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Evidence Project Final Report

 Note In line with the Freedom of Information Project identification Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. 1. Defra Project code SEO313 The Evidence Project Final Report is designed to capture the information on 2. Project title the results and outputs of Defra-funded Improved molecular tools applicable to Brucella research in a format that is easily surveillance. publishable through the Defra website An Evidence Project Final Report must be completed for all projects. 3. Contractor Department of Bacteriology  This form is in Word format and the organisation(s) Animal Health and Veterinary boxes may be expanded, as appropriate. Laboratories Agency Woodham Lane  ACCESS TO INFORMATION New Haw The information collected on this form will Addlestone, KT15 3NB be stored electronically and may be sent to any part of Defra, or to individual

researchers or organisations outside 54. Total Defra project costs £ 655,025 Defra for the purposes of reviewing the (Agreed fixed price) project. Defra may also disclose the information to any outside organisation 1st April, 2009 acting as an agent authorised by Defra to 5. Project: start date ...... process final research reports on its behalf. Defra intends to publish this form st end date ...... 31 March, 2012 on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 28 6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...... YES NO (a) When preparing Evidence Project Final Reports contractors should bear in mind that Defra intends that they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow. Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the Evidence Project Final Report can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer. In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. (b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary 7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 2 of 28 Brucellosis remains a zoonotic disease of great global significance that causes reproductive problems (abortion and sterility) in many livestock species and a potentially chronic and debilitating disease readily transmitted to humans. Great Britain has official Brucellosis free status for sheep, pigs and cattle, with Official Bovine Brucellosis Free status being granted by the OIE in 1985 following a lengthy and expensive eradication campaign. This has been successfully maintained as a number of introductions have been identified speedily and stamped out successfully. However, with brucellosis still endemic in much of the world, including significant parts of the EU, it is crucial that vigilance is maintained to reduce the risk of reintroduction and that appropriate tools are in place to ensure the organisms is rapidly identified and controlled in the case of future incursions. Much of the research carried out within the Brucella group at AHVLA supports the maintenance of disease freedom by working to develop and implement the most modern detection, diagnostic and molecular typing tools.

The project described here included a number of seemingly discrete areas of work but all supported the overall strategy of the development of improved tools for detection and characterisation of Brucella.

As part of this work we have undertaken the following.

(i) Investigated whether the inclusion of additional compounds to selective media might be effective in reducing the growth of confounding non-Brucella organisms. This work identified two compounds 4- hydroxycoumarin and manganese chloride that might be useful additional components in currently used selective media.

(ii) Investigated whether rapid molecular approaches (PCR) might offer a useful additional tool for confirming a true Brucella infection from serum samples. While this approach appeared specific results suggested very disappointing sensitivity and the tool as stands would be of limited value as a confirmatory diagnostic. As a result of these findings we have suggested that further studies should focus on investigating approaches that might enrich DNA both physically and involve the use of alternative components from blood samples.

(iii) Extended a previously developed typing scheme based on single nucleotide polymorphisms (SNPs) that identified Brucella isolates to the species level and differentiated vaccine isolates. As well as being extended to include all newly described species the scheme resolution has also been extended to divide isolates at the subspecies level. Because the classical epidemiological subdivision of biotype applied to Brucella, and based on phenotype alone, was shown not to be wholly congruent with relationships based on genotype the concept of ‘genomovar’ was introduced to divide Brucella groups into epidemiologically significant clusters. This approach offers an exemplar for future developments – the involvement of AHVLA as a partner in ongoing genome sequencing projects and the rapid technological developments in whole genome sequencing promise that genome sequencing and SNP typing based on comprehensive assessment of genomes will become a mainstay of Brucella epidemiology in the near future. As a result of these findings we have suggested that future work should focus on assuring that AHVLA remains at the forefront of Brucella whole genome sequencing and ensures it has suitable embedded skills to continue to utilise these data.

(iv) Developed new molecular assays for both detection and typing, particularly a LAMP assay, representing an emerging molecular amplification approach that offers a potentially more robust, rapid and cost-effective alternative to traditional PCR based approaches.

(v) Continued to develop and exploit multi-locus sequence typing (a tool based on the DNA sequence of multiple fragments of the bacterial genome) as a tool to understand the relationship between Brucella strains at a global level and to provide a framework for characterisation of newly emerging or atypical isolates and for understanding the evolution and emergence of such strains. As part of the work described here we have developed an entirely novel approach that enables us to characterise not only Brucella but its nearest genetic neighbours in a single approach. This is important as many novel and atypical isolates of both Brucella and the nearest genetic neighbours are currently being described that are closing the ‘genetic space’ that traditionally separates these groups. This tool provides a unique approach to empirically establishing the relationship of any new isolates to both Brucella and nearest neighbours and will help maintain an accurate understanding of the relationships within this important group of bacteria at a time when there is potential for significant confusion. Within this study we have also used MLST to more widely characterise the emerging Brucella from marine mammals and to add to the body of evidence that suggests that a genotype previously associated with zoonotic infection of

EVID4 Evidence Project Final Report (Rev. 06/11) Page 3 of 28 humans may not be a significant threat in European waters. MLST has also been a crucial tool in the completed and ongoing descriptions of novel groups of Brucella associated with novel hosts. All these studies are crucial in furthering understanding of the group and allow us to maintain awareness and rapidly assess whether any newly emerging group may represent a threat to the UK.

(vi) Undertaken studies that allow comparison of data obtained from a high resolution typing scheme based on variable numbers of tandem repeats (VNTR) in our laboratory with data from other laboratories. VNTR typing developed at AHVLA and elsewhere has revolutionised Brucella molecular epidemiology for the first time providing a tool with sufficient discriminatory capacity to allow trace-back and outbreak investigation. However a number of schemes using have been developed in different laboratories that are not fully compatible. Here we have harmonised some of our existing data to a recently agreed ‘common’ scheme to allow comparison of this and future data with data from other laboratories. This strengthens that value of this approach by allowing comparison of any profiles obtained with a potentially much more comprehensive global database.

(vii) Examined whether a specialised class of VNTRs, Single Nucleotide Repeats (SNRs) may be of additional value in high resolution genotyping. Here we show that there is diversity in some of these types of loci even within isolates in a highly geographically restricted endemic area and suggest some tools that could be implemented to rapidly index such diversity To our knowledge this represents the first examination of these elements in Brucella. As with section (iii) extensive genome sequencing projects in which the AHVLA is now a partner will shortly facilitate a fuller assessment of the value of these markers.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 4 of 28

Project Report to Defra 8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include:  the objectives as set out in the contract;  the extent to which the objectives set out in the contract have been met;  details of methods used and the results obtained, including statistical analysis (if appropriate);  a discussion of the results and their reliability;  the main implications of the findings;  possible future work; and  any action resulting from the research (e.g. IP, Knowledge Exchange).

The objectives (and associated milestones) as laid out in the SID3 form for SEO313 were as follows:

1. Development of an improved selective media for isolation of Brucella, based on previous molecular studies identifying compromising organisms, that reduces the number of isolates that are currently recognised as suspect Brucella in UK surveillance.

Milestone 8 (Phenotypic array typing complete for identification of potential selective media components)

Introduction to topic and experimental approach: The gold standard approach for confirmation of brucellosis is through the culturing of aborted material, milk and vaginal swabs for the presence of Brucella. However, with the organism taking up to seven days to grow up, in a non selective medium, any pathogen present would be out competed by faster growing contaminants. To this end, a few selective solid media such as Thayer-Martin (Thayer et al., 1965) and Farrell’s (Farrell and Robertson, 1972) have been used for the selective isolation of Brucella. However, it has been noted that Farrell’s medium can actually inhibit the growth of certain Brucella species (De Miguel et al., 2011). In addition, it has also been observed through routine work done within the surveillance remit of AHVLA that a number of non-Brucella organisms (NBOs) grow on selective media and in turn cause a number of avoidable enquires. The aim of this work was to investigate whether the current Farrell’s selective media for the isolation of Brucella species could be improved by inhibiting the growth of organisms commonly confused as Brucella.

To undertake this work, the approach taken was to use the Biolog phenotype microarray™ (PM) system, which gives the possibility of screening 96 different chemical combinations per run. Using redox chemistry through tetrazolium dye colour change and employing cell respiration as a universal reporter it was possible to monitor growth in different conditions. For this study, over 330 different compounds (spread over eleven 96-well plates) were screened as potential additives for Farrell’s media. Digital images and quantitative colour change values are recorded by the Biolog OmniLog® reader-incubator several times in an hour.

The panel used for testing comprised of all Brucella spp. type strains and representatives of NBO isolates submitted to AHVLA Weybridge and identified by 16SrRNA sequencing (Figure 1 for a list of NBO isolates used). There was an issue in the work involving B. abortus biovar 2, which is capnophillic whilst the Biolog system is aerobic. Nevertheless, although growth was weaker for this Brucella in comparison with the others, the data was still usable and was included in the analysis. However through an issue of contamination and insufficient time (the Biolog apparatus could only be spared and held in Brucella containment laboratories for a defined period), it was not possible to obtain a full phenotypic profile for B. abortus biovar 5. However this biovar is rarely reported today and unlikely to represent a major threat to the UK. Furthermore even though a number of attempts at culture were made, it was not possible to obtain adequate growth to perform phenotypic analysis of three NBOs; UK10/07, UK18/07 and UK3/09.

For the remainder of strains, these were grown from lyophilised storage for three days on Serum Dextrose Agar (SDA) plates. Growth was then subbed onto two Tryptic Soy Agar (TSA) plates for 3 days. The Brucella spp strains were grown at 37°C supplemented with 10% CO2 whilst NBOs were initially grown at 37°C aerobically. Cells from the TSA plates were harvested into IF-10b GNGP Base inoculating fluid with the cell suspension turbidity read in a Biolog turbidimeter and adjusted to a level of 4% T (transmission) for subsequent use. From this suspension, 100µl per well was added onto a number of plates measuring osmotic/ ionic response and bacterial chemical sensitivity. Plates were incubated in the Biolog Omnilog

EVID4 Evidence Project Final Report (Rev. 06/11) Page 5 of 28 Incubator-Reader set at 37°C for 72 hours and results were analysed using Biolog software.

Results: The Biolog system generated a number of growth curve graphs for each strain and each condition tested. A representative of the data generated is shown in Figure 2. Analysis of graphical data and plate digital images showed that there was no single well on any of the 11 plates that grew all of the Brucella isolates but inhibited the growth of the panel of non-Brucella strains tested. However it was observed that a combination of two chemicals, 4-hydroxycoumarin (C9H6O3 also known as 2- hydroxychromen-4-one or Benzotetronic acid) and Manganese (II) chloride (MnCl2) allowed growth of all Brucella tested but inhibited all but two rarely isolated NBOs, UK6/08 and UK34/07-5 (Table 1).

Milestone 17 (Complete development and testing of improved selective media)

Introduction to the topic and experimental approach: Continuing on from work done to achieve the previous milestone, maximal concentrations of MnCl2 and C9H6O3 that still allowed for growth of Brucella but inhibited all NBOs were determined. Solutions of MnCl2 and C9H6O3 were made in methanol and initially added separately at various two-fold titrations to a combination of SDA media and 10% horse serum. Media plates containing no additional chemicals were also set up at all stages of all tests to act as isolate growth controls.

For the Brucella work, B. abortus Bv2 was selected as a representative of the Brucella genus as it has the strictest environmental conditions for growth and Brucella reference strain 544 (B. abortus bv1) was included at a later stage in order to introduce a strain representative of those considered most likely to be isolated from abortive material in the UK. In terms of NBOs, three isolates were taken from the initial panel for subsequent testing. These were UK34/07-1, UK06/08 and UK14/09 with this final isolate (UK14/09) being chosen as it was already shown to be sensitive to both agents in the Biolog testing.

