18. c7. elisa wang et al. aquaculture elisa for spiroplasma

8/2/2019 18. C7. ELISA Wang Et Al. Aquaculture Elisa for Spiroplasma

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

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

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

Spiroplasmas are emerging pathogens in commercially exploited freshwater crustaceans from China. The

bacteria infect Chinese mitten crab (Eriocheir sinensis), crayfish (Procambarus clarkii) and pacific 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 (3105 CCUml1 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 coefficient 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.

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 significantly 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 fresh-

water 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). Thespecies 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

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 ofSpiroplasma ex Eriocheir, or a similar agent, have also

occurred in the American crayfish (Procambarus clarkii) and fresh-

water-cultured Pacific white shrimp (Litopenaeus vannamei) in Jiangsu

and Zhejiang Provinces in China (Wanget al., 2005). At the sametime,

a spiroplasma was associated with mortalities in Pacific 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 efficient 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 moleculardiagnostics (Ding et al.,

2007), have been used for detection of the spiroplasmas from

crustaceans and other aquatic animals. However, it is difficult to

implement these methods in aquaculture farms due to the require-

ment 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 field diagnostics (Chen et al., 2007). The ELISA methodology

Aquaculture 292 (2009) 166171

Abbreviation: CCU, colour change unit.

Corresponding author. Tel.: +86 25 85891955; fax: +86 25 85891526.

E-mail address: [email protected] (W. Wang).

0044-8486/$ see front matter 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2009.04.022

Contents lists available at ScienceDirect

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
mailto:[email protected]://dx.doi.org/10.1016/j.aquaculture.2009.04.022http://www.sciencedirect.com/science/journal/00448486http://www.sciencedirect.com/science/journal/00448486http://dx.doi.org/10.1016/j.aquaculture.2009.04.022mailto:[email protected]


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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 field (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) andestablished 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

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. Washsolution (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 ml1 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 (BIO-

RAD 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 confirmed that

the spiroplasma was pathogenic, causing TD in E. sinensis (Wang et

al., 2004b) and crayfish (Procambarus clarkii) (Wang et al., 2005).

Other aquaculture-associated bacterial isolates (Acinetobacter spp,

Bacillus subtilis, Streptomyces sp., Pseudomonas aeruginosa, Escher-

ichia 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

Threedifferent preparation methods(viableorganism, formaldehyde-

killed 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 five-fold dilutions of the

antisera from 1:50 to 1:156,250. Coating antigen for the indirect ELISA

was 106 CCUml1 pathogens in the previously mentioned coating

solution. The best working concentration of antisera was monitored

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 thecoating solution andincubated for30 min at

37 C followed by three washes with PBS. Positive and negativecontrols 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 andthe color reactionwas 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, specificity and reproducibility

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. Specificity test

Seven common bacteria foundin aquaculture weretested 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 flowers(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, trialwas 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 ofSpiroplasma ex Eriocheirin serial densitiesfrom 105 to

108 CCU/mL were administered to crabs via injection directly into thehemolymph 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 werediluted 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 andeach of these samples was retested five times to examine variability

of the ELISA procedure. Infections were confirmed 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, crayfish 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

167J. Wang et al. / Aquaculture 292 (2009) 166171


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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 specificity of the ELISA test was also estimated

in terms defined by Pestal et al. (2003). The crab samples were

examined by both diagnostic methods, and the results were

summarized in a cross-classified 22 table (Table 3). From this, theepidemiological sensitivity, Se, and specificity, Sp, of the ELISA method

could be estimated as:

Se infected determined by both methods/infected determined

by electron microscopy

Sp 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 final protein concentration was 0.9 mg/mL or an

approximate protein content per bacteria of 107 g. According to the

OD value and protein content from the analytical sensitivity test, the

relationship between theELISAtest and theserial dilutionsof 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 thatthe antigenthat consistentlyproduced

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

Fig.1. Determination of thetitervalueof antisera.The highestreadabletiterof 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. 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.

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.

Table 1

Analytical specificity of the antiserum against Spiroplasma ex Eriocheir at OD450.

