An enzyme linked immunosorbent assay for the detection of Kudoa thyrsites in Atlantic salmon Salmo salar

Aquaculture 250 (2005) 8 – 15 www.elsevier.com/locate/aqua-online

An enzyme linked immunosorbent assay for the detection of Kudoa thyrsites in Atlantic salmon Salmo salar Kimberley Taylor, Simon JonesT Fisheries and Oceans Canada, Pacific Biological Station, 3190 Hammond Bay Road, Nanaimo, British Columbia, Canada V9T 6N7 Received 28 June 2004; received in revised form 14 December 2004; accepted 2 February 2005

Abstract An antigen capture enzyme linked immunosorbent assay (ELISA) using high-titre rabbit polyclonal antiserum was developed to detect soluble Kudoa thyrsites antigen in skeletal muscle of Atlantic salmon. Raw serum provided capture antibodies and biotinylated polyclonal immunoglobulins served to detect the captured antigen. The optical density (OD) regressed significantly with the number of purified, glutaraldehyde-fixed K. thyrsites spores. Higher OD values were obtained with antigen extracted from the supernatant of an infected muscle homogenate using cold buffer containing Tween 20 compared with Triton X-100 or without detergent. OD values were also higher from cold supernatants compared with boiled supernatants and with uncentrifuged homogenates. OD values from unexposed, freshwater-reared salmon were negligible. Agreement between ELISA OD values and the results of polymerase chain reactions derived from 25 exposed Atlantic salmon was good compared with the agreements between ELISA or PCR and histological analysis. The relationship between a histologically determined K. thyrsites plasmodium score and ELISA OD value suggested the presence of histologically undetectable extrasporogonic stages in some samples. Serological detection of soluble K. thyrsites antigen may form the basis of a quantitative, field-based diagnostic assay. D 2005 Elsevier B.V. All rights reserved. Keywords: Kudoa thyrsites; Atlantic salmon; Quantitative diagnosis; ELISA

1. Introduction Kudoa thyrsites (Myxozoa: Multivalvulida) occurs in the skeletal muscle of several species of marine fishes and has a global distribution (Moran et al., T Corresponding author. Tel.: +1 250 729 8351; fax: +1 250 756 7053. E-mail address: [email protected] (S. Jones). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.02.040

1999). The parasite develops as spore-filled plasmodia within myocytes and is associated with an economically important post-mortem myoliquefaction (softflesh) of Atlantic salmon (Salmo salar) that are farmed on the Pacific coast of North America (Whitaker and Kent, 1991). Infections with K. thyrsites are diagnosed by microscopic examination of fresh or histological preparations of skeletal muscle that have been either stained (Moran et al., 1999) or

K. Taylor, S. Jones / Aquaculture 250 (2005) 8–15

treated with a labelled DNA probe (Jones et al., 2003). Parasite DNA is also detected in muscle by using a polymerase chain reaction (PCR) (Hervio et al., 1997). Histological examination and PCR provide only a limited ability to quantify the severity of infection, thus limiting the capacity of these methods to predict the development of soft-flesh. On the other hand, spore counts obtained from known masses of skeletal or hyohyoideus ventralis muscle have been used to provide quantitative severity data and have confirmed that the extent of soft-flesh correlates with the severity of infection (St-Hilaire et al., 1997a,b; Dawson-Coates et al., 2003). Serological characterisation of K. thyrsites spores (Chase et al., 2001) and the demonstration that an immuno-blot method could be used to quantify spores purified from salmon muscle (Chase et al., 2003) suggested serological approaches may prove useful in estimating the severity of infection. The present study describes an antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of K. thyrsites in Atlantic salmon.

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USA) as described in Harlow and Lane (1999). Igcontaining fractions were eluted with glycine buffer (pH 3.0), neutralized with 1.0M Tris (pH 8.0) and the protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Fractions containing 500 Ag/ml or more of protein were pooled. Immunofluorescence on purified spores and histological sections of infected salmon muscle were used to confirm the presence of K. thyrsitesspecific antibody in the affinity-purified Ig solution using the methods of Chase et al. (2001). 2.3. Biotinylation of purified polyclonal antibodies The concentration of the affinity-purified Ig solution was increased by dialysis against polyvinylpyrolidone (PVP; Sigma). The concentrated Ig was incubated for 3 h with biotinamidohexanoic acid Nhydroxysuccinimide ester (Sigma) in the presence of 5% DMSO (Harlow and Lane, 1999). Uncoupled ester was removed through extensive dialysis against 100 mM Tris containing 0.1% NaN3 (pH 8.0). The biotinylated Ig was stored in 20 Al aliquots at 20 8C.