After a purity check for Brucella and NBOs, a stock 1x1010 suspension of each isolate was prepared in 0.1M Phosphate Buffered Saline (PBS) and a 10ul loopful of this suspension was streaked a total of 60 times over each type (SDA control plates, SDA + MnCl2 and SDA + C9H6O3) of media plate (twenty times per plate over three media plates of each type of media) with the growth from the last twenty streaks (last plate) being recorded as a percentage of growth – 5% growth being assigned to each of the potential 20 streaks of confluent growth. Following on from this, both MnCl2 and C9H6O3 were combined at various concentrations (checkerboard titration) initially on SDA media (non-selective) and then on Farrell’s media (selective) in order to ascertain optimal concentrations. In addition to this, to quantify the effect of both additives to Brucella and NBOs, serial dilutions of the stock suspension were prepared (1x107 cfuml-1 to 1x103 cfuml-1 dilutions used), with 100 l of each dilution used for plating. For counting purposes, on plates where practical, individual cfus were counted. However, where there was much growth, counting was impractical and results were recorded as either greater than 200 colonies or as confluent growth.

Results: From the work using SDA and titrations of either MnCl2 or C9H6O3, it was not possible to find an optimal concentration for either compound that would both inhibit NBOs but support Brucella growth. A concentration of 800ug/ml MnCl2 did not inhibit the growth of B. abortus Bv2 but at the same time had no effect on the growth of two of the three NBOs. For the remaining NBO, growth was reduced, but only by -1 60%. However, increasing the MnCl2 concentration to 1600 gml , inhibited NBO growth and reduced B. -1 abortus Bv2 by 40%. In the case of C9H6O3, a concentration in excess of 87.5 µgml inhibited the growth -1 of B. abortus Bv2, but NBOs required a concentration of C9H6O3 in excess of 700 µgml before growth was sufficiently impeded. It was also found that combining both MnCl2 and C9H6O3 with SDA media did not lead to a selective media for optimal Brucella isolation and growth. At the concentrations determined individually for each compound, there was no inhibitory effect on the NBOs whilst a slight increase in -1 -1 concentration to 100 µgml of MnCl2 and 105 µgml of C9H6O3 reduced growth of NBOs by on average, 28%, but repeatedly and completely inhibited B. abortus Bv2 growth.

When the selective Farrell’s media was used as a basis for the titrations, it was found that optimal concentrations for a combination of MnCl2 and C9H6O3 allowing Brucella growth but inhibiting that of NBOs -1 -1 had dropped dramatically to 43.8gml C9H6O3 and 600 gml MnCl2. In the case of the Brucella 544 strain, it was observed that at all but the 1 x103 cfuml-1 dilution on this modified Farrell’s, where there was a 19% drop in growth, there was no inhibition in the growth of this strain. It should, however, be noted that there was slight inhibition of the poor growing B. abortus Bv2 with a concentration of 1x 104 cfuml-1 giving only 24 colonies on the modified Farrell’s medium. Nevertheless the effect on NBOs was much more dramatic. Illustrating this, plating 1x104 cfuml-1 of UK14/09 reduced the colony count from in excess of 200 colonies on a commercial Farrell’s plate to only 2 colonies on the modified Farrell’s media. For UK06/08 an in-plate 1 x 103 cfuml-1 concentration on SDA resulted in 184 colonies were counted which was reduced to 3 colonies on commercial Farrell’s media and to zero with the introduction of MnCl2 and 6 -1 C9H6O3. Finally, isolate UK34/07-1 at an on-plate 1x 10 cfuml concentration on both commercial Farrell’s and in-house Farrell’s gave in excess of 200 colonies. This was then reduced to only 3 colonies

EVID4 Evidence Project Final Report (Rev. 06/11) Page 6 of 28 with the introduction of MnCl2 and C9H6O3.

Another interesting observation seen with the modified Farrell’s medium was the change in the morphology of Brucella strain 544, but interestingly not B. abortus Bv2, at 1 x104 cfuml-1 (growth above this concentration was confluent and individual colonies could not be visualised). At this particular concentration, colony morphology was no longer characteristic of Brucella with colonies being slightly matt orange in colour and lacked the rainbow iridescence when observed by obliquely reflected light. This was not seen at weaker dilutions.

-1 -1 Conclusions and future work: In conclusion the introduction of 43.8 µgml C9H6O3 and 600 µgml MnCl2 to commercial Farrell’s media both impedes the growth of the NBOs used with no to slight inhibition of Brucella growth depending on the strain used. It should however be stated that in light of the B. abortus strain 544 results where there was a change in colony morphology at certain dilutions with the inclusion of the two compounds to Farrell’s medium, there still remains a number of experiments that need to be undertaken. This would include the testing of a far larger panel of smooth Brucella and NBO organisms and maybe the inclusion of supplemental CO2 to see if this has any effect on the growth of Brucella and NBOs on this modified media.

This work is to our knowledge the first to identify C9H6O3 and MnCl2 as possible selective agents for the isolation of Brucella. Further, whilst there have been recent publications on the development of novel media for the selective isolation of Brucella (De Miguel et al., 2011; Ferreria et al., 2011), neither publication has mentioned the growth of NBOs on Farrell’s medium but rather have approached the topic from the inhibition of certain Brucella strains viewpoint. The work undertaken in this project, namely to trial additional chemicals with a pre-existing selective media, is an ideal introduction to the preparation of a next generation of selective media. Farrell’s media was conceived as Brucella selective agent in the 1970s but in addition to adversely affecting the growth of certain Brucella isolates, growth of other non Brucella is not impeded. A complete “strip down and rebuild” of a novel media based on Farrell’s but with the incorporation of the two agents found in this work would be of benefit not only for our own use but also internationally. This might reduce the number of isolates incorrectly submitted as suspect Brucella although it should be noted that the numbers of these have dropped significantly in recent years.

2. Examination of the potential of serum based real-time PCR as an additional tool used to assess suspect Brucella cases.

Milestone 9 (Serum panel for PCR evaluation assembled)

Milestone 18 (Complete validation of real-time PCR of serum as a surveillance tool)

Introduction to topic and experimental approach: If PCR from serum can be shown to be a reliable tool for the detection of Brucella it would be a welcome addition to the toolbox of tests used to assess a potential outbreak. Since the development of the initial PCR assay for the detection of Brucella in aborted material (Fekete et al., 1992), there have been a number of publications and assays describing direct brucellosis diagnosis from clinical samples. In cases of human brucellosis there is correlation between PCR positives from serum testing and infection (Zerva et al., 2001). However, results from this type of testing have been variable. Leal- Klevezas et al. (1995) reported that it was possible to obtain positive correlation of serology, culture and blood PCR from animals that had been artificially infected with Brucella. However, in this very same paper there are discrepancies between PCR, serological and culture positive results when looking at naturally infected animals (Leal-Klevezas et al., 1995). Other publications in a variety of animals have also demonstrated the inconsistency between classical and molecular diagnostic results. To add some clarity to this topic, it was decided to test sera isolated from culture positive and negative bovines by PCR using two Brucella specific targets; bscp31 (Mayfield et al., 1988) and the multiple copy insertion sequence, IS711 (Halling et al., 1993). Our reasoning for using two Brucella markers rather than one target was that although both have been shown to be Brucella specific, there is no biological function assigned to IS711 in Brucella and there may be instances where there are strains that lack this target (as shown for example in Mycobacterium tuberculosis where some strains lack the diagnostic insertion sequence, IS6110 (Das et al., 1995; Radhakrishnan et al., 2001)

During the planning stages for this current ROAME, we had hoped to look at an extensive panel of culture positive blood samples that would be obtained through collaboration with colleagues at the Pendik Veterinary Centre and Research Institute (PVCRI). Blood sampling was targeted to animals that had aborted recently and were taken from the Marmara region of Turkey (the locality of PVCRI). However, although hundreds of blood samples were taken and sent back to AHVLA for culture analysis, blood culture yielded only one positive sample which was later found to be negative by PCR when tested. Culturing of Brucella from blood is known to be extremely difficult and issues such as transportation of blood samples and the time taken from blood isolation to culturing may be contributing factors to the poor

EVID4 Evidence Project Final Report (Rev. 06/11) Page 7 of 28 performance of culture. In addition to this there had been attempts to source serum from culture positive bovines through targeted requesting. However, the problem of this approach was that whilst many serologically positive samples were received, there was no provenance in terms of culture that could determine whether the animal was truly infected.

To overcome this problem, it was decided to look within the serum bank held at AHVLA for bovine serum samples with matched confirmed positive cultural status. To this end, the positive material tested (20 samples in all) came from outbreaks in Cornwall (2004) and Anglesey (1993) as well as from four laboratory infected cows from a study performed in the 1990s.

In total 170 serum samples were used to test the possibility of direct detection of Brucella from serum. Of this panel, 20 came from blood samples that were culture positive for Brucella whilst the remaining 150 sera came from serological/ culture negative bovines from Great Britain provided by Laboratory Services, AHVLA. Extractions were performed using the Roche Hi Pure Ultraclean PCR kit. This kit was selected over the Qiagen DNeasy Blood and Tissue kit based on the findings (numbers of PCR positive animals) generated by a German group looking at camel sera (Gwida et al., 2011). Although the kit protocol suggested that 200 µl serum was processed for extraction the scarcity of many of the samples meant we were only able to extract from l of serum from these culture positive cases. To compensate, 2 l of the resulting extraction instead of 1 µl was used in testing. For real time PCR, each extraction was run in duplicate using bcsp31 (Probert et al., 2005), IS711 (Celebi-Court, 2005) and 18SrRNA PCR Inhibition Amplification Control (IAC) (K. Gopaul, Unpublished). For the purposes of this study, a Brucella PCR positive sample was one that generated reproducible positive results within 35 cycles for IS711 and within 40 cycles for bcsp31.

Results: The results of this study showed that 168 samples tested as negative using the two Brucella directed PCR assays (bcsp31 and IS711). All 170 samples tested positive using the 18SrRNA IAC indicating that the negative Brucella results were attributable to lack of target rather than PCR inhibition. No culture negative serum samples tested positive by PCR. In comparison, only 2 out of 20 culture positive samples tested positive by both PCR methods used (Table 2). It should be noted that both positive samples had high cycle threshold values for both Brucella assays, indicating the presence of very low amounts of Brucella DNA in both serum samples.

Discussion and conclusions: In this study, the sensitivity of PCR was poor with only 2 out of 20 culture positive animals being PCR positive with both Brucella targets. In comparison, specificity was very good with all 150 culturally and serologically negative samples being PCR negative. It could be argued that the poor recovery of positive PCR results from the sera of culture positive cattle could be attributed to repeat freeze thawing of these samples. However, in a study of DNA stability in whole blood subjected to repeat freeze thawing (up to 40 repetitions), whilst DNA recovery yield decreased by 25% after the first round of freezing, over the next 40 rounds yield only dropped another 5% whilst there were no signs of DNA degradation (Ross et al.,1990). Another study, looking at the stability of Hepatitis B Virus DNA in serum undergoing repeat freeze thawing found that over eight repeat freeze thaw cycles, there was little loss in viral load (Krajden et al., 1999).

In addition to the issues of freeze thawing, one other criticism of these results could be that the age of the sample has had some effect on the sensitivity of the PCR. However, it has been shown, albeit not in a clinical context, that it is possible to detect Brucella DNA by PCR in ancient material, namely skeletal remains (Mutalo et al., 2012). In this study, the authors demonstrated the presence of Brucella DNA by PCR using bcsp31 and IS711 in bone lesions from skeletons that were over 700 years old, showing the robustness of this material in the absence of viable organisms.

It is likely that bacterial load and the preferred location of the organism may be important factors in the diagnosis of brucellosis by molecular methods. The limit of reproducible detection of both assays used is around 100fg (approximately 30 genome equivalents) in the volume used for PCR (2 µl). Extrapolating this figure back would mean that anything less than 1.5 x104 genome equivalents per ml of serum would not be detectable by PCR. Little is known about the magnitude or extent of bacteraemia in bovine infection. Further, as Brucella organisms are intracellular pathogens, if these were in whole blood a more likely location would be within circulating macrophages rather than in cell free serum, with the period of bacteraemia being limited and variable between animals. To this end, the findings of the work presented here, indicating that direct PCR from serum is of limited diagnostic value, has helped to direct our approach to future work where we intend to both examine both DNA enrichment approaches and different blood fractions (namely peripheral blood mononuclear cells and plasma) to improve PCR detection levels.

3. Taking advantage of forthcoming genomic sequence data we will continue development of our front line molecular typing tool, real-time PCR based SNP typing, to characterise isolates at the

EVID4 Evidence Project Final Report (Rev. 06/11) Page 8 of 28 subspecies level and to ensure it remains up-to-date with current Brucella taxonomy.

