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2007 Annual Report

Mission Statement:

ounded in 1994, WINDREF seeks to advance health and environmental development through multidisciplinary

research and education programs. WINDREF strives for program excellence by promoting collaborative relationships between internationally recognized scholars and regional scientists, and by adhering to the highest ethical and academic standards in the design and conduct of research.

WINDREF’s primary goals include: • To provide a scientific resource center capable of coordinating international collaborative research of the highest caliber in the areas of medical and veterinary public health, anthropology, ecology, marine and terrestrial biology, and ethics.

• To provide a first rate academic opportunity to scientists from the Caribbean and around the world offering unique research opportunities to enhance the knowledge and welfare of local and international communities.

• To conduct applied scientific research for the benefit of community and health development at the local, national and international levels.

• To share relevant scientific information with local and international communities.

Cover page legend: Kevin Neill, a WINDREF Research Scientist, taking sand temperatures on Grand Anse Beach, Grenada, to examine the potential for transmission of infective nematode larvae from dogs to humans in this environment. His report starts on page 51.

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WINDREF Research Institute Annual Report 2007 i

/0 TABLE OF CONTENTS

1.0. Director’s Report: The WINDREF Research Institute’s Activities in 2007 (Calum N.L. Macpherson, PhD, DIC) ................................................................................ 1

2.1. WINDREF Research Institute Board of Directors .............................................................. 3

2.2. WINDREF St. Vincent and the Grenadines Board of Directors .......................................... 3

2.3. WINDREF Research Institute Scientific Advisory Board................................................... 3

2.4. WINDREF Research Fellows............................................................................................. 3

2.4.1. Senior Research Fellows......................................................................................... 3

2.4.2. Research Fellows.................................................................................................... 3

2.4.3. New Research Fellows 2007................................................................................... 4

2.5. WINDREF Research Institute Research Scientists ............................................................. 5

2.6. WINDREF Research Institute Administration .................................................................... 5

3.0. WINDREF (USA).............................................................................................................. 5

4.0. WINDREF (UK)................................................................................................................ 5

4.1. WINDREF (UK) Board of Trustees........................................................................ 5

4.2. WINDREF (UK) Administration ............................................................................ 6

5.0. Institutional Review Board (IRB)....................................................................................... 6

6.0. Institutional Animal Care and Use Committee (IACUC) .................................................... 7

7.0. Current Research Projects .................................................................................................. 8

7.1. St. Vincent Streptococcal Project (Trevor P. Noël, MPH)................................................... 8

7.2. Photovoltaic Power Generation Program (Calum N.L. Macpherson, PhD, DIC)................. 9

7.3. Guyana Lymphatic Filariasis Elimination Program (Shamdeo Persaud, MD, Chief Medical Officer and National Epidemiologist, Guyana) ... 10

7.4. Dr. Martin Forde part of CAN$1.6 Million Dollar TeasdaleCorti Grant .......................... 12

7.5. Dr. Martin Forde Completes Survey of Biomedical Waste Management Practices in the OECS Countries....................................................... 13

7.6. Graduate and Undergraduate Research Education Becomes an International Effort (Svetlana Kotelnikova, PhD) ......................................... 15

7.7. Microbial Diversity in the Ironoxidizing Biofilms of Soda Springs in Grenada (Ravinda Naraine, et al.)....................................................... 22

WINDREF Research Institute Annual Report 2007 ii

7.8. Circadian cycle of IronOxidation in Warm Soda Springs in St Andrew’s, Grenada, West Indies (Svetlana Kotelnikova, PhD, et al.)........................... 26

7.9. Do the Microorganisms in the Soda Spring Water Derive Energy from the Oxidation of Manganese? (Maurissa Walker and Teresa Cains) ......................... 31

7.10. Novel Marine Bacteria and their Antagonistic Properties Against Medically Relevant Biofilms (Cynthia Bruno, MSc) ........................................... 33

7.11. Physiological Characterization of Novel Marine Bacterial Species Isolated off Grenada (Jason Johnson et al.).......................................................... 37

7.12. The Ecological Distribution of Viable Indicator Organisms and Associated Health Risks from St. George’s Bay in Grenada (Rakesh Patel)...................... 39

7.13. Characterisation of Marine Spongeassociated Bacteria and Cytotoxic Activity of Sponge Extracts towards Human Cancer Cells (Hillary Crane) ....................... 43

7.14. Antimicrobial Properties of Skin Secretions from Eleutherodactylus johnstonei on Bacteriological Isolates (Jonathan Ashcroft).................. 46

7.15. The Surgical Anatomy of the Occipital Nerves’ Contributions to the Development and Treatment of Occipital Neuralgia (Abraham ElSedfy)......................... 47

7.16. Evaluation and Analysis of Prostate Cancer in Grenada (Megan Kaminskij) .................... 49

7.17. The Public Health Importance of Dogs, Grand Anse, Grenada ......................................... 51

7.18. Research Activities (Theresa McCann) ............................................................................ 53

8.0. Acknowledgements.......................................................................................................... 54

9.0. Grants .............................................................................................................................. 56

10.0. Past, Present, and Future Research Projects...................................................................... 57

10.1. Noncommunicable Diseases ................................................................................ 57

10.2. Infectious Diseases ............................................................................................... 58

10.3. Unique Projects .................................................................................................... 60

11.0. Conferences/Meetings/Workshops Sponsored in Grenada ................................................ 61

12.0. Abstracts/Presentations at International Conferences Invited Plenary/Workshop/Roundtable/Professional Meetings/CME ................................ 61

13.0. Publications ..................................................................................................................... 61

14.0. Reviewers for Journals and Boards................................................................................... 66

15.0. Thesis Defenses ............................................................................................................... 66

16.0. Seminars .......................................................................................................................... 66

17.0 Contact Information ......................................................................................................... 68

WINDREF Research Institute Annual Report 2007 1

1.0. The WINDREF Research Institute’s Activities in 2007

Calum N.L. Macpherson, Director

2007 saw the completion of a number of existing research projects and the development of new programs and projects. We report with sadness that Dr. Bob Sturrock, a longterm Senior Research Fellow, passed away early in 2007. Dr Sturrock worked with WINDREF research scientists on schistosomiasis in St. Lucia and was until 2006 a senior lecturer at the London School of Hygiene and Tropical Medicine. It was fitting that partly as a result of his work on the snails in St. Lucia with us that PAHO decided to use WINDREF\SGU as their venue for a recent meeting to plan for the elimination of schistosomiasis from this region of the world. We have also lost the considerable services of Sir Malcolm (Calum) Macnaughton, who was an inaugural member of the scientific advisory board until his resignation during the year. We would like to take this opportunity to thank him for all his help, enthusiasm and inputs during the 13 years that he served on the board, and wish him and Margaret Anne all the very best.

In recognition of her outstanding contributions to the understanding of the natural history and ecology of the mona monkey (Cercopithicus mona) in Grenada, Dr. Mary Glenn was appointed as a Senior Research Fellow in 2007. Drs. Grant Burgess, Dirk Burkhardt, Francis Fakoya, Martin Forde, Bonnie Rusk, Samina Rutrecht, Esperance Schaffer, and Karin Schiøler and were appointed as Research Fellows. A number of new research scientists were appointed during the year.

The Youth Centre Program, which is directed by Dr Karen Lawson, continued to examine ways to enhance the health and wellbeing of children in Grenada. Discussions were continuously held with the relevant Ministries of the Government. Mrs. Alexandra OtwayNoël was appointed as the assistant director of this program.

The Caribbean Environmental Research Initiative Program, directed by Dr. Svetlana Kotelnikova, continued its excellent and productive research program during the year involving a number of research scientists. An expansion of the CERI program occurred in 2007 with the development of new collaborative initiatives in research and education with two European Universities: the University of Gothenburg, Sweden, and the University of Newcastle, UK.

A new Health Promotion Research Program (HPRP) was developed and will be directed by Dr. Forde, a professor within the Department of Public Health and Preventive Medicine at St. George’s University. Dr. Forde, together with two coprinciple investigators, Dr. Eric Dewailly and Dr. Neela Badrie, and the multinational and organizational team they put together, was one of the teams to successfully secure a Teasdale Corti grant. Their proposal is entitled “Caribbean Eco health Programme: Public and Environmental Health Interactions in Food and Waterborne Illnesses (CEP)”. The CEP has three major components: (1) Research, (2) Regional Capacity Strengthening, and (3) Training. There are three main research areas that will be pursued: 1) Prevalence of persistent organic pollutants in the Caribbean region; 2) Burden of illness study; and 3) Microbial contamination of harvested rainwater.


A new WINDREF program, the Photovoltaic Power Generation Program, was proposed and will be directed by Dr. Dirk Burkhardt, who has a wealth of experience in this field in the Caribbean region. The timing of the introduction of this new initiative is optimal given the global focus on global warming and its potential and current impact on the world today. It is widely accepted that global warming and the resulting environmental changes will be one of the main threats to health in the coming decades. The need to address this threat is the responsibility of all, and any small change that can be made to reduce this potential threat will be important to take.

Collaborative Centre Research Programs and projects continued in Grenada (dengue, prostate cancer, antimicrobial properties of skin secretions from frogs, birth rate study in Grenada and St Vincent, genetic correlation of addictive diseases, cocaine, alcohol and marijuana), St. Vincent and the Grenadines (Rheumatic fever), Guyana (lymphatic filariasis), the Dominican Republic (sanitation and intestinal parasitoses), and the USA (genetic correlation of addictive diseases). Further details of all the ongoing research activities can be found in the WINDREF 2007 Annual Report which is also available on the www.sgu.edu website.

During the year, a number of invited and submitted oral conference presentations were delivered and publications were published in international journals. The 8 th Annual WINDREF lecture was presented by Dr. John David, MD, the Richard Pearson Strong Professor of Immunology and Infectious Diseases, Department of Immunology and Infectious Diseases, Harvard School of Public Health, Harvard University. His lecture was entitled

“Leishmaniasis: A novel approach to control visceral leishmaniasis and another to treat cutaneous leishmaniasis”. Keith Taylor became the second recipient of the Mike Fisher memorial award. This is an annual award presented to an individual who has made an outstanding contribution to health (veterinary, medical or both) in memory of the late Dr. Mike Fisher. It was in Dr. Fisher’s laboratory where the drug ivermectin was first isolated.

Lord Soulsby will host a fundraiser at the House of Lords on June 17 th 2008, which will be focused on the Neglected Infectious Diseases. Professor David Molyneux, the current president of the Royal Society of Tropical Medicine and Hygiene, has agreed to be the speaker at this event.

I would like to take this opportunity to thank everyone, board members, fellows, research scientists, faculty, collaborators, the Minister of Health, the Environment, Social Security and Ecclesiastical Relations, the Hon Ann DavidAntoine, members of the communities in many countries, and donors for their contributions to WINDREF’s activities during 2007.

Participants at the PAHO/WHO Conference on Plans to Eliminate Schistosomiasis in the Caribbean at WINDREF at SGU, 1314 December 2007.

http://www.sgu.edu/


2.1. WINDREF Research Institute Board of Directors

• Lord Soulsby of Swaffham Prior, MRCVS, DVSM, MA, C.Biol., F.I.Biol., DSc(Hon), (President)

• Dr. Calum N.L. Macpherson, PhD, DIC (Vice President)

• Ms. Margaret Lambert, MA (Secretary / Treasurer)

• Dr. Karen Lawson, PhD • Mr. Trevor P. Noël, MPH • Dr. Allen Pensick, PhD

2.2. WINDREF St. Vincent and the Grenadines Board of Directors

• Dr. Ed Johnson, MD, Director • Sir Fredrick Ballantyne, MD,

Deputy Director

2.3. WINDREF Research Institute Scientific Advisory Board

• Sir Frederick Ballantyne, MD • John R. David, MD • John J. Ferguson, MBChB, FRCGP • Malcolm FergusonSmith, MBChB,

FRCP, FRCPath • Edmond Fischer, DSc • C. James Hospedales, MB, BS, MSc • Mary Jeanne Kreek, MD • Sir Malcolm Macnaughton, MD,

LLD, FRCPG, FRAC • Calum Macpherson, PhD, DIC • Thomas W. Meade, CBE, DM, FRS • Graham Serjeant, MD, FRCP, CMG • Sir Kenneth Stuart, MD, DSc (Hon) • M.S. Swaminathan, DSc • John B. Zabriskie, MD

2.4. WINDREF Research Institute Research Fellows The following investigators have

been appointed to the Windward Islands Research Institute as Research Fellows and are currently conducting collaborative research projects.

2.4.1. Senior Research Fellows • Paul Fields, PhD, Brigham Young • Paul Garner, MBBS, PhD, Liverpool

School of Tropical Medicine • Mary Glenn, PhD, Humboldt State

University • Duane Gubler, ScD, Hawaii

University • Ruth Milner, MSc, Vancouver

Hospital • Stephen Morse, PhD, Columbia

University • Leslie Ramsammy, PhD, DSC (Hon)

Minister of Health, Guyana • Dr. Douglas Slater, MD, MPH,

Minister of Health, St. Vincent and the Grenadines

• Stanley Weiss, MD, UMDMJ

2.4.2. Research Fellows • John Adamski, MD, MSc, MPH • Zuri AmuleruMarshall, PhD • Glennis Andall, PhD • Charles Avgeris, MD, MSc • Grant Burgess, PhD • Francis Fakoya, PhD • Martin Forde, DSc • Orazio Giliberti, MD • Richard Kabuusu, DVM, MPH • Svetlana Kotelnikova, PhD • Matthias Lorenz, PhD • Marios Loukas, MD, PhD


• Theresa McCann, MPH, PhD • Barrymore McBarnette, MD • Craig McCarty, PhD • Clare Morrall, PhD • Shamdeo Persaud, MD, MPH • Bonnie Rusk, MSc • Samina Rutrecht, PhD • Shanti Singh, MD, MPH

2.4.3. New Research Fellows 2007

Grant Burgess

Dirk Burkhardt

Francis Fakoya

Martin Forde

Bonnie Rusk

Samina Rutrecht


2.5. WINDREF Research Institute Research Scientists

Research Scientists include: Sadiq AlTamini, Jonathan Ashcroft, Sumita Asthana, Yitzhack Asulin, Bishara Baddour, JeanPierre Barakat, Matthew Beeson, Keith Bensen, Matthew Boles, Karen Brennan, William Brown, Matt Browne, Ella Cameron, Nicholas Caputo, Jessica Clayton, Mmakgomo Coangae, Rae Connolly, Abraham ElSedfy, Daniel Firer, Scott Forman, Vamsi Guntur, François Hallé, John Holleran, Anthony Junck, Megan Kaminskyj, Sebastian Kreitzschitz, Erik Lacy, Richard Lehman, Setshidi Makwinja, Paul Mancuso, Baher Maximos, John McCormack, David Melamed, Kirk Minkus, Jessica Morlok, Kevin Neill, Bayela Nfila, Yolanda Ng, Michael Nillas, Steve Nimrod, Andre Panagos, Rakesh Patel, Barry Politi, Sandeep Pulim, Sean Ramsammy, Justin Rebo, Tarek Refaie, Alan Rhoades, Laura Robinson, Karin Schioler, Corey Schwartz, Christopher Skaff, David Steinberg, Derrick Tlhoiwe, Sarah Treter, Nghia Truong, James Tsai, Dan Twyan, Frank Van Natta, RuAmir Walker, Juliette Williams, David Winokur, Colleen Wunderlich, Elliot Yung.

2.6. WINDREF Research Institute Administration Mrs. Isha English continued as

Administrative Assistant, Ms. Meg Conlon as Executive Secretary and Mrs. Rachel Paul King as Secretary.

Mrs. Isha English, Ms. Meg Conlon and Mrs. Rachel PaulKing.

3.0. WINDREF (USA) WINDREF (USA) was

established to facilitate coordination of the USA activities and to administer charitable donations from the United States to the WINDREF Research Institute. As a nonprofit organization, its goal is to enhance the development of WINDREF’s research and educational programs. The offices are located on Long Island in New York to provide administrative and logistical support for the WINDREF Research Institute. Ms Sherryl Murray continues as program coordinator in the New York Office.

4.0. WINDREF (UK) WINDREF (UK) was setup in

Winchester, England in 1999 to promote collaboration between WINDREF scientists and academic centers of research in the United Kingdom. It is hoped that by reaching out to a larger scientific community, WINDREF will broaden its research opportunities by forming collaborations with scientists from the European community.

4.1. WINDREF (UK) Board of Trustees A Board of Trustees was

appointed in 1999 to oversee the activities of WINDREF (UK). • Lord Soulsby of Swaffham Prior,

MRCVS, DVSM, MA, C.Biol., F.I.Biol., DSc(Hon), (Chairman)

• Sir Kenneth Calman, KCB, FRCSE • Baroness Howells of St. David, OBE • Sir Kenneth Stuart, MD, DSc • Richard Summerfield, MB, BChir, • Keith B. Taylor, DM, FRCP • Calum Macpherson, PhD, DIC

(Ex Officio)


4.2. WINDREF (UK) Administration Ms. Sue Huntington continues as

Executive Secretary. Ms. Huntington provides the administrative support and expertise that is central to WINDREF’s (UK) fundraising, administrative and collaborative activities.

5.0. Institutional Review Board (SGU IRB)

The IRB is registered with the US Office of Human Research Protection, Department of Health and Human Services, and conforms to international standards. It meets four times a year, usually the 3rd Friday of February, April, October and November. The deadline for the receipt of proposals to be reviewed is two weeks before the meeting. Proposals that meet the criteria for Expedited Review are reviewed within approximately two weeks by the Executive Committee.

IRB EXECUTIVE COMMITTEE: • Dr. Robert Hage (Chair), Anatomical

Sciences Department, SGU • Dr. Cheryl Cox Macpherson (Vice

Chair) Bioethics Department, SGU • Fr. Charles Dominique (Secretary),

Roman Catholic Church • Sir Paul Scoon, GovernorGeneral of

Grenada 19781992 • Mrs. Jean Robinson, Charity

Foundation Director • Dr. Theresa McCann (Past Chair),

Behaviorial Sciences Department, SGU

• Dr. Calum Macpherson (Ex Oficio), Director of Research at SGU

• Ms. Meg Conlon (Administrator). Office of Research, SGU

BOARD MEMBERS: • Dr. Subbarao Chamarthy, Clinical

Skills Department, SGU • Rev. Raphael Osbert James,

Presbyterian Church • Mr. Alban John, BarristeratLaw • Dr. Bradford Noel, Internal Medicine • Mr. Peter Slinger, Department of

Educational Services, SGU • Dr. Timothy Ayliffe, School of

Veterinary Medicine, SGU • Mr. Terry Charles, Director, Grenada

Red Cross Society • Dr. Brian Curry, Anatomical Sciences

Department, SGU • Mr. Winston Duncan, Director,

Grenada Family Planning Association • Mr. Basil Harford, Retired Civil

Servant • Dr. Vibart Yaw, Dentist

NEW BOARD MEMBERS Three SGU faculty members were appointed to the IRB in October 2007: • Dr. Jennifer Durst, Counseling and

Psychological Services • Dr. Angela Gomez, Public Health and

Preventive Medicine • Dr. Marios Loukas, Anatomical

Sciences Department

PROJECTS REVIEWED: 18 projects were submitted for the IRB for review; nine were approved by expedited review and three were formally exempted as they did not involve human subjects. None were disapproved, and four were pending at the end of 2007.


PROJECTS APPROVED BY EXPEDITED REVIEW: 1. Jennifer Durst, Assistant Professor,

Counseling and Psychological Services “Survey of Substance Abuse at St. George’s University”, 12 March 2007.

2. Satesh Bidaisee, Term 2 MSPH student, “An Exploration of Perceptions towards Knowledge of Zoonotic Diseases by Adult, Non Health Professional, Grenadian Citizens from the Grand Anse and Carenage Areas in the Parish of St. George”, 11 May 2007.

3. Dr. Angela Gomez, Assistant Professor, Department of Public Health and Preventive Medicine, “Analysis of the Effects of Displacement on People’s Agency Capacity”, 25 April 2007.

4. Matthew Browne, MD/MSc Student, Microbiology, “Biofilm Analysis of Body Piercings”, 13 July 2007.

5. Mr. Navin Dargani,, First Year SOM Student, “Psychological and Occupational Impact of PersonTrain Collisions on Subway Operators”, 7 September 2007

6. Dr. Robert Hage, Professor of Anatomical Sciences, Ear, Nose and Throat Specialist, “Hearing Health Care project”, 5 September 2007.

7. Dr. Robert Hage, Professor of Anatomical Sciences, Ear, Nose and Throat Specialist, “Noise Induced Hearing Loss in Grenada. Is it a Problem? A Pilot Study”, 14 September 2007.

8. Mathangi RajaramGilkes, MD, Instructor in Histology in MSc program, Anatomiocal Sciences Department, “Otoacoustic emission (OAE) testing in infants in the tri island state of Grenada, Carriacou and

Petite Martinique”, 10 September 2007.

9. Ms. Sonja Antoine. MSc Student, Department of Social Sciences, University of Northampton, “‘Jus’ how me pick up me roof and put me house back up, just so they want me to pick up me life too. You hear dat?’ A thematic analysis of the experiences of adolescents following Hurricane Ivan”, 10 September 2007.

