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J. Comp. Path. 2006,Vol.135,108^115

0021-9975/$ - seedoi:10.1016/j.jcpa.

Correspondence

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Experimental Coxiella burnetii Infection inPregnant Goats: a Histopathological and

Immunohistochemical Study

J. Sanchez*, A. Souriauy, A. J. Buendıa*, N. Arricau-Bouveryy, C. M. Martınez*,J. Salinasz, A. Rodolakisy and J. A. Navarro*

*Departamento deAnatom|¤ a yAnatom|¤ a PatoloŁ gica Comparadas andzDepartamento de Sanidad Animal, Facultad deVeterinaria,

Universidad deMurcia, Campus de Espinardo, 30100Murcia, Spain, and yPathologie Infectieuse et Immunologie, INRA,

Tours-Nouzilly, 37380 Nouzilly, France

Summary

Pregnant goats were inoculated subcutaneosly with Coxiella burnetii and the course of infectionwas studied. Abor-tion in the last third of pregnancy occurred in all infected animals. Tissues from the placenta and other organswere studied before and after abortion by immunohistochemistry and PCR analysis. After infection, mild lesionswere observed in severalmaternal organs, mainly themammaryglandbut also the lungand the liver.The tropho-blast cells of the choriallantoic membrane were the ¢rst target cells of the placenta; there was, however, a substan-tial delay between initial infection andplacental colonization. In the last weeks of pregnancy, just before abortion,massive bacterial multiplicationwas detected in the placenta. In this stage of infection a necrotic and suppurativeplacentitis separated the fetal trophoblast cells frommaternal syncytial epithelium.Vasculitis was observed in thefetal mesenchyme. A strong maternalT-cell response was detected in the inter-placentomal areas but not in theplacentomes, where only neutrophils and smaller numbers of macrophages were associated with the lesions.Neither lesions nor C. burnetiiDNAwere found in maternal organs in animals maintained until day120 post-abor-tion.

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Keywords: abortion; bacterial infection; Coxiella burnetii; goat; placenta; Q fever

Introduction

Coxiella burnetii is a gram-negative intracellular bacter-ium responsible for Q fever, a zoonosis of worldwidedistribution. Human infection may be acute (£u-likesyndrome, pneumonia or hepatitis) or chronic (mainlyendocarditis). Many species of mammals, birds andticks are reservoirs of C. burnetii (Maurin and Raoult,1999), but ruminants, especially goats (McQuistonand Childs, 2002), are the major sources of human in-fections. C. burnetii-infected animals remain symptom-less in the acute phase of infection and clinical signsare shown only by pregnant females, inwhich infectionmay induce abortion in late pregnancy, stillbirths, peri-

front matter2006.06.003

to: J. SaŁ nchez (e-mail: jsanchez@um.es).

natal mortality, retained placenta, endometritis andinfertility (Palmer et al., 1983). Ruminants appear notto develop endocarditis as a result of chronic infection,the lesions being con¢ned to the uterus and mammarygland (Maurin and Raoult, 1999). A model of chronicendocarditis was established in mice by Stein et al.(2000).C. burnetii is a signi¢cant cause of abortion inWestern

countries. As such, in goats in theUSAandEurope, it issecond only to Chlamydophila abortus (Moeller, 2001;Chanton-Greutmann et al., 2002). Shedding of the mi-cro-organism after abortion has been studied in natur-al and experimental infections (Arricau-Bouvery et al.,2003a,b; Berri et al., 2005), but the pathogenesis of C.burnetii placental infection in ruminants is poorly un-derstood. An experimental model of abortion in mice

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Coxiella burnetii Infection in Pregnant Goats 109

was described by Baumg�rtner and Bachmann (1992)and Baumg�rtner et al. (1993), but data on cattle, sheepand goats have been derived only from post-abortionstudies of naturally infected animals (Moore et al.,1991;Van Moll et al., 1993; Bildfell et al., 2000; Moeller,2001).The aim of this study in goats was to investigate (1) C.

burnetii colonization in certain organs, and especially indi¡erent areas of the placenta, and (2) possible chronicinfection and lesions after abortion.

