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Emerging and Endemic Pathogens

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Advances in Surveillance, Detection and Identification

Kevin P O’Connell

Aberdeen Proving Ground, MD, USA

Evan W Skowronski

U.S Army Edgewood Chemical Biological Center

Aberdeen Proving Ground, MD, USA

Alexander Sulakvelidze

University of Florida

Gainesville, FL, USA

Lela Bakanidze

National Center for Disease Control

Tbilisi, Republic of Georgia

Pathogens

U.S Army Edgewood Chemical Biological Center

Emerging and Endemic

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Proceedings of the NATO Advanced Research Workshop on

Advances in Surveillance, Detection and Identification of Emerging

and Endemic Pathogens

v

Preface

It is a truism among biologists that an organism’s phenotype is the product of both its genotype and its environment An organism’s genotype contains the total informational potential of the individual, while its environment shapes the expression of the geno-type, influences the rate of mutation and occurrence of modifications, and ultimately determines the likelihood that the genotype (or fractions thereof) will survive into the next generation In the relationship between host and pathogen, therefore, each forms a part of the environment of the other, mutually influencing the biology of both partners

on scales ranging from the life history of individuals to the fate of populations or entire species

Molecular biologists working on problems in pathogenesis generally think of the host organism as the pathogen’s environment and perhaps occasionally consider the pathogen as part of the host’s environment However, because “environment” can be defined at many scales, so, too, can phenotypes: if a pathogen, as a species, is con-sidered to exist in a host, as a species, then among its phenotypes is the nature of the pandemic disease it can cause within the host community The contributors to the proceedings of this NATO Advanced Research Workshop have treated the interplay of environment and genotype in the host–pathogen relationship and its relationship to the problem of emerging infectious disease at both the macroscopic and microscopic/ molecular levels along this continuum of scale (with some human history thrown in at times for good measure)

Keynote Chapter

The contribution from the meeting’s keynote speaker highlights the importance of understanding the underpinnings of pathogen phenotypes at both scales The example

of Vibrio cholerae is considered macroscopically and genetically in an examination of

the factors influencing the emergence and spread of new strains of human bacterial

pathogens Citrus greening, caused by the bacterium Liberibacter asiaticus and vectored by the Asian citrus psyllid Diaphorina citri, is discussed to illustrate the effect

of a vector species’ biology on disease emergence and spread An unfortunate lesson from these examples is that diseases that have already emerged and have spread rapidly may be difficult to control; however, any hope of disease control will be founded on an understanding of the genetic and molecular basis for pathogenesis and the environmental factors (including vectors) that contribute to the transmission of the microorganism

Section I: Surveillance

The next four chapters treat country-specific approaches, and their results, in one of the most fundamental tasks in combating emerging infectious disease: detecting and

vi PREFACE

describing the incidence of disease in a geographic region By its very nature, this effort

is labor-intensive in terms of fieldwork (both human and environmental) and in the subsequent laboratory analysis of samples In the Balkans, the Caucasus, and the Central Asian republics, like elsewhere in the developed and developing worlds, surveillance work ranges from the basic (trapping and culturing from members of a reservoir species) to the complex (use of sensitive laboratory molecular methods, such

as PCR) and the application of the resulting data to forestalling and controlling the outbreak of endemic diseases Akimbayev et al., from Kazakhstan, and Gurbanov and Akhmedova, from Azerbaijan, provide a description of surveillance efforts in recent years that highlight the human and economic factors that influence disease transmission From the Republic of Georgia, Bakanidze et al provide a historical perspective that demonstrates the role that militaries have played in the development of public health methods and practices, born of necessity: throughout history, armies over time have lost more soldiers to disease than to violence Complementing the paper by Bakanidze and colleagues, the chapter by Zhgenti et al reports on the use of modern molecular bio-logical techniques to differentiate closely related strains of pathogenic bacteria isolated from both environmental and clinical samples in Georgia and throughout the Caucasus Stikova describes a syndrome-based, nationwide effort deployed in the Republic of Macedonia to report priority communicable diseases that is complementary to the routine surveillance system that reports individual confirmed disease cases This system, called ALERT, aided in forecasting and detecting the start of the influenza season

The goal of surveillance always has been actionable information that would allow public health workers to forestall the spread of disease “Classical” surveillance and epidemiologic reporting as described in these first four chapters, however, now also provides data that are being analyzed by advanced computational and geographic methods known collectively as Geographical Information Systems Blackburn rounds out Section I by describing new tools that enable the fusion of climatologic, geographic, and epidemiologic data with concepts in ecological niche theory to construct models

that may predict the future incidence, prevalence, and transmission of Bacillus anthracis,

but the methods are generalizable to other diseases

Section II: Molecular Analysis and Tools

At the scale of the bacterium and bacterial genome, the contributors to this section each provide an example of how cutting-edge molecular biological methods are being applied to answer key questions in the study of emerging infectious disease

How did the pathogens we observe in the world come to their present state?

Technical challenges abound in the analysis of biological specimens for evidence of ancient infections Aboudharam et al describe the development of dental pulp as a target material for isolation of bacterial DNA and the diagnosis of ancient bacteremias,

including Yersinia pestis infections Key to their methodology is the development of

single-use primer pairs for the detection and amplification of ancient target sequences

in a method they term “suicide PCR.”

What determines the severity of disease a pathogen may cause? Perry et al

demonstrate the utility of comparative genomics in identifying a putative hemagglutinin

gene (“Region E”) that is present in Brucella melitensis 16M and absent in Brucella

PREFACE vii

abortus The data suggest that “Region E” has a host-specific influence on virulence,

and the authors speculate that expressing the hemagglutinin in certain Brucella strains

may improve their performance as vaccines

What genome-wide adaptations predispose a pathogen to cause severe disease?

Rakin examines the contributions of both gain-of-function genetic changes (via lateral

gene transfer) and negative selection (favoring what is termed pathoadaptive mutations)

in the evolution of pathogenic bacteria His analysis points out the importance of

single-nucleotide polymorphisms that, besides being markers for strain identification,

can have significant effects on the functions of virulence and pathogenicity genes The

implication of these results is apparent: in a selective environment or host, mutations

can occur that lead to a sudden emergence of a virulent bacterial strain

What tools are available for practical studies when containment is not available or

practical, but safety must be maintained? Researchers have long used non-pathogenic

surrogates, or “simulants” in place of pathogens and protein toxins for reasons of

con-venience, safety, reduction of expense, and speed of work Such simulants have included

benign enteric bacterial species, bacteriophage (especially MS2), and proteins such as

ovalbumin Ouellette et al review here information that suggest that baculoviruses, long

used in organic agriculture and widely regarded as having no ill effects on humans,

animals or plants, may serve as a new class of simulants for some viral pathogens

How do recent advances in sequencing affect the genetic analysis of pathogens?