Milestone 2 (Complete scanning of genomes for SNPs potentially useful in distinguishing biovars)

Milestone 13 (Completion of real-time PCR SNP typing scheme to type at subspecies level and to include all new species)

Introduction to topic and experimental approach: In a previous ROAME (SEO311), we described the use of single nucleotide polymorphisms (SNPs) to identify Brucella to the species level (Scott et al., 2007; Gopaul et al., 2008) and used the same approach to confirm identity as a vaccine strain (Gopaul et al., 2010). Another possible use for SNPs is bacterial identification to sub-species level and in a previous ROAME SNP defining assays using a real time PCR platform that were congruent with B. suis biovars were described. A similar group of assays used to characterise B. suis to the biovar level has since been published in conjunction with another workgroup (Fretin et al., 2008). The development of SNP based discrimination assays was relatively straight forward for species and B. suis biovars as the multi-locus sequence analysis (MLSA) data obtained by this department revealed genetic clusters congruent with biovars (Whatmore et al., 2007). In contrast there appeared to be little relationship between biovar and genotype in B. melitensis and B. abortus occupies an intermediate position in which there is some relationship between biovar and genotype. It was therefore decided that one of the objectives in this current ROAME was to take this work forward and develop molecular assays that would subtype B. abortus and B. melitensis along the lines of genomic differences as an exemplar along the road to more comprehensive SNP based typing that is likely to become a method of choice as availability of whole genome sequences to identify such SNPs increases. However, where scientifically valid and justifiable, markers should divide into groups congruent with classical biovars given the historical data associated with the biovar approach and the likelihood that an approach based around these traditional epidemiological divisions would be more acceptable to the field. Clearly this is at least in part possible for B. abortus where, for example, all isolates of biovars 5 and 9 are known to constitute a single genotype but impossible for B. melitensis given the absolute lack of relationship between biovar and genotype seen by MLSA (and an increasing number of other studies in the literature).

It was therefore decided to employ two strategies to develop sub-species typing. For B. melitensis where it was evident that there was no congruence of genotype to biovar, this involved the direct use of existing MLSA data to define ‘genomovars’ (clusters identified on the basis of sequence) and develop assays based on these. However, in the case of B. abortus biovars it was decided to employ a wider screen using data beginning to emerge from the Brucella whole genome sequencing projects in which AHVLA is a partner and which involved sequencing all B. abortus type strains (corresponding to all seven biovars and vaccine strain B. abortus S19). We have worked extensively with collaborators at the University of Northern Arizona (Dr Jeff Foster) to exploit these data and generate a file of the locations of all SNPs unique to the various type strains. On first inspection, it was seen that although there were two B. abortus “biovar 1” examples (2308 and 9-941), there were no common SNPs (other than those that were associated with B. abortus species) indicating that there was no obvious genotypic correlation for this phenotypic group. There were also issues concerning the validity of B. abortus biovars 3 and 6 which gave us reason to avoid the generation of assays towards these groups. Nevertheless, for the remaining biovars (2, 4, 5 and 9), primers were designed based on genome sequence findings to facilitate the sequencing of regions specific to the biovar example of interest (Table 3 for list of primers and targets) in a panel of field isolates selected at AHVLA that comprised of every B. abortus biovar (Table 4 for list of strains used).

Depending on the outcome of this work, multiple outcome Minor Groove Binding (MGB) real time PCR assays would be developed for the determination of both B. melitensis and B. abortus ‘genomovar’. These were similar to those we had already developed in a previous ROAME for the identification of Brucella isolates to species level (Gopaul et al., 2008). Basically, for each target there were two target probes (called wild-type and specific), differing in only the target SNP. Both probes were labelled with different fluorophores to allow for easy identification. Based on the sequence, one of the two probes would preferentially bind to the target and be cleaved. This would manifest itself as an increase in the fluorescence reading of the marker associated with this probe. Assays would be generated in such a way that all wild-type probes would be labelled with VIC® fluorophore (Applied Biosystems, Warrington, UK) whilst ‘genomovar’ directed assays were all labelled with 6-FAM. Thus if a green curve was generated from the reaction (as detected by the Agilent MX3005p real time PCR machine), the sample did not have the specific target for that ‘genomovar‘/ biovar directed assay whilst a blue curve would represent the converse.

Results: From the MLSA scheme developed by Whatmore et al., (2007), it was seen that B. melitensis isolates could readily be divided into three ‘genomovars’ that have distinct geographical associations (Figure 3), with the branches of these ‘genomovars’ (MGV) corresponding to specific SNPs. These targets

EVID4 Evidence Project Final Report (Rev. 06/11) Page 9 of 28 are shown in Table 5.

For B. abortus, sequencing results validating the presence of SNPs identified as potential biovar specific markers by comparative whole genome sequence analysis were, in the main, disappointing. All but one of the identified SNPs had no correlation with biovar from the data obtained through sequencing of the targets mentioned in Table 3 in strains shown in Table 4. The one exception to this was BAB2_1005_which was identified as specific to B. abortus Bv5 (interestingly biovar 5 and biovar 9 isolates comprise a distinct ST within B. abortus based on MLSA, see Figure 3). Passaging of biovars 5 and 9 earlier in this project had shown that the phenotypic characteristics of both biovars were stable. This biovar-specific SNP was of interest as the target SNP change (G in wild type →A in biovar 5) introduces a stop codon that would lead to a truncated protein. Based on these results the same approach already applied to B. melitensis was taken with groups assigned based on data obtained by MLSA that divides isolates into distinct phylogeographical lineages with, for example, ‘genomovars’ 3, 4 and 5 associated exclusively with isolates from Africa (Figure 3). The one exception is biovar 5 which does appear to correspond well to a distinct genetic grouping. These targets are shown in Table 5 separating B. abortus into 5 ‘genomovars’ plus an additional assay corresponding to B. abortus biovar 5 isolates.

Validation of sub-species defining assays: To validate these subspecies defining assays, a number of B. melitensis and B. abortus isolates that had not been previously typed by MLSA were tested blindly. Our assumption was that aside from B. abortus that were either ‘genomovar’ 3 (which is a subset within ‘genomovar’ 4) or biovar 5 (which is a subset within ‘genomovar’ 1), each B. melitensis or B. abortus isolate tested would only be identified by one assay. If this were not the case it would suggest that the classification on which this work is based is not robust. In addition, to further characterise ‘genomovar’ 3, we obtained 14 B. abortus Bv3 isolates taken from cattle in the Gambia as part of an unrelated Belgian study (Bankole et al., 2010).

In total, 120 strains were used to validate the B. abortus multiple outcome ‘genomovar’ defining assay (including five non B. abortus Brucella strains). In the case of the B. melitensis work, 104 isolates (including two non B. melitensis Brucella strains) were used to validate the ‘genomovar’ assays. It was found that in all cases barring the non-B. melitensis samples for the B. melitensis ‘genomovar’ assays and the non-B. abortus samples for the B. abortus ‘genomovar’/ bv5 assays, there was identification to the biovars previously defined (Figure 4 for example of B. melitensis assay). In the case of B. abortus biovar 5, these were a subset of the more extensive ‘genomovar’ 1 group and so gave a result for this target in addition to that for BAB2_1005. In the case of B. abortus ‘genomovar’ 3, these are a subset of the ‘genomovar’ 4 group and so an isolate from ‘genomovar’ 3 would contain both this specific marker and “genomovar” 4 targets (Figure 3).

Discussion and conclusion: The use of phenotypic characteristics/ biovars to sub-type B. abortus and B. melitensis isolates is widely undertaken and accepted as a means of identification. However, there are many pitfalls to this methodology. There may be differences in the media used and certain dyes are known to degrade quite quickly, leading to a false result. Interpretation of growth characteristics is in the main part subjective and this may lead to inconsistencies between laboratories. It is for this reason that we have taken the approach of trying to find a genetic basis (in the form of SNPs) that would allow the development of assays for Brucella strains that defined clusters that correspond to classical biovars. However, aside from BAB2_1005 in B. abortus biovar 5, SNPs of this nature could not be found for the remaining B. abortus and B. melitensis biovars and so a purely genetic basis to defining clusters was taken for most of the work.

The clusters identified on the basis of MLSA (referred to as ‘genomovars’) do correspond to meaningful phylogeographic groups, Thus for example B. abortus Group 4 isolates all come from Africa with Group 3 isolates being associated exclusively with a more confined geographical region within West Africa. In comparison B. abortus Group 1 isolations tend to be from Europe but encompass three biovars. For B. melitensis isolates, Al Dahouk et al., (2007) proposed three geographic groups based on VNTR data, called “American”, “West Mediterranean” and “East Mediterranean”. With harmonisation of our VNTR data with other schemes (see section 6), one piece of future work that could be undertaken could be to determine whether the three ‘genomovars’ proposed in this ROAME correlate with these VNTR groups.

In the case of BAB2_1005, the sole SNP that could be linked with biovar, an interesting observation is that the protein truncated (iron sulphur cluster binding/ ferrodoxin protein) may be linked with the production of H2S. Interestingly, in addition the biovar 5 sulphite reductase gene, BAYG_00484 (cysI or BAB1_0181) has a 9bp deletion within its coding sequence. Oliveria et al., (2008) described a structure involving the combination of ferrodoxin and sulphite reductase for the respiration of sulphate leading to production of H2S. Hydrogen sulphide production is characteristic of all B. abortus isolates with the exception of members of biovar 5. Whilst it is hard to determine whether the deletion in BAYG_00484 allowed for the toleration of the SNP in the BAB2_1005 biovar 5 homolog or vice versa, these changes might suggest that

EVID4 Evidence Project Final Report (Rev. 06/11) Page 10 of 28 the pathway of sulphate respiration is redundant at some point in biovar 5 which, in turn can explain the specific phenotype this biovar has. However, a more thorough study of the genes involved with this pathway in biovar 5 would establish the point at which this pathway became redundant.

The results from the testing of the B. abortus and B. melitensis ‘genomovar’/ biovar assays conclusively assigned isolates to known clusters during validation. We did not see any instance (aside from B. abortus biovar 5 and ‘genomovar’ 3) where an isolate gave more than one profile. This gave confidence in the discriminatory power of the targets chosen. In conclusion, it is more reliable, beneficial and informative to base the groupings within species on genetic traits. It is a consistent and robust method to understand the way in which strains are related globally, and can show where and when potential divergences occur.

A major finding of this work is that there is a correlation between Brucella genotype (in terms of polymorphisms rather than tandem repeats) and geography. This observation has previously been described in a number of important pathogens including Mycobacterium tuberculosis (Gagneux and Small, 2007), Bacillus anthracis and Yersinia pestis (Keim and Wagner, 2009). For example, it was found that out of 14 isolates provided from the Gambia, 13 isolates shared the same ‘genomovar’ (‘Genomovar’ 3) profile as three existing isolates in the AHVLA collection, proving our hypothesis concerning a West African ‘genomovar’. The remaining isolate was identified a member of ‘genomovar’ 1, which interestingly is a grouping of strains largely of European origin.

Currently, a number of whole genomes of various Brucella species taken from a wide spatial and temporal area are being sequenced as part of a global collaboration in which the AHVLA represent the major partner. We believe based on the findings of this project, that the analysis of these sequences will inevitably define additional markers that can further subtype Brucella and that many of these clusters will correspond to groups with a clear phylogeographic basis. These SNPs could provide the basis for rapid real time PCR assays that could be used to quickly determine the source of infection in an outbreak scenario. To this end we have suggested that genome sequencing and subsequent SNP discovery and exploitation form a major component of future research work at AHVLA.

4. The assessment of a number of novel tools for both detection and SNP based typing of Brucella. The common theme of these tools is that they are simpler, cheaper and require less investment in expensive capital equipment. Thus such tools may be much more applicable in developing countries with endemic brucellosis issues as well as being tools that could potentially be applied to UK surveillance.

