Rickettsial infections in Slovenia 
Clinical and laboratory study 
RICKETTSIAL INFECTIONS IN SLOVENJA 
H. Hren-Vencelj, T. Avšič-Župane and D. Keše 
ABSTRACT 
Using indirect immunofluorescence, complement fixation, and enzyme immuno assay Rickettsia conorii and Coxiella bumetii 
infections were followed in suspected cases of rickettsial disease. 
The study of patients sera for Rickettsia conorii antibodies elucidated two positive cases with tache noire, and nine cases 
with pathological skin changes and lymphadenopathy. The immune response to rickettsia antigens was found also in 10 out of 
150 patients with an episode of tick bite. 
Patients with a respiratory disease suffered from Q fever in 25 to 58%. In 30% of patients sera with symptoms of a viral 
disease Coxiella bumetii was involved as well as in 19% of those with hepatitis and in 24% of endocarditis cases. 
KEYWORDS 
Rickettsia conorii, Coxiella burnetii, Mediterranean spottedfever, Qfever 
INTRODUCTION 
The epidemic typhus (typhus exanthematicus) appears 
from tirne to tirne in an epidemic form, mostly during war-
time. It is caused by an intracellular bacteria Rickettsia 
prowazekie and transmitted by the body louse. During the 
2nd World War epidemics were observed in certain regions 
of former Yugoslavia. 
The medica! doctors are less aware of the tick-mediated 
Mediterranean spotted fever (MSF) (fievre boutonneuse) 
appearing mostly in the Mediterranean countries, in India 
and Africa (1,2) . This rickettsiosis is caused by Rickettsia 
conorii and transmitted through bites by a variety of ticks 
(dog ticks and Ixodes species). The ethiologic agent ofMSF 
belongs to the "spotted fever group" (SFG) agents, which 
acta dermatovenerologica A.P.A. Vol 5, 96, No 1 
include also the Rocky Mountain Spotted Fever (RMSF), 
caused by Rickettsia rickettsii, and numerous other species 
spread all over the world (3). 
After the inoculation of rickettsia into the skin a small 
ulcer with black necrotic centre and ared areola (tache noire) 
develops on the site of the tick bite within 5 to 7 days . Later 
on the systemic symptoms may develop: headache, malaise, 
joint and stornach pains and, rarely even mental confusions. 
The elevated body temperature (39-40°C) lasts far 7-14 days. 
During this tirne, maculopapular or purpuric eruptions of 
the skin appear. In the past, MSF was considered a benign 
disease but severa! recent studies have shown a mortali_ty 
rate of approximately 2% to 6% (4,5,6,7,8). 
The other rickettsial disease, Q fever, appears in Slovenia 
accidentally when infection breaks out in domestic animals . 
15 
Rickettsial infections in Slovenia 
It is caused by Coxiella bumetii. This zoonosis is widespread 
in severa! domestic animals. The organism is being found in 
animals' placenta, mammary gland, and milk, through ticks 
can be involved in the transmission of agent as well (9). Cases 
of Q fever can often be traced to farms, tanneries, and 
slaughterhouses. The disease is well-known in Southern 
Europe, Northern Africa and Middle East (10). 
Q fever bas acute and chronic forms. The diagnosis of 
acute Q fever is seldom established except in setting of an 
epidemiologically investigated common source outbreak. 
Acute disease is characterized by the sudden onset offever, 
headache and chills. Usually there is no rash. The incubation 
period is 2 to 4 weeks. The disease lasts for 5 do 30 days if 
patient is not properly treated. Many cases are subclinical, 
and appear as a mild or moderate self-limited respiratory 
disease but more often presented as atypical pneumonia and 
hepatitis. Hepatitis is evaluated and diagnosed usually in 
patients with granulomas seen in hepatitic biopsy specimens 
or in patients with culture-negative endocarditis. Rarely the 
Table 1. Patients sera tested for Rickettsia conorii infection 
Clinical diagnosis 
Febris mediterranea susp. 
Rickettsioses susp. 
Erythema nodosum 
Exanthema maculopapulare 
Lymphadenopathia 
Borreliosis Lyme (tick bite) 
St. febrilis 
Others 
No. 
417 
2 
62 
15 
58 
38 
150 
17 
75 
Table 2: Serological analyses for Coxiella specific antibodies 
16 
Clinical diagnosis 
Bronchitis 
Bronchopneumonia 
Pneumonia 
Pneumonia atypica 
St. febrilis 
Virosis i.o. 
Endocarditis, myocarditis 
Hepatitis 
Acute renal failure 
Others 
No 
262 
2 
24 
2 
113 
6 
27 
17 
16 
1 
54 
affected person develops chronical infection, e.g. subacute 
endocarditis or other complications (11). The mortality rate 
in such cases may be as high as 60%. 
In this study the results of the serological evaluations of 
patients suspected to be affected by MSF or Q fever are 
presented. 