Molecular biologists are relying on the rapidly decreasing cost per base of DNA

sequencing to support the continuing effort to detect and identify the genes (as is

discussed by Perry et al.) or gene variants (as in Rakin) that influence bacterial

patho-genicity and virulence Khan briefly reviews the procession from Sanger dideoxy

sequencing (and the dye-coupled PCR-driven variant) to so-called next-generation

sequencing (NGS) methods NGS methods have a much higher throughput than the

Sanger methods but with generally smaller average read lengths Concurrent increases

in computational power allow the rapid querying of databases for bacterial identification

However, although faster computation also speeds contig formation from unique

sequences, short read lengths can result in more contigs that require more effort to

assemble into finished whole bacterial genomes Fortunately, complementary technologies

such as whole genome optical restriction mapping are emerging that very rapidly

provide the scaffolding data needed to match the increased rate at which NGS produces

contigs

Bacterial genome sequencing that 15–20 years ago required years of effort now

takes weeks The rate at which sequencing technology is accelerating has been compared

with Moore’s Law in computing power, except that the rate of improvement for

sequencing has proven to be steeper than the drop in the cost of memory and clock

cycles over time The “next” in NGS likely is ready to become dated in use as single

molecule sequencing methods are being commercialized by at least two companies and

faster methods still are certain to follow

No sequencing technology currently is employed widely outside of laboratories or

core facilities However, entrepreneurs are fervently seeking the right combinations of

technology and business models that will put NGS (and beyond) into the hands of

nonlaboratory end users (clinicians, epidemiologists, law enforcement, and first

responders) The eventual goal is to provide a user with an encyclopedic understanding

viii PREFACE

of the DNA sequences present in a sample, breaking the barrier that currently separates sensitivity plus specificity from speed of analysis The possibility of a technology that will permit fast, accurate, complete data from genuinely unknown samples (unlike PCR) may at last be on the horizon

ix

Acknowledgements

We gratefully acknowledge the assistance of the staff members of the Edgewood Chemical Biological Center, the Georgian National Center for Disease Control, and the University of Florida for their contributions to the success of this Advanced Research Workshop, whose speakers contributed to this volume We also thank the members of the staff of ARW Secretariate for their tireless help with the logistical details of the conference In particular, we thank Geoff Doyle of SAIC, for his outstanding organizational skills, and Rebecca Bryan for fellowship and her good humor as well as expert assistance with innumerable tasks during the conference in Tbilisi

In the production of the conference proceedings, the NATO Science Series staff, in Jean McHale for the many hours spent editing, formatting and compiling the papers included in this book Without her expert help the publishing of this book of conference proceedings would not have been possible

particular Ms Wil Bruins, provided invaluable advice and assistance Lastly, we thank

xi

Organizers

Kevin P O’Connell Evan W Skowronski

U.S Army Edgewood Chemical Biological Center Aberdeen Proving Ground, Maryland, USA

Alexander Sulakvelidze

Emerging Pathogens Institute University of Florida Gainsville, Florida, USA

Lela Bakanidze

National Center for Disease Control Tbilisi, Republic of Georgia

Roger Hewson

Centre for Applied Microbiology and Research

Health Protection Agency Porton Down, Salisbury, United Kingdom

Kazakh Scientific Center for

Quarantine and Zoonotic Diseases

Almaty, Kazakstan

Andrey Anisimov

State Research Center for Applied

Microbiology and Biotechnology

Obolensk, Russia

Giorgi Babuadze

National Center for Disease Control

Tbilisi, Republic of Georgia

Lela Bakanidze

National Center for Disease Control

Tbilisi, Republic of Georgia

Nelli Barnabishvili

National Center for Disease Control

Tbilisi, Republic of Georgia

School of Veterinary Medicine Lousiana State University Baton Rouge, Lousiana, USA

Robert Esler

US Civilian Research and Development Foundation Arlington, Virginia, USA

Jason Farlow Arizona State University Tempe, Arizona, USA

Tbilisi, Republic of Georgia

Tatiana Gremyakova International Science and Technology Centre

National Center for Disease Control

Tbilisi, Republic of Georgia

Akbar Khan

Defense Threat Reduction Agency

Fort Belvoir, Virginia, USA

Defense Threat Reduction Agency

Fort Belvoir, Virginia, USA

Tinatin Onashvili

Laboratory of Ministry of Agriculture

Tbilisi, Republic of Georgia

Daniel Rock Department of Pathobiology College of Veterinary Medicine University of Illinois

Urbana, Illinois, USA

Elisaveta Stikova Faculty of Medicine University of St Cyril and Methodius Skopje, Republic of Macedonia

Nikoloz Tsertsvadze National Center for Disease Control Tbilisi, Georgia

Natalya Vydayko Central Sanitary-Epidemiological Station

Ministry of Health Kiev, Ukraine

Eka Zhgenti National Center for Disease Control Tbilisi, Georgia

xv

Other Participants

Wallace Buchholz

US Army Research Office

Research Triangle Park, North

Defense Threat Reduction Agency

Fort Belvoir, Virginia, USA

Health Protection Agency Porton Down, Salisbury, United Kingdom

Tbilisi, Georgia

Alexander Sulakvelidze Emerging Pathogens Institute University of Florida

Gainsville, Florida, USA e-mail: asulakvelidze@ufl.edu e-mail: ros@1000.pvt.ge e-mail: evan.skowronski@us.army.mil

e-mail: Roger.Hewson@hpa.org.uk e-mail: wallace.buchholz@us.army.mil

xvii

Contents

Acknowledgements ixOrganizers xiSpeakers xiii

Global Effect and Prevention of Emerging and Epidemic Pathogens: Cholera

J Glenn Morris, Jr

Section I: Surveillance

The Epidemiological Surveillance of Highly Pathogenic

Alim M Aikimbayev, Jumabek Y Bekenov, Tatyana V Meka-Mechenko,

and Gulnara A Temiraliyeva

Lela Bakanidze, Paata Imnadze, Svetlana Chubinidze, Nikoloz Tsertsvadze,

Gela Mgeladze, Irakli Shalutashvili, Shota Tsanava, Merab Shavishvili,

Julietta Manvelyan, Nana Ninashvili, and Guram Katsitadze

Application of Modern Techniques for Studying Bacterial Pathogens in Georgia 29

Ekaterine Zhgenti, Gvantsa Chanturia, Mariam Zakalashvili,

and Merab Kekelidze

Sh Gurbanov and S Akhmedova

Strengthening the Early-Warning Function of the Surveillance System:

Elisaveta Stikova, Dragan Gjorgjev, and Zarko Karadzovski

Integrating Geographic Information Systems and Ecological Niche Modeling

into Disease Ecology: A Case Study of Bacillus anthracis in the United States

Jason K Blackburn

13

CONTENTS xviii

Section II: Molecular Analysis and Tools 89

Applications of Paleomicrobiology to the Understanding of Emerging

Gérard Aboudharam, Michel Drancourt, and Didier Raoult

Characterization of a Putative Hemagglutinin Gene in the Caprine Model

Quinesha L Perry, Sue D Hagius, Joel V Walker, Lauren Duhon,

and Philip H Elzer

Alexander Rakin

Detection of Pathogens Via High-Throughput Sequencing 119

Akbar S Khan

Environmental Influences on the Relative Stability of Baculoviruses

Gary D Ouellette, Patricia E Buckley, and Kevin P O’Connell

Keynote Contribution

J Glenn MORRIS, Jr

Professor and Director, Emerging Pathogens Institute, University of Florida,

Gainesville, Florida

Abstract Emerging and epidemic infectious diseases have had a major effect on

human history We are just now coming to appreciate the mechanisms by which

new strains emerge and the factors that permit their rapid spread within human

populations Cholera is a classic epidemic disease that causes periodic pandemics

(possibly reflecting genetic changes in surface antigens of the microorganism) and

seasonal epidemics that appear to be triggered by environmental factors Citrus

greening is a plant disease that kills citrus trees; a vector-borne bacterial disease, it

is currently spreading rapidly across Florida Control of these and other epidemic

and emerging diseases may be difficult, particularly if the infection is already

widespread in target populations Any chance of successful control requires a

comprehensive understanding of the pathogenesis and transmission pathways of

the microorganism

Emerging and epidemic pathogens have played a major role in human history The concept of plagues – infectious and otherwise – is deeply enmeshed in the Biblical story

of Moses, Pharaoh, and the departure of the Jewish people from Egypt in approximately