Materials and Methods

Inoculum

C. burnetii strain CbC1 was used. This strain had beenisolated as follows from the placenta of an aborted goat(Arricau-Bouvery et al., 2003a). Mice were inoculatedintraperitoneally withplacental homogenate. At 9 dayspost-infection (p.i.) the mice were killed and spleenhomogenate was used to infect speci¢c pathogen-freefertile eggs. At the third egg passage, egg material wastitrated for infectivity and dispensed in small volumes,whichwere frozen at�70 1C until used.Themethod oftitration consisted in inoculating mice intraperitone-ally with decimal dilutions and detecting C. burnetii inthe spleen by the polymerase chain reaction (PCR)(Arricau-Bouvery et al., 2003a,b); the titre was ex-pressed as infective mouse doses (IMD).The inoculumobtained was used both in previous studies (Arricau-Bouvery et al., 2003a, b) and in the present study.

Animals, Infection and Experimental Design

The animal experimentation was carried out in a level3 biosecurity building. Sixteen healthy pregnant goats,aged1year, were obtained from herds serologically ne-gative for C. burnetii and Chlamydophila abortus. Twelvegoats were infected subcutaneously in the shoulder atday 90 of pregnancy with 104 IMD of the CbC1strain.The other four animals remained uninfected, their tis-sues (1) serving as negative controls for the di¡erenttechniques used in this study, and (2) enabling compar-ison to be made with tissue changes seen in infected an-imals. Goatswere kept in separate rooms and inspecteddaily for clinical signs. In the infected group, two goatswere killed on day 26 p.i. (day 116 of pregnancy) andtwo were killed on day 40 p.i. (day 130 of pregnancy).The other eight infected goats were maintained untilthe time of abortion; subsequently, two animals werekilled on day 8 post-abortion (p.a.) and six on day 120p.a.Two of the four uninfected controls were killed onday130 of pregnancy and two 8 days after parturition.

Necropsy and Sampling

Goats were killed with an intravenous overdose of so-dium pentobarbital and subjected to necropsy. Fiverandomly selected placentomes and ¢ve interplacento-mal areas were collected from animals killed duringpregnancy (at 26 or 40 days p.i.). In these animals, sam-ples were also collected from the maternal liver, lung,spleen and mammary gland and from the fetal liver,lung and spleen. In animals maintained until the timeof the abortion, samples of cotyledons and fetal organswere collected from aborted material.When these ani-mals were subsequently killed (at 8 or 120 days p.a.),samples were collected from the uterus, liver, lung,spleen, heart and mammary gland. All samples weretaken in duplicate, one part being ¢xed in 10% buf-fered formalin and the other part frozen at �70 1C forsubsequent PCR analysis.

Histopathology and Immunohistochemistry (IHC)

Samples ¢xed in formalin were embedded in para⁄nwax and sectioned (4 mm) before being stained withhaematoxylin and eosin (HE) for histopathological ex-amination. Adjacent sections were examinedby an avi-din^biotin^peroxidase complex (ABC) technique, aspreviously describedby Buend|¤ a et al. (1999), with a pri-mary antibody consisting of an anti-Coxiella polyclonalantibody obtained from experimentally infected mice.When an in£ammatory response was observed withthe HE stain, an ABC immunohistochemical techni-que with an anti-CD3 polyclonal antibody (Dakopatts,Denmark) was used on adjacent sections to detect Tcells.The immunohistopathological ¢ndings in the pla-centa were ranked as follows: Level 1, when there wasno in£ammatory response and little antigen; Level 2when there was a mild in£ammatory response andmoderate quantity of antigen; Level 3 when there wasa severe in£ammatory responsewith extensive necroticfeatures, together with abundant antigen.

