INFECTION AND IMMUNITY, Dec. 1992, p. 5232-52410019-9567/92/125232-10$02.00/0Copyright X) 1992, American Society for Microbiology

Vol. 60, No. 12

Histological and Immunocytochemical Characterization ofCoxiella burnetii-Associated Lesions in the Murine Uterus

and PlacentaWOLFGANG BAUMGARTNER* AND SABINE BACHMANN

Institut fiir Veterinar-Pathologie, Justus-Liebig-Universitat, FrankDfurter Strasse 96, 6300 Giessen, Germany

Received 7 July 1992/Accepted 8 September 1992

The fetoplacental units and the postgravid uterus of BALB/cJ (H-2d) mice inoculated intraperitoneally withCoxiella burnetii (Nine Mile isolate, phase I) on day 6 of pregnancy were examined histologically andimmunocytochemically at 1 to 160 days postinoculation. Clinically, abortions, stillbirths, and perinatal deathswere observed. Histological lesions in the placenta were characterized by severe necrosis of the decidua basalisand the labyrinth, fibrinoid degeneration of decidual vessels, and microthrombosis. Pyometra and endometritisat the sites of previous placental attachment, characterized by ulceration, central necrosis, and moderatecellular infiltration consisting of neutrophils and macrophages, were observed postpartum. Pups sacrificed atthe age of 9 days exhibited interstitial pneumonia with few granulomas and granulomatous hepatitis andsplenitis. Immunocytochemically, antigen-bearing cells were first detected in the decidua 9 days postconcep-tion, and single immunopositive cells were detected in the fetal placenta 4 days later. Thereafter, until abortionor parturition, abundant accumulation of C. burnetii antigen was observed in the maternal and fetalcompartments of the placenta. Up to 28 days postinoculation, many immunopositive cells were demonstratedat the sites of previous placental attachment, whereas the adjacent endometrium contained only a fewantigen-positive cells. C. burnetii antigen was demonstrated in decidual cells, trophoblasts, and macrophagesand extracellularly within the sinuses of the labyrinth and in the uterine lumen but not in granulated metrialgland cells. Fetuses in utero and aborted, stillborn, or perinatally dying offspring were immunocytochemicallynegative for C. burnetii antigen; however, pups killed 9 days after birth showed lesion-associated positiveimmunoreaction in the lung, liver, and spleen. The present study shows that infection with C. burnetii duringpregnancy results in uncontrolled growth of the organism in the murine uteroplacental unit and that associatedlesions are characterized by necrosis of placental tissues, fibrinoid degeneration of decidual vessels, andmicrothrombosis.

Coxiella burnetii, the etiological agent of Q fever, is anobligate, gram-variable, intracellular pathogen of the familyRickettsiaceae (49). Infection with C. bumetii has beendemonstrated in arthropods, numerous free-living and do-mestic animal species, and humans (1, 40). In general,natural infection of animals is described as unapparent ordormant, and organisms are not excreted until parturitionoccurs (1, 40). Humans are usually infected by inhaling dustfrom dried ruminant fetal fluids. In humans, infection can beasymptomatic or result in acute or chronic disease (4, 5, 40).Acute disease is characterized by a febrile illness which isoften diagnosed clinically as influenza or atypical pneumonia(4, 37). Chronic infection in humans results in valvularendocarditis, osteomyelitis, and hepatitis (4, 5, 15).

C. bumetii has a marked affinity for mononuclear phago-cytes, and organisms can be demonstrated readily in thespleen, liver, and bone marrow during the acute phase ofinfection (5, 6a, 22, 40). Furthermore, C. burnetii has beendetected in the gravid uterus and placental tissue of manyspecies, including mice, guinea pigs, cattle, goat, sheep, andhumans (1, 24, 25, 28, 30, 40, 44). In addition, Q feveroutbreaks in humans were associated with parturient carni-vores (26, 35). In animals, as in humans, aerosol infectionrepresents the main route of infection; however, in contrastto humans, hepatic, cardiac, and respiratory tract lesions arenot observed in livestock animals. Although infection is

* Corresponding author.