EXEMPTED PROJECTS: 1. Hilary Crane, MSc Student,

Newcastle University, “Phylogenetic diversity and biogeography of marine spongeassociated bacteria and the potential anticancer properties of secondary metabolites”, 23 February 2007.

2. Dr. Jonathan Kibble, “Analysis of Online Quiz Use”, 11 May 2007

3. Lindonne Glasgow, MSPH Student, Department of Public Health and Preventive Medicine, “Analysis of chemical and microbial contaminants in harvested rainwater cisterns in Carriacou and Petite Martinique”, 14 September 2007.

Submitted by Meg Conlon IRB Administrator

6.0. Institutional Animal Care and Use Committee (IACUC)

The use of animals for research or teaching purposes must be pre approved by the SGU Institutional Animal Care and Use Committee. The IACUC forms are available on the Office of Research website, forms and policies page. IACUC members are: • Chair: Dean, School of Veterinary

Medicine: Dr. Raymond Sis


• School of Veterinary Medicine (two members): Dr. Desmond Baggot and Dr. Eugene Rennie

• School of Medicine (one member): Dr. Randall House

• Director, WINDREF: Dr. Calum Macpherson

• University Veterinarian: Dr. Buxton Nyack

The IACUC approved the use of animals for teaching as well as one research project proposed by Dr. Harry Hariharan, entitled “Bacteria associated with mucous membranes, skin wounds, and ears of feral cats in Grenada”.

Submitted by Meg Conlon Office of Research

7.0. Current Research Projects

7.1. St. Vincent and the Grenadines Streptococcal Project

Rheumatic Fever is as a result of exposure to Streptococcus pyogenes. The sequelae of this can lead to Rheumatic heart disease. Rheumatic heart disease is a debilitating condition that is usually brought on by stenosis of the mitral or aortic valves.

The St. Vincent and the Grenadines project has begun its initial sensitization project by first holding dialogue with the community nurses for all the clinics in St. Vincent and the Grenadines. Nurses Donette Cunningham and Ynolde Smart have been selected as the St. Vincent coordinators to oversee the implementation stages of the project.

Nurse Cunningham with the community nurse team and representative from the Ministry of Education.

Sensitization to Principal Godfrey James and students at Kingstown Government School, Stoney Grounds, St. Vincent and the Grenadines.

Trevor Noël discusses the protocol with the St. Vincent and the Grenadines Community Public Health Nurses.

3,000 informed consent forms and questionnaires were distributed in the schools in the districts of St. Vincent and the Grenadines. Work has begun on creating brochures and a powerpoint presentation to be used in all 66 schools in St. Vincent and the Grenadines.


The consent forms have already begun to be returned to the schools. The ten schools that were selected were Pageat Farm, Bequia, Mustique Government, Questelles, Chateaubelair, Sandy Bay, Georgetown, Stubbs Government, Mesopotamia, Stony Grounds, CW Prescodd. These schools were included in an initial Echocardiogram study that used ECHO only without a provision for the Duckett Jones Criteria. The study will involve drawing blood samples and throat swabs. The blood samples will be subjected to Anti streptolysin O titres and the throat swabs to cultural isolation for beta hemolytic streptococci. All positive cases will be treated at the Cardiology clinic or at decentralized clinics in the community. Positive beta hemolytic streptococci samples will be sent to Rockefeller University and the University of Minnesota for further subtyping. The study has been approved by the Research Oversight Committee in St. Vincent and the Grenadines. This study will begin in January 2008 and will be carried out in collaboration with WINDREF, Rockefeller University, University of Minnesota and the Ministries of Health and Education, St. Vincent and the Grenadines.

Submitted by Trevor P Noël, MPH Deputy Director, WINDREF

7.2. Photovoltaic Power Generation Program

This new WINDREF program was proposed and will be directed by Dr. Dirk Burkhardt, who has a wealth of experience in this field in the Caribbean region. The timing of the introduction of this new initiative is optimal given the

global focus on global warming and its potential and current impact on the world today. It is widely accepted that global warming and the resulting environmental changes will be one of the main threats to health in the coming decades. The need to address this threat is the responsibility of all, and any small change that can be made to reduce this potential threat will be important to take.

There are a number of reasons as to why such a program in the Caribbean and in particular in Grenada makes a lot of sense including: 1) the reliable source of sunlight; 2) the extremely high (and constantly

rising) electricity prices; 3) the fact that the local electricity

production comes from non renewable energy sources which contribute to the global warming problem.

Technical and scientific data, for example, irradiation data or empiric data on Photovoltaic (PV) Systems utilizing various types of PV Systems, are not yet available for realistic applications for the Caribbean. Therefore this will be the first project in the region to set up a PV test field where different types of Photovoltaic Modules, particularly the recently developed Thin Film Technology, will be tested against a standard crystalline silicone PV System under Caribbean conditions.

The Standard System, a 5.04 kWp System (representing a value of US$ 37,000.00), is a donation of Grenada Solar Power Ltd. The generated electricity will be used by WINDREF.

The new program’s goals are to: 1) provide a forum to answer technical

questions about photovoltaic power generation;


Three 2,88 kWp PV Systems on Maca Bana Villas in Grenada.

2) create a public awareness of the problem of CO2 emissions, generated by the burning of fossil fuels;

3) provide a forum to explain the existence of alternative renewable energy sources;

4) create a '100 roof program' where 100 small houses are to be equipped with small PV Systems (1.1 kW). The program is planned to be financed through donations, and each installation will be accompanied by research on solar mapping and appropriate education. It is hoped that this particular goal will improve the quality of life for many families who will benefit from a reduced electrical bill delivered in an environmentally friendly way.

5) organize poster exhibitions and a conference on the use and advantages of photovoltaic systems.

6) launch a capacity building training program for engineers and electricians in the region to acquire a certificate as ‘Solateur’, a specialist for this new type of power generation.

7) One of the longterm goals is to make St. George’s University the first ‘Green University’ in this region of the world and Grenada the leading country in using the sun as source for sustainable energy generation.

By Dr. Calum Macpherson, Director, WINDREF Research Institute

7.3. Guyana Lymphatic Filariasis Elimination Program

INTRODUCTION The Lymphatic Filariasis Elimination

Program was developed in Guyana in 2001 following the WHO decision to work towards global elimination of LF by 2020. The initial assessment done in 2001 established filarial transmission in eight of the ten health units (regions) of Guyana at a rates between 37% in the urban areas of Georgetown to 4% in rural areas in Region #2, with an average of about 10% nationally with an atrisk population of about 690,000 of the 750,000 population.

The Government of Guyana decided to implement the recommendations of WHO: a) The mass treatment program for the at

risk population using the WHO recommended strategy of fortification of table salt with diethylcarbamazine (DEC) and promoting the uses of the DECsalt nationally;

b) The establishment of a morbidity control program to manage cases of Lymphatic Filariasis resulting in elephantatisis, hydrocelel and other clinical manifestations.

The program has also developed the capacity to consciously monitor and evaluate the implementation plan and to support all the program activities by training and research.

WINDREF, from the early developmental stages of the program, provided vital assistance in conducting several key research and training for technical officers involved in implementation. Since then, six different research papers were published, two operational research protocols were developed, and field work commenced. Additionally, two candidates completed Masters in Public Health and two are reading for Masters in Sciences and PhD degrees at SGU. WINDREF has also been involved in the strategic plan development and the monitoring program.


This report reviews the activities of the two main components of the program and provides the summary of the results of a spot check evaluation done in November 2007. The program continues to receive technical support from PAHO/WHO, WINDREF and the Emory LF Support Centre. Financial resources are provided by the Government of Guyana from national funds.

The research component of the program allows for continuous evaluation of the DECsalt program and strategies used to achieve the target of elimination. The project to examine the impact of DECsalt on the reduction of LF parasites in high burdened population has been able to demonstrate the positive correlation between use of DECsalt and absence of LF parasites in the body. The researchers have already studied about 1,200 persons with about 950 participating in a cohort looking at use and no use of DECsalt. The second part of the study deals with tadditional benefits of the use of albendazole along with DECsalt and its concomitant positive impact on the adult parasites.

Preliminary results indicate a decrease in the overall prevalence of LF in Guyana, but with pockets of infection in high burden areas. Currently, the technical team is examining the possibilities of introducing intermittent rounds of MDA using DEC tablets and albendozole to complement the slow gains of DECsalt in interruption of LF transmission in these areas.

COMPONENT 1: INTERRUPTION OF TRANSMISSION

DEC FORTIFIED SALT PROGRAM

1. Imports of DECsalt (September 2006 September 2007) by Quarter

Agency

1 st Quarter (Sept Nov)

2 nd Quarter (Dec Feb )

3 rd Quarter (Mar Jun)

4 th Quarter (Jul Sept)

Grand Total

New GPC

1,600 bags

800 bags

−− −− 2,400 bags

R&R Int.

4,000 bags

800 bags

800 bags

1,600 bags

7,200 bags

Total 9,600 bags

Total 480.000 Kgs

Each bag contains 50Kg DEC fortified salt

2. Amount of DECsalt purchased for Social Mobilization

Agency MOH/ PAHO

New GPC 500 bags R&R Int. 120 bags

Grand Total 620 bags 31,000 Kg

3. Number of persons trained in DECsalt marketing between Sept 2006Sept 2007: 13 persons in Region 5.

4. Number of sessions held for social mobilization officer: one, in Dec 2006.

5. Number of people reached (door to door promotion): 2007 approximately 25,000.

6. Public events DECsalt promoted at: • Guy Expo 2006; • Linden Town Expo Week 2007; • Ministry of Education Health Fair

2007; • US Embassy Health Fair, 2007; • Guyana Defence Force Health Fair

2007; • Guy Expo 2007; • The Guyana telephone directory

carried a 4page section on LF and DECsalt.

• Television/Radio Daily sponsorships of 30 seconds

add at prime time on one national channel;

Sponsorship of Cricket World Cup matches played in Guyana and the West Indies in March/April 2007 (96 sessions sponsored);

Daily PSA on national radio at 08.15 Hrs and 18:45 Hrs.

SENTINEL SITE

Spot Checks were done in two sentinel sites (random “Grab” sample): 1. Lodge:

a. Number of persons tested ICT and microfilaria smears: 122.

b. Number of ICT positives: 8 (6.5%). c. Number of smears positive: 6 (4.9%). d. Number using DECsalt: 96 (78%).


2. Tucber: a. Number of persons tested ICT and

microfilaria smears: 102. b. Number of ICT positives: 7 (6.8%). c. Number of smears positive: 1 (0.9%). d. Number using DECsalt: 93 (91.1%).

COMPONENT 2: MORBIDITY CONTROL PROGRAM

ACTIVITIES SEPTEMBER 2006 – AUGUST 2007 1. Training of Health Workers

a. Introduction workshops: 4 (in Regions 4, 10, 6 and 5).

b. Follow–up training: 9 (Regions 2, 7, 4, 6, 10 and 6).

c. On the job mentoring 32 (Region 4 – East Bank, Georgetown, and East Coast, Region 5 and Region 10).

2. Treatment Sites a. Establishment of New CARE Sites:

Georgetown: 2 sites developed. East Coast: 1 site developed. Region 5:1 sites developed. Total New: 4 sites.

b. Maintenance of Existing Sites: Region 4 – 9. Region 5 – 2. Region 2 – 2 sites. Region 10 – 3 sites. Region 6 – 3 sites. Total: 19 sites

c. Total Treatment Sites: 23 active sites. 3. Support Groups

a. Existing Groups: Region 4 (Georgetown, East

Bank, East Coast): 3 (Total 41 Members).

Region 10 (Wismar): 1 (16 members).

Region 6 (New Amsterdam): 1 (12 members).

Region 5 ( Bellabrum) – 1 (28 members).

Total Active – 6 b. New Groups:

Region 5: 1 (7 members). Total Members – 104 Members.

4. Hydrocele: 3 Hospitals conducting surgery: 211 (66%) surgeries done out of 319 registered.

Shamdeo Persaud, MD, MPH, Chief Medical Officer and

National Epidemiologist, Guyana, WINDREF Research Fellow

7.4. Dr. Forde part of CAN$1.6 Million Dollar TeasdaleCorti Grant

Established in 2001, the Canadian Global Health Research Initiative (GHRI) brought together four federal agencies to maximize Canada’s support for health research and its application in low and middleincome countries (LMICs). One major initiative being managed by GHRI is the TeasdaleCorti (TC) Program. This Program is named in honor of Drs. Lucille Teasdale and Pierro Corti, a Canadian husband and wife team who devoted their lives to improving health care in Africa until Dr. Teasdale’s death from occupationally acquired AIDS in 1998. The TC program is designed to strengthen the research and research capacity of LMICs through a series of grants and awards that has already exceeded $25 million over the past five years.

Early in 2006, more than 250 teams partnering Canadian and LMIC researchers, managers and healthsystem policymakers, responded to a call for proposals by submitting a letter of intent. From these LOIs, less than 40 teams were asked to further develop their proposals. Out of this subset, 14 teams were finally selected by an international peer review panel to receive fouryear grants averaging $1.5 million each.

Dr. Forde, a professor within the Department of Public Health and Preventive Medicine at St. George’s University, along with his two other coprinciple investigators, Dr. Eric Dewailly and Dr. Neela Badrie, and the multinational and organizational team they put together was one of the teams to successfully secure one of these grants. Their


proposal, entitled “Caribbean Ecohealth Programme: Public and Environmental Health Interactions in Food and Waterborne Illnesses (CEP)”, was successful in securing the maximum amount of funds under this initiative, $1.6 million over the 4year length of this initiative.

The CEP has three major components: (1) Research, (2) Regional Capacity Strengthening, and (3) Training. There are three main research areas that will be pursued: (1) Prevalence of persistent organic pollutants in the Caribbean region; (2) Burden of illness study; and (3) Microbial contamination of harvested rainwater.

The CEP teams are comprised of seven primary members: the Caribbean Epidemiology Centre (CAREC), the Caribbean Environmental Health Institute (CEHI), the Public Health Agency of Canada (PHAC), the PanAmerican Health Organization (PAHO), and the Universities of Laval (Canada), St. George’s University (SGU), and the University of the West Indies (UWI). Laval, PAHO, and WINDREF located at SGU will manage the funds of this grant.

TeasdaleCorti Proposal Workshop, 78 September 2006. Dr. Forde is standing, fourth from right.

Submitted by Martin Forde, PhD WINDREF Research Fellow

7.5. Dr. Forde Completes Survey of Biomedical Waste Management Practices in the OECS Countries

Dr. Martin Forde was recently awarded a US$11,100.00 grant to look at biomedical waste management practices in the Organization of Eastern Caribbean States (OECS) region. His proposal, one of 53 grants for health projects awarded across 20 countries by the Pan American Health Education Foundation’s (PAHEF) Small Grants Program in 2006, was titled, “A Review of Current Biomedical Waste Management Practices in the Organization of Eastern Caribbean States (OECS) Countries.” The grant was awarded to the Health Promotion Research Program which is located within WINDREF.

From July to November 2007, Dr. Forde traveled to all ninemember OECS island states. In each island, he met with Ministry of Health officials, solid waste management personnel, and toured the major healthcare facilities and landfill sites. His findings have been documented in a report which he has submitted to PAHEF and each OECS Ministry of Health.

Overall goals of this research project was to provide a better picture of biomedical waste management practices among OECS countries that can be useful for the development of a regional biomedical waste management policy and infrastructure, such as a standardized training program within public health facilities within the OECS region. Previously, in 2005, Dr. Forde was funded by the World Bank to develop a biomedical waste management manual for healthcare personnel in Grenada, which resulted to the development of a manual titled Biomedical Waste Management Manual for use in Grenada. This earlier work was the catalyst for examining biomedical waste practices in the wider OECS region.


St. Lucia: Victoria Hospital, sharps container, lid open.

Antigua: Bio Health Medical Lab.

St.Kitts: JNF Hospital.

Anguilla: Princess Alexandra Hospital.

Anguilla: Princess Alexandra Hospital.

Anguilla: Corito Landfill, old cars.


Grenada: General Hospital incinerator.

SUPPLEMENTARY INFORMATION The term “biomedical waste

(BMW)” refers to any waste from healthcare, research, and laboratory facilities that pose a hazard to the environment and human health. According to the Caribbean Environmental Health Institute (CEHI), the challenge of ensuring effective management of biomedical wastes in the Caribbean is a major source of concern to the policy makers and operatives who have been charged with the responsibility for environmental management and the protection of human health. Caribbean island states are very much aware that there are potential negative impacts related to workers’ occupational health and safety, environmental contamination, and containment of contagious diseases if such wastes are not managed properly.

Submitted by Martin Forde, PhD, WINDREF Research Fellow

7.6. Graduate and Undergraduate Research Education Becomes an International Effort

Vigorous development and fruitful collaboration in research and education took place in collaboration between CERI

WINDREF and two European Universities: the University of Gothenburg, Sweden, and the University of Newcastle, UK, in 2007. The cooperation in the education of microbiological research took place in a number of areas.

STUDENTEXCHANGE BETWEEN THE UNIVERSITY OF GOTHENBURG, SWEDEN AND SGU, GRENADA

In February 2007, Dr. Pedersen brought with him three research students from Gothenburg, Maria Ulnes, Kajsa Kroneld and Maria Hallbeck, who have been working on the project “Characterization of biofilms and water in Warm Soda Springs at St Andrew, Grenada” since January 2007. The students were introduced to the biosafety regulations for biosafety level 2 microbiological laboratories at the Microbiology Department, SGU. They participated in two field trips to the St. Andrew Soda Springs in February 2007. Dr. Pedersen used his Swedish research funds to finance the flight, accommodations costs, and all the research expenses and shipment of equipment via air cargo for his students, including the transportation to the sampling site.

The research group (from left): Teresa Caines, Anna Hallbeck, Bjorn Hallbeck, Maria Hallbeck, Karsten Pedersen, Lotta Hallbeck, Maria Ulnes, Breon Samuel, Kajsa Kroneld, Marisa Walker, Danielle Garraway, and Svetlana Kotelnikova.


SGU premedical student Danielle Garraway working on her research project in the Laboratory in the Microbiology Department.

Teresa Cain and Maurisa Walker presenting the results of their research project during the final poster session, 27 April 2007, SGU.

Breon Samuel and Danielle Garraway presenting the results of their research project during the final poster session, 27 April 2007 at SGU.

During two of the field tours, Saturday, the 10 th and Sunday, the 18 th of February 2007, Prof. Pedersen took four premedical students working on the research project under Dr. Kotelnikova’s supervision to visit the “Sulfur” Warm Soda Springs in the rainforest of St Andrew’s. We collected a number of specimens including biofilms, groundwater, gas and physical field data. The equipment for the measurements, such as an illuminometer, conductivity, oxygen, pH and temperature sensors, was temporarily imported by Dr. Pedersen from Sweden. A flow cell was also brought from Sweden and used to collect fresh biofilms (48 days old). Samples collected have been conserved and transported to Sweden for further analysis including microscopy, DNA extraction and molecular analysis of the microbial community. The field data confirmed data which was collected a year earlier. The mineral composition and the chemistry in the collected specimens of water, gases and biofilms were analyzed in Sweden in April 2006. The gas content was also analyzed. Dr. Pedersen has shown that it contains CO2, CH4 N2, H2 and CO. The mineral chemistry of the biofilms was analyzed and compared to the groundwater and minerals. Light microscopy was performed on the bacterial communities from the biofilms from this warm spring (Kotelnikova et al, 2006). All reports are available from Dr. Kotelnikova. Posters “New ironoxidising bacteria in St. Andrew’s Warm Springs” by Breon Samuel and Danielle Garraway, and “Manganeseoxidation in St. Andrew’s Warm Springs” by Teresa Caines and Maurissa Walker have been presented during the course BIOL390 in Research Methods in May 2007.


The project has been supported by SGU Graduate Studies Program Small Research Grant Initiative funding and the Swedish Research Council.

More chemical analyses will be performed on both spring water and biofilms. Molecular biological characterization of the microbial community has been performed by Dr. Sara Jagevall in the laboratory of Microbial Analytic Inc. in Gothenburg, Sweden. We found genes of microorganisms that are distantly related to ironoxidising Gallionella ferruginea (93 and 95%) and irondepositing Siderooxidans litheroautotrophicus (93%) along with photosynthesizing new forms of uncultured Cyanobacteria (90%). Here at SGU, we are culturing ironoxidizing, manganeseoxidizing and photosynthesizing microorganisms isolated from the Soda Springs. The study is currently under development in both laboratories.

INPUT TO THE PUBLICITY OF SGURESEARCH

The project “New ironoxidizing microbial community in St Andrews, Warm Springs in Grenada, West Indies” was presented at the SGU Panel on Research and Scholarly Activities, 15 February 2007.