1300 BC Cholera, with an ancestral home in the delta of the Brahmaputra and Ganges Rivers, is noted in the Sushruta Samhita, written about 500–400 BC; this includes the Sanskrit term generally used to refer to cholera as well as the description of a representative case Reports of cholera can be found in the Arab literature by 900 AD, with descriptions subsequently appearing in European, Indian, and Chinese literature

[1] Plague (caused by Yersinia pestis) was responsible for the Black Death of 1348–

1349, which has been called the greatest biomedical disaster in European and possibly world history [2] Epidemics of yellow fever in the United States were a major driver for the creation of departments of public health beginning with the formation of the Philadelphia Board of Health in 1794

Although there is general familiarity with these “classic” epidemic diseases, recent work by Jones and colleagues [3] suggests that the emergence of new pathogens is a constant, ongoing process Based on a literature review, these authors identified 335 infectious disease emergence “events” occurring between 1940 and 2004 Zoonotic patho-ens (i.e., those that have a nonhuman animal source) predominated, accounting for 60.3%

of events; vector-borne diseases were responsible for 22.8% of events The majority of pathogens identified were bacteria or rickettsia (54.3%) Although some of these events reflected small numbers of actual cases, it is clear that the potential for new disease emergence – with the threat of subsequent epidemic spread – is always present

K.P O’Connell et al (eds.), Emerging and Endemic Pathogens,

DOI 10.1007/978-90-481-9637-1_1, © Springer Science + Business Media B.V 2010

Global Effect and Prevention of Emerging and Epidemic Pathogens: Cholera and Citrus Greening as Examples

4 J.G MORRIS

There are a variety of factors that underlie the emergence of new pathogens Emergence of a pathogen may reflect genetic changes that result in increased virulence, the ability to avoid immunologic detection by the host or killing by antibiotics, or the ability to survive in new ecologic niches Independent of genetic changes, there may

be changes in opportunities for pathogen growth and spread, often as the result of anthropogenic (human-created) changes This may include environmental changes (such as temperature change), changes in ecologic niches, changes in host behavior, and/or introduction of a pathogen into a new geographic area (intentional or otherwise) Finally, pathogen emergence may be influenced by changes in host behavior, including loss of herd immunity (such as the diphtheria epidemics that occurred in parts of the former Soviet Union as a result of decreases in immunization rates) and immuno-suppression (which may be related to disease, aging, and malnutrition) [4]

1 Cholera: A Classic Epidemic/Pandemic Pathogen

Cholera remains the one disease that consistently can cause dehydrating diarrhea in a healthy adult The symptoms of cholera are caused by cholera toxin (CT), a protein enterotoxin that elicits profuse diarrhea [5, 6] Clinically, patients with the most severe form of the disease can pass in excess of 1 L of diarrheal stool per hour; if fluid losses are not replaced by oral or intravenous fluids, this can result in severe dehydration, shock, and death in 12–24 h With appropriate therapy, mortality rates for cholera should be less than 1% However, in the absence of an adequate public health infrastructure to provide treatment, mortality rates may reach or exceed 40% This is reflected in the 2005 World Health Organization’s cholera-surveillance data (the most recent available): 131,942 cholera cases were reported in 52 countries, the majority of which had case-fatality rates below 1%; rates in excess of 1% occurred almost exclusively in sub-Saharan Africa, with multiple countries in this region reporting rates

Why do pathogens emerge?

– Appearance of new/genetically different strains

– Changes in opportunities for pathogen growth and spread (often

anthropogenic)

– Changes in host susceptibility

ability to shift to a rugose phenotype (Fig 1), involving production of an amorphous

GLOBAL EFFECT AND PREVENTION 5

Figure 1 Smooth (left) and rugose (right) colony morphologies for Vibrio cholerae

More than 200 O-groups have been identified for V cholerae, with epidemic cholera cases traditionally linked with O-group 1 (V cholerae O1) Key virulence

factors necessary for occurrence of cholera include CT and associated genes (carried by

the CTX phage, which is capable of transfer among V cholerae strains) and the vibrio

pathogenicity island, which includes genes for toxin-coregulated pilus, a key attachment factor (and the receptor for the CTX phage) However, it appears that the ability to cause epidemic disease is dependent on additional and still poorly charcterized factors In studies conducted in our laboratories using multilocus sequence typing (MLST), all clinical cholera strains clustered into a single MLST clonal complex, consistent with the hypothesis that strains capable of causing disease are closely related phylogenetically

In contrast, there may be striking sequence divergence between epidemic cholera

strains and V cholerae strains from other O-groups In work that we have done with V

cholerae strain NRT-36S, an O31 strain, we found only 89% sequence homology with

epidemic cholera isolates, with absence of a number of putative virulence genes [9] Epidemiologically, cholera tends to occur in two patterns: it spreads in pandemic form, moving across continents, and, after introduction into an area, it may settle into

an “endemic” pattern marked by seasonal epidemics From the perspective of tanding emergence of pathogens, this leads to two basic questions: what mechanisms underlie occurrence of pandemic disease, and, once the pandemic wave has passed, what are the triggers for recurrent seasonal epidemics?

under-1.1 Pandemic Cholera

The modern history of cholera begins in 1817 with the occurrence of what has been designated as the first of seven cholera pandemics It was during the spread of the third pandemic to London in 1854 that John Snow demonstrated the association between illness and consumption of sewage-contaminated water His work established the role

of epidemiology in public health and highlighted the efficacy of simple interventions –

in this case the removal of the handle of the Broad Street pump, which had been linked with illness The seventh (and most recent) cholera pandemic began in 1961, with an

outbreak of disease in the Celebes The strain responsible for this outbreak (V cholerae

O1 biotype El Tor) subsequently has spread through Asia, Africa, Europe, and the Americas, resulting in substantial global morbidity and mortality and leaving behind an endemic pattern of seasonal epidemics (Fig 2) [10]

6 J.G MORRIS

Figure 2 Global spread of the seventh pandemic of cholera

In 1992, against this background and outside of normal seasonal epidemic patterns, cholera began to spread rapidly across India and Bangladesh, with subsequent spread to other parts of Asia In contrast to the traditional endemic pattern of cholera in these areas, all ages were affected, suggesting a lack of preexisting immunity within the population [11] In subsequent studies, we and others found that the strain responsible for this “new” epidemic was from a different O-group (O139), was encapsulated, and had undergone a genetic substitution/deletion with the introduction of 35 kb of “new” DNA encoding the O139 capsule, replacing 22 kb of “original” DNA encoding the O1 antigen [12, 13] Aside from this one substitution, the epidemic strain appeared to be

identical to seventh pandemic V cholerae O1 El Tor strains

Further studies from our laboratory have shown that the gene cluster controlling

expression of the O-antigen and capsule is bounded consistently by two genes – gmhD and rjg Genetic substitutions within this region are not uncommon and may account

for the diversity of O-groups within the species [14, 15] Although the initial epidemic

due to V cholerae O139 did not progress to pandemic disease (i.e., with involvement of

multiple continents), it is clear that this new strain had pandemic potential, and there were suggestions that its appearance should be designated as the beginning of the eighth pandemic Based on our findings with this strain, we would hypothesize that new cholera pandemics result from genetic changes leading to expression of new surface antigens (O-group and capsule), permitting rapid spread of the disease through populations that are immunologically naive to the new antigens

