.018393 0.029416 0.066085 0.049429 0.028813 0.041873 0.005238 0.044584 0.031044 0.066418 0.001815 0.003673 0.037597 0.080353 0.080738 0.061977 0.073424 0.062027 0.058198 0.053093 0.096553 0.005844 0.01598 0.00486 0.017247 0.01448 0.020145 0.016638 0.019919 0.017459 0.03009 0.067344 0.01458 0.023184 0.02773 0.052378 0.012298 0.036893 0.049622 0.049622 0.010323 0.013217 0.094281 0.058926 0.097532 0.067344 0 S CV Between series Mean 0.655 0.026756 0.040849 S CV

II

0.5405

0.028818

0.053318

III

0.4035

0.020727

0.051369

Moderate infection 0.10.3

IV

0.2505

0.019368

0.077318

0.251

0.024708

0.098439

VI

0.1617

0.015193

0.093957

Light infection 00.1

VII

0.0415

0.004138

0.099717

VIII

0.0544

0.004992

0.091774

IX

0.0587

0.005707

0.097231

Negative control

0.0242

0.0248

0.0245

CV: coefcient of variation.

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the same as that in the best working concentration test (antiserum concentration was 1:400, and for enzyme labelled antibody, 1:3000). The antisera produced from Spiroplasma ex Eriocheir showed no reaction when exposed to any of the seven different bacterial species commonly found in aquaculture. Further, there was only a weak color reaction between the antigen from Spiroplasma ex Eriocheir and those spiroplasmas from cole owers and honeybees (Table 1), indicating little cross reactivity. However, the weak reaction observed with these latter two spiroplasmas would not be expected to interfere with the utility of the antigen and ELISA as a diagnostic tool in these aquaculture systems. The hemolymph samples collected during the infection/inoculation trial with E. sinensis were tested several times for the presence of Spiroplasma ex Eriocheir by indirect ELISA. Hemolymph samples from healthy, uninfected crabs served as negative controls. All crabs were tested in the Dose Response trial. As shown by the change in color in the ELISA reaction (OD), hemolymph samples were positive for the spiroplasma 3 6 days after inoculation. Samples remained positive throughout the course of the infection. Negative controls remained negative and had negligible color change during the course of the experiment. The coefcient of variation in the ELISA was low. Each of the samples selected from the doseresponse trial was tested 5 times (Table 2). The coefcient of variation was around 5% in heavily infected animals, but as much as 10% in lightly and moderately infected animals. The coefcient of variation, either in the same series (within) or between series, was less than 10%, indicating that the ELISA had low variability within treatment groups and high reproducibility. Moreover, the coefcient of variation was highest in the light and moderate infections, which may be the result of more highly variable bacterial densities in these categories of infection. 3.4. Detection for spiroplasma from naturally infected mitten crabs Over 70 crabs from aquaculture farms were examined for disease by both indirect ELISA and the TEM negative staining method; 14 crabs were positive and 56 negative by both methods (Table 3). Epidemiological sensitivity was calculated as the number infected according to both methods divided by the number infected according to electron microscopy. The epidemiological sensitivity of the test was 77.8% (14/18), indicating that the test was quite comparable to the use of electron microscopy for diagnostic purposes. The epidemiological specicity was calculated as the number not infected according to both methods divided by the number not infected according to electron microscopy. The epidemiological specicity was 100% (56/56) indicating that the number of potential false negatives should be very low using the ELISA technique. 3.5. Testing the ELISA in serial dilutions of cultured spiroplasma Given that the original protein content of antigen in the analytical sensitivity test was 183 g/mL (10 7 g/bacterium), the number
Table 3 Epidemiological sensitivity and specicity of the ELISA method versus negative staining by TEM. Data are from naturally infected crabs sampled from a culture pond in Jiangsu Province. Source Electron microscopy test Negative 0 56 56 Total % infected 14 60 74 100.000 6.667 24.324

Fig. 4. The relationship between the ELISA test and the serial dilutions of cultured spiroplasmas. The original protein content of antigen in sensitivity test was 183 g/mL and protein content per bacteria was 10 7 g.