often unapparent in sheep, cattle, and goats, there is increas-ing evidence that C. burnetii infection in these species isassociated with abortion, stillbirth, and delivery of weakoffspring (1). However, studies of naturally occurring rumi-nant coxiellosis are hampered by species-specific differ-ences. Sheep, in contrast to goats and cattle, rarely becomechronically infected with C. burnetii, and abortion duringcoxiellosis epizootics have been described for goat andsheep, whereas the pathogenic potential for cattle is stillcontroversial (24, 25, 40). Furthermore, investigations ofsporadic outbreaks of C. burnetii-associated abortion arefrequently complicated by concurrent infections and by thelatent and subclinical course of the disease. Clinical analysisof reports on human C. burnetii infection during pregnancyrevealed that perinatal infection occurs, but no abortion riskfor humans was demonstrated (25). Investigations of theabortifacient potential of C. bumetii are further complicatedby the fact that the organism can be isolated from placentaltissue and birth products of animals and humans followingabortion and normal birth (24, 25, 36, 39, 40, 44, 46, 50).

Despite different placental interhemal barriers, which aresyndesmochorial for ruminants, endotheliochorial for dogsand cats, and hemochorial for rodents and humans (3), C.burnetii shows a high tropism for the gravid uterus, indicat-ing a species-independent affinity for placental tissue. So far,most reports have focused on the epidemiological aspects ofabortion- or birth-associated shedding of the organism,whereas only a few studies have investigated the morpho-genesis of C. bumetii-associated lesions in the placenta.

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C. BURNETII-ASSOCIATED MURINE PLACENTAL LESIONS 5233

Therefore, the objectives of this study were to characterizethe histological lesions in the placenta of mice infected onday 6 of pregnancy and to immunocytochemically identifysites infected with coxiellae.

MATERIALS AND METHODS

Organism and cell culture. The Nine Mile isolate of C.bumetii, phase I (originally, kindly provided by Dr. Re-hacek, Bratislava, Czechoslovakia), was used for thepresent study. The propagation, purification (kindly per-formed by N. Schmeer), and in vivo virulence of this strainhave been described previously (6, 6a). Briefly, C. burnetiiwas propagated after 10 passages in mice in the yolk sacs ofembryonated chicken eggs, harvested, and purified byRenografin gradient centrifugation. Titration of the stocksuspension was performed in Buffalo green monkey cells,and titers are expressed as inclusion-forming units as de-scribed before (41).

Mice. Female and adult male BALB/cJ (H-2d) mice, 8 to 12weeks old, were used in this study. The animals werepurchased from the Zentralinstitut fir Versuchstiere, Han-nover, Germany. They were housed at 25°C and were givencommercial-laboratory food and water ad libitum. Mice weretested serologically and microbiologically for commonmouse pathogens, including mouse hepatitis virus and Sen-dai virus. Primigravid pregnancies were obtained by cagingone or two females with one adult male. The time ofconception was determined by the presence of a vaginal plugand designated day 0 of pregnancy.

Infection of mice and clinical studies. Animals were inocu-lated on day 6 of pregnancy. Sixty-three pregnant mice,selected at random, received 3.85 x 106 inclusion-formingunits of C. bumnetii intraperitoneally in 400 ,ul of phosphate-buffered saline (PBS). Seventeen pregnant animals weremock-infected with 400 pl of PBS. Mice were examineddaily for clinical signs of infection. Animals were weighed ondays 0 and 10 of gestation and daily thereafter until day 22postconception (p.c.), and when they were killed. Rates ofabortion, stillbirth, and birth (number of litters with abor-tions, stillbirths, and normal births as a percentage of alllitters), pregnancy rate (percentage of actually pregnant miceamong all animals with a vaginal plug), number of live-bornpups per dam, and number of offspring surviving for 9 daysafter birth were determined.

Histopathology and immunocytochemistry. At various in-tervals after inoculation or when the animals were moribund(days 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 20, 28, 48, 78, and 160),the animals were killed by inhalation of CO2 and decapita-tion. Offspring born alive were sacrificed 9 days postpartum.At necropsy, the spleen and uterus with the fetoplacentalunits of the dam and various organs, including the liver,lung, spleen, and bones, of the pups were collected. Theuterus was fixed with pins on cardboard. All organs wereimmersed in Bouin's fluid, and mineralized tissues weredecalcified in 10% (wt/vol) EDTA disodium salt dissolved indouble-distilled water, pH 7.4, for 72 h at room temperature(6). For routine histology, tissues were dehydrated, embed-ded in paraffin at 53°C, and stained with hematoxylin andeosin.The methods used for immunocytochemical staining of C.