Treatment or species Absorbance

Positive control 0.779, 0.765Negative control 0.041, 0.04

Blank control 0.003, 0.004

Escherichia coli 0.021, 0.029

W22 0.05, 0.05

VC 0.005, 0.008

BS 0.035, 0.049

AY 0.011, 0.015

SDD 0.039, 0.039

CR 0.197, 0.202

XcZ 0.036, 0.037

CH 0.055, 0.057

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.

168 J. Wang et al. / Aquaculture 292 (2009) 166171


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that produced by inactive bacteria injection. Therefore, the antigen chosen

for further testing was that of the whole bacteria inactivated formalde-

hyde. 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);andtheOD valueof the

best workingconcentration of antiserawas read at 450nm (Fig.2):the

antigen to distilled water ratio was 1:20; theworking concentration of

antiserum was 1:400, and for enzyme labelled antibody, 1:3000.

3.3. Determination of sensitivity, specificity 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 figure

(Fig. 3). For determination of analytical sensitivity and specificity, the

working concentrationof antiserum and enzyme labeled antibody was

Table 2

Intra- and inter-assay variability of the indirect ELISA for Spiroplasma ex Eriocheir.

Series Sample OD OD Within series S CV Between series S CV

Mean Mean

Heavy infection

0.30.8 1 I 0.633 0.673 0.653 0.028284 0.043314 0.655 0.026756 0.040849

2 0.655 0.684 0.6695 0.020506 0.030629

3 0.623 0.648 0.6355 0.017678 0.027817

4 0.683 0.701 0.692 0.012728 0.018393

5 0.612 0.638 0.625 0.018385 0.029416

1 II 0.56 0.51 0.535 0.035355 0.066085 0.5405 0.028818 0.053318

2 0.497 0.533 0.515 0.025456 0.049429

3 0.601 0.577 0.589 0.016971 0.028813

4 0.539 0.508 0.5235 0.02192 0.041873

5 0.538 0.542 0.54 0.002828 0.005238

1 III 0.409 0.384 0.3965 0.017678 0.044584 0.4035 0.020727 0.051369

2 0.419 0.401 0.41 0.012728 0.031044

3 0.416 0.457 0.4365 0.028991 0.066418

4 0.389 0.39 0.3895 0.000707 0.001815

5 0.386 0.384 0.385 0.001414 0.003673

Moderate infection

0.10.3 1 IV 0.238 0.251 0.2445 0.009192 0.037597 0.2505 0.019368 0.077318

2 0.279 0.249 0.264 0.021213 0.080353

3 0.256 0.287 0.2715 0.02192 0.0807384 0.262 0.24 0.251 0.015556 0.061977

5 0.21 0.233 0.2215 0.016263 0.073424

1 V 0.238 0.218 0.228 0.014142 0.062027 0.251 0.024708 0.098439

2 0.233 0.253 0.243 0.014142 0.058198

3 0.304 0.282 0.293 0.015556 0.053093

4 0.232 0.266 0.249 0.024042 0.096553

5 0.243 0.241 0.242 0.001414 0.005844

1 VI 0.175 0.179 0.177 0.002828 0.01598 0.1617 0.015193 0.093957

2 0.145 0.146 0.1455 0.000707 0.00486

3 0.166 0.162 0.164 0.002828 0.017247

4 0.148 0.145 0.1465 0.002121 0.01448

5 0.178 0.173 0.1755 0.003536 0.020145

Light infection

00.1 1 VII 0.042 0.043 0.0425 0.000707 0.016638 0.0415 0.004138 0.099717

2 0.035 0.036 0.0355 0.000707 0.019919

3 0.041 0.04 0.0405 0.000707 0.017459

4 0.046 0.048 0.047 0.001414 0.030095 0.04 0.044 0.042 0.002828 0.067344

1 VIII 0.049 0.048 0.0485 0.000707 0.01458 0.0544 0.004992 0.091774

2 0.06 0.062 0.061 0.001414 0.023184

3 0.052 0.05 0.051 0.001414 0.02773

4 0.056 0.052 0.054 0.002828 0.052378

5 0.058 0.057 0.0575 0.000707 0.012298

1 IX 0.056 0.059 0.0575 0.002121 0.036893 0.0587 0.005707 0.097231

2 0.059 0.055 0.057 0.002828 0.049622

3 0.059 0.055 0.057 0.002828 0.049622

4 0.068 0.069 0.0685 0.000707 0.010323

5 0.053 0.054 0.0535 0.000707 0.013217

Negative control 1 0.021 0.024 0.0225 0.002121 0.094281 0.0242 0.0248 0.0245

2 0.023 0.025 0.024 0.001414 0.058926

3 0.031 0.027 0.029 0.002828 0.097532

4 0.02 0.022 0.021 0.001414 0.067344

5 0.026 0.026 0.026 0 0

CV: coefficient 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 flowers 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 theutility of theantigenand ELISAas a diagnostic tool in theseaquaculture

systems.