PATIENTS AND METHODS 
Patients: 
Sera of 679 patients suspected to be infected with Rickettsia 
conorii and Coxiella burnetii or sent to exclude rickettsial 
infections were collected. Out of these, 417 specimen were 
taken of the patients who might have had Mediterranean 
spotted fever and 262 for the evaluation of the Coxiella 
burnetii lgG antibodies. Additionally, we analyzed also 40 
patients sera from the region where the coxiella zoonosis 
outbreak occurred. 
Rickettsia conorii 
CF IIF-IgG 
(titer > 1:16) (titer > 1: 160) 
2 2 
o o 
2 3 
o 4 
1 2 
5 10 
o 1 
2 
Coxiella burnetii 
CF (%) IIF-IgG (%) 
(titer > 1:16) (titer > 1:160) 
o o 
14 (58) 14 (58) 
o o 
26 (23) 28 (25) 
o o 
7 (26) 8 (30) 
3 (18) 4 (24) 
3 (19) 3 (19) 
NT o 
NT o 
acta dermatovenerologica A.PA. Vol 5, 96, No 1 
Rickettsial infections in Slovenia 
Methods: 
All sera were tested according to procedures accepted for 
laboratory diagnosis of rickettsial infections. We tried to 
detect specific IgG antibodies with the indirect immuno-
fluorescence test (IIF-IgG) and with complement fixation 
test (CF). Some sera were tested for specific IgM and IgG 
antibodies to Coxiella burnetii phase I and phase II antigen 
by enzyme-immuno assay (ELISA). 
The analyses of 40 patients' sera for phase II and phase I 
antibodies in ELISA test showed that 5 specimen taken from 
1 O patients with endocarditis and hepatitis reacted in IgG 
immune response to phase I. Specific IgM directed to phase 
II was detected in 3 cases of atypical pneumonia and in 1 
case with clinical suspicion ofvirosis but IgG was proved in 
13 out of 30 patients. 
DISCUSSION 
RESULTS 
The results of the analyses of patients sera assayed for 
antibodies to Rickettsia conorii ar in whom this infection 
was to be excluded are shown in Table 1. 
Routinely we looked for Coxiella burnetii specific IgG 
antibodies in the case of a suspected acute or chronic infection 
(Table 2) . 
The ecosystem of Rickettsia in the Subalpine region of 
Slovenia is not yet completely understood. In the past we 
collected data on rickettsial infections among rodents and 
domestic animals in three Slovene regions: Vipava, Novo 
mesto and Ilirska Bistrica. The results showed that the 
prevalence of antibodies to Rickettsia conorii was high in 
the house mouse Mus musculus and field mouse Apodemus 
agrarius species (70 -90% ). Antibodies to Coxiella burnetii 
Table 3. Mediterranean Spotted Fever Diagnostic Score* 
Epiderniologic criteria 
Life or recent travel in endernic area 2 
Onset between May and September 2 
Contact with ticks 2 
Clinical criteria 
Temperature higher than 39 °C 5 
&~ar~~oo~ 5 
Maculopapular or purpuric eruption 5 
Two or three clinical criteria 3 
Three clinical criteria together 5 
Unspecific biologic criteria 
Platelet count <150 x 109 / L 1 
Liver enzymes >50 IU / L 1 
Bacteriologic criteria 
Isolation of Rickettsia conorii from blood 25 
Detection of Rickettsia conorii in skin biopsy using IFA 25 
Serolo gic criteria (immunofluorescence) 
Sole serum with to tal Ig > 1: 128 5 
Sole serum with IgG > 1: 128 and IgM > 1: 64 10 
Two sera with fourfold titer elevation within 2 weeks 20 
Total 
* Tota/ score > 25 is consistent with a presumptive diagnosis of Mediterranean spotted fever (14 ) 
acta dermatovenerologica A .P.A. Vol 5, 96, No 1 17 
Rickettsial infections in Slovenia 
were investigated in sheeps and goats (12,4% and 4%). The 
data on rickettsial infections in vectors (ticks) are scarce. In 
a small group ofticks found on animals the rickettsia-infected 
Ixodes species prevailed (12) . 
As the isolations of rickettsia is a particularly hazardous 
for the laboratory personnel, serologic tests are mostly used 
for the confirmation of the clinical diagnosis. The tests 
currently used detect antibodies to rickettsial surface 
antigens. As the SFG rickettsias share common group 
antigens of the cell wall with other species, the tests like CF 
are less specific when compared to the tests detecting 
antibodies to species-specific S-protein or the rickettsial 
genome (13). The most commonly used serologic procedures 
are the enzyme-immuno assay and the indirect immuno-
fluorescence test. The latter is the reference method. All 
rickettsial tests suffer from a lack of specificity due to the 
small amount of S-protein in antigen preparation as well as 
to cross-reactivity that may occur between SFG and typhus 
group of rickettsias (14). 
IgM and IgG rickettsial antibodies appear between day 5 
and 10 after the onset of fever. The IgG antibodies can persist 
for years. Detection of IgG and IgM antibodies in patients 
sera helps to prave a recent infection and to avoid false 
positive results due to cross-reactivity with antibodies 
(essentially IgM) to the lipopolysaccharides of other bacteria. 
