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Research Article
6 September 2013

Application of Variable-Number Tandem-Repeat Typing To Discriminate Ralstonia solanacearum Strains Associated with English Watercourses and Disease Outbreaks


Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks.


The broad-host-range plant pathogen Ralstonia solanacearum causes serious crop losses around the world. Potato (Solanum tuberosum) is especially vulnerable to the pathogen, which induces severe wilt and tuber rot symptoms. One particular subgroup of R. solanacearum (historically known as race 3 biovar 2) is a high-concern quarantine pest, the accidental introduction of which has serious economic and political consequences. The pathogen is therefore the subject of statutory control measures in the European Union, the United States, and many other countries worldwide, which aim to monitor and prevent its spread and direct eradication measures where needed. The characteristics of the pathogen and its hosts and pathology have been extensively reviewed (13). Aspects of race 3 biovar 2 biology that concern cold tolerance relative to other taxa in the species have also been studied, and these factors may be important in predicting the geographical range of the pathogen (4, 5). Phylogenetic analysis of the species, based on comparison of partial endoglucanase (egl) and other gene sequences, found considerable intraspecies diversity, and four major phylogroups with distinct geographical centers of origin have been identified (68). Phylogroup II strains were found to have a center of origin in South America. R. solanacearum race 3 biovar 2 isolates are represented by a single egl sequevar within phylogroup IIB, designated PIIB sequevar 1 (abbreviated as PIIB-1 here) (8). This strain has been disseminated around the world (6, 7) in infected potatoes and has been isolated on potatoes in several European countries over the last 2 decades (3, 8, 9). In Europe, R. solanacearum isolates from field crops of potato and tomato (Solanum lycopersicum) have all been identified as PIIB-1, and in this region, non-PIIB-1 strains are rare and restricted to various greenhouse crops (10). The same PIIB-1 strain was also introduced into the United States and Europe on infected geranium (Pelargonium) cuttings (11, 12).
Diversity within the taxon has been widely studied using a range of other analyses, such as whole-cell fatty acid profiling (13), metabolic profiling (14), 16S rRNA sequencing (15, 16), and various genetic fingerprinting methods, including amplified fragment length polymorphism (AFLP) (16, 17), rare-cutting pulsed-field gel electrophoresis (RC-PFGE) (18), repetitive sequence-based PCR (rep-PCR) (17), restriction fragment length polymorphisms of pathogenicity genes (16, 19), and multilocus sequence typing (20). All the studies concluded that there is little or no diversity within PIIB-1 isolates from worldwide sources, and the taxon has been described as nearly clonal. This conclusion is supported by the extremely high level of homology between independently sequenced genomes of two PIIB-1 isolates (21, 22). The lack of discrimination among PIIB-1 isolates collected from a wide geographic distribution has been attributed to their relatively recent international dissemination through centralized trade of vegetatively propagated potatoes infected with the strain.
To date, there have been seven confirmed outbreaks of brown rot disease caused by R. solanacearum PIIB-1 in potato crops in England; two in different locations in the Thames Valley (in 1992 and 1995), two in Northamptonshire in 1999, one in Kent in 2000, one in Nottinghamshire in 2005, and one in 2009 where an infected imported seed stock was planted in Somerset. The PIIB-1 strain has also caused two outbreaks of tomato bacterial wilt in heated greenhouse crops grown at one locality in Bedfordshire (in 1997 and 1998). The tomato crops in which there were outbreaks were also directly irrigated from a watercourse contaminated with R. solanacearum. The pathogen is known to persist in rivers and other watercourses by infecting riparian woody nightshade, or bittersweet (Solanum dulcamara), with aquatic roots growing out from the river banks (9, 2325). The plant is common along the banks of the Thames and other rivers in southern England, and mature plants of the perennial can form a dense tangle of semisubmerged stolons. Infection of bittersweet is therefore considered to be an important means of establishment and persistence of the PIIB-1 strain following introduction into ecosystems in northern Europe (5, 2325).
The high mutation rate associated with tandemly repeated DNA sequences has been exploited in producing highly discriminatory variable-number tandem-repeat (VNTR) profiles, which are used extensively in the forensic identification of individuals and discrimination among close relatives (26). Prokaryote VNTR structure and diversity has been reviewed (27, 28). Analysis of sequenced bacterial genomes has found that tandem-repeat sequences are frequent and widespread. VNTR analysis has resolved very closely related strains to facilitate many epidemiological studies of human and animal diseases, including anthrax, tuberculosis, and salmonellosis (29, 30, 31). Studies using VNTR-based analyses to differentiate among strains of phytopathogenic bacteria and to provide epidemiological information have not been extensively reported. Two studies on the occurrence of VNTR-containing loci in Xylella fastidiosa (32) and Pseudomonas syringae (33) have been reported. At the time of writing, a comparative analysis of the occurrence of VNTR-containing loci within the phylotypes of R. solanacearum, including PIIB-1 strains, had become available (34).
There were three main objectives of this first application of VNTR to study R. solanacearum PIIB-1 associated with disease outbreaks. The first was to produce VNTR profiles from a panel of 12 PIIB-1 reference isolates from diverse sources and to evaluate them as a means to produce a highly discriminatory strain identification scheme among these closely related isolates. The second objective was to establish the utility of VNTR profiling in source tracing. To accomplish this, VNTR profiles for isolates from infected crops and for environmental isolates collected upriver on watercourses used to irrigate the crops, which were implicated as sources of the pathogen, were compared. The third objective was to ascertain whether VNTR profile analysis would provide additional epidemiological information on the pathogen's colonization, survival, and spread along the River Thames catchment. This study involved comparing VNTR profiles of isolates collected from bittersweet growing along the River Thames following the first known brown rot outbreaks in potato during 1992 and 1996. To investigate the longevity of infection over time in bittersweet, the site of earlier isolations was revisited and new strains were isolated. The VNTR profiles of the newly isolated strains were then compared with those isolated 15 years previously from the same stretch of river.


