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Polymerase chain reaction detection of Renibacterium salmoninarum in fish: Validation of a modified protocol
Aquaculture 287 (2009) 35–39
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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
Polymerase chain reaction detection of Renibacterium salmoninarum in fish: Validation of a modified protocol Edel M. Chambers a, David A. Nagel b, Edward A. Elloway b, Kelly L. Addison b, Gavin A. Barker a, David W. Verner-Jeffreys a,⁎, David M. Stone a a b
Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK Department of Biological Sciences, Aston university, Aston Triangle, Birmingham, B4 7ET, UK
a r t i c l e
i n f o
Article history: Received 2 April 2008 Received in revised form 22 September 2008 Accepted 17 October 2008 Keywords: Renibacterium salmoninarum PCR Validation
a b s t r a c t A PCR protocol (McIntosh et al., 1996. Appl. Environ. Microbiol. 62, 3929–2932), designed to detect the P57 gene of Renibacterium salmoninarum, causative agent of Bacterial Kidney Disease, was modified slightly by redesign of the forward primer and recommended conditions to prevent possible false positives observed when tested against pure cultures of Yersinia ruckeri. The modified PCR, in combination with an improved DNAzol™-based DNA extraction technique, was very specific and sensitive (detecting between 5 and 72 R. salmoninarum cfu per mg head kidney tissue). It could detect infected fish using only 50 mg head kidney material, making the technique suitable for sampling small fish. It was also shown that samples could be pooled from up to five rainbow trout prior to PCR without noticeably affecting the sensitivity of the assay, providing at least 50 mg head kidney tissue was included from each of the fish that were pooled. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.
1. Introduction Bacterial Kidney Disease (BKD) caused by the bacterium Renibacterium salmoninarum (R. salmoninarum) is regarded as one of the most persistent bacterial diseases of salmonids worldwide (Evenden et al., 1993). BKD can be detected using a variety of test methods (Bruno et al., 2007), such as culture on specialised media (Chambers and Barker, 2006), direct and indirect fluorescent antibody test DFAT, IFAT, (Bullock et al., 1980; Cvitanich, 1994), as well as the more rapid ELISA (Gudmundsdottir et al., 1993; Jansson et al., 1996; Pascho and Mulkahey, 1987). However the sensitivity (Bandin et al., 1996), specificity (Dixon, 1987) and reproducibility (Scott and Johnson, 2001) of some of the ELISA tests has been questioned (Anon, 1999). More recently there has been an increase in the use of probe (Mattson et al., 1993; Leon et al., 1994a) and PCR-based assays to look for the presence of R. salmoninarum DNA and RNA in head-kidney, ovarian fluid and eggs (Brown et al., 1994; Leon et al., 1994b; Magnusson et al., 1994; McIntosh et al., 1996; Miriam et al., 1997; Chase and Pascho, 1998; Cook and Lynch, 1999). However, before using such proxy-based detection methods it is important to thoroughly validate them (Hiney and Smith, 1997).
⁎ Corresponding author. Tel.: +44 1305 206725; fax: +44 1305 206601. E-mail address: [email protected] (D.W. Verner-Jeffreys).
In this study, a modification of a PCR protocol McIntosh et al. (1996), designed to detect the P57 protein encoding gene of Renibacterium salmoninarum, causative agent of Bacterial Kidney Disease, hereinafter referred to as the modified PCR procedure, was developed. This modified protocol was tested for its ability to detect the presence of R. salmoninarum derived nucleic acid in a panel of test bacteria and fish both artificially and naturally infected with R. salmoninarum. 2. Materials and methods 2.1. Testing of McIntosh et al. (1996) recommended PCR procedures against a panel of bacteria A range of bacterial isolates, representing organisms that may be encountered during sampling of wild or farmed fish for BKD (Table 1), were grown in suitable broth media and resuspended in PBSa to an OD550 of 1.0. DNA was then extracted from undiluted and 1:1000 dilutions of each isolate, and tested for the presence of R. salmoninarum specific P57 protein encoding gene (Chien et al., 1992) by PCR, as described by McIntosh et al. (1996). 2.2. Modified PCR The protocol recommended by McIntosh et al. (1996) was modified to prevent coincidental binding of the forward primer to the Yersinia ruckeri genome (see Results). The PCR protocol developed, following a period of optimisation (data not shown), consisted of 10 μl reaction
0044-8486/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.10.031
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Table 1 Strain panel isolates used to test the specificity of the two PCR protocols [McIntosh et al., 1996; modified PCR protocol (this study)] designed to detect R. salmoninarum DNA from fish head kidney Strain
Identification
Origin
Aerococcus viridans Aeromonas caviae Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila sub sp. formicans Aeromonas hydrophila sub sp. sobria Aeromonas salmonicida Aeromonas salmonicida Aeromonas salmonicida sub sp. achromogenes Aeromonas salmonicida sub sp. achromogenes Arthrobacter globiformens Bacillus cereus Bacillus polymyxa Bacillus subtilis Bacillus sphaericus Brevibacterium linens Corynebacterium aquaticum Corynebacterium aquaticum Corynebacterium aquaticum Carnobacterium piscicola Escherichia coli Escherichia coli Flavobacterium psychrophilum Flavobacterium psychrophilum Flavobacterium psychrophilum Flavobacterium maritimus Klebsiella aerogenes Lactobacillus brevis Micrococcus luteus Pseudomonas aeuruginosa Pseudomonas aeuruginosa Pseudomonas anguilliseptica Pseudomonas fluorescens Pseudomonas maltophilia Renibacterium salmoninarum Renibacterium salmoninarum Renibacterium salmoninarum Renibacterium salmoninarum Renibacterium salmoninarum Rothia dentocariosa Salmonella typhimurium Serratia marcescens Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Vibrio anguillarum Vibrio ordalii Vibrio salmonicida Yersinia ruckeri Yersinia ruckeri Yersinia ruckeri Yersinia ruckeri Yersinia ruckeri
930681 NCIMB 1330 NCIMB 72 NCIMB 9241 NCTC 10362
Lobster, England Freshwater fish Moribund goldfish (C. auratus) Human myositis Sewage
NCIMB 37
No information available
NCIMB 833 MT423 NCIMB 1109
Brook trout (S. fontinalis) Atlantic salmon (Salmo salar L) Brown trout (Salmo trutta L.)