bumnetii and production of the rabbit anti-C. bumetii serumhave been described previously (6). Briefly, 4-pm-thicktissue sections were mounted on gelatin-covered slides,dried, deparaffinized, and rehydrated. After quenching of theendogenous peroxidase, sections were sequentially incu-

bated with rabbit anti-C. bumnetii serum, biotinylated linkantibody, and the avidin-biotin-peroxidase complex (ABC)(rabbit immunoglobulin G; Vectastain Kit; Vector Labora-tories, Inc., Burlingame, Calif.). The polyclonal rabbit an-ti-C. burnetii antibody was used at a dilution of 1:3,000. Apositive reaction was demonstrated by diaminobenzidinetetrahydrochloride (DAB)-H202 precipitation. Previousstudies showed that blocking of endogenous biotin was notnecessary for tissues fixed in Bouin's fluid (6). Control slidesof tissues from C. burnetii-infected and uninfected micewere prepared. To confirm that staining was specific for C.burnetii, normal rabbit serum or PBS was used instead ofprimary and bridge antibody and the ABC reagent. Effectiveblocking of endogenous peroxidase and absence of reactivetissue biotin were demonstrated by incubation of the sec-tions with DAB and ABC. The intensity and distribution ofthe immunoreaction were scored subjectively as negative,minimal, moderate, or strong staining. The results for eachorgan are expressed, if not stated otherwise, as the meanscore for all animals examined on the same day.

Statistical analysis. Differences between control and in-fected animals in survival of offspring to 9 days of age,occurrence of external vaginal bleeding, and pregnancy,abortion, stillbirth, and birth rates were determined byFisher's one-sided exact test. The two-tailed Wilcoxon ranksum test was applied to analyze the number of offspring bornalive in each group. The statistical significance of the differ-ences between groups in time to abortion, stillbirth, andnormal birth was evaluated by the nonpaired Student's t test.To determine whether the difference in body weight betweencontrol and infected animals was significant, two-way anal-ysis of variance with unbalanced repeated measures wasperformed. Results were regarded as significant when P was<0.05.

RESULTS

Clinical signs and gross findings. Infected animals showedlethargy, dehydration, and ruffled fur between 3 and 15 daysafter infection. One animal was found dead 12 days postin-oculation (p.i.), and two were moribund at 9 and 14 days p.i.following abortion and stillbirth. In pregnant control mice,the body weight increased from 26.2 ± 1.5 g to 49.2 + 6 guntil parturition, compared with 34.2 ± 6.5 g for C. bumnetii-infected animals at day 17 of pregnancy (Fig. 1). Aftergestation, body weights returned to prepregnancy values inboth groups. Necropsy findings (fetoplacental unit) andclinical observations (increase in body weight and occur-rence of abortion, stillbirth, and birth) for C. burnetii-infected animals revealed that 39 of 63 mice (62%) with avaginal plug were pregnant, whereas in control animals thepregnancy rate was 82% (14 of 17). Eleven infected animalsand seven control animals were necropsied before day 18 ofpregnancy, and therefore, the possible reproductive failureof these animals could not be determined with certainty. C.bumetii infection-associated effects on reproductive perfor-mance and the significance of the findings for the remaining28 infected and 7 control animals are summarized in Table 1.Abortion, stillbirth, and normal birth were observed for 12,14, and 2 infected pregnant mice, respectively. Abortion wasnot demonstrated until day 15 of pregnancy, and externalvaginal bleeding, an indication of in utero fetal death ordemise, occurred between days 13 and 18 of pregnancy.Among infected animals, 7 of 13 offspring born alive diedwithin 24 h after birth, whereas 45 of 47 pups born to controlanimals were still alive at day 9 after birth.

