P. Imnadze 1, L. Bakanidze 1, M. Manrikyan 2, T. Kukhalashvili 1, E. Zangaladze 1, M. Kekelidze 1, D. Tsereteli 1, M. Nadiradze 1, Sh. Tsanava 1, N. Tsertsvadze 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

Summary
In the last years a significant increase in the number of anthrax cases was detected in the whole South Caucasus region. Anthrax in Georgia and Armenia is characterized by marred seasonal prevalence – the end of spring and in early autumn register 93 per cent of cases. The disease cases were registered in cattle and humans as well as an appearance of recently infected fields has been observed. After 1989 only one patient was registered with the intestinal form of the disease, all others had cutaneous form.

During the last 13 years in countries of Southern Caucasus 35 strains of B. anthracis were isolated (among them 17 from humans, others from beef, cattle skin, animal hair and soil). Isolated strains are of high virulence. AP-PCR identified a relationship among the tested isolates. The genetic profiles of 23 B. anthracis strains were identical to each other. One strain isolated in 1989 from the burial site has a distinguished genetic profile.

Zusammenfassung
In den letzten Jahren musste im ganzen südlichen Kaukasus ein deutlicher Anstieg der Inzidenz von Anthrax festgestellt werden. In Georgien und Armenien zeigt das Auftreten von Anthrax eine charakteristische jahreszeitliche Verteilung: 93% der Fälle finden sich im späten Frühling und im frühen Herbst. Mit Ausnahme eines Falles von Darmmilzbrand manifestierten sich alle Humanerkrankungen (Auswertung seit 1989) als Hautmilzbrand.

In den letzten 13 Jahren wurde im südlichen Kaukasus 25-mal Bacillus anthracis isoliert (in 17 Fällen aus humanem Untersuchungsmaterial;
die restlichen Isolate stammen aus Rindfleischproben, Rinderhäuten, Tierhaaren und Erdproben). Alle Isolate erwiesen sich im Mäusetierversuch als virulent. Mittels AP-PCR wurden 24 Bacillus anthracis-Isolate subtypisiert. Mit Ausnahme eines Stammes, welcher im Jahr 1989 aus Bodenproben von einem bekannten Wasenplatz kultiviert wurde, zeigten alle Stämme ein einheitliches genetisches Profil.

Anthrax is an acute infectious disease, and its eradication or control is one of the main goals of Public Health and veterinary. Recent events in US, wide use of anthrax, as the agent of biological terrorism, increased the interest in this pathogen. Combating the infection, reducing potential sources of it, and carrying out timely measures is very important worldwide.

Anthrax in Georgia is well known from the middle of the centuries. Even in 1697 the famous Georgian writer Sulkhan-Saba Orbeliani describes this disease as very acute and dangerous. During the 20th century increase in anthrax morbidity was seen in the years 1924-1925 and 1941-1945.

Anthrax was diagnosed based on clinical, epidemiological and laboratory data. Laboratory investigation consists of microbiological methods [1]. Skin lesions, materials from cattle, environmental samples were tested. Main method for laboratory diagnostics of anthrax was microbiological (microscopy, seeding on media, bioprobes on laboratory animals), serological and allergic methods were used as well. B. anthracis was isolated by growth on beef-peptone agar and beef-peptone broth pH 7,2. After incubation in 37ºC for 24 hours typical for B. anthracis growth was seen both on solid and liquid media. B. anthracis was identified by a number of specific characteristics: morphology of microbial cells, character of growth in broth, mobility, hemolitic activity, coagulation of egg protein and pathogenicity for laboratory animals (white mouse). AP-PCR was performed using a RAPD Kit containing ready-to-go analysis beads („Amersham“). Primer # 5 of the kit (5-AAC GCG CAAC-3) was superior to the other primers examined. Therefore all subsequent AP-PCR was performed using this primer. Bacterial DNA for AP-PCR was derived by the standard methodology. AP-PCR was performed using the following cycle profile: 1 cycle at 95°C for 5 minutes, 45 cycles (95°C - 1 min, 36°C - 1 min, 72°C - 2 min), 1 cycle at 72°C for 5 min.

In the last years a significant increase in the number of anthrax cases was detected in the whole South Caucasus region. During 1996-2001 in Georgia 153 cases of anthrax were registered, in Armenia 41 cases. 64% of patients with anthrax are men, the disease prevails in age groups 31-40 years (31%) and 41-50 years (26%). Anthrax in Southern Caucasus is characterized by marred seasonal prevalence – the end of spring and in early autumn register 92,6 per cent of the cases. The disease cases were registered in cattle and humans as well as appearance of recently infected fields has been observed. After 1989 only one patient was registered with the intestinal form of the disease, all others had cutaneous form. Two patients died (one of them had intestinal form). Incubation period in 95% of cases lasted 2-4 days. In cutaneous form ulcers were located predominantly on hands (97% of cases) (Fig. 1). In one case the skin lesion was on the nose (Fig. 2). The main source of infection with anthrax is infected live-stock (among them cattle in 84,3%, small cattle 14,2%, horses 0,8% and pigs 0,7% of cases). The disease cases were registered in cattle and humans as well as appearance of recently infected fields has been observed [2]. In 2001 17 cases of anthrax were registered in humans in western Georgia. During the same year in Armenia 18 human anthrax cases were registered. All were cutaneous forms.

Fig. 1: Skin lesion on the hand

Fig. 2: Skin lesion on the nose

The main reason of infecting humans and cattle with anthrax today is:
1) incomplete vaccination of cattle;
2) lack of effective methods of sanitation of soil foci;
3) insufficient veterinary and sanitary conditions of burial sites.
During the last years permanent soil foci arose on the territories of several regions in Georgia as well as in Armenia. During the last 13 years in countries of Southern Caucasus 35 strains of B. anthracis were isolated (among them 17 from humans, others from beef, cattle skin, animal hair and soil). Isolated strains are of high virulence.

24 B. anthracis-strains were tested in this study. Isolates were collected from different sources during 1989-2001 and identified by the NCDC Anthrax Laboratory. AP-PCR identified relationship among the tested isolates. The genetic profiles for 23 B. anthracis-strains were identical to each other. One strain (No. 2) isolated in 1989 from the burial site has a different genetic profile (Fig. 3).

Fig. 3: AP-PCR, B. anthracis-strains

1. „Diagnostics of particularly Dangerous and Less Known Infections (Methods of Laboratory investigation).“ Rostov University Press, (1970) 193-214.

2. Turnbull P.C.B.: „Guidelines for the Surveillance and Control of Anthrax in Humans and Animals, WHO Department of Communicable Diseases Surveillance and response.“ (1998).