NCIMB 1110
Brown trout
NCIMB 8907 NCIMB 8012 ATCC 12321 NCIMB 11232 NCIMB 9370 NCIMB 9909 970798 1.3.4 970805 NCIMB 9460 NCIMB 2264 NCTC 10418 NCIMB 9482 NCIMB 1947 980070 18 970804 3.2.2 NCIMB 2158 NCTC 9528 NCIMB 11973 NCIMB 8553 NCIMB 13066 NCIMB 8295 NCIMB 1949 930029 NCIMB 9203 NCIMB 1111 NCIMB 2235 970083 970153 970419 NCTC 10917 NCTC 12484 NCTC 1377 NCTC 8532 NCIMB 12702 NCIMB 12721 NCIMB 2286 NCIMB 2167 NCIMB 2262 NCIMB 1316 NCTC 10476 NCTC 10477 NCTC 10478 NCIMB 2194
No information available No information available No information available No information available No information available Good harzerkase cheese Rainbow trout (O. mykiss) UK Farmed rainbow trout No information available Cutthroat trout (S. clarkii) No information available Torry research station (K12) Coho salmon (O. kisutch) Rainbow trout England Rainbow trout England Dover sole (Solea solea L.) Water Human faeces No information available Freshwater fish No information available Eel (Anguilla anguilla) O.mykiss eggs England Human mouth swab Atlantic salmon, Scotland O. tschawytscha Rainbow trout England Grayling (T. thymallus), England Atlantic salmon (S. salar) England Carious teeth No information available No information available Pleural fluid Clinical isolate Nose Pathogenic for fish Coho salmon (O. kisutch) Atlantic salmon (S. salar) Rainbow trout, England Rainbow trout, England Rainbow trout, England Rainbow trout, England Rainbow trout, England
buffer IV (ABgene), 1.5 mM MgCl2 (ABgene), 0.25 mM each dNTP (ABgene), 100 pmol of each primer (Pharmacia biotech), 10 μl DNA template, made up to 95 μl with distilled water, overlaid with mineral oil (Sigma) and heated to 94 °C. To this was added 5 μl Red hot™ Taq polymerase (ABgene) at 0.025 U/μl to give a total reaction volume of 100 μl. Cycling conditions consisted of 35 cycles of 94 °C for 1 min, 57 °C for 1 min, 72 °C for 2 min, followed by a single cycle of 94 °C for 2 min, 55 °C for 2 min and 72 °C for 10 min. The primers used in the modified PCR were DN1 and DN2 (Table 2). For all PCR procedures, the strictest attention was paid to minimise the risks of contamination. Initial harvesting of lymphocytes prior to DNA extraction (see below) was done in contained sterile bijoux, rather
then in open Petri dishes, all DNA extractions and preparation of PCR reactions were done in separate filtered airflow PCR cabinets in a separate controlled room to where PCR amplification, and subsequent agarose gel visualisation of resultant amplicons took place. The risk of contamination was monitored by inclusion of negative water controls during PCR preparation. An open container of water was also placed on the bench during lymphocyte harvesting and tested by PCR to confirm the risk of cross-transfer of infected material during this part of the process was also negligible. Sterile, molecular grade, reagents and plastic consumables were used at all times, including use of barrier pipette tips designed for PCR-based procedures. 2.3. Sensitivity testing The sensitivity of the modified technique was first determined in peptone/saline with serial tenfold dilutions laboratory cultures of R. salmoninarum NCIMB 2235, 970083, 970153 and 970419 cultured in KDM (kidney disease medium; Evelyn 1977 ) broth with shaking at 15 °C for 10 d. Contents of the broths were poured into 2 × 50 ml centrifuge tubes and centrifuged at 2500 g for 20 min, with pellets resuspended in peptone saline to an OD540 of 1.25. Serial tenfold dilutions were prepared in peptone saline and spot plates were prepared from these dilutions on each of SKDM (selective kidney disease medium; Austin and Austin, 1983) and 10-Met (Teska et al., 1994) plates. KDM, SKDM and 10-Met media were prepared in house using ingredients sourced from either Oxoid (Basingstoke, England) or Sigma (Poole, England). 250 μl DNA template for PCR from each dilution was prepared by centrifuging 1 ml volumes of each dilution at 2500 g for 20 min, resuspending the resultant pellet in 100 μl PBSa, adding 1 ml DNAzol™ (Invitrogen). DNAzol™ extraction was carried out according to the manufacturers instructions, except the resultant DNA pellet was suspended in 250 μl deionised molecular grade water and heated to 65 °C for 10 min to encourage it to dissolve before freezing at −20 °C until required. Previously frozen 50 mg head kidney samples of rainbow trout and grayling, previously determined to be PCR-negative, were also spiked with similarly prepared 100 μl serial tenfold dilutions of NCIMB 2235, 970083, 970153 and tested using the modified PCR with 10 μl volumes of template DNA used in all reactions. The developed PCR was also validated using material from clinically unapparent, approximately 500 g, disease free rainbow trout that were sampled eight weeks after being i.p. injected with 3.12 × 103 colony forming units (cfu) R. salmoninarum NCIMB 1111 cells (based on plate counts on SKDM), that had been grown in EPC (Epithelioma papillosum cyprini) cells (Chambers, 2005). Fish were reared in freshwater in 350 l tanks at 11 ± 1 °C for the duration of the experiment. Template DNA from spiked and experimentally infected fish head kidney material was prepared as described below. Amplification for all sensitivity testing samples was carried out as previously described using the modified primers, and comparing the use of either 5 μl or 10 μl of the different template preparations, with PCR products visualised following electrophoresis through 2% agarose
Table 2 PCR Primers used in the study to target a 376 bp region of the P57 protein encoding gene in R. salmoninarum Name
Direction
Primer sequence
Reference or source
G6481
Forward
5′-GCGCGGATCGAAAATAAAAAAAATTTTAGCGCTG-3′
G6480
Reverse
5′-GCGCGGATCCTTGGCAGGACCATCTTTGT-3′
DN1 DN2
Forward Reverse
5′-GAAAATAAAAAAAATTTTAGCGCTG-3′ 5′-CTTGGCAGGACCATCTTTGT-3′
McIntosh et al., 1996 McIntosh et al., 1996 This study This study
The underlined portions refer to published sequences of the target gene (GenBank accession number S46378).
E.M. Chambers et al. / Aquaculture 287 (2009) 35–39
Fig. 1. Agarose gel showing evidence of cross-reaction when high concentrations of Y. ruckeri isolates were tested using the McIntosh et al. (1996) R. salmoninarum PCR. Every isolate yielded a product indistinguishable in size from that of R. salmoninarum. Lanes 1 and 10) 100 bp ladders. 2) Y. ruckeri NCIMB 2194. 3) Y. ruckeri NCTC 10476. 4) Y. ruckeri NCIMB 1316 5) Y. ruckeri 961156 farmed rainbow trout. 6) Y. ruckeri 980070 farmed rainbow trout 7) Y. ruckeri 980051 Mirror carp. 8) negative water control. 9) R. salmoninarum positive control.
gels. In all experiments appropriate negative (water only) and positive controls were included.
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of another study (Chambers and Barker, 2006). After taking swabs of head kidney material for culture on a variety of different media for that study, the head kidneys of the fish were then removed, placed into separate vials, and stored at −20 °C until required. Likely detection limits were determined by testing pooled samples containing either four positive kidney samples, with one negative sample, or four negative samples with one positive kidney sample using the modified PCR. Samples were selected from a range of fish that had previously been characterised by culture-based methods as strongly positive (+++), positive (++), weakly positive (+), and borderline positive (less than 10 R. salmoninarum colonies recovered; +/−) (Chambers and Barker, 2006). DNA was extracted from head kidney material from the pools of five fish using two minor modifications of the method described above. For the first method, 10 mg of head kidney from each of 5 fish was pooled prior to extraction. The effect of removing 50 mg head kidney from each fish, and adding it to a proportionately larger volume of diluent prior to Dnazol™ extraction, was also tested. 3. Results
