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The First Epidemy of Tularemia in
FR Yugoslavia
Lako Branislav*, Ristanovic Elizabeta*, Spasic Miroslav**,
Prodanovic Radivoje***, Djuric Roman.*
* Military Medical Academy,
Crnotravska 17, Belgrade
** Institute of Public Health, NIS
*** Faculty of Chemistry, Belgrade
INTRODUCTION
Tularemia
is zoonotic disease caused by Francisella tularensis. The reservoirs
of disease are the mamals of geni Lagomorpha and Rodentia and vectors
are ixodic ticks and other haematophagic insects. Tularemia is predominantly
disease of the northern hemisphere, and the form of the disease depends
on the route of entry of F.tularensis in the organism. The ulceroglandular,
tonsilopharyngeal, gastrointestinal and pulmonary form of disease occur
most often. F.tularensis can be used as a weapon in biological warfare.
Immunodiagnostic procedures, based on detection of specific antibodies
on F.tularensis in sera are most often used in diagnostic of tularemia.
Although
FR Yugoslavia is an endemic region, the first epidemic of tularemia
in our country broke out in 1998, in the region of mountain Rtanj, near
Sokobanja. In that period thirty-eight people, between 7 and 77 years,
had the disease. The most often appeared tonsopharyngeal form and rarely
ulceroglandular form of tularemia. The epidemic lasted from 1999 through
2000, in the wider region of Sokobanja, including villages near Pirot
and Aleksinac. During 1999. and 2000, tularemia appeared in the southern
Serbian province, Kosovo and Metohia, but unfortunately we have not
evidence nor any date about disease from that region. For the first
time in FR Yugoslavia, in the beginning of 1999, F.tularensis was isolated
from dead individuals of the genus Apodemus in the infected region.
This work is devoted to biochemical, genetic and immunochemical characterization
of that isolate and the use of our isolate as antigen for preparation
and standardization of homemade immunodiagnostic procedures, such as
agglutination, immunofluorescence and immunoenzyme tests.
MATERIALS AND METHODS
We
developed the microagglutination test using the reference strain Schu
(S84) of F.tularensis tularensis. This was used for examination of the
sera of people from infected region.
The
biological essay on experimental mice was used for isolation of F.tularensis
from dead animals of the genus Apodemus from infected region. F.tularensis
was cultivated on the Francis medium enriched with cysteine. The virulence
of isolated strain of F.tularensis, its erythromycin sensitivity, the
metabolism of glycerol, glucose and cytrulin-ureidase activity were
examined.
rRNA
hybridization was used for confirmation of the isolate and for its genetic
typing.
SDS-PAGE
was used for immunochemical characterization of the isolate and its
comparison to other F.tularensis strains.
The
domestic isolate of F.tularensis was used as antigen for preparation
and standardization of homemade immunodiagnostic procedures, such as
agglutination test, immunofluorescence test.
RESULTS AND DISCUSSIONS
Examination
of the sera of people from contaminated region by the microagglutination
test showed that 38 persons (between 7-77 years) gave positive reaction.
All of them had typical clinical signs of tonsylopharyngeal tularemia,
except two who had the ulceroglandular form of disease. Agglutination
titers ranged between 1:80-1:2560.
Using
biological assays from dead individuals of the genus Apodemus from the
contaminated region, F. tularensis was isolated at the Institute for
Microbiology, Military Medical Academy, Belgrade (Lako B., Ristanovic
E.) in the beginning of 1999. It was the first isolate of F.tularensis
in FR Yugoslavia. Although our country is in the endemic region for
tularemia, there has not been an epidemic of this disease in FR Yugoslavia
before, and the disease appears only rarely, so we had not isolated
strain of F.tularensis. Until now, for diagnosis of tularemia in our
country, we used only agglutination tests made by use of reference strain
Schu (S84) of F.tularensis as the antigen.
The
domestic isolate of F.tularensis is high virulent for experimental animals,
erythromycin sensitive, it metabolizes glucose and it has no cytrulin-ureidase
activity. It is cultivated and stored on Francis media enriched with
cysteine, which is necessary for growth of F.tularensis. Hybridization
with fluorescent-labeled probes specific for rRNA of F.tularensis confirmed
that isolated strain belongs to Francisella tularensis subsp.palaearctica.