Milestone 4 (LAMP developed and assessed, compared with existing methods, need for sample purification assessed)

Introduction to topic and experimental approach: In terms of molecular diagnostics for brucellosis, the majority of published assays have relied upon the amplification of DNA using a PCR based approach. These assays are rapid, easy to use and interpret, sensitive and specific. However, the cost of equipment, particularly thermocylers to allow for the rapid changes in temperature required for PCR, means that the use of these techniques has been limited particularly in the developing world where brucellosis is most pervasive. Furthermore, it is well known that PCR is prone to inhibition from components within clinical samples such as haem in whole blood and calcium ions in milk (Akane et al., 1994; Bickley et al., 1996). Therefore it is necessary to have an extraction protocol in place to remove such inhibiting agents prior to PCR. If these problems could be overcome then there would be the potential to widen the use of molecular methods in routine diagnosis and even attempt pen side testing.

In this project we have investigated alternative technologies to facilitate the amplification of DNA. One technique of great interest was Loop-mediated AMPlification (LAMP) which allows amplification through two pairs of primers referred to as Internal (IP) and External (EP) (Notomi et al., 2000 for a description of the method). The addition of a third set of primers based on the loop structures (loop primers or LPs) formed as amplification proceeds has been shown to accelerate the LAMP reaction further (Nagamine et al., 2002). As the name implies, in isothermal amplification, the entire reaction proceeds at one temperature and there is no need for a denaturing step to allow primers to bind (Nagamine et al., 2001). This means that a costly thermocycler can be replaced by a much cheaper hot block or water bath. Further, there have been publications where this technique has been used to test clinical material (whole blood, swab washes and serum) without an extraction protocol (Poon et al., 2006; Minami et al., 2006; Ihira et al., 2007). One point of interest was the lack of published data on the direct testing of milk using LAMP. With these points in mind, we set about the development of an assay for the detection of Brucella in whole milk.

Results: At the commencement of this project, there was only one publication where LAMP had been described in relation to Brucella using the bscp31 gene as target (Ohtsuki et al., 2008). However, for the

EVID4 Evidence Project Final Report (Rev. 06/11) Page 11 of 28 purposes of the current ROAME, it was decided to use a novel target for the ROAME assay as it has been shown that Ochrobactrum anthropi has a homologue of bcsp31 and we were concerned that novel isolations of Ochrobactrum may also contain this target. We believed that a target that may play a role in infection would be preferable. To that end, omp25d gene was chosen as it has been suggested to have a role in Brucella intracellular replication (Martín- Martín et al, 2008). Primers (both IPs and EPs) were designed using primer explorer software (http://primerexplorer.jp/elamp4.0.0/index.html). Initially six sets were tested using a number of known Brucella isolates and from this, one set taken forward. Subsequently, LPs were generated by eye (there is currently no software available that can design these primers) and were found to have a positive effect on the rapidity of result generation (see Figure 5).

Specificity: In terms of specificity testing, a number of non-Brucella boilates (see Table 6 for list) were tested to determine specificity of the LAMP assay. In this testing, whilst there was sporadic amplification in a few samples, this was firstly not reproducible and secondly occurred much later (after 18 minutes) in comparison with genuine Brucella which were positive within 10 minutes (Figure 6)

Sensitivity: To determine sensitivity, B. melitensis Rev1 DNA was quantified using a spectrophotometer and dilutions made in water from 1ngµl-1 – 1fgµl-1. Replicates of 6 individual runs for each dilution were made and the results generated by using the real time PCR thermo-cycler/ EvaGreen showed that the minimum reproducible sensitivity was 100fg, comparable to real time PCR work described in the previous ROAME, SEO311 (P. Celebi-Court, 2005). However, unlike the real time assay, LAMP generated this sensitivity within 16 minutes in comparison with 1½ hours with real time PCR (Figure 7).

Robustness: To determine robustness of the LAMP technique, assays were run at 65°C, 63°C and 60°C and it was found that whilst there was no drop in activity as determined by the time required to generate a result at the two higher temperatures, the assay was slower when the incubation temperature was reduced to 60°C. The LAMP assay was run at 65°C for an hour for the remainder of the work undertaken.

Direct testing: To determine the usefulness of LAMP in direct testing of milk samples, dilutions in 0.1M PBS of 1x 109 to 1x 101 cfuml-1 heat inactivated B. abortus strain 544 were used to spike full fat milk resulting in final concentrations of between 1x 108 to 1x 100 cfuml-1. Testing these samples directly, without an extraction protocol, the detection threshold was 1x 105 cfuml-1. When this value was compared with that derived from LAMP with the same material but extracted with two commercial kits, DNeasy Blood and Tissue (Qiagen) and Instagene (Bio-Rad) it was found that the limits of detection were the same. However, reaction proceeded much more quickly when applied to extracted material versus the equivalent crude material, (Figure 8 shows an example of DNeasy extraction versus un-extracted spiked material). In terms of improvement in rapidity, an extraction with Instagene will take around 40 minutes, with a DNeasy extraction taking around 30 minutes. Further, there are added manipulation steps and costs with extraction and so, from data presented here, even though the assay is improved in terms of time it would not be beneficial to incorporate such a step given that neither sensitivity or overall result generation time are improved.

Comparison with RT-PCR: To compare the LAMP assay developed here with real time PCR, a Taqman assay was developed based on omp25d using Beacon design software. Cycling conditions were similar to other real time PCR assays run within the department, running for 40 cycles (1½ hours). Based on titrations of B. abortus strain 544 DNA, the limit of reproducible detection of the Taqman assay was 100fg in 35 cycles. To compare omp25d real time PCR and LAMP assays un-extracted whole milk spiked with various titrations of Brucella was compared with matching extractions obtained using the DNeasy Blood and Tissue kit. The limit of detection for the real time PCR assays was 1x108 cfuml-1 in un-extracted milk and 1x 105 cfuml-1 with the matched extractions (Figure 9). The limit of detection of real time PCR in extracted milk was the same as that of LAMP with un-extracted material.

Detection approach: Another major aspect of this work was the examination of approaches to visualise the LAMP reactions. Whilst using the EvaGreen/ real time PCR machine set up was successful for assay design and validation this platform would not be ideal for use in the field. The same is true of agarose gels that, even in the case of E-gels, require time to set up and run and some equipment burden. Turbidity as suggested by Mori et al., 2001 (the precipitation of magnesium pyrophosphate in a positive reaction leading to cloudiness) was ruled out as the addition of milk to the reaction mix during testing gave a similar appearance. Using Picogreen/ Wood’s lamp was also ruled out as it was found that high concentrations of degraded non- target DNA also fluoresce. Another avenue explored was the use of a colorimetric assay based on magnesium ion titration based on the work by Goto et al., (2009) involving hydroxyl-napthol blue (HNB), an agent used to measure magnesium ion concentration in water. In their work using a concentration of 120 µM HNB, a colour change from violet to sky blue indicated a positive LAMP reaction. However, using HNB with our reaction set up generated false positive results where as soon as the dye made contact with the mix in the absence of target, there was a colour change. Later work determined the cause of the colour change as deoxyribonucleotide triphosphate, which is required for the LAMP reaction

EVID4 Evidence Project Final Report (Rev. 06/11) Page 12 of 28 to progress.

Lateral Flow Devices: The approach that ultimately proved most attractive to visualise LAMP reactions was that taken by Hayman et al., (2011). This approach relies upon the incorporation of biotin and fluorescein into the LAMP product (through labelled LPs) with subsequent detection on a Lateral Flow Device (LFD). Incorporating the relevant tags LAMP reactions with and without genomic DNA (1ng B. suis Bv2 DNA) were set up and run at 65°C for 20 minutes. These reactions were then diluted 1 in 100 (1 µl in 99 µl) using the buffer provided with the LFDs and then added to the devices confirming specific detection of samples containing Brucella DNA. This work was successfully repeated using 1x 108 cfuml-1 heat inactivated B. abortus spiked milk although the incubation time had to be extended to 40 minutes (Figure 10). To check for specificity, we used heat inactivated non Brucella cultures (see Table 1) to spike milk in a ratio of 1:10 and test these after an incubation period of 1 hour at 65°C. No non-Brucella organism from the panel previously used to ascertain specificity gave a result using the LFD in this time frame.

Discussion, conclusions and future work: Findings concerning the testing of isothermal amplification as an alternative to PCR are encouraging, particularly as it has been shown that the limit of detection using LAMP directly on spiked milk is equivalent to that of real time PCR on extracted material. Although, at the time of writing this report, there are already two published Brucella LAMP assays (Ohtsuki et al., 2008; Lin et al., 2011), neither of these have been applied directly to milk samples but on extracted material. The coupling of this technique to LFDs to visualise the reaction result instead of using gel based methods is another encouraging development of this work as it brings this technique one step closer to a field application rather than just another molecular diagnostic for the laboratory. In comparison with other field systems such as ruggedized PCR/ RAZOR™ (Idaho Technology, Salt Lake City, Utah, USA), LAMP/ LFD is much cheaper to implement, with LFD units cost about £1 each and hot blocks costing significantly less that thermocyclers.

However, there is still much validation required and room for improvement in the application of LAMP. There is no definitive study on Brucella bacterial load in milk and therefore it is still unclear whether the current detection limit for the LAMP assay is relevant to clinical samples. In addition to the testing of field material (which is being facilitated through connections with Turkey) another aspect that should be investigated in future will be the concentration of sample through filtration of large volumes of whole milk (typically, 5mls), with the filtrate being eluted in a much smaller volume for subsequent LAMP testing.

Furthermore, as with all DNA amplification methods, negative results need to be accounted for as the absence of target rather than a problem with amplification of sample. To this end, AHVLA has developed an internal amplification control (IAC) based on a synthetic target which can be multiplexed with other LAMP assays. It is our intention to multiplex the IAC with our Brucella LAMP assay and then aim to publish our findings.

Milestone 11 (Assessment of alternative SNP typing approaches).

Milestone 14 (Complete development of full SNP typing scheme to species level for selected alternative approach).

Introduction to topic and experimental approach: In a previous ROAME (SEO311) the development of multiple outcome species and vaccine defining assays based on discriminatory SNPs and using a real time PCR platform was described (Gopaul et al., 2008; Gopaul et al., 2010) and these assays have been extended in this project as described in Section 3.. Whilst these assays are simple to use and offer better discrimination than other assays published at that time (Koylass et al., 2010), the cost of reagents (particularly the Taqman™ MGB probes which are only available from Applied Biosystems) make these assays unattractive for use for many laboratories. Therefore, the primary aim of this work was to look alternatives to Taqman™ real time PCR for assaying SNPs. To this end, it was decided to look at three alternative and rather experimental strategies; one using real time PCR but employing cheaper melt chemistry and two looking at adaptations in isothermal amplification. These methodologies were High Resolution Melt (HRM) curve analysis (Winchell et al., 2010), SMart Amplification Process (SMAP) analysis (Mitani et al., 2007) and SNP-LAMP (Fukuta et al., 2006). Using melt analysis is not novel in the identification of SNPs. However, HRM is unique in that fluorescence measurements are read over very small temperature increments (0.01°C), allowing changes in melting temperature (Tm) to be detected that have been caused by a single base pair change in the sequence of the target. As for the methods involving isothermal amplification, as mentioned earlier using these (particularly if results could be read by LFD) would open up the possibility of pen side testing. In the case of HRM, whilst this would require a specialist real time thermocycler (in our case the Rotorgene Q platform from Qiagen was used) to perform the melt analysis, the actual PCR and labelling of products could be undertaken in any laboratory with a conventional thermocycler and products could then be transported for analysis remotely.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 13 of 28 Both isothermal amplification based techniques rely upon the premature termination of amplification where there is a mismatch in the 3’ end of the primer. However in SMAP, this is taken one step further with the addition of the protein MutS, which binds the mismatch between the primer and target, stopping even minute amounts of non-specific amplification (for a full description of the technique see Mitani et al., 2007). In addition to this, the use of an un-labelled probe directed towards the alternate sequence state to compete for binding with SMAP amplification primers has also been used in this technique to prevent non- specific reactions (Watanabe et al., 2007).