2. Materials and methods

2.4. Development of the antigen capture ELISA

2.1. Kudoa thrysites spore purification

Wells in 96-well polystyrene plates (Sarstedt) were sensitized by overnight incubation (room temperature) with the raw immune serum (capture antibody) diluted 1:100 to 1:25,600 in carbonate–bicarbonate coating buffer (pH 9.6). Free binding sites were blocked with a milk-based solution (KPL, Gaithersburg, MD, USA), diluted 1:20 in distilled water at room temperature. Two-fold dilutions of glutaraldehyde-fixed spores (100,000 to 781 spores) or muscle antigen extract (see below) were then incubated in the sensitized wells. Wells receiving no spores or antigen extract served as blanks. The biotinylated Ig (detector antibody), two-fold serially diluted from 1:100 to 1:25,600, was then applied to the plate so that each dilution of capture antibody was incubated with a series of detector antibody dilutions. Alternatively, in some assays, monoclonal antibodies (MAbs) raised against K. thyrsites spores (1H2, 2F4, 3E8, 4H2; described in Chase et al., 2001) were substituted alone or pooled, undiluted or diluted 1:10, for the biotinylated Ig. All wells were then incubated with alkaline phosphatase-conjugated streptavidin diluted 1:1000

Spores were purified from the skeletal muscle of a naturally infected Atlantic salmon by discontinuous gradient centrifugation using Percollk as previously described (Chase et al., 2001). The purified spores were resuspended in 0.5% glutaraldehyde in Sfrensen’s phosphate buffer (pH 7.4) and stored at 4 8C. 2.2. Purification of polyclonal antibody Polyclonal antibodies were raised in rabbits against fresh K. thyrsites spores that had been purified as described earlier (Chase et al., 2003). Immunoglobulins (Ig) were precipitated from the raw serum with 100% saturated ammonium sulphate. The precipitate was pelleted, dissolved in phosphate buffered saline (PBS, pH 7.4) and desalted in PD-10 columns (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Desalted Ig was affinity purified using protein A-sepharose chromatography (Sigma, St. Louis, MO,

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K. Taylor, S. Jones / Aquaculture 250 (2005) 8–15

(SA-AP, Sigma) (AP-conjugated goat anti-mouse Ig in the case of MAbs) followed by Blue Phos Microwell Phosphatase Substrate System using buffers provided by the manufacturer (KPL). Unless otherwise indicated, all ELISA data are based on triplicated wells (100 Al per well) per treatment and all incubations including chromogen development were for 60 min at 37 8C. Antibodies, spores and the SA-AP were diluted in PBS containing 0.05% Tween 20 (PBS/20). Wells were washed between incubations with PBS/20 using an automated plate washer (Dynatech Laboratories, Alexandria, VA, USA). Plates were read at 630 nm using a microplate reader (Dynatech Laboratories). Net mean optical density (OD) values were calculated by subtracting antigen blank OD values from observed OD values.

natant retained. The resulting 24 extracts were then applied to the ELISA. Antigen was extracted from an additional 48 Atlantic salmon skeletal muscle homogenates prepared as described above using the cold centrifugation method with PBS/20. Twenty of these fish (mean weight ~1236 g) were from a commercial seawater salmon farm near Vancouver Island and 25 (mean weight ~225 g) had been exposed to seawater for 12 months (mean temperature ~10 8C) at the Fisheries and Oceans Canada West Vancouver Laboratory. Antigen was also extracted by the same method from skeletal muscle of three exclusively freshwater-reared Atlantic salmon that served as negative controls. Positive control antigen was the cold centrifuged (PBS/20) supernatant from the infected muscle described above.

2.5. Antigen extraction and validation of the ELISA 2.6. Polymerase chain reaction (PCR) A 2 g skeletal muscle sample was collected from an Atlantic salmon post-smolt that had been exposed for approximately 6 months to seawater from Departure Bay, British Columbia, Canada. The muscle was dissected from the left fillet adjacent to the lateral line between the dorsal and adipose fins. K. thyrsites occurs in this Bay and microscopic examination of the muscle confirmed infection. A similar sample was also collected from an exclusively freshwater-reared Atlantic salmon. The samples were processed separately by finely mincing using a sterile scalpel blade and combined with 8 ml of Hank’s balanced salt solution (HBSS; Sigma). They were further homogenised for 1 min in a Stomacher Lab-Blender 80 (Seward Laboratory UAC House, London, England) and filtered through a 1 mm stainless steel mesh. Triplicate 1:1 mixtures of the two filtrates were made with each of the following buffers: PBS, PBS/20, PBS/80 (0.05% Tween 80), and PBS/100 (0.05% Triton X-100). Each buffer mixture was subjected to the following extraction methods: the mixed method—mixed by vortexing for 5 s, stored overnight at 4 8C and mixed again; the cold centrifugation method—mixed for 5 s, stored overnight at 4 8C, centrifuged for 5 min at 11,000 RPM and the supernatant retained; or the boiled centrifugation method—mixed for 5 s, incubated in a 100 8C water bath for 15 min, stored at 4 8C overnight, centrifuged as above and the super-

