2016 Secondment and Technical Reports

a-tskhvediani-secondment-reports-uhoh-2016

williams-secondment-report-eig-2016

zakalashvili-secondment-report-kas-nov-2016

abazashvili-secondment-report-kas-jun-0216

abazashvili-secondment-report-nov-2016

askilashvili_-secondment-report-cardiff-2016

bolkvadze_-secpndment-report-cardiff-2016

brown-secondment-report-eig-2016

l-leshkasheli-secondment-report-to-uhoh-2016

martashvili-secondment-report-kas-nov-2016

martashvili-secondment-report-kas-jun-2016

rhiannon-probert-secondment-report-eig-2016

Technical reports

d4-17-month-30-internal-technical-report

d4-21-month-36-internal-technical-report-completed

milestone-7-progress-report-2016

modebadze-secondment-report-kas-jun-2016

m8-report-on-the-efficacy-of-field-trials-progress-report

m9-report-on-the-genetic-diversity-of-banthacis-isolates-in-the-region-progress-report

2015 Secondment Updates

Summary and technical reports of 2015 secondments

Technical Report Mariam Zakalashvili form NCDC to KAU 2015

NCDC ro KAU 03-07-2015-report-of-research-activity-nino-modebadze

Bolotin and Koshheliev from NSC IECVM to UHOH 2015

Beyer from UHOH to NCDC Sept 2015

Emanuele Campese to NCDC 2015

Baillie from CU to NCDC 03-07-2015

Coskun from KAU to NCDC 2015 (1)

Technical Report of trip in Cardiff-Adam Kotorashvili DEcember 14-27[1]

Technical Report of trip in Cardiff-Adam Kotorashvili

Report of Research Activity – Lia Askilashvili

Report of Research Activity – Darejan Bolkvadze

Technical ReportE.Khmaladze from NCDC to WiHiE 2015

03-07-2015-report-of-research-activity-from-eig-to-izspb-natia-skhirtladze-2

Technical Report T. Imnadze to NCDC from WiHie 2015

Technical Report K. Sidamonidze from NCDC to WiHiE 2015

Secondment report Beyer

Secondment reporrt Rohmer from UHOH to EIG 2015

Report AEDNet Project- Emanuele Campese

Report Buyuk and Akca KAU to EIG 2015

Technical Report of trip in Cardiff-Adam Kotorashvili-1

Anthrax decontamination Ukrainian

 

Abstracts and Posters

Recent Conference abstracts and Posters

Investigation on Bacillus anthracis circulating in Bangladesh through the analysis of soil samples in areas at risk and imported animal feed –  Anthrax in Bangladesh 

Anthrax Environmental Decontamination Network Abstract Abstract Anthrax ADEnet-4 and Poster Poster_Chakvetadze_NCDC_BACT_2015_20151020-2

Modern Turkish approaches to soils ‘decontamination from anthrax’ agent with attention to Ukraine. Biosafety and Biosecurity 2015. Ukraine paper

 

 

AEDnet update; Tbilisi Workshop 2015 presentations

Below are the recent presentations giving updates from each group working within the AEDnet project. 

  1. Cardiff University, Wales, UK- CU presentation
  2. NCDC, Georgia – AEDnet-Anthrax NCDC presentation-LM
  3. Bundeswher Institute for Microbiology, Germany – Bundeswher-Ecology of Bacillus anthracis
  4. National Centre for Anthrax Research, Italy – IZPSB
  5. University of Kafkas, Turkey –Kafkas presentation
  6. Eliava Institute, Georgia – Kutateladze (Eliava Institute)
  7. University of Hohenheim, Germany – Tiflis 2015 
  8. Military Institute of Hygiene and Epidemiology, Poland – WiHIE

Milestone 7 Report

Romuald Gryko – Military Institute of Hygiene and Epidemiology – Poland

Milestone 7:  A germanant/phage lysin decontamination formulation suitable for field trials- (WiHE)- progress report April 2015

Colleagues at the Military Institute of Hygiene and Epidemiology  in Poland (WiHE) are working to identify bacteriophages which contain lysins capable to targeting the cell wall of B.anthracis.   They  first isolated bacteriophages from environmental samples with activity against B.anthracis 34F2.

Environmental isolation method

In brief, 20 gram of  environmental samples (agricultural soils, sewages) was added to 40ml of Tryptose Soy Infusion  supplemented with 5 mM MgSO4  (TSIMg).   The suspension was then inoculated with  100 µl of a  24 h. culture of B. anthracis 34F2  and incubation   for 24 hr at 370C. Following incubation  the sample was treated with chloroform and centrifuged at 4000rpm for 30 min (2952 x g).

To determine if any phages were present  sample supernatant was dropped onto the surface of a  Tryptose Soy Infusion Agar  plate supplemented with 5 mM MgSO4  (TSAMg)  in which the  upper layer of agar had been inoculated  with  B. anthracis 34F2.