Results:

SMAP/SNP-LAMP: Here, it was initially decided to perform a proof of concept SMAP based assay using the SNP in BMEI0752 (rpsL) which gives B. melitensis Rev1 streptomycin resistance. Our reasons for doing this were that if this work was successful, this would be of sufficient impact for immediate publication. Primers were designed using online software provided by the Riken Company of Japan (http://www.smapdna.com). Two assay pairs (each pair has one assay targeting the SNP of interest whilst the other targets general sequence) were initially designed and ordered. However, upon testing with B. melitensis Rev1 and B. abortus strain 544 (which does not contain the target SNP) both at 1ng, although there was preferential amplification from the correct assay/ target combination, there was also non-specific amplification after a short time lag. The use of competitive probes, changes in the concentrations of primers used, and varying incubation temperatures were all trialled in the attempt to improve specificity. However, it was found that none of these changes could improve the specificity of amplification. The same lack of specificity was seen using SNP-LAMP, where assays were designed using the same software as the molecular diagnostic earlier for specific SNPs in B. abortus and B. melitensis. Again, the same variables looked at for SMAP were altered for SNP-LAMP but without success. In addition, it was found that the enzyme preferred for this reaction, Aac polymerase was only available from one source and was around ten times more expensive than BstI polymerase (the enzyme used for LAMP). The lack of success in obtaining specific amplification with either isothermal method as well as the costs of ordering reagents for SMAP meant neither of these methods were taken forward and, as outlined in section 4.8 of the proposal, HMRC analysis, which proved much more promising in preliminary studies, was selected and pursued in greater depth in the later stages of the project.

HMRC: Information provided from both the AHVLA Brucella MLSA scheme and from the international whole genome sequence project hosted by the Broad Institute (Cambridge, Massachusetts, USA) was used to identify SNPs (and associated flanking sequence of 100bp) specific to five most important Brucella species; B.abortus, B. melitensis, B. ovis, B. suis and B. canis. The criteria for target choice were that there should be only one SNP per target, that there should be no other SNPs for different species in the same region, and that target sequences should be free of secondary structure that would interfere with the melt. To determine this third point, secondary structure of the potential product strands were screened for eligibility by assessing the secondary structure of the sequence surrounding the SNP, using the DINAMelt programme available freely online (http://mfold.rna.albany.edu/?q=DINAMelt). Primers were then designed around the targets meeting the required criteria using the Primer 3 software (http://frodo.wi.mit.edu/primer3/ (Table 7 for targets used).

Reactions were initially run in simplex to determine the optimal primer concentrations using a combination of highly purified (through phenol-chloroform extraction) and crudely extracted (through boiling or storage in methanol) Brucella DNA samples. One interesting observation at this point was that the concentration of DNA template added had an effect on the melting temperature of the PCR product formed. This was worrying as this change in dynamics could change the outcome of the species call. Subsequent titration work determined that 1ng of template was the minimum amount of DNA that could be added for a conclusive result. For this reason, the remainder of the work undertaken was done with highly purified DNA extractions diluted to 1µgml-1 (1µl of this corresponding to 1ng). In addition each reaction contained final concentrations of 1x HRM Type-it™ mix (Qiagen) and 0.7µM forward and reverse primer (for each target). Temperature cycling parameters for the amplification stage were a hold of 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds, 72°C for 20 seconds. For the HRM stage, fluorescence recordings were made over the range of 65-95°C by increments of 0.01°C.

Results of simplex reactions for the initial five species (in terms of species discrimination) were promising (see Figure 11 for examples) and to improve the utility of the assay attempts were made to multiplex the five individual assays into one run, something that had not been attempted in published HRM work. To do this the melting temperatures of the products of each assay had to be manipulated to a certain temperature by adjusting product size (altering primer positions, primer sizes, or extensions) to ensure no overlap between each individual melt peak that would render sample identification impossible. The concentrations of primers added to the primer mix were meticulously optimised to ensure high quality and equally sized melt curves were generated for each species defining assay in the multiplex. Final optimised concentrations of primers within the primer mix were 12.5 µM B. abortus primers, 10 µM B. ovis primers,

EVID4 Evidence Project Final Report (Rev. 06/11) Page 14 of 28 11.25 µM B. suis primers, 5 µM B. melitensis primers and 2.5 µM B. canis primers (equating respectively to a ratio of 10:8:9:4:2 abortus: ovis: suis: melitensis: canis). By successfully combining these into a multiplex, the resulting melt curve contained 5 discriminatory differences, each able to define their respective species according to the melting temperature of their amplified products (Figure 12).

In addition to the five species mentioned, HRM assays were also developed and tested for B. inopinata (Scholz et al., 2010), B. microti (Scholz et al., 2008), B. neotomae and the novel Brucella recently isolated from baboons (Schlabritz-Loutsevitch et al., 2009) and a novel human isolate (BO2) (Table 7 for targets and primers). However, given difficulty in re-adjusting the 5-plex assay already developed along with issues concerning interpretation of data where there were more than 5 melts occurring per sample meant that these assays were not multiplexed further. Also, although we had initially hoped to include SNP assays for the three most common Brucella vaccine strains (B. abortus S19, B. abortus RB51 and B. melitensis Rev1), there were issues with the SNP targets previously used (Gopaul et al, 2010) concerning secondary structure which made this assays impractical to design and test. With the multiplex fully optimised and reproducible, DNA from a panel of isolates was quantified and diluted into a concentration of 1 µg/ml, and then tested. A total of 75 samples were successfully tested and characterised with the multiplex HRM assay (see Table 8).

Discussion and conclusions: The aim of this section of work was to develop an alternative SNP based tool to the Taqman™-based multiple outcome species defining assay developed in the previous ROAME. To this end, the three approaches studied were initially selected as they were thought have the potential to be quicker, cheaper and simpler than the currently employed assays. It was hoped that one of the isothermal methods would have proved successful in testing. However both SMAP and SNP-LAMP were found to have poor specificity regardless of the optimisation strategies taken. The very nature of the primers used for amplification in these techniques (requiring two regions for binding with the target in comparison with one for PCR) may be the explanation for these observations. In addition the cost of reagents for SMAP added to the unattractiveness of this technique.

In comparison, HRM was feasible in this study and 10 simplex assays were developed to all known terrestrial Brucella species. In addition, it was possible to multiplex five of the more economically relevant species in one assay. In testing a panel of 75 isolates, it was found that each isolate was identified correctly (including B. suis and B. canis isolates known to be problematic in some assays). Further, another interesting observation made as part of this work was that in the Brucella 5-plex assay, it was found that B. inopinata generated a novel profile (Figure 12) even though there was no assay directed towards that species in the multiplex.

As mentioned earlier, during the course of this work, another group published a set of HRM assays for the determination of Brucella species (Winchell et al., 2010). In this paper, a number of simplex assays for the identification of seven Brucella species and Brucella of marine mammal origin are described. However, where this publication differs from the work described here is that there is no mention of multiplexing of reactions. Further, and more disappointingly, although the publication describes the use of HRM, in the case of B. suis the assay described is not HRM (not based on a SNP) but is a simple PCR based on a “conserved” IS711 insertion seen in only B. suis isolates. In addition, the authors of the published work mention that their assay could not distinguish B. suis biovar 4 from B. canis, which is something that the assay described here is capable of doing.

The HRM method offers a simple solution to genotyping, with little downstream processing and a closed tube excluding the hazards of product manipulation in terms of subsequent cross-contamination. Reagent prices are relatively low, as only primers and a generic dye are needed, rather than expensive probes as with real time methods. Data acquisition is rapid, with a melt only taking a few minutes post amplification. Further, if run in simplex, amplification products can be taken directly for sequencing to confirm the identity of novel profiles.

However there are a number of issues with this methodology that diminish the usefulness of HRM for typing. Although the design process for HRM primers is straightforward, target choice is hampered by the need for minimum secondary structure. In situations where the number of SNPs available is small, this could be a major problem. This technique cannot identify the location of target SNP and so if there is a similar base substitution elsewhere in the fragment but not in the region of interest, this will be miscalled. Further, although one run on the Rotorgene Q (Qiagen, Hilden, Germany) can accommodate up to 100 reactions, there is currently no software available that will allow for the comparison of profiles from previous runs with those from current runs. These means that some slots are always lost through the addition of reference strains for comparison and identification. Even if it were possible to compare the results of different runs, it was also noted that there were slight variations in melting temperatures between runs, exacerbating the need for controls to be incorporated into each run. A further drawback is the fact that the melting temperature of DNA in this technique is not only dependant on the concentration

EVID4 Evidence Project Final Report (Rev. 06/11) Page 15 of 28 of template pre-amplification but also on ionic conditions. In turn this means not only does the sample need quantification pre-amplification but that the extraction methods and how the sample is stored have to be consistent. This is difficult to achieve with field samples, or those that are sent from other work groups.

In conclusion, the HRM technique is a highly accurate method for typing Brucella to species level based on a single SNP. It is a simple and rapid closed tube test with minimal risks of cross-contamination. The fact that we were able to multiplex five of the assays allows for a much higher throughput than with the individual assays, and also gives the assay the ability to identify potentially new and emerging strains of Brucella. The assay would not, however, be appropriate for direct testing from clinical material as there are too many constraints with regard to the conditions that the DNA samples need to be in prior to amplification. . 5. The continuing development of our Brucella multilocus sequence analysis (MLSA) scheme will involve setting up a publically accessible database in order to encourage other laboratories to use the scheme and submit their own data and continuing use of the scheme to address controversial issues in Brucella taxonomy and nomenclature. Furthermore, given ongoing debate about the potential merger of the genus Brucella with other genera within the family Brucellaceae, we intend to attempt to develop a pan-Brucellaceae MLSA scheme that will allow direct comparison of the genetic and evolutionary relationships within this family. This will contribute to the debate by unambiguously clarifying the position of Brucella in relation to other related bacteria and allowing rapid placement of any newly discovered organisms within the family.

Milestone 3 (MLSA database made publically available)

Milestone 5 (Molecular comparison of North American marine mammal Brucella isolates complete)

The focus of this area was the application of an existing MLSA scheme and the development of a pan- family MLSA scheme that, rather than just allowing typing and placement of Brucella spp. allowed the placement of isolates within the wider context of the Brucellaceae family. These schemes will assist in attempts to categorise many emerging potentially new Brucella species and be crucial in understand the global diversity of the group.

It has not yet proven possible to make our existing Brucella MLST scheme available in the ideal format of an interrogable database against which isolates can be compared. However we have endeavoured to make the data publically available by depositing relevant sequence data in GenBank (Accession numbers AM694191 to AM695630). Further where laboratories have requested we have freely provided sequence data files formatted for use in phylogenetic software into which colleagues can insert their own data for comparison with the extant database. This has allowed other laboratories to independently make use of the outputs of ROAME research to describe new Brucella and contribute to understanding of the group (Tiller et al., 2010a; Tiller et al., 2010b). It remains a longer term ambition to make data available in web- based interrogable database (such as that at http://www.emlsa.net) covering both our classical Brucella scheme developed previously and the pan-family scheme that allows placement emerging Brucella in a wider context, developed in this project, and described below. However, as the hosting of such sites is currently incompatible with the Defra network, we will remain dependent on the goodwill of other institutes to ultimately achieve this.