DNA extracted from approximately 50 Al of each of the 48 filtered homogenates described above (prior to mixing with the PBS/20 buffer) was used as template in polymerase chain reactions (PCR) following the methods described in Jones et al. (2003). Amplification of a 531 base pair (bp) segment of the K. thyrsites 18S rRNA gene was considered a positive result. 2.7. Histological examination A 1 cm3 piece of skeletal muscle was dissected from three locations on each of the 45 seawater exposed salmon: immediately anterior to and immediately posterior to the dorsal fin and adjacent to the adipose fin. The muscle samples were fixed overnight in Davidson’s solution, processed for routine histology and stained with hematoxylin and eosin stains (Dawson-Coates et al., 2003). The mean number of plasmodia observed in 50 microscopic fields (160) from each of the three muscles samples from each fish was calculated and expressed per mm2. 2.8. Statistical analysis ELISA OD data were presented as net mean OD Fstandard error. The significance of differences of mean OD values among treatments from the tissue

K. Taylor, S. Jones / Aquaculture 250 (2005) 8–15

extraction experiment was estimated by two-way ANOVA (with interactions) and linear regression analysis. In the latter, the main effect variables (buffer, extraction method) were transformed to a series of binary variables and each compared to reference values (PBS/20, cold centrifuge). Differences were assumed to be significant when P b 0.05. The agreement among ELISA, PCR and histological analyses was estimated by calculating the kappa statistic (k = (observed agreement agreement by chance) / (total agreement agreement by chance)).

3. Results Fluorescence was associated with spores and histological sections of K. thyrsites-infected muscle following incubation with the affinity-purified immunoglobulin. A 1:800 dilution of capture antibody combined with a 1:100 dilution of biotinylated detector antibody resulted in the highest net mean optical density (OD) value when 25,000 purified fixed spores were used as antigen. Using these antibody dilutions, OD values regressed significantly with the number of spores (Fig. 1) and the relationship between OD and spore number was estimated by: OD =0.025  ln(spore number) 0.173. The 12 methods of extracting antigen from infected muscle generated OD values that ranged from 0.160 to 0.273 (Fig. 2) whereas the 12 OD values generated from the uninfected sample ranged from 0.005 to 0.012 (not shown). Significant differences in OD values were found among buffers (ANOVA, P = 0.0009) and extraction methods ( P = 0.0002).

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Statistical interactions between buffers and extraction methods were not significant ( P = 0.45). Optical density values generated using PBS/20 were significantly higher than those obtained using PBS (regression analysis, P =0.0005) and PBS/100 ( P = 0.03) but were not different from PBS/80 ( P = 0.92). Similarly, OD values generated using cold centrifugation extraction were significantly higher than the mixed (regression analysis, P = 0.0001) and the boiled centrifugation method ( P =0.04). The cold centrifugation extraction method with PBS/20 was adopted for ELISA validation. Of the 20 commercial salmon samples, no PCR products were amplified and there was no histological evidence of infection. Mean net OD values ranged from 0.023 to 0.035. In contrast, a 531-bp PCR product was amplified from 16 of the 25 samples obtained from the laboratory-exposed salmon (Table 1). Histological evidence of K. thyrsites infection was detected in 17 of 25 samples and among these, the mean number of plasmodia per mm2 (PM) of skeletal muscle ranged from 0.006 to 2.849 (Table 1). A kappa value of 0.73 was obtained when PCR and histology (infected-uninfected) scores were compared. The OD values among the 25 samples ranged from 0.010 to 0.628. The OD values of the three freshwater-exposed samples were all 0.015 and no PCR amplification product was produced. Based on these observations, an OD threshold was established such that samples with OD values N 0.035 were scored positive (infected). Thus, kappa values of 0.91 and 0.63 were generated when OD values were compared with PCR and histology, respectively. The relationship between the histological severity of infection and ELISA

Optical Density

0.140 0.120 0.100

R2 = 0.945

0.080 0.060 0.040 0.020 0.000 0

20000

40000

60000

80000

100000

Number of Spores Fig. 1. Relationship between net mean optical densities (630 nm) obtained in an antigen capture enzyme-linked immunosorbent assay and numbers of glutaraldehyde-fixed K. thyrsites spores.