Bacteria used in the study.

Isolate designations are listed in Table 1. Reference isolates (prefixed NCPPB) were obtained from the National Collection of Plant Pathogenic Bacteria, Food and Environment Research Agency (FERA), Sand Hutton, York, United Kingdom. Other R. solanacearum isolates (prefixed P) are maintained at −80°C in a research collection at FERA. All strains were identified as PIIB-1 using egl sequencing and real-time PCR analysis (6, 35). The research was conducted under Plant Health license PHSI 23/6501.
Table 1
Table 1 VNTR profiles of R. solanacearum reference strains
StrainCountryaHostYrTandem-repeat copy no.VNTR profile
NCPPB 173KenyaPotato19454713856P1
NCPPB 2505SwedenPotato19735712857P2
NCPPB 1584CypressPotato19635712857P2
NCPPB 3980AustraliaPotato19975712856P3
NCPPB 4153EgyptPotato19985712856P3
N1B1EuropePotato 5710856P4
NCPPB 4159EuropePotato19977712866P5
P5518ScotlandRiver water20005712847P6
NCPPB 2796SwedenBittersweet19755712866P8
NCPPB 4212KenyaGeranium20015712956P10
IPO 1609bNetherlandsPotato199557121166P17
The isolates are historical and may have been intercepted in imported material; they do not reflect the current status of the pathogen in any country.
VNTR profiles derived from genome sequences (GenBank accession numbers NC_003295.1 and CU914168).

Genome analysis for identification of tandemly repeated sequences.

The genome sequence of PIIB-1 strain UW551 (21) was analyzed for VNTR sequences using the MREPS program (36). The program has settings to screen the genome for repeats of repeat frequency (size), minimum length (period), and tolerance of repeats (resolution); the last feature allows identification of repeated sequences that have minor sequence differences within them. Searches used a minimal size of 25, a period value of 5, and a resolution value of 1 (note that the parameters do not have units). Using these settings, repeated sequences that were less than 5 bases in length or were repeated less than five times were not identified. Additional selection of loci was done by rejecting repeats that did not contain at least three different bases or that contained sequences of limited diversity, e.g., single-base repeats.