From the standpoint of disease control, these findings underscore the need for rapid vaccine development capabilities to permit the creation of new vaccines (for cholera as well as for other possible emergent pathogens) to match whatever new antigenic combination may appear As a case study, based on our research, we were able to develop a polysaccharide conjugate vaccine rapidly that was protective in animals

GLOBAL EFFECT AND PREVENTION 7

against the O139 cholera strain [16] However, there was inadequate infrastructure and funding to move on to human trials, leaving us, to date, with no available vaccine for this new pandemic strain

1.2 Endemic Cholera with Seasonal Epidemics

As noted above, after passage of a pandemic wave, cholera tends to shift to an endemic pattern of seasonal epidemics We undertook a series of studies in Lima, Peru, in the mid-1990s [17] to try to gain a better understanding of why such epidemics occur The seventh pandemic had entered South America in 1991, appearing first in Peru and then moving across South and Central America In subsequent years, illness settled into an endemic pattern, with epidemics occurring each summer (December–February) Over a 2-year period, we collected monthly samples from eight environmental sites in the

Lima area Detection of CT-producing V cholerae (i.e., strains capable of causing

epidemic disease) in the environment correlated significantly with occurrence of disease in the community 2 and 3 months later; the increase in counts in the environ-ment, in turn, correlated with increases in water temperature associated with the beginning of summer These data support a model of cholera seasonality in which

initial increases in number of V cholerae in the environment (triggered by temperature)

are followed by “spillover” of illness into the human population, with these human for the concept of temperature being an important element in triggering the epidemics has come from other investigators working with data from Peru as well as Bangladesh, with particular attention being given to the potential role of the El Nino-southern oscillation as a driver of the process [18]

Figure 3 Model of cholera transmission

Cholera infections in humans

8 J.G MORRIS

In further studies in two rural communities in Bangladesh (Bakerganj and

epidemic V cholerae strains tend to be closely related phylogenetically, making it

difficult to separate strains by MLST (or other standard molecular typing methods) In contrast, we found that variable numbers of tandem repeats provided us with excellent discrimination among strains Using this technique, we evaluated 68 environmental and 56 clinical isolates from the two communities We found that there was minimal crossover between environmental and clinical strains as well as minimal crossover between strains in Bakerganj and Mathbaria We also found that “epidemics” in any one location, rather than being caused by a single strain, appeared to reflect the sequential appearance of different strain subsets in the human

epidemiologic-population Although environmental V cholerae may serve as a trigger for an epidemic,

these data suggest that subsequent transmission among humans is more likely to be person-to-person The data also suggest that distinct locales have their own strains and strain subsets; that is, an epidemic is not due to a single strain sweeping across the countryside but, rather, reflects the appearance of local strains in human populations

To further explore questions relating to person-to-person transmission, we developed a mathematic model of cholera transmission [20] Interestingly, the best fit for the model was obtained when we incorporated the concept of a “hyperinfectious

state.” This follows from laboratory experiments suggesting that passage of V cholerae

through the intestinal tract results in a short-lived increase in infectivity that decays in a matter of hours into a state of lower infectiousness

These observations help to highlight possible control strategies Although it is unlikely that triggers for environmental proliferation of the microorganism (such as

transmission during high-risk time periods when temperatures are elevated Given the clear importance of person-to-person transmission, efforts also should be focused on minimizing the risk of such transmission within households, with a particular emphasis

on minimizing risk of transmission of the short-lived, hyperinfectious form of the microorganism present in recently passed fecal material

2 Citrus Greening

Citrus greening is a recently emergent infectious disease that currently is estimated to infect 30% of the citrus trees in Florida, and it is spreading rapidly Although not a human disease, it is causing major economic losses and provides some interesting, and different, perspectives on disease emergence

Citrus greening first was reported in the late 1800s in China, where it is known as huanglongbing, or yellow dragon, reflecting the pattern of leaf-yellowing within affected trees It now has spread through citrus-growing areas in much of the world There is no effective control once a tree is infected Infected trees produce less fruit, and the fruit that is produced tends to be bitter and misshapen The etiologic agent for the disease is

thought to be the bacterium Liberobacter asiaticum, transmitted by an insect, the Asian

Mathbaria), we used variable numbers of tandem repeats as a means of typing

V cholerae strains from clinical and environmental sources [19] As previously noted,

temperature) can be blocked, an awareness of the role of environmental V cholerae

in initiating epidemics may permit the focusing of resources on preventing such

GLOBAL EFFECT AND PREVENTION 9

psyllid Although we have a basic understanding of the transmission pathways, a great deal remains to be learned about both the bacterium and the vector [21]

The Asian psyllid first was identified in Florida in September 2005, and, as shown

Figure 4 Spread of citrus greening in Florida

the disease have focused on quarantining infected orchards and bulldozing and burning

in Fig 4, it has spread rapidly across citrus-growing areas of the state Efforts to control

10 J.G MORRIS

infected trees and on using insecticides to kill the psyllid vector Neither approach has been overwhelmingly successful, with the disease continuing to spread rapidly across the state and with reports of psyllids being identified in Louisiana and, as of July 2008,

in California

3 Conclusions

Emerging and epidemic pathogens have been an ongoing cause of human disease since the dawn of recorded history Factors that drive their emergence include genetic changes, changes in opportunities for pathogen growth and spread, and changes in host susceptibility Prevention and/or control of emergent pathogens is possible but requires early recognition and intervention; mathematic modeling that we have done [22] underscores the fact that, by the time new diseases are recognized, they often have spread to the point that control is difficult if not impossible Citrus greening provides an excellent example: traditional control strategies are, at this point, largely ineffective owing in part to the high percentage of trees that already are infected Prevention and control also require a comprehensive understanding of pathogenesis and transmission Cholera provides an example of a pathogen for which better and better data are becoming available, allowing focusing of control strategies At the same time, cholera demonstrates the complexity of these natural systems and the difficulties inherent in designing interventions even with a reasonable knowledge base

3 Jones, K.E., Patel, N.G., Levy, M.A., Storeygard, A., Balk, D., Gittleman, J.L., Daszak, P 2008 Global

trends in emerging infectious diseases Nature 451:990–993

4 Morris, J.G., Potter, M 1997 Emergence of new pathogens as a function of changes in host

susceptibility Emerg Infect Dis 3:435–441

5 Kaper, J.B., Morris, J.G., Levine, M.M 1995 Cholera Clin Microbiol Rev 8:48–86

6 Morris, J.G 2003 Cholera and other vibriosis: a story of human pandemics and oysters on the half

shell Clin Infect Dis 37:272–280

7 World Health Organization 2006 Cholera 2005 Wkly Epidemiol Rec 81:297–308

8 Morris J.G., Jr., Sztein, M.B., Rice, E.W., Nataro, J.P., Losonsky, G.A., Panigrahi, P., Tacket, C.O., Johnson, J.A 1996 Vibrio cholerae O1 can assume a chlorine-resistant rugose survival form that is

virulent for humans J Infect Dis 174:1364–1368

9 Chen, Y., Johnson, J.A., Pusch, G.D., Morris, J.G., Stine, O.C 2007 The genome of non-O1 Vibrio

cholerae NRT36S demonstrates the presence of pathogenic mechanisms that are distinct from O1