The existing 21 locus MLSA scheme was applied to 51 North American isolates of Brucella from marine mammals largely to extend understanding of this group as virtually all work in the past had been focused on isolates from Northern European waters. These isolates were also subject to an extensive array of complementary genotypic (omp typing, IS711 typing, Bruce-ladder, IRS PCR) and phenotypic (full biotyping) typing that will form the basis of a comprehensive publication (in preparation) providing the first comparative analysis of European and North American strains of Brucella from marine mammals. Full data is not presented here due to lack of space but a summary of relationships based on MLSA data in the context of existing data from European isolates is shown in Figure 13. The main points of interest are as follows: • Most isolates correspond to genotypes (and corresponding phenotypes) previously seen in Europe but two novel genotypes were seen – minor variant of ‘porpoise complex’ and major variant of ‘dolphin complex’. • In general host specificity appears to hold (i.e. isolates fall into three groups corresponding to isolates predominantly from seals, porpoises and dolphins as described previously in Europe (Groussaud et al., 2007)) although the North American collection was dominated by pinniped isolates with few cetacean isolates. • ST27 is of particular interest as we have previously described this ST associated with all naturally acquired human infections with marine mammal Brucella reported to date (Whatmore et al., 2008). ST27 had never been seen in European waters and we had previously suggested, based

EVID4 Evidence Project Final Report (Rev. 06/11) Page 16 of 28 on observed distribution of human cases, that this might be geographically restricted to Pacific waters. However it had also only previously been seen in a single marine mammal – a captive bottlenose dolphin so the natural host of this type was unclear. As predicted ST27 was seen among isolates in this collection and, in support of the above, only in isolates from Pacific rather than Atlantic coasts. Californian sea lions, which are geographically restricted to the West Coasts of USA and Mexico appear to be a natural host for this genotype although how the bacteria then transmit to humans remains unclear – human cases were loosely associated with raw seafood so there may be intermediary hosts. Importantly these observations add to a body of evidence indicating that ST27 is not present in European waters and therefore that there may be limited threat of zoonotic infections originating from the marine environment in Europe. • The variant (B. pinnipedialis?) genotypes previously reported as unique to hooded seals – ST53 and ST54 - (Groussaud et al., 2007, Maquart et al., 2007, Nymo et al., 2011) are also present in harp seals which share a similar geographical range. • First characterisation of isolates from beluga whales – surprisingly these are B. pinnipedialis. • This work continues to support previous observations (Whatmore, 2009) that the taxonomy of the marine mammal Brucella is not consistent with phylogeny i.e. B. ceti is a paraphyletic group. This is not merely a semantic issue but has implications for any molecular test as, because of the evolutionary history of the group, there are highly unlikely to be markers common to both B. ceti groups that distinguish them from other groups. This is reflected in all our molecular assays which define only to the level of ‘marine mammal’ Brucella. Efforts will be made in forthcoming work to suggest revisions to the taxonomy of this group and help resolve this issue.

Milestone 6 (Development of pan-family MLSA scheme complete)

Milestone 15 (Complete testing of all isolates using pan-family MLSA establishing picture of genetic relationships)

The aim of this workpackage was to develop an extended MLSA scheme applicable to all members of the family Brucellaceae (for clarity the genus Brucella is within the family Brucellaceae within the order Rhizobiales within the class Alphaproteobacteria).This was driven by the increasing emergence of novel Brucella isolates and increasing confusion with the nearest neighbour Ochrobactrum, both phenotypically and genetically, as the known diversity of the Brucella continues to expand. Thus there is an urgent need for a single robust mulltilocus scheme that can be applied to the wider family – primers used for our existing 21 locus MLSA scheme had been found to amplify only a small subset of strains within the group and then often very poorly. This work therefore aimed to develop a seven locus MLSA scheme applicable to the wider Brucellaceae family but based on loci already included in the existing Brucella specific 21- locus scheme such that data are directly comparable with the existing Brucella MLSA database with no need for additional characterisation of these isolates.

Substantial development work was required to set up such a scheme with degenerate PCR primers needing to be designed and validated as capable of amplifying equivalent loci across the Brucellaceae family (consisting of the genera Brucella, Ochrobactrum, Pseudochrobactrum. Paenochrobactrum, Daeguia and Mycoplana). Other than for Brucella, whole genome sequences of remaining members of the family have yet to be described with the exception of for two Ochrobactrum species. Because of this it was impossible to design primers based on comprehensive sequence alignments within the family and alignments across the wider order Rhizobiales which allowed inclusion of additional genome sequences from isolates such as Rhizobium and Agrobacterium had to be considered. This approach also had the advantage that if a primer pair could be designed based on the wider diversity of the order Rhizobiales it would theoretically be more likely to be able to amplify successfully all isolates within the less diverse family Brucellaceae.

In an iterative process of design, testing and reoptimisation of primers (using both software and manual approaches) attempts were made to design degenerate primers targeting all 21 loci used in the existing Brucella MLSA scheme. In four of 21 cases it was clear that there was too much diversity to allow design of primers. For the remaining 17 loci a range of primers were designed and tested against a panel representing all 25 type strains of species within the family (including several new species of Ochrobactrum, Pseudochrobactrum and Paenochrobactrum that were described during the course of this work). In order that the ultimate scheme was robust and simple to perform all primer pairs were designed to amplify under identical PCR conditions. After substantial validation work the best performing seven loci/primer pairs that could be successfully amplify from all 25 type strains were selected as the basis of the novel pan-Brucellaceae family MLSA – acnA, csdB, dnaK, gap, leuA, prpE and trpE. Once all data was collated individual phylogenetic trees were constructed for each of the genes with generally good congruence in clustering pattern between genes with the exception of two newly described species O. daejeonense and O. hematophilum (data not shown). Sequences from these strains were particularly closely related to Brucella at some loci (dnaK and prpE) but more distantly related at others emphasising

EVID4 Evidence Project Final Report (Rev. 06/11) Page 17 of 28 the value of a multilocus rather than a single locus approach for assessing the true relationships between strains.

In order to provide an overall assessment of the relationship between strains sequence data were concatenated and used to construct a phylogenetic tree as shown in Figure 14. These data demonstrate that the new scheme gives a comprehensive picture of the relationships between isolates. Importantly there is good separation between even the emerging divergent Brucella groups and the closest Ochrobactrum species implying that the scheme will be of value in helping classify newly emerging and/or atypical isolates. Ochrobactrum clearly comprise two distinct clusters and, as mentioned above, placement within these clusters is largely consistent between genes with the exceptions already mentioned. Contrary to much literature these data suggest that overall O. intermedium is no longer, as is widely accepted, the closest relative of the Brucella (Velasco et al, 1998) with the newly isolated O. daejeonense from sewage sludge (Woo et al., 2011) appearing to now represent the most closely related taxa. Future comparative analysis of whole genome sequences of these organisms may help in understanding how the preferentially intracellular and pathogenic Brucella emerged from these free-living soil organisms. In agreement with recent taxonomic revisions Pseudochrobactrum comprises a distinct well defined group related to, but distinct from, the recently described Paenochrobactrum.

In order to further validate the universal applicability of the scheme it was then applied to two distinct cohorts of field isolates. One cohort represented strains submitted to Weybridge in recent years as suspect Brucella but discounted and thought to represent confounding members of the Brucellaceae. The other represents a diverse panel of environmental Brucellaceae previously described (Scholz et al, 2008) and obtained from collaborators in Germany. Figure 15 shows the placement of 41 isolates from Gemany (labelled D), obtained to validate the procedure, and 12 isolates from the UK (labelled UK), obtained from abortion enquiries as suspect Brucella, relative to type strains. The data reveal a number of interesting observations but the crucial point is that there remains good separation between even the newly emerging divergent Brucella such as B. inopinata, BO2 and 83/13 and the closest non-Brucella such that the tool appears useful in accurately placing any new isolate in the context of the family. Interestingly most of the UK isolates submitted as suspect Brucella appear to fall within the Pseudochrobactrum although most do not comfortably fall within existing species (note that the labelling of some of these isolates as Ochrobactrum relates to the, now clearly erroneous, nearest neighbour description at the time of isolation and before the genus Pseudochrobactrum was described). Similarly an isolate of Ochrobactrum obtained in the same circumstances (VLA07/50) again likely represents a novel species – these findings illustrate how this tool will be useful in understanding the relationship of Brucella with other Brucellaceae and as a tool for categorically confirming the identity of an isolate as Brucella in the context of an understanding of extant diversity of the group.

Milestone 16 (Have completed descriptions and characterisation of novel Brucella species to allow publication)

Multilocus sequencing has played a crucial role in describing a number of emerging new species during the course of this work. The group contributed to the description of a new species from a human case, B. inopinata, in which MLSA was crucial (Scholz et al, 2009) as well as undertaking a more detailed description of the diversity of the novel species from voles, B. microti, which we jointly described during a preceding ROAME (Al Dahouk et al, 2012). MLSA has also been used to characterize a number of emerging Brucella groups including isolates from foxes (Hofer et al, 2012) and frogs (Eisenberg et al., 2012) by us as well as by others as described above. Work is ongoing to complete a formal taxonomic description of novel Brucella isolates previously described by us from a baboon colony and MLSA should continue to be central to the description of the increasing isolations from novel sites contributing to the rapid expansion of the genus Brucella.

6. We will continue to build up our VNTR typing database and use the data, in conjunction with MLSA data, to address ongoing issues in Brucella taxonomy. We will also seek to harmonise our existing data with an internationally agreed scheme that will allow the ongoing development of an international database of VNTR profiles.

Milestone 10 (Harmonisation of VNTR data complete)

Introduction to topic and experimental approach: The usefulness of variable number tandem repeat (VNTR) analysis as a molecular tool to aid in epidemiological trace-back/ outbreak control for brucellosis has been described by a number of research groups (Abril et al., 2011; Alvarez et al., 2011; Gwida et al., 2012) following pioneering work in this area from AHVLA. There are now several VNTR schemes described although an attempt was made by a number of groups (including AHVLA) to agree a common scheme which would ensure that data are comparable between studies This scheme, known as MLVA16, has become the most widely used scheme and an interrogable website is available where laboratories can

EVID4 Evidence Project Final Report (Rev. 06/11) Page 18 of 28 compare their own data with a freely available website. The standard VNTR scheme used at the AHVLA makes use of 21 loci (Whatmore et al., 2006) although there is considerable overlap with MLVA16. Although within the AHVLA panel there was some overlap in the targets used (9 loci are conserved between AHVLA and MLVA-16), this still left 7 loci which the AHVLA scheme did not examine (Bruce 06, 11, 12, 19, 21, 42 and 43 according to MLVA16). This in turn makes it difficult to compare data generated by AHVLA with other workgroups using MLVA16 to track transmission. To resolve this issue, in this ROAME, work was undertaken to generate data for the seven remaining loci from a database of VNTR profiles already held at AHVLA. Primers for the seven mentioned loci were ordered based on the work done by Le Fleche et al., 2006. To ensure that the sizes generated in this study were comparable with those of Le Fleche et al., (2006) for subsequent automated annotation of repeat size, 48 Brucella strains with published repeat sizes were examined (Table 9). In the case of the remaining nine loci that were common to both the European and AHVLA schemes, an in silico analysis of type strains facilitated a harmonisation of allele calls.

With this done, work then progressed to process the large isolate bank of highly purified (through phenol chloroform extraction) and crudely lysed either by boiling or fixation in 66% methanol as described by Whatmore et al., 2006 but with the fragment analysis being performed using the Applied Biosystems 3130XL genetic analyzer. Aside from Bruce 06 reactions were multiplexed for amplification as follows: Bruce 21 & Bruce 43, Bruce 11 & Bruce 42, Bruce 12 & Bruce 19. However, for subsequent fragment analysis on the sequencer Bruce 06 was combined with Bruce 11 & Bruce 42. Results for the seven new loci were combined with harmonised calls for the remaining nine loci on a new database created using BioNumerics™ software (Applied Maths NV, St- Martens-Latem, Belgium).

Results: In total, full MVLA-16 profiles were obtained for over 800 strains held within the AHVLA database. To demonstrate the benefits of harmonising VNTR schemes subsets of AHVLA data were compared with that published by European colleagues. To this end we compared a subset of Brucella profiles obtained from isolates taken in Turkey with the profiles generated by Gwida et al., 2012 in a study of Brucella isolations in Germany and Turkey taken both from humans and livestock. Using a similar approach to that published in the paper, a dendrogram of the 57 German and 36 AHVLA isolates was created (Figure 16). In un-weighted analysis, it was shown that four AHVLA isolates had identical profiles to six German isolates in four distinct clusters (Figure 16).

Discussion and conclusion: The harmonised scheme described above revolutionises the way that Brucella molecular epidemiology is performed at AHVLA. Previously, although the 21-loci scheme used provided exceptional resolution of cases, the data generated could not easily be interpreted in a wider setting. It now allows AHVLA the opportunity of collaborating on cross-border schemes similar to that published by Gwida et al., (2012) where VNTR was used to show that strains isolated in humans of Turkish origin from Germany had origins in Turkish livestock. It will also allow us the opportunity to collaborate with European colleagues to create in the future an information repository of VNTR profiles that will allow tracking of the disease throughout the continent rather than just within our own national borders. This will be of great use as although many countries within the EU have the OIE status of being officially brucellosis free, a number of countries including Spain, Portugal and Greece still have on-going problems with the disease in cattle. In addition, it has been reported that two B. abortus isolations recently made in Belgian cattle were similar to those found in the country pre-eradication many years previously

In terms of how work will now be undertaken at AHVLA, it is envisaged that VNTR 21 will be retained as a useful tool for internal studies but that samples will additionally be tested using the remaining seven loci to allow for wider comparisons in future.