Following overnight incubation at 370C clear zones of inhibition  were  harvested from the agar and  placed in 8 ml of TSIMg.  Following agitation the solution was treated with chloroform and  centrifuged after which the  supernatants were diluted and  plated onto a  bilayer TSAMg  as described above. Following overnight incubation clear plaques were again harvested and the process repeated on a further four occasions. The host range of the resulting phages and there lysins were determined using the methods  described below.

Method  to determine the bacterial host range

A 5 ml of suspension of  test cells of Bacillus spp. strains  in 0,7% TSAMg was laid over the surface of 15ml of TSAMg. Once the agar had solidified tested phage lysates were dropped (about 25 µl) on the surface of plates and  were incubated for 24 h in 370C and zones of lysis were noted. The gamma and  Fah phages were included as positive  controls.

Screening method  to determine of phage produced  lysins

A 5 ml  1% agarose overlay contained heat inactivated cultures of the test bacteria.  This was  achieved by  harvesting the growth from 50ml of a 24 hr culture of the test bacteria  at 370C and resuspending it in 5ml of water. This was then mixed with 10 ml of  1.5% agarose and boiled for 15 mins.  The agarose was then overlaid onto  TSAMg and after solidifing 6 mm diameter holes were cut and the bottom of each hole was sealed with a drop molten TSAMg.  For each hole 25 µl of 24h liquid culture of B. anthracis 34F2 (host strain for all phages) were dropped. After 3 h of incubation in 370C lysates of tested phages (25 µl) in the rate 1.8 – 3.2 x 106pfu were dropped to individual holes. Dishes were incubated for 24 – 48 h in 370C and zones of brightening around holes were noted. The gamma and  Fah phages were included as positive  controls.

In the case of poorly visible of the zones  plates were dipped on 15 min in the water containing 1 – 5 µg/ml of active  chlorine. 

Results

An example of the results achieved using this method can be seen in figure 1 and a summary of all of the results is presented in table 1.  As expected the control phages, gamma and Fah  were only able to infect the B.anthracis isolates include in this study. They also  produced lysins which  were specific to B.anthracis.    While the majority  of environmentally isolated bacteriophages were highly specific for B.anthracis  there were exception such B.cereus ATCC 23261 and B.sp. Ba 813. We saw a different picture for the lysins and observed that a number of phages produced lysins with activity against B.anthracis  other members of the Bacillus test panel.

figure 1

 

Figure 1. A example of the activity of phage lysates on the lysis of dead cells of B.thuringiensis T7-030 and Bacillus sp. B.a. 813. #7.  K1- is a negative control in that it only contains  B.anthracis  34F2 and no phage

Table 1. The activity of  15 environmentally isolates bacteriophages and two control bacteriophages (gamma and Fah)  propagated in B.anthracis  4342 against living and dead cells of test bacteria.  A= activity of phages against living bacteria, B= activity of lysins against dead cells.

Strains   Bacteriophages
 

F3

 

F4 F7 F8 F9 F12 F13 F14 F15 F16 F17 F18 F19 F20 F22 Gamma Fah  
   B. anthracis

   1584

A + + + + + + + + + + + + + + + + +  
B + + + + + + + + + + + + + + + + +  
   B. anthracis

   211

A + + + + + + + + + + + + + + + + +  
B + + + + + + + + + + + + + + + + +  
   B. anthracis

   SL 1809

A + + + + + + + + + + + + + + + + +  
B + + + + + + + + + + + + + + + + +  
   B. anthracis

   Sterne 34F2

A + + + + + + + + + + + + + + + + +  
B + + + + + + + + + + + + + + + + +  
   B. thuringiensis

   ATCC 33679

A  
B  
   B. thuringiensis

   ATCC 35646

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. thuringiensis

   ATCC 10792 T

A  
B +

 

+ + + + + + + + _- _- nd Nd nd nd  
   B. thuringiensis

   T7 – 128

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. thuringiensis

   T7 – 019

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   ATCC 19637

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   UW 85

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   F 17202

A  
B + + + + + + + + + + + +/- + +/- +  
   B. cereus

   ATCC 14579 T

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   F 16959

   A  
B +

 

+ + + + + + + + nd Nd nd nd  
   B. cereus

   F – 1728 S

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. cereus

   ATCC 23261

A + + + +  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. mycoides

  ATCC 21929

A  
B +

 

+ + + + + + + + + + +/- + +/- +  
   B. subtilis

   ATCC 6633

A  
B +

 

+ + + + + + + + + + nd Nd nd nd  
   B. sp. Ba 813

   15 (11614-2)

 

A nd Nd nd nd  
B + + + + + + + + + + + nd Nd nd nd  
   B.sp. Ba 813 #

   6 (I/2)

A + + + + nd Nd nd nd  
B +

 

+ + + + + + + + + + nd Nd nd nd  

To further understand the nature of the environmental bacteriophages three isolates, F9, F15 and F17 were selected for further characterization by electron microscopy, PFGE and restriction enzyme digestion. Analysis of the  images  of F15 and F17  (figures 2 and 3) revealed that these phages belonged to the Siphoviride family. The morphology of F9 has yet to be determined.

