Originalia V. Preac-Mursic, B. Wilske, E. Patsouris, S. Jauris, G. Will, E. Soutschek, S. Rainhardt, G. Lehnert, U. Klockmann, P. Mehraein

Active Immunization with pC Protein of Borrelia burgdorferi Protects Gerbils against B. burgdorferi Infection Summary: Serious infection due to Borrelia burgdorferi and the disseminated infection characteristic of the disease possess unique treatment problems. The wide and still increasing incidence of Lyme borreliosis as well as the problems in treatment call for effective prevention strategies by active immunization. Vaccination experiments were done to determine if active immunization of gerbils with recombinant OspA and pC protects against infection with strains of B. burgdorferi. Gerbils were vaccinated with recombinant OspA and pC (20 kDa protein) and challenged four weeks later with a clone (derived from B. burgdorferi

strain PKo) which expresses an abundant amount of pC but only little OspA. Non-immunized gerbils challenged with the same B. burgdorferi strain were used as controls. Both groups of immunized gerbils developed antibodies against the recombinant vaccines. The pC vaccinated group was protected against infection, whereas the OspA vaccinated group showed signs of infection. The non-vaccinated group developed generalised infection. These results show that pC should be considered as a further vaccine candidate and probably needs to be combined with OspA for an efficient vaccine against B. burgdorferi.

Zusammenfassung: Aktive Immunisierung mit pC-Protein yon Borrelia burgdorferi hat bei Gerbils protektive Wirkung gegen die B. burgdorferi-Infektionen. Weite Verbreitung und zunehmende Inzidenz der Lyme Borreliose sowie Therapieprobleme bei schweren Erkrankungsformen besonders im Sp/itstadium der Infektion sind Gr/jnde f/jr die Entwicklung einer m6glichen Alternative zur Antiobitikatherapie wie zum Beispiel Schutzimpfung. Die Schutzwirkung einer aktiven Immunisierung mit rekombinanten OspA und pC gegen die Infektion mit Borrelia burgdorferi wurde bei Gerbils gepr/jft. Die Tiere wurden mit rekombinanten OspA und pC vom B. burgdorferi-Stamm PKo immunisiert; die Infek-

tion erfolgte vier Wochen nach der Immunisierung mit dem PKo-Stamm (schwache OspA, gute pC Expression). Als Kontrollgruppe dienten infizierte, nicht immunisierte Gerbils. Die immunisierten Tiere bildeten Antik6rper gegen rekombinante Vakzine. Die mit pC immunisierten Tiere waren vor der Infektion geschiitzt, die Kontrollgruppe zeigte eine generalisierte Infektion. Die Immunisierung mit OspA sch/jtzte nicht, die Tiere zeigten, wie die Kontrollgruppe Merkmale der Infektion. Die Ergebnisse dieser Studie zeigen, dab pC f/jr eine Immunisierung in Frage kommt. Eine Kombination von OspA und pC scheint f/Jr eine effektive Vakzine gegen Borrelia burgdorferi n(~tig zu sein.

Introduction Borrelia burgdorferi, the causative agent of Lyme borreliosis [1], an illness that includes a number of different clinical conditions [2], has been extensively investigated. Immunological and molecular heterogeneity have been demonstrated amongst isolates from the USA and Europe [3-12]. Gerbils, hamsters, mice and rats are susceptible to experimental infection by B. burgdorferi. Infected animals develop clinical infection with histopathological alterations in most of the major organ systems [13-16]. The chronic manifestations, the wide geographic distribution of the vectors of the disease and the relatively large endemic area have made studies on prevention of B. burgdorferi infection important. Vaccination, especially of risk groups, could probably prevent infection and reduce the burden of severe chronic manifestations. No such vaccine is as yet available. Several types of passive and active immunization have been tested to protect animals from infection. Johnson et al. [17,18] observed that passive 40 / 342