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FIG. Ipregnantgestationweight u:expresse

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Histoluterus iidifferen(23, 32).embryoby the 1

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5234 BAUMGARTNER AND BACHMANN

formed by hormonally modified endometrial cells. The de-cidua capsularis is found antimesometrially, and the lateralcompartment is termed the decidua parietalis. The outer-

most region of the placenta, subjacent to the decidua basalis,represents the metrial gland, with granulated metrial gland

UT T (GMG) cells.---o------ Control animals had no significant microscopic lesions and

showed physiological degenerative changes of the fetopla-l lo cental unit, especially of the decidua basalis, between days

15 and 18 of pregnancy, as described by others (18, 32). Until4 days postpartum, sites of previous placental attachmentwere characterized by focal necrosis, a few macrophages,,.neutrophils, and single persistent GMG cells. Focal accumu-

0 10 20 30 40 50 lation of hemosiderin-laden macrophages was observed sub-Days post conception jacent to areas of previous placental attachment until 84 days

after conception.1. Body weights of control (0) and C. bumetii-infected (0) Histological evaluation of spleen and uteroplacental units in

BALB/cJ mice. Animals were inoculated on day 6 of infected mice. Splenic lesions consisted of venous micro-i. Note significantly (P < 0.05) reduced increase in body in e earl phseof infectin Betwen iado4intil parturition (19 days p.c.) in infected mice. Results are thrombi in the early phase of infection. Between 3 and 48d as mean standarddeviation.days p.i., inflammatory changes were characterized by gran-

ulomas and pyogranulomas in the red pulp.At days 9 and 11 of pregnancy (3 and 5 days p.i.),

perivascular accumulations of neutrophils and macrophagesavid females, gross findings such as hepatospleno- were observed in the mesometrium and between the innerand multifocal hepatic necrosis were prominent and outer layer of the myometrium in the stratum vasculare.

n 3 and 28 days after infection. Until day 9 of Single vacuolated mononuclear cells were present in theon, no macroscopic differences in the gravid uterus decidua parietalis, decidua capsularis, and the metrial gland.bserved between control and C. bumetii-infected At 13 days p.c. (7 days p.i.), infiltrating neutrophils were

,. Thereafter, fetoplacental resorption and in utero found in the decidua basalis, the metrial gland, and the-ath, characterized by smaller embryo size, fetal endometrium. In addition, macrophages, erythrocytes, ands, and placental hemorrhage, were demonstrated in cellular debris were observed in the uterine lumen. Between-ted animals necropsied. 15 and 20 days p.c. (9 and 14 days p.i.), severe diffuselogical evaluation of the placenta and postgravid necrosis and moderate cellular infiltration consisting of neu-

n control mice. The following terms are used for the trophils and macrophages were present in the decidua basa-it compartments of the gravid murine uterus (Fig. 2A) lis (Fig. 2B and C). In some animals, there was an area ofThe innermost area of the fetal placenta, close to the necrosis without inflammatory cells subjacent to the giant-represents the chorionic plate; this area is followed cell layer, followed by a second layer consisting of degener-

labyrinth with maternal sinuses and fetal capillaries, ated neutrophils and macrophages in the decidua basalis. In?hospongium, and the giant-cell layer. The latter is contrast, changes in the metrial gland were minimal, andthe maternally derived decidua basalis, which is only a few infiltrating neutrophils and macrophages were

observed. In addition, fibrinoid degeneration of maternalvenules was a prominent finding in the decidua basalis (Fig.

E 1. Reproductive performance of BALB/cJ mice after 2C). Furthermore, microthrombi were observed in decidualinfection with C. burnetii on day 6 of pregnancy vessels.

The earliest histological lesions in the fetal placenta wereC. bumetii- demonstrated on day 13 of pregnancy (7 days p.i.). Changes

Parameter Control mice infected especially prominent in the labyrinth were characterized bymice multifocal necrosis, mild neutrophilic infiltration, and vacu-

cy rate (%) 82 62a olation of trophoblasts. Between days 15 and 20 p.c. (9 andi rate (%) 0 42.9b 14 days p.i.), inflammatory lesions and necrosis increased inrate (%) 0 50b severity. In addition, the labyrinth had a spongy appearance

te (%) 100 7.1 due to ectatic sinuses (Fig. 2B), and microthrombi were

of abortionc d 17.6 ± 0.9 observed in the labyrinthine blood spaces (Fig. 2D). Numer-of stillbirthc -19.1 ± 0.5aof birth 19.6 ± 0.53c 19.5af ous organisms, subsequently identified as coxiellae byIvaginal bleeding (%) 14 75b immunocytochemistry, were demonstrated in the cytoplas-ffspring born alive 7 (5, 9) o (0, g)" mic vacuoles of trophoblasts in the giant-cell layer, tropho-Lnig spongium, and labyrinth. Furthermore, ectatic sinuses of thespring surviving for 9 96 46b labyrinthine placenta were focally engorged with neutrophils

and coxiella-laden macrophages. In addition, numerous

~ignificantly different from control group value.extracellular coxiellae, forming emboli, were present in the

icantly different from control group value (P < 0.05). maternal sinuses. Single-cell necrosis was also observed in-+standard deviation. the chorionic plate. In some animals, complete necrosis ofne observed. the uteroplacental unit but not of the metrial gland wasicantly different from day of birth for control animals or day of observed.for infected mice (P < 0.05). Histoloical evaluation of the postgravid uterus in infectedfor two litters.s are shown as median (minimum, maximum). mice. Until 26 days p.c. (20 days p.i.), following abortion and