                        Figure 2. An electron Microscopy image of the F15  bacteriophage

Measurement  of the phage revealed a head  size of 36.9-38nm and a tail of  172 nm.

                     Figure 3. An electron Microscopy image of the F17  bacteriophage

Measurement  of the phage revealed a head size of 43.3-41nm and a tail of 154.35 nm.

To further characterize these phages their  DNA  was extracted and  subjected to  pulse field gel electrophoresis (PFGE) and to restriction enzyme digestion.

Pulsed field gel electrophoresis (PFGE)

Following dialysis a 50 µl  suspension of phage was mixed with 50 µl of 2 % (w/v) plug agarose (CleanCut, Bio-Rad) and dispended into a plug mold were it was left to solidified.  The plug was then  removed from the  mold,  suspended in phage lysis buffer (50mM EDTA, 50mM Tris pH 8.0, 1% w/v SDS and 1mg/ml of proteinase K) and incubated overnight at 54 ºC, with shaking.  The digestion buffer was then decanted, and the samples were washed three times using TE buffer (10mM Tris, 1 mM EDTA, pH 8)  after which it was incubated in TE buffer (4 ºC, 1 h).   The plug was  then placed into  a well cut into a 1% Pulsed Field Certified agarose (Bio-Rad)  made using  0.5 % TBE. Lambda Ladder PFG Markers (New England Biolab) were used as the MW standards. The samples were electrophoresed using a CHEFDRII System (Bio-Rad) at 6Vcm/1 with pulse ramps from 1 to 25 s  for 20 h at 14 ºC in 0.5 % TBE buffer.   Following electrophoresis, nucleic acids were stained with ethidium bromide (1 µg/ml) for 30 min [Clokie MRJ, Kropinski AM (2009) Bacteriophages: Methods and Protocols.Volume 2: Molecular and Applied Aspects. Humana Press, Totowa, NJ.]

eee

Figure 4. The DNA extracted from the phages F9, F15 and F17 were subjected to pulse field gel electrophoresis (PFGE)

Isolation of the phage DNA for restriction digestion

Following incubation the phage infected culture was treated with chloroform    and then centrifugation at 25 000 rpm ( max 115900 x g) for 2 hours at 4 ºC in  Sorvall T-890 rotor the   phage pellet was  suspended in SM buffer and  sodium dodecyl sulfate (SDS) (final concentration of 0.5 %) and proteinase K (final concentration 50 µg/ml) were  then added. The  suspension was then incubated at 55 °C for 1 hour.  An equal volume of phenol-chloroform (1:1) was then added to remove the proteinaceous material. This  extraction was repeated twice, and the DNA was precipitated according to the standard procedures [Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]   Restriction enzyme digestions of the phage DNA were carried out according to the instructions provided by the suppliers. Restriction endonucleases tested for digestibility of the F9,F15 and F17 phages  DNA were: EcoRV, HaeIII and Bsp. After the enzymatic digestion electrophoresis of the samples in 0.4 % (w/v) agarose with addition of ethidium bromide (1 µg/ml) was performed.

As can be seen in Figure 5 the restriction digestion profiles on all three phages differed suggesting  that they were unrelated phages.

Picture5.png

 

AEDnet FP7 project management meeting, NCDC Tbilisi, Georgia, 22nd April

Present
Les Baillie (CU)
Mzia (EIG)
Lile (NCDC)
Mitat Sahin (KAU)

Agenda items
1. Missed secondments from 2014
2. Moving secondments from one institution to another
3. Secondment reports
4. Project related presentations
5. Progress on project deliverables
6. Project workshop in Tbilisi, Sept 2015
7. Project website
8. Any other business.

Notes
1. Missed secondments from 2014
During the first year of the project we only completed 16 of the proposed 50 secondments. We need to reschedule the missed secondments over the remaining period of the project and send a revise a copy of the plan to our EU project officer. Lile and Mzia will send there plans to reschedule the secondments from there institutions which were not completed.

2. Moving secondments from one institution to another.

1. A request was made by Lile to investigate the possibility of moving secondment from NCDC to KAU (3 months in year 1 and 1 month in year 2) to another EU partner institution within the project. Les will contact the EU project officer to determine if this is possible as it will involve changes to individual budgets.

2. There was also a request to determine if a secondment from UHOH to NCDC planned for year 1 which could not be completed by UHOH could be reallocated to NCDC. Les will contact the EU project officer to determine if this is possible.

3. Jason Farlow has not been able to resolve his position at ISU and as consequence it has been proposed that we reassign all of the secondment that will be going to ISU to NCDC. Les will contact the EU project officer. We will also have to reassign any secondments which we planned to take place from ISU.

4. The group from IZPSB are concerned about sending there start to Ukraine and have request that there secondments be shifted from the Ukrainian institution to the Intitution in Georgia. I will contact the EU project officer to see if this is possible.

5. The group in IZSPB would like to break a one month secondment to Georgia into two blocks of 15 days and to send two researchers to Georgia at the same time, both for 15 days. The total funding for this secondment would add up to 1 month as budgeted. I will approach the EU project officer to determine if this is possible.