immunization of hamsters with antiserum to B. burgdorferi as well as active immunization with whole inactivated strain 297 protected animals. Fikrig et al. [19] demonstrated that antiserum to 13. burgdorferi N 40 from mouse and rabbit was protective in the C3H/He mouse model and active immunization with recombinant OspA protein protected animals from challenge with several strains of B. burgdorferi. Simon et al. [20] reported on the immunogenicity of both native and recombinant OspA in mice. B. burgdorferi specific immune mouse sera as well as Received: 28 July l~Revision accepted: 12 Octot~r 1992 Dr. rer. nat. Vera Preac-Mursic, Dr. med. Bettina Wilske, Dr. rer. nat. SigridJauris, Dipl.-Biol.Gitta Will, Dr. rer. nat. SylviaReinhardt, Dr. rer. nat. Gisela Lehnaert, Max v. Pettenkofer-lnstitut, LudwigMaximilians-Universit/~t, Pettenkoferstr. 9a, Dr. med. E. Patsoum, Prof. Dr. med. P. Mehraein, Institut fiir Neuropathologie, Ludwig-Maximilians-Universitat,Thalkirchnerstrage36; Dr. rer. nat. E. Soutschek, MikrogenGmbH, WestendstraBe125, W-8000 M~nchen2; Dr. rer. nat. U. Klockmann, BehringwerkeAG, W-3550 Marburg, Germany.

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PKo PGau

monoclonal antibody to OspA protein were able to prevent the development of disease in scid mice [21]. These studies provided evidence for the therapeutic efficacy of active and passive immunization with OspA antigens. However, in consideration of immunological [3,7] and molecular [5,22] heterogeneity of OspA the search for other proteins is indicated. Furthermore, OspA might be a poor immunogen in humans because antibodies against OspA appear late if ever during human disease

[23-25]. OspA negative strains have been cultured from patients with Lyme borreliosis and OspA can be lost by B. burgdorferi during subculturing [26,24]. About 40% of European B. burgdorferi strains express an abundant amount of a 22 kDa protein designated pC [7]. As analyzed so far pC is expressed by OspA negative strains (about 10% of European isolates) [7]. In contrast to OspA, this protein is a major immunogen in the early immune response of patients with Lyme borreliosis [24]. The pC component has been cloned and efficiently expressed in Escherichia coli [27]. The aim of this study was to investigate whether active immunization with recombinant pC protein protects gerbils from the challenge with B. burgdorferi expressing pC as a major protein.

60 kDa

41 kDa OspB OspA

pC

Materials and Methods

Animals: Gerbils between seven to eight weeks of age weighing 70--80 g were obtained from an animal colony at the Max v. Pettenkofer-Institute Munich, Germany. A total of 15 animals were divided into three groups. The animals of all groups were fed a standard diet and observed for illness during 42 days. Challenge strain: B. burgdorferi PKo, clone 25 was obtained from strain PKo [7] by cloning on a solid medium [28]. The stock culture was grown in MKP-medium [29] and analyzed by SDS-PAGE followed by Coomassie blue staining as described [25]. Vaccination: The gerbils were immunized with recombinant OspA and pC derived from strain PKo. Cloning and expression of OspA and pC has been described previously [22,27]. Recombinant OspA and pC were purified by anion exchange chromatography as described [24]. The gerbils were randomly assigned to three groups of five animals. Group 1 was immunized with recombinant OspA, group 2 with recombinant pC and group 3 served as nonimmunized control. Each gerbil was vaccinated subcutaneously with 500 Ixl of vaccine containing 100 p.g OspA or pC precipitated with 0.2% AI(OH)3 (Behringwerke, Marburg). AI(OH)3 was chosen as an adjuvant because - in contrast to Freund's adjuvant - AI(OH)3 may also be used in humans. Gerbils were immunized on days 0 and 21, respectively. Challenge infection: One week after the second immunizing injection all animals, including untreated controls, were infected intraperitoneally with 0.5 ml suspension containing 1 x l&/ml B. burgdorferi PKo clone 25, as described previously [15,30]. PKo clone 25 is characterized try abundant expression of the pC protein, no major OspA is visible in SDS-PAGE (Figure 1). At 14 days after infection, gerbils were sacrificed. Borrelia reisolation: The reisolation of B. burgdorferi from blood and several organs of infected gerbils was made in MKP medium 14 days after infection and analysed by darkfield microscopy as

Figure 1: SDS-PAGE of strains PKo/clone 25 and PGau. (Coomassie brilliant blue staining).