The Swedish students, Maria Ulnes, Kajsa Kroneld and Maria Hallback, presented their results at the regional scientific contest “Young Research” in Gothenburg. Their research project was selected to represent young researchers of Gothenburg in Stockholm during the country contest, and the visiting students from Gotherburg, Sweden, Maria Ulnes, Maria Hallback and Kajsa Kroneld Viktorsson, have been awarded for the Best Undergraduate Research in

Sweden. They will represent Sweden at the EU competition for Young Scientists with the project “Ironoxidising bacteria in a new environment”, which was performed in Grenada in collaboration with St. Georges University under the supervision of Dr. Kotelnikova, Dr. Hallback and Dr. Pedersen in February 2007.

The project became one of the projects selected to represent Sweden during the European contest, which took place in Valencia, Spain, 14 September 2007. St. George’s University, Grenada, was represented as one of the originating research institutes along with the University of Gothenburg, Sweden, on the poster presented during the EU Best Yong Scientist Contest, and the contribution is published as a paper in the WINDREF Annual Report 2007.

There are several students who have been taking SASBIOL 390 research methods course at Microbiology Research Lab. They have performed their premedical research under the supervision of Dr. Kotelnikova, Cynthia Bruno and Dr. Hallbeck. They studied the iron oxidation and manganese oxidation in the springs and practiced methods for isolation of pure cultures that are responsible for the iron and manganese oxidation. The abovementioned bacteria are extremely fastidious. and therefore special techniques should be used for their isolation and culturing. Very few people in the world govern the culturing technique for the metal oxidizing microorganisms. Dr. Hallbeck is a “world’s expert” in cultivation of the neutrophilic ironoxidizing Gallionellalike microorganisms. She is the first author of the chapter on Gallionella in the prestigious Bergey’s Manual of Systematic Bacteriology 2 nd


edition. She kindly advised our students on how to approach this complicated scientific research task under two occasions, February the 13 th and February 15 th , 2007. The results will be reported in December 2007.

DEVELOPMENT OF RESEARCH FACILITIES, STUDENT, FACULTY AND STAFF TRAINING

A graduate research seminar was delivered on the 14 th of February 2007 at 12.00 noon in WINDREF Conference Room by Prof. Karsten Pedersen, PhD in Microbiology. The title was: “Molecular solution of problems in microbial ecology Basic rules of the game”.

The seminar was attended by a number of SGU graduate students and research faculty including our new instructor Adria Tiller, and undergraduate research students. Dr. Pedersen informed us that we could equip our microbiological laboratory with the basic tools needed for approximately US $30,000, which would enable our graduate students to produce results that can be published in peerreviewed scientific journals. He is supporting our proposal which would enable him to assist with the training of personnel from SGU in molecular biology techniques using BIORAD equipment such as optimization of Polymerase Chain Reaction (PCR), primer selection, template concentration optimization, primer design, and Denaturing Gradient Gel Electrophoresis (DGGE) and agarose gel electrophoresis. The personnel should be pretrained in molecular biology in order to be trained in the practical cloning and genetic engineering techniques, and be able to provide the technical assistance to our

SGU research students. I suggested that Dr. Pedersen bring his molecular biology staff scientists, Dr. Sara Eriksson and Dr. Sara Jägevall, to Grenada to arrange this training in Grenada after we have purchased the equipment. Similar equipment, including DNA isolation kits, have been requested by the scientists from the University of Newcastle to be used in May 2007.

RESEARCHCOOPERATION TO TRAIN SGU STUDENTS ABROAD AND EXTERNALACCOMMODATION OF GRADUATE RESEARCH

Dr. Pedersen provided Ms. Cynthia Bruno and Dr. Kotelnikova with the opportunity to use two Internet wired offices and a research laboratory (including equipment and reagents) at the Institute of Cell and Molecular Biology, at the Gothenburg University, Sweden, from 23 May to 20 June 2007. Ms. Bruno and Dr. Kotelnikova used the laboratory to perform molecular biology experiments on 11 bacteria from Grenada. The techniques included: DNA isolation, % (G+C) composition, and DNADNA hybridization experiments. Dr. Pedersen facilitated the purchase of five type reference cultures needed for the experiments from the Culture Collection of the University of Gothenburg. In addition, Dr. Pedersen met with PhD candidate Ms. Bruno and advised her on her thesis. Since then, Ms. Bruno presented her PhD research proposal in January 2006 and has been approved as a PhD candidate.

ACADEMIC COMMUNICATION AND EXCHANGE OF IDEAS

Dr. Pedersen met with all faculty members involved in research and teaching at the Microbiology


Department, including Dr. Lennon. Dr. Qureshi, Dr. Rayner and Dr. Kotelnikova. He also has met with the newly appointed instructor from the School of Veterinary Medicine, Ms. Adria Tiller, and assisted her with consultations on requirements for GC and MS and the purchasing list. He met multiple times with Dr. Kotelnikova and advised on molecular biology equipment to be purchased. He also advised on the safety of the subsurface well which is currently providing water for two desalination plants at SGU for the production of drinking water.

SUPERVISION OF SGU MD/MSC RESEARCH STUDENTS

Dr. Pedersen took the responsibility of being an external supervisor for MDMSc candidate Rakesh Patel, otherwise supervised by Dr. Kotelnikova, Dr. Lennon and Dr. Champney, with the research topic titled “Distribution of indicator organisms in the St. John’s Estuary in Grenada, West Indies”. In the results of his research, Rakesh found that the sewage is discharged into St. John's River upstream from the sampling sites in St. George’s Estuary and Bay. The river effluents are affecting the water quality in St. George’s Bay. St. John's River and St. George’s Bay is designated for recreational use. The concentrations of the organisms indicating the fecal contamination have not been studied before at this site. The main objective was to determine whether a significant health risk exists within the bay, and whether the bay is a potential source of antibiotic resistant strains. The distance of travel and concentrations of the indicators Enterococcus faecalis and Escherichia coli were assessed in the surface water and sediments of the St.

George’s Bay estuary. The antibiotic susceptibility of these indicators was tested using the KirbyBauer disk diffusion method. By measuring distances, current rates and direction, oxygen and salinity gradients, the establishment of approximate health risk zones was deciphered and applied to the particular ecological dynamics of the system.

Dr. Pedersen has also advised MD/MSc candidates John Holleran, Richard Kabuuso, Jonathan Ashcroft and Mattew Brown.

In conclusion, one of the major contributions by Dr. Pedersen has been filling in the equipment and reagent gaps at SGU which allow our graduate research students to be successful. Aside from his visit, he has been spending his time back home in Sweden advising and reading drafts, and kindly facilitated the PhD research opportunity for the SGU PhD candidate at the University of Gothenburg, Sweden.

Thesis defense “Distribution of indicator organisms in the St. John’s Estuary in Grenada, West Indies” was presented by Mr. Rakesh Patel. His supervisors, from the left, Dr. Champney, Dr. Lennon, Dr. Kotelnikova. Dr. Pedersen is not pictured.


STUDENTEXCHANGE BETWEEN THE UNIVERSITY OF NEWCASTLE, UK, AND SGU, GRENADA

Drs. Burgess and Kotelnikova have been cosupervising MSc student Hillary Crane, who has been working with a research project, “Phylogenetic diversity and biogeography of marine spongeassociated bacteria and the potential anticancer properties of secondary metabolites”, studying the diversity of microorganisms associated with marine sponges from UK and Grenada. She has been isolating pure cultures of spongesassociated bacteria and elucidating their phylogenetic relatedness with the cloned metagenome associated with the sponges. Information of the microbial diversity of the tropical and temperate sponges will be established. Culture collection consisting of 80100 isolates remained at the SGU microbial culture collection. She demonstrated that our research lab is an uptodate facility and may be used for the most advanced research using molecular biology methods.

The aim of this study was to increase our understanding of the biodiversity of marine sponge associated bacteria and to determine whether these bacterial communities show biogeographic variability. Marine sponges harbour large numbers of bacteria that may perform various symbiotic functions such as nutrient acquisition, stabilization of the sponge skeleton and production of secondary metabolites of potential use to the host. Only a small percentage of these bacteria are growing in lab media, but recent advances in molecular techniques have contributed greatly to our understanding of spongeassociated microbial diversity and have lead to the discovery of novel species and even

suggested a new phylum. However, there is still much debate regarding the stability of spongeassociated bacterial communities and their geographic variability. This project aims to investigate whether these bacterial communities show stability between individual hosts and whether they display biogeographic variability both on a regional and global scale. It also aims to increase our understanding of sponge microbial diversity so as to aid marine conservation and management programs working under the remit of the Convention on Biological Diversity, as well as aiding the search for new marine natural products by the pharmaceutical industry. The potential cytotoxic activity of sponge secondary metabolites towards human cancer cell lines will also be investigated to demonstrate whether they have potential for use in anticancer drugs.

Hilary Crane in the research lab of SGU’s Microbiology Department, working on her project “Microbial diversity of epiphytic microflora on marine sponges”, June 2007.

Hilary received a distinction (highest award) for her Master’s course and was awarded a prize for the best mark of her year at the University of Newcastle, UK. We are happy that our help for her at SGU contributed to this achievement.


Dr. Burgess, professor of Marine Biotechnology, was invited to the Department of Microbiology as a representative of the University of Newcastle and director of Dove Marine Laboratory, UK, an affiliate with SGU and WINDREF, in August 2007.

Our plan is to continue research based education of graduate degree students of Grenadian origin at the Department of Microbiology at SGU, and to test the cultures at different growth phases, which will be shown to be associated with sponges, for anti biofilm activity, by using the microplate assay. To cover expenses, a major proposal entitled “Academic: Using Marine Biotechnology to Strengthen the Knowledgebased Economy of the African and Caribbean Regions by Capacity building in Higher Education”, has been jointly submitted by Drs. Kotelnikova and Burgess in cooperation with Professor Mavuti, PhD, Director for International Programs and Links, University of Nairobi, Kenya, to the EU Commission EDULINK Programme in September 2007. Making scientific research and education directly relevant to society is of critical importance in the regions of Africa and the Caribbean, where pressure from society to establish RTD requires the unambiguous linkage of RTD programs to key societal objectives, economic, environmental or social. Heterogeneity in scientific priorities, resources and partnerships characterize each of the three regions. This requires the adoption of differentiated approaches to thematic priorities that enable the identification of broad areas of convergence of interests common to a large majority of countries.

In Grenada it is important to look for synergies and complementary issues between activities supported by public

funding and private sector investments. The cooperation framework that will emerge from this dialogue intends to create synergistic opportunities for specific sectors, generating knowledge for the public good as well as promoting an enabling environment for the participation by the private sector in knowledgebased products and services. A key area for the Caribbean, and African coastal countries such as Kenya, are their rich marine resources.

The grant would potentially provide educational funds from the European Community for cooperation of European Universities with universities and NGOs in Africa (University of Kenya) and the Caribbean (SGU). Dr. Burgess and Professor Polunin will act as partners at the European Institution, Newcastle. Dr. Mark Haman at the University of Mississippi, USA, is a specialist in natural product chemistry who may be eventually involved in supervision. Dr. Pedersen expressed willingness to supervise MSc (Microbiology) student Ravindra Naraine with the project “Microbial diversity in the biofilms in Soda Springs of Grenada” while Ravindra works with molecular techniques in the lab in the University of Gothenburg in Sweden in May and June 2008. Our graduate research students will have access to the advanced chemical and molecular biological labs at the University of New Castle, UK, and Gothenburg, Sweden.

I would like to express my gratitude and appreciation to SGU’s Chancellor, Dr. Charles Modica, for the funding of travel and accommodation for Dr. Pedersen and his wife. The visit was very successful, dynamic and beneficial for the graduate students and faculty. I appreciate that I have been given an opportunity to have multiple


opportunities for scientific interactions and planning of the graduate research with Dr. Pedersen and Dr. Hallbeck.

Dr. Svetlana Kotelnikova WINDREF Research Fellow

7.7. Microbial diversity in the Ironoxidizing Biofilms of Soda Springs in Grenada

The microbial diversity in the “Fountain of Youth” has finally been discovered and analyzed. This “Fountain of Youth” is an iron spring located on the wall of a caldera, close to the Mount St. Catherine region in St. Andrew’s, Grenada (NO12 o 08’870”, W61 o 40’562”) (Kotelnikova, 2007).

The biofilm samples were collected from the bottom of the pool and compared to the biofilms grown on the sterile glass slides during seven days exposure to the pool water at the outlet point from the pool. The same organisms Pseudoananbeana lonchoides dominated both in the biofilms sampled from the bottom of the pool and in the biofilm grown on the sterile glass slides exposed to the water in the pool for seven days. The same organisms prevailed in both settings, which means that we could reproduce the environment quite closely and still visualize it. The microbial succession to 100% of Pseudoananbeana lonchoides on the slides in comparison to 65% in the bottom biofilm would indicate that Pseudoananbeana lonchoides is a pioneer attaching to the surface. Even if there were more photosynthesizing organisms detected in the bottom biofilms than on the glass surface, the soda spring’s algal community was not very diverse. Seven algal species were

identified from the algal mat matrix. Cyanobacteria were the dominant group, represented by five species (71% of the reported species) and two diatoms species (29%) were identified. Pseudoananbeana lonchoides Anagnostidis was the overall dominant species with 65% of the cells counted in the community and 65% in biovolume. Pseudoananbeana lonchoides has a thin mucilage sheath but is responsible for developing the algal mat matrix. Rhabdoderma zygnemicolum Copeland was common with 33% of the relative percentage and 20% in biovolumes. Rhabdoderma zygnemicolum was very inconspicuous in the samples; as being small (1 μm in width and length), mostly coiled, and in chains. Further investigation needs to be accomplished to determine if this is a new species. Leptolyngbya gelatinosa (Voronichin) Anagnostidis et Komárek was not dominant (1% relative percentage) and found sporadically but provided a higher percentage in biovolume (15%). The other four species (2 cyanobacteria and 2 diatom) identified provided less than 1% of the relative percentage and biovolume; Chroococcus membraninus (Meneghini) Nägeli, Calothrix contarenii (Zanard.) Borent et. Flahault, Fragilaria virescens Ralfs, and Nitzschia fonticola Grunow (Figure 1).

Figure 1. Phase contrasted image of Soda Springs biofilm.


Calothrix contarenii has terminal heterocyst for nitrogen fixation, which undoubtedly also supplies the mat community with usable nitrogen for cellular growth.

To isolate and identify the microbial population in the spring, both culturing and molecular techniques were used. To identify these microorganisms, a series of processes were carried out to obtain their bacterial sequences.

Firstly, samples of biofilm were obtained from the spring. A portion of the biofilm was cultured using specialized broth to isolate iron oxidizing bacteria. Specifically, Gallionella ferruginea medium (Hallbeck, L. 2007), and sterilized spring water with supplemented iron sulphide.

DNA was then extracted from both biofilm and cultured samples using GeneEluteTm Bacterial Genomic DNA kit, SIGMAALDRICH. The extracted DNA was then amplified using bacterial universal primers (27F, 1492R) (Jägewall, S. 2007), for 30 cycles.

The amplified DNA was subsequently separated using Thermal Gradient Gel Electrophoresis (TGGE). This technique separates DNA according to their individual nucleotide composition. The separated DNA was exercised and purified using quitech kit (Invitrogen). The purified DNA was afterwards cloned using TOPOTA cloning kit from Invitrogen.

The cloned 16S rDNA were sequenced in MWG Biotech, Germany. The sequenced DNA was then compared to the National Center of Biotechnology Information (NCBI) database using the BLAST program, which is software that compares your DNA sequence with the sequences from a large database of microbes. Matches that are greater than 99% signify microorganisms of the same

genus but different species, while matches less than 99% signify novel microorganisms.

Molecular analyses on the biofilm showed the presence of ten microorganisms, while molecular analysis on cultured samples, indicated the presence of 17 microorganisms.

These organisms ranged from 91%99% match with NCBI database, and therefore signify that all the organisms found in the spring were novel. Table 1 shows the amount of microorganisms found from both cultured and biofilm samples, and their closest culturable relative.

Table 1: Microbial Diversity present in Iron Spring and their closest relatives

Closest Relative % Identical

Cultured Match*

Biofilm Match**

Sideroxydans 9296 4 4

Gallionella 9395 0 6

Cyanobacterium 90 0 1

Nitrospirae 9396 6 0

Rhizobiales 98 1 0

Labrys 98 1 0

Hyphomicrobiaceae 97 1 0

Finegoldia 99 1 0

Mariprofundus 94 1 0

Planctomycete 91 1 0

Candidatus 96 1 0

Uncultured Bacteria 9397 5 0

* Microorganisms discovered via molecular analyses on cultured organisms

** Microorganisms discovered via molecular analyses on biofilm


Though almost all of the clones matched less than 99% with the organisms in Table 1, by studying these relatives of the clones, it is possible to get an idea about the preference of electron acceptors and carbon source of the clones.

Sideroxydans, Mariprofundus and Gallionella are iron oxidizing bacteria. They are preferentially, chemolitothrophic, microaerophilic neutrophiles. They oxidize ferrous iron (Fe 2+ ) to ferric iron (Fe 3+ ), and use the energy captured via this process, to produce organic carbon (Figure 2).

Figure 2. Gramstained cells on the glass slide of flow cell after seven days of exposure.

The iron associated with the biofilm was 100,000 times greater than iron dissolved in the spring water (Kotelnikova, S. 2007). Microcosm experiments (Kotelnikova, S. 2007), indicated that there were iron oxidizing bacteria present in the iron spring. It is possible that the iron oxidizing bacteria in the spring is utilizing similar metabolic pathways as their closest relatives for the oxidation of iron.

Nitrospirae and Candidatus are microbes that utilize nitrite oxidation as a source of energy and reducing power. In the process of oxidation, nitrate is produced.

Gallionella and Nitrospirae are not only lithotrophic organisms, but also chemoorganotrophic microbes. Such organisms can switch between two different sources of energy and are usually referred to as mixotrophes. They are capable of using organic carbon like acetate and carbohydrates as their source of energy.

Rhizobiales, Labrys, Hypomicrobiacea, and Finegoldia are heterotrophic microbes. Finegoldia is the only bacteria of the four that is anaerobic. It is usually part of the natural human flora associated with the mucocutaneous surfaces of the body. They are opportunistic pathogen and can result in several diseases including endocarditis.

Rhizobiales, Labrys, Hypomicrobiacea, and Finegoldia are aerobic chemoorganothrophs. Some Rhizobiales strains are capable of reducing atmospheric nitrogen to nitrates when associated with the roots of plants.

Several cultured organisms from the spring displayed similar budding and ciliated morphologies like the microorganisms Hyphomicrobiaceae and Labrys (Figure 3).

Figure 3. Cultivation of biofilm organisms in Galionella medium dilution 8.


Similarly, isolates in the spring were seen with comparable twisted, plait like structures that are usually associated with Gallionella.

However, since no pure isolates were obtained, the sequences isolated could not be compared to the morphologies of the isolates.

The data retrieved from the BLAST search can also be used to develop phylogenetic trees. Phylogenetic trees are graphical representations showing the highest probable route of evolutionary migration and relation to other microorganisms. By producing such trees, it is possible to get an idea about the level of relationship between different microorganisms and from where these microbes diverged.

Figure 4: Phylogenetic Tree for Clone 9.

Figure 4 is a phylogenetic tree for the isolate clone 9. Each immediate vertical branch indicates sister organisms while nodes indicate areas of speciation. From Figure figure, it can be seen that clone 9 is not closely related to its closest identified relatives. Furthermore, the closest relatives are comprised of mainly uncultured bacteria, which indicates the absence of morphological and biochemical information for such isolates, and thus stresses the importance of culturing technique.

The amount and diversity of organisms isolated via culturing was strikingly greater (17) than those isolated from sole molecular analysis of biofilm

(10). This is due to the biases that are present in each technique. Culturing techniques create a bias representation of the microbial environment by over supplementing with excess nutrients. In this case, it was over supplementation of ferrous iron. As a result, the microbes grown via culturing may have given a representation of the nondominant microbial population in the iron spring.

On the other hand, molecular analysis of biofilm results in isolation of probably the most dominant microbes. Furthermore, limitations like primer specificity, PCR and cellular lysis inhibitors, and even sampling, all limit the ability to obtain a true representation of microbial diversity. Nevertheless, by utilizing both of these techniques, it is possible to obtain a better representation of the spring’s microbial population.

Molecular technique results in the identification of organisms via genetic analysis. However, when novel organisms are discovered, as is the case with the iron spring, this technique is lacking; there is no visual or biochemical information linking the sequences to a particular identified isolate. Thus, future goals of this research are to match these new microbes with their morphologies.

Such future endeavors may utilize Fluorescent in situ Hybridization (FISH) probes made using the sequence isolated. These probes can then be used to adhere to the microbe from which the sequence originate, fluoresce, and indicate a possible match.

Another option is to culture and isolate the microbes, then extract their DNA, and compare it for a 100% match with the previously obtained sequences.

At the end of this research, it is expected that the physiology, morphology, biochemistry and genomic functions of all these novel


microorganisms will be discovered and understood in greater depth. As a result it is anticipated that such knowledge may lead to a greater understanding of Grenadian springs and that perhaps novel microbial metabolic products may be of biotechnological and pharmacological significance.

Ravinda Naraine during his seminar presentation at WINDREF.