TABL

Pregnan(AbortionStillbirthBirth ratDay p.c.Day p.c.Day p.c.ExternalNo. of o

per da% of offsdaysa Not sib Signific Mean-, nc

' Signifistillbirth ff Meang Value

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5236 BAUMGARTNER AND BACHMANN

FIG. 3. Lesions in the liver (A) and lung (B) of a 9-day-old offspring from a C. bumetii-infected dam. (A) Single granuloma (arrow) in theliver and foci of extramedullary hematopoiesis (arrowhead). (B) Mild interstitial pneumonia and focal accumulation of macrophages andneutrophils. Hematoxylin and eosin stain. Bars, 35 ,um.

parturition, pyometra was a common finding in C. burnetii-infected animals. Lesions, prominent at sites of previousplacental attachment, were characterized by superficial ul-ceration, central necrosis, and moderate cellular infiltrationconsisting of neutrophils, macrophages, and persistent GMGcells. Fibrin thrombi were occasionally demonstrated insmall intralesional venules. The necrosis extended throughthe still disrupted inner layer of the myometrium into thestratum vasculare. Between 26 and 54 days p.c., sites ofprevious placental attachment were separated from theadjacent endometrium by several layers of fibroblast-likecells; the number of inflammatory cells declined, and GMGcells were no longer detectable. No significant microscopiclesions were observed on days 78 and 160 after infection.

Histological evaluation of fetuses and offspring. No inflam-matory lesions were demonstrated in fetal tissues in utero orin aborted, stillborn, or perinatally dying offspring. All sixpups killed at 9 days of age showed mild to moderategranulomatous hepatitis (Fig. 3A); granulomatous splenitiswas also demonstrated in one offspring. Mild interstitialpneumonia with a few granulomas was observed in fouranimals (Fig. 3B). The remaining organs, including thealimentary tract, central nervous system, bone marrow, andkidneys, had no significant inflammatory lesions.Immunocytochemistry. DAB precipitation products varied

from coarse to fine grains and were found intra- and extra-cellularly. Intracellularly, C. bumetii antigen was foundpredominantly in cytoplasmic vacuoles. These inclusionswere either packed with coarse granular dark brown immu-nopositive material or exhibited membrane-bound immuno-reactivity. In some cells, C burnetii antigen exhibited dif-fuse cytoplasmic distribution. Extracellularly, dark brownimmunopositive material, coccobacillary in shape and 1 to 2p,m in length, presumably representing single organisms,was demonstrated. In areas of necrosis, C burnetii antigenstained light brown and had a fine granular appearance, mostlikely representing residual particular antigenic debris.

C. burnetii antigen in spleen and uteroplacental units. Thespread and distribution of C. bumetii antigen in the uterusand spleen are outlined in Table 2. In the spleen, numerousimmunopositive cells were observed in the red pulp between3 and 15 days p.i. (Fig. 4A). Thereafter, the number ofimmunopositive cells decreased, and few C. bumetii anti-gen-bearing cells were still present in one animal at day 78after infection. C. burnetii antigen was observed in vacuo-

lated phagocytes, granuloma-forming macrophages, and ex-tracellularly.At 9 days p.c. (3 days p.i.), few immunopositive cells were

present in the decidua parietalis and decidua capsularis (Fig.4B). Single C. bumnetii antigen-bearing cells were found inthe decidua basalis and the adjacent endometrium. Morpho-logically, immunopositive decidual cells varied from poly-gonal to spindle-shaped. At 11 days p.c. (5 days p.i.), thenumber of immunopositive cells in the decidua increased.Furthermore, at days 11 and 13 p.c., diffuse positive stainingwas demonstrated in areas with necrosis in the deciduacapsularis and parietalis.At 13 and 15 days p.c. (7 and 9 days p.i.), strong diffuse