3.Secondment reports- At the completion of the secondment the secondee must write a short report of there visit describing the new skills and/or knowledge that they have gained from the visit. Please include pictures if possible. Send the report to Cardiff. Please also send copies of airline tickets as confirmation of the secondment dates. Attached is an example of a recent secondment report for a visit to IZSPB. Thesae reports will be posted on the website.

4.Project related presentations- Please send details of any presentations/ publication which have arisen from the project to Cardiff so that they can be included in the progress report to the EU and posted on the website. Also for future presentations please ensure that the Marie Curie and EU logos are included.

5. Progress on project deliverables for 2015. We have the following deliverables scheduled for the end of 2015. The lead institution for each activity is shown in brackets.
i) Training to characterise phages from field experiments (EIG)

ii) Training in the cloning and expression of B.anthracis specific lysins (WiHE)

iii) Identification and optimization of site specific spore germinants (CU)

iv) Full characterization anthrax spore contaminated sites in Georgia (NCDC), Turkey (KAU) and Ukraine (NSC IECVM)

v) A germinant/ phage decontamination formulation suitable for field trials- (CU)

vi) A germinant/phage lysin decontamination formulation suitable for field trials- (WiHE)

We are making good progress in all of these areas and we call review progress during our meeting in Tbilisi in Sept 2015.
6. Project workshop to be held in Tbilisi towards the end of Sept 2015, date and details to be finalised. – To reduce organizational costs this will be a combined meeting of two anthrax related projects both of which involve partners in Tbilisi.- Outline programme is attached- we need to agree presentations for the AEDnet session. The suggestion is for each institutional lead to give an overview as to there activities and then for individual young researchers who have been on secondment to give a brief presentation of their experience an the knowledge they have gained.

7. Project website.- https://aednetproject.wordpress.com/
Project deliverables, reports and papers arising from the project will be posted on the website as they are generated. Please check the partner institution section and confirm that you are happy with the text which as been posted, if not please send us new text.

7. Any other business
The next meeting will be held in Tbilisi in Sept before or after the workshop on the 29th Sept 2015

PIRSES-GA-2013-612309 AEDnet Project Mid-Term Report December 2014

ANNEX 1: MID-TERM REPORT

Marie Curie Actionsseventh framework programme

Marie Curie Actions – International Fellowships

Project n°: _PIRSES-GA-2013-612309________
Project Acronym: ___AEDnet_________
Project Full Name: __Anthrax Environmental Decontamination Network___
Marie Curie Actions
IRSES Mid-term Report
Period covered: from ___01.01.14_________ to ____31.12.16____
Period number: _______1_____
Start date of project: _____1st Jan 2014_______
Project coordinator name: ___Prof Les Baillie_________
Project coordinator organisation name: __Cardiff University UK__________
Date of preparation: ____24th Dec 2014________
Date of submission (SESAM): ___24th Dec 2014_________
Duration: ____12 months________
Version: _____1______

1. GENERAL PROGRESS OF THE PROJECT
Please indicate if the project:
a) has fully achieved its objectives and technical goals for the period;
b) has achieved most of its objectives and technical goals for the period with relatively minor deviations;
c) has failed to achieve critical objectives and/or is not at all on schedule.

If you answered b) or c) please include a detailed description of the modifications in the report
While the project has achieved all of its deliverables, we have not completed all of the planned secondments. On reflection, front loading the distribution of the secondments to the first 12 months (50 months in total) of the project was unrealistic. We were not helped by the fact that it took several months to complete the contractual arrangements between the project coordinator and the individual partners. It also took some time for all of the partners to fully understand the process and their respective responsibilities. Finally the on going problems in Ukraine meant that only 5 of the planned 13 secondments from that institution took place.
It is our intention to re-arrange the missed secondments so that they are completed during the lifetime of the project. A summary of the planned and completed secondments is shown in the attached GNATT chart.

Qualitative indicators of progress and success in line with workplan and milestones (description of progress towards the milestones and deliverables)
The project has met all but one of its 6 deliverables within the 12 month period and the 6th deliverable, the Month 12 meeting of the Project Management Group will be completed early in the new year.

D1.1, Month 12: A standardised method for the isolation of B.anthracis specific bacteriophages- (see attached).

D4.1, Month 3: Meeting of the Project Management Group
Held at the project meeting at the Anthrax workshop in Cardiff in March 2014. At this meeting, the contractual arrangements between the partners and Cardiff University were explained, as was the process by which funds would be transmitted to all the institutes. The reporting requirements were also explained. Due to the unrest in Ukraine, our colleagues from NSC IECVM were unable to attend the meeting in Cardiff. Information with transmitted to them via email.

We held a second informal project meeting in Tbilisi, Georgia in June 2104, to coincide with a congress on Chemical, Biological, Radiological, Nuclear and explosives (CBRNe) Science and Consequence Management which was attended by a number of the partners.