reported before [15]. Immunological examination: Pre- and post immunization as well as postinfection sera were collected from gerbils and B. burgdorferi specific antibodies were determined by indirect immunofluorescence (IFT) and by ELISA. IFT and ELISA were modified from procedures described earlier [25]. The IFT antigens were prepared from strain PKo clone 25 and strain PGau [7] (Figure 1). The gerbil sera were tested in doubling dilutions beginning at 1:16. Immune complexes were detected with anti-mouse immunoglobulin FITC-conjugate 1:50 (Dakopatts, Denmark). For the ELISA purified recombinant OspA and pC preparations were umd as antigens. Microtiter plates were coated with 50 ~tl antigen solution containing 1 p.g/ml recombinant OspA and 1 Ixg/ml recombinant pC, respectively. Sera were tested in doubling dilutions beginning at 1:250 and sera from mice immunized with OspA and pC respectively served as controls. Immune complexes were detected with anti-mouse immunoglobulin HRPO conjugate 1:4(~ (Dakopatts, Denmark). Immunoblots were performed as described [25] with the modification that recombinant OspA, pC and pl00 [31] were used as antigens.

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Histopathology:The animals were observed daily for illness for a

burgdorferi was isolated from all gerbils which had received

period of 42 days post immunization. On day 14 after infection and day 42 after immunization, gerbils were sacrificed and tissue samples were obtained from most major organs and examined for histopathological alterations, as de~ribed previously [15]. Four ti~ue sections of each organ were examined under blind conditions, coded and scored (- none, + slight, + + moderate, + + + severe) for the presence and extent of inflammatory infiltrates.

recombinant OspA. No significant differences were noted between the culture findings seen in the OspA immunized animals and the nonimmunized control group.

Histopathological Findings B. burgdorferi inoculation induced significant histopathologic changes in most major organs and their surrounding adipose and fibrous connective tissues in the group of animals immunized with OspA as well as in the nonimmunized control group. In the group immunized with pC minimal to moderate changes were observed in some organs of three animals (Table 2).

Results

In this immunization study challenge infection was performed with a clone derived from strain PKo whose virulence and pathogenicity had been confirmed in gerbils in previous experiments [15,30,32]. The results of the reisolation, i.e. persistence of B. burgdorferi, are presented in Table 1 and those pertaining to inflammatory infiltrates in several organs in Table 2. The antibody response to vaccination with recombinant OspA and pC and antibody response after challenge is presented in Tables 3, 4 and 5.

The inflammatory infiltrates consisted mainly of lymphocytes and histiocytes, admixed with some plasma cells and some eosinophils and mast cells. The perivascular pattern of lymphohistiocytic infiltrates was a constant finding in almost all the involved organs (Figure 2).

Cultural Reisolation of Borrelia burgdorferi

Eyes and orbital contents, peripheral nerves, the lungs, the gastrointestinal tract, liver, skin and striated muscle of the thighs were slightly to severely involved in most animals of the nonimmunized control group and the group immunized with OspA. Furthermore, most of them revealed a follicular hyperplasia of the spleen and in the examined thoracic and abdominal lymph nodes. In the group immunized with pC slight to moderate inflammatory infiltrates were observed in three animals in the lungs, gastrointestinal tract, liver, striated muscle (Figure 3); lymphnodes and spleen revealed a follicular hyperplasia.

As shown in Table 1 no borreliae could be isolated from gerbils immunized with recombinant pC. In contrast, B. Table 1: Detection of Borrelia burgdorferi after infection and active immunization with OspA and pC (from strain PKo - clon 25) and nonimmunized gerbils.

I

Osp A pC Control

5/3 5/0 4/4

5/5 5/0 4/1

5/1 5/0 4/0

5/5 5/0 4/3

5/4 5/0 4/3

5/5 5/0 4/3

5/3 5/0 4/1

The rest of the examined organs of the infected animals, including the central nervous system and the knee joints were intact, without histological signs of inflammation.

No, of gerbils tested / no. o f positive gerbils,

Table 2: Protection of gerbils from infection with Borrelia burgdorferi by active immunization with OspA and pC. Histopathologic evaluation 14 days after infection, Inflammatory infiltrates: + slight, + + moderate, + + + severe.