immunostaining was present in the decidua basalis, whereasonly a few foci of immunoreactivity were demonstrated inthe fetal placenta (Fig. 4C). Between 17 and 20 days p.c. (11and 14 days p.i.), strong immunoreactivity was observed inthe maternal and fetal compartments of the placenta (Fig.4D). Immunostaining was especially prominent in the ectaticmaternal vessels of the labyrinthine placenta. The labyrin-thine blood spaces contained many immunopositive mono-cytes (Fig. 5A). In addition, extracellular positively stainingcoccobacillary organism-like material was found in the lab-yrinthine sinuses and the uterine lumen. In the fetal pla-centa, C. bumetii antigen was found in trophoblasts of thegiant-cell layer, trophospongium, and labyrinth. However,unequivocal discrimination between immunopositive largecytoplasmic vacuoles and slightly dilated labyrinthine bloodspaces was not always possible (Fig. SB). Few immunopo-sitive cells were observed in the endometrium, metrial gland,chorionic plate, visceral wall of the yolk sac, Reichert'smembrane, uterus epithelium cells, and uterine lumen. Inthese compartments, C. burnetii antigen was demonstratedin resident and granuloma-forming macrophages, epithelialcells, and extracellularly but not in GMG cells.

C. burnetii antigen in the postgravid uterus. Sites of previ-ous placental attachment exhibited strong immunostainingpostabortion and postpartum until 28 days after infection(Fig. 6). Only few immunopositive mononuclear cells werepresent in the other compartments of the uterus. At 28 and48 days p.i., animals whose fetuses were aborted or stillbornwere still immunocytochemically positive at the sites ofprevious placental attachment, whereas both animals whogave birth normally lacked C. bumetii antigen. At the sites ofprevious placental attachment, positive immunoreaction was

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C. BURNETII-ASSOCIATED MURINE PLACENTAL LESIONS 5237

TABLE 2. Spread and distribution of C. bumnetii antigen in the uteroplacental unit and spleen of BALB/cJ (H-2d) mice afterintraperitoneal inoculation on day 6 of gestation

C bumetii antigen immunoreactmtya on day p.i.:Site

lb 3b 5b 7b 9c llb 12c 13b 14c 15b 20b 28b 48b 78b 160b

Spleen - +++ +++ ++ ++ ++ ++ ++ ++ ++ + + +d +dUterusEndometrium - + + + + + + + ++ + + + + + + + +dEpithelium - - - - + + + + + + - +Lumen - - + ++ - + + + + + - +

Maternal placentaMetrial gland - - + + + ++ + Ne + N N N N N NDecidua basalis - + ++ ++ +++ +++ +++ N +++ N N N N N NDecidua parietalis - + + + + + N N N N N N N N N N NDecidua capsularis - + + + + + + N N N N N N N N N N N

Fetal placentaGiant-cell layer - - - + + + + + + + N ++ N N N N N NLabyrinth - - - + ++ +++ +++ N +++ N N N N N NChorionic plate - - - - + + + N + N N N N N N

Sites of previous placental N N N N N N N +++ N + + ++ ++ - - Nattachment

Fetus - - - - - - - N - N N N N N NI Scoring: -, negative; +, weak, single focus; + +, moderate, multifocal; ++ +, strong, multifocal.b Values represent the mean score for all animals killed on the same day after infection.c Values are for one animal only.d Demonstrated in only one animal.I N, tissue not present at time of examination.

restricted to macrophages and few fibroblast-like cells. NoC. burnetii antigen was demonstrated in GMG cells. In theuterine lumen, positive immunostaining was observed until15 days after infection. The DAB precipitation product waspresent in macrophages and sloughed epithelial cells andextracellularly.

C. burnetii antigen in fetuses and offspring. C. bumnetiiantigen was not found in fetal tissues in utero or in aborted,stillborn, or perinatally dying offspring. In contrast, positivestaining was observed in the liver, lung, and spleen of six,four, and one offspring, respectively, at 9 days of age. Inthese organs, C. burnetii antigen was present in migrating orgranuloma-forming macrophages. The remaining organs, in-cluding the bone marrow, kidney, and central nervoussystem, were immunocytochemically negative for C. bur-netii antigen.