The
results of SDS-PAGE show that both strains (isolated and standard laboratory
strain) of F.tularensis have protein bands of the same electrophoretical
mobility. Because of that, this method cannot be used for gene typing
of F.tularensis strains (Fig 1).
The
immunodiagnostic procedures made by use of domestic isolate of F.tularensis-Rtanj
as the antigen are more sensitive and more specific than procedures
based on use of laboratory strains of F.tularensis-Schu (S84).
The
sensitivity of microagglutination test (MAT) made by use of isolated
strain F.tularensis-Rtanj as the antigen is 86.36% and its specificity
is 94.12%. The sensitivity and specificity of the same test (MAT) made
by use of referent strain-Schu F.tularensis as antigen are lower, their
values are 81.82% and 92.16%, respectively.
The indirect immunofluorescence test (IIF) is generally more sensitive
and more specific than microagglutination test. IIF test enables us
to detect and determine each immunoglobulin class (not only IgM antibodies),
and because of that it is better than agglutination tests. The sensitivity
of IIF test using isolated strain F.tularensis-Rtanj, as antigen is
88.64%, and its specificity is 94.34%. The sensitivity and specificity
of IIF test using the reference strain-Schu F.tularensis as antigen
were the same. The preparation and standardization of homemade immunoenzyme
(ELISA) test is in progress.
CONCLUSIONS
The
first epidemic of tularemia in FR Yugoslavia broke out at the end of
1998 in the region of mountain Rtanj, near Sokobanja. The epidemic lasted
from 1999 through 2000, in the wider region of Sokobanja, including
the villages near Pirot and Aleksinac. A detailed examination of these
natural foci of tularemia is in progress.
For
the first time in our country, F.tularensis was isolated from dead individuals
of the Apodemus genus. This isolate was confirmed by rRNA hybridization
and identified as F.tularensis subsp. palaearctica.
Immunodiagnostic procedures (agglutination and im- munofluorescent)
using the isolated strain F.tularensis-Rtanj as the antigen are more
sensitive and specific than procedures using the referent strain Schu
(S84). The preparation and standardization of homemade immunoenzyme
(ELISA) test is in progress.
SUMMARY
Tularemia
is zoonotic disease caused by Francisella tularensis. The reservoirs
of disease are the mammals of geni Lagomorpha and Rodentia, and vectors
are mainly ixodic ticks and other haematophagic insects. F.tularensis
can be used as a weapon in biological warfare. Immunodiagnostic procedures,
based on detection of specific antibodies on F.tularensis in sera are
most often used in the diagnosis of tularemia.
Although
FR Yugoslavia is in an endemic region, the first epidemic of tularemia
in our country broke out in 1998 in the region of mountain Rtanj, near
Sokobanja. In that period thirty-eight people, between 7 and 77 years,
had the disease. The epidemic lasted from 1999 to 2000. The detailed
examination of these natural foci of tularemia is in progress.
For
the first time in FR Yugoslavia F.tularensis was isolated from dead
individuals of the genus Apodemus from infected region in the beginning
of 1999. This isolate was confirmed by rRNA hybridization with probes
specific for genus Francisella, species F.tularensis, subs. F.tularensis
palaearctica. The domestic isolate of F.tularensis is high virulent
for experimental animals, erythromycin sensitive, it metabolizes glucose
and it has no citrulin-ureidase activity. This isolate is used as antigen
for preparation and standardisation of home-made immunodiagnostic procedures,
such as agglutination (rapid- and microagglutination test), immunofluorescent
(IIF) and immunoenzyme (ELISA) test.
REFERENCES
- Ristanovic E., (1999) Imunodijagnostika tularemije koriscenjem izolata
Francisella tularensis sa naseg geografskog podrucja kao antigena.
Master's thesis, Faculty of Biology, Belgrade.
- Jacobs R., (1997) Tularemia. Adv.Ped.Infect. Dis. Vol.12,55.
- Morner T., (1992) The ecology of tularemia. Rev.Sci,Tech.11(4):1123
- Morner T., et al. (1993). Identification and classification of different
isolates of Francisella tularensis.Zentralbi.Veterinarmed.B.40(9-10):613
- Sato et al., (1990) Microagglutination test for early and specific
serodiagnosis of tularemia.J.Clin.Microbiol. 28(10):2372
KEY WORDS
tularemia, Francisella tularensis, isolation, rRNA typisation, immunodiagnostic
procedures.
FIGURES
Figure 1. SDS-PAGE proteins of F.tularensis
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