A potentially significant issue emerged during the course of this work when it was observed that some template preparations gave better results than others with isolates fixed in methanol being the most troublesome. Bricker et al., (2003) in their work to evaluate the Brucella species defining AMOS multiplex PCR assay described fixation in methanol as a good means of long term storage of DNA. It should be noted that a number of methanols were made and stored over 5 years ago and this may have contributed to their poor performance in terms of amplification and fragment analysis. Nevertheless, this work has raised some issues about the long term storage of genetic material and whether it may be better to use heat inactivated material for routine VNTR work in the future.

7. Building on existing work examining the potential of VNTR as a tool applicable to local epidemiology and disease control we will assess whether single nucleotide repeats (SNR) offer an additional tool suitable for typing at a local level and address the develop of rapid assays to type such markers.

Milestone 1 (Scanning of genome sequences to identify SNR tracts that may be useful for B. abortus typing).

EVID4 Evidence Project Final Report (Rev. 06/11) Page 19 of 28

Milestone 7 (Identification of SNRs with suitable balance of stability/discriminatory capacity for use in typing)

Milestone 12 (Development of SNR typing methods and assessment of value of such markers as epidemiological tools)

Introduction and experimental approach: Simple Sequence Repeats (SSR) are repetitive DNA elements that have been detected in all sequenced bacterial genomes to date in both coding and non- coding regions (van Belkum et al. 1998, You-Chun et al. 2002). There are two broad categories of SSR’s namely microsatellites and minisatellites, with microsatellite repeats being between 1 to 6 bps long and minisatellites being over 6 bps (Kashi & King 2006). One such element family that falls into this latter group is that of Single Nucleotide Repeats (SNRs). This class of repeats involves the repeat of a single nucleotide base several times and are recognised for their high mutation rates (Keim et al. 2004, Stratillo et al. 2006). It has been shown SNR loci are also highly variable between strains, providing discriminatory power to differentiate genetically homogenous taxa (Diamant et al. 2004). They have also been used to investigate intra-specific variation in several bacterial genera and species (Keim et al. 2004, Stratillo et al. 2006, Garofolo et al. 2010). The advantage offered by SNRs as a molecular marker is that they may be applied to species or isolates with a very low level of genetic diversity and be able to provide and increased level of resolution specifically in an outbreak situation (Keim et al. 2004).

Based on this perception, this study was undertaken to provide a preliminary evaluation of the potential use of SNRs to provide increased resolution to distinguish between isolates of B. abortus that had previously been analysed and ordered by VNTR. The hypothesis was that the addition of SNR to VNTR data would rapidly add further resolution which would be beneficial in interpretation for molecular epidemiology. As an exemplar we have used isolates from Northern Ireland as the kind of restricted sample set across a geographically contained area where SNRs might be of value in understanding transmission events. The purpose of this work was only to prove principle both in terms of SNRs being variable in such a population and in terms of the kind of assay that could be used to index this diversity.

Three genome sequences of B .abortus were investigated for the presence and distribution of SNRs: B. abortus 9-941, B. abortus S19, and B. melitensis biovar abortus 2308. These sequences were used as input files for SSR Table software programme (http://www.technion.ac.il~anne/choice3.html ) (Gur-Arie et al. 2000) to identify SNRs. Specific search criteria implemented in SSR Table for SNR searches included a) a minimum number of repeats = 3, b) maximum motif length = 1 and c) minimum length of repeats = 1. These specifications resulted in the identification of SNR tracts with a minimum of 3 bps. repeats and a maximum motif length of 1 bp (Table 10). Repeats with a tract length of 7 bps or greater, identified were selected for further investigations.

Flanking sequences of approximately 250 bp. upstream and downstream of the SNR tracts were included to obtain genomic sequence fragments of approximately 500bp. These sequence fragments were used to compare with B. abortus genomes from all known biovar type strains made available on the Broad Institute website (http://www.broadinstitute.org/annotation/genome/brucella_group/MultiHome.html) as part of the whole genome sequencing project. From this, 16 SNR loci that were identified as being polymorphic amongst at least B. abortus biovars 1 and 2 were taken forward for subsequent sequencing. Primers (Table 11) were designed using Primer 3 software (http://frodo.wi.mit.edu/primer3/) and were applied to sequence from a test panel of 46 B. abortus isolates originating from cattle in Ulster. In addition a sub set of the five most polymorphic loci were investigated for stability by sequencing six serial sub cultures of B. abortus 544 taken over six years.

In addition to determination of SNRs as tools for added resolution to VNTR, alternative strategies were explored to rapidly assay SNR length (i.e. without the need for sequencing). To this end, two alternative approaches were taken; fragment analysis (Stratilo et al., 2006) and High Resolution Melting (HRM) (Ricchi et al., 2011). In the case of fragment analysis, to prove principle for this technique primers were ordered for one target, BMBA2308-C2-974305 with the forward primer being 5’ labelled with 6-FAM. Amplification and analysis proceeded as for VNTR with sizes being compared to the ROX-500 ladder. For HRM, sequences from each of the five loci chosen were subjected to secondary structure analysis as described in section 3. From this analysis, two loci were taken forward with primers being designed as described in section 3. Subsequent HRM reactions were run in simplex using conditions outlined in section 3.

Results: Results from the SNR table analysis of the three B. abortus genomes indicated that the most frequent SSR repeat among the three genomes were SNR tracts with a length of 3 bp. However the number of these repeats varied in the all three strains (Table 10). It was noticed that the frequency of SNRs was inversely proportional to the length of the SNR repeat motif but that there was still an

EVID4 Evidence Project Final Report (Rev. 06/11) Page 20 of 28 appreciably high frequency of SNRs of 6bp (Table 10). However, there was a significant decrease in frequency once SNR motifs increased in length from 7-10bp (Table 10).

Based on the frequency of elements from the three B. abortus strains, SNR loci with repeat lengths of seven base pairs or more were taken forward for further investigations of polymorphisms. Comparing these regions to those found in the type strains of the remaining B. abortus biovars, it was found that 16 SNR loci exhibited polymorphisms between two or more of the B. abortus biovars (Table 12 and Figure 17). From the analysis of a panel of 41 Ulster B. abortus isolates using these 16 loci, five SNRs were found to be polymorphic: BA9941-C1-647987, BA9941-C1-705121, BAS19-C1-703439, BA9941-C1- 481826 and BA9941-C1-1091176 (Table 13 and Figure 18). However it should be noted that two SNR regions, BA9941-C2-979548 and BMBA2308-C2-974305, were abandoned due to repeated difficulties in amplification for sequencing. The remaining nine SNR loci proved to be monomorphic among the B. abortus isolates from the test panel. In stability testing using serial passages of B. abortus, B. suis and B. melitensis taken over six years, it was found that all SNR repeat loci were stable for the three strains through all the serially sub-cultured strains exhibiting no alteration in the repeat length of the SNR loci (Table 14).

In terms of annotation, for each of the 41 Ulster isolates a numeric profile was generated based on the number of repeats in each of the five SNRs (Table 13). These profiles were then uploaded in BioNumerics® (Applied Maths, Gent, Belgium) as character types for cluster analyses. Dendogram construction included pairwise similarities with multi-state coefficient as categorical data and the Unweighted Pair Group Method with Arithmetic Means (UPGMA) as the dendogram type (Figure 18). This analysis arranged the B. abortus isolates into six distinct clusters (A-F) (Figure 18). Based on the observed alleles the clusters represent 6 multi-locus haplotypes of B. abortus. Cluster B accommodated the greatest number of isolates (23) followed by cluster A (12), D (3) and C, E and F all with a single isolate. Cluster A and B represented sister clusters that were most similar. Isolates within these two clusters had identical alleles for four of the five SNR loci and were only divergent for locus BA9941-C2- 1091176. Genotypes D & F were also similar in shared alleles and grouped in a well supported cluster with differences in loci BA9941-C1-647987 and BA9941-C2-1091176 leading to differences observed between these genotypes. Genotype C clustered alone but sister to genotypes A and B and only differed from these two genotypes by a one step change in two loci BA9941-C1-481826 and BA9941-C2-1091176 (Table 13, Figure 18). Clustering of B. abortus isolates from the test panel did not exhibit any defined geographic association, however there is extensive cattle movement in Ulster and, without firm supplementary epidemiological data for these isolates, no conclusive associations based on the clustering can be proposed.

For comparative purposes dendograms were constructed of the same 41 B. abortus isolates using VNTR data from 11 loci (Hoofprint1, 2, 3, 4, 6, 8, VNTR 2, 5a, 5b, 16, Bruce 72) (Bricker et al. 2003b, Whatmore et al. 2006, Le Fleche et al. 2006). Analyses and dendogram preparation of the isolates from the test panel for the 11-loci VNTR followed that analyses method as outlined by Groussaud et al. (2007). It was found that a greater degree of resolution in separating the B. abortus isolates was achieved when using the 11- loci VNTR than 5-loci SNR (Figure 19).

To demonstrate alternatives to sequencing in determining SNR profile, two methods, fragment analysis and High Resolution Melting (HRM) were examined. Using two loci, BA9941-C1-705121 and BAS19-C1- 703439 HRM was found to reproducibly differentiate strains based on the number of repeats within the particular locus tested (Figure 20). In comparison, fragment analysis using one locus, BMBA2308-C2- 974305 was not as successful in differentiating strains as, there was no consistency in the sizes of supposedly identically sized fragments in different strains (Table 15). However the differences are small and this technique may become suitable for use with appropriate controls. Nevertheless, further work involving the sequencing of this locus in our B. abortus C68 and B. abortus 86/8/59 DNA preparations would be required to determine whether the size difference seen was real (from whole genome sequencing, both strains have ten repeats in this locus).

Discussions and Conclusions: This study has described the isolation and characterisation of SNR loci for the pathogenic bacterium B. abortus. This study represents the first to isolate and characterise SNRs within Brucella and consider their application as additional phylogenetic and population markers within this genus. The rationale behind this study was to determine if SNR markers could add a further degree of molecular resolution when combined with other relatively fast evolving markers such as VNTR loci. This could be particularly advantageous in a brucellosis outbreak where allele scores of SNR’s and VNTR’s could be combined to investigate the molecular epidemiology of B. abortus.

Unfortunately, whilst 16 loci of 7bp and above were initially identified using the specialist software, only 5 polymorphic SNR loci were identified for B. abortus. However, viewing this in light of the well-documented genetic homogeneity of species within this genus it would be unlikely to identify many more SNR loci with

EVID4 Evidence Project Final Report (Rev. 06/11) Page 21 of 28 long repeat motifs. Nevertheless, with the incorporation of more B. abortus field strains from a wider geographic area into the whole genome sequencing project, it may be worthwhile revisiting the remaining 11 loci at a later date to determine if these regions can be polymorphic.

A limited number of variations were identified across the five polymorphic loci for the test panel of B. abortus isolates (41 out of a possible 800 isolates). Looking at a larger sample size from this population may increase the number of variations seen at each locus. If this does not prove to be the case, examination of the flanking sequences of the SNR amplicon for additional SNP’s may prove useful. In this way additional parsimony-informative sites may be identified and included in the analyses as has been achieved for Vibrio cholerae (Danin-Poleg et al. 2007). However, the effect of homoplasy should be taken into consideration if SNP’s within flanking sequences are considered (Stratilo et al. 2006, Kenefic et al. 2008).

From results generated from the SSR Table software it is clear that a significant amount of SNR’s with motif lengths of less than seven base pairs exist within the genome of B. abortus (Table 8). There is therefore a strong possibility that these additional SNR motifs of shorter lengths may represent additional polymorphic loci. Clearly, to identify and test these loci is time consuming and less cost effective. However, recently developed Perl programmes that have been developed which quickly scan genome sequences and identify promising SNR tracts with concomitant PCR design and validation for future data mining (Stratilo et al. 2006, de Maia et al. 2008).