Submitted by Ravinda Naraine* Dr. Thomas Smith**, Lotta Hallback***

S. Jagevall***, K. Pedersen*** and S. Kotelnikova*

* SGU, CERI/WINDREF ** University of Arkansas, and

***Microbial Analytic, Gothenburg,

7.8. Circadian cycle of Iron Oxidation in the Warm Soda Springs in St Andrew’s, Grenada, West Indies

The target of our study was the warm soda springs, which is located on the wall of the caldera of an old volcano next to the Mountain of St. Catherine (406 m above the sea), in the rain forest of St. Andrews, Grenada (N12 o 08’870”, W61 o 40’562”). The springs contain bright yellow, brown and greenish colored biofilms, both in the water and on the stones. The water is clear and saturated with gas. The gas that bubbles

up from deep inside consists mainly of carbon dioxide (“Fountain of Youth” in the iron springs in St. Andrews, Grenada: preinvestigation stage” by Kotelnikova, Pedersen and Hallback, 2006). The springs’ microbial biofilms contained about 1,000 times more iron than the water (Kotelnikova et al., 2006). Therefore the microbial iron cycling was studied using the original biofilms and the spring water.

The water from the springs is used by Grenadians as source of a “healthy” and “youthgiving” drink. Elderly people bathe in the pools to get rid of joint pains caused by arthritis or rheumatism. People come up the mountain to sit in the warm pool and rub their bodies with the brownish sediments from the pool. They also drink this water after diluting it into whisky as a traditional medicine for pains and aches. In other words, the St. Andrew’s Soda Springs so called “Sulfur Springs”, because of the yellow coloration of the biofilms, are considered to be a “Fountain of Youth” in Grenada (Kotelnikova et al., 2006). The research carried out in Grenada proves useful in that upon identification of the chemical composition of water, gas and biofilms as well as microorganisms responsible for iron oxidation in the springs.

Grenadians bathing in the pool of warm water, 10 February 2007.


The site is one of the major tourist attractions on the island. More information about the chemical and biological processes taking place in the water would attract even more visitors and allow the scientific guidance. More information about potential effects of the water, gas, biofilms, and microorganisms on the water quality and public health will be beneficial for the public and private sectors, and in turn has potential to assist the island’s economy and Grenada as a whole.

Identifying members of the biofilmassociated bacterial community is important as it may provide insights into microbial contributions to the biofilm growth based on totally autorophic way of life and their general physiology and ecology. As a result, studies into ironoxidizing biofilm associated microbial diversity also have important implications for the management of natural spring ecosystems. The difference in findings between research groups regarding the biogeographic variability of iron oxidizing biofilmassociated bacteria highlights the need for further study in this area, and this is one of the primary research aims of the current project.

Effective environmental management strategies are dependent upon knowledge of patterns in microbial diversity in different environments and the threats to their existence as well as the potential uses of these resources. This project contributes to this knowledge through an increased understanding of biofilmassociated microbial communities based on iron oxidation, and therefore has significant relevance for research into renewable sources of carbon and energy and environmental management. Biological diversity may help maintain ecosystem

functioning by increasing resilience to disturbances and enhancing productivity and stability. Biodiversity, therefore, may well provide a large number of goods and services (e.g. food supplies, opportunities for recreation and tourism, and sources of medicines and energy) to human populations and it is essential that it be conserved (CBD 2000). Conservation of the microbial gene pool is also a critical issue due to their direct value, through biotechnological development, as major pharmaceutical and industrial resources.

Studies concerning the taxonomic characterization of biofilm associated microorganisms are of interest as the microorganisms are involved in the cycling of elements, carbon sequestration, heavy metal binding to the iron hydroxides, negatively charged exopolymeric substances and cells in the biofilms, and decrease of concentrations of the heavy metals in the spring water. In addition, these organisms may be involved in the production of secondary metabolites of pharmaceutical relevance. There is a need to develop a better understanding of the biology of unique microorganisms for effective explorations for new metabolic pathways. Phylogenetic data on microbial community composition in the biofilms are of biotechnological importance since these data assist in the rational selection of culture conditions to increase the diversity of bacteria available for natural product screening. In addition, increased understanding of the symbiotic relationships between ironoxidizing and photosynthetic bacteria in biofilms in general provide a better understanding of the ecological function of some natural microbial communities, which may lead to the discovery of potential mechanism of


carbon sequestration based on new metabolic interactions between chemolithotrophic and photolithotrophic microorganisms.

The aim of this research was to study, using culturing and molecular techniques, the microbial diversity of the biofilmassociated community found in the Warm Soda Springs in St. Andrews, Grenada. We hoped to determine whether these microbial communities show biogeographic variability. Our project dealt with further characterization of the warm soda spring and determination of ironoxidation rates in the in vitro experiment. In addition, we attempted enrichment and isolation of ironoxidizing microorganisms from the warm soda spring. We designed and performed our experiments in order to determine what species of bacteria are present in tropical ironoxidizing biofilm community (the results are present elsewhere: R. Naraine, T. Smith, L. Hallback, S. Jagevall, K. Pedersen. and S. Kotelnikova “Microbial diversity in the ironoxidizing biofilms of soda springs in Grenada” in WINDREF Annual Report, 2007) .

The current article describes what the rate of ironoxidation in the biofilm was and how the sunlight affected the iron cycling and the microbial diversity in the biofilms in the soda springs.

SETUP OF THE MICROCOSM EXPERIMENT In addition to molecular

characterization and isolation, photosynthesizing ironoxidizing community microcosms were set up to mimic the cycling of iron in the spring. To do this, 5 ml of the biofilm was added to two different sets of 13 ml tubes filled with aseptically collected spring water. The microcosms were

incubated on the porch of SGU’s Marine Station; one set was exposed to both day and night light while the other set was kept in tight dark box. The microcosm systems contained only naturally present ferrous iron as an electron donor. In addition, the system contained carbon dioxide as a carbon source for autotrophic microorganisms. The setup aimed at creating conditions that mimic the temperature, pH, chemical composition, level of the sunlight illumination and diurnal cycle typical for the spring’s natural environment and promote microbial growth based on its selective need for ferrous iron. Half of each set of tubes was added with formaldehyde (2.5% final) and used as a control. Furthermore, filter sterilized spring gas was added into the bottom of the tubes with syringes on a weekly basis. The tubes were locked, therefore unless produced de novo oxygen could be limited. Concentrations of ferrous iron were analyzed by using the ferrozine method. Calibration curve was produced. Individual microcosms were removed from the incubation space every week in duplicates, half of them were reduced chemically and concentrations of ferrous iron measured. The rates of iron oxidation were calculated in mg/g/day by using the extrapolation of the charts produced and based on the experimental data. The variable rates under the light were compared to the rates in darkness using Ttest (p<0.005). Phasecontrast microscopy was used to determine whether there was any microbial growth associated with oxidation of iron in the biofilms of the spring.

EFFECT OF SUNLIGHT ON IRON CYCLING AND STRUCTURE OF THE BIOFILMS


The rate of iron of oxidation in the microcosms exposed to daylight alternated with the night exposure were significantly higher than the iron oxidation rates in the microcosms incubated in darkness all the time. In addition, the dark microcosms presented an active reduction of iron which took place at the constant rate during the experimental period. These results indicates that oxidation of iron is enhanced in the biofilm which is exposed to the daytime light (Figure 2).

Iron oxidation, mg/l/day

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Figure 2. Chart presenting average amount of ferric iron in the respective microcosms exposed to the light/dark (1) and darkness only (3). 2 and 4 are control microcosms.

Nighttime darkness promoted ironreduction and provided the iron oxidizing microorganisms with electrons from ferrous iron. Therefore the microbial community observed in the spring is using iron as an electron shuttle between different groups of microorganisms. The redox potential is affected by the temperaturedependant solubility of oxygen (33°C, 0.5 mg/l of oxygen), microbial respiration and the illumination. The mechanism responsible for the enhancement of iron oxidation under the daylight is not known, however we observed cells and morphologies typical for photosynthesizing microorganisms in the light exposed microcosms. A large

number of heterocystcontaining cells were observed (Figure 3).

Figure 3. Morphological identification suggested the presence of Nostoc, Pseudoanabena and Rhaboderma.

The rate of ironoxidation ranged between 1.15 and 7.08 mg/l/day with standard deviation of 0.5% between replicates in the microcosms exposed to the light (Figure 4).

Iron oxidation under light

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Figure 4. Iron oxidation in the light microcosms. The rate of iron oxidation was calculated as 1.15 mg/l/d after the inoculation.

The light exposed microcosms presented a photosynthesizing community of slimy green biofilm with mucilage forming a gelatinous matrix. This community consisted from at least 10 different cyanobacterial partners forming large heterocysts, chains of cells, including Nostoc, Phormidium, Pseudoanabena like morphology; Scitonema, Rhaboderma, Nostoc like cells, threads of photosynthesizing


organisms of 50100 µm long similar to Heliotrix, Oscillochloris, Oscilatoria and large cocci of organisms similar to Chlorococcus 56 µm in diameter. Diatoms resembling Nitzschia, Naricula and Anthautheus were also present in the original biofilms. All the cells that showed green coloration under the phasecontrast microscope were inspected under UV light and 1,000X magnification. The red epifluorescence of the cells indicated that they contained chlorophyll A. This consortium was actively producing oxygen. There was no black reduced iron present in the system. Large oxygen bubbles were trapped in the biofilm mucilage. Carbon dioxide was produced as a result of respiration and consumed by autotrophic microorganisms. Supposedly, the photosynthetic microorganisms were producing oxygen, which might have been oxidizing iron chemically; however the process was not sufficient to compensate the respiration already after 20 days of incubation (Figure 5).

Iron reduction in the biofilms in the dark microcosms, n=3.

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Fe 2+, m

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Figure 5. Iron reduction in the dark microcosms. The rate was calculated as 3.2 mg/l/d after the inoculation. SD was 34%.

Microscopically detected exopolymeric substances produced by the photosynthetic organisms were inferred to be oxidized, oxygen was not anymore sufficient; therefore microbial succession shifted the community to the iron reduction (Figure 4).

The dark microcosms presented the ongoing reduction of iron already after a day of incubation (Figure 5). The microscopic observations showed completely different cellular morphologies being associated with the dark microcosms in comparison to the light ones (Figure 6). There were no cyanobacteria present. The dark experiment resulted in the cell morphologies similar to the observed in the flow cell exposed to the open system with cystlike cells and iron aggregated, long filaments reminding Toxotrix and no Galionella stalks. The tube contained dark precipitate covered with brown biofilm on the top. There were ring forming biofilms developed in the microcosm. Small grampositive rods and budding irregular cells developed around the aggregates of ferric iron (Figure 6).

Figure 6. Cellular morphologies in the dark microcosm. Magnifications of 1,000X.

Rate of iron oxidation, mg/l/day

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Figure 7. Observe the lack of iron oxidation in the dark microcosms.


The circadian rhythm was affecting the oxidation and reduction of iron, which was the compound shuttling electrons between different members of the microbial community. This process contributed to the efficient energy conservation, observed efficiency of carbon sequestration and biofilm growth in the spring. In addition to the energy of the photosynthetically produced biomass, mixotrophic iron oxidizers could have used the energy of reduced iron. Galionella is known as neutrophilic mixotrophic organism capable autotrophic carbon dioxide fixation and ferric iron oxidation. The photosynthetic organisms identified in the biofilm are also known to be mixotrophs. Therefore, the whole microbial community may be reshuffling between autotrophic way of life (photosynthesis and iron oxidationbased carbon fixation) in the daylight to the oxidation of organic matter and respiration of oxygen and ferric iron in the nighttime. This is an interesting example of the natural system and microbial community that amazingly adapted to the low energy and carbon ecosystem. Such a selfindependent system could have been an extraterrestrial environment or an original pool where the life was born and developed. In any case, the soda spring presented an excellent example of an ecosystem recycling carbon, nitrogen and iron. The natural system, which possesses enormous potential of carbon and energy sequestration, may potentially be used as a model for the source of renewable energy.

Submitted by Svetlana Kotelnikova* B. Samuel,* D. Garraway *

and Thomas Smith** * SGU, CERIWINDREF

** University of Arkansas, USA

7.9. Do the Microorganisms in the Soda Spring Water Derive Energy from the Oxidation of Manganese?

The water in the Grenadian soda springs contained bright yellow, and greenish coloured biofilms. Gas bubbles were seen primarily near the green biofilmcovered rocks where photosynthesis was evident, and it was noted that this occurred on the west and south side of the spring which received more sun during the day. (Figure 1).

Figure 1. Soda springs with bright yellow sediments due to the presence of ferric iron.

Oxygen content recordings were much higher in these areas of the spring. The spring’s gas content was analysed by Microbial Analytics Sweden AB in a previous study (Kotelnikova et al, 2006). The gas that bubbles up from deep within Grenada is primarily carbon dioxide and nitrogen. Other gases were evident in smaller amounts and they included argon, methane, helium hydrogen and carbon monoxide. The water content of the spring was also analyzed and high concentrations of the following physiologically important metals were found: manganese, iron, cobalt, copper, aluminum, zinc, cadmium, chromium, molybdenum and nickel. Biofilm samples for DNA analysis were collected using a


syringe to ensure human microflora did not contaminate the sample. We collected our sample of biofilms from the southern end of the spring. Samples for microscopy were also collected in plastic bottles from the southern end of the spring. The water containing the biofilms was scooped up into the plastic bottles. In a previous study (Kotelnikova et al, 2006) the concentration of manganese in the water was detected at 672 mg/l compared to 235 mg/kg of dry biofilm collected from the same spring. There is no information available on the presence of manganese oxidation in the biofilms of the spring.

Do the microorganisms in the soda spring water derive useful energy from the oxidation of manganese?

Two set of tubes containing JAC medium differential and enrichment for Mn oxidizing bacteria were inoculated by freshly collected biofilms from St. Andrew’s Soda Spring, Grenada in February 2007. The enrichment cultures were incubated at 30C for two weeks and serially diluted onto the agar plated containing JAC agar. Twelve pure cultures have been isolated and tested for the ability to oxidize manganese.

After a number of tubes had been inoculated, control tubes were inhibited with 2% formaldehyde.

Both sets of tubes were incubated in light conditions at the Marine Station along with standard control tubes. The Leucoberbelin blue (LLB) method was used to quantify the relative amounts of oxidized manganese in both sets of tubes. The curve of absorbance and hence concentrations of a representative sample of these tubes is shown below as well as the calibration curve which was prepared from standard solutions of MnO4.

After the four day period, growth was observed on the agar plates. To

ascertain whether the colonies grown on the media were actually capable of manganese oxidation, tubes of liquid JAC medium were inoculated with each of the different colonies. These tubes, along with an uninoculated control tube, were incubated at 30 degrees Celsius for a period of 13 days. After this period, the LBB assay was used to determine the level of oxidized manganese present in the tubes. In addition, the morphologies of the different colonies were documented from direct observation as well as microscopy (Figures 2 and 3). Each of the distinct colonies was taken off the plate with a sterile bacterial loop and placed on a slide with a small amount of water. The slides were air dried and then heat fixed and Gramstained.

In general the shape, size and features of the microbes were unusual and they were not easily classified. Some of them were cocci, which are round, and some were bacilli, which are rod shaped. There were others that were not easily categorized as round or rod shaped, and a few had produced endospores due to the stressful and unfamiliar environment to which they had been subjected. There were Grampositive as well as Gram negative microbes seen.

The LBB assay of the JAC tubes that were incubated with the different colonies did not prove that the organisms oxidized manganese since the absorbance measured by the spectrophotometer was insignificant when compared with the absorbance of the control tube. Therefore we were not able to prove that our cultures oxidized manganese.

LIMITATIONS OF RESEARCH: 1. Difficulty encountered in attempting

to reproduce the natural growth conditions of the microorganisms under study.


Figure 2. Grampositive and spore producing cells of strain Mn3 grown in the presence of MnO4. Magnification of 1000X.

Figure 3. Cells of strain Mn9 surrounding MnO4 particals. Magnification of 1000X.

2. The number of studies previously done is small.

3. Knowledge of the manganese oxidizing microbes is limited.

4. The microorganisms responsible for manganese oxidation are only capable of doing so in their natural habitat or one very similar to it.

Submitted by Maurissa Walker and Teresa Cains

7.10. Novel Marine Bacteria and their Antagonistic Properties Against Medically Relevant Biofilms

Biofilms, complex microbial communities enclosed in a polymer matrix, are involved in more than half of all chronic infections. We are currently unable to treat these infections successfully because biofilms are resistant both to the cells of the immune system and the existing antimicrobials. The goal of this study was to characterize novel marine isolates from Parrotfish (Sparasoma viridae) (designated as strains P32, P44, P52 and P 66) and a marine rock (strain PB711B); screen crude ether and supernatant extracts from the marine isolates for antibiofilm activities against human pathogens (Staphylococcus aureus, S. epidermidis, and Pseudomonas aerugionsa); determine the nature of the active substances in the extracts (as far as, broadspectrum, Gram negative quorum sensing inhibitory, enzymatic, proteinaceous, and antibacterial); choose one to study in more detail; and determine the mutagenicity and toxicity of that extract. Together, the marine isolate crude extracts showed inhibitory or stimulatory effects on pathogenic biofilms. Supernatant extracts from isolates P44, P52, P66, and PB711B resulted in broadspectrum antibiofilm activity. Genetic strains reporting quorum sense interfering compounds were used to test all extracts. None of the extracts resulted in antibacterial activity against the pathogens. P32, P44, P52 and P 66 ether extracts did not interfere with Gram negative quorum sensing. Additionally, none of the extracts contained measurable amounts of proteins in accordance with the methods used. The strongest antibiofilm activity was observed for P32


stationary phase supernatant extract (n=36) against 24 hour S. aureus biofilm, significantly reducing its biofilm formation by 51.84% (p=5.35x10 13 ) compared to negative controls (n=24). Thus, it was chosen for further testing which resulted in a 28.43% (p=3.33x10 11 ) reduction (n=252) of 48 hour S. aureus biofilm when compared to negative controls (n=168). The P32 supernatant extracts did not prove to be mutagenic in accordance with the Ames test. Only one percent of microorganisms were cultured while the remainders are bacteria known by their gene signatures only and being a part of a gene library associated with certain environments. The most common practice of microbiologists today regarding the study of microbial diversity is to extract DNA from an environment, amplify a gene of choice with PCR, clone it, sequence the genes from the clone library, compare them using the database, only to find out that the majority of the most closely related genes belong to organisms that have never been cultured therefore, with nothing determined about their physiology. The physiological and biochemical information about cultured microorganisms is extremely important for the future reference, future applications of the organisms in biotechnology, optimization of cultivation process, as well as for the identification of the most related organism, defining its unique nature, predicting metabolites associated with certain activities, and exploiting its metabolism as a potential source of novel therapeutics. Finally, physiological characterization is important in regards to the study of microbial ecology, which is the relationship of the microorganisms with their physical and biological environment. Microbial ecological approaches are used to identify and explore the role the

organism plays in the ecosystem and identifies the mechanisms of interaction with other members of the community and/or a host.

A polyphasic taxonomic approach was used to study the taxonomy of the marine isolates. 16S rRNA gene sequencing demonstrated that isolates P3 2, P44, P52, and PB711B represent new sublines within the genera Aerococcus, Psychrobacter, Erythrobacter and Micrococcus, respectively. The sequencing of the 16S rRNA gene demonstrated that isolate P6 6 represents a new genus. On the basis of phylogenetic and phenotypic evidence, it is proposed that the novel marine bacteria (P32, P44, P52, P66, PB711B) be classified as Aerococcus piscidermidis sp. nov.; Psychrobacter piscidermidis sp. nov.; Erythrobacter piscidermidis sp. nov.; Micrococcus piscidermidis sp. nov.; and Brunonia piscidermidis gen. nov., sp. nov. The type strain of Aerococcus pisciepidermidis is PTA6763 T (=CCUG T ); Psychrobacter pisciepidermidis is PTA6682 T (=CCUG T ); Erythrobacter pisciepidermidis is PTA6764 T (=CCUG T ); Micrococcus piscidermidis is PB711B T (=CCUG T ); and Brunonia pisciepidermidis is PTA6766 T (=CCUG T ). The antagonistic properties and nature of the antibiofilm stubstances were studied further by determining if the substances: 1. have antibacterial activity against the

pathogens S. aureus, S. epidermidis, and/or P. aeruginosa because the presence of antibacterial substances indicates that the antibiofilm activity observed may have been due to the killing or inhibition of growth of the pathogens reducing the number or organisms able to form a biofilm;


2. have Gramnegative quorum sensing inhibitory molecules similar to those which include analogs of SAM that inhibited AHL signal generation by competitive inhibition or brominated furanones that are molecular analog of AHLs;

3. have enzymatic activity that has the potential to target the EPS of the biofilm or signaling molecules which would inhibit biofilm formation as enzymatic biofilm reducers include bismuth dimercaprol that reduced polysaccharide production and biofilm density in Pseudomonas aeruginosa ERC1 biofilms, DNAse that has the potential to target biofilms with DNA in the matrix, and AiiA, that has been shown to inactivate Gramnegative AHLs; and

4. contain proteins or polypeptides because their presence may indicate Grampositive QS AIP molecular analogs. which include RNA III inhibiting peptide that inhibits RAP which is part of the RAPTRAP QS system in Staphylococcus spp. and the protein produced by Pseudoalteromonas tunicata with a broad range of biofilm inhibition.