The hemolymph samples collected during the infection/inoculation

trial with E. sinensis were tested several times for the presence ofSpiro-

plasma ex Eriocheir by indirect ELISA. Hemolymph samples fromhealthy,

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 positivefor 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 coefficient of variation in the ELISA was low. Each of the

samples selected from the doseresponse trial was tested 5 times

(Table 2). The coefficient of variation was around 5% in heavily

infected animals, but as much as 10% in lightly and moderately

infected animals. The coefficient 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 coefficient 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; 14crabs were

positive and 56 negative by both methods (Table 3). Epidemiological

sensitivity was calculated as the number infected according to bothmethods divided by the number infected according to electron micro-

scopy. 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 specificity was

calculated as the number not infected according to both methods divided

by the number not infected according to electron microscopy. The

epidemiological specificity 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 (107 g/bacterium), the number

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 confirm 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 significance 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 anddiagnose the presence of the agent. This is particularly crucial in light of

the expanded hostrange 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 (Wanget al.,2004b) andPCR-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

diagnosingthe spiroplasma. In China, newplantspiroplasmas and insect

(bees) spiroplasmas have been isolated and confirmed as pathogens in

several commercially important host species (Chen et al., 1988).

Enzyme-linked immunosorbent assay (ELISA) is an ideal method for

field or small support laboratory applications because the color change

that results from the presence of the spiroplasma can be visuallyidentified with minimal training or with the use of hand-held spectro-

photometers. This study makes the first step in the development of a

commercially available field 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 labora-

tory 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 thebufferingcapacityof 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.

Table 3

Epidemiological sensitivity and specificityof the ELISA method versus negativestaining

by TEM. Data are from naturally infected crabs sampled from a culture pond in Jiangsu

Province.

Source Electron microscopy test Total %

infectedIndirect ELISA

test

Positive Negative

Positive 14 (8 heavy infections; 6 moderate

infections)

0 14 100.000

Negative 4(light infections) 56 60 6.667

Total 18 56 74 24.324

Se=number infected according to both methods/number infected according to

electron microscopy=14/18=77.8%.

Sp=number not infected according to bothmethods/number not infected according to

electron microscopy=56/56=100%.

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 107 g.

170 J. Wang et al. / Aquaculture 292 (2009) 166171


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Additionally the DDW was more suitable for rapid detection of

spiroplasmas, particularly given its applicability to field situations.

Further optimization was also provided by using a coating solution at

1:20 dilution,the polyclonal antiserum at 1:400, andthe 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 coefficient

of variation was around 5% in heavily infected animals, but as much as

10% in lightly and moderately infected animals. This is quite reason-

able for field 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 field 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 thanwhole-body sampling,

and will not lead to the death of crab. The ELISA method could quicklydetect 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 epidemiolo-

gical specificity 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 confirmed infection,

potentially some 23% of animals that are infected may not be

diagnosed as a positive with this methodology. We are currently

undertaking refinements in the methodology to improve its sensitiv-

ity for further application.

Acknowledgements

This work was supported by grants from the Natural Sciences

Research Foundation of China (NSFC No.30771649), Jiangsu Key

Scientific 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.

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Table 4

The comparison of the traditional ELISA method and our method optimized for quick

reading of the plates.

The traditional method The optimized method

Coating solution Carbonic acid buffer(ph9.6) Distilled water

Antigen proportion 1:100 1:20

Coating time 4 C overnight 2527 C 30 min

Incubation time 37 C 2 h 2527 C 30 min

Antisera proportion 1:2000 1:400

Second antibody proportion 1:3000 1:3000Cumulative time 16.5 h 2.3 h


18. c7. elisa wang et al. aquaculture elisa for spiroplasma

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