2.4. DNA extraction from head kidney samples 3.1. McIntosh et al. (1996) PCR Various methods for preparation of the template were tried, including maceration by micropestle, FastDNA tubes (Anachem) and the preparation of a crude lymphocyte lysate as published by McIntosh et al. (1996; results not shown). A slightly modified version of the crude lymphocyte lysate was found to give the most sensitive and clean results. Briefly 50 mg head-kidney samples were added to individual sterile 5 ml bijou containing 1 ml ice-cold PBSa and disrupted by serial passage through a 2 ml syringe. The contents were then diluted in 1 ml ice-cold PBSa, cell debris separated by centrifugation (275 g for 6 min) and 1.5 ml of the supernatant transferred to a microcentrifuge tube and centrifuged (10,000 g for 2 min). The pellet was washed twice with ice-cold PBSa, resuspended in 100 μl of ice-cold PBSa, and vortexed well. DNA extraction was carried out by adding 1 ml of DNAzol™ to each tube. DNAzol™ extraction was carried out as described earlier for peptone saline dilutions, in all cases the resultant DNA pellet was finally dissolved in 250 μl of deionised molecular grade water which was used as template for subsequent PCR assays. 2.5. Effect of pooling The effect of pooling different combinations of infected and uninfected samples on the sensitivity of the modified PCR was also tested using naturally-infected fish, collected from a fish farm as part Table 3 The sensitivity of the modified PCR assay to detect R. salmoninarum DNA from spiked peptone/saline samples Test 1 2 3 4 5 6 7 8
Isolate NCIMB 2235 970083 970083 970083 970083 970153 970153 970419
PCR lower limit of detection
The R. salmoninarum isolates generated 376 bp products when tested using the PCR protocol recommended by McIntosh et al. (1996). No member of the strain panel, other than Y. ruckeri, generated any product with this PCR protocol. All Y. ruckeri isolates generated products indistinguishable in size from that expected for R. salmoninarum when undiluted DNA extractions were tested (Fig. 1). When 1:1000 dilutions were tested, occasional cross-reactions were observed in repeated testing, not specific to any particular Y. ruckeri strain tested. Sequence analysis of the false positive amplicons generated from the Y. ruckeri isolates, showed that there was coincidental binding of the forward primer to both the 5′ and 3′ termini of the Y. ruckeri product (Results not shown). The sequence had 75.5% homology at the nucleotide level to a 5-enolpyruvylshikimate 3-phosphate synthase (aroA) gene-derived sequence of Yersinia enterocolitica (Gen Bank Accession number M32213). 3.2. Modified PCR The modified PCR protocol routinely detected less than 100 viable R. salmoninarum cells per reaction in peptone saline [limit of detection (L.O.D.) range 0.1–82 cfu/PCR reaction; Table 3], while results from spiked kidney samples suggested that the presence of kidney tissue may inhibit the PCR slightly (L.O.D range 9.82–
Table 4 The sensitivity of the modified PCR assay to detect R. salmoninarum DNA from spiked kidney samples
cfu/ml
cfu/PCR reaction
Test
Isolate
82.8 5.5 48.8 23.3 0.1 0.6 71.2 37.5
Head kidney source
PCR lower limit of detection
2070 137 1220 582 3.0 14.1 1780 938
cfu/mg
cfu/PCR reaction
1 2 3 4 5 6
970083 NCIMB 2235 NCIMB 2235 970153 970153 NCIMB 2235
Grayling Grayling Grayling Grayling Grayling Rainbow trout
12.6 4.6 6.5 72.4 22.0 63.2
25.2 9.1 13.0 145 44.0 126
Limit of detection results are expressed as both the minimum number of colony forming units of R. salmoninarum per ml of peptone saline that resulted in a PCR positive result, and the number of colony forming units of R. salmoninarum detected per PCR reaction when 10 μl of DNA template was used per reaction. Peptone/saline solutions were spiked with serial dilutions of R. salmoninarum, with DNA template for PCR prepared using the DNAzol™ based DNA extraction method, with resultant DNA pellets dissolved in 250 μl of deionised water each time (Section 2.3). Template DNA was PCR-amplified using the modified primers and PCR reaction conditions (Section 2.2).
Limit of detection results are expressed as both as the minimum number of colony forming units of R. salmoninarum per mg of kidney that resulted in a PCR positive result, and the minimum number of colony-forming units of R. salmoninarum detected per PCR reaction when 10 μl of DNA template was used per reaction. 50 mg head kidney samples were spiked with serial dilutions of R. salmoninarum. DNA template for PCR was prepared from the spiked head kidney samples using the DNAzol™ based DNA extraction method, with DNA pellets dissolved in 250 μl of deionised water each time (Section 2.3). Template DNA was PCR-amplified using the modified primers and PCR reaction conditions (Section 2.2).
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144.8 cfu/PCR; Table 4). However, when expressed in terms of detectable colony forming units per mg of head kidney tissue the PCR was shown to be capable of detecting between 4.56 and 72.4 cfu/mg fish head kidney (Table 4). There was no evidence that the modified PCR produced false positive amplicons with any of the panel of bacteria tested (Table 1). For the template preparation methods involving Dnazol™ extraction, more PCR positive samples were detected following the preparation of the crude lymphocyte lysate from tissue samples than with other disruption methods tested (e.g. direct homogenisation of head kidney material in DNAzol™; and FastDNA (Anachem) results not shown). In particular, templates produced by the other methods needed to be diluted 1:10 to ensure the production of a clear band, reducing test sensitivity. The use of 10 μl rather than 5 μl of template also detected more positive samples without any perceptible increase in inhibition (results not shown). DNAzol™-based DNA extraction of a crude lymphocyte fraction from clinically unapparent fish that had previously been exposed to R. salmoninarum allowed the detection of more positive samples than the Instagene™-based DNA extraction method (Table 5), even though the Instagene™ DNA extraction procedure required 500 mg of head kidney material, compared to 50 mg for the DNAzol™ DNA extraction procedure. None of these experimentally infected fish were positive by culture when tested. When tested with field-infected material, the modified PCR detected individual fish that were also shown to be culture positive (Table 6).
Table 6 The effect of pooling and fish head kidney sample amount on the sensitivity of the PCR to detect R. salmoninarum p57 protein encoding gene DNA Fish no.
Infectivity level
Combination (+:−)
Pooling 10 mg each
Pooling 50 mg each
82 52 62 69 20 87 80 50 59 4,52,82,3 81,53,8,84 20,62,69,89
+++ +++ + + + +/− +/− +/− +/− +++ ++ +
01:04 01:04 01:04 01:04 01:04 01:04 01:04 01:04 01:04 04:01 04:01 04:01
+ + − + + − − + + + + +
+ + + + + + + + + + + +
A comparison was made of the difference in sensitivity between pooling 5 × 10 mg, or 5 × 50 mg samples of fish head kidney in a proportionally higher amount of diluent, prior to DNA extraction and PCR. 10 and 50 mg samples of head kidney were taken from farm fish that were previously demonstrated to be both PCR and culture positive when tested individually (based on 50 mg samples) and DNA was extracted. Samples were prepared in a ratio of one positive to four negative, or one negative to four positive fish, then tested by PCR. When preparing the four positive fish mixes, an attempt was made to combine templates from fish that all had apparently similar infection levels, based on numbers of R. salmoninarum colonies cultured on SKDM inoculated with head kidney material. +/−: grew less than 10 R. salmoninarum colonies. +: grew between 10–20 R. salmoninarum colonies. ++: grew between 20–100 R. salmoninarum colonies. +++: grew more than 100 R. salmoninarum colonies.
3.3. Pooling Results showed that sensitivity was reduced when 10 mg head kidney was taken from each fish and pooled (Table 6). Of the fish assessed by culture and PCR as of low infectivity levels, three out of seven gave false negative results when they were each mixed with head kidney material from four other fish that were culture negative (Table 6). However when using 50 mg from each fish and altering the Table 5 Detection of R. salmoninarum DNA by the modified PCR following extraction by two different methods: the preparation of a crude lymphocyte lysate from 500 mg head kidney followed by Instagene™ extraction, and the preparation of a crude lymphocyte lysate from 50 mg head kidney followed by DNAzol™ extraction Fish no. C1 T1.1 T1.2 T1.3 M1 C2 T2.1 T2.2 T2.3 C4 T4.1 T4.2 T4.3 C5 T5.1 T5.2 T5.3 C6 T6.1 T6.2 T6.3 T9.1
Extraction method Instagene 500 mg
DNAzol 50 mg
− − − + + − − + w+ − − w+ − − − − − − − vw+ − +
− + + + + − − + + − − w+ w+ − − w+ − − − vw+ vw+ +
A selection of the results generated is shown. C: control fish. T: test fish (injected with 3.12 × 103 cfu R. salmoninarum previously). All test and control fish were from a laboratory infection experiment. No test fish showed clinical signs of disease or were culture positive. M: mortality; + represents a bright band following electrophoresis; w+ represents a weak band following electrophoresis; vw+ represents very faint bands.