Vibrio cholerae Infect Immun 75:2645–2647

10 Morris, J.G Cholera and other vibrioses In: Encyclopedia of public health Amsterdam: Elsevier; 2008

11 Nair, G.B., Ramamurthy, T., Bhattacharya, S.K., Mukhopadhyay, A.K., Garg, S., Bhattacharya, M.K.,

Takeda, T., Shimada, T., Takeda, Y., Deb, B.C 1994 Spread of Vibrio cholerae O139 Bengal in India

J Infect Dis 169:1029–1034

12 Johnson, J.A., Salles, C.A., Panigrahi, P., Albert, M.J., Wright, A.C., Johnson, R.J., Morris, J.G 1994

Vibrio cholerae O139 synonym Bengal is closely related to Vibrio cholerae El Tor but has important

differences Infect Immun 62:2108–2110

GLOBAL EFFECT AND PREVENTION 11

13 Comstock, L.E., Maneval, D., Jr., Panigrahi, P., Joseph, A., Levine, M.M., Kaper, J.B., Morris, J.G.,

Johnson, J.A 1995 Capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a

genetic region not present in Vibrio cholerae O1 Infect Immun 63:317–323

14 Sozhamannan, S., Deng, Y.K., Li, M., Sulakvelidze, A., Kaper, J.B., Johnson, J.A., Nair, G.B., Morris,

J.G 1999 Cloning and sequence of the genes downstream of the wbf gene cluster of Vibrio cholerae

serogroup O139 and analysis of the junction genes in other serogroups Infect Immun 67:5033–5040

15 Chen, Y., Bystricky, P., Adeyeye, J., Panigrahi, P., Ali, A., Johnson, J.A., Bush, C.A., Morris, J.G.,

Stine, O.C 2007 The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae

NRT36S: genes are embedded in the LPS region BMC Microbiol 7:20

16 Johnson, J.A., Joseph, A., Morris, J.G 1995 Capsular polysaccharide-protein conjugate vaccines

against Vibrio cholerae O139 Bengal Bull l’Institut Pasteur 93:285–290

17 Franco, A.A., Fix, A.D., Prada, A., Paredes, E., Palomino, J.C., Wright, A.C., Johnson, J.A., McCarter,

R., Guerra, H., Morris, J.G 1997 Cholera in Lima, Peru, correlates with prior isolation of Vibrio

cholerae from the environment Am J Epidemiol 146:1067–1075

18 Pascual, M., Rodo, X., Ellner, S.P., Colwell, R., Bouma, M.J 2000 Cholera dynamics and El

Nino-southern oscillation Science 289:1766–1769

19 Stine, O.C., Alam, M., Tang, L., Nair, G.B., Siddique, A.K., Faruque, S.M., Huq, A., Colwell, R., Sack, R.B., Morris, J.G 2008 Seasonal cholera from multiple small outbreaks, rural Bangladesh Emerg

Infect Dis 14:831–833

20 Hartley, D.M., Morris, J.G., Smith, D.L 2006 Hyperinfectivity: a critical element in the ability of V

cholerae to cause epidemics? PLoS Med 3:e7

21 Bove, J.M 2006 Huanglongbing: a destructive, newly emerging, century-old disease of citrus J Plant

Pathol 88:7–37

22 Smith, D.L., Harris, A.D., Johnson, J.A., Silbergeld, E.K., Morris, J.G 2002 Antibiotic use in animals has an early but important impact on antibiotic resistance in humans Proc Natl Acad Sci USA

99:6434–6439.

Section I Surveillance

The Epidemiological Surveillance of

Alim M AIKIMBAYEV1, Jumabek Y BEKENOV2, Tatyana V

1M Aikimbayev’s Kazakh Scientific Centre for Quarantine and Zoonotic Diseases,

Almaty, Kazakhstan

2Aktobe Plague Control Station, Aktobe, Kazakhstan

Abstract The Central Asian deserts’ plague focus occupies vast zones of desert

and semidesert in Central Asia and Kazakhstan The differentiation of plague

strains on virulence from the plague foci of Kazakhstan testifies to its high

epidemic virulence From 1990–2003, 23 cases of human plague were registered

From 2004 to 2007, no cases human plague were registered The growth of human

plague has been caused not only by an increase in epizootic activity of the natural

foci but also by the crises of social, economic, and health protection conditions in

the Republic of Kazakhstan during the period of Perestroika The same conditions

challenged the increase in human anthrax, tularaemia, and brucellosis during the

same period Annually, 70,000–100,000 people are vaccinated and revaccinated

with live vaccine strain tularemia Kazakhstan is not endemic for cholera;

therefore, all initial cases of cholera were imported from places such as Pakistan,

Uzbekistan, Iran, Turkey, and Indonesia For epidemiologic supervision of anthrax,

the cadastre of anthrax foci is transferred in electronic format using a Geographical

Information System (GIS) For Kazakh samples, 12 unique MLVA subtypes (KZ-1

through KZ-12) were used.

1 Geographical Epidemiology of Plague

A considerable portion of the Republic of Kazakhstan is located in the territory of one

of the biggest plague foci in the world: the Central Asian desert plague focus, which occupies vast zones of desert and semidesert in Central Asia and Kazakhstan In Kazakhstan, the plague enzootic area covers 1,007,350 km2, that is 39% of Republic territory or 70% of the Commonwealth of Independent States’ natural plague foci The

M Aikimbayev’s Kazakh Scientific Centre for Quarantine and Zoonotic Diseases (10 plague-control stations, 19 local plague branches, and 30 temporary antiepidemic divisions) carries out plague surveillance More than 1,500 people work in the plague-control services of Kazakhstan, including 400 people with higher and middle specialized education All medical and biological employees are certified to do laboratory work after 2–3 months of special training

The main reservoir species of plague in the Central Asian desert plague focus are gerbils, susliks, and marmots Marmots and the yellow suslik are hunted, and people usually are infected by fleas For preventive measures and epidemiologic monitoring, the most important plague flea vectors were determined The initial stages of the flea’s physiological development are the best period for using insecticides

K.P O’Connell et al (eds.), Emerging and Endemic Pathogens,

DOI 10.1007/978-90-481-9637-1_2, © Springer Science + Business Media B.V 2010

Highly Pathogenic Diseases in Kazakhstan

Veterinary surveillance of camels is an important prevention measure because infection of these domestic animals could cause an epidemic [1] Camels are only rarely infected by plague; on average, a plague-infected camel is registered once every 10 years However, the meat of a slaughtered camel can cause disease not only during the slaughter, but human plague has been diagnosed in purchasers of infected meat long distances from the location of slaughter Wintertime human plague infections caused by plague-infected camels have been connected to latent infection in the camels as a result

of poor feeding and a decrease in the animals’ resistance to infection

Plague surveillance and prophylaxis consists of several synergistic elements: the control of plague transmission, epidemiologic investigation, field disinfection, settle-ment disinfection and deracination, vaccination of humans, work with a medical network, work with a veterinary service on camel plague prophylaxis, and sanitary and educational work with the population Those at risk for plague infection include cattle breeders and members of their families, railway-communication workers, participants in expeditions, workers at meteorologic stations, field workers, fur-trade workers, veterinary workers, medical workers in the countryside, and inhabitants of small cities having cattle grazing

on the enzootic territories

The natural factors of Aral Sea regression and Caspian Sea transgression were

Caspian Sea rose 2 m, changing the contours of the coastal site Raised subsoil in the waters has changed the microclimate in rodent holes, which has resulted in the dying off of fleas and the sanitation of foci