The nature of the antibiofilm substances is a very important aspect in regards to the practical applications of antibiofilm substances which include: topical applications for wound infections (e.g. burn victims), ear drops (e.g. otitis media), incorporation into indwelling medical devices (e.g. catheters), probiotics (e.g. fish and poultry farms), antifouling nontoxic paint (e.g. boats), and pipe coatings (e.g. water systems). For example, the isolation and usage of enzymes that target the EPS of a biofilm could be used in industrial applications, however, they would not be ideal to use in the human body upon which the

Closest Related Species

P32 %

similarity

P44 T %

similarity

P52 T %

similarity

PB711B T %

similarity A. viridans T 67 − − − P. pacificensis T − 71 − − P. immobilis T − 76 − − P. phenylpuruvicus T − 84 − − E. citreus − − 65 − C. alkalitolerans T − − − 54

Table 1. Percent similarities of marine isolates with closest related species comparing all expressed features (morphology, physiology, and enzymology). A percent similarity was determined for each pair by dividing the number of shared characteristics by the total number of characteristics tested for.

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Figure 1. Bar graph of mean absorbance (490nm) representing the percent reduction of 24 hour S. aureus biofilm formation by supernatant extract from stationary phase of isolate P32 grown in TSB (n=36) as compared to negative control (consisting of pathogen and sterile media) (n=24), and positive control consisting of pathogen and penicillin (final concentrations 1000 μg/ml) (n=24). Bars represent standard deviation values for each mean.

enzymes could have a harmful effect. Quorum sensing signaling inhibitors could be used in the human body (in conjunction with antibiotics) to prevent or treat biofilms on skin, tissues, or indwelling medical devices. The detachment properties should be studied first because if the compounds has this activity it may cause detachment of existing biofilms, clogging of blood capillars and vessels, disruption of blood circulation, release of virulent toxins and other catastrophic events leading to the death of patients. Please warn about risks


associated with the usage of QS for non topical applications for human patients. The use of probiotics in fish farms may lead to colonization of fish surfaces with the bacteria which produce enzymes that target pathogenic biofilm EPS or QS signaling and by doing so prevent fish pathogens from attaching to the surface.

Marine microbes that prevent biofilm formation have extremely important biotechnological application because 65% of antibiotic and immune resistant pathogens grow in biofilms on human organs and medical devices. New antibiofilm strategies can be divided into categories by their target (e.g. quorum sensing or EPS matrix) and these methods can be preventative (e.g. probiotics) or used to treat an existing biofilm infection in conjuction with antibiotics. The most feasible practical applications for isolate A. piscidermidis P32 antibiofilm supernatant extracts against Staphylococcal bacterial settlement would be as a topical treatment of Staphylococcal skin infections and/or incorporation in indwelling medical devices for prevention of Staphylococcal biofilm formation. These therapeutic application choices are supported by the results that there were no enzymatic or toxic properties of the extracts. Prevention of biofilm formation would allow for antibiotics and the immune system to remove the planktonic cells.

Before novel therapeutics can be used in humans to target antibiotic and immune resistance of biofilms, it is necessary to test them in vivo to find out if they trigger release of toxins by the pathogens or massive detachment of pathogenic biofilms. Before these applications could be used in humans, the active antibiofilm substance in A. piscidermidis P32 supernatant extracts should be isolated using activity

fractionation, structurally identified via advanced mass spectrometry, and studied to determine the in vivo effects. A method that can be used to determine the in vivo effects of A. piscidermidis P32 extracts is with the induction of graft associated infections that was previously used by Balaban and colleagues (Balaban et al., 2003). In short, rats are anesthetized and an incision is made in their back. A graft, previously soaked in the antibiofilm substance or control, is implanted in the back. Subsequently, S. aureus would be innoculated onto the graft surface along with controls. Grafts would be assessed after their removal by sonicating the graft and plating serial dilutions to obtain the numbers of viable bacteria that attached to graft (Balaban et al., 2003). A risk exists that the extracts which are used to prevent biofilms will detach clumps of biofilms after they have already developed. Even if we showed that the extracts from the marine bacteria prevented the attachment of pathogens on polysterol surface, there is still a potential that the pathogens bacteria may attach on adhesive tissue surface and they would still be impossible to be removed by antibiotic or our immune system.

The broadspectrum antibiofilm substance produced by isolates P. piscidermidis P44, E. piscidermidis P52, B. piscidermidis P66, and M. piscidermidis PB711B may be incorporated into coatings as antifouling agents which could be applied to boats to reduce marine fouling, as an antibiofilm coating on water pipes to prevent bacteria from contaminating the inside of the pipes, or as probiotic cells on the surface of fish protecting the fish from the settlement of pathogens on their surfaces. The marine applications are supported by the results previously obtained for isolates A. piscidermidis P32, P. piscidermidis


P44, and E. piscidermidis P52, which were previously screened for in situ anti biofilm and antifouling activity using gels on glass slides exposed to the seawater (Bruno, 2004). The in situ attachment of marine biofilms was inhibited 84.5% (p=0.024) by ether extracts of isolate E. piscidermidis P52 and those extracts were not bacteriocidal. In regards to marine fouling, in situ photosynthesizing eukaryotic colonization was inhibited 41.7% (p=0.013) and 78.2% (0.009) by ether extracts from A. piscidermidis P32 and living cells of isolate P. piscidermidis P44, respectively and in situ nonphotosynthesizing eukaryotic colonization was inhibited 45.4% (p=0.009) by ether extracts of isolate, A. piscidermidis P32.

Submitted by Cynthia K. Bruno, MSc

7.11. Physiological Characterization of Novel Marine Bacterial Species Isolated off Grenada

The sea is home to many bacteria. Some have been fully characterized, however many have either not been isolated or have only been characterized by RNA and not by physiological properties. Our samples were collected from biofilms on marine rocks in many different bays near St. George’s University, Grenada. It was shown by Cynthia Bruno that one of the species showed potential as an anti biofilm agent against common medically and industrially harmful bacteria. This finding prompted further characterization of the bacteria, including its physiological characteristics, to accompany the 16S RNA sequences already elucidated. Bacteria with antibiofilm properties may be key in controlling otherwise difficult to control bacteria.

Grenada Reef

The collected species were grown at varying pH and temperatures, as part of an effort to characterize them. The samples were also subject to API testing to differentiate metabolic properties, and distinguish them from other known species.

The API testing proved differences from known species, and it was found that the isolates grew at an optimum pH of 78.

METHODS: In order to test the most active pH,

a range of pH values were chosen from very acidic (pH 2), to very basic (pH 12). Growth media was made from malt, yeast extract, sodium chloride, and potassium chloride. This media was then autoclaved and separated into test tubes for pH buffering.

The buffer for each acidic pH was created using varying amounts of citric acid and sodium citrate. These mixtures were then brought to equal volume by adding a needed amount of distilled, autoclaved water. The basic pH buffers were created using varying amounts of sodium hydroxide, and each was brought to the same volume as the acidic pH, using distilled, autoclaved water. Buffer and media were mixed in a one to one ratio, and each was tested for pH. A buffer and


media solution aliquot was then added to test tubes, two tubes for each bacterial sample, for each pH being tested. The tubes were then covered and autoclaved. A sample of tubes from each pH were tested for maintenance of postautoclave pH.

A small amount of bacteria from each culture was then incubated for 24 hours in a shaker bath. Samples of bacteria from these colonies were then added to their respective tubes in each pH category. The tubes were tested for initial turbidity using a spectrometer.

The tubes were tested again for turbidity every hour after the initial test, and were incubated at 30 degrees Celsius between each test. The results were recorded, and then after 24 hours, they were tested again several times.

After curves were completed of time versus number of cells were completed, and generation time was calculated, curves of generation time to pH were completed. Throughout the experiment, periodic checks for pH stability were taken. These pH tests revealed that in two of the organisms under study, PB 521, and P32, the bacteria were actually producing an acid, and lowering each pH to around 5. These organisms were not concluded in the final study due to this fact. Organism P52 showed optimum growth at pH 8, and the other three organisms, PB711, P44, and P66, showed optimum at 7, with a range up to 8 for P66 and PB 711 and up to 10 for P4 4. (See appendix) The pH was also tested for pH 12, but the absorbance data were inconsistent with data obtained from plating the tubes.

API TESTING: The API tests revealed the

percentage of differences between the novel organisms, PB 711, and PB 521, and closely related vibrio species. The

P44 Optimum Growth Curve

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results of the tests showed that PB 711 and PB 521 were only 53.21% similar to each other. Also, PB711 was 75% similar to V. Harveyii, and PB 521 was 87.5%


similar to V. Alginolyticus. These similarity percentages indicate that the species under study could be novel.

CONCLUSION: Optimum pH testing showed that

the bacterial strains collected from Grenada were similar to the closest known species. API testing for other physiological characteristics also concluded similarites with known organisms that ranged from 59.4%87.5%, showing that the species in question could be novel. Further studies should be undertaken to determine characteristics so that the species can be considered novel bacteria.

Submitted by Jason Johnson, Jayson Weir, and Cynthia Bruno

7.12. The Ecological Distribution of Viable Indicator Organisms and Associated Health Risks from St. George’s Bay in Grenada

The coastal environment is an invaluable aspect for the thriving human civilization on earth. This complex ecosystem, with its numerous and unique organism interactions, plays a critical role in the development of its exceptional features. The coastal ecosystem is also a significant link between the terrestrial environment and the vast oceanic environment, incorporating both of their respective ecological properties.

The large value and dependence of humans on the coastal environment is demonstrated by the fact that nearly two thirds of the world’s observed population claims residence within 150 kilometers of the shore. The numerous advantages of living near coastal waters include

moderate climate, ease of transportation, available food source, job opportunities, and the development of unique social and cultural values. Following comfortable settlement of coastal regions, people begin to recognize the aesthetic value and recreational potential of living near the marine environment (Fujioka, 2002). Coastal waterbased recreation has become an important component of leisure activities and tourism growth throughout the world.

In order to compensate for the increased residential population and tourism development near coastal environments, there has been a dramatic increase in the building of cities, homes, industries, hotels, restaurants, shops, harbors, and many other population dense facilities. Unfortunately, with the increase in population density and its associated activities, there is the inevitable increase in the production of point source pollution, discharges of sewage and industrial wastes, as well as nonpoint pollution sources of agricultural and urban runoff, with concomitant discharge into the coastal environment.

The coastal marine environment can be seen as the ultimate sink for the by products of human activity. The larger coastal populations and increasing development have led to a greater loading of toxic substances, nutrients, and pathogens leading to subsequent stress on the coastal ecosystem. The main sources of the polluting substances are river inputs of domestic agricultural and industrial effluents and direct sewage discharges from the local residential population.

A significant effect of the increased loading of these polluting substances is the resulting nutrient enrichment, mainly attributable to the increase in nitrate and phosphate concentrations, leading to localized


eutrophication of the coastal region. Sewage, which is primarily organic in nature, accounts for the greatest volume of waste discharged to estuaries and coastal marine environments. Since coastal waters are typically less oligotrophic than open ocean waters and support more dense populations of phototrophic microorganisms, coastal regions receiving high levels of nutrients from sewage or industrial wastes can lead to very high phytoplankton and bacterial populations. The shallow marine waters can become anoxic from the consumption of oxygen by bacteria, causing the creation of a toxic environment for the marine life in the vicinity, resulting in the production of hydrogen sulfide by sulfatereducing bacteria that quickly develop in anoxic seawater. Long periods of dissolved oxygen values below 5mg/L can harm the life stages for many fish and shellfish species (EPA, 2000). As a result, sewage wastes have caused the eutrophication and dissolved oxygen depletion of coastal environments in many regions of the world. A more pressing concern from the human standpoint is the increase of pathogen density from the influence of sewage discharge into the coastal waters. This not only has a negative impact on the coastal environment by making it less attractive for recreation and causing economic loss as a result from the prohibition in the sale of shellfish from the region, but also leads to the development of an increase in health risks.

It has become increasingly apparent that the ecosystem and human health are intricately linked. The United Nations Group of Experts on the Scientific Aspects of Marine Pollution (GESAMP), in a 1990 survey of the health of the world’s oceans, placed sewage discharges to the marine environment at the top of its list of

concerns. This was not due to the fact that sewage causes damage to the marine environment, but instead the concern for public health risk. In developing countries, with lower standards of public health and nutrition, enteric disease transmitted through coastal marine water was responsible for many deaths, especially among children.

It was previously believed that bacterial and viral pathogens did not survive in marine water and that sea bathers were at little risk from sewage contamination unless the pollution in the waters was great enough to be visible. However, this is most certainly not the case as bacteria can enter a dormant phase becoming devoid of detection by normal methods, and viruses can be very persistent in marine water. As a result, cuts and skin abrasions may become infected, and accidental swallowing of marine contaminated water may result in acute diarrhea caused by bacteria such as Salmonella or Shigella, or lead to an infection by polio or hepatitis viruses. Diseases associated with waterborne infections include mild to lifethreatening gastroenteritis, hepatitis, skin and wound infections, conjunctivitis, respiratory infections, and more generalized infections.

Waterborne transmission is a very effective way of spreading pathogens to a large portion of the population. Numerous pathogens can be transmitted by the ingestion of, inhalation of, or contact with sewage polluted waters. The microbial waterborne pathogens of great concern are those that originate in the enteric tract of humans or animals and enter the coastal environment through sewage effluents. Pathogenic enteric bacteria causing gastrointestinal illness include pathogenic Escherichia coli, Shigella, and Salmonella, while those causing


dermatitis and skin infections include Staphylococcus, Aeromonas, Vibrio algynolyticus and Pseudomonas. There are also over 100 enteric viruses in human sewage, including adenoviruses, Norwalk virus, picornaviruses, reoviruses, caliciviruses, astroviruses, hepatitis A, and unclassified small round structured viruses (SRSV). There are also some more serious illnesses transmitted through sewage contamination of marine water, including hepatitis, cholera, typhoid, and poliomyelitis, but these are significant only in a population where an epidemic is present.

The effectiveness of transmission of these pathogens depends on the amount of fecal contamination in the water, the survival of the pathogenic organism in the water, the infectious dose of the organism, and the level of exposure to an individual in the coastal environment. The determination of pathogenicity is further complicated by the dilution, survival, and resuspension of sedimentassociated pathogens, which are affected by continual environmental fluctuations.

N 12 o 03’ W061 o 45’

N 31.16’’

W 16.01’’

31’’

30’’

29’’

28’’

27’’

26’’

25’’

24’’

32’’

33’’

34’’

35’’

36’’

North Latitude

18’’ 20’’ 22’’ 24’’ 26’’ 28’’ 30’’ 32’’ 34’’ 36’’ 38’’ 40’’ 42’’ 44’’ 46’’ 48’’ West

Longitude

Average Directional Current Vector

Health Risk Based on a 95% Confidence Interval of E. FaecalisMPN Values/100ml at Specific Distance Intervals River: 95% CI : 0 – 39,415 ; Health Risk : >10%GI ; >3.9% AFRI 4070 m: 95% CI: 0 – 229 ; Health Risk: <1% GI ; <0.3% AFRI to 510% GI ; 1.93.9% AFRI 70100 m: 95% CI: 0 – 67 ; Health Risk: <1% GI ; <0.3% AFRI to 15% GI ; 0.31.9% AFRI 100200 m: 95% CI: 0 – 337 ; Health Risk : <1% GI ; <0.3% AFRI to 510% GI ; 1.93.9% AFRI 200400 m: 95% CI: 0 – 190 ; Health Risk : <1% GI ; <0.3% AFRI to 15% GI ; 0.31.9% AFRI 400700 m: 95% CI: 0 – 373 ; Health Risk: <1% GI ; <0.3% AFRI to 510% GI ; 1.93.9% AFRI 7001000 m: 95% CI: 0 – 7 ; Health Risk: <1% GI ; <0.3% AFRI

The hazardous pathogens encountered include bacterial, viral, and protozoan members. The density and variety of these pathogens depend on the size of the human population from which the sewage is discharged, the seasonal incidence of the specific illnesses, and

distribution of pathogens within the community.

The distance of travel and concentrations of the indicators Enterococcus faecalis and Escherichia coli were assessed in the surface water and sediments of the St. John’s River Estuary in Grenada. The antibiotic susceptibility of these indicators was also tested. The main objective was to determine whether a significant health risk exists within the bay, and whether or not the bay is a potential source of antibiotic resistant strains. By measuring distances, the establishment of approximate health risk zones was deciphered and applied to the particular ecological dynamics of the system. Sampling was conducted once per week over the course of six weeks in conjunction with the resulting sample analysis. A Most Probable Number (MPN) method was used for the enumeration of the indicators and the KirbyBauer disk diffusion method was applied for antibiotic susceptibility testing. The St. John’s River MPN levels were equivalent to that of raw sewage. A drastic decrease (~99%) in MPN levels was observed in the surface water approximately 40 m from the coastline and fluctuating levels were found within the bay itself. Sediment sample results were inadequate to effectively determine health risk zones based on possible pathogen resuspension. E. faecalis samples were susceptible to 79% of the antibiotics tested, while E. coli samples responded to 64% of the antibiotics tested. By applying the results observed to water movement dynamics within the estuary, it can be concluded that the St. John’s River estuary is capable of managing the incoming pathogen load, and therefore does not categorize as a significant health hazard zone or a source of dangerous antibiotic resistant strains.


Based on the results of this project, the main hypothesis can be rejected, which initially stated that the large contamination load from the St. John’s River would cause a significant health hazard of a >10% GI (Gastrointestinal) illness risk; >4% AFRI (Associated Health Risks) risk within 100 meters from the coastline. The results showed that St. George’s Bay can effectively dilute and remove the majority of the incoming pathogen load from the St. John’s River effluent. However, a health risk of <1% GI; <0.3% AFRI to 510% GI; 1.93.9% AFRI with a lowest probability of 1 case of gastroenteritis in 100 exposures and a negligible AFRI illness rate to a highest probability of 1 in 10 to 1 in 20 of gastroenteritis for a single exposure and a range of approximately 1 in 50 to 1 in 25 AFRI risk, according to WHO guidelines. There was an indication of hypoxic conditions throughout the bay, with the lowest values in the 95% confidence interval in the hypoxic range in all distance intervals. This supports the hypothesis that hypoxic conditions would be found in the bay. However, since a greater percentage of dissolved oxygen values were above the chronic criterion for growth at most distance intervals, it can be concluded that nonhypoxic conditions are observed most of the time.

The project’s results should prove to be valuable to the various ministries and fisheries of Grenada, providing them with information that can be of importance when considering daily activities in the St. George’s Bay area. Both the government and general public should be made aware of the significant health hazard that the St. John’s River potentially poses to people who may come into contact with its water. As for bathing, fishing, or water recreation within St. George’s Bay, though the project results

indicate a lower than expected health hazard. EPA guidelines would not recommend such activities in this area. However, ultimate discretion should be made by the Ministry of Health in Grenada after consideration of this project’s results and the possible use of future water treatment before discharging contaminated water into the bay.

Recommendations for future research include a greater number of samples to be collected during each sampling date, where each sample site should be resampled during each subsequent sampling date based on the GPS (Global Positioning System) location. The sample sites taken should span the entire bay during each sampling date so that correlations can be formed between the environmental conditions present on the particular sampling date with MPN values obtained. This would provide for more accurate conclusions based on ecological dynamics.

Possibilities for future research include a comparison of other bays around Grenada in diluting and removing land based runoff and determining the reasons for such differences, especially in more shallow bays and estuaries which may not have as much of a dilution effect. Another possibility would be to compare indicator settlement in the sediments of other bays to that of St. George’s Bay and possible health risks due to resuspension as well as the determination of sediment survival rate. Though less feasible, another possible project would be to obtain surface water samples from St. George’s Bay during the early morning hours in order to compare indicator MPN values between the hours of no sunlight influence to that of this project’s results during mid day sunlight presence. This could indicate whether UV radiation plays a significant role in managing indicator concentrations


in the bay in conjunction with the dilution effects. These possibilities for future research would greatly supplement the research conducted in this project and provide for a better understanding of coastal ecological management to human waste influences.

Submitted by Rakesh Patel

7.13. Characterisation of Marine Spongeassociated Bacteria and Cytotoxic Activity of Sponge Extracts towards Human Cancer Cells

ABSTRACT The use of high throughput

cultivation techniques is believed to be an important factor in our ability to assess a broad range of microorganisms associated with a sponge host, hostmicroorganism interaction and microbial functions. The diversity of culturable bacterial communities in four Caribbean sponges, Aplysina fulva, Ircinia strobilina, Ulosa ruetzleri and Xestospongia muta, and one temperate European sponge, Halichondria panicea, was examined. Phylogenetic analysis of 16S rDNA gene sequences from bacteria isolated from these sponges indicated that the majority were related to the Alphaproteobacteria (40%) and Gammaproteobacteria (47%) divisions, with remaining isolates clustering within the Bacteroidetes. Several isolates were closely affiliated to other spongederived sequences from different geographic regions and different host species. However, some isolates appeared to be unique to their particular sponge hosts, suggesting that some isolates may be host specific whilst others are universal to many sponge species regardless of location. In addition, crude extracts from two sponges,

A. fulva and U. ruetzleri, showed cytotoxic activity against ML1 and/or LoVo human cancer cells as measured by the colorimetric XTT cell proliferation assay. This suggests that these sponges may produce compounds as potential anticancer drug leads.