diluent volumes, there was no apparent loss of sensitivity, with even the combination with template derived from a single fish with only an apparent low level infection mixed with kidney material from four negative fish yielding PCR product bands of equal intensity to the infected kidney alone (Table 6). When using combinations of four positive fish to one negative fish, amplicons were easily visible when using 50 mg from each fish, with no evidence of inhibition or obscuring by smears 4. Discussion The purpose of this study was to validate and adapt, if necessary, a simple PCR method for the detection of R. salmoninarum in both wild and farmed fish from selected river catchments in England and Wales (Chambers et al., 2008). The technique chosen needed to be reliable, reproducible, quick, and relatively inexpensive. Before utilising a new method one must look at the intended application and purpose of the application (Hiney, 1997). The importance of validating methods, before a specific use, has been demonstrated. The original authors of the PCR-based R. salmoninarum detection technique (McIntosh et al., 1996) tested the technique with a low concentration of Y. ruckeri and did not see any evidence of crossreaction. However, as R. salmoninarum can be detected in fish with coexisting heavy Y. ruckeri infections (personal observations), high concentrations of bacteria were deliberately used in the bacterial strain panels. High concentrations consistently produced crossreactions with the original primers. Lower concentrations produced occasional cross-reactions. Redesign of both primers produced a combination which removed the cross-reaction. Another important modification, that may also be relevant to other published PCR-based detection protocols, was the finding that R. salmoninarum positive fish could be reliably detected using as little as 50 mg head kidney tissue if a crude lymphocyte lysate was prepared from the tissue followed by a DNAzol-based DNA extraction even from laboratory infected fish that were clinically inapparent or positive by culture. The McIntosh et al. (1996) Instagene matrix™-based protocol requires 500 mg head kidney tissue, a quantity readily produced from
E.M. Chambers et al. / Aquaculture 287 (2009) 35–39
farmed fish near table-size. However, for other purposes, such as wild fish surveys (e.g. Chambers et al., 2008) much smaller quantities of head kidney may be available from individual fish. In the early stages of R. salmoninarum infection the bacterium is not evenly distributed throughout the head kidney, but present in discreet lesions or granulomas (Bruno, 1986). This could possibly explain why at least 50 mg of tissue needed to be sampled from each fish in the pooling experiment to reliably detect R. salmoninarum in the pool. The results also show that it is possible to pool samples of fish into groups of 5 without significantly reducing the sensitivity of the PCR test, saving time and resources. The developed PCR method was used in a companion study (Chambers et al., 2008) and was able to detect a high prevalence of R. salmoninarum DNA in wild grayling sampled from two river catchments close to rainbow trout farms in Southern England. That study also showed there were likely false positives generated from a number of pike (Esox lucius) samples tested using the modified PCR protocol (Chambers et al., 2008). Sequencing cloned amplicons from the pike positive samples showed that a 348 bp DNA fragment of unknown origin (that may have been pike chromosomal DNA) was amplified from those samples using the modified primers (Chambers, 2005). It is therefore recommended that presumptively positive results from pike, as well as other species, generated using this and other PCR protocols, are confirmed as of R. salmoninarum P57, or other target gene origin, by sequencing the resultant amplicons. As this and the Chambers et al. (2008) studies have demonstrated, false positives can be produced using even well-validated PCR-based protocols. The OIE recommended PCR method (OIE, 2006) is based on a nested PCR protocol that targets part of the P57 encoding gene, developed by Chase and Pascho (1998). Nested protocols are inherently less prone than single round PCRs to false positives, produced as the result of coincidental primer annealing with host genomic, or other non-R. salmoninarum DNA. Conversely, they are more laborious and prone to potential laboratory contamination. It is recommended that, where possible, complementary techniques, such as use of ELISA (e.g. Jansson et al., 1996), as well as direct culture of the organism and confirmatory biochemical and immunoassay identification and other well-validated methods, are used in addition to PCR assays. This is particularly important if the resultant data is used to help inform BKD control programmes. Acknowledgements This work was funded by Defra through project number C0280. References Anon, 1999. Report of the Scientific Committee on Animal Health and Animal Welfare European Commission Unit B3, Bacterial Kidney Disease, Sanco/B3/AH/R14/1999 p. 11. Austin, B., Austin, D.A., 1983. Selective isolation of Renibacterium salmoninarum. FEMS Microbiol. Lett. 17, 111–114. Bandin, I., Heinen, P., Brown, L.L., Toranzo, E., 1996. Comparison of different ELISA kits for detecting Renibacterium salmoninarum. B. Eur. Assoc. Fish Pat. 16, 19–22. Brown, L.L., Iwama, G.K., Evelyn, T.P.T., Nelson, W.S., Levine, R.P., 1994. Use of the polymerase chain reaction (PCR) to detect DNA from Renibacterium salmoninarum within individual salmonid eggs. Dis. Aquat. Org. 18, 165–171. Bruno, D.W., 1986. Histopathology of bacterial kidney disease in laboratory infected rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L., with reference to naturally infected fish. J. Fish Dis. 9, 523–537. Bruno, D., Collet, B., Turnbull, A., Kilburn, R., Walker, A., Pendrey, D., McIntosh, A., Urquart, K., Taylor, G., 2007. Evaluation and development of diagnostic methods for Renibacterium salmoninarum causing bacterial kidney disease (BKD) in the UK. Aquaculture 269, 114–122. Bullock, G.L., Griffin, B.R., Stuckey, H.M., 1980. Detection of the Corynebacterium salmoninus by direct fluorescent antibody test. Can. J. Fish. Aquat. Sci. 37, 719–721.
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Chambers, E., 2005. Development of methods to assess the prevalence of Renibacterium salmoninarum in selected river systems in England and Wales. MPhil thesis, University of Plymouth, UK. Chambers, E., Barker, G., 2006. Comparison of culture media for the isolation of Renibacterium salmoninarum from naturally infected rainbow trout (Oncorhynchus mykiss). B. Eur. Assoc. Fish Pat. 26, 137–142. Chambers, E., Gardiner, R., Peeler, E.J., 2008. An investigation into the prevalence of R. salmoninarum in farmed rainbow trout (Oncorhynchus mykiss) and wild fish populations in selected river catchments in England and Wales between 1998 and 2000. J. Fish Dis. 31, 89–96. Chase, D.M., Pascho, R.J., 1998. Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney. Dis. Aquat. Org. 34, 223–229. Chien, M.S., Gilbert, T.L., Huang, C., Landolt, M.L., O'Hara, P.J., Winton, J., 1992. Molecular cloning and sequence analysis of the gene coding for the 57 kDa major soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum. FEMS Microbiol. Lett. 96, 259–266. Cook, M., Lynch, W.H., 1999. A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.). Appl. Environ. Microbiol. 65, 3042–3047. Cvitanich, J.D., 1994. Improvements in the direct fluorescent antibody technique for the detection, identification, and quantification of Renibacterium salmoninarum in salmonid kidney smears. J. Aquat. Anim. Health 6, 1–12. Dixon, P.F., 1987. Detection of Renibacterium salmoninarum by the enzyme-linked immunosorbent assay (ELISA). J. Appl. Ichthyol. 3, 77–82. Evelyn, T.P.T., 1977. An improved growth medium for the kidney disease bacterium and some notes on using the medium. Bull. Off. Int. Epizoot. 87, 511–513. Evenden, A.J., Grayson, T.H., Gilpin, M.L., Munn, C.B., 1993. Renibacterium salmoninarum and bacterial kidney disease — the unfinished jigsaw. Ann. Rev. Fish Dis. 1, 87–104. Gudmundsdottir, S., Benediktsdottir, E., Helgason, S., 1993. Detection of Renibacterium salmoninarum in salmonid kidney samples: a comparison of results using doublesandwich ELISA and isolation on selective medium. J. Fish Dis. 16, 185–195. Hiney, M., 1997. How to test a test: methods of field validation for non-culture-based detection techniques. Bull. Eur. Assoc. Fish Pathol. 