D4.2, Month 6: First Internal technical report- see attached

D4.3, Month 6: Establish project website- A project website was established and can be viewed at https://aednetproject.wordpress.com/ We are still waiting for all of the partners to provide information about their institutions which we will post on the site.
D4.4. Month 12: Internal technical report- see attached

D4.5. Month 12: Meeting of the Project Management Group- This meeting is yet to be held and will be hosted in the new year by Cardiff via Skype. The meeting will address the problem of identifying staff willing to go on secondment and will identify how the secondments which were not completed in the first 12 months are reassigned across the rest of the project period.

D4.6, Month 12: Preparation of mid- term review report to the EU Commission
Completed and submitted to EU- completed

2. PROJECT ACHIEVEMENTS
Scientific highlights and research achievements
1. With support from researchers from CU, KAU and EIG we developed A standardised method for the isolation of B.anthracis-specific bacteriophages

2. We have isolated novel B.anthracis-specific bacteriophages which will be assessed for their suitability as potential decontamination agents

3. As a consequence of the interaction between KAU and NCDC, and with support from CU, we have established a new project which started in Nov 2014 and is funded by the US Biological Engagement Program to genetically type the pathogenic strains of B.anthracis which cause anthrax in Georgia and Turkey. The project will reinforce the link formed as part of this EU funded project and will provide a foundation for future joint research efforts. The project aims to provide short-term training by WRAIR and NCDC Lugar Center collaborators to Turkish personnel in the areas of bacterial DNA extraction and molecular analysis of anthrax isolates.

Transfer of knowledge and Training activities (workshops…)
Secondment activities
WP1- Development and characterisation of phage-based decontamination agents: Nino Modebadze, Ketevan Sidamonidze from NCDC visited the group at CU for 1 month for training in the isolation and characterization of B.anthracis specific bacteriophages (task 1.2). They isolated phages form soil and observed the effect of different phages on a vaccine strain of B.anthracis. As part of this process, they employed a number of different methods which included enrichment from soil, propagation of phage in liquid broth, determination of bacteriophage titre and purification of bacteriophage.
Ms Aleksandra Nakonieczna, from WiHe visited the group at EIG for 2 months for training in the isolation and characterisation of B.anthracis-specific bacteriophages (task 1.1). During the visit she worked in a Molecular Biology laboratory led by Dr. Mzia Kutateladze. The aim of this exchange was to share and extend the knowledge about bacteriophages, including B. anthracis phages. During the secondment she took part in bacteriophage isolation from sewage samples, purification and propagation of bacteriophages, phage DNA isolation, DNA restriction analysis, PFGE analysis, phage characterization assays (adsorbtion rate, one step growth experiments, phage host range), phage adaptation according to the Appelman’s method, media preparation, and API testing.

WP2- Development of methods to enhance the activity and stability of sporicidal agents: Dr Ekaterine Tevdoradze from EIG visited the group at CU for 1 month for training in how to develop a standardised soil microcosm with which to assess germinant efficacy (Task 2.1). Studies suggested that we need to optimise the ratio of phage to target bacteria to achieve effective killing of the pathogen in soil.
Ekaterine Khmaladze, Mariam Zakalashv from NCDC visited the group at CU for 1 month for training in how to develop a standardised soil microcosm with which to assess germinant efficacy (Task 2.1). The were introduced to the method that shows the effect of germinants alone on spore reduction in soil and the principle of adding germinants to soil to trigger the conversion of spores to vegetative form. They observed that B. anthracis failed to persist in vegetative form after it had been introduced to germinate.
WP3- Field trials, efficacy testing and assessment of environmental impact
Dr Andriy Buzun from NSC IECVM visited the group at KAU for 1 month to receive training in the identification and characterisation of potential anthrax spore decontamination test sites (task 3.1). He visited a number of potential decontamination sites in Turkey with a view to identifying similar sites in Ukraine.

Dr Fatih Buyuk, Ms Aliye Gulmez and Ms Elif Tazegul from KAU visited the group at NCDC for 1 month to receive training in characterization of the genetic diversity of B.anthracis isolates (task 3.2) and safely working with B.anthracis biosafety 2 and 3 (task 3.5). During the visit they spent two weeks in the Genome Laboratory where they were introduced to sequencing methods and the platform which is used at NCDC to genotype bacterial isolates. The final two weeks were spent visiting the biosafetly level 2 and 3 facilities for training in the various aspects of operating safely in such faciltities.

Dr Leila Kvachadze from EIG visited the group at CU for 1 month for training in how to work safely in a UK containment level 2 laboratory (task 3.5).

The following researchers Dr Anatoliy Paliy,Dr Olena Kolchyk, Dr Oleksandr Rula and Mr Mykola Kalashnyk from NSC IECVM visited IZSPB Italy for one month for training in the following topics: characterization of the genetic diversity of B. anthracis (genotyping by 15 loci Multi Locus VNTR Analysis -MLVA) isolates from environmental samples via G.A.B.R.I. technique (task 3.2) and training on safely working with B. anthracis in laboratory Biosafety Level 3. (task 3.5).

