D. Tsereteli 1, L. Bakanidze 1, I. Velijanashvili 1, L. Beridze 1, M. Manrikyan 2, M. Shakhikyan 2, M. Kekelidze 1, E. Zangaladze 1, N. Tsertsvadze 1, P. Imnadze 1
1 National Center for Disease Control, Tbilisi, Georgia
(Director: P. Imnadze, Ph.D., M.D.)
2 Center for Prevention of Particularly Dangerous Infections, Yerevan, Armenia
(Director: Dr. M. Manrikyan)

• Key-words
• Summary
• Schlüsselwörter
• Zusammenfassung
• Introduction
• Materials and Methods
• Results and Discussion
• References

Yersinia pestis is one of the most devastating infectious agents in the world. Cases of plague were described starting from the pre-Christian era. Despite of major advances made in the knowledge of the disease, plague has not been eradicated yet. Today, in the perspective of the last events the threat of bioterrorism made the society more sensitive to those infectious agents, which can be used as a biological weapon. And plague is one of the main threats of bioterrorism.
Plague in Georgian manuscripts first is mentioned in the XI century. Here clinical manifestations – bubonic form, epidemic character in densely populated areas are described. Later, in the XV century, in another Georgian manuscript, the „Book of Medical Treatment-Karabadini“, more detailed information on plague is given, particularly, the clinical manifestation of bubonic form is described. In 1616 cases of plague were described in western Georgia, probably brought in by catholic missioners. In Georgian folklore plague is named „zhami“ (in old Georgian it means „bad time“). Historically it was well-known that the population from different mountainous regions of Georgia had schistose tombs, very characteristic for given places – mud-huts, towers, etc. These tombs were used for the isolation of people with plague. If the isolated person died, his remains stayed in the tomb unburied, while the other sick persons could stay there at the same time. According to folklore, these tombs, so-called „anatoras“, first were built by inhabitants of the village Anatori, and were emptied by plague in the beginning of the XIX century. The last outbreak of plague took place in 1910, in Batumi – one of the main ports of Georgia, at the Black Sea. In the 1970s four tombs from Khevsureti (Southeast Caucasus) were investigated on the existence of a specific plague antigen. Y. pestis antigen F1 was found in two samples of bone remains from these „anatoras“ [3]. In 2001 we have found some anatoras at the altitude of 2.500 m above sea level on the south slope of Big Caucasus, near the border with Chechnya (Fig. 1). Most of the anatoras (characteristic not only for Georgians, but other Caucasian ethnic groups as well) were destroyed during military actions in the region.

Fig. 1.: Tomb „Anatora“ in Caucasian mountains (altitude 2.500 m above sea level)

Materials for the investigation were taken from live rodents and rodent remains from locations of epizootics: parts of spleen, liver, lungs, pathologically changed lymph nodes, bone marrow and brain. Fleas and ticks were combed out, and collected in burrows. Suspected on plague, material was investigated by microscopy, cultivation on elective media, serology and infecting laboratory animals. Isolated cultures were investigated by bacteriological, biochemical and biological methods. All the strains were analyzed by PCR that was performed using ready-to-go analysis beads („Pharmacia Biotech“). Bacterial DNA for PCR was derived by the standard methodology [1]. The primer pairs used for identifying F1 capsular antigen gene and for V antigen gene were as described [2]. PCR for F1 capsular antigen gene was performed using the following cycle profile:
1 cycle at 95°C for 5 minutes, 35 cycles (95°C - 1 min, 55°C - 1 min, 72°C - 5 min), 1 cycle at 72°C for 5 minutes. PCR for V antigen gene used the following cycle profile:
1 cycle at 94°C for 10 minutes, 30 cycles (94°C - 1 min, 60°C - 1 min, 72°C - 1 min), 1 cycle at 72°C for 10 minutes. PCR for pPLA antigen gene was performed using the following cycle profile:
1 cycle at 95°C for 5 minutes, 35 cycles (95°C - 1 min, 55°C - 1 min, 72°C - 5 min), 1 cycle at 72°C for 5 minutes.

There are three natural foci of plague on the territory of Georgia and Armenia: plain–foothills, high-mountainous and Araks-side. Plain–foothills focus includes the eastern part of Georgia and Azerbaijan territories near the Azerbaijan-Georgia border. Epizootics of plague in the Georgian part of this focus were registered in 1966 and 1968-1971 [3, 4]. The main reservoir of plague in these foci is Meriones erythrourus (libicus), the main vector – Xenopsylla conformis and Ceratophyllus laeviceps. Totally 83 strains were isolated: 30 strains from rodents and 53 strains from the vectors. High-mountainous focus includes the southern part of Georgia and the eastern part of Armenia. Conventionally by state border it is divided into two parts. High-mountainous focus of southern Caucasus first was identified in 1958, when the Y. pestis strain was isolated from Microtus arvalis in the Giumri neighborhood. In the Armenian part of this focus every year in the period of 1958-1988 there were epizootics of plague of different intensity. It must be mentioned that after the earthquake in Armenia in 1988 and until the end of the 1990s epizootic processes decreased in the Armenian part of the focus, and reservoirs and vectors of plague were heavily depressed. Beginning from the year 2000, epizootic processes are activated. In the Georgian part of the focus epizootics of plague were identified in the years 1979-1983 and 1992-1997. The main reservoir in this focus was Microtus arvalis, the main vector – Callopsylla caspia, Nosopsyllus consimilis. From 39 strains isolated 5 strains were from rodents and 34 from vectors. In 1971 Araks-side focus of plague was identified. The main reservoir in this focus was Meriones vinogradovi, the main vector – Xenopsylla conformis and Callopsylla iranicus. Strains of Y. pestis isolated from high-mountainous focus differ from strains of Y. pestis isolated in plain–foothills and Araks-side foci by several characteristics: they ferment ramnose during 48 hours, are less virulent or avirulent for guinea pigs, do not produce pesticin, but are sensitive to it.