Determination of VNTR profiles.

Initially, a PCR and sequencing approach was used to determine the number of repeated sequences at each selected VNTR locus. This protocol is described below. PCR and sequencing primers are listed in Table SI1 in the supplemental material. One locus (L578) comprises two different tandem-repeat sequences. DNA was prepared from cultures grown on nutrient dextrose medium for 48 h at 25°C. Cells were suspended in water (optical density at 650 nm [OD650], 1 to 1.2) and heated at 100°C for 6.5 min. Two microliters of boiled suspension was added per 50 μl of PCR mixture. A PCR enzyme mixture (Long PCR; Thermo Scientific, United Kingdom) was used according to the manufacturer's instructions. Each locus was amplified separately. The PCR conditions were as follows: denaturation at 94°C for 2.0 min, then 34 cycles of 94°C for 45 s, 55°C for 30 s, and 72°C for 30 s, followed by extension at 72°C for 10 min. The amplicons were then processed to remove primers using the PCR Clean Up Kit (Promega Ltd.) and then sequenced by a commercial sequencing service using the sequencing primers specified in Table SI2 in the supplemental material. The tandem repeats were then counted from the determined sequences. Standard start and finish points, specified in Table SI2, were used to count the repeats.
Subsequently, except for locus L578, which contains two distinct repeat sequences, a capillary column method was used to determine the number of tandem repeats. The primers used for amplification are listed in Table SI2 in the supplemental material, which also indicates the fluorochrome with which they were labeled. Fluorochrome labeling was done through a commercial oligonucleotide synthesis service. The PCR conditions were the same as described above for the sequencing-based method. After PCR, the amplicons were diluted 1/60 in water. One microliter of amplicon was added to 9 μl of formamide and 0.3 μl of size standard labeled with the ROX fluorochrome (Gene Scan 350; Applied Biosystems Ltd.) and applied to a capillary column (ABI 3130 Genetic Analyzer). The determined amplicon size was then used to derive a tandem-repeat number for each locus using the reference data provided in Table 2, which relates a known profile (from strain P6001) determined by sequencing to an amplicon size determined using the capillary column method.
Table 2
Table 2 Reference data for determination of VNTR numbers using capillary column size determination with data from strain P6001
VNTR locusFluorescent labelbDetermined length (bp)Repeat length (bp)Repeat no.c
L578aFAM 6 + 65, 6
The VNTR number for L578 was determined by sequencing, since the locus comprises two distinct tandemly repeated sequences.
HEX, 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein, succinimidyl ester; FAM, 6-carboxyfluorescein.
Tandem-repeat numbers were derived from the size of the locus using this reference table. For amplicon sizes that differed from those in the table, the repeat number was deduced as a multiple of the size of the repeat.

Description of the Thames Valley study area and outbreak history.

River locations of bittersweet from which the pathogen was isolated and two potato farms where the first United Kingdom outbreaks of potato brown rot caused by R. solanacearum were detected are indicated in Fig. 1. The first outbreak occurred in 1992 (farm 1) on potatoes irrigated from the River Thames in Oxfordshire. In the subsequent year, the organism was isolated from bittersweet growing on the banks of the Thames nearby and upriver from the farm. Isolates were also obtained from bittersweet growing in a small ditch (ditch 1) that drained through farm 1 and that, in times of flood, back-flowed from the main river, filling an on-farm reservoir used for irrigation. Strains from bittersweet were also isolated from a second, minor upstream tributary (stream 1) approximately 2 km upriver from farm 1 and also from the more distant River Ray, which enters the River Thames approximately 45 km upstream (Fig. 1). During 1996, a second potato outbreak was identified in Berkshire at a location (farm 2) bordering the River Colne, another tributary joining the River Thames approximately 60 km downstream from farm 1. In addition to isolates collected from the potato farms, extensive isolations were subsequently made from bittersweet close to the farms and more widely encompassing the Thames Valley study area from Slough to the River Ray, including the River Colne, River Ray, and stream 1. In a third study involving an outbreak on tomatoes in 1997 outside the Thames Valley study area, a single isolate recovered from infected tomatoes at farm 3 in Bedfordshire was compared with an isolate from the River Ivel, water from which had been used to irrigate the farm, located approximately 5 km downriver.
Fig 1
Fig 1 Diagram of the study area in the Thames Valley indicating locations of historical R. solanacearum isolates and VNTR profiles. The annotations S1 to S7 indicate the river stretches in which strains were isolated (Table 3). For clarity, only major profiles P3, P11, and P12 are annotated. (Contains Ordnance Survey data [Crown copyright and database right 2011].)