As expected, we were able to confirm the clinical diagnosis 
ofMSF in both patients with a skin ulcer. In all other patients 
this rickettsiosis was rarely confirmed serologically. Out of 
150 sera assayed for presumed Lyme borrelioses 10 tumed 
to be positive for rickettsia (6,6%). This fact stresses the 
observation that in areas endemic for Lyme borreliosis 
infections with Rickettsia conorii are underdiagnosed. 
Clinical manifestations of the MSF differs from RMSF in 
its less severe symptomatology and the presence of tache 
noire, an inoculation eschar at the site of the tick bite, but 
this occurs only in 70% of infected persons (15). 
Beside this there are also subtypes of Rickettisa conorii 
causing Israelian tick typhus and Astrakhan fever in which 
the skin ulcer is uncommon. Therefore the diagnosis of 
rickettsioses is rather difficult and complex. There are severa! 
epidemiologic, clinical and other criteria to be considered 
for the correct diagnosis of infection. In this aspect we agree 
with Raoult and Brouqui (14) who proposed the diagnostic 
score for MSF (Table 3). 
Lyme borrelisosis is endemic in Subalpine regions of 
Slovenia where during the 50s a serologic survey for 
arboviruses showed a high prevalence of antibodies to tick-
born meningoencephalitis virus (TBE) (16). As rickettsia 
(according to our findings in the past), borrelia and TBE use 
the same vector for the transmission to the new host the agents 
must compete for their ecologic niche. This competition is 
18 
reflected as a reduction of prevalence of TBE antibodies 
during the last decade in regions where borrelia has 
predominated (personal communication) and a very low 
prevalence of antibodies to Rickettsia conorii. 
Coxiella burnetii, the etiologic agent of Q fever has a 
different way of spreading. Although the microbe can be 
transmitted from species to species (and also to men) by 
direct contact or through tick bite, however this occurs only 
very rarely. People become usually infected by inhaling the 
organism which is able to survive for months in the environ-
ment (10). It is obvious that the Q fever diagnosis is easier 
when epidemiologic data point to the disease (17). 
Specific laboratory diagnosis is possible most often by 
serologic methods. Although Coxiella burnetii can be 
propagated in some laboratory animals or tissue cultures the 
isolation could be utilized in laboratories with P-3 level 
biohazard containment facilities . The risk of infection by 
handling specimen is high because as small an inoculum as 
one organism is capable of initiating infection. 
Serologic methods currently used are complement fixation 
(CF) and indirect immunofluorescent antibody assay (IIF). 
Coxiella burnetii exhibits an antigenic variation with the 
appearing of antigens designated as antigens phase I and 
phase II. In the case of primary infection the patient develops 
both IgM and IgG antibodies to Coxiella burnetii phase II 
antigen which appear earlier than antibodies to phase I 
antigen. Antibodies to phase II antigen can be detected 
between the second and fourth weeks of infection. Later on 
the titers decrease. In the patients who develop chronic 
infection the IgG and IgA antibodies to Coxiella burnetii 
phase I antigen prevail and it can be provoked in high titers. 
Therefore chronic infection can be diagnosed with the single 
serum antibody titer to Coxiella bumetii phase I antigen (14). 
Out of 262 patients included in the study in cases with 
bronchopneumonia more than one half of sera (58%) 
appeared to have significant IgG titres. When the leading 
symptom was atypical pneumonia 25% of sera were reactive. 
The unpu blished data on 146 cases of coxiella contacts during 
a zoonosis outbreak in Ilirska Bistrica (personal commu-
nication) showed that more than one half of persons (54%) 
had been infected with coxiella organism but only 24% of 
infected people developed respiratory disease. 
CONCLUSION 
Previous laboratory studies have demonstrated that 
reservoirs and vectors of Rickettsia conorii do exist in certain 
areas ofSlovenia. In the presentmanuscript two typical cases 
of tache noire are mentioned. It has also to be stressed that 
in 10 out of 150 sera from patients initially diagnosed as 
Lyme borreliosis a diagnostic titer for antibodies to Rickettsia 
acta dermatovenerologicaA.P.A. Vol 5, 96, No 1 
Rickettsial infections in Slovenia 
conorii were detected. The same observation was made in 3 
out of 15 cases diagnosed as erythema nodosum, in 4 cases 
of maculopapular rash, and in 2 with lymphadenopathy. 
These results should make dermatologists, infectologists as 
well as general practitioners aware of a possible Medi-
terranean spotted fever infection. 
Sirnilar conclusion is valid for infections with Coxiella 
burnetii causing the so-called Q fever. 
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AUTHORS' ADDRESSES 
Helena Hren-Vencelj PhD, Professor of rnicrobiology, Institute of Microbiology Medical Faculty, 
1000 Ljubljana, Zaloška 4 
TatjanaAvšič-ŽupancPhD, Assistant Professor, same address 
Darja Keše, Msc, biologist, same address 
acta dermatovenerologica A.P.A. Vol 5, 96, No 1 19