Purification of C: burnetii DNA from Samples

Vaginal swabs were sampled as previously described(Berri et al., 2001). Placental tissues and other tissuesfrom does and fetuses were homogenizedwith a Stoma-cher (Seward Medical, Worthington, UK) in sterilebu¡ered saline. DNAwas extracted from 50 ml of tissuehomogenate or100 ml of milk with theQUIAampDNAmini kit (Qiagen, Hilden, Germany) according to themanufacturer’s instructions.Vaginal and milk sampleswere taken only from the six animals killed on day 120p.a., the samples being taken at days 1, 2, 3 and 7 p.a.and thenweekly.

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J. Sanchez et al.110

PCR Assay

PCR assays were performed as previously described(Berri et al., 2000), with 2.5 ml of DNA extract. ThePCR reaction was carried out in an automated DNAthermal cycler (UNOThermobloc; Biometra, Gottin-gen, Germany); PCR products (687 base pairs, 10 ml)were analysed by electrophoresis on a 1% agarose gel,stained with ethidium bromide, ‘‘visualized’’ under anultra-violet transilluminator and photographed.Whenno DNA was ampli¢ed, 1in10 and 1in100 DNA dilu-tions were made and a new PCR test was performed.If the PCR was positive at one of these dilutions, thesample was considered positive.

Results

Goats Killed on Day 26 p.i. (Day 116 of Gestation)

Placenta. Most of the placentomes examined (80%)showed no histopathological changes or C. burnetii anti-gen.The ¢ndings in two placentomes (20%) were rankedas Level 1, antigen being found mainly in the trophoblastcells of the chorioallantoic membrane (Fig. 1A) and ap-pearing as discrete granules of positive intracytoplasmicmaterial or as a weak di¡use immunoreaction in vacuo-lated cells. In the erythrophagocytic trophoblast, occa-sional cells near the chorioallantoic membrane were alsoa¡ected. In only one placentome was antigen detected incell debris within the maternal crypts at the periphery ofthe placentome. All of the interplacentomal areas showedoedema in the endometrial stroma and a mild subepithe-lial leucocyte in¢ltration, but no C. burnetii antigen. ThePCR (Table 1) showed that 50% of the placentomes ex-amined were negative; the immediately adjacent chorial-lantoic membrane, however, was invariably positive.

Maternal organs. Mild periportal hepatitis, manifestedas an in¢ltration of lymphocytes, and small foci of inter-stitial pneumonia were observed. C. burnetii antigen wasnot detected in liver or lung. PCR analysis (Table 1) wasnegative in all organs, except in one mammary glandand one lung sample.

Fetal organs. There were no relevant immunohisto-pathological ¢ndings, but PCR analysis gave positive re-sults in the liver and spleen of two fetuses; all samples offetal lung were negative.

Goats Killed on Day 40 p.i. (Day 130 of Gestation)

Placenta. Di¡erent degrees of pathological change anddistribution of C. burnetii antigenwere observed in the pla-centomes. One placentome (10%) showed no lesions orantigen.The ¢ndings in three of the10 placentomes exam-ined were ranked as Level 2. In these placentomes the

trophoblast of the chorioallantoic membrane appearednecrotic and ulcerated, and some vacuolated cells con-tained C. burnetii antigen. This area of the trophoblastwas covered by an exudate composed of cell debris, neu-trophils and extracellular antigen. The erythrophagocy-tic trophoblast was more severely a¡ected than at day 26p.i.The remaining placentomes examined (60%) showedsevere placentitis (Fig. 1B), with widespread necrosis andhaemorrhage of the placentomal base. Necrotic materialwas composed of desquamated trophoblast cells and hya-linized andmineralized maternal septa. A substantial de-gree of neutrophil in¢ltrationwas observed in the necroticareas, together with abundant extracellular antigen.Thebases of some of themain chorionic villi were partly ulcer-ated, while others were surrounded by swollen tropho-blast cells with C. burnetii antigen in the cytoplasm.Adjacent maternal syncytial epithelium lining the septaalso showed C. burnetii antigen (Fig.1C). Neutrophils wereobserved in the chorionic mesenchyme adjacent to the ul-cerated trophoblast. However, margination or diapedesiscould not be observed in the blood vessels of the chorion,suggesting that the neutrophil in¢ltration originated inthe maternal placenta, where a substantial number ofsuch cells could be observed in the small vessels and con-nective tissue of the septa (Fig.1C).Severe endometritis and oedema were observed in