INTRODUCTION Large numbers of extracellular

bacteria populate the mesohyl matrix of many sponges, sometimes contributing up to 50% of the sponge biomass (Vacelet & Donadey 1977). The detailed roles that spongeassociated microorganisms play within sponges are still unclear, but various symbiotic functions have been proposed (Wilkinson & Fay 1979, Ebel et al. 1997, Webster et al. 2001). Surveys of sponge microbial communities have described a diverse range of heterotrophic bacteria, cyanobacteria, facultative anaerobes, unicellular algae and archaea (Hentschel et al. 2001, Taylor et al. 2004, Webster et al. 2004, Li et al. 2006). Many of these include novel species, and one study has reported the presence of a formerly unrecognised evolutionary lineage of the domain Bacteria suggesting a new phylum, Poribacteria (Fieseler et al. 2004). However, there is still some debate regarding host specificity of sponge associated bacterial communities and whether or not they show temporal and spatial stability (Hentschel et al. 2002, Taylor et al. 2004). Several authors have reported that different species of marine sponges, separated by large geographic and habitat differences, harbour closely related microbial communities (Hentschel et al. 2002, Enticknap et al. 2006, Hill et al. 2006). Conversely, research into the relationship between microbial diversity and host specificity of three cooccurring temperate sponges from Australia found that microbial communities varied little


within each species of sponge, but variability among species was substantial (Taylor et al. 2004). Spongeassociated bacteria were also found to be hostspecific in the South China Sea where four sponges from the same geographical location had differing predominant bacterial diversity and community structures (Li et al. 2006).

Sponges are also known to be a rich source of bioactive secondary metabolites that are of biotechnological interest as well as ecological interest, because of their antiviral, antimicrobial, antifungal or antitumour properties (Faulkner 2000). Accumulating evidence has shown that bacteria associated with sponges are the origin of many of these compounds (Unson et al. 1994, Schmidt et al. 2000, Hill 2004). Culturing the symbionts could help to solve the supply problem for spongederived drugs. At present, even excellent drug candidates from sponges are often not developed because the sponges are rare and/or difficult to collect (Faulkner 2000). A number of cytotoxic products have been isolated from marine sponges, but advanced clinical studies have been conducted for only a few of these substances due to the problems outlined above. There are currently two drugs (Ara A and AraC) that were synthetically derived from sponge metabolites in long term clinical use for the treatment of cancer (Newman & Cragg 2004).

The aim of this study was to determine the bacterial species present in one temperate and four tropical sponges and to establish whether spongeassociated bacterial communities vary between different sponge species (i.e. are host specific) or whether uniform communities exist across host species. The study also examined the cytotoxic effects of crude extracts from the five species of marine sponges on two human cancer cell lines.

SUMMARY OFRESULTS AND DISCUSSION

Overall, 70 bacterial isolates were cultured from the Grenadian sponges Aplysina fulva (16 isolates), Ircinia strobilina (17 isolates), Ulosa ruetzleri (20 isolates) and Xestospongia muta (17 isolates) and have been stored in triplicate in a culture collection at St. George’s University, Grenada. In addition to the Grenadian culture collection, 15 isolates were also cultured from the temperate sponge Halichondria panicea. Genomic DNA was successfully extracted and amplified from 44 Grenadian sponge isolates (11 from each sponge species studied) and 14 H. panicea isolates. Altogether, partial 16S rDNA sequences were obtained for 15 isolates from the sponges A. fulva (3 sequences), I. strobilina (5 sequences), H. panicea (2 sequences), U. ruetzleri (1 sequence) and X. muta (4 sequences).

Phylogenetic analysis of the bacterial 16S rDNA gene sequences of the sponge isolates indicates that the majority are related to the Alphaproteobacteria (40%) and Gammaproteobacteria (47%) divisions. Two isolates, from only one sponge (I. strobilina), belong to the phylum Bacteroidetes and these are both clustered with 93% bootstrap confidence in the order Flavobacteriales. Three of the Alphaproteobacteria isolates, XM10, AF5 and AF4, are grouped solely with other marinesponge derived species with 100% bootstrap confidence. The Gammaproteobacteria isolates are clustered within three orders: Alteromonadales, Vibrionales and Pseudomonadales, with isolates XM12, AF6 and IS1 grouped solely in sponge specific clusters. The closest relatives of isolates HP1, HP6, IS8, IS10, IS16, UR3,


Figure 1

XM6, and XM18 are not other known spongeassociated bacteria.

In the present study, 47% of 16S rDNA sequences obtained from the sponges Aplysina fulva, Halichondria

Figure 1. Effect of (a) Aplysina fulva 50% ethanol extract, (b) Aplysina fulva EtOAc:IPA extract, (c) Ulosa ruetzleri 50% ethanol extract and (d) Mitoxantrone (a known Topoisomerase II poison) on the proliferation of human cancer cell lines ML1 and LoVo measured using the colorimetric XTT assay. Note: No concentration values are available for the U. ruetzleri 50% ethanol extract (dilution values are given instead) since too small an amount of this extract was obtained to weigh out. Therefore the extract was dissolved in 1ml DMSO in the flask used for rotary evaporation, which was weighed before and after removal of the dissolved extract to give an approximate weight of extract dissolved in 1ml DMSO stock solution of <10mg.

panicea, Ircinia strobilina, Ulosa ruetzleri and Xestospongia muta were related to other spongederived sequences, lending further support to the theory of a highly diverse, yet specifically spongeassociated bacterial community proposed by previous authors (Hentschel et al. 2002, Hill et al. 2006). The close affiliation of several isolates from the Grenadian sponges in the current study with previously published strains of spongeassociated bacteria from different geographic regions and different host species is consistent with the findings of Hentschel et al. (2002). These authors suggested uniformity among sponge associated microbial communities irrespective of host or geographic location. However, bacteria were also isolated from sponges in the present study that do not fall into spongespecific clusters or were not closely related to any other sponge bacterial sequences. These isolates appear to be unique to their particular sponge hosts and contradict the theory of uniformity in sponge bacterial communities. Altogether, the results of the


present study suggest that some members of the spongeassociated bacterial community are hostspecific whilst others are universal to many sponge species from all regions of the globe. This is similar to the findings of Taylor et al. (2004) who identified three types of spongeassociated bacteria: (1) ‘specialists’ – found only on one host species; (2) ‘sponge associates’ – found on multiple hosts but not in seawater; and (3) ‘generalists’ from multiple hosts and seawater.

Three sponge extracts, Aplysina fulva 50% ethanol extract, Aplysina fulva 1:1 EtOAc:IPA extract and the Ulosa ruetzleri 50% ethanol extract, showed an inhibitory effect on the ML1 cell line (Figure 1). The A. fulva 50% ethanol extract and U. ruetzleri 50% ethanol extract also inhibited the growth of the LoVo cell line. IC50 values for these extracts were in the range of 2.2 to 21.0 μg/ml for A. fulva extracts and a dilution of 3.8 x 10 5 to 6.0 x 10 5 for U. ruetzleri extracts, in comparison to IC50 values of 0.0017 to 0.028 µM for Mitoxantrone (a known topoisomerase II poison). These results suggest that the sponges A. fulva and U. ruetzleri collected from Grenadian coastal waters hold potential for identifying possible drug leads for the development of human anticancer treatments.

Submitted by Hilary Crane School of Marine Science and

Technology, Newcastle University, Newcastle upon Tyne, UK

7.14. Antimicrobial Properties of Skin Secretions from Eleutherodactylus johnstonei on Bacteriological Isolates

Since the discovery of penicillin by Dr. Fleming about 80 years ago and the discoveries of subsequent antibiotics, we have become complacent with regard to infectious diseases. In 1967, the Chief Medical Officer of the United States, then SurgeonGeneral William Stewart, reported to the White House that “we can close the book on infectious disease.” With antibiotics to treat acute infections and our reclassification of tuberculosis from the category of “extremely dangerous” to that of “easily managed infections,” this declaration was well supported. During this “golden age of antibiotics”, the eradication of diseases seemed to be only a matter of time. However, misuse and injudicious use of antibiotics have led to the emergence of antibiotic resistant bacterial strains, with methicillin resistant Staphylococcus aureus (MRSA) receiving most of the attention.

Figure 1. Death rates from infectious diseases: 19001992 (Lederberg, 1997).

We must once again turn our attention back to nature if innovative chemotherapeutic treatments with new modes of action are to be found. Notwithstanding the tremendous diversity of species found in nature, one


often notices that the same basic principles and techniques for solving distinct, but arguably related problems such as defense against infection by microorganisms, are often employed. Recently, in spite of many intrinsic organizational differences that exist between species and even kingdoms, similarities have been discovered in the way these species resist attacks by the numerous microorganisms they encounter daily. The innate immunity of vertebrates to microbial invasion is mediated by an intricate network of host defense mechanisms involving, in part, a nonspecific chemical defense system that includes a series of broadspectrum antimicrobial peptides. Thus far, antimicrobial peptides have been isolated from a wide variety of organisms including insects, plants, bacteria, mammals and amphibians.

Table 1. Activities of certain cationic peptides and their respective activities (Lederberg, 1997).

Although not fully understood, the mechanisms of action of antimicrobial peptides are so different from conventional antibiotics, it is strongly believed that resistant strains will not easily arise. These aspirations are further supported by the observation that such antimicrobial peptides have, most probably, already been in use by higher organisms against pathogens for millions of years, yet few incidences of resistance have been found to date.

Numerous peptides exhibiting antimicrobial properties have been

Figure 2. Eleutherodactylus johnstonei.

isolated from many species of amphibians from around the world. These peptides offer an innate chemical defense system against a broad range of microbial agents that may exist in the amphibian’s environment (fungus, bacteria, parasites and enveloped viruses). The purpose of our study is toexamine the antimicrobial potential of peptides isolated from Eleutherodactylus johnstonei (a species found in Grenada and throughout most of the Caribbean) to act against human pathogens – an area which has been relatively neglected in the scientific community. This project, if successful, has the potential to open new avenues towards the development of novel chemotherapeutic agents and, at the very least, will increase our understanding of the amphibian innate immune system.

Submitted by Jonathan W. Ashcroft MD/MSc Student

7.15. The Surgical Anatomy of the Occipital Nerves’ Contributions to the Development and Treatment of Occipital Neuralgia

Occipital neuralgia is a common cause of headache and is characterized


by paroxysmal stabbing pain. Examination of patients suffering from occipital neuralgia reveals pain and tenderness along the topographic locations of the greater occipital nerve (GON). In addition, patients usually complain of continuous unilateral pain (bilateral during severe attacks) throughout the occipital and parietal scalp. Furthermore, hyperalgesia, dysthesia and paroxysmal vertigo are conditions often associated with occipital neuralgia and are similar to symptoms that arise during migraines. Diagnosis of occipital neuralgia is made when disease or injury to the GON or its roots is believed to be the cause of symptoms.

Possible zones of GON irritation and entrapment include the point at which the nerve emerges from the C2 dorsal ramus between the atlas and the axis, the point where the GON courses between obliquus capitis inferior and semispinalis capitis, the point where it pierces the belly of semispinalis capitis, and at the point of exit from the tendinous aponeurosis of trapezius.

The aim of this study was to identify topographic landmarks for the accurate identification of the GON, lesser occipital (LON) and third occipital nerves (TON), which will facilitate their anesthetic blockade.

The course and distribution of the GON, TON and LON and their relation to the aponeuroses of the trapezius and semispinalis capitis were examined in 36 formalinfixed cadavers. In addition, the relative position of the nerve on a line between the external occipital protuberance (EOP) and the mastoid process, as well as between the mastoid processes was measured.

The GON was found bilaterally in all specimens. It was located at a

mean distance of 3.9 cm (range 1.7 to 7.1 cm) lateral to a vertical line through the EOP and the spinous processes of the cervical vertebrae. It was also located approximately 40% of the distance along the intermastoid line (medial to a mastoid process) and 20% of the distance between the EOP to the mastoid process. The location of the GON for anesthesia or any other neurosurgical procedure has been established as one thumb breadth lateral to the external occipital protuberance (2 cm lateral) and approximately at the base of the thumb nail (2cm inferior) (Figure 1).

Figure 1 demonstrates the relationship of the right and left greater occipital nerves (marked *) at their exit point with the vertical and intermastoid lines.

The TON was found bilaterally in all specimens. It was located at a mean distance of 0.5 cm (range 0.1 – 1.7 cm) lateral to a vertical line through the EOP and the spinous processes of the cervical vertebrae. It was also located approximately 48% of the distance along the intermastoid line (medial to a mastoid process) and 3% of the distance between the EOP to the mastoid process (Figure 2).


Figure 2 shows the TON piercing the semispinalis capitis muscle.

The LON was found bilaterally in all specimens. It was located at a mean distance of 7 cm (range 6 – 9 cm) lateral to a vertical line through the EOP and the spinous processes of the cervical vertebrae. It was also located approximately 11% of the distance along the intermastoid line (medial to a mastoid process) and 92% of the distance between the EOP to the mastoid process (Figure 3).

Figure 3 shows how the LON courses along the posterior edge of the SCM.

This is the first study proposing the use of landmarks in relation to anthropometric measurements. Based on these observations, we propose a target zone for local anesthetic injection that is based on easily identifiable landmarks and suggest that injection at this target point could be of benefit in the relief of occipital pain. It is hoped that this study

will provide clinicians, researchers and anatomists with valuable data regarding the variable peripheral course of the GON in treating occipital neuralgia. Clinically, extensive knowledge of all possible anatomical variations as well as identifiable landmarks would prove useful for the administration of local anesthetic nerve block. The application of this data may optimize GON, LON and TON block procedures by taking into account varying skull sizes and thus improving efficiency as well as decreasing recurrence of occipital neuralgia.

Submitted by Abraham ElSedfy MD/MSc Student

7.16 Evaluation and Analysis of Prostate Cancer in Grenada

Prostate cancer is the second most common cause of cancer related deaths in men above 50 years of age. In the United States it is the most common cancer in men. Risk factors for the disease include age, race, diet, and family history. Eight out of ten patients diagnosed with prostate cancer are above the age of 65. An increased incidence of prostate cancer has been reported amongst Caribbean nationals of African decent and African Americans while Asians have shown to have a lower risk.

Currently in the USA annual screening is performed on men over the age of 50. The screening tests entailed are prostate specific antigen (PSA) serum levels, digital rectal exam (DRE), and transrectal ultrasound (TRUS). The higher the PSA level, the more likely the chance of prostate cancer. Levels above 4 ng/ml are suspicious, however, PSA is an organ specific antigen, not cancer


specific. This means the results from PSA screening do not provide a definitive diagnosis. Increased PSA values are produced in 75% of prostate cancer cases and can potentially catch the disease in an asymptomatic man in its most curable stage. With early stage disease, which is the stage that prostate cancer screening is meant to detect, the 5 year survival rate is 80% and the 10 year survival rate is 60%. In advanced disease, which occurs once symptoms present, the five year survival drops to only 20% signifying the need for screening. Unfortunately, PSA values are also increased in other benign pathologies of the prostate such as nodular hyperplasia, prostatitis, ejaculation, extensive exercise, and infarct. Iatrogenic causes include cystoscopy and biopsy. This leads to an increased occurrence in false positives and poor statistical significance (46% sensitivity and specificity) of PSA screening instigating the debate on whether prostate cancer screening should continue in the USA. After a suspicious result is received on a screening test, a biopsy is taken of the prostate. The biopsy is not only an invasive and painful procedure, but it requires resources such as money and time. False positives create unnecessary pain to the patient and result in wasted resources. In Grenada there is no screening program. Prostate cancer is only diagnosed in men who go to their doctor once symptoms present, or upon request by men with a strong family history of the disease. Without screening there is no waste of resources on false positives, unfortunately this also means the men in this high risk population are missing the opportunity to catch the disease in its most curable stage.

World wide, once prostate cancer is Gleason Grade value, it is graded depending on the biopsy of the cancerous cells that is taken and is given a score known as the Gleason Grade. The histological differentiation of the cells. Two Gleason Grades are then combined to give the Gleason Score which correlates to the aggressiveness and stage of the cancer and depicts the treatment options. Standard protocol is that men in both extremes of the spectrum will not receive treatment. Because prostate cancer is one of the least aggressive types of cancer, men with a low Gleason Score, meaning they have a very low stage of cancer, will not undergo treatment and usually participate in “watchful waiting”. This is due to the idea that the men will die first of natural causes before the disease ever affects them; the treatment will also cause unnecessary negative effects to their quality of life. Men who receive a high Gleason Grade are beyond a cure and any hope of surviving the disease and treatment again will only negatively affect the quality of life that is remaining. Midrange scores are of the treatment receiving, although men on the lower end of the scale can opt for treatment as well.

The prognostic parameters (such as Gleason score, prostate specific antigen, and digital rectal exam) correlate to the survival rate and Gleason scores that fall in the midrange show high survival with treatment. In Grenada, however, men with the same prognostic parameters as men in the United States do not respond equally to the standards of care. They have identical values on tests that were performed and receive the same treatment but yet do not have the same outcome. Without ignoring the


confounding data present in my research, a statistical comparison is being made between the Grenadian population, Jamaican population, and the United States. This research has the possibility of showing that survival of prostate cancer in the Grenadian population cannot be correlated to the Gleason score. The data will be analysed to examine the suggestion that Grenadian men should undergo annual screening and that the standard clinical predictors for these men do not reflect the same meaning as they do in other populations.

Megan Kaminskyj outside the Birth and Death Registry where she collected data on the males who have died and have had prostate cancer.

Submitted by Megan E. Kaminskyj, MD/MSc candidate

7.17. The Public Health Importance of Dogs, Grand Anse, Grenada

Zoonoses are diseases that are naturally transmitted between vertebrate animals and humans. Zoonotic diseases are caused by various pathogenic agents including viruses, bacteria, protozoa, fungi and helminthes. Transmission routes for zoonotic diseases are varied for each specific agent. Infection may occur through fecal oral routes, sexual

transmission, blood contamination, vector borne transmission, physical contact and skin penetration by the infectious agent.

Animals that can transmit zoonotic diseases are found all over the world and in many different climates. One particular area of public health importance concerns animals that may facilitate the spread of zoonoses in popular human recreation areas.

Grand Anse, Grenada, West Indies, is one of the most popular human recreation areas in Grenada. The local beach, park and surrounding area is visited by numerous local and visiting residents of Grenada each year. The Grand Anse area is special due to its dual nature. It is a popular recreation area and is also a highly populated residential area. Grand Anse is also home to a local free roaming dog population.

The free roaming dog population in Grand Anse is comprised of dogs that can be categorized into three separate groups: privately owned, community owned, and stray. Privately owned free roaming dogs are dogs that have a full time caregiver and a permanent residence. The free roaming abilities of privately owned dogs are determined by owners and the restrictive nature of home residences. Community owned dogs are dogs that are not privately owned and share a noncommittal relationship with various caregivers within a select community. Community owned dogs do not have a permanent residence and may free roam when they are in search of sustenance, taking on a temporary caregiver in the process. Strays are dogs that have neither a fulltime nor parttime caregiver and have no permanent residence. These dogs


Figure 1. A free roaming dog enjoys a meal from a tipped over garbage can.

survive solely through their free roaming activities (Figure 1).

The health of free roaming dogs is often very poor and the life expectancy of such animals is about three years (Fielding et al., 2005). The poor health of free roaming dogs may increase the rate of canine acquisition and transmission of zoonotic diseases to the local human population, making them a potential public health threat (Figure 2).

Figure 2: A sickly dog rests in a parking lot next to Grand Anse beach during the daytime.

This research project is an observational study of dog ecology, which was developed in order to generate a hypothesis on the public health importance of free roaming dogs in Grand Anse, Grenada.

The methods used in this study include observation of dog ecology in Grand Anse, public health surveillance

of zoonoses in local clinics, door to door surveying of local dog ownership practices and public opinion of free roaming dogs in Grand Anse and laboratory analysis of free roaming dog stool samples for four common zoonotic diseases (hookworm, Cryptosporidium, Giardia and Toxocara canis). In conjunction with the laboratory work, a surveillance system for hookworm was set up at the Grand Anse Medical Center and the True Blue Health Clinic at St. George’s University.

The preliminary results of this study showed that 94% of fecal samples tested (N=18) contained hookworm eggs and 38% contained Cryptosporidium. Toxocara canis was identified in one sample. All samples were negative for the presence of Giardia.

During the surveillance period of 17 November 2006 to 13 July 2007, there were zero cases of cutaneous larva migrans caused by hookworm infection at the Grand Anse Medical Center and True Blue Health Clinic.