In summary these data demonstrate that SNR loci are variable even within a very localised bacterial population and may therefore be a useful additional tool to examine for local level molecular epidemiology. Surprisingly, at least in the case of the loci examined here, VNTR of larger sequence repeats actually appeared to provide more resolution that the SNRs that conventionally one would expect to evolve extremely rapidly. The rapid advance of genome sequencing in the last few years and the central role of the AHVLA in such studies for Brucella mean that many of these isolates are now in the process of being examined by this approach. This will provide a more comprehensive assessment of the generation of diversity (both SNPs and deletions) in a localised endemic bacterial population and provide a more extensive assessment of how these markers evolve and thus how they can be exploited for molecular epidemiology.

Work looking at alternative methodologies has demonstrated that HRM is a platform that could be utilised in place of sequencing to determine SNR variability. In the event that more polymorphic loci that are capable of resolving current VNTR clusters are identified, this system could be used for future analysis. However, it should be stated that the same pitfalls mentioned in section 3 for the use of HRM for SNP typing are equally valid for using this technique for SNR analysis.

References:

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EVID4 Evidence Project Final Report (Rev. 06/11) Page 24 of 28 (2006) Identification and characterization of variable-number tandem-repeat markers for typing of Brucella spp. J Clin Microbiol 44: 1982-1993 Whatmore, A. M. (2009). Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infect Genet Evol 9: 1168-1184. Winchell, J. M., Wolff, B. J., Tiller, R., Bowen, M. D. , and Hoffmaster, A. R. (2010) Rapid identification and discrimination of Brucella isolates by the use of real-time PCR and high-resolution melt analysis. J Clin Microbiol 48: 697-702 Woo, S. G., Ten, L. N., Park, J., and Lee, M.. (2011) Ochrobactrum daejeonense sp. nov., a nitrate-reducing bacterium isolated from sludge of a leachate treatment plant.Int J Syst Evol Microbiol. 2011 Nov;61: 2690-2696. You-Chun, L., Korol, A. B., Fahima, T., Avigdor, B., and Nevo, E. (2002). Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review. Mol Ecol. 11: 2453-2465. Zerva, L., Bourantas, K., Mitka, S., Kansouzidou, A., and Legakis, N. J. (2001) Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR. J Clin Microbiol.39:1661-1664.

References to published material 9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 25 of 28

Peer- reviewed publications/book chapters generated during course of this project.

1. Sriranganathan, N., Seleem, M.N., Olsen, S.C., Samartino, L.E., Whatmore, A.M., Bricker, B., O’Callaghan, D., Halling, S.M., Crasta, O.R., Wattam, R.A., Purkayastha, A., Sobral, B.W., Snyder, E.E., Williams, K., Yu, G.-X., Ficht, T.A., Roop, C.M., de Figueirido, P., Boyle, S.M., He, O., & Tsolis, R.M. 2009. Chapter 1 – Brucella. In ‘Genome Mapping and Genomes in Animal-Associated Microbes’, Springer-Verlag, Berlin. Nene, V. & Kole, C. (Eds.). ISBN 3540740406.

2. Scholz, H., Hofer, E., Vergnaud, G., Le Fleche, P., Whatmore, A.M., Al Dahouk, S., Pfeffer, M., Krüger, M., Cloeckaert, A. & Tomaso, H. (2009). Isolation of Brucella microti from mandibular lymph nodes of red foxes, Vulpes vulpes, in Lower Austria. Vector Borne Zoonotic Dis. 9:153-156.

3. Schlabritz-Loutsevitch, N., Whatmore, A.M., Quance, C.R., Koylass, M.S., Cummins, L.B., Dick, E.J., Snider, C.L., Capelli, D., Ebersole, J.L., Nathanielsz, P.W., and Hubbard, G.B. (2009). A novel Brucella isolate in association with two cases of still-births in non-human primates – first report. J Med Primatol 38:70-73.

4. Whatmore, A.M. (2009). Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infect Genet Evol. 9:1168-1184.

5. Jenner, D.C., Dassa, E., Whatmore, A.M. and Atkins, H.S. (2009). ATP-binding cassette systems of Brucella. Comp Funct Genomics. Article ID 354649.

6. Scholz, H.C., Nöckler, K., Göllner. C., Bahn, P., Vergnaud, G., Tomaso, H., Al Dahouk, S., Kämpfer, P., Cloeckaert, A., Maquart, M., Zygmunt, M.S., Whatmore, A.M., Pfeffer, M., Huber, B., Busse, H.J. and De, B.K. 2010. Brucella inopinata sp. nov., isolated from a breast implant infection. Int J Syst Evol Microbiol 60:801-808.

7. Koylass, M.S., King, A.C., Edwards-Smallbone, J., Gopaul K.K., Perrett, L.L. and Whatmore, A.M. 2010. Comparative performance of SNP typing and 'Bruce-ladder' in the discrimination of Brucella suis and Brucella canis. Vet Microbiol 142:450-454.

8. Gopaul, K.K., Sells, J., Bricker, B.J., Crasta, O.R., and Whatmore, A.M. (2010). Rapid and reliable SNP based differentiation of Brucella live vaccine strains from field strains. J Clin Microbiol 48:1461- 1464.

9. Lucero, N.E., Tenenbaum, M., Jacob, N.R., Escobar, G.I., Groussaud, P., and Whatmore, A.M. (2010). Application of VNTR typing to describe familial outbreaks of brucellosis in Argentina. J Med Microbiol 59:648-652.

10. Godfroid, J., Scholz, H.C., Barbier, T., Nicolas, C., Wattiau, P., Fretin, D., Whatmore, A.M., Cloeckaert, A., Blasco, J.M., Moriyon, I., Saegerman, C., Muma, J.B., Al Dahouk, S., Neubauer, H., and Letesson, J.J. (2011). Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Prev Vet Med 102:118-131.

11. Whatmore, A.M and Gopaul, K.K. (2011). Chapter 4: Recent advances in molecular approaches to Brucella diagnostics and epidemiology. In ‘Brucella: Molecular Microbiology and Genomics’. Horizon Press. Lopez-Goni, I. and O’Callaghan, D. (Eds). pp. 57-88.

12. Godfroid, J., Nymo, I., Tryland, M., Cloeckaert, A., Jauniaux, T, Whatmore, A., Moreno, E and Foster, G. (2012). Marine mammal brucellosis. In ‘Conservation Medicine: Applied Cases of Ecological Health’. Alonso Aguirre, Richard S. Ostfeld and Peter Daszak (Eds) ,Oxford University Press, In press.

13. Wattiau, P., Whatmore, A.M., Van Hessche, M., Godfroid, J., and Fretin, D. (2011). Nucleotide polymorphism-based single-tube test for robust molecular identification of all currently described Brucella species. Appl Environ Microbiol 77: 6674-6679.

14. Kämpfer P, Scholz HC, Lodders N, Loncaric I, Whatmore A, Busse HJ. Camelimonas abortus sp. nov. isolated from placental tissue of cattle. Int J Syst Evol Microbiol. 2011 Jul 1. [Epub ahead of print]

15. O’Callaghan, D and Whatmore, A.M. (2011). Brucella genomics as we enter the multi-genome era. Brief Funct Genomics 10: 334-341.

EVID4 Evidence Project Final Report (Rev. 06/11) Page 26 of 28 16. Cloeckaert, A., Bernardet, N., Koylass, M.S., Whatmore, A.M., and Zygmunt, M.S. (2011). Novel IS711 chromosomal location useful for identification of marine mammal Brucella genotype ST27, which is associated with zoonotic infection. J Clin Microbiol 49:3954-3959.

17. Al Dahouk, S. , Hofer, E., Tomaso, H., Vergnaud, G., Le Flèche, P, Cloeckaert, A., Koylass, M. S., Whatmore, A. M, Nöckler, K., Scholz, H. C.. (2012). The genetically homologous species Brucella microti reveals intra-species biodiversity. Appl Environ Microbiol 78: 1534-1543

18. Eisenberg T, Hamann HP, Kaim U, Schlez K, Seeger H, Schauerte N, Melzer F, Tomaso H, Scholz HC, Koylass MS, Whatmore AM, Zschöck M. Isolation of potentially novel Brucella spp. from frogs. Appl Environ Microbiol. 2012 Mar 9. [Epub aheadof print]

Major Presentations

1. Whatmore, A. Brucellosis: New Tools to understand an old disease. From animals to humans: A multi- disciplinary approach to the study of zoonotic disease, University of Surrey, UK, 21st-22nd May, 2009 (Invited Speaker)

2. Whatmore, A. The application of multilocus sequencing to characterisation of the genus Brucella..Medical Biodefence Conference 2009, Bundeswehr Institute of Microbiology, Munich, Germany , 20th-22nd October, 2009 (Invited Speaker)

3. Wakeley, P., North. S., and Gopaul, K. Isothermal Amplification Methods, InterAct Isothermal Genetic Testing workshop, First Intuition, Conway Mews, London, UK. 27th January, 2010 (Oral presentation)

4. Gopaul, K., Sells, J., Brew, S., Whatmore, A. M., and Stack, J. The development of a rapid and economical diagnostic for the detection of Brucella in milk. 416th Scientific Meeting of the Veterinary Research Club, AHVLA Weybridge, UK, 30th April, 2010 (Oral presentation)

5. Sobrai, B., Wattam, A., Williams, K., Mane, S., Foster, G., Shukla, M, Dickerman, A., Scott, M., Boyle, S., O’Callaghan, D., and Whatmore, A. Comparative Genomics of Brucella spp., 63rd Annual Brucellosis Research Conference, Chicago, Illinois, USA 4th-5th December, 2010 (Oral presentation)

6. Whatmore, A. Brucellosis activities at AHVLA- contributions to the changing face of Brucellaceae. Federal Institute for Risk Assessment (BfR), Berlin, Germany, 23rd November, 2011. Invited speaker.

7. Whatmore, A., Dawson, C., Perrett, L., Koylass, M., Groussaud, P., Hunter, G., Shankster, S., Brew, S., King, A., Muchowski, J., Quance, C., and Harris, B. Molecular and phenotypic characterization of North American Isolates of Brucella from marine mammals and comparisons with European Isolates. Brucellosis 2011, International Research Conference including the 64th Brucellosis Research Conference, Buenos Aires, Argentina, 21st-23rd September, 2011 (Oral presentation).

8. Whatmore, A. NRL UK- An update on the application of multilocus sequence typing to Brucella. 4th Annual Workshop of the EU NRLs for brucellosis, Sliema, Malta, 12th-13th May, 2011 (Oral presentation)

9. Randall, L. P., Mappley, L., Lemma, F., Chrchward, C. P., Ayling, R. A., Brew, S., Stubberfield, E., Fenner, J., Smith, L., La Ragione, R., Fielder, M., Whatmore, A. M., and Coldham, N. Speciation of Brachyspira, Brucella, Leptospira and Mycoplasma by MALDI-Tof, 21st European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 27th International Congress of Chemotherapy, Milan, Italy, 7th -10th May, 2011 (Oral presentation)

10. Whatmore, A. VNTR typing: Principles and applications in veterinary microbiology. Bacteriology Workshop, AHVLA Sutton Bonington Regional Laboratory, 4th-5th May, 2011 (Oral presentation)

11. Whatmore, A. Genetic relationship of Brucella strains isolated from the African Continent with global isolates based on extensive multilocus sequence analysis. Bacterial Waterborne and Emerging Infectious

EVID4 Evidence Project Final Report (Rev. 06/11) Page 27 of 28 Diseases: Collaborative Research Opportunities in North Africa and the Middle East (NIAID conference), Nicosia, Cyprus, 30th January- 3rd February, 2011 (Invited speaker)

13. Foster, G., Whatmore, A.., Koylass, M., Perrett, L., Dalgleish, M., Reid, B., and Brownlow, A. Smooth and rough strains of Brucella ceti recovered from different tissues in a stranded harbour porpoise (Phocoena phocoena) Brucellosis 2011, International Research Conference including the 64th Brucellosis Research Conference, Buenos Aires, Argentina, 2ist-23rd September, 2011 (Poster presentation)

14. Foster, J. T., Beckstrom-Sternberg, S., Keim, P., O’ Callaghan, D., and Whatmore, A. A global look at Brucella through whole genome sequencing. Brucellosis 2011, International Research Conference including the 64th Brucellosis Research Conference, Buenos Aires, Argentina, 2ist-23rd September, 2011 (Oral presentation)

EVID4 Evidence Project Final Report (Rev. 06/11) Page 28 of 28