the interplacentomal areas, the substantial in¢ltrate ofleucocytes (mainly subepithelial but also epithelial)being composed of neutrophils and lymphocytes, withsome plasma cells and occasional macrophages. Mostof the lymphocytes were shown by IHC to be Tcells(Fig. 1E). Some uterine glands were dilated and ¢lledwith neutrophils and cell debris. No C. burnetii antigenwas detected in these interplacentomal areas, but a po-sitive immunoreaction was observed in the uterine lu-men. PCR analysis (Table 1) was positive in all theplacental samples, both in the placentomes and inter-placentomal areas.

Maternal organs. Lesions similar to those seen at day 26p.i. were observed in the maternal livers and lungs. Onemammarygland showedan extensive interstitial accumu-lation of lymphocytes. C. burnetii antigenwas not detected.PCR analysis (Table 1) gave positive results in the livers,lungs and mammary glands of both animals killed. Onlyone spleen sample was negative.

Fetal organs. IHC gave negative results, but PCRanaly-sis was positive in most of the samples examined (Table1).

Material from Abortions

The two uninfected goats had a normal parturition atday15072, but all the infected animals maintained untilthe end of gestation aborted at day 13274. Examination

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Fig.1 A-H. Immunohistochemical study of C. burnetii infection in placental tissues. (A) C. burnetii antigen in the placentome at day 26 p.i.Theonly cells that showed antigen were the trophoblast cells of the chorioallantoic membrane and the allantoic epithelium (arrows). No in£am-matory response was observed. ABC. �50. (B) Severe necrotic and haemorrhagic placentitis with substantial neutrophil in¢ltration andulceration of the trophoblast layer at day 40 p.i. HE. �50. (C) C. burnetii in trophoblast cells in the chorionic villi at day 40 p.i. Mesenchymeof the villi shows neutrophilic in¢ltration. ABC. �630. (D) C. burnetii in maternal syncytial cells (arrows) at day 40 p.i. Antigen also detectedin neutrophils and cell debris between maternal septa (asterisk).Vacuolated trophoblast cells contain C. burnetii antigen (arrowheads). ABC.� 400. (E) Tcells in leucocyte in¢ltrate located in the intercaruncular area at day 40 p.i. Uterine epithelium is vacuolated and cell debris isseen in the uterine lumen. ABC. �200. (F) C. burnetii antigen in a cotyledon from an aborted fetus. Antigen is located in trophoblast cells andin the in£ammatorymaterial (neutrophils andcell debris) in thebase of chorionic villi. ABC. �50. (G) Chorioallantoicmesenchyme fromanaborted fetus showing a blood vessel with thrombotic lesions and mononuclear cell in¢ltrate. HE. �100. (H) Tcells in the chorioallantoicmesenchyme from an aborted fetus. Note the predominance of Tcells in the in£ammatory in¢ltrate. ABC. �200.

Coxiella burnetii Infection in Pregnant Goats 111

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Table 1Detection of C. burnetiiDNA in the infected goats and their fe-

tuses by PCR analysis

Tissues examined PCR results at days

26 p.i. 40 p.i. 8 p.a. 120 p.a.