Eyes and orbital contents Sciatic nerves Particles of other peripheral nerves In the striate muscle of the thighs Heart Lun~ Lymph nodes Stomach Intestine Liver Spleen Kidneys Skin (site of inoculation) Striate muscle of the thighs

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V. P r e a e - M u r s i e et al.: L y m e B o r r e l i o s i s - Active I m m u n i z a t i o n

Table 3: Immune response to recombinant Osp A and pC measured by EUSA prior to immunization (day O) and at the time of challenge (day 28).

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

Osp A

0.000 0.005 0.006 0.120 0.005

<1:250 <1:250 <1:250 <1:250 <1:250

1.705 2.463 2,159 1.~5 2.536

pC

0.004 0.008 0.003 0.002 0.005

< 1:250 <1:250 < 1:250 <1:250 < 1:250

0.000 0.003 0.028 0.~4 0.114

0.004 0.006 0.002 0.006 0.003

< 1:250 <1:2.50 <1:250 <1:250 <1:250

0.~2 0.008 0.(105 0.004 0.005

none (control)

1:2000 1:16 ~ 1:8000 1:1000 1:32 000

0.001 0.002 0.004 0.002 0.000

<1:250 <1:250 <1:250 <1:250
0.000 0.054 0.006 0.001 0,112

< 1:250 <1:250 < 1:250 <1:250 < 1:250

0.003 0.006 0.004 0.004 0.007

< 1:250 <1:250 < 1:250 <1:250 < 1:250

0.371 1,019 1~185 0.735 0.690

< 1:250 <1:250 <1:250 <1:250 <1:250

0.003 0.010 0.000 0.002 0.002

< 1:250 <1:250 <1:250 <1:250 <1:250

0.(103 0.012 0.(105 0.~ 0.005

<1:250
OD = Optical density measured in sen~m dilution 1:200. Antibody titers are defined as the highest dilution with an OD of >0.200 in case of OspA and an OD of >0,100 in the case of pC.

Table 4: Immune response to recombinant OspA and pC measured by IFT prior to immunization (day 0) and at the time of challenge (day 28).

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

OspA

pC

none (control)

< < < < <

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< < < < <

16 16 16 16 16

< 16 16 16 16 16

64 64 128 16 128

< < < < <

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< 16 16 16 16 16

< 16 16 16 16 16

< < < < <

16 16 16 16 16

< < < < <

< 16 16 16 16 16

< 16 16 16 16 16

16 16 16 16 16

~ SDS-PAGE pattern of the strains are shown in Figure 1.

Antibody Response to Vaccination with OspA All animals vaccinated with OspA developed antibodies to OspA demonstrable by OspA-EL1SA (Table 3). The pC-ELISA (Table 3) remained negative. Using a skin isolate PGau (the same OspA serotype as the OspA positive variant of PKo) as antigen antibody titers of 1:64 or 1:128 were demonstrable before challenge (Table 4) (except in one gerbil with an antibody titer of only 1:16). Infection 20 (t992) No. 6

Table 5: Immune response to the challenge strain: antibodies delected by IFT using PKo/clone 25 as antigen; antibodies to recombinant pl00 delected by.immunoblot.

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

OspA

pC

none (control)

< < < < <

16 16 16 16 16

512 2048 512 1024 2048

+ + + +

512 1024 2048 256 1024

512 2048 4006 nd 2048

nd -

< < < < <

2048 2048 1024 1024 2048

+ + + + +

16 16 16 16 16

11 day of challenge; :~ day of sacrifice.

This gerbil, however, had a significant antibody response detected by ELISA. As expected, strain PKo clone 25 did not react with sera from OspA vaccinated gerbils. Antibody Response to Vaccination with pC All gerbils vaccinated with pC developed antibodies to pC demonstrable by pC ELISA (Table 3). By contrast, the OspA ELISA remained negative (Table 3). The pC positive strain PKo clone 25 was reactive in the IFT (antibody titers ranged from 1:256 to 2,048), whereas the pC negative (but OspA positive) strain PGau was negative (Table 4). As expected, the OspA positive (and pC

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V. Preac-Mursic et al.: Lyme Borreliosis - Active Immunization

A

B

C

plO0

p41

OspA

pC

Figure 3: Inflammatory infiltration of the striate muscle of the thigh. Animal immunized with pC.

negative strain) PGau did not react in the IFT with sera from pC vaccinated gerbils.