Dissemination of results (conferences, publications)
To date, we have not disseminated the results of our work outside of the network. It is our intention to disseminate our results and activities to the wider scientific community and to advertise our activities on the project website at https://aednetproject.wordpress.com/
3. PROJECT MANAGEMENT
Overview of the activities carried out by the partnership, identification of problems encountered and corrective action taken.
This project involves the co-ordination of 9 institutions, five based in EU member or associate states, three based on Georgia and one based in Ukraine. Co-ordination and control of such a disparate group is a challenge. The project coordinator has been greatly assisted by the efforts of a part time project administrator who in addition to coordinating details of secondments, is also responsible for managing the project website.

We held a kickoff meeting of the Project Management Group in Cardiff in march 2014. A second informal project meeting was held in Tbilisi, Georgia in June 2104 and was attended by representatives of the majority of the groups. Our 12 month meeting is yet to be held and will be hosted in the new year by Cardiff via Skype. The meeting will address the problem of identifying staff willing to go on secondment and will identify how the secondments which were not completed in the first 12 months will be reassigned across the rest of the project period.
4. ADDITIONAL INFORMATION
Please indicate any additional information, which may be considered useful to assess the work done during the reporting period. The socio-economic aspects of the project may be addressed in this section.
Ethical issues
As stated in the proposal, no exchange of viable strains of pathogens between laboratories and between countries has or will take place (monitoring safety issues).

Also there have been no anthrax cases in the environment of the research sites.

All of the research which has been undertaken as part of this project complies with the ethical standards and guidelines of the European Union.

There is no intension to transport samples containing B.anthracis from Georgia or the Ukraine.

Statements from members of the external, independent Advisory Board stating that the project has not created any concerns with regards to bioterrorism, misuse and dual use are attached.
Appendix A: D4.2. Month 6: First Internal technical report- delayed- will be same as phage report
Appendix B: D4.4. Month 12: Internal technical report- summary of project reports
Visits to date
Attachment: Deliverable 1.1.A standardised method for the isolation of B.anthracis-specific bacteriophages

Person in charge of the project for the beneficiary/consortium
Name: Les Baillie
Date 24th Dec 2014
Signature

Appendix A: D4.2. Month 6: First Internal technical report- delayed- will be same as phage report

For administrative reason there was a delay in completing the contractual arrangements between the institution of the project coordinator and the individual partners. It also took some time for all of the partners to fully understand the process and their respective responsibilities. As a consequence, there was a delay in the start of the secondments.

In the first six month there were secondments between EIG and Cardiff and KAU and NCDC.

Dr Ekaterine Tevdoradze from EIG visited the group at CU for 1 month for training in how to develop a standardised soil microcosm with which to assess germinant efficacy (Task 2.1). Studies suggested that we need to optimise the ratio of phage to target bacteria to achieve efficient killing of the pathogen in soil.

Dr Leila Kvachadze from EIG visited the group at CU for 1 month for training in how to work safely in a UK containment level 2 laboratory (task 3.5).

Dr Fatih Buyuk, Ms Aliye Gulmez and Ms Elif Tazegul from KAU visited the group at NCDC for 1 month to receive training in characterization of the genetic diversity of B.anthracis isolates (task 3.2) and safely working with B.anthracis biosafety 2 and 3 (task 3.5). During the visit they spent two weeks in the Genome Laboratory were they were introduced to sequencing methods and the platform which is used at NCDC to genotype bacterial isolates. The final two weeks were spent visiting the biosafetly level 2 and 3 facilities for training in the various aspects of operating safely in such faciltities.

Appendix B: D4.4. Month 12: Internal technical report- summary of project reports visits to date

WP1- Development and characterisation of phage based decontamination agents

Isolation of B.anthracis specific phages using the Sterne vaccine strain of B.anthracis

1. The group at CU with help from colleagues from EIG and NCDC isolated novel B.anthracis specific phages from environmental samples and determine there biological activity. Electron microscopy images of the phages can be seen below.

micro 1micro 2micro 3
2. Ms Aleksandra Nakonieczna, from WiHe with support form the the group at EIG isolated and characterisation B.anthracis specific bacteriophages

Researchers from NCDC received training in the isolation and characterisation of B.anthracis specific bacteriophages (task 1.2) and as a consequence should be better equipped to isolate bacteriophages from there own B.anthracis contaminated sites.
WP2- Develop of methods to enhance the activity and stability of sporicidal agents

Researchers from Georgia have received training in how to set up and run soil microscosms. They were introduced to the method that shows the effect of germinants on spore reduction in soil and the principle of adding germinants to soil to trigger the conversion of spores to vegetative form. They observed that B. anthracis failed to persist in vegetative form after it had been introduced to germinate.
WP3- Field trials, efficacy testing and assessment of environmental impact
Dr Andriy Buzun from NSC IECVM visited the group at KAU for 1 month to receive training in the identification and characterisation of potential anthrax spore decontamination test sites with a view to identifying similar sites in Ukraine.