MREPS analysis of the R. solanacearum PIIB-1 (UW551) genome for identification of VNTR loci.

The genome search identified a total of 562 loci containing tandemly repeated sequences. From these loci—avoiding those with minimal sequence diversity—five were arbitrarily selected to produce a VNTR profile for typing R. solanacearum PIIB-1 strains (Table 2). The locus designations were derived from locus identifiers produced by the MREPS program.

VNTR profiles identified.

A total of 17 VNTR profiles (P1 to P17) were identified from all PIIB-1 reference strains and isolates analyzed (including the reference strains UW551 and IPO 1609 analyzed from published genome sequences), and they are listed in Table 1. Eleven profiles (P1 to P10 and P17) were identified among the reference strains that had been isolated around the world from potato, river water, soil, bittersweet, and geranium (Table 1). Among these reference strains, profiles were shared between potato isolates from Sweden and Cyprus (P2), as well as from Australia and Egypt (P3).
River stretches in the Thames Valley from which R. solanacearum isolates were collected are indicated in Fig. 1, and the VNTR profiles determined for the isolated strains from each river stretch (S1 to S7) are listed in Table 3. Isolates from the first United Kingdom brown rot outbreak in 1992, at potato farm 1 in the Thames Valley, comprised two profile types (P11 and P12). Both of these profiles were also isolated from bittersweet taken from ditch 1, which was the irrigation source for farm 1. Bittersweet isolates from the River Thames close to and upriver of farm 1 (at stretch S3) were P11 (6 isolates), P12 (1 isolate), P13 (7 isolates), and P14 (9 isolates). VNTR profiles of R. solanacearum isolates obtained from stream 1, a small River Thames tributary upriver of farm 1 at stretch S4, had different profiles (P8, P14, and P15) from isolates associated with farm 1, suggesting that this contaminated stream was not the source of the pathogen. VNTR profile P8 was shared with an isolate from bittersweet collected in Sweden in 1975.
Table 3
Table 3 VNTR profiles from Thames Valley and farm 3
Stretch of Thames ValleyIsolate sourceaNo. of isolatesbVNTR profileRiver stretch length (km)
S1R. Ray5320
S2R. Thames (east of Oxford)135
S3R. Thames (Oxford to Abingdon) and farm 161116
Farm 1 potato111
Farm 1 potato112
Farm 1 ditch 1111
Farm 1 ditch 1112
S3cR. Thames (Oxford to Abingdon)41316
S4Stream 1182
S5R. Thames (Abingdon to Wallingford)61113
S6R. Thames (Sonning to Henley)1210
S7R. Colne9320
Farm 2 potato13 
Not in Thames ValleyR. Ivel1165
Farm 3 tomato116 
All isolates were from bittersweet unless otherwise indicated. R, River.
Total number of isolates, 60.
Isolates from bittersweet made during a return sampling trip in 2008.
Isolates from river water at stretch S5, immediately downriver of the outbreak, were PII, which was also found at the potato outbreak on farm 1. However, isolates from further downriver at stretch S6 were found to have different profiles. They were mostly profile P3 strains but with single representatives of profiles P2 (shared with a potato isolate from Sweden collected in 1973) and P9 (shared with a Kenyan geranium strain isolated in the United States). VNTR profile P3 strains were also isolated from river water taken from the River Ray and bittersweet sampled from the River Colne tributaries at the up- and downriver extremes of the study area. Four bittersweet isolates collected in 2008 close to farm 1 at stretch S3 all shared the P13 profile, which was also isolated from bittersweet in the same stretch of the Thames in 1993 some 15 years earlier.
The tomato isolate (P6017) from farm 3, located outside the Thames Valley study area, had a VNTR profile (P16) identical to that of the bittersweet isolate (P6106) sampled from the watercourse that had been used to irrigate the tomato nursery.
Several of the reference isolates had profiles (P1, P4, P5, P6, P7, P10, and P17) that were not shared with any of the other isolates studied. Of these profiles that had only a single strain representative, five (P1, P4, P7, P10, and P17) were not isolated from the United Kingdom.