Infection Two weeks after infection, a considerable increase of antibody titers against the challenge strain (from < 1:16 to 1:512-1:2,048) in nonvaccinated and OspA vaccinated animals was observed. In pC vaccinated animals no significant increase of antibody titers was observed after challenge. However, prechallenge titers had been high due to immunization (Table 5). As shown by immunoblot, all nonimmunized and four out of five OspA vaccinated gerbils developed antibodies to pl00, an immunodominant and highly specific protein of B. burgdorferi [24,31] which is not contained in the vaccine. In contrast, no pl0t) specific immune response was observed in pC vaccinated gerbils (Table 5). Three representative blots are shown in Figure 4. Discussion

In view of the wide and increasing incidence of Lyme disease successful antibiotic therapy together with

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Figure 4: Recombinant immunoblot: (A) nonvaccinated gerbil; (B) gerbil vaccinated with OspA; (C) gerbil vaccinated with pC. Sera were obtained 14 days after infection with Borrelia burg-

dorferi. efficient control of the disease including active immunization is indicated. Preliminary immunization experiments in hamsters, mice, scid mice and gerbils demonstrate the difficulties in the development of an animal model and implementation of a vaccine for Lyme disease. Immunodeficient mice [21] as well as hamster are not useful as animal models for active immunization. Hamsters infected with B. burgdorferi lack morphologic organ damage and severe inflammation [13]. Results of active immunization experiments demonstrate protection with recombinant OspA from B. burgdorferi. Animals actively immunized with OspA were protected from subsequent infection, whereas the control animals readily developed infection [18-20]. In the first experiments the protective effect of OspA was obtained by challenge with homologous organism and strains with a high degree of similarity [18-20]. However, very recently Fikrig et al. reported that crossprotection was not achieved using strains with different OspA genotype [33]. Analysis of B. burgdorferi isolates from Europe and the USA has shown immunological [3,7] and molecular heterogeneity of OspA

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[5,22]. This finding and the fact that the antibodies against infiltrates. Johnson et al. [13] also observed variable OspA are rarely detected in sera from patients with Lyme pulmonary interstititial infiltrates in B. burgdorferi infected borreliosis [23-25] must be taken into consideration. In hamsters. Until now, we could not find any report of lung contrast to OspA, the pC is a immunodominant protein in or gastrointestinal involvement in patients with Lyme the early immune response in patients with Lyme diseases. On the contrary', in all of the examined animals borreliosis. Antibodies to pC were detected in 35-36%, in our series, the central nervous system and the knee antibodies to OspA only in 8-9% of patients with joints remained intact. Both sites are involved in human erythema migrans [24]. Only 40% of European isolates Lyme disease [45-48]. Positive findings in these tissues are expressed pC but 90% of the isolates expressed OspA in observed later than in other organs, mostly by day 21 after culture [7]. infection and later. It is known that the reaction of These observations do not exclude that OspA is infected animals after infection with B. burgdorferi varies; immunogenic in humans; they could also be explained by different animals react to infection with different expression of pC in the host tissue by primarily symptoms [13-16,19,49]. non-expressing strains. Variable expression of OspA and Immunization of gerbils with pC antigen led to a pC has been observed among subcultures or clones substantial change in the course of B. burgdorferi infection. derived from the same isolate [7,28]. This was reflected in the demonstration of significantly In the present study the gerbil was used as an animal smaller lesions and the absence ofB. burgdorferi persisters, model. Gerbils were chosen, because they had proven to compared with those of control animals and animals be a practicable animal model in our previous studies, in immunized with OspA. These animals showed 100% which all animals became infected and antibody titers symptomatic infection with significant histopathologic increased considerably as well as histopathologic changes changes and persistence of B. burgdorferi in many organs. were observed after infection [15,30,32]. As was expected, These results suggest that immunization with pC vaccine not only the involved organ systems but also the pattern of prevents more effectively the damage caused by B. the inflammatory infiltrates observed in this series are burgdorferi not expressing a major OspA. Furthermore, the quite similar to those seen in our previous experiments significant increase of antibody titers after infection which with B. burgdorferi infected gerbils [15]. was observed in non protected animals confirms this The high number of organs involved in a slight to severe finding. inflammation in this series resembles the multiorgan Interestingly, antibodies to pl00 (an antigen not contained in the vaccines) have been demonstrated in the involvement found in human Lyme disease [34]. Conjunctivitis, episcleritis and panophthalmitis have been non-protected animals but not in the pC vaccinated group. described in patients with Lyme disease [35-37] and many Thus the presence of antibodies against pl00 could be a cases of human peripheral nerve biopsies revealed valuable and specific marker of infection with B. inflammatory involvement [38-40]. Liver involvement in burgdorferi. The pl00 has previously shown to be a highly human Lyme disease has been also reported [41]. specific antigen of B. burgdorferi [24]. Furthermore, the infected striated muscles from the thighs Certainly, extended studies with much more different B. and the skin specimens of the infected gerbils in these burgdorferi variants (serot}qaes) are needed to provide series revealed a similar histological pattern of additional information on the breadth of protection. involvement to that seen in human Lyme disease in the Although previous experiments indicate protection with OspA and pC, further studies must clarify whether a striated muscles and the skin of the patients [34,42-44]. The almost constant finding of the perivascular combined vaccine is effective in the prevention of Lyme inflammatory infiltrates observed in the involved organs of borreliosis. However, in view of the immunological the infected gerbils was also described in cases of human heterogeneity of OspA and pC [4] extended Lyme disease [34,38-40,42--45]. In these series, as crossprotection studies will be necessary for the previously reported [15], the lungs and the gastrointestinal development of an effective vaccine. tract of many infected animals revealed inflammatory