Researchers from KAU in Turkey visited the group at NCDC in Georgia for training in the use of molecular methods to characterize the genetic diversity of B.anthracis. Researchers from NSC IECVM in Ukriane underwent similar molecular characterization training at IZSPB Italy in the method of genotyping using 15 loci Multi Locus VNTR Analysis –MLVA.

Researchers from NCDC, EIG, KAU and NSC IECVM received training in how to operate safely 8in Biosafety level 2 and 3 facilities

Deliverable 1 1 A standardised method for the isolation of B anthracis specific bacteriophages

Introduction: One of the aims of this project is to harmonise and standardize methods across the network so that results obtained from different laboratories can be compared. We have developed a B.anthracis specific bacteriophage isolation method for the recovery of phages from B.anthracis contaminated animal burial sites (WP1.1). The method shown below is a composite of the approaches developed by researchers from Kafkas University in Turkey (KAU), the Eliava Institute in Georgia (EIG) and Cardiff University in the UK (CU). Training in this combined method has already been provided to researchers from the National Center for Disease Research in Georgia.

Composite B.anthracis specific Bacteriophage isolation and production methods

Bacteriophage isolation from soil
Environmental phage isolations were performed by adding 25 g of soil to 25 ml of TSB followed by the addition of 10 ml of mid-log phase culture of the Sterne strain of Bacillus anthracis. The resulting suspension was mixed thoroughly and was incubated overnight at 37°C with shaking at 125 RPM.

Each incubated mixture was centrifuged at 5,000 x g for 20 mins and the supernatant fraction was filtered using syringe filter of pore sizes 0.45 μm and then 0.22 μm. Each filtrate was screened for the presence of bacteriophages against B. anthracis Sterne using the agar method described below.

To recover the phage a plug of agar which included the plaque was taken and
suspended in 500 µl phage buffer to which a small drop of chloroform was added to lyse any remaining bacterial cells. The suspension was vortexed and then left overnight at 4°C to allow any phage particles to diffuse out of the agar. The samples were centrifuged at 5,000 x g for 10 minutes and the supernatant fraction decanted into a sterile microcentrifuge tube.

This solution was then retested for the presence of phage using the agar method and if plaques were seen, the extraction was repeated a minimum of three times to ensure that the phage preparation was pure. If plaques were not seen after an initial round of purification and testing against B. anthracis, no further testing was performed.
Bacteriophage plaque morphology: The plaque morphology of each phage was determined using an agar overlay method. The phage preparation at a range of dilutions was added to overnight cultures of the propagation strain of bacteria in 5ml molten TSA agar. The agar was poured over the surface of a TSA plate and allowed to dry. The plate was then incubated overnight at 37°C for 18 – 24 hours and examined for the presence of plaques.

Bacteriophage propagation
Bacteriophage propagation in broth was performed by inoculating fresh TSB with 50 μl of an overnight culture of the bacterium spore and 50 μl bacteriophage which was then incubated at 37°C and 180 RPM overnight. Following overnight incubation Vortex mix. Add a drop of chloroform at a ratio of 1:1000 chloroform: broth and Vortex mix again. Leave the suspension for 5 minutes and then centrifuge at 5,000 x g for 20 minutes to pellet the bacterial debris. The supernatant is then filtered using a double stacked syringe filter (0.45um and 0.22um filter joined together). The resulting phage suspension was stored at 4°C.

Bacteriophage enumeration (drop count method to determine PFU/ml): A version of the Bacteriophage agar propagation method was used.
1. Prepare a fresh culture of the bacteria (inoculate fresh media with 1ml of overnight culture of bacteria – usually will result in exponentially growing bacteria after 6 hr)
2.Prepare 8 eppendorfs filled with 900 μl of sterile PBS/water
3.Serial dilute bacteriophage in the 8 eppendorfs ensuring vortexing between each dilution using 100 μl neat bacteriophage culture
4.Add 0.1ml of exponentially growing bacterial host to 4.9ml of 50% TSA (or TSB with 0.7% agar-agar): pour agar as an overlay onto set TSA plate. Prepare two agar plates in total, allow to dry and divide into four sections.
5.Drop 10 μl of each bacteriophage dilution in triplicates onto one of the four sections.
6.Allow the drops to dry on the plate, then incubate overnight.
7.Phage titre determined as above (multiply by dilution and amount spotted to determine PFU/ml).

Spore production methods
The spore production method is adapted from a method developed by Curtis Thorne (J. Virol. 2(7):657.1968) The method is as follows;
1 Prepare an overnight broth and Incubate broth at 37oC
2 overnight with shaking at 250RPM
3 Remove broths from incubator and centrifuge at 3800 x g for 10 min at 20oC
4 Discard the supernatant and re-suspend in 10 mL Sterile distilled water
(SDW)
5.Innoculate 1 mL of this suspension into 50 mL NBYS broth. Incubate at 28 oC for 3 days with shaking at 250 RPM
6.Following incubation transfer broth to centrifuge tubes and centrifuge at 3800 x g for 15 min at 4oC.
7.Discard supernatant and re-suspend in 30 mL SDW
8.Store in the fridge at 4oC for 3 days
9. Remove suspensions from the fridge and centrifuge at 4oC for 15 min at 3800 x g.
10. Discard the supernatant and re-suspend pellet in 30 mL SDW
11. Heat shock suspensions at 65oC for 30 min
12. A 1 mL aliquot of the suspension will then be removed and perform pre and post heat shock viable counts. Aliquots are also looked at under x100 magnification phase contrast to see phase bright numbers.