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K. Taylor, S. Jones / Aquaculture 250 (2005) 8–15

0.35 0.30

PBS PBS-20 PBS-80 PBS-X100

Optical Density

0.25 0.20 0.15 0.10 0.05 0.00 Mix

Centrifuge

Boil

Muscle Treatment Fig. 2. Optical densities (OD) from capture enzyme-linked immunosorbent assays using soluble antigen extracted from K. thyrsites infected Atlantic salmon skeletal muscle with PBS, PBS m 0.05% Tween 20, PBS m 0.05% Tween 80 or PBS m 0.05% Triton X-100. Antigen was sampled from the buffer mixtures directly (Mix), or from the supernatant following centrifugation in which the mixture was cold (Centrifuge) or boiled (Boil). See text for details. Net mean OD (F standard error) at 630 nm of triplicate reactions.

analyses is illustrated in Fig. 3 and is estimated by PM= 9.136(OD)2 1.566(OD)F 0.085.

4. Discussion An antigen-capture enzyme-linked immunosorbent assay (ELISA) was shown to be a quantitative and sensitive method for the detection of K. thyrsites infection in Atlantic salmon. The assay utilised uncharacterised soluble parasite antigens and therefore differed from previous methods in which the severity of K. thyrsites infection was determined by microscopic examination of fresh or histological muscle preparations or by the enumeration of purified spores (St-Hilaire et al., 1998; Dawson-Coates et al., 2003). The latter studies reported considerable heterogeneity in the severity of infections among fish in a sample population. In addition, the consistency with which the infection is detected among samples from an individual fish varies directly with the severity of infection probably due to an over-dispersed distribution of the parasite in less-severe infections. Increasing the number of samples collected from an

individual may compensate for the poor agreement among samples (Dawson-Coates et al., 2003). False negative ELISA and PCR scores were obtained in two samples that were weak-positive by histological analysis suggesting that the consequences of an over-dispersed parasite distribution also apply to these assays. Among the three diagnostic methods compared, k-values were 0.63 (ELISA versus histology), 0.73 (histology versus PCR) and 0.91 (ELISA versus PCR). The kappa statistic provides an estimate of agreement between two sets of observations and kvalues greater than 0.7 indicate the reliability of the agreement (Silcocks, 1983). Kappa, however, can be biased by symmetrical imbalance (unequal numbers of infected and uninfected observations) (Feinstein and Cicchetti, 1990) such as occurred in each of the assay scores examined. Nevertheless, the kappa scores suggested that agreement between ELISA and PCR was better than between ELISA or PCR and histology and this may be related to the relative sensitivities of these tests. While modifications to the sampling design may reduce the rate of false-negative scores in low- or moderately infected fish, increased sampling may be impractical due to cost or time

K. Taylor, S. Jones / Aquaculture 250 (2005) 8–15 Table 1 Comparison of Atlantic salmon muscle samples examined for K. thyrsites by enzyme linked immunosorbent assay, histological examination and polymerase chain reaction Samplea 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

ELISAb 0.01 0.008 0.002 0.001 0.005 0.006 0.025 0.03 0.045 0.056 0.059 0.06 0.062 0.077 0.099 0.125 0.177 0.198 0.228 0.262 0.275 0.301 0.315 0.375 0.628

Histologyc 0 0.007 0 0 0 0 0.006 0 0.359 0 0.027 0.052 0 0.04 0.228 0.256 0.272 0.266 0.224 0.125 0.801 0.106 0.196 0.521 2.849

PCRd

+ + + + + + + + + + + + + + + +

a Muscle samples from Atlantic salmon exposed to infective seawater at the West Vancouver Laboratory for approximately 12 months. b Net mean enzyme linked immunosorbent assay optical density (630 nm). Antigen extracted from muscle homogenates using the cold centrifugation method with PBS/20. Optical density values from the muscle of three freshwater-reared Atlantic salmon parr were all 0.015. c Number of K. thyrsites plasmodia per mm2 of hematoxylin and eosin-stained skeletal muscle. Approximately 150 microscopic fields (200) were examined per fish. d Amplification of 531 bp of the K. thyrsites 18S rRNA gene by polymerase chain reaction from DNA extracted from ~35 mg of the same homogenate used to prepare the ELISA antigen. +, positive reaction; , negative reaction.