Use of landscape epidemiologic principles has allowed us to reduce epizootologic inspection of plague foci tenfold and to concentrate our field work in areas where the main part of the rodent population is located Informative inspection in difficult economic conditions was developed and used as the reconnaissance method for inspections of gerbil foci During a 10-day tour, the zoologist (parasitologist) collects flea probes for testing in the central laboratory The territory around the settlements was subject to inspection; during this period, sparsely populated areas were not surveyed Positive results in the epizootics in any part of the autonomous focus were extrapolated

to the entire territory and were indications for prophylaxis This has allowed us to reduce the number of exposed antiepidemic groups [2]

2 Analysis of Plague Isolates

Differentiation of Yersinia pestis strains from the plague foci of Kazakhstan by genetic

analysis [3] suggests that the most of the strains are likely to be highly virulent in humans The high epidemic virulence of Central Asian plague-focus isolates is proved

by modern methods of genetic analyses [4] on the variability of the nucleotide

sequences of the genes of rha-locus Yersinia pestis strains of the basic and nonbasic

subspecies The same research shows an evolutionary antiquity of Caucasian strains

and their similarity to Yersinia pseudotuberculosis, which explains the low epidemic

potential of Caucasian foci plague strains

zone enzootics in the shoaled parts of the Aral Sea was revealed The water level in the considered when determining plague-focus epidemic potential The expansion of

16 A.M AIKIMBAYEV ET AL

THE EPIDEMIOLOGICAL SURVEILLANCE OF HIGHLY PATHOGENIC DISEASES 17

3 Incidence of Human Cases of Plague

As a result of the epizootologic investigations of the past two decades, new plague foci

or sites have been discovered in the Central Asian desert plague focus From 1990 to

2003 in Kazakhstan, 23 cases of human plague were diagnosed in 17 geographical foci

of human plague Morbidity increased fourfold in comparison with the foregoing period (1977–1989), during which six cases of human plague were registered Of the cases diagnosed from 1990 to 2003, 11 cases of human plague were caused by flea bites The main causes of mortality in these cases were delayed medical attention, incorrect primary diagnosis, and accompanying chronic disease [2] The growth of human plague has been caused not only by an increase in epizootic activity of the natural foci but also

by the crises of social and economic conditions in the Republic of Kazakhstan, which did not allow adequate funds for preventive action

The negative social effects during the period of Perestroika reduced the immune status of the population and the resistance of the inhabitants of the Commonwealth

of Independent States not only to plague but also to other infectious diseases The decreasing immune status was caused by stressful living conditions, including unemploy-ment and a falling standard of living The stress accompanied by an increase in the hormone level of corticosteroids resulted in an immune-depressive action and a decrease in organisms’ resistance to infections For example, earlier patients were infected with bubonic plague by multiple flea bites; in 2003, one trace of flea bite was found in a child who died of plague Approximately 25,000 bacteria – the quantity of plague microbe delivered by one flea bite – was enough for plague transmission From 2004 to 2007, no cases of human plague were registered Since 2006, the medical service has used a definition of cases of especially dangerous infection regulated by the Order of the Ministry of Health RK #623 (15.12.2006), in which stages of the diagnosis are subdivided into suspect, presumptive, and confirmed plague

4 Treatment of Plague Infections

The first stage in the treatment plan for plague patients [1] is detoxification by introduction a 0.5-L salt solution with diuretic The use of bacteriostatic antibiotics then

is preferred It allows avoiding the Jarisch-Herxher reaction The daily dose of biotic should not exceed 2.0 g (3 g in combination) The antibiotics and the salt solution should be administered in a 1:1 ratio (1.0 g of antibiotic to 1.0 L of solution) We prefer gentamycin for replacement bacteriostatic antibiotics for bactericidal treatments on the second day This antibiotic penetrates well through the hematoencephalic barrier and prevents infection with meningoencephalitis Meningoencephalitis complicates 50% of cases in children younger than 14 years [1] This antibiotic also counteracts microbe endotoxin In cases of bubonic plague, 80.0 mg of gentamycin was injected three times per day; in cases of pneumonic and septic plague, 80.0 mg of gentamycin was injected three to four times per day

anti-The use of oxacillin is not recommended because it can cause necrosis at the point

of injection Doxycycline in combination with ciprofloxacin has no expected synergistic action It is recommended to replace doxycycline with amikacin or rifampicin or, in their absence, cefotaxim

The use of corticosteroids in indicated where the collapse of the patient’s condition

is progressive, but corticosteroids suppress phagocytosis [5] The antibiotic-resistant cells multiply, which may cause a relapse in 2–3 days In this case, we recommend using other group reserve antibiotics For treatment of the plague-related skin ulcers,

we recommend applying polyphytoleum (“Kyzylmay,” manufactured in Kazakhstan)

We have proved the safety of discharging patients diagnosed with plague from the hospital after treatment and one negative test instead of three This allows closing the epidemic foci 4 days earlier, and it already is regulated by the new Ministry of Health standard

5 Surveillance and Treatment of Cholera

Kazakhstan is not endemic for cholera; therefore, all initial cases of cholera were imported from places such as Pakistan, Uzbekistan, Iran, Turkey, and Indonesia [6] The cholera infection then spread through household contact To determine whether

Vibrio cholerae was in the water of the Syr-Darya River, we tested the rivers upstream

and discovered a camp of nonlegal immigrants – workers from Kara-Kalpak (Uzbekistan) The bacteriological tests were negative because the workers took antibiotics, but cholera infection was confirmed by the presence of vibriocidal antibodies [7] The highest case rate occurred in 1993, when 65 cholera patients came by air from Karachi (Pakistan) to Almaty over the course of 3 days For the temporary isolation of passengers arriving from countries with cholera outbreaks, an area was set up in Almaty airport with room for 500 people Those who had contact with people who were sick were sent to city hospitals, where they received preventive treatment with the antibiotic Ciflox Travellers leaving Kazakhstan received a certificate of epidemiologic safety for their countries of residence A total of 119 patients were isolated in 1993 Kazakh tourists and migrants were infected with cholera in Pakistan, Indonesia, Tajikistan, and Uzbekistan In 1996, Kazakh tourists who were travelling to countries with cholera outbreaks were given the licensed intestinal antiseptic Intetrix (Beaufour IPSEN International, France) for chemoprophylaxis if there was a likelihood of secondary transmission These measures prevented the introduction of cholera into Kazakhstan in 1996 [8] In Almaty in 2000,

a strain of V cholerae O-139 Bengal was isolated from a patient Kazakh Scientific

Centre for Quarantine and Zoonotic Diseases (KSCQZD) immunoglobulin erythrocyte

diagnosticum is used to test for V cholerae O-139

6 Surveillance and Prophylaxis for Tularemia

The natural tularemia foci in Kazakhstan occupy 552,400 km2 (26% of the territory of the Republic) The most effective method of prophylaxis is vaccination by live vaccine

strain Francisella tularensis holarctica (Russian), which provides reliable immunity for

5 years Annually, between 70,000 and 100,000 people are vaccinated and revaccinated

We have patented the strain F tularensis mediasiatica KA-29 for creation of a domestic vaccine that is highly immunogenic, non reactogenic, and will induce cross-

immunity [9]