The first objective of this study was to establish whether the technique could be used to discriminate different VNTR profile types among reference isolates of R. solanacearum PIIB-1. The 17 distinct VNTR profiles that were identified clearly provide an efficient means for discriminating between PIIB-1 strains. Considering the recognized lack of diversity among PIIB-1 strains (1320), the technique provides a very high level of strain discrimination.
This VNTR analysis presents a valuable opportunity to identify individual populations within the PIIB-1 group of R. solanacearum. This VNTR study of PIIB-1 strains used a limited set of loci, and future studies using additional loci may extend the discriminatory power of the technique still further. Recently, a VNTR scheme for discriminating Listeria monocytogenes has been optimized by combining tandem-repeat-containing loci from four previously reported schemes (37). At the time of writing, a study had been reported (34) that provides an analysis of VNTR loci throughout R. solanacearum. In agreement with our study, this study also found that VNTR profiles could discriminate PIIB-1 strains. Four discriminative loci identified 10 VNTR profile types among 31 PIIB-1 strains analyzed. Additionally, the study found VNTR loci were strongly associated with phylogroups and that PIIB-1 loci were rarely present in genomes of the other phylotypes. The availability of the genome sequence (21) from the representative PIIB-1 strain (UW551) was an essential step in identifying the discriminatory VNTR loci in our study. This confirms the importance of identifying VNTR-containing loci using a genome sequence derived from a strain very closely related to those under epidemiological investigation.
The second aim of this study was to evaluate the use of VNTR profiling in source tracing. This was done by investigating three isolated disease outbreaks for which R. solanacearum isolates were available from affected crops, as well as associated river water and bittersweet previously implicated as sources of the pathogen. This provided a functional evaluation of the suitability of VNTR profiling in source tracing. For successful source tracing, the VNTR scheme should be sufficiently discriminatory to identify clonal types, but at the same time, the VNTR profiles should be sufficiently stable to provide consistent profiles within an outbreak. VNTR profiles obtained from strains isolated at the three outbreaks were specific to each outbreak. In each case, the profiles of isolates from bittersweet growing upriver matched those from the affected crops, thus supporting the potential utility of VNTR profiling in source tracing. This is consistent with the utility of VNTR analyses in epidemiological studies with human pathogens (29, 30, 31). VNTR profile stability within an outbreak of Salmonella enterica was studied using 190 isolates, and only small changes in profiles were found during the course of the outbreak (37). Similarly, the optimized L. monocytogenes VNTR scheme (38) used to analyze strains from an outbreak found one of the nine loci was variable. In our study, two similar profile types (P11 and P12) were associated with the outbreak on farm 1, both of which were found in bittersweet at ditch 1 on the farm and immediately upstream from farm 1 in the Thames. Source-tracing studies should therefore examine multiple isolates to gain a clear picture of the diversity of types involved in an outbreak.
The third objective of the study was to determine whether VNTR profiling could provide epidemiological information on PIIB-1 colonization of bittersweet in the Thames Valley to further previous studies on the pathogen's survival and spread (39, 40, 41). All four bittersweet isolates from stretch S3 sampled during 2008 were identified as P13, which was the predominant profile in this stretch of the Thames when they were isolated during 1993 and 1994 some 14 or 15 years earlier. This suggests long-term associations of strains with specific VNTR profiles with bittersweet during this period and also that VNTR profiles may be stable over many years in the Thames environment. The shared (P8) profile common to bittersweet strains isolated from stream 1 in 1993 and from the same host from Sweden (1975) approximately 18 years earlier also points to long-term stability of the profile. However, the possibility remains that strains with common profiles could be derived independently by mutation from strains with different profiles and origins.