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21. Schaible, U. E., Kramer, M. D., Eichmann, K., Modoleil, M., Museteanu, C., Simon, M. M.: Monoclonal antibodies specific for the outer surface protein (OspA) prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc. Natl. Acad. Sci. USA 87 (1990) 3768-3772. 22. Zumstein, G., Fuchs, R., Hofmann, A., Preac-Mursic, V., Soutschek,

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Ixodes dammini spirochete. J. Clin. Invest. 72 (1983) 504-515. 24. Wilske, B., Preae-Mursic, V., Fuchs, R., Bruckbauer, H., Hofmann, A., Zumstein, G., Jauris, S., Soutschek, E., M o ~ M.: Immundominant proteins of Borrelia burgdorferi; implications for improving serodiagnosis of Lyme borreliosis. In: Neu, tt. C. (ed.): New antibacterial strategies. Churchill Livingstone, London 1990, pp. 47-63. 25. Wilske, B., Preae-Mursic, V., Schierz, G., Liegl, G., Gueye, W.: Detection of IgM- and lgG antibodies to Borrelia burgdorferi using

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Book Review W. E. Farrer, M. J. Wood, J. A. Innes, H. Tubbs Infectious Diseases: Text and Color Atlas, 2nd ed. 392 pages, 471 figures Gower Medical Publishing, London-New York 1991 Price: $175.The author's aim was to provide a comprehensive survey in words and pictures of frequent and more rare infectious diseases, including tropical diseases, and those of special importance. This is fully realized. Numerous illustrations - clinical pictures supplemented by microbiological, parasitological and histological colour presentations, radiographs and C~- pictures as well as charts comprise more than half of the book. It is divided into 14 chapters which deal with both, body regions and organ systems (lower respiratory tract, urinary tract, gastrointestinal tract, etc.). In a separate contribution systemic infections are presented (from septicaemia via malaria, tularaemia, to disseminated fungai infections ). Fourteen pages on HIV infections and AIDS follow.

The depicted infectious diseases are explained in a concise text. The information on pathogenic agents, clinical forms of progress, hints on diagnostics and therapy, in some cases in the form of dosage schemes, are scientifically up-to-date. The presentation of clinical pictures according to body regions and organ systems is in part repetitious but the synopsis has the advantage of a detailed index. The layout of the book is striking and the presentations are technically excellent. Thus, this book not only reaches the interested specialist in infectious diseases but will also address many representatives in other fields for whom a quick visual orientation with a concise presentation of diagnosis and therapy indications is important. This book is a broad overview, and lacks references, which would have been useful for students. Despite some shortcomings, it is an excellent book and a pleasure to read. One can only wish that it will have a wide readership.

Infection 20 (1992) No. 6 © MMV Medizin Verlag GmbH M~inchen, Mtinchen 1992

tl. Padelt ~rlin-Buch

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