18 A.M AIKIMBAYEV ET AL

THE EPIDEMIOLOGICAL SURVEILLANCE OF HIGHLY PATHOGENIC DISEASES 19

7 Surveillance of Anthrax and Analysis of Bacillus anthracis Strains

In Kazakhstan, there are 2,598 anthrax soil foci; 1,767 stationary anthrax settlements are registered in a 2,100 km2 area For epidemiologic supervision of anthrax the cadastre of anthrax foci is transferred in electronic format The geographic distribution

of Bacillus anthracis strains throughout Kazakhstan are monitored using GIS methods

For Kazakh samples, MLVA was performed on 93 anthrax strains from the KSCQZD collection using eight VNTR markers developed in the laboratory of Р Keim A

UPGMA dendrogram of 88 isolates from the KSCQZD B anthracis collection revealed

12 unique MLVA subtypes: KZ-1 through KZ-12 Genotyping of Kazakh anthrax strains according to Keim et al [10] and Pazylov et al [11] has shown that they fall under genetic group “A,” which is widely spread throughout the world The majority of isolates (n = 79) belong to the previously described A1a genetic cluster Similar strains

were isolated in China, Turkey, and Europe Nine isolates belong to the A3b, A4 clusters and novel genetic lineages A5, A6

In accordance with veterinary laws in the Republic of Kazakhstan, the owners of livestock suspected of having anthrax receive financial compensation at market price if they give the ill animals to the veterinary service This has been the practice in Kazakhstan since1968 and has had high preventive efficiency

8 Brucellosis

The problem of brucellosis is caused by animal industries and the prevalence of small

cattle (such as sheep and goats), which are the carriers of the most pathogenic Brucella species, Brucella melitensis A total of 3,000 people are sickened annually Lambing-

time work attracts many teenagers, and it could result in their becoming disabled Therefore, the first step in preventing infection is explaining safety measures to those involved in lambing-time work The most important measure is immunization of livestock

9 Outlook

By 2015, the government of the Republic of Kazakhstan is planning to spend up to 4%

of the gross national product on public health services The steps for preventing highly pathogenic diseases are the scientific development of bases of epidemiologic super-vision, the introduction of new technologies, and the perfection of normative documents and legislations

References

1 Aikimbayev, A The plague [Russian] Almaty: Kazinformcenter; 1992

2 Aikimbayev, A., Atshabar, B.B., et al The Kazakh natural plague foci epidemic potential [Russian] Almaty: DOIVA; 2006

3

4 Kukleva, L.M., Eroshenko, G.A., Kuklev, V.E., Shavina, N.Iu., Krasnov, Ia.M., Guseva, N.P., Kutyrev,

V.V 2008 A study of the nucleotide sequence variability of rha locus genes of Yersinia pestis main

and non-main subspecies [Russian] Mol Gen Microbiol Virusol 2:23–27

5 Aikimbayev, A., Temiralyeva, G Cortisones for plague diagnostic and treatment [Russian] Almaty:

9 Aikimbayev, A., Chimirov, O The strain Francisella mediaasiatica 240, attenuated, candidate of

tularemia vaccine Patent #312 Astana: Committee on intellectual property rights of the Ministry of Justice of Republic Kazakhstan; 2002

10 Keim, P., Price, L.B., Klevytska, A.M., Smith, K.L., Schupp, J.M., Okinaka, R., Jackson, P.J., Jones, M.E 2000 Multiple-locus variable-number tandem repeat analysis reveals genetic relationships

Hugh-within Bacillus anthracis J Bacteriol 182:2928–2936

11 Pazylov, Y., Meka-Mechenko, T., Easterday, W., Van Ert, M., Keim, P., Hadfield, T., Francesconi, S., Blackburn, J., Hugh-Jones, M., Aikimbayev, A , Lukhnova, L., Zakaryan, S., Temiraliyeva, G Molecular

diversity of Bacillus anthracis in Kazakhstan 17th European Congress of Clinical Microbiology and

Infectious Diseases Oxford: Blackwell Publishing; 2007 p 155

Aikimbayev, A., Kisselev U., et al The effect of human plague isolates on eukaryotic cells [Russian]

Temiralyeva, G., Meka-Mechenko, T., et al Dangerous zoonotic diseases control and biosafety system

20 A.M AIKIMBAYEV ET AL

The plague epidemiological surveillance and prophylaxis measures Almaty: Kazakhstan, 1992, pp 45–48

Aikimbayev, A., Temiralyeva, G Chemoprophylaxis and diagnostics of cholera [Russian] Health

Kazakhstan, 1994

Kazakhstan, 2002 pp 106–110

protection of Kazakhstan Almaty: Kazakhstan, 1996; pp 26–29

21

K.P O’Connell et al (eds.), Emerging and Endemic Pathogens,

DOI 10.1007/978-90-481-9637-1_3, © Springer Science + Business Media B.V 2010

Surveillance on Plague in Natural Foci

in Georgia

Lela BAKANIDZE, Paata IMNADZE, Svetlana CHUBINIDZE,

Nikoloz TSERTSVADZE, Gela MGELADZE, Irakli SHALUTASHVILI,

Shota TSANAVA, Merab SHAVISHVILI, Julietta MANVELYAN,

Nana NINASHVILI, and Guram KATSITADZE

National Center for Disease Control and Public Health and Medical Statistics

of Georgia, Tbilisi, Georgia

Abstract Plague is one of the oldest and most devastating recorded human

diseases Several epidemics of plague have occurred in the territory of Georgia In

1933, the Transcaucasian Anti-Plague Center was established in Tbilisi There are

two natural foci of plague in the territory of Georgia: plain–foothill and high

mountainous The Georgian Anti-Plague Station carried out active surveillance on

natural foci In the plain–foothill focus, plague epizootics were established in 1966

and in 1968–1971 In the high-mountainous focus, plague epizootics were

established in 1979–1983 and in 1992–1997 A total of 122 strains of Yersinia

pestis were isolated in Georgia – 83 in the plain–foothill focus and 39 in the

high-mountainous focus; 46 strains are kept at the National Center for Disease Control

and Public Health’s Microbial Library Although no new isolates were obtained in

recent years, the plague foci in Georgia are so close to populated areas that they

must be under permanent control to be able to respond rapidly to emergencies

1 Introduction

Plague is one of the oldest recorded human diseases, and it likely originated in the

Himalayan region during the pre-Christian era One of the first recorded outbreaks of

plague was described in Athens in 430 BC during the Peloponnesian War The outbreak

caused an estimated 300,000 deaths During the Christian era, epidemics of plague

occurred in 5- to 12-year cycles grouped in three pandemics The first pandemic, called

Justinian plague (AD 541–750), spread from Egypt through the Middle East and

Mediterranean basin Population loss was 50–60% The second pandemic, called The

Black Death, started in central Asia in AD 1330 The pandemic killed an estimated

17–28 million Europeans The third pandemic, which is currently ongoing, started in

1855 in the Yunnan region of China and spread to Hong Kong, India (where it killed an

estimated 12.5 million people from 1898 to 1918), Africa, South and North America,

and much of the rest of the world

An estimated 200 million deaths have been attributed to Yersinia pestis infection

throughout recorded history It is the first bacterium used as a biological warfare agent,

when the Tatars catapulted plague-infected corpses into the sieged Black Sea port of

Kaffa (currently Feodossia in the Ukraine) in 1346 During World War II, the Japanese