considerations. However, since soft-flesh is associated with the more severe K. thyrsites infections, poor agreement in the diagnosis of pre-harvest salmon with low or moderate infections may be of lesser importance. The quantitative capacity of the antigen-capture ELISA, documented using spore dilutions, suggested that the assay will be useful not only in identifying infected individuals but also in estimating the severity

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of infection. Furthermore, the evident similarity in sensitivity between PCR and ELISA suggested that the latter assay, with an appropriate sampling design, will recognize infections before they become patent and thus serve as an early diagnostic tool. A high-titre polyclonal serum, raised against K. thyrsites spores, formed the basis of the ELISA. In this assay, immunoglobulins in the rabbit immune serum captured the antigen and following biotinylation, they served to detect the captured antigen. The capture design is similar to that described earlier for the detection of soluble Renibacterium salmoninarum antigens (Pascho and Mulcahy, 1987, Pascho et al., 1991). Soluble antigens were extracted in cold phosphate buffered saline from the purified pseudocysts of an unidentified Kudoa sp. from Chilean hake (Merlucius gayi gayi), supporting the observations made here (Martı´nez de Velasco and Cue´llar, 2003). Although the present ELISA used soluble antigens extracted from muscle with Tween 20, unlike that of R. salmoninarum, the K. thyrsites antigen was partially heat labile as observed OD values were significantly reduced in heated extracts. This suggested that the soluble antigen contained a mixture of heat stable and heat labile parasite epitopes, consistent with earlier observations on the apparent fragility of some soluble Kudoa sp. antigens (Martı´nez de Velasco and Cue´llar, 2003). One or more carbohydrate is the dominant epitope of the K. thyrsites spore surface antigen (Chase et al., 2001) and lectin analyses suggest carbohydrates are common constituents of the valve wall of numerous myxosporean species (Mun˜oz et al., 1999). Unfortunately the antigenic determinants of myxosporeans are poorly understood and the epitopes recognized by the polyclonal serum used here (Chase et al., 2003) are not known. However, the ability of the ELISA to detect pre-sporogonic stages is suggested from the relationship between histological scores and OD values (Fig. 3). A relatively wide range of OD values was observed over a narrow range of histological scores indicating perhaps, the greater occurrence of extrasporogonic stages in some samples than were detected histologically. This antiserum recognized epitopes in K. thyrsites stages in muscle preparations from 60-day-exposed salmon (Young and Jones, 2005), further supporting the potential of this reagent as the basis of an early diagnostic tool. The negligible

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K. Taylor, S. Jones / Aquaculture 250 (2005) 8–15

Plasmodia/mm2

3 2.5 2 1.5 1 0.5 0 -0.1

R2 = 0.895 0.1

0.3

0.5

0.7

Optical Density Fig. 3. Plasmodia per mm2 of K. thyrsites in histological preparations of Atlantic salmon skeletal muscle and the net mean optical density (630 nm) obtained from the same muscle samples using the antigen capture enzyme-linked immunosorbent assay.

ELISA values obtained when monoclonal antibodies raised against K. thyrsites spores were substituted for the biotinylated antibody (data not shown) was consistent with earlier studies using dot-blot assays (Chase et al., 2003). The available biotinylated Ig generated sufficient data to document the feasibility of an antigen capture ELISA to measure levels of soluble K. thyrsites antigen in salmon muscle. The cross-reactivity among myxosporean species of some diagnostic antisera (Bartholomew et al., 1989; Martı´nez de Velasco and Cue´llar, 2003) indicates, however, that additional reagent will be required to answer important questions relating to the specificity and to the development of sampling designs that maximise sensitivity of this assay. Finally, the development of a quantitative and field-friendly enzyme immunoassay that incorporates the present soluble antigen capture design may be envisioned. Such an assay, once calibrated with muscle texture, would prove useful in screening preharvest salmon for K. thyrsites infections that are sufficiently severe that the risk of soft-flesh is high.

Acknowledgements We thank Mr. Mahmoud Rowshandeli, Fisheries and Oceans Canada, West Vancouver Laboratories and Dr. Val Funk, Pan Fish Canada for providing salmon and Dr. Terry Pearson, University of Victoria for donating the antiserum. Thanks also to Dr. Marc Trudel, Pacific Biological Station for assisting with the statistical analysis. This study was funded by the

Fisheries and Oceans Canada ACRD Program and the British Columbia Salmon Farmers Association.

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