DISCUSSION

The present study demonstrates the high affinity of C.burnetii for the gravid uterus and its abortifacient potential.Experimental infection of pregnant mice with C. burnetiiresulted in abortion, stillbirth, and perinatal death. Antepar-tum, necrosis of placental tissues, fibrinoid degeneration ofdecidual vessels, and microthrombosis were observed, andC. burnetii antigen was simultaneously demonstrated to bepresent in abundance in the placenta. Surprisingly, fetaltissues were devoid of C. burnetii antigen. However, off-spring killed at day 9 of age exhibited lesion-associatedpositive staining for C. burnetii antigen in the lung, liver, andspleen.

Clinically, C. burnetii-infected pregnant mice displayed avariety of failures in reproductive performance but also gavebirth normally. Similar observations have been reported forrodents and other species infected with C. bumnetii (14, 24,28, 30, 40). Initial infection of the placenta occurred in thedecidua, and then the fetal compartment was invaded.Localization of C. bumnetii antigen in the labyrinthine pla-

centa indicates hematogenous dissemination of the organismfrom the decidua to the fetal placenta. Once the organismshad invaded the decidua and labyrinth, infection resulted inuncontrolled growth and/or accumulation of C. bumnetii inthe placenta. Comparative evaluation of the spleen andplacenta revealed that the former showed strong immuno-reactivity as early as 3 days after infection. In contrast, inthe uteroplacental unit, a plateau of strong immunoreactivitywas present between 9 and 14 days after infection. More-over, the intensity of the immunostaining in the uteroplacen-tal region exceeded the intensity of staining in the spleen.Despite strong immunoreactivity in the uteroplacental unit,C. burnetii antigen was not demonstrated in fetal tissues,indicating that the fetoplacental unit resisted vertical infec-tion. Similarly, lack of fetal infection despite heavy placentalcolonization was reported for mice inoculated with Chla-mydia trachomatis (47).The failure to find C bumetii antigen in fetal tissue was

not due to the inadequacy of the method used, because C.burnetii antigen was readily demonstrated in other organs ofthe dam and in offspring at 9 days of age. However, lack offetal infection needs to be confirmed by more sensitivetechniques, such as the mouse infectivity test or polymerasechain reaction. Neonatal infection with C. burnetii of theoffspring of latently infected guinea pigs and mice has beendescribed (44). The fact that C. burnetii antigen was found inthe lung, liver, and spleen of the offspring suggests thataerosol infection occurred by inhalation of C. burnetii re-leased from birth products. However, alimentary infectionafter ingestion of contaminated milk (mammary glands werenot investigated) or fetal fluids followed by hematogenousdissemination cannot be completely ruled out.The role of the metrial gland and the GMG cells during

pregnancy is still undetermined, but it appears that they areimportant in the immunology of the fetomaternal relation-ship (12). Several studies suggested that GMG cells, descen-dants of bone marrow cells morphologically resembling largegranular lymphocytes, may be natural killer-like cells (34).

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C BURNETII-ASSOCIATED MURINE PLACENTAL LESIONS 5239

FIG. 4. Spread and distribution of C. bumetii in the spleen and uteroplacental unit. (A and B) at 3 days p.i., numerous immunopositivecells in the splenic red pulp (A) and few antigen-bearing cells in the decidua parietalis (arrows) (B). (C) At 9 days p.i., strong immunostainingin the decidua basalis and few foci of immunoreactivity in the labyrinthine placenta. (D) At 11 days p.i., strong immunoreactivity in the fetaland maternal compartments of the placenta. Abbreviations: D, decidua basalis; E, endometrium; G, giant-cell layer; L, labyrinth; M,myometrium; MG, metrial gland. ABC staining technique. Bars: (A) 150 p,m; (B) 162 pm; (C and D) 200 pm.