22 L BAKANIDZE ET AL

infected fleas with Y pestis and released them into several Chinese cities, causing small

epidemics of plague

Y pestis also was the key component of biological weapon development programs

in the United States, the former Soviet Union, and other countries, and it is also likely

to be one of the preferred agents to be used during possible bioterrorism attacks in the United States and elsewhere

2 History of Plague in Georgia

Georgia is located at the crossroads of Europe and Asia, and it was a major link in the chain of the Great Silk Road It always was a vivid transition point for voyagers, thus it was not protected from the spread of different epidemics – among them plague

In Georgian folklore, plague was named zhami (in old Georgian, it means misfortune) Plague first was mentioned in Georgian manuscripts in 11th century, when clinical manifestations (bubonic form, epidemic character in densely populated areas) were described Later, in the 15th century, more detailed information on plague was given in another Georgian manuscript – “Book of Medical Treatment-Ustsoro Karabadini” (“Peerless Karabadini”); particularly, the clinical manifestation of the bubonic form was described

While information on individual cases of plague in the 16th and 17th centuries can

be found, official registration of plague cases started in the 19th century after Georgia was joined to Russia Three plague epidemics were registered in Georgia during the 19th century: 1803–1807, 1811–1812, and 1838–1843 The epidemics started mainly in the south of Georgia – Akhaltsikhe and surrounding territories – and later spread east, north, west, and to Tbilisi There were special quarantine checkpoints arranged at the entrances to the capital city, and disease surveillance was also conducted in military units

On February 2, 1804, after several cases of plague had been identified, the Russian Tsar’s representative in Georgia issued an order containing special measures that were

to be carried out against plague epidemics These measures were not effective, however, and did not prevent the spread of epidemics that had a devastating effect on the population of the northern part of Georgia Many villages were emptied because of the disease In 1807, in the Larsi citadel in the north of the country near the border with Russia, 1,596 cases of plague were registered, out of which 1,144 (71.7%) people died The population of the mountainous regions in the north of Georgia built tombs to isolate those who showed signs of plague In Khevsureti and Tusheti – regions in the north of the country – we still find such tombs today, called anatora after the village Anatory, the entire population of which died of plague

In 1811, the epidemics spread to Tbilisi The city major reported to the Tsar’s representative the necessity of taking anti-epidemic measures Shortly thereafter, the disease spread to neighboring villages Later plague epidemics expanded to the central part of Georgia and, despite quarantines, reached many villages not only in the central but also in the eastern part of the country

Later in 19th century, plague epidemics were registered in Georgia from 1803 to

1807, from 1811 to 1812, and from 1838 to 1843 Usually they started in the southern

SURVEILLANCE ON PLAGUE IN NATURAL FOCI IN GEORGIA 23

regions, particularly in Akhaltsikhe The population of Akhaltsikhe, especially the adults, was more protected from plague compared with those in other parts of Georgia There was a tradition of gathering and saving necrotic parts of plague pustules; during the subsequent epidemic, they were ground and drunk with water We speculate that partial protection from plague might have resulted from this practice

The first officially documented plague outbreak in western Georgia was in 1836 in Batumi There were subsequent plague outbreaks in Batumi in 1901, 1910, and 1920

In 1927, by decision of the Narkomzdrav (People’s Commissariat of Public Health) of the Georgian Soviet Socialist Republic, a specialized anti-plague (AP) laboratory was set up at the Batumi port to carry out the quarantine monitoring of the ships coming from countries considered to be risks with regard to plague In 1934, the Batumi port

AP laboratory joined the centralized AP system of the USSR as a department of especially dangerous infections The reason for the repeated occurrences of plague was believed to be the poor sanitary conditions in the city The fact that the initial cases of plague always were discovered in proximity to the port facilities led Soviet epidemiologists

to conclude that the plague was brought to Georgia by foreign naval vessels from Turkey and other Middle Eastern countries where unsatisfactory epidemiological conditions prevailed The last officially registered human plague case in Georgia also was registered in Batumi, in 1924 It was imported by a sailor on a foreign ship

3 Establishing an Anti-plague System in Georgia

In 1933, under the initiative of Professor Giorgi Eliava, the Transcaucasian AP Center was created in Tbilisi at the Institute of Bacteriology In 1937, the Transcaucasian AP Center became an independent organization and was renamed the Tbilisi AP Monitoring Station The main function of this organization was to carry out epidemiological monitoring of the Tbilisi city territories and surrounding districts In 1939, under the leadership of Nikoloz Abashidze, the functions of the Tbilisi AP Monitoring Station expanded Georgian AP specialists began to study epidemiological outbreaks of unknown etiology and undertook the epizootic monitoring of areas near the Turkish border Later, the Georgian AP Station became an integral part of the centralized AP system, controlled by the Main Department of Quarantinous Diseases of the Ministry of Health of the USSR The AP system had a very well-defined hierarchy; and the supervisor for the Georgian AP Station was the Stavropol AP Institute All new isolates

of especially dangerous pathogens (if any) had to be sent to Stavropol for confirmation, after which the isolates were to be destroyed

In 1953, plague epizootics was discovered on Apsheron Peninsula in Azerbaijan

among Libyan jirds (Meriones libicus erythrourus) The Georgian AP system organized

and sent the first epidemiological team to look for a natural plague focus in Eastern Georgia on the then-administrative border with Azerbaijan In 1956, the continuous plague epizootics in neighboring Armenia and Azerbaijan prompted the reorganization

of the Tbilisi AP Monitoring Station into the Georgian Republic AP Station, and active surveillance on plague had started In 1958, by decision of the Ministry of Health (MOH) of the USSR, the Batumi port AP laboratory was upgraded into a field AP station and placed under the administrative control of the Georgian Republic AP

24 L BAKANIDZE ET AL

Station in Tbilisi In 1979, another field AP station was established in Tsitelitskaro (now Dedoplistskaro) to conduct epidemiological monitoring in eastern Georgia The Georgian AP system consisted of the Georgian AP Station in Tbilisi, two field AP stations in Batumi and Tsitelitskaro, and four seasonal AP laboratories in Aspindza, Dmanisi, Jandara, and Ninotsminda

As a result of the activities at the stations and laboratories of the Georgian AP system, the existence of two natural foci of plague in the territory of Georgia – plain/

Figure 1 Natural foci of plague in eastern Georgia

The main reservoir in the plain/foothill focus is the jird Meriones erythrourus; the main vectors between rodents are fleas (most commonly Xenopsylla conformis and

Ceratophyllus laeviceps) This information was determined by sampling not only

High-mountainous focus first was identified in 1958 The main reservoir here is

the common vole Microtus arvalis; main vectors are Callopsylla caspia, Nosopsillus

consimilis, and Ctenophthalmus teres

As was mentioned above, all isolates of Y pestis were sent to the Stavropol AP

Institute Later, however, at the request of the head of the Georgian AP Station, Professor

Levan Sakvarelidze, several strains of Y pestis, including isolates from Georgia, Dagestan,

Kyrgyzia, and Armenia, were returned to the Museum of Live Cultures at the Georgian

AP station in Tbilisi for research

After the collapse of the Soviet Union, independent Georgia went through economic difficulties, and there was a considerable decrease in state funding Limited financial resources forced the Georgian AP Station to cut back on epizootic surveillance and epidemiological monitoring of natural plague foci Remaining seasonal field work was limited, and field-team size was reduced

foothill and high mountainous – was established (see Fig 1)

rodents themselves, but also from analysis of the contents of rodent burrows (Fig 2)