17, 245–250. Hiney, M.P., Smith, P.R., 1997. Validation of polymerase chain reaction-based techniques for proxy detection of bacterial fish pathogens: framework, problems and possible solutions for environmental applications. Aquaculture 162, 41–68. Jansson, E., Hongslo, T., Hoglund, J., Ljungberg, O., 1996. Comparative evaluation of bacterial culture and two ELISA techniques for the detection of Renibacterium salmoninarum antigens in salmonid kidney tissues. Dis. Aquat. Org. 27, 197–206. Leon, G., Martinez, M.A., Etchegaray, J.P., Vera, M.I., Figueroa, J., Krauskopf, M., 1994a. Specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum. World J. Microb. Biotechnol. 10, 149–153. Leon, G., Maulen, N., Figueroa, J., Villanueva, J., Rodriguez, C., Ines, V.M., Krauskopf, M., 1994b. A PCR-based assay for the identification of the fish pathogen Renibacterium salmoninarum. FEMS Microbiol. Lett. 115, 131–136. Magnusson, H.B., Fridjonsson, O.H., Andresson, O.S., Benediktsdottir, E., Gudmundsdottir, S., Andresdottir, V., 1994. Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish, detected by nested reverse transcriptionPCR of 16S rRNA sequences. Appl. Environ. Microbiol. 60, 4580–4583. Mattson, J.G., Gersdorf, H., Jansson, E., Hongslo, T., Gobel, U.B., Johansson, K.-E., 1993. Rapid identification of Renibacterium salmoninarum using an oligonucleotide probe complementary to 16S rRNA. Mol. Cell. Probes 7, 25–33. McIntosh, D., Meaden, P.G., Austin, B., 1996. A simplified PCR-based method for the detection of Renibacterium salmoninarum utilizing preparations of rainbow trout (Oncorhynchus mykiss, Walbaum) lymphocytes. Appl. Environ. Microbiol. 62, 3929–3932. Miriam, A., Griffiths, S.G., Lovely, J.E., Lynch, W.H., 1997. PCR and Probe-PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.) ovarian fluid and kidney tissue for the presence of DNA of the fish pathogen Renibacterium salmoninarum. J. Clin. Microbiol. 35, 1322–1326. OIE, 2006. Bacterial Kidney Disease. Manual of Diagnostic Tests for Aquatic Animals. . Chapter 2.1.11 http://www.oie.int/eng/normes/fmanual/A_summry.htm. Pascho, R.J., Mulkahey, D., 1987. Enzyme-linked immunosorbent assay for a soluble antigen of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Can. J. Fish. Aquat. Sci. 44, 183–190. Scott, R., Johnson, K., 2001. Inconsistency of Kirkegaard and Perry BKD ELISA antibody lots. Fish Health News 29, 4–6. Teska, J.D., 1994. In vitro growth of the bacterial kidney disease organism Renibacterium salmoninarum on a nonserum, noncharcoal-based ‘homospecies-metabolite’ medium. J. Wildl. Dis. 30, 383–388.
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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
Polymerase chain reaction detection of Renibacterium salmoninarum in fish: Validation of a modified protocol Edel M. Chambers a, David A. Nagel b, Edward A. Elloway b, Kelly L. Addison b, Gavin A. Barker a, David W. Verner-Jeffreys a,⁎, David M. Stone a a b
Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK Department of Biological Sciences, Aston university, Aston Triangle, Birmingham, B4 7ET, UK
a r t i c l e
i n f o
Article history: Received 2 April 2008 Received in revised form 22 September 2008 Accepted 17 October 2008 Keywords: Renibacterium salmoninarum PCR Validation
a b s t r a c t A PCR protocol (McIntosh et al., 1996. Appl. Environ. Microbiol. 62, 3929–2932), designed to detect the P57 gene of Renibacterium salmoninarum, causative agent of Bacterial Kidney Disease, was modified slightly by redesign of the forward primer and recommended conditions to prevent possible false positives observed when tested against pure cultures of Yersinia ruckeri. The modified PCR, in combination with an improved DNAzol™-based DNA extraction technique, was very specific and sensitive (detecting between 5 and 72 R. salmoninarum cfu per mg head kidney tissue). It could detect infected fish using only 50 mg head kidney material, making the technique suitable for sampling small fish. It was also shown that samples could be pooled from up to five rainbow trout prior to PCR without noticeably affecting the sensitivity of the assay, providing at least 50 mg head kidney tissue was included from each of the fish that were pooled. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.
1. Introduction Bacterial Kidney Disease (BKD) caused by the bacterium Renibacterium salmoninarum (R. salmoninarum) is regarded as one of the most persistent bacterial diseases of salmonids worldwide (Evenden et al., 1993). BKD can be detected using a variety of test methods (Bruno et al., 2007), such as culture on specialised media (Chambers and Barker, 2006), direct and indirect fluorescent antibody test DFAT, IFAT, (Bullock et al., 1980; Cvitanich, 1994), as well as the more rapid ELISA (Gudmundsdottir et al., 1993; Jansson et al., 1996; Pascho and Mulkahey, 1987). However the sensitivity (Bandin et al., 1996), specificity (Dixon, 1987) and reproducibility (Scott and Johnson, 2001) of some of the ELISA tests has been questioned (Anon, 1999). More recently there has been an increase in the use of probe (Mattson et al., 1993; Leon et al., 1994a) and PCR-based assays to look for the presence of R. salmoninarum DNA and RNA in head-kidney, ovarian fluid and eggs (Brown et al., 1994; Leon et al., 1994b; Magnusson et al., 1994; McIntosh et al., 1996; Miriam et al., 1997; Chase and Pascho, 1998; Cook and Lynch, 1999). However, before using such proxy-based detection methods it is important to thoroughly validate them (Hiney and Smith, 1997).
⁎ Corresponding author. Tel.: +44 1305 206725; fax: +44 1305 206601. E-mail address: [email protected] (D.W. Verner-Jeffreys).
In this study, a modification of a PCR protocol McIntosh et al. (1996), designed to detect the P57 protein encoding gene of Renibacterium salmoninarum, causative agent of Bacterial Kidney Disease, hereinafter referred to as the modified PCR procedure, was developed. This modified protocol was tested for its ability to detect the presence of R. salmoninarum derived nucleic acid in a panel of test bacteria and fish both artificially and naturally infected with R. salmoninarum. 2. Materials and methods 2.1. Testing of McIntosh et al. (1996) recommended PCR procedures against a panel of bacteria A range of bacterial isolates, representing organisms that may be encountered during sampling of wild or farmed fish for BKD (Table 1), were grown in suitable broth media and resuspended in PBSa to an OD550 of 1.0. DNA was then extracted from undiluted and 1:1000 dilutions of each isolate, and tested for the presence of R. salmoninarum specific P57 protein encoding gene (Chien et al., 1992) by PCR, as described by McIntosh et al. (1996). 2.2. Modified PCR The protocol recommended by McIntosh et al. (1996) was modified to prevent coincidental binding of the forward primer to the Yersinia ruckeri genome (see Results). The PCR protocol developed, following a period of optimisation (data not shown), consisted of 10 μl reaction
0044-8486/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.10.031
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E.M. Chambers et al. / Aquaculture 287 (2009) 35–39
Table 1 Strain panel isolates used to test the specificity of the two PCR protocols [McIntosh et al., 1996; modified PCR protocol (this study)] designed to detect R. salmoninarum DNA from fish head kidney Strain
Identification
Origin
Aerococcus viridans Aeromonas caviae Aeromonas hydrophila Aeromonas hydrophila Aeromonas hydrophila sub sp. formicans Aeromonas hydrophila sub sp. sobria Aeromonas salmonicida Aeromonas salmonicida Aeromonas salmonicida sub sp. achromogenes Aeromonas salmonicida sub sp. achromogenes Arthrobacter globiformens Bacillus cereus Bacillus polymyxa Bacillus subtilis Bacillus sphaericus Brevibacterium linens Corynebacterium aquaticum Corynebacterium aquaticum Corynebacterium aquaticum Carnobacterium piscicola Escherichia coli Escherichia coli Flavobacterium psychrophilum Flavobacterium psychrophilum Flavobacterium psychrophilum Flavobacterium maritimus Klebsiella aerogenes Lactobacillus brevis Micrococcus luteus Pseudomonas aeuruginosa Pseudomonas aeuruginosa Pseudomonas anguilliseptica Pseudomonas fluorescens Pseudomonas maltophilia Renibacterium salmoninarum Renibacterium salmoninarum Renibacterium salmoninarum Renibacterium salmoninarum Renibacterium salmoninarum Rothia dentocariosa Salmonella typhimurium Serratia marcescens Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Vibrio anguillarum Vibrio ordalii Vibrio salmonicida Yersinia ruckeri Yersinia ruckeri Yersinia ruckeri Yersinia ruckeri Yersinia ruckeri
930681 NCIMB 1330 NCIMB 72 NCIMB 9241 NCTC 10362
Lobster, England Freshwater fish Moribund goldfish (C. auratus) Human myositis Sewage
NCIMB 37
No information available
NCIMB 833 MT423 NCIMB 1109
Brook trout (S. fontinalis) Atlantic salmon (Salmo salar L) Brown trout (Salmo trutta L.)
