PREVALENCE OF COXIELLA BURNETll INFECTION IN MILITARY TRAINING AREAS IN THE CZECH REPUBLIC AND SLOVAKIA

Lit era k I.: Prevalence of Coxiella burnetii Infection in Military Training Areas in the Czech Republic and Slovakia. Acta vet. Brno 1995, 64: 179-186. Between 1987 and 1992, the presence of antibodies to the Q fever agent was investigated in seven military training areas (MTA) on the territory of the Czech republic and Slovakia. Using a complement fixation test (CFT), antibodies in cattle were confirmed (Coxiella burnetii, phase II, titer ~ 8) in two MTA, and in small ground mammals in five MT A. In one MT A, the C. burnetii strain was isolated from cow milk. No antibodies were found in enlisted soldiers, horses, sheep, dogs or game. The presence of C. burnetii strains under the specific MTA conditions has been demonstrated and it is possible that people in those areas may be infected. The risk of infection, however, is the same as in a civilian environment. It seems that the virulence of C. burnetii strains circulating in MTA is relatively low. Coxiella burnetii, antibodies, complement fixation test, military training areas, soldiers, cattle, sheep, small mammals, game Military training areas (MT A) are large areas that have been used by the army forces for a long time. Some parts of those facilities have been extensively damaged by military activities, while others are well-preserved because they have been practically off-limits for the non-military, with farming and forest exploitation restricted to a minimum. The specific uses ofMT A bring soldiers to a close contact with the environment inhabited by wild and, in some cases, domestic animals. This exposes the soldiers and animals to the risk of a contact with circulating zoonosis agents. These may occur in the environment, or may have been brought there recently by incoming soldiers or farm animals. Q fever is a zoonosis, the causative agent of which, Coxiella bumetii, occurs in Central Europe in the nature, e.g. in ticks and reservoir wild mammals, as well as in places related to human activities, namely in herds of domestic animals, mainly cattle and sheep CR e hac e k 1987). The aim of the present paper was to investigate a number of military training areas for the presence of the rickettsiae of Coxiella bumetii, indirectly by serological examination and by direct isolation, and to assess the risks of infection for people in this environment. Materials and Methods The study was carried out in seven military training areas: I. Mimoi'l MTA in northern Bohemia. Area over 30 thousand ha, about half of which covered with forests. Cattle and horses production. Mimoi'l MT A is extensively poluted with oil products as a result of a long presence of the ex-Soviet Union troops. 2. Ubava MTA in northern Moravia. Area over 30 thousand hectares (ha), with almost 90% covered with forests. Cattle production. 3. Jince MTA in central Bohemia Area about 25 thousand ha. Almost 90% of the area are forests. No animal production.

Military training areas (MT A) are large areas that have been used by the army forces for a long time.Some parts of those facilities have been extensively damaged by military activities, while others are well-preserved because they have been practically off-limits for the non-military, with farming and forest exploitation restricted to a minimum.The specific uses ofMT A bring soldiers to a close contact with the environment inhabited by wild and, in some cases, domestic animals.This exposes the soldiers and animals to the risk of a contact with circulating zoonosis agents.These may occur in the environment, or may have been brought there recently by incoming soldiers or farm animals.
Q fever is a zoonosis, the causative agent of which, Coxiella bumetii, occurs in Central Europe in the nature, e.g. in ticks and reservoir wild mammals, as well as in places related to human activities, namely in herds of domestic animals, mainly cattle and sheep CR e hac e k 1987).The aim of the present paper was to investigate a number of military training areas for the presence of the rickettsiae of Coxiella bumetii, indirectly by serological examination and by direct isolation, and to assess the risks of infection for people in this environment.

Materials and Methods
The study was carried out in seven military training areas: I. Mimoi'l MTA in northern Bohemia.Area over 30 thousand ha, about half of which covered with forests.Cattle and horses production.Mimoi'l MT A is extensively poluted with oil products as a result of a long presence of the ex-Soviet Union troops.
2. Ubava MTA in northern Moravia.In MTA 1 to 6, which are now situated on the territory of the Czech Republic, the study was made from 1987 to 1989.In MT A 2 and 7, tests were made in 1991 and 1992.MTA 7 is now situated on the territory of the Slovak Republic.
Enlisted soldiers were examined in MTA 2 and 7. Domestic and farm animals examined were cattle, sheep, horses and dogs.Cattle was examined in MTA 2, 4, 6 and 7, sheep and dogs in MT A 7, and horses in MT A 2.
Small mammals were caught using spring traps arranged in lines per 100 traps each.Seven such lines were usually placed at different locations in training areas for two nights in May, September and November.Antibodies to C. burnetii were examined in the so-called eluates, substances separated by elution from split hearts in 1 ml buffered physiological saline.The eluate was used like serum in a 1: 10 dilution.Small mammals were examined in MT A 1-6.
In game from military training areas, blood was collected from individual animals immediately after they were killed and tests were made of their blood serum.Game animals were examined in training areas 1-6.