of spiroplasmas in each titration well could be estimated based on protein content and color change (Fig. 4). Clearly, the color reaction in the ELISA test was stronger in relation to the number of pathogenic spiroplasma present in the test sample. Using a hemacytometer and light microscopy we can further estimate the concentration of the spiroplasmas from both cultured and naturally infected crab, which when combined with the ELISA data allow us to conrm the degree of infection in samples used in the ELISA test. For example, a heavily infected crab with tremor disease showed approximately 1 108 spiroplasmas/mL under light microscopy, which corresponded to an ELISA reading of OD 1.76 (Fig. 4). 4. Discussion 4.1. The signicance of ELISA for detection of pathogenic spiroplasmas Given the emergence of the important pathogen, Spiroplasma ex Eriocheir, it is critical that we have the appropriate tools to detect and diagnose the presence of the agent. This is particularly crucial in light of the expanded host range of this spiroplasma, or other similar pathogens into cultured and native crustaceans in China (Wang et al., 2005) and elsewhere (Nunan et al., 2004, 2005). Although light microscopy, electron microscopy (Wang et al., 2004b) and PCR-based methods (Ding et al., 2007) are effective laboratory detection methods for the spiroplasma, in practical aquaculture operations there is a growing need to have a simple, inexpensive, reliable detection method for diagnosing the spiroplasma. In China, new plant spiroplasmas and insect (bees) spiroplasmas have been isolated and conrmed as pathogens in several commercially important host species (Chen et al., 1988). Enzyme-linked immunosorbent assay (ELISA) is an ideal method for eld or small support laboratory applications because the color change that results from the presence of the spiroplasma can be visually identied with minimal training or with the use of hand-held spectrophotometers. This study makes the rst step in the development of a commercially available eld kit to detect Spiroplasma ex Eriocheir. 4.2. Current situation and development of indirect ELISA for the detection of aquatic spiroplasmas Combined with traditional hemolymph testing method, our laboratory indirect ELISA method for pathogen detection has been optimized from previous studies. Sodium carbonate buffer was replaced with double-distilled water (DDW) as the coating liquid from a standard ELISA method (Small et al., 2002). Although the buffering capacity of the DDW was slightly lower than that of the carbonate buffer, it was easier to use and was not altered by preservation period or other factors.

Indirect ELISA Positive test Positive Negative Total 14 (8 heavy infections; 6 moderate infections) 4(light infections) 18

Se = number infected according to both methods/number infected according to electron microscopy = 14/18 = 77.8%. Sp = number not infected according to both methods/number not infected according to electron microscopy = 56/56 = 100%.

J. Wang et al. / Aquaculture 292 (2009) 166171 Table 4 The comparison of the traditional ELISA method and our method optimized for quick reading of the plates. The traditional method Coating solution Antigen proportion Coating time Incubation time Antisera proportion Second antibody proportion Cumulative time Carbonic acid buffer(ph9.6) 1:100 4 C overnight 37 C 2 h 1:2000 1:3000 16.5 h The optimized method Distilled water 1:20 2527 C 30 min 2527 C 30 min 1:400 1:3000 2.3 h

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Scientic Project on Key Problems (BE2007343) and the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 07KJD240105). JDS would like to thank Nanjing Normal University for travel support. References
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Additionally the DDW was more suitable for rapid detection of spiroplasmas, particularly given its applicability to eld situations. Further optimization was also provided by using a coating solution at 1:20 dilution, the polyclonal antiserum at 1:400, and the goat anti-rabbit IgG at 1:3000 dilution. Other changes coating time included an overnight at 4 C with a reduced incubation time from 2 h to only 30 min (2527 C, room temperature). Hence the entire time for preparation and detection can be greatly reduced from 16.5 h to 2.5 h (Table 4). 4.3. Field testing the ELISA In the laboratory, the assay was robust and reliable. The coefcient of variation was around 5% in heavily infected animals, but as much as 10% in lightly and moderately infected animals. This is quite reasonable for eld applications as the primary interest is whether crabs have or do not have the disease. Further, the ELISA can be used to detect infections of the spiroplasma in crabs 36 days after inoculation, but before they exhibit signs of TD. This further supports its potential use to monitor or survey for infections in eld situations. The newly developed ELISA was tested in a sample of crabs collected from an aquaculture pond with satisfactory. Hemolymph samples were used for the on-site testing of the ELISA on breeding crabs and water samples from an aquaculture farm because hemolymph collection is more convenient than whole-body sampling, and will not lead to the death of crab. The ELISA method could quickly detect the presence of spiroplasmas in the hemolymph of infected crabs. However, in epidemiological terms, the ELISA was not as sensitive as the negative staining method using TEM, as it had an estimated sensitivity of 77.8%, which indicates that low level infections may not be detected. However, the estimated epidemiological specicity was excellent at 100%, indicating that that there should be few false negatives or false positives with the method. Unfortunately, while a positive ELISA result is a conrmed infection, potentially some 23% of animals that are infected may not be diagnosed as a positive with this methodology. We are currently undertaking renements in the methodology to improve its sensitivity for further application. Acknowledgements This work was supported by grants from the Natural Sciences Research Foundation of China (NSFC No.30771649), Jiangsu Key