NCIMB 1110
Brown trout
NCIMB 8907 NCIMB 8012 ATCC 12321 NCIMB 11232 NCIMB 9370 NCIMB 9909 970798 1.3.4 970805 NCIMB 9460 NCIMB 2264 NCTC 10418 NCIMB 9482 NCIMB 1947 980070 18 970804 3.2.2 NCIMB 2158 NCTC 9528 NCIMB 11973 NCIMB 8553 NCIMB 13066 NCIMB 8295 NCIMB 1949 930029 NCIMB 9203 NCIMB 1111 NCIMB 2235 970083 970153 970419 NCTC 10917 NCTC 12484 NCTC 1377 NCTC 8532 NCIMB 12702 NCIMB 12721 NCIMB 2286 NCIMB 2167 NCIMB 2262 NCIMB 1316 NCTC 10476 NCTC 10477 NCTC 10478 NCIMB 2194
No information available No information available No information available No information available No information available Good harzerkase cheese Rainbow trout (O. mykiss) UK Farmed rainbow trout No information available Cutthroat trout (S. clarkii) No information available Torry research station (K12) Coho salmon (O. kisutch) Rainbow trout England Rainbow trout England Dover sole (Solea solea L.) Water Human faeces No information available Freshwater fish No information available Eel (Anguilla anguilla) O.mykiss eggs England Human mouth swab Atlantic salmon, Scotland O. tschawytscha Rainbow trout England Grayling (T. thymallus), England Atlantic salmon (S. salar) England Carious teeth No information available No information available Pleural fluid Clinical isolate Nose Pathogenic for fish Coho salmon (O. kisutch) Atlantic salmon (S. salar) Rainbow trout, England Rainbow trout, England Rainbow trout, England Rainbow trout, England Rainbow trout, England
buffer IV (ABgene), 1.5 mM MgCl2 (ABgene), 0.25 mM each dNTP (ABgene), 100 pmol of each primer (Pharmacia biotech), 10 μl DNA template, made up to 95 μl with distilled water, overlaid with mineral oil (Sigma) and heated to 94 °C. To this was added 5 μl Red hot™ Taq polymerase (ABgene) at 0.025 U/μl to give a total reaction volume of 100 μl. Cycling conditions consisted of 35 cycles of 94 °C for 1 min, 57 °C for 1 min, 72 °C for 2 min, followed by a single cycle of 94 °C for 2 min, 55 °C for 2 min and 72 °C for 10 min. The primers used in the modified PCR were DN1 and DN2 (Table 2). For all PCR procedures, the strictest attention was paid to minimise the risks of contamination. Initial harvesting of lymphocytes prior to DNA extraction (see below) was done in contained sterile bijoux, rather
then in open Petri dishes, all DNA extractions and preparation of PCR reactions were done in separate filtered airflow PCR cabinets in a separate controlled room to where PCR amplification, and subsequent agarose gel visualisation of resultant amplicons took place. The risk of contamination was monitored by inclusion of negative water controls during PCR preparation. An open container of water was also placed on the bench during lymphocyte harvesting and tested by PCR to confirm the risk of cross-transfer of infected material during this part of the process was also negligible. Sterile, molecular grade, reagents and plastic consumables were used at all times, including use of barrier pipette tips designed for PCR-based procedures. 2.3. Sensitivity testing The sensitivity of the modified technique was first determined in peptone/saline with serial tenfold dilutions laboratory cultures of R. salmoninarum NCIMB 2235, 970083, 970153 and 970419 cultured in KDM (kidney disease medium; Evelyn 1977 ) broth with shaking at 15 °C for 10 d. Contents of the broths were poured into 2 × 50 ml centrifuge tubes and centrifuged at 2500 g for 20 min, with pellets resuspended in peptone saline to an OD540 of 1.25. Serial tenfold dilutions were prepared in peptone saline and spot plates were prepared from these dilutions on each of SKDM (selective kidney disease medium; Austin and Austin, 1983) and 10-Met (Teska et al., 1994) plates. KDM, SKDM and 10-Met media were prepared in house using ingredients sourced from either Oxoid (Basingstoke, England) or Sigma (Poole, England). 250 μl DNA template for PCR from each dilution was prepared by centrifuging 1 ml volumes of each dilution at 2500 g for 20 min, resuspending the resultant pellet in 100 μl PBSa, adding 1 ml DNAzol™ (Invitrogen). DNAzol™ extraction was carried out according to the manufacturers instructions, except the resultant DNA pellet was suspended in 250 μl deionised molecular grade water and heated to 65 °C for 10 min to encourage it to dissolve before freezing at −20 °C until required. Previously frozen 50 mg head kidney samples of rainbow trout and grayling, previously determined to be PCR-negative, were also spiked with similarly prepared 100 μl serial tenfold dilutions of NCIMB 2235, 970083, 970153 and tested using the modified PCR with 10 μl volumes of template DNA used in all reactions. The developed PCR was also validated using material from clinically unapparent, approximately 500 g, disease free rainbow trout that were sampled eight weeks after being i.p. injected with 3.12 × 103 colony forming units (cfu) R. salmoninarum NCIMB 1111 cells (based on plate counts on SKDM), that had been grown in EPC (Epithelioma papillosum cyprini) cells (Chambers, 2005). Fish were reared in freshwater in 350 l tanks at 11 ± 1 °C for the duration of the experiment. Template DNA from spiked and experimentally infected fish head kidney material was prepared as described below. Amplification for all sensitivity testing samples was carried out as previously described using the modified primers, and comparing the use of either 5 μl or 10 μl of the different template preparations, with PCR products visualised following electrophoresis through 2% agarose
Table 2 PCR Primers used in the study to target a 376 bp region of the P57 protein encoding gene in R. salmoninarum Name
Direction
Primer sequence
Reference or source
G6481
Forward
5′-GCGCGGATCGAAAATAAAAAAAATTTTAGCGCTG-3′
G6480
Reverse
5′-GCGCGGATCCTTGGCAGGACCATCTTTGT-3′
DN1 DN2
Forward Reverse
5′-GAAAATAAAAAAAATTTTAGCGCTG-3′ 5′-CTTGGCAGGACCATCTTTGT-3′
McIntosh et al., 1996 McIntosh et al., 1996 This study This study
The underlined portions refer to published sequences of the target gene (GenBank accession number S46378).
E.M. Chambers et al. / Aquaculture 287 (2009) 35–39
Fig. 1. Agarose gel showing evidence of cross-reaction when high concentrations of Y. ruckeri isolates were tested using the McIntosh et al. (1996) R. salmoninarum PCR. Every isolate yielded a product indistinguishable in size from that of R. salmoninarum. Lanes 1 and 10) 100 bp ladders. 2) Y. ruckeri NCIMB 2194. 3) Y. ruckeri NCTC 10476. 4) Y. ruckeri NCIMB 1316 5) Y. ruckeri 961156 farmed rainbow trout. 6) Y. ruckeri 980070 farmed rainbow trout 7) Y. ruckeri 980051 Mirror carp. 8) negative water control. 9) R. salmoninarum positive control.
gels. In all experiments appropriate negative (water only) and positive controls were included.
