Borrelia transfer by ticks during their life cycle

Zbl. Bakt. Hyg. A 263, 29-33 (1986)

Borrelia Transfer by Ticks During Their Life Cycle Studies on Laboratory Animals GEROLD STANEK 1 , INGRID BURGERI, ALEXANDER HIRSCHL 1 , GUNTHER WEWALKA 1, ALFRED RADDA 2 I 2

Hygiene Institute (Director: Prof. Dr. H. Flamm) and Institute of Virology (Director: Prof. Dr. Ch. Kunz) of the University of Vienna, Austria

Summary Ticks of the species Ixodes ricinus were cultured in the laboratory. Yellow silver rabbits, gerbils and white mice served as blood hosts. Borrelia burgdorferi could be detected by means of an IFA test in homogenates of female ticks, their eggs as well as the respective larval and nymphal ticks. Blood infection of splenectomized gerbils and ordinary white mice or of ordinary white mice alone has been demonstrated after feeding of larval or nymphal ticks on them, respectively. Spirochetemia started 5 to 8 days after feeding and lasted for ca 3 weeks. Two distinct peaks in the cell count of spirochetal organisms per ml blood plasma could be observed on days 11-13 (5 X 10 5 to 2 X 10 6 cells,rnl) and 17-19 (10 5 cells,rnl), regardless whether splenectomized gerbils or white mice were used. The results display that B. burgdorferi is vertically from the female ticks to their eggs and transstadially transmitted. The transmission-rate from larval to nymphal ticks is 100%. These findings show the tick itself as a main reservoir of B. burgdorferi. The established mouse-model appears to be a useful tool to detect Borrelia carrying ticks.

Introduction Since the discovery of the etiologic agent of Lyme disease (5) physicians of many countries of Europe have become to discover the frequency of tick-borne Borrelia infections in their own area. It was suspected that small and large wild animals of the forests might act as a reservoir for Borrelia. Studies in the USA showed that Borreliae are harboured by rodents and deer species (1,2,3). In Austria we have demonstrated antibodies against Borrelia burgdorferi in about 20% of wild hare (unpublished data). On the other hand we collected ticks and searched for Borrelia in them by immunofluorescence and culture technique. The percentage of Borrelia carrying ticks amounted to 40% in some geographical areas of Austria (6). From these facts it was not surprising to find that in each state of Austria Borrelia infections were discovered (7). The question arises as to what is the most effective way of harbouring an infectious agent to distribute it so effectively. The countrywide distribution of cases strongly suggests that ticks harbour and transfer the spirochetal organisms to their hosts by all stadia. The following study was set up to prove under laboratory conditions 1) if Borreliae are transmitted from the female

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tick vertica lly to its eggs, 2 ) if Borreliae are transmitted transstadiall y, 3) if feeding ticks in any of the va rios sta di a are infectious to their blood ho st s, a nd 4 ) if Borreliae are demonstrable in an imals u sed as blood host s.

Material and Methods

Laboratory animals 2 yellow silver rabb its, 3 gerbils (meriones ungu iculatus, kindly provided by Pro f. Dr. H . Krarnpitz, Munich ), 23 white mice (M us musculu s: inbred strain of the Hygiene Institute of the University of Vienna )

Culture of ticks (T able 1) Adult ticks of the species Ixodes ricinus were collected by flagging in th e countryside of Low er Austria. They were put onto the ears of 2 rabbits for feedig. After engo rgement , S to 6 days, the female ticks were collected and sepa rated in cotton plugged glass tubes at room-temperature and saturated humidity. After 10 days eggs were produced from the separ ated females. After a further incubation period of 8 to 12 weeks lar val ticks emerged from the eggs. Th e larval ticks stemming from one female tick were used for the further examinations. Groups of 30 to 50 larv al ticks were put onto the skin of 3 wh ite mice as well as onto 3 splencto mized gerbils to feed on them. The engorged larval ticks were left to molt into nymph al ticks for a further 4 to 6 weeks. The nymph al ticks were put for feeding singly onto 20 white mice and isolated in glass tub es after engo rgement.