Decidual tissue is composed of leukocytes and stromal,macrophage-like, and GMG cells (20, 21). In the presentstudy, C. bumnetii antigen was found predominantly in mac-rophage-like decidual cells and labyrinthine trophoblasts.Interestingly, GMG cells were immunocytochemically neg-ative for C. bumetii antigen, and only a few macrophage-likepositive cells were demonstrated in the metrial gland. Thesignificance of these observations and their relevance for thepathogenesis of C. burnetii-associated lesions in the graviduterus remain to be determined.The fetoplacental unit represents an immunologically priv-

ileged site, and several factors could account for the uncon-trolled growth of C. burnetii at this location. Survival of theconceptus has been attributed to active systemic and localimmune suppression as well as to the placental barrier (8).Luft and Remington (27) showed that, during pregnancy,peritoneal macrophages are defective in their ability to killintracellular pathogens. However, in the present study,

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there was no evidence of systemic disturbance in macro-phage function. C. burnetii organisms disseminated to andwere cleared from the spleen as described for nonpregnantBALB/cJ mice (6a). Numerous suppressor substances andsuppressor cells have been detected in the pregnant murineuterus, especially in the decidua (9, 23). Decidual cells maydeliver inhibitory signals to macrophages, T cells, or both(8). Several immunosuppressive mediators, including a mol-ecule related to transforming growth factor beta-2 that isreleased by non-T small lymphocytic suppressor cells andimpairs the mobility of interleukin-2 receptors, have beendescribed (10). Furthermore, decidual cells and macro-phages suppress T-cell activity by release of prostaglandinE2 (9). Accordingly, in vitro studies revealed that macro-phage-like cells in uterine cell suspensions inhibit T-cellproliferation (19). In addition, nonspecific intrauterine sup-pression and differential regulation of cell proliferation mightbe a function of the metrial gland (12). Furthermore, active

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AFIG. 5. Appearance of C. burnetii antigen in the labyrinthine placenta 11 days after infection. (A) Numerous immunopositive mononuclear

cells within dilated maternal vessel. (B) C burnetii antigen in cytoplasmic vacuoles of trophoblasts (arrow) and within blood spaces (S). ABCstaining technique. Bars, 12 pLm.

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5240 BAUMGARTNER AND BACHMANN

FIG 6 Distribution of C bumeti antigen in the postgraviduterus. At 28 days p.i., immunoreactivity is restricted to sites ofprevious placental attachment. Abbreviations: E, endometrium; M,myometrium; U, uterine lumen. ABC staining technique. Bar, 190

local immunosuppression could be enhanced by the phase Icell-associated immunosuppressive complex of C. bumetii(48). Studies investigating infection of the murine uteropla-cental region by Listeria monocytogenes found evidencethat local immunoregulation, which normally prevents ma-ternal antifetal response, also inhibits an effective antiliste-rial response (38).The uterine lesions can be described as self-limiting, with

complete resolution. Although the connection is highly spec-ulative, the observed placental lesions are reminiscent ofchanges described as a local Schwartzman-like reactionfollowing administration of endotoxin (13, 17, 29, 51). Somebiological activities associated with gram-negative bacterialendotoxin are elicited by C. burnetii lipopolysaccharide(LPS) (5), but compared with other pathogens, Coxiella LPScan be classified as a poor endotoxin (2, 16, 33, 42, 43), andlarge quantities of purified C. burnetii LPS are necessary toinduce endotoxic changes (31). Many biological activitiesonce attributed directly to LPS are now known to bemediated by LPS-induced cytokines. Recent studies demon-strated production of tumor necrosis factor alpha by murinespleen and peritoneal exudate cells within 48 h after inocu-lation of a killed C. bumnetii phase I whole-cell preparation(48). Since many macrophage-like decidual cells were immu-nocytochemically positive for C bumetii antigen, it remainspossible that cytokines secreted by these cells play a role inthe development of C. bumnetii-associated lesions in theplacenta. Accordingly, in vitro studies with human decidualcells showed that these cells synthesize and secrete tumornecrosis factor alpha and prostaglandin F2a, (7). Further-more, application of LPS, tumor necrosis factor alpha, orinterleukin-la during pregnancy results in placental necrosisfollowed by fetal death and abortion (45). In addition, mice

primed with C. bumetii extracts are highly susceptible totumor necrosis factor-induced abortion (11).However, further studies are necessary to investigate the

pathogenesis of C. burnetii-associated lesions in the pla-centa. The susceptibility of different placental cell popula-tions to this pathogen and infection-induced release of cy-tokines by these cells need to be evaluated.

ACKNOWLEDGMENTS

This workwas supported by grants from the Deutsche Forschungs-gemeinschaft (Ba 815/2-1 and 2-2). Sabine Bachmann was therecipient of a research award from the Justus-Liebig-Universitat,Giessen, Germany.We thank Annette Artelt for excellent technical assistance and

Ute Zeller for photography.

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