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of another study (Chambers and Barker, 2006). After taking swabs of head kidney material for culture on a variety of different media for that study, the head kidneys of the fish were then removed, placed into separate vials, and stored at −20 °C until required. Likely detection limits were determined by testing pooled samples containing either four positive kidney samples, with one negative sample, or four negative samples with one positive kidney sample using the modified PCR. Samples were selected from a range of fish that had previously been characterised by culture-based methods as strongly positive (+++), positive (++), weakly positive (+), and borderline positive (less than 10 R. salmoninarum colonies recovered; +/−) (Chambers and Barker, 2006). DNA was extracted from head kidney material from the pools of five fish using two minor modifications of the method described above. For the first method, 10 mg of head kidney from each of 5 fish was pooled prior to extraction. The effect of removing 50 mg head kidney from each fish, and adding it to a proportionately larger volume of diluent prior to Dnazol™ extraction, was also tested. 3. Results
2.4. DNA extraction from head kidney samples 3.1. McIntosh et al. (1996) PCR Various methods for preparation of the template were tried, including maceration by micropestle, FastDNA tubes (Anachem) and the preparation of a crude lymphocyte lysate as published by McIntosh et al. (1996; results not shown). A slightly modified version of the crude lymphocyte lysate was found to give the most sensitive and clean results. Briefly 50 mg head-kidney samples were added to individual sterile 5 ml bijou containing 1 ml ice-cold PBSa and disrupted by serial passage through a 2 ml syringe. The contents were then diluted in 1 ml ice-cold PBSa, cell debris separated by centrifugation (275 g for 6 min) and 1.5 ml of the supernatant transferred to a microcentrifuge tube and centrifuged (10,000 g for 2 min). The pellet was washed twice with ice-cold PBSa, resuspended in 100 μl of ice-cold PBSa, and vortexed well. DNA extraction was carried out by adding 1 ml of DNAzol™ to each tube. DNAzol™ extraction was carried out as described earlier for peptone saline dilutions, in all cases the resultant DNA pellet was finally dissolved in 250 μl of deionised molecular grade water which was used as template for subsequent PCR assays. 2.5. Effect of pooling The effect of pooling different combinations of infected and uninfected samples on the sensitivity of the modified PCR was also tested using naturally-infected fish, collected from a fish farm as part Table 3 The sensitivity of the modified PCR assay to detect R. salmoninarum DNA from spiked peptone/saline samples Test 1 2 3 4 5 6 7 8
Isolate NCIMB 2235 970083 970083 970083 970083 970153 970153 970419
PCR lower limit of detection
The R. salmoninarum isolates generated 376 bp products when tested using the PCR protocol recommended by McIntosh et al. (1996). No member of the strain panel, other than Y. ruckeri, generated any product with this PCR protocol. All Y. ruckeri isolates generated products indistinguishable in size from that expected for R. salmoninarum when undiluted DNA extractions were tested (Fig. 1). When 1:1000 dilutions were tested, occasional cross-reactions were observed in repeated testing, not specific to any particular Y. ruckeri strain tested. Sequence analysis of the false positive amplicons generated from the Y. ruckeri isolates, showed that there was coincidental binding of the forward primer to both the 5′ and 3′ termini of the Y. ruckeri product (Results not shown). The sequence had 75.5% homology at the nucleotide level to a 5-enolpyruvylshikimate 3-phosphate synthase (aroA) gene-derived sequence of Yersinia enterocolitica (Gen Bank Accession number M32213). 3.2. Modified PCR The modified PCR protocol routinely detected less than 100 viable R. salmoninarum cells per reaction in peptone saline [limit of detection (L.O.D.) range 0.1–82 cfu/PCR reaction; Table 3], while results from spiked kidney samples suggested that the presence of kidney tissue may inhibit the PCR slightly (L.O.D range 9.82–
Table 4 The sensitivity of the modified PCR assay to detect R. salmoninarum DNA from spiked kidney samples
cfu/ml
cfu/PCR reaction
Test
Isolate
82.8 5.5 48.8 23.3 0.1 0.6 71.2 37.5
Head kidney source
PCR lower limit of detection
2070 137 1220 582 3.0 14.1 1780 938
cfu/mg
cfu/PCR reaction
1 2 3 4 5 6
970083 NCIMB 2235 NCIMB 2235 970153 970153 NCIMB 2235
Grayling Grayling Grayling Grayling Grayling Rainbow trout
12.6 4.6 6.5 72.4 22.0 63.2
25.2 9.1 13.0 145 44.0 126
Limit of detection results are expressed as both the minimum number of colony forming units of R. salmoninarum per ml of peptone saline that resulted in a PCR positive result, and the number of colony forming units of R. salmoninarum detected per PCR reaction when 10 μl of DNA template was used per reaction. Peptone/saline solutions were spiked with serial dilutions of R. salmoninarum, with DNA template for PCR prepared using the DNAzol™ based DNA extraction method, with resultant DNA pellets dissolved in 250 μl of deionised water each time (Section 2.3). Template DNA was PCR-amplified using the modified primers and PCR reaction conditions (Section 2.2).
Limit of detection results are expressed as both as the minimum number of colony forming units of R. salmoninarum per mg of kidney that resulted in a PCR positive result, and the minimum number of colony-forming units of R. salmoninarum detected per PCR reaction when 10 μl of DNA template was used per reaction. 50 mg head kidney samples were spiked with serial dilutions of R. salmoninarum. DNA template for PCR was prepared from the spiked head kidney samples using the DNAzol™ based DNA extraction method, with DNA pellets dissolved in 250 μl of deionised water each time (Section 2.3). Template DNA was PCR-amplified using the modified primers and PCR reaction conditions (Section 2.2).
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E.M. Chambers et al. / Aquaculture 287 (2009) 35–39
144.8 cfu/PCR; Table 4). However, when expressed in terms of detectable colony forming units per mg of head kidney tissue the PCR was shown to be capable of detecting between 4.56 and 72.4 cfu/mg fish head kidney (Table 4). There was no evidence that the modified PCR produced false positive amplicons with any of the panel of bacteria tested (Table 1). For the template preparation methods involving Dnazol™ extraction, more PCR positive samples were detected following the preparation of the crude lymphocyte lysate from tissue samples than with other disruption methods tested (e.g. direct homogenisation of head kidney material in DNAzol™; and FastDNA (Anachem) results not shown). In particular, templates produced by the other methods needed to be diluted 1:10 to ensure the production of a clear band, reducing test sensitivity. The use of 10 μl rather than 5 μl of template also detected more positive samples without any perceptible increase in inhibition (results not shown). DNAzol™-based DNA extraction of a crude lymphocyte fraction from clinically unapparent fish that had previously been exposed to R. salmoninarum allowed the detection of more positive samples than the Instagene™-based DNA extraction method (Table 5), even though the Instagene™ DNA extraction procedure required 500 mg of head kidney material, compared to 50 mg for the DNAzol™ DNA extraction procedure. None of these experimentally infected fish were positive by culture when tested. When tested with field-infected material, the modified PCR detected individual fish that were also shown to be culture positive (Table 6).
Table 6 The effect of pooling and fish head kidney sample amount on the sensitivity of the PCR to detect R. salmoninarum p57 protein encoding gene DNA Fish no.