It is also possible that some VNTR profile diversification has occurred within the Thames Valley. Three VNTR profiles (P11, P12, and P13) were isolated from contiguous stretches of the Thames (stretches S3 and S5), and each contained the same 4- and 6-repeat combination for the double-tandem-repeat-containing locus L578. These profiles, which have so far been found only in this region of the Thames, may have diversified here, which would explain why two profile types were associated with the outbreak at farm 1. However, the diversity of VNTR profiles identified along the whole stretch of the Thames suggests multiple contamination events have probably occurred. The possibility of multiple contamination events in the River Thames catchment is consistent with the theory that PIIB-1 strains could have been introduced through discharge into the drainage system from contaminated potato waste originating from domestic use (9, 24). Shared VNTR profiles among R. solanacearum isolates (e.g., P2 and P3) from the Thames Valley and from potatoes originating from other countries also support this possible means of introduction.
In contrast to the heterogeneity of VNTR profile types found in R. solanacearum isolates from the Thames, colonization of bittersweet along the River Colne and Ray tributaries upriver from their confluence with the River Thames exclusively comprised a single (P3) VNTR type. Considering the long distance separating the two tributaries, it is possible that independent contamination of the tributaries occurred from a common infection source, e.g., by discharge of the same VNTR profile strain from the same source of contaminated potatoes through independent drainage systems.
The two examples of contiguous river stretches contaminated with strains of only a single VNTR profile (Rivers Colne and Ray) suggests sequential infections of bittersweet from infected plants upriver. Long-term infections of bittersweet and transmission of the pathogen to bittersweet hosts located downriver are factors that are likely to have contributed to this host serving as an intractable infection reservoir.
The lack of diversity within the PIIB-1 group at the sequevar level indicates that these strains have only recently been disseminated around the world and that insufficient time has elapsed for strains to diversify. Intensification and centralization of potato production, together with transport of infected potatoes across international boundaries, would have contributed to the dissemination of PIIB-1 strains. This study provides evidence for the potential value of VNTR profile analysis for source tracing of PIIB-1 outbreaks and for epidemiological analysis of the pathogen in the environment. New information has been gained on the possible pathways of introduction and transmission of PIIB-1 strains from the environment to crops. The application of the technique could assist in the elimination of pathways of introduction of this quarantine pathogen and in the management of future R. solanacearum eradication programs.


This research was funded by the Department for Environment, Food, and Rural Affairs (DEFRA).

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Information & Contributors


Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 79Number 191 October 2013
Pages: 6016 - 6022
PubMed: 23892739


Received: 26 April 2013
Accepted: 17 July 2013
Published online: 6 September 2013


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Neil Parkinson
Food and Environment Research Agency (FERA), Sand Hutton, York, United Kingdom
Ruth Bryant
John Innes Centre, Norwich Research Park, Norwich, United Kingdom
Janice Bew
Food and Environment Research Agency (FERA), Sand Hutton, York, United Kingdom
Christine Conyers
Food and Environment Research Agency (FERA), Sand Hutton, York, United Kingdom
Robert Stones
Food and Environment Research Agency (FERA), Sand Hutton, York, United Kingdom
Michael Alcock
Food and Environment Research Agency (FERA), Sand Hutton, York, United Kingdom
John Elphinstone
Food and Environment Research Agency (FERA), Sand Hutton, York, United Kingdom


Address correspondence to Neil Parkinson, [email protected].

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