A survey of 51 Grand Anse households showed that 82% of dog owners surveyed (N=23) allowed their dog to roam free in Grand Anse unsupervised. When asked about taking care of stray dogs, 42% of nondog owners (N=28) and 46% of dog owners reported that they had taken care of stray dogs in the past. When asked if they considered stray dogs a problem in their neighborhood, 75% of nondog owners believed that stray dogs represented a problem compared to 65% of dog owners (Figure 3). Both groups reported that the number one problem caused by free roaming dogs was garbage tipping and rummaging. This response correlates with the survey results in Fielding et al., 2005.


Figure 3. A local boy eats his dinner while a hungry free roaming dog looks on and waits for his chance to get, or steal, some food.

The final results of this study hopes to determine the public health importance of dogs in Grand Anse, Grenada. The preliminary data shows that hookworm infection in free roaming dogs is high, but there were zero documented cases of cutaneous larva migrans at the clinical sites during the surveillance period. This indicates a potential disruption in the zoonoses transmission cycle. This disruption could possibly be environmental, mechanical, or pharmacological in nature. This will be investigated and the results will be incorporated into the final submission of this study.

Submitted by Kevin Neill

7.18. Research Activity for 2007

We continued our community based projects in Grenada during 2007. The first is “A Churchbased intervention to improve hypertension prevention and control among women in Grenada.” This proposal was competitively reviewed and funded for US$10,000 from the Caribbean Health Research Council (CHRC) in May 2004.

Although sidelined by hurricane Ivan, the project finally started data collection in 2007.

The objectives of the study are to screen for high blood pressure in a church population; and to test the feasibility and effectiveness of different intensities of a behavioral intervention to control blood pressure, improve dietary intake, reduce obesity and increase physical activity. The project is a partnership with the Catholic Church in Grand Anse, and several other churches in the south St. George’s area. To date, nearly 150 persons have been enrolled and contributed baseline data. Participants then attend an educational workshop that emphasizes physical activity, nutrition and stress management as ways to manage blood pressure. Once randomized to one of two intensities of intervention, the women are followed for six months. At that point they are invited to for another assessment to measure any changes. A final recruitment of new participants will take place in early 2008.

Many persons have contributed to making the study run smoothly. Father Charles Dominique of the Blessed Sacrament Parish in Grand Anse and Bishop Darius have supported the project from inception, and provide space to conduct interviews, hold educational workshops, and conduct facilitated small group discussions in the intensive intervention group. Several clinical tutors, medical students, partners of students, Arts and Sceinces students, and other volunteers have worked on the study as their time permits and contributed to making the study go forward over the year.

Preliminary findings from the study were written up as three presentations and submitted to the


Caribbean Health Research Council (CHRC) for consideration of acceptance at the upcoming meeting in Suriname in April. To date, 57 women have completed the six month followup assessment. So far the results are encouraging. Women who completed the intervention have shown an increased amount of physical activity, modest weight loss in some cases, and beneficial changes in blood pressure. Considering that this methodology has been successful in other sites and is feasible for Grenadian communities, this approach has potential to reach many high risk persons and family members.

Norma Purcell nutrition session, “Hypertension Prevention and Control among Women” project.

Dr. Vijayarahavan in counseling session.

Another area of research involves HIV/AIDS. With colleagues from Florida State University and the University of the West Indies, we have explored several key constructs relating to risk behaviors, stigma and

discrimination by health care providers in three Caribbean countries.

We found that providers whose roles were more likely to involve touch were less comfortable and more likely to show distancing from and to condemn persons living with HIV/AIDS than were providers having less direct contact. A paper: “Abell N, Rutledge SE, McCann TJ, Padmore J. ‘Examining HIV/AIDS provider stigma: assessing regional concerns in the islands of the Eastern Caribbean’”, was published in AIDS Care, a highlycited journal, first electronically, and later in print.

On the basis of this work we were contacted by CHRC, who requested permission to disseminate our research findings using media such as fact sheets, policy briefs and press releases. With CHRC, the Caribbean Institute of Media and Communication (UWI) has developed dissemination products citing our study — both a press release and a fact sheet — concerning HIV/AIDS stigma among health care providers. Our participation is part of a regional effort to increase the impact of Caribbean HIV/AIDS research on policy, practice and program development.

Theresa McCann, PhD Professor, Department of Behavioral Sciences, and

WINDREF Research Fellow

8.0. Acknowledgements

WINDREF and researchers work in close collaboration with a number of local and internationally based institutions and individuals. In Grenada, we would like to thank the Ministry of Health, the Ministry of Education, the


Ministry of Agriculture and the National Parks and Protected Areas Department, the Ministry of Tourism and the Forestry Department for their help and cooperation with the research projects. We would specifically like to thank Senator Ann DavidAntoine, the Minister of Health, for her considerable input during 2007. We would like to thank everyone, board members, fellows, research scientists, faculty, collaborators, members of the communities in many countries and donors for their contributions to WINDREF’s activities during 2007, including the following: Dr. Bjorn Akerman, Institute of

Biophysical Chemistry, Chalmers University, Gothenburg, SwedenCERI

Tristano BacchettideGregoris...... CERI Mr. Andre Blackman..................... CERI Board of Trustees, SGUSOM............All Dr. Feisal Brahim.................A. El Sedfy Marilyn Bruno .........................C. Bruno Dr J. Grant Burgess, Professor,

Marine Biotechnology and Director, Dove Marine Laboratory, School of Marine Science and Technology, Newcastle University............... CERI

Linden Burzell .............................. CERI Mr. Roy Campbell......................... CERI Dr. Thomas Champney ................. CERI Dr. Vijaya R. Chellapilla ............K. Neill Ms. Meg Conlon ...............................All Ms. Camile Coomansing ............K. Neill Clark Crawford, NICR, U.K.......... CERI Dr. Brian Curry....................A. El Sedfy Mrs. Grace Dolphin....................... CERI Dove Marine Laboratory, U.K....... CERI Mrs. Isha English ..............................All Mr. Peter Giesler ........................... CERI Dr. Robert Hage...................A. ElSedfy Ms. Suzanne Hall .......................... CERI

Dr. Lotta Hallback, Microbial Analytic, Sweden .... CERI

Mark Hamann ............................... CERI Russell Hill ................................... CERI Dr. Svetlana Kotelnikova ..................All Dr. Lee

University of Nebraska LincolnCERI Mr. Leslie LeBarrie ....................R. Patel Dr. David Lennon .............................All Dr. Per Lincoln, Institute of


Dr. Marios Loukas ...............A. El Sedfy Dr. Calum Macpherson .....................All Dr. Cristophre Martin.................... CERI Dr. Eugene Martin

University of Nebraska ........... CERI Professor A.M Mavuti, PhD, Director,

International Programs and Links, University of Nairobi, Kenya... CERI

Mrs. Lana McPhail, Permanent Secretary, Ministry of AgricultureCERI

Microbiology Dept., SGU ............. CERI Dr. Theresa McCann ........M. Kaminskyj Dr. Charles Modica, SGU .................All Dr. Clare Morrall .......................... CERI Mrs. Grace Narine, SGU ............... CERI Brent Nelson ................................. CERI Mr. Trevor Noel................................All Dr. Nicholas H. Oberlies,

Research Triangle Institute (RTI), North Carolina, USA ............... CERI

Bjulah Paterson............................. CERI Mrs. Rachel PaulKing ......................All Dr. Karsten Pedersen, Professor,

Microbiology, Department of Cell and Molecular Biology, Göteborg University and Director, Microbial Analytics, Sweden ................... CERI

Dr. Allen Pensick ..............................All Dr. Rhonda Pinkney...................K. Neill Dr. Ateef Qureshi..............................All


Dr. Thomas Rasmussen, Center for Biomedical Microbiology, Denmark, SwedenCERI

Dr. Joanna Rayner.............................All Mr. and Mrs. Rennie,

landlords, Soda Springs ........... CERI Mr. Justin Rennie, Chief Fisheries

Officer, Min. Agriculture......... CERI Dr. Rooney.......................M. Kaminskyj Dr. Diana Stone..........................K. Neill Dr. Chamarthy Subbarao ............K. Neill Ms. Vasilla Sylvester, Grand Anse

Health Clinic ........................K. Neill Mr. Trevor Thompson, NAWASA

.............................................R. Patel Mr. John Trumpane, Institute of


Dr. Marcus Wilhelmsson, Institute of Biophysical Chemistry, Chalmers University, Gothenburg, SwedenCERI

Mr. Nicholas Winkler, SGU.......... CERI Dr. Robert Yearwood.......M. Kaminskyj

9.0. Grants

We would like to thank all of the donors who have made WINDREF’s work possible in 2007. These include:

• The Bartholomew J. Lawson Foundation for Children for the Youth Center Program.

• Bayer HealthCare AG, Germany, for the project “Assessment of the efficacy of 10% imidacloprid and 2.5% moxidection spoton (Advocate ® ) in the therapy of generalized demodicosis”.

• The Caribbean Health Research Council (CHRC) for the support of the Close to Heart project.

• Grensol for the support of the new Photovoltaic Power Generation Program.

• Dr. MaryJeanne Kreek, Rockefeller University, NY, for the support of the Genetic Correlation of Addictive Diseases project.

• The Liverpool Support Center for their continued support of the Lymphatic Filariasis Elimination Project in Guyana.

• The Lounsbery Foundation for their continued support of the Rheumatic Fever Prevention Program.

• Pan American Health Organization (PAHO) for the support of the Survey of Biomedical Waste Management Practices in the OECS Countries.

• The Robert Wood Johnson Foundation in Princeton, New Jersey, in collaboration with the Department of Public Health and Preventive Medicine, for funding a Childhood Obesity Symposium to be held at SGU in the spring of 2008.

• St. George’s University for supporting the Soothing Touch Massage Clinic.

• The TeasdaleCorti Program for the grant of 1.6 million Canadian dollars which will support the Caribbean Ecohealth Programme (CEP) for the duration of the 4year program.

10.0. Past, present and future research projects (present research projects bolded)


10.1. Noncommunicable diseases

• Angiotensin converting enzyme and angiotensinogen gene polymorphisms in the Grenadian population: relation to hypertension

• Development of a decision rule for screening Obstructive Sleep Apnea and its epidemologic relevance to the people of Grenada

• Prevalence and associated risk factors of hypertension in a sample population of native Caribbean’s in Grenada, West Indies

• Assessing the prevalence of diabetic complications by examining type I and type II adult diabetics for signs of retinopathy, neuropathy, nephropathy and dermatological changes associated with poor glucose control within the native Caribbean population of Grenada

• Hypertension management and control in two Caribbean countries

• Assessment of the effectiveness of broadspectrum treatment to children with protozoan and nemathelminthic parasitic infections on diarrhea and school attendance

• The effects of irondeficiency anemia on cognition and behavior in infants

• Diurnal variation of urinary endothelinI and blood pressure: related hypertension

• Alcohol consumption in Grenada

• The incidence and mortality of cancer in Grenada over the ten year period: 19901999

• The prevalence of abnormal haemoglobin traits in Grenadian secondary school adolescents

• Knowledge, attitudes, beliefs and practices of Sickle Cell Anemia in Grenadian Primary and Secondary school children

• Decompression sickness among the indigenous fishing population in Grenada: Assessing the burden of disease

• WINDREF / SGU Hurricane Relief

• Spice Research Program

• SulfateReducing Bacteria in Oxidized Freshwater of Tropical Mangroves

• Novel Antibiotics from Tropical Marine Environments: Drug Development in Grenada

• Study of the Mutacin C7A

• Gramnegative bacteria isolated from aquatic environments of Grenada (61.4°W, 12.0°N), West Indies

• Identification of bacteria producing antibiotics isolated from deep marine biofilms of Grenada

• SGU Environmental Testing Unit (ETU)

• Posthurricane water surveillance in problematic areas of Grenada

• Evaluation of the relocation potential for villagers residing in Queen Elizabeth National Park, Uganda

• Study of the Calls of the Spotted Hyena at Feeding

• Survey on the attitude of villagers in Queen Elizabeth National Park, Uganda towards the threat of lions, leopards and hyenas

• Epidemiology of Human Injuries Resulting From Wildlife in Ten


Villages within Queen Elizabeth National Park, Uganda

• Rural Ugandan Village Perspective on Lion, Leopard and Hyena Conservation

• Epidemiology of Human Injuries by Wildlife in Six Villages within Queen Elizabeth National Park, Uganda

• Prevalence of Campylobacter fetus subspecies venerealis and other Microorganisms in the Reproductive Tracts of Cattle from the Southern Region of Santo Domingo, Dominican Republic

• Antimicrobial properties of skin secretions from Eleutherodactylus johnstonei on bacteriological isolates

• Examination and analysis of prostate cancer in Grenada

• A Churchbased intervention to improve hypertension prevention and control among women in Grenada

10.2. Infectious diseases

• Investigation of the prevalence of SIV in the mona monkey (Cercopithcus mona) in Grenada

• Seroprevalence of HIVI and HIVII in pregnant women in Grenada, W.I. Their knowledge of AIDS and their exposure hazards to the virus

• A cross sectional study of the current status of Schistosoma mansoni in St. Lucia by field surveys and supplementary data collection

• Identification and characterization of hantaviruses among the mammal population of Grenada

• HIV/AIDS health education and evaluation program in Grenada

• The seroprevalence of Toxoplasma gondii in a population of pregnant women and cats in Grenada, West Indies

• The efficiency of diagnosing women of Toxoplasma gondii using PCR techniques in comparison with ELISA

• Dengue virus in Grenada: seroprevalence and associated risk factors

• A current appraisal of dengue virus in Grenada serotype analysis and vector assessment

• A site receptivity study determining the threat of reintroduction of malaria into Grenada through the study of Anopheline spp. mosquito vectors

• Chlamydial infection among STD clinic attenders in Grenada

• Fever in Grenada

• Mosquitoes and Tourism in Grenada

• Effectiveness of a formula feeding/weaning intervention program in preventing transmission of HTLV1 from seropositive mothers to newborns in Grenada

• A multicenter longitudinal research study of the behavioral significance of the prevalence of HIV1 infection in pregnant women and their babies on the islands of Grenada and St. Vincent


• A multicenter longitudinal research study of the ethical analysis of informed consent of the prevalence of HIV1 infection in pregnant women and their babies on the islands of Grenada and St. Vincent

• Determining the role of IL15 in mediating function of viralspecific CD8+ T cells in the myelopathogenesis of HTLV1: Symptomatic versus asymptomatic patients

• Intestinal protozoan infections in 6 12 year old children in Grenada

• Intestinal helminth infections in 612 year old children in Grenada

• The prevalence of intestinal parasites in school children in rural Guyana

• The prevalence of filariasis and its effects on children aged 814 in the central corentyne region of rural Guyana

• The prevalence of streptococcal infection in school children aged 5 – 15 years in Grenada, Carriacou and Petit Martinique

• Studies examining the elimination of lymphatic filariasis as a public health problem in Guyana

• Seroprevalence of heartworm infection in dogs in Grenada.

• Dengue in Grenada

• Assessing the potential risk factors of dengue and dengue hemorrhagic fever in the triisland state of Grenada, Carriacou and Petit Martinique

• A comparative study to find out if there is an association between sexual practices and knowledge in adult populations of Botswana and

Grenada with the prevalence of HIV/AIDS

• HIV/AIDS in rural Botswana differentiating between informing and educating

• Evaluating the level of perceived fear and desensitization towards HIV/AIDS in Botswana

• Rheumatic Fever in Grenada

• Streptococcal program in St. Vincent

• Isolating T cells from Rheumatic Fever positive blood: Immunofluorescent assay of T lymphocytes via fluorescently labeled monoclonal antibodies

• Possible genetic predisposition to Rheumatic Fever: Demonstrating the inheritance fashion of nonHLA B lymphocyte alloantigen D8/17, a marker for Rheumatic Fever

• ELISA antibody titres against group A streptococcal M protein moiety and cell wall NAcetylD Glucosamine in Grenadian Rheumatic Fever patients

• Evaluating the effectiveness of educational methods in the prevention of Rheumatic Fever and Knowledge, Awareness and Practices

• Prevalence of intestinal helminth infections in rural Grenadian school children

• Cystic echinococcosis in Morocco and Uganda

• Elimination of Lymphatic Filariasis in Guyana Program


10.3. Unique projects

• Characterization of five amphibians inhabiting Grenada and subsequent isolation and antimicrobial assay of potential antibiotics derived from their skin

• Mona Monkey studies in West Africa

• Investigation of medicinal plants in Grenada

• Use of medicinal plants in Grenada

• Medicinal drugs from the sea. What do Grenada’s waters have to offer?

• Beekeeping in Grenada: Effects of the mite Varroa jacobsoni and its control

• Effects of Grenadian Medicinal Plants on Endemic Microbial causes of Diarrhoeal Diseases

• The neurobiological basis of hypoglycemiaassociated autonomic failure

• Stimulation of angiotensin 4 in cardiac fibroblasts activates matrix metalloproteinases through MAP kinases pathways: A model for astrocytes

• REM sleep and memory

• End of life care in Grenada

• Novel antibiotics from tropical marine environments

• Genetic Correlates of the Addictive Diseases: Cocaine, Alcohol, and Marijuana Addiction– Grenada

• An Investigation of Pediatric Botanical Medicine for Acute Respiratory Infections

• Efficacy of phage therapy using an in vitro biofilm wound model system

• Degradation of 7 keto cholesterol by Xenohydrolases

• Ecological survival properties of pelagic and benthic indicator microorganisms from the St. John’s river outflow in Grenada

• The Public Health Importance of Dogs, Grand Anse, Grenada

• Greater occipital nerve zones for treatment of occipital neuralgia

• Photovoltaic Power Generation Program

• Review of Current Biomedical Waste Management Practices in the Organization of Eastern Caribbean States (OECS) Countries

• Caribbean Ecohealth Programme: Public and Environmental Health Interactions in Food and Waterborne Illnesses (CEP)

• Microbial Diversity in the Iron oxidizing Biofilms of Soda Springs in Grenada

• Circadian Cycle of Ironoxidation in Warm Soda Springs in St. Andrew’s, Grenada, West Indies

• Do the Microorganisms in the Soda Spring Water derive Energy from the Oxidation of Manganese?

• Novel Marine Bacteria and their Antagonistic Properties Against Medically relevant Biofilms

• Physiological Characterization of Novel Marine Bacterial Species Isolated off Grenada


• Characterization of Marine Spongeassociated Bacteria and Cytotoxic Activity of Sponge Extracts towards Human Cancer Cells

• Examining HIV/AIDS Provider Stigma: Assessing Regional Concerns in the Islands of the Eastern Caribbean

11.0. Conferences, meetings, workshops sponsored in 2007

PAHO/WHO Meeting on Epidemiological Data Needed to Plan Elimination of Schistosomiasis in the Caribbean, 1314 December 2007.

12.0. Abstracts, Presentations at International Conferences, Invited Plenary / Workshop / Roundtable / Professional

Abell N., Rutledge S.E., McCann T.J., Padmore J. Examining HIV/AIDS provider stigma: assessing regional concerns in the islands of the Eastern Caribbean. AIDS Care 19(2): 242247.

Coomansingh, C., Pinckney, R.D., Macpherson, C.N.L., Stone, D. Helminth parasites in dogs in Grenada, West Indies. Annual meeting of the American Association of Veterinary Parasitologists (AAVP), 1417 July 2007, Washington, D.C. Cox Macpherson, C. Professionalism: What is It? First Annual Bioethics Forum of the Bioethics Society of the Englishspeaking Caribbean (BSEC), 4 7 May 2007, Montego Bay, Jamaica. Dr. Cox Macpherson is the Vice President of the BSEC.

Cox Macpherson, C. Medical student views about medical ethics: Preliminary data, David Thomasma International Retreat, 1822 June 2007, Cambridge, UK. Loukas, M. Invited lecture, “Anatomical Research, ‘Quo Vadis’?” October 2007. 12 th National Anatomical Congress, Denizli. Turkey Loukas, M. Moderator, Scientific Platform Session 4 – Head and Neck and Teaching. 24 th Annual Meeting of the American Association of Clinical Anatomists, June 1620, 2007 Las Vegas, NV. USA. Macpherson, C.N.L. Ultrasound Classification of Cystic Echinococcosis. WHO Informal Working Group Meeting on Upgrading the WHOIWGE Guidelines, September 2007, France.

Williams, S., Dunaway, D., Ranasinghe, N., Kulkarni R., Ongcapin E., Extracranial Spread of Glioblastoma Multiforme Presenting as a Parotid Abscess, poster presentation, College of American Pathologists Conference, 30 September3 October 2007, Chicago, Illinois. Abstract published in Archives of Pathology and Laboratory Medicine, 2007, Sept; 131(9):1425, the official Journal of the College of American Pathologists. Cited in PubMed, PMID: 17824799.

13.0. Publications Cox Macpherson C. Global Bioethics: Did the Universal Declaration on Bioethics and Human Rights Miss the Boat? Journal of Medical Ethics. 33 (10); 588590. 2007. Cox Macpherson, C. Editor, Newsletter of the Bioethics Society of the English


speaking Caribbean, Issue 1 No. 1, July 2007.