Infectivity level
Combination (+:−)
Pooling 10 mg each
Pooling 50 mg each
82 52 62 69 20 87 80 50 59 4,52,82,3 81,53,8,84 20,62,69,89
+++ +++ + + + +/− +/− +/− +/− +++ ++ +
01:04 01:04 01:04 01:04 01:04 01:04 01:04 01:04 01:04 04:01 04:01 04:01
+ + − + + − − + + + + +
+ + + + + + + + + + + +
A comparison was made of the difference in sensitivity between pooling 5 × 10 mg, or 5 × 50 mg samples of fish head kidney in a proportionally higher amount of diluent, prior to DNA extraction and PCR. 10 and 50 mg samples of head kidney were taken from farm fish that were previously demonstrated to be both PCR and culture positive when tested individually (based on 50 mg samples) and DNA was extracted. Samples were prepared in a ratio of one positive to four negative, or one negative to four positive fish, then tested by PCR. When preparing the four positive fish mixes, an attempt was made to combine templates from fish that all had apparently similar infection levels, based on numbers of R. salmoninarum colonies cultured on SKDM inoculated with head kidney material. +/−: grew less than 10 R. salmoninarum colonies. +: grew between 10–20 R. salmoninarum colonies. ++: grew between 20–100 R. salmoninarum colonies. +++: grew more than 100 R. salmoninarum colonies.
3.3. Pooling Results showed that sensitivity was reduced when 10 mg head kidney was taken from each fish and pooled (Table 6). Of the fish assessed by culture and PCR as of low infectivity levels, three out of seven gave false negative results when they were each mixed with head kidney material from four other fish that were culture negative (Table 6). However when using 50 mg from each fish and altering the Table 5 Detection of R. salmoninarum DNA by the modified PCR following extraction by two different methods: the preparation of a crude lymphocyte lysate from 500 mg head kidney followed by Instagene™ extraction, and the preparation of a crude lymphocyte lysate from 50 mg head kidney followed by DNAzol™ extraction Fish no. C1 T1.1 T1.2 T1.3 M1 C2 T2.1 T2.2 T2.3 C4 T4.1 T4.2 T4.3 C5 T5.1 T5.2 T5.3 C6 T6.1 T6.2 T6.3 T9.1
Extraction method Instagene 500 mg
DNAzol 50 mg
− − − + + − − + w+ − − w+ − − − − − − − vw+ − +
− + + + + − − + + − − w+ w+ − − w+ − − − vw+ vw+ +
A selection of the results generated is shown. C: control fish. T: test fish (injected with 3.12 × 103 cfu R. salmoninarum previously). All test and control fish were from a laboratory infection experiment. No test fish showed clinical signs of disease or were culture positive. M: mortality; + represents a bright band following electrophoresis; w+ represents a weak band following electrophoresis; vw+ represents very faint bands.
diluent volumes, there was no apparent loss of sensitivity, with even the combination with template derived from a single fish with only an apparent low level infection mixed with kidney material from four negative fish yielding PCR product bands of equal intensity to the infected kidney alone (Table 6). When using combinations of four positive fish to one negative fish, amplicons were easily visible when using 50 mg from each fish, with no evidence of inhibition or obscuring by smears 4. Discussion The purpose of this study was to validate and adapt, if necessary, a simple PCR method for the detection of R. salmoninarum in both wild and farmed fish from selected river catchments in England and Wales (Chambers et al., 2008). The technique chosen needed to be reliable, reproducible, quick, and relatively inexpensive. Before utilising a new method one must look at the intended application and purpose of the application (Hiney, 1997). The importance of validating methods, before a specific use, has been demonstrated. The original authors of the PCR-based R. salmoninarum detection technique (McIntosh et al., 1996) tested the technique with a low concentration of Y. ruckeri and did not see any evidence of crossreaction. However, as R. salmoninarum can be detected in fish with coexisting heavy Y. ruckeri infections (personal observations), high concentrations of bacteria were deliberately used in the bacterial strain panels. High concentrations consistently produced crossreactions with the original primers. Lower concentrations produced occasional cross-reactions. Redesign of both primers produced a combination which removed the cross-reaction. Another important modification, that may also be relevant to other published PCR-based detection protocols, was the finding that R. salmoninarum positive fish could be reliably detected using as little as 50 mg head kidney tissue if a crude lymphocyte lysate was prepared from the tissue followed by a DNAzol-based DNA extraction even from laboratory infected fish that were clinically inapparent or positive by culture. The McIntosh et al. (1996) Instagene matrix™-based protocol requires 500 mg head kidney tissue, a quantity readily produced from
E.M. Chambers et al. / Aquaculture 287 (2009) 35–39
farmed fish near table-size. However, for other purposes, such as wild fish surveys (e.g. Chambers et al., 2008) much smaller quantities of head kidney may be available from individual fish. In the early stages of R. salmoninarum infection the bacterium is not evenly distributed throughout the head kidney, but present in discreet lesions or granulomas (Bruno, 1986). This could possibly explain why at least 50 mg of tissue needed to be sampled from each fish in the pooling experiment to reliably detect R. salmoninarum in the pool. The results also show that it is possible to pool samples of fish into groups of 5 without significantly reducing the sensitivity of the PCR test, saving time and resources. The developed PCR method was used in a companion study (Chambers et al., 2008) and was able to detect a high prevalence of R. salmoninarum DNA in wild grayling sampled from two river catchments close to rainbow trout farms in Southern England. That study also showed there were likely false positives generated from a number of pike (Esox lucius) samples tested using the modified PCR protocol (Chambers et al., 2008). Sequencing cloned amplicons from the pike positive samples showed that a 348 bp DNA fragment of unknown origin (that may have been pike chromosomal DNA) was amplified from those samples using the modified primers (Chambers, 2005). It is therefore recommended that presumptively positive results from pike, as well as other species, generated using this and other PCR protocols, are confirmed as of R. salmoninarum P57, or other target gene origin, by sequencing the resultant amplicons. As this and the Chambers et al. (2008) studies have demonstrated, false positives can be produced using even well-validated PCR-based protocols. The OIE recommended PCR method (OIE, 2006) is based on a nested PCR protocol that targets part of the P57 encoding gene, developed by Chase and Pascho (1998). Nested protocols are inherently less prone than single round PCRs to false positives, produced as the result of coincidental primer annealing with host genomic, or other non-R. salmoninarum DNA. Conversely, they are more laborious and prone to potential laboratory contamination. It is recommended that, where possible, complementary techniques, such as use of ELISA (e.g. Jansson et al., 1996), as well as direct culture of the organism and confirmatory biochemical and immunoassay identification and other well-validated methods, are used in addition to PCR assays. This is particularly important if the resultant data is used to help inform BKD control programmes. Acknowledgements This work was funded by Defra through project number C0280. References Anon, 1999. Report of the Scientific Committee on Animal Health and Animal Welfare European Commission Unit B3, Bacterial Kidney Disease, Sanco/B3/AH/R14/1999 p. 11. Austin, B., Austin, D.A., 1983. Selective isolation of Renibacterium salmoninarum. FEMS Microbiol. Lett. 17, 111–114. Bandin, I., Heinen, P., Brown, L.L., Toranzo, E., 1996. Comparison of different ELISA kits for detecting Renibacterium salmoninarum. B. Eur. Assoc. Fish Pat. 16, 19–22. Brown, L.L., Iwama, G.K., Evelyn, T.P.T., Nelson, W.S., Levine, R.P., 1994. Use of the polymerase chain reaction (PCR) to detect DNA from Renibacterium salmoninarum within individual salmonid eggs. Dis. Aquat. Org. 18, 165–171. Bruno, D.W., 1986. Histopathology of bacterial kidney disease in laboratory infected rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L., with reference to naturally infected fish. J. Fish Dis. 9, 523–537. Bruno, D., Collet, B., Turnbull, A., Kilburn, R., Walker, A., Pendrey, D., McIntosh, A., Urquart, K., Taylor, G., 2007. Evaluation and development of diagnostic methods for Renibacterium salmoninarum causing bacterial kidney disease (BKD) in the UK. Aquaculture 269, 114–122. Bullock, G.L., Griffin, B.R., Stuckey, H.M., 1980. Detection of the Corynebacterium salmoninus by direct fluorescent antibody test. Can. J. Fish. Aquat. Sci. 37, 719–721.
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