D4 4 Month 12- Internal technical report

Progress in the first six months
For administrative reason there was a delay in completing the contractual arrangements between the institution of the project coordinator and the individual partners. It also took some time for all of the partners to fully understand the process and there respective responsibilities. As a consequence there was a delay in the start of the secondments.

In the first six month there were secondments between EIG and Cardiff and KAU and NCDC.

Dr Ekaterine Tevdoradze from EIG visited the group at CU for 1 month for training in how to develop a standardise soil microcosm with which to assess germinant efficacy (Task 2.1). Studies suggested that we need to optimise the ratio of phage to target bacteria to achieve efficacy killing of the pathogen in soil.

Dr Leila Kvachadze from EIG visited the group at CU for 1 month for training in how to work safely in a UK containment level 2 laboratory (task 3.5).

Dr Fatih Buyuk, Ms Aliye Gulmez and Ms Elif Tazegul from KAU visited the group at NCDC for 1 month to receive training in Characterization of the genetic diversity of B.anthracis isolates (task 3.2) and – Safe working with B.anthracis biosafety 2 and 3 (task 3.5). During the visit they spent two weeks in the Genome Laboratory were they were introduced to sequencing methods and the platform which is used at NCDC to genotype bacterial isolates. The final two weeks were spent visiting the biosafetly level 2 and 3 facilities for training in the various aspects of operating safely in such faciltities.

Progress by month 12

WP1- Development and characterisation of phage based decontamination agents

Isolation of B.anthracis specific phages using the Sterne vaccine strain of B.anthracis

1. The group at CU with help from colleagues from EIG and NCDC isolated novel B.anthracis specific phages from environmental samples and determine there biological activity. Electron microscopy images of the phages can be seen below.

micro 1 micro 2 micro 3
2. Ms Aleksandra Nakonieczna, from WiHe with support form the the group at EIG isolated and characterisation B.anthracis specific bacteriophages

Researchers from NCDC received training in the isolation and characterisation of B.anthracis specific bacteriophages (task 1.2) and as a consequence should be better equipped to isolate bacteriophages from there own B.anthracis contaminated sites.
WP2- Develop of methods to enhance the activity and stability of sporicidal agents

Researchers from Georgia have received training in how to set up and run soil microscosms. They were introduced to the method that shows the effect of germinants on spore reduction in soil and the principle of adding germinants to soil to trigger the conversion of spores to vegetative form. They observed that B. anthracis failed to persist in vegetative form after it had been introduced to germinate.
WP3- Field trials, efficacy testing and assessment of environmental impact
Dr Andriy Buzun from NSC IECVM visited the group at KAU for 1 month to receive training in the identification and characterisation of potential anthrax spore decontamination test sites with a view to identifying similar sites in Ukraine.

Researchers from KAU in Turkey visited the group at NCDC in Georgia for training in the use of molecular methods to characterize the genetic diversity of B.anthracis. Researchers from NSC IECVM in Ukriane underwent similar molecular characterization training at IZSPB Italy in the method of genotyping using 15 loci Multi Locus VNTR Analysis –MLVA.

Researchers from NCDC, EIG, KAU and NSC IECVM received training in how to operate safely 8in Biosafety level 2 and 3 facilities

D4 2 Month 6- First Internal technical report

Progress in the first six months

For administrative reason there was a delay in completing the contractual arrangements between the institution of the project coordinator and the individual partners. It also took some time for all of the partners to fully understand the process and their respective responsibilities. As a consequence, there was a delay in the start of the secondments.

In the first six month there were secondments between EIG and Cardiff and KAU and NCDC.

Dr Ekaterine Tevdoradze from EIG visited the group at CU for 1 month for training in how to develop a standardised soil microcosm with which to assess germinant efficacy (Task 2.1). Studies suggested that we need to optimise the ratio of phage to target bacteria to achieve efficient killing of the pathogen in soil.

Dr Leila Kvachadze from EIG visited the group at CU for 1 month for training in how to work safely in a UK containment level 2 laboratory (task 3.5).

Dr Fatih Buyuk, Ms Aliye Gulmez and Ms Elif Tazegul from KAU visited the group at NCDC for 1 month to receive training in characterization of the genetic diversity of B.anthracis isolates (task 3.2) and safely working with B.anthracis biosafety 2 and 3 (task 3.5). During the visit they spent two weeks in the Genome Laboratory were they were introduced to sequencing methods and the platform which is used at NCDC to genotype bacterial isolates. The final two weeks were spent visiting the biosafetly level 2 and 3 facilities for training in the various aspects of operating safely in such faciltities.