Serological examination
The complement fixation micromethod with antigen to phase II of the C. bumetii Nine Mile strain was used in all serologic examinations (Bodibion, MEVAK Nitra, Slovak Republic).

Isolation assay of Coxiella burnetii
Cows in MTA 7 were tested by injecting 1 ml of milk from selected cows intraperitoneaily to serologically negative laboratory mice of a Swiss strain.Two of four mice were sacrificed three weeks after the milk had been injected, and impression preparations of their spleens were made and stained for a coxiella assay according to Gimenez.Spleens were then frozen to -18°C.The remaining two mice were sacrificed 6 weeks after the milk was injected, and their blood was collected at that time for serological examination.Impression preparations from spleens of these mice were also stained.Spleens of the first two mice were thawed, mixed with the spleens of the other two mice and a suspension was made of them and buffered physiological saline.This was injected intraperitoneaily (second passage) to two laboratory mice (1 ml each).The mice were sacrificed one week later, impression preparations from their spleens were stained, and the passage repeated.Repeated passages were made of samples which had caused seroconversion in mice after injection.

Military Personnel
E~tions were made in the Libava and Plierovce training areas, with a total of 72 and 30 soldiers respectively.No antibodies to C. humetii were detected in any of the tests (Tab. 1 and 2).

Cattle
In the Boletice and Hartmanice training areas, a total of 202 and 336 head of cattle respectively were examined, and no antibodies were found.
In the Libava MT A, a 5.1 % prevalence of antibodies was ascertained in a production herd of the Czech Spotted Cattle.No antibodies were found in a Hereford herd, quarantined there after arrival from Canada (Tab. 1).
In 1991, when cattle was not vaccinated against Q fever, antibodies were found in 21.3% of the Pliewvce MTA cattle, a local breed reared in traditional cowsheds.When, independently of our investigation, the high seroprevalence of antibodies to Q feverin the Plierovce area was ascertained by the State Veterinary Institute in Zvolen, local veterinary authorities decided that all the cattle should be vaccinated against Q fever in early 1992 (Bodibion vaccine, i.e. C. bumetii in phase I).In 1992, antibodies were found in 24 % of the cows tested (rab.2).In the tests, eFT witha phase IIantigen was used (the eFT used does ootdetect the postvaccination antibodies to phase I antigen).In the Plierovce MTA, oostatistically significantdifference wasfoundbetween the 1991 and 1992 incidence of antibodies (:x2test, X} = 0.36, 1 df, P > 0.5).A statistically highly significant difference was, on the other hand, found in seroprevalence of antibodies to Q fever between the Ubava and the Plierovce MT A cattle (:x2 test, X} = 20.7,1df,P<O.OOl).
In isolation tests of milk from 24 cows, the C. burnetii strain was detected in 4 cows by a seroconversion in laboratory mice which had been injected with the milk tested.The C. burnetU strain was directly detected in the spleen of experimental mice in two cases (Tab.3).She e p, H 0 r s e san d Dog s A total of 53 sheep and 11 dogs from the Plie §ovce MT A and 14 horses from the Libava MTA were tested.In all these cases, results were negative (Tabs 1 and 2).
Small Mammals A total of 1.486 small mammals were tested.In 12 cases (0.8 %), antibodies to C. burnetii were detected (Tab. 4).In small mammals, antibodies were found in five of the six mili- tary training areas surveyed.The seroprevalence ranged from 0.3 % to 1.9 %.Antibodies were detected in 11 specimens of the Apodemus sp. and 1 specimen of Microtus arvalis (Tab.5).Small mammals with antibodies to C. bumetii were usually trapped in places used by man or domestic animals.

Game animals
No antibodies to C. bumetii were detected (Tab. 6) in any of the 169 animals hunted in MT A 1 to 6 (Tab.6). in mice (b)  in mice (c) 1 K.V.