Cox Macpherson C., Kenny N. Professionalism and the basic sciences: An untapped resource. Medical Education. Accepted 2007. Gomez, A., Franco, R. Survival Always Trumps Ideology: A Case Study. Human Organization 66 (3). Liechty, P., Tubbs, R.S., Loukas, M., Blount, J.P., Wellons, J.C., Acakpo Satchivi, L., Oakes, W.J., Grabb, P.A. Spinal Accessory Nerve Meningioma in a Pediatric Patient: Case Report. Folia Morphologic (2007) 45:2325. Loukas, M., Louis, R.G., Wartmann, C. T2 Contributions to Brachial Plexus. Neurosurgery, (2007) 60 (2 suppl 1) 13 18. Loukas, M., Louis, R.G., Black, B., Pham, D., Fudalej, M. False tendons: an endosopic approach to fill the gaps left by echocardiography. Clinical Anatomy (2007) 20:163169

Loukas, M., Louis, R.G., Wartmann, C. Tubbs, R.S., Esmaeili, E., Bagenholm, A.C., Merbs, W., Curry, B., Jordan, R. Superior phrenic artery, an anatomic study. Surgical and Radiologic Anatomy (2007) 29:97102.

Loukas, M., Tobola, M.S., Tubbs, R.S., Louis, R.G., Karapidis, M., Khan, I., Spentzouris, G., Linganna, S., Curry, B. The clinical anatomy of the Internal Thoracic Veins Folia Morphologica (2007) 66: 2532.

Loukas, M., Tubbs, R.S., Bright, J., Bartczak, A., Fudalej, M., Wagner, T., Anderson, R.H. The anatomy of the tendon of Infundibulum, a follow up study and a review of the literature. Folia Morphologica (2007) 66: 3338.

Loukas, M., Louis, R.G., Stewart, L., Hallner, B., DeLuca, T., Morgan, W., Shah, R., Mlejnek, J. The clinical anatomy of ulnar and median nerve communications in the palmar surface of the hand. Journal of Neurosurgery (2007) 106:887893 Loukas, M., Wartmann, C.T., Louis, R.G., Tubbs, S.R., Ona, M., Curry, B., Jordan, R., Colborn, G.L. The Surgical Anatomy of the Posterior Gastric Artery. Surgical and Radiologic Anatomy (2007) 29:361366. Loukas, M., Thorsell, A., Tubbs, R.S., Louis R.G., Vulis, M., Hage, R., Jordan, R. The Ansa Cervicalis Revisited. Folia Morphologica (2007) 66:120125

Loukas, M., Wartmann, C.T., Louis, R.G., Tubbs, R.S., Salter, G.E., Gupta, A.A., Curry, B. Cisterna Chyli: A Detailed Anatomic Look. Clinical Anatomy (2007) 20:683688. Loukas, M., Tubbs, R.S., ElSedfy, A., Jester, A., Polepalli, S., Kinsela, C., Wu, S. The Clinical Anatomy of the Triangle of Petit. Hernia (2007) 11:441 444.

Loukas, M., Tubbs, R.S., Jordan, R. Aneurysm of the greater cardiac vein caused by an arterial branch. Surgical and Radiologic Anatomy (2007) 29:169 172. Loukas, M., Tubbs, R.S. An accessory muscle within the suboccipital triangle. Clinical Anatomy (2007) 20:962–963.

Loukas, M., Pinyard, J., Kinsella, C., Vaid, S., Tariq, A., Tubbs, R.S. The clinical anatomy of the celiac artery compression syndrome, a review. Clinical Anatomy (2007) 20:612617. Loukas, M., Louis, R.G., Wartman, C.T. Letter to the Editor, Response to


Fernando Martínez. Clinical Anatomy (2007) 23:474. Loukas, M., Jordan, R. Comment on “A Portrait of Aristotle as an Anatomist” Clinical Anatomy (2007) 20:712. Loukas, M., Tubbs, R.S., Wartmann, C.T., Louis R.G. Comment on “Superficial Palmar Communications between the Ulnar and Median Nerves in Turkish Cadavers” Clinical Anatomy (2007) 20:849850. Loukas, M., Linganna, S., Jordan, R. Jean Casimir Félix Guyon. A Glimpse from the Past. Clinical Anatomy (2007) 20:12. Loukas, M., Linganna, S., Jordan, R. St George’s University, an anatomist experiences during Grenada’s revolution and intervention. Clinical Anatomy (2007) 23:350356

Loukas, M., Clarke, P., Tubbs, R.S. Accessory Breasts, a historical and current perspective. American Surgeon (2007) 73:525528

Loukas, M., Tubbs, R.S., Louis R.G., Pinyard, P., Vaid, S., Curry, B. The cardiovascular system in the pre Hippocratic era. International Journal of Cardiology. (2007) 120:145149. Loukas, M., Spentzouris, G., Tubbs, R.S., Kapos, T., Curry, B. Ruggero Ferdinando Antonio Guiseppe Vincenzo Oddi Glimpse of our past, World Journal of Surgery (2007) 31:22602265

Loukas, M., Clarke, P., Tubbs, R.S., Kapos. T. Raymond de Vieussens. Anatomical Science International (2007) 82:233236 Nature (Peer to Peer), 22 May 2007. Loukas, M., Grabska, J., Tubbs, R.S., Louis R.L. An unusual union of the Intercostobrachial Nerve and the Medial

Pectoral Nerve. Folia Morphologica (2007) 66:356359.

Loukas, M., Klaassen, Z., Tubbs, R.S., Wartmann, C.T., Apaydin, N. Popliteal Artery Aneurysms: A Review. Folia Morphologica (2007) 66:2726. Martin, C.C., Storey, K.B. Ensuring anonymity in the internet age.

Rutrecht, S.T., Klee, J., Brown, M.J.F. Horizontal transmission success of Nosema bombi in its adult bumble bee hosts: effects of dosage, spore source and host age. Parasitology 134, 1719 1726.

Ryan T.D., Dimmitt R.A., Tubbs R.S., Roberts O.A., Choi H., Loukas M., Blount J.P. Contribution of surgical evacuation of a large subgaleal hematoma to the resolution of severe conjugated hyperbilirubinemia in a neonate. Journal of Neurosurgery (2007) 106:131133.

Tubbs, R.S., Jackson, P., Shoja, M.M., Loukas, M., Oakes, W.J. Foramen Arcuale: Anatomical Study and Review of the Literature. Journal of Neurosurgery Spine (2007) 6:3134. Tubbs, R.S., Loukas, M., Shoja, M.M., Oakes, W.J. Observations at the Craniocervical Junction with Simultaneous Caudal Traction of the Spinal Cord. Child’s Nervous System (2007) 23:367369. Tubbs, R.S., Loukas, M., Slappey, J.B., Linganna, S., Shoja, M.M., Oakes, W.J. Surgical and Clinical Anatomy of the Interclavicular Ligament. Surgical and Radiologic Anatomy (2007) 29:357360. Tubbs, R.S., Loukas, M., Louis, R., Shoja, M.M., Askew, C.S., Phantana angkool, A., Salter, E.J., Oakes, W.J. Surgical Anatomy and Landmarks for


the Basal Vein of Rosenthal. Journal of Neurosurgery (2007) 106:900902

Tubbs, R.S., Bui, C.J., Rice, W.C., Loukas, N., Naftel, R.P., Holcombe, M.P., Oakes, J. Critical Analysis of the Chiari I Malformation Found in Children with Lipomyelomeningocele: A Radiological and Anatomical Study. Journal of Neurosurgery. (2007) 106:196200.

Tubbs, R.S., Bui, C.J., Loukas, M., Shoja, M.M., W. Oakes, W.J. Partial Dorsal Rhizotomy for Spasticity in Children with Congenital Brain Malformations Journal of Neurosurgery (2007) 106:4079.

Tubbs, R.S., Stetler, W., Kelly, D.R., Blevins, D., Shoja, M.M., Loukas, M., Oakes, W.J. Acrel’s Ganglion. Surgical and Radiologic Anatomy (2007) 29:379 381. Tubbs, R.S., Loukas, M., Louis, R.G., Shoja, M.M., AcakpoSatchivi, L., Blount, J.P., Salter, G., Oakes, W.J., Wellons, J.C. Anatomy of the Falcine Venous Plexus. Journal of Neurosurgery (2007) 107:1557. Tubbs, R.S., Loukas, M., Slappey, J.B., Shoja, M.M., Oakes, W.J., Salter, G.E. Clinical Anatomy of the C1 Dorsal Root, Ganglion, and Ramus: A Review and Anatomic Study. Clinical Anatomy (2007) 20:624627. Tubbs, R.S., Loukas, M., Shahid, K., Judge, T., Pinyard, J., Slappey, J.B., McEvoy, W.C., Oakes, W.J. Anatomy and Quantitation of the Subscapular Nerves, Clinical Anatomy (2007) 20:656659 Tubbs, R.S., Loukas, M., Allsion, R., Mohammadali, S., Salter, G., Oakes, W. The Vertebral nerve revisited. Clinical Anatomy (2007) 20:644647.

Tubbs, R.S., Loukas, M., Louis, R., Shoja, M.M., Hansasuta, A., Salter, G.E., Oakes, W.J. The Basilar Venous Plexus, Clinical Anatomy (2007) 20:755 759. Tubbs, R.S., Louis, R.G., Wartmann, C.T., Loukas, M., Shoja, M.M., Ardalan, M.R., Oakes , W.J. The Suprascapular Nerve as a Donor for Extracranial Facial Nerve Reanimation Procedures: Cadaveric Feasibility Study. Journal of Neurosurgery (2008) 108:14.

Tubbs, R.S., Stetler, W., Kelly, D.R., Blevins, D., Humphrey, R., Chua, G.D., Shoja, M.M., Loukas, M., Oakes, W.J. Human Spinal Arachnoid Villi Revisited: Immunohistological Study and Review of the Literature. Journal of Neurosurgery (2007) 7:328 331.

Tubbs, R.S., Loukas, M., Phantana angkool, A., Shoja, M.M., Oakes, W.J. Posterior Distraction Forces of the Posterior Longitudinal Ligament Stratified According to Vertebral Level. Surgical and Radiologic Anatomy (2007) 29:667670 Tubbs, R.S., Hill, M., Loukas, M., Shoja, Oakes, W.J. Volumetric Analysis of the Posterior Cranial Fossa in a Family with Four Generations of the Chiari I Malformation. Journal of Neurosurgery Pediatrics (2008) 1:2428 Tubbs, R.S., Wartmann, C.T., Louis R.G., Shoja, M.M., Loukas, M., Cormier, J., Oakes, W.J. Use of the scapular spine in lumbar fusion procedures: Cadaveric feasibility study. Journal of Neurosurgery Spine (2007) 7:554557

Tubbs, R.S., Loukas, M., Shoja, M.M., Apaydin, N., Oakes, W.J., Salter, E.J. Anatomy and Potential Clinical Significance of the Vastoadductor


Membrane. Surgical and Radiologic Anatomy. (2007) 29:569573

Tubbs, R.S., Bui, C.J., Loukas, M., Shoja, M.M., Oakes, W.J. The horizontal sacrum as an indicator of the tethered spinal cord in spina bifida aperta and occulta. Neurosurgical Focus (2007) 23, E11, 14

Tubbs, R.S., Kelly, D.R., Pugh, J.L., Loukas, M., Oakes, W.J. Benign Fibrous Histiocytoma of the skull base: case report. Journal of Neurosurgery, (2007) 106:6567. Tubbs, R.S., Shoja, M.M., Loukas, M., Oakes, W.J. Simultaneous Lateral and Posterior Ponticles Resulting in the Formation of a Vertebral Artery Tunnel of the Atlas: Case Report Folia Neuropathologica (2007) 45:4346. Tubbs, R.S., Shoja, M.M., Shokouhi, G., Loukas, M., Oakes, W.J. Retroscalenic Course of the Transverse Cervical Artery with the Suprascapular Artery Traveling Through the Suprascapular Notch. Folia Morphologica (2007) 66:8082

Tubbs, R.S., Loukas, M., Shoja, M.M., Salter, E.G., Oakes, W.J. Indirect Inguinal Hernia of the Urinary Bladder through a Persistent Canal of Nuck: Case Report. Hernia (2007) 3:287288. Tubbs, R.S., Winters, R.G., Naftel, R.P., Acharya, V.K., Conklin, M., Shoja, M.M., Loukas, M., Oakes, W.J. Predicting Orthopedic Involvement in Patients with Lipomyelomeningoceles Child Nervous System (2007) 8:835838. Tubbs, R.S., Loukas, M., Shoja, M.M., Blevins, D., Humphrey, R., Chua, G.D., Kelly, D.R., Oakes, W.J. An Unreported Variation of the Cervical Vagus Nerve: Anatomical and

Histological Observations. Folia Morphologica (2007) 66: 155157

Tubbs, R.S., Loukas M., Shoja, M.M., Oakes, W.J. Accessory Venous Sinus of Hyrtl. Folia Morphologica (2007) 66: 198199.

Tubbs, R.S., Stetler, W., Shoja, M.M., Loukas, M., Salter, E.G., Oakes, W.J. An Unusual Muscular Variation of the Infratemporal Fossa Folia Morphologica (2007) 66: 200202. Tubbs, R.S., Hansasuta, H, Loukas, M., Louis, R., Shoja, M.M., Salter, G., Oakes, W.J. Branches of the Petrous and Cavernous Segments of the Internal Carotid Artery: A Review. Clinical Anatomy (2007) 20:596601. Tubbs, R.S., Lyerly, M.J., Loukas, M., Shoja, M.M., Oakes, W.J. The Pediatric Chiari I Malformation: A Review. Childs Nervous System (2007) 23:12391250.

Tubbs, R.S., Louis, R.G., Loukas, M., Gupta, A.A., Shoja, M.M., Oakes , J., Oakes, W.J. The First Description of the Palmaris Brevis Muscle R. J Hand Surg European (2007) 32E:382383. Tubbs, R.S., Loukas, M., Salter, E.G., Oakes, W.J. Wilhelm Erb and Erb’s Point. Clinical Anatomy (2007) 20:486 488 Tubbs, R.S., Shoja, M.M., Loukas, M., Oakes, W.J. Abubakr Muhammad Ibn Zakaria Razi, Rhazes (865925 AD) Child’s Nervous System (2007) 23:1225 1226

Tubbs, R.S., Loukas, M., Shoja, M.M., Oakes, W.J. Matthew Baillie (1761 1823) and His Early Detailed Descriptions of Childhood Hydrocephalus in his Morbid Anatomy JNS (2007) 107:33841.


Tubbs, R.S., Linganna, S., Loukas, M. Jacobus Sylvius 14781555. Physician teacher and anatomist. Clinical Anatomy (2007) 20:868870

Tubbs, R.S., Loukas, M., Spinner, R., AcackpoSatchivi, L, Wellons, J.C., Blount, J.P., Oakes, W.J. Endoscopically assisted decompression of the suprascapular nerve: A cadaveric feasibility study. Journal of Neurosurgery (2007) 107:11641167 Shoja, M.M., Tubbs, R.S., Loukas, M., Shokouhi, G., Oakes, W.J. A complex duralvenous variation in the posterior cranial fossa: A triplicate falx cerebelli and an aberrant venous sinus. Folia Morphologica (2007) 66: 148151 Yabsley, M.J., McKibben, J., Macpherson, C.N.L., Cattan, P.F., Cherry, N.A., Hegarty, B.C., Breitschwerdt, E.B., O’Conner, T., Chandrashekar, R., Paterson, T., Perea, M.L., Ball, G., Friesen, S., Goedde, J., Henderson, B., Sylvester, W. Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, and Hepatozoon canis in dogs from Grenada tested in 2004 and 2006, Veterinary Parasitology.E Publication.

14.0. Reviewer for Journals and Boards

Dr. Svetlana Kotelnikova: • FEMS Microbiology Ecology • Geomicrobiology Journal (2006) • Journal of Environmental Science

and Technology

Dr. Marios Loukas • Associate Editor, Clinical Anatomy • Associate Editor, Neuroanatomy • American Journal of the Medical

Sciences • Clinical Anatomy

• Clinical Rehabilitative Tissue Engineering Research

• European J. of Surgical Research • Indian Journal of Plastic Surgery • Journal of Anatomy • Journal of Brachial Plexus and

Peripheral Nerve Injury • Life Sciences • Medical Science Monitor • Pain Practice • International Advisory Board,

Surgical and Radiologic Anatomy

Dr. Calum Macpherson: • Acta Tropica • Transactions of the Royal Society of

Tropical Medicine and Hygiene • Journal of Infection • Member, Editorial Team, The Africa

Journal for Animal and Biomedical Sciences

• Member editorial team: West Indian Medical Journal

15.0. Thesis Defenses Brown III, William C. (2007) A case for Sanitation. Patel, Rakesh. (2007) “Ecological survival properties of pelagic and benthic indicator organisms from the St. John’s estuary in Grenada.”

16.0. Seminars Research Ethics and the Food and Drug Administration. Dr. Ruth Macklin, 24 th January 2007. Novel marine bacteria with antagonistic properties against human biofilms. Cynthia Bruno, 31 st January 2007.

Molecular solutions of problems in microbial ecology – basic rules of the


game. Dr. Karsten Pederson, 14 th February 2007.

An overview of Giardia. Camille Coomansingh, 21 st February 2007.

Identification of the Greater Occipital Nerve landmarks in the treatment of occipital neuralgia. Abraham El Sedfy, 28 th February 2007.

An overview of research at the University of Birmingham, Alabama. Dr. RS Tubbs, 14 th March 2007. Research in Anatomy, Quo Vadis. Dr. Marios Loukas, 21 st March 2007. Medical Bioremediation. Justin Rebo, 28 th March 2007. Evaluation and analysis of prostate cancer in Grenada. Megan E. Kaminskyj, 4 th April 2007.

Ecological survival distribution of pelagic & benthic indicators from the St. Johns river estuary in Grenada. Rakesh Patel, 2 nd May 2007.

Antimicrobial properties of skin secretions from Eleutherodactylus johnstonei on bacteriological isolates. Jonathan Ashcroft, 16 th May 2007.

The Public Health Importance of Dogs, Grand Anse, Grenada. Kevin Neill, 23 rd May 2007. Marine microbial biotechnology: biomedical applications of marine bacteria. Dr. Grant Burgess, 15 th August 2007. Trauma. Dr. John Adamski, 22 nd August 2007. Are we spending too much money on HIV/AIDS? Roger England, 29 th August 2007.

The legacy of chemical weapons. The truth unfolded. Robert Johnson, 5 th September 2007.

Straight talking Ugandan Youth: the role of behavior change education in the prevention of HIV/AIDS. Bilal Rahim, 12 th September 2007.

Status of the critically endangered endemic Grenada Dove, Leptotila wellsi. Bonnie Rusk, 19 th September 2007. Benefit sharing in International Research. Dr. Udo Schucklenk, 26 th September 2007.

Opossum: Care and management. Dr Kunwar K. Srivasatava, 3 rd October 2007. The state of the world’s animal genetic resources for food and agriculture. Dr Bowen Louison, 10 th October 2007.

Relationships between macroalgae cover and grazer biomass on coral reefs in southwest Grenada. Steve Nimrod, 17 th October 2007.

Developing community health through sporting partnerships: a global perspective. Ian Elvin, 24 th October 2007.

The hidden microbial world in the “Fountain of Youth”. Ravindra Naraine, 21 st November 2007. The Biological fuel cell – electrical energy from wastewater.“The Power of Poop“. Dr. Dave Lennon, 28 th November 2007. Subversion of cellular signal transduction pathways by human papillomavirus oncoproteins. Dr. Amy Baldwin, 5 th December 2007. Incredible Dissemination in poor rural and agricultural communities – An overview of schistosomiasis.Victor Amadi, 7 th December 2007.


17.0. Further Information

Dr. Calum Macpherson Director WINDREF (Grenada) P.O. Box 7, St. George’s Grenada, West Indies Tel: 1 (473) 4443068 Fax: 1 (473) 4443041 Email: [email protected]

Mr. Trevor Noel Assistant Director WINDREF (Grenada) P.O. Box 7, St. George’s Grenada, West Indies Tel: 1 (473) 4443997 Fax: 1 (473) 4443041 Email: [email protected]

Mrs. Isha English Administrative Assistant WINDREF (Grenada) P.O. Box 7, St. George’s Grenada, West Indies Tel: 1 (473) 4443997 Fax: 1 (473) 4443041 Email: [email protected]

Ms. Meg Conlon Executive Secretary WINDREF (Grenada) P.O. Box 7, St. George’s Grenada, West Indies Tel: 1 (473) 4444175 x 2221 Fax: 1 (473) 4394388 Email: [email protected]

Ms. Sheryll Murray Administrative Assistant WINDREF (USA) 1 East Main Street, Suite 228 Bay Shore, New York, 11706 United States of America Tel: 1 (800) 8996337 Fax: 1 (631) 6652796 Email: [email protected]

Mrs. Sue Huntington Executive Secretary WINDREF (UK) Kingdon’s Yard, Parchment Street Winchester, Hampshire, SO 23 8AT United Kingdom Tel: 01962 850650 Fax: 01962 850567 Email: [email protected]

Dr. Svetlana Kotelnikova Director CERI P.O. Box 7, St. George’s Grenada, West Indies Tel: 1 (473) 4444175 ext. 2465 Fax: 1 (473) 4391845 Email: [email protected]

mailto:[email protected]




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