T able 1. Scheme for culturing Ixodes ricinus ticks using lab orato ry animals as blood hosts Host

Ixodes ricinus Female ticks

- - - -- -7, Rabbi t (Silver Yellow)

! Eggs

! Larval ticks

! N ymphal ticks

! Adult ticks

30-S0/each

- -----7, White mou se, Gerbil !leach

- - - - - - 7, White mo use 1/each

- - - ---7, White mous e

Detection of spirochetal organisms To dete ct wether spirochet al o rganisms are present in the different sta dia of the tick 1) an immunofluorescen ce assay (IFA) was used to examine homogenat es o f engor ged female ticks and homogenate s of the ir eggs. 2) Blood was tak en by mean s o f hepa rinized Pasteur pipettes fro m the retroorbital plexu s of the lab orator y ani mals whi ch were used as blood hos ts. Blood was exa mined by both the Giesma stai n and dark field microscopy. T he treshold -Ievel of the Giesma stain for detecting or ganisms in the peripher al blood is a bout 10 5 cells per ml blood . Plasma was obta ined from the blood by centri fugation of heparini zed-b lood at 2500 rp m in a micro fuge (Beckmann). Th e plasma was exami ned by dar kfield micro scopy (x 200). Ten fields of 0.011 mrn' volumes of each plasma sample were exa mined for spiroche ta l organisms. The mean

Borrelia Transfer by Ticks

31

of such a count was multiplied with the factor to achieve the number of spirochetal organisms per 1 ml plasma. With this method the threshold level could be reduced to ca 5 x l O''cells per ml. 3) Blood of gerbils and white mice was cultured in Barbour-Sroenner-Kelly (BSK, 4) medium, which was prepared without rabbit serum (BSK-S).

Results The homogenates of 3 of 10 engorged female ticks as well as their eggs were positive for spirochetal organisms examined by IFA-test. The infectiousness of larval ticks was tested on white mice and gerbils. Feeding of larval ticks (feeding period 4-5 days) on white mice resulted in blood infection which was demonstrable on day 8 by dark-field microscopy (x 200). The cell count increased to 5 X 105 cells/ml (mean) on day 11. A second, minor peak of spirochetemia was observed on day 17 in all investigated animals. Beyond day 21 no further spirochetemia could be detected in the white mice by dark field microscopy. Spirochetemia was also observed in splenectomized gerbils after feeding of larval ticks. A first peak of spirochetemia was observed on day 12 (mean: 2 X 10 6 spirochetal organisms/ml plasma). The second peak was observed on day 21. The spirochetemia lasted longer in the splenectomized gerbils (32 days) than in the ordinary white mice (Figure 1).

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Figure 1. Blood infection of 3 splenectomized gerbils (+) and 3 ordinary white mice (e) after feeding of larval ticks (groups of 30 to 50) of the species Ixodes ricinus,

The nymphal ticks were put singly onto the white mice. After a feeding period of 3 (16 mice), 4 (3 mice) and 5 (1 mouse) days spirochetemia was observed on day 5 in all animals investigated. The cell count per ml was 1.5 X 10 4 (mean) and amounted to 2 x 10 6 (mean) on day 13. Another peak in the cell count was observed an day 19 (mean: 10 5 cells/ml plasma). After a period of 3 weeks no further spirochetemia could be observed (Figure 2).

32

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Figure 2. Blood infection of 20 ordinary whitemice after feeding of nymphal ticks,one each white mouse, of the species Ixodes ricinus.

From the blood of 1 gerbil and 1 white mouse spirochetal organisms were cultured in BSK-S after an incubation period of 10 days. The strains were examined by IFA test using a high-titered patient's serum and the monoclonal antibody H 5332 (kindly provided by Dr. A. Barbour, Hamilton, Montana, U.S.A.) reactive with the 31K outer membrane protein of B. burgdorferi. With the patient's serum a strong positive reaction could be observed. No reaction has been detected with the monoclonal antibody. Electrophoretic analysis (SDS-PAGE, Larnmli-rnethod) of the strains, however, showed the typical pattern of B. burgdorferi.