Discussion
The prevalence of antibodies to Q fever in the population of the Czech Republic is at present only about 1 % (Re h ac ek etal: 1985; Li terak 1994a).1twas not at all surprising that no antibodies were found in the 72 people from the Libava MTA or the 32 people from the Plie~ovce MT A, particularly because these people (draftees doing their national service) did not get into any close contact with potentially infected cattle.
In recent years, a generalized prevalence of antibodies to C. burnetii in cattle herds in the Czech Republic and Slovakia has been observed.Antibodies to C. burnet;; have also been found in small mammals in several areas (R e hac e k et al. 1979, 1985, 1987ab et al. 1993).For that reason, we really cannot consider the finding of antibodies to C. burnet;; in cattle and small mammals in Czech and Slovak military training areas as something unusual.The prevalence, however, is a proof that the agent of the zoonosis circulates both in cattle herds, i.e. close to human settlements, and in the nature.The importance of these findings depends mainly on the virulence of autochthonous strains of C. burnetii.In the herds in the training facilities investigated, no clinical symptoms of Q fever were found.In cattle herds with seroprevalence of antibodies to C. burnetii on civilian farms, we observe, for the most part, latent infections without any clinical manifestations.No relationship was confirmed, for example, between abortions in the cattle in southern Moravia and seroprevalence of antibodies to C. burnetii (L i t era k and Rod rig u e z 1994).An indispensable prerequisite for an epidemiological and epizootiological evaluation of antibody findings is an isolation of C. burnetii strains and testing their virulence in experimental infections.It seems that strains of C. bumetii currently circulating in Moravia are less virulent for laboratory animals than the standard C. burnetii strain, i.e. the Nine Mile strain (Valkova etal.I994).
The fact that prevalence of antibodies in the Libava MT A cattle was significantly lower than in the Pliesovce MT A can be explained by objective differences between Q fever in cattle in the Czech Republic and Slovakia (R e hac e k 1987).In the Czech Republic, prevalence is lower than in Slovakia, and so is probably virulence of autochthonous circulating strains of C. burnetii.It is assumed that the reason is the incidence of Dermacentor marginatus, the tick that is an important vector and reservoir of coxiellas.Dermacentor marginatus occurs mainly in southern areas of central Slovakia, including also the Zvolen district where the Pliesovce MT A is situated.In northern Moravia, where the Libava MT A is, Dermacentor marginatus does not occur.
In cattle herds with a level of seroprevalence of antibodies to C. burnetii, coxiellas are excreted in milk.In the Pliesovce MT A, coxiellas were found in 4 out of 24 milk samples.However, normal excretion of C. burnetii by latently infected cows was reported by, e.g., R e usc h (1982) or S c h a a I and S c h a fer (1989).This fact, however, is not assumed to have much importance for human health CD u ran d and Lim 0 u z i n 1983).
The prevalence of antibodies to C. burnetii in small mammals deserves particular attention.After all, infected specimens were found in five out of six military training areas investigated at places which, like, e.g., log cabins, rubbish heaps, farm, shooting range, were related man's activities.In unaffected areas of the countryside, however, antibodies to C. burnetii were absent not only from small mammals but also from game.Very similar results in small mammals at municipal waste dumps in southern Bohemia were reported by V lee k (1991).The sources of infection, which might playa role in human infections, as well as the origin and virulence of individual strains of C. burnetii, are some of the questions to which answers must be sought.
; R e hac e k 1987; Lis a k et al. 1989; V 0 St a et aI.1988, 1989; C em p iT k 0 v a et al. 1993; Lit era k 1994b, 1995; Lit era k and Calvo Rod rig u e z 1994).Similar situation also exists in neighbouring central European countries (C y g a n et aI.1983; S c h wei g h a r d t et al. 1984; R a d y et al. 1985; K r a us s et al. 1987; R e hac e k . Boletice MTA in southern Bohemia.Area over 20 thousand ha, almost 60% of which are forests.Cattle and sheep production. 5. Vy §kov MT A in southern Moravia.Area about 15 thousand ha, more than 13 thousand ha covered with forests.No animal production.6. Hartmanice MT A in southern Bohemia.Area almost 20 thousand ha, forests about 17 thousand ha.Cattle production, some pigs and sheep.7. Plie §ovce MTA in central Slovakia.Large area partially covered with forests.Cattle and sheep P,l"oduction. Area over 30 thousand hectares (ha), with almost 90% covered with forests.Cattle production.3.Jince MTA in central Bohemia Area about 25 thousand ha.Almost 90% of the area are forests.No animal production.4

Table 1
Results of serologic examinations for antibodies to C. bumetii in Libava MTA in 1991 and 1992

Table 2
Results of serologic examinations for antibodies to c. bumetii in Plieiovce MTA in 1991 and 1992

Table 3
Results of isolation surveys of cow milk from two farms in PlieSovce MTA to assay the strain of C. bumetii (K.V. -Kamenny Vrch Farm, V.V. -Vidov Vrch Farm)

Table 4
Antibodies to c. burnetii in small mammals in individualMTA between 1987 and 1989

Table 6
Results of game suney Cor antibodies to C. burnetii (results of all tests were negative)