Maternal

Placentomes +* + y y

Interplacentomal areas + + y y

Mammary gland +(1/2) + + �

Liver � + � �

Spleen � +(1/2) � �

Lung +(1/2) + + �

Fetal

Liver +(2/4) + y y

Spleen +(2/4) + y y

Lung � +(3/4) y y

y, No entry.Figures in parentheses indicate number of positive animals/numbertested. Absence of parentheses indicates that all animals tested gave po-sitive results.*Although 50% of placentomes gave negative results, the immediatelyadjacent smooth chorion (chorioallantoic membranes) was positive.Cotyledons from aborted material and caruncles from animals killed onday 8 p.a. gave positive results.

J. Sanchez et al.112

of the cotyledons and organs from the fetuses gave thefollowing results.

Fetal placenta. All cotyledons examined showed level 3changes, characterized by multifocal necrosis of the chor-ionic epithelium; numerous chorionic villi appeared ul-cerated and tended to coalesce (Fig. 1F). All cotyledonsshowed strong immunolabelling for C. burnetii. The loca-tion of antigenwas closely similar to that described in thefetal placentas from animals killed at day 40 p.i. (seeabove); however, antigenwas seldomdetected in the chor-ionic mesenchyme adjacent to ulcerated trophoblast orwithin cell debris in blood vessels. Severe suppurative in-£ammation was observed at the base of the villi, the in-£ammatory exudate being composed mainly ofneutrophils, with occasional macrophages. Desquamatedmononuclear andbinuclear trophoblast cells andminera-lized cell debris were observed in the exudate. A mild-to-moderate in¢ltrate of mononuclear cells was sometimesobserved in the allantoic blood vessel walls, and intravas-cular ¢brin thrombi were associated with these lesions(Fig. 1G). In the chorionic mesenchyme, there was oede-ma with haemorrhagic foci and ¢brin deposits. A signi¢-cant number of mononuclear cells, appearing in focalclusters or as a di¡use in¢ltrate, occurred in most cotyle-dons. These cells were characterized by IHC as T cells(Fig. 1H). PCR analysis (Table 1) of samples was positivein all cotyledons.

Fetal organs. The only organ in which lesions were ob-served was the liver, which usually showed mild-to-mod-erate perivascular hepatitis, with neutrophils andlymphocytes surrounding the vessels. Neutrophils some-times formed foci or appeared as a di¡use in¢ltrate inthe hepatic parenchyma.Thrombotic lesions and necroticfoci were seen only rarely. No C. burnetii antigen was de-tected in the lesions, but PCR analysis showed that mostof the spleen, liver and lung samples contained C. burnetii

DNA (Table1).

Goats Killed on Day 8 p.a.

Maternal placenta. The caruncles showed Level 2changes, characterizedbymultifocal necrosis of the septa,which were congested and in¢ltrated by numerous neu-trophils; between the septa, haemorrhages and extensivein¢ltration of neutrophils, with some macrophages,epithelial cells and mineralized cell debris, were ob-served. Granulation tissue with numerous macrophagesoccurred at the base of the caruncles. C. burnetii antigen,present in super¢cial areas of the caruncles, was either lo-cated within neutrophils and macrophages, or was extra-cellular. In the intercaruncular areas the in£ammatoryin¢ltrate was substantially less than that seen at day 40p.i. PCRanalysis (Table1) gave positive results in all sam-ples.

Maternal organs. No relevant immunohistopathologi-cal ¢ndingswere observed in anyorgan, but PCRanalysisgave positive results in lung and mammary gland sam-ples.

Goats Killed on Day 120 p.a.

Excretion of C: burnetii. PCR analysis of milk and va-ginal secretions showed that all the animals excreted C.

burnetii after abortion. The presence of C. burnetii in milksamples occurred16.879.6 days after abortion, and in va-ginal swabs 21.478.2 days after abortion.

Maternal organs. No signi¢cant immunohistopatholo-gical ¢ndings were observed in any organ, and all the tis-sue samples subjected to PCR analysis were negative.