Pathogenic Y. pestis-isolates are characterized by the presence of three different plasmids. The Y. pestis - specific 9,5 kbp plasmid pPla carries the plasminogen activator/coagulase and pesticin genes [2, 9]. The other Y. pestis-specific plasmid is pFra, 110 kbp in size. The highly immunogenic fraction F1-capsular antigen and the murine toxin are well-studied proteins encoded by this plasmid. The third plasmid is 70 kbp in weight and is shared by all pathogenic members of the species Y. pestis, Y. enterocolitica and Y. pseudotuberculosis. It contains a number of highly conserved virulence marker genes, and codes for the V antigen, a factor in Yersinia supposed to influence the inflammatory response [1, 8]. Only the 70 kb plasmid is shared among the pathogenic yersinia [5, 7].

In this study 21 strains of Y. pestis, isolated from high-mountainous focus, 1 strain of Y. enterocolitica and 2 strains of Y. pseudotuberculosis were tested by PCR. All 21 strains of Y. pestis produced amplicons in the V antigen gene PCR (Fig. 2). 19 of them produced amplicons in the F1 capsular antigen gene PCR, 2 strains were negative (Fig. 3). All strains lacked pPLA antigen gene. This finding correlates with the data that strains from Transcaucasian Alpines and Daghestan Mountains are exceptions – they do not consist of 9,5 kbp pPLA [6]. 2 strains of Y. pseudotuberculosis and the strain of Y. enterocolitica did not produce amplicons in any of three – V, F1 and pPLA antigens gene PCR. Further studies of these strains (PFGE) will show the correctness of these findings.

Fig. 2: V antigen gene PCR. 1-20 and 22 are Y. pestis-strains, 21 and 23 are Y. pseudotuberculosis-strains, 24 Y. enterocolitica-strain

Fig. 3: F1 antigen gene PCR. 1-20 and 22 are Y. pestis-strains, 21 and 23 are Y. pseudotuberculosis-strains, 24 Y. enterocolitica-strain

1. Brubaker B.: „The V antigen of Yersiniae: an overview.“ In: Une T., Maruyama T. and Tsubokura M. (eds), Current Investigations of the Microbiology of Yersiniae. Karger, Basel (1991) 127-133.

2. Campbell J., Lowe J., Walz S., Ezzell J.: „Rapid and specific identification of Yersinia pestis by using a nested polymerase chain reaction procedure.“ J. Clin. Microbiol. 31 (1993) 758-759.

3. Cherchenko I., Dzebisashvili Iu., Nersesov V., Oganyan E., Magradze G., Mchedlidze G., Velijanashvili I.: „Detection of the Specific Plague Antigen F1 in the Soil from the Tombs of the 19th Century in the Caucasus. Problems of Particularly Dangerous Infections (annals of the Plague Prevention Institutions of the USSR.“ Saratov, 6 (1973) 107-108.

4. Chkheidze G., Dzebisashvili Iu., Nersesov V., Jmukhadze I.: „Natural Foci of Plague on the Territory of Georgia. J. Particularly Dangerous Infections in Caucasus.“ Stavropol. 1 (1974) 70-72.

5. Chu M.C.: „Laboratory Manual of Plague Diagnostic Tests.“ CDC – WHO. (2000) 10-40.

6. Filippov A.A., Solodovnikov N.S., Kookleva L.M., Prostenko O.A.: „Plasmid Content in Yersinia pestis-strains of different origin.“ FEMS Microbiol. Lett. 67 (1990) 45-48.

7. Neubauer H., Meyer H., Prior J. et al.: „A Combination of Different Polymerase Chain Reaction (PCR) Assays for the Presumptive Identification of Yersinia Pestis.“ J. Vet. Med. B 47 (2000) 573-580.

8. Price S.B., Leung K.Y., Brave S.S., Straley S.C.: „Molecular analysis of lcrGVH, the V antigen operon of Yersinia pestis.“ J. Bacteriol. 171 (1989) 5646-5653.

9. Podzorsli R.P., Kukuruga D.L., Long P.M.: „Introduction to Molecular Methodology.“ Manual of Clinical Immunology, 5th Ed., Washington, DC; ASM Press (1997) 77-107.

Corresponding author:
D. Tsereteli Ph. D., M.D.
National Center for Disease Control
9, Asantiani St, Tbilisi 380077, Georgia
E-Mail: rabdato@yahoo.com