Discussion The results of this study give answers to the serveral points that came into question. 1) Borreliae are transmitted vertically from the female tick to its eggs. It is proven by both the IFA results on egg-, maternal- and larval tick-smears as well as by the blood infection of white mice and splenectomized gerbils after feeding of larval ticks. 2) Borreliae are transmitted transstadially. They could be detected in larval and nymphal ticks by IFA tests as well as in white mice that served as blood hosts. 3) The various stadia of ticks originating from a Borrelia harbouring mother tick are infectious to laboratory animals as gerbils and white mice. Feeding of larval and nymphal ticks on gerbils and white mice resulted in blood infections of all animals used. The spirochetemia appeared 5 to 8 days after the ticks started to feed. As groups of larval ticks were put onto white mice or gerbils the percentage of infectious larval ticks cannot be calculated. The nymphal ticks, however, were put singly onto white mice. After their feeding blood infection was regularly observed in all of the white mice used. The transfer of Borreliae by adult ticks is under study. Up to date we have no relevant data about the infectiousness of adult ticks. 4) Another constant findings are the 2 peaks of spirochetemia on days 11-13 and 17-19 after feeding of larval and nymphal ticks.

Borrelia Transfer by Ticks

33

These 2 peaks were regularly observed regardless whether larval or nymphal ticks were feeding and regardless whether gerbils or white mice were used as blood hosts. These findings remind of relapsing fever and indicate that B. burgdorferi may undergo antigenic variation. This hypothesis is a matter of further investigation. Interestingly, spirochetemia started within a relatively short period of time after feeding of ticks and lasted for about 3 weeks. It could not be observed again later on. This does not necessarily implicate that the spirochetal organisms are eradicated from the blood of the host. Their number might be below the threshold levels of the Giemsa or dark-field method to detect them. On the other hand, we know from other studies (3) that Borreliae could be isolated from the organs of rodents. It is very likely that the spirochetal organisms are cleared from the blood and are persisting in the organs of the hosts. The question arises if the spirochetal organisms in the blood or in organs do cause any sign of illness. In the present study we could not observe any change in the behaviour of the gerbils or white mice during the period of blood infection. There was no change in body temperatu re by feeling. After several weeks, however, when there were no longer organisms demonstrable in the blood the fur of some of the infected animals become shabby. Hair loss could be observed especially on the margin of the ears. Demonstration of spirochetal organisms in removed ears gave evidence that these phenomena are due to persisting spirochetal organisms. A study is on the way to elucidate these findings. From the results of this study we conclude that the ticks themselves are not only most important vectors but, moreover, the main reservoir of B. burgdorferi. This hypothesis is supported by studies of Anderson and coworkers who did not find B. burgdorferi in feral rodents from areas where ticks are rare (3). The mouse-model seems to be a useful tool for identifying Borrelia harbour ing ticks.

References 1. Anderson, ] . E , L. A. Magnare//i, W. Burgdorfer, A. G. Barbour: Spiroc hetes in Ixodes damm ini and mamm als fro m Co nnecticut. Am. ] . T rop . Med. Hyg. 32 (1983) 8 18-824 2. Anderson, ]. F., L. A. j\llagnare//i: Avian and mamm alian hosts for spirochete infected ticks and insecrs in a Lyme d isease focus in Connecticut. Yale ] . BioI. Med. 57 (1984) 62 7-64 1

3. Anderson, J. F., R. C. johnson, L. A. Magnarelli, F. W. Hyde: The culturing of spirochetes 4. 5. 6. 7.

from organs of feral rod ent s. Co mpo Abstracts 2nd Int. Symp. Lyme Disease and Relat ed Disorders. Vienna , Sept. 17-19 (1985) 11 Barbour, A. G.: Isolatio n and Cultivation of Lyme Disease Spiroche tes. Yale J. BioI. Med. 57 (1984) 521-525 Burgdorjer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwald, J. P. Davis: Lyme disease - a tick-borne spirochetosis? Science 216 (1982 ) 131 7-1 319 Burger, I., A. Radda, G. Wewalka, G. Stanek: Unter suchungen zur Oko logie von Borrelia bur gdorferi. Mitt. Osrerr . Ges. T rop . Paras irol. 7 (1985) 51-54 Stanek, G., H . Flamm: Epidemiologie von Borrelia-Infekt ionen in Osterreich, O sterr, Arzrezeitung 4 (1985) 19-22 Dr. G. Stanek, Hygiene-Institu t der Universirat , Kinder spitalgasse 15, A-1095 Wien

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Zbl. Bake Hyg. A 263, 1-2