Discussion

The experimental infection induced abortion towardsthe end of pregnancy, as in naturally infected animals.Abortion appeared to coincide with striking multipli-cation of C. burnetii in the fetal trophoblast cells; multi-plication was, however, much less in the cells of thematernal placenta.These ¢ndings suggest that themul-tiplication of C. burnetii in the placenta does not signi¢-cantly a¡ect other maternal organs. Althoughsecondary C. burnetii dissemination from the foci of

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Coxiella burnetii Infection in Pregnant Goats 113

placental multiplication cannot be ruled out, the ma-ternal immune response seemed able to preventchronic lesions and clear the infection.Trophoblast cells are themain target for themultipli-

cation of C. burnetii.The part of the trophoblast locatedin the chorioallantoic membrane is the ¢rst to be af-fected, infection then extending to the adjacent ery-throphagocytic trophoblast at the hilar region of theplacentome.Trophoblast cells are also the targets of in-tracellular ruminant pathogens such as Brucella abortusand Chlamydophila abortus (Anderson et al.,1986; Buxtonet al.,1990; Navarro et al., 2004); in these two infections,however, the ¢rst trophoblastic area to be invaded wasthe erythrophagocytic trophoblast, followed by the ad-jacent chorioallantoic membrane. In experimentalmurinemodels ofC. burnetii, B. abortus, C. abortus andTox-oplasma gondii infections (Baumg�rtner and Bachmann,1992; Tobias et al.,1993; Buend|¤ a et al.,1998; Ferro et al.,2002), infection of the trophoblast (in these instances,the trophoblast giant cell layer between decidua and la-byrinth) was also the ¢rst event in the colonization ofthe placenta. There are several possible explanations.Firstly, haemorrhages from maternal vessels occur inthe central area of the placentomes of small ruminantsfrom day 60 of pregnancy onwards (Wimsatt, 1950).These haemorrhages lead to contact between maternalblood and placental trophoblast cells and consequentinfection of these cells byblood-borne agents. Secondly,trophoblast cells from ewes and goats do not expressMHC I between days 9 and 125 of pregnancy (Gogo-lin-Ewens et al.,1989;Mart|¤ nez et al., 2005).This is prob-ably exploited by intracellular bacteria to prevent theMHCI-dependent lytic action of cytotoxic lymphocytesubsets. Furthermore, MHC II is not expressed in thetrophoblast cells of ewes, cattle and goats (Gogolin-Ewens et al., 1989; Low et al., 1990; Mart|¤ nez et al.,2005). Finally, it is tempting to hypothesize that tropho-blast cells produce metabolites that act as growth fac-tors for C. burnetii. Indeed, for optimal multiplication,species of the genus Brucella need the erythritol andthe progesterone produced by the fetal placenta (Kep-pie et al.,1965; Misra et al.,1976).The main microscopi-cal lesion observed in the placenta was a pronouncedin¢ltration by neutrophils, with relatively few macro-phages, seen mainly at the base of the placentome. Si-milar in¢ltration is a common feature of placentalinfection by other intracellular pathogens (Andersonet al.,1986; Baumg�rtner and Bachmann,1992; Montesde Oca et al., 2000;VaŁ zquez-Boland et al., 2001; Buxtonet al., 2002).C. burnetii infection in animals may be accompanied

by various lesions, such as (1) necrotizing purulent pla-centitis, as seen in ewes and cattle (VanMoll et al.,1993;Bildfell et al., 2000) and in the present study, (2) mixedleucocyte in¢ltration (Moore et al., 1991), or (3) necro-

tizing non-suppurative placentitis (Moeller, 2001). Inthe present study, macrophages did not play an obviousrole in the in£ammatory response; such cells have beenregarded as target cells for C. burnetii in the lung andliver (Maurin andRaoult,1999), and a recent study im-plicated macrophages in the pathogenesis of C. burnetiiinfection, suggesting that they transport the organismto the fetus (Bildfell et al., 2000).No lymphocytes were observed in the maternal por-

tion of the placentome in the present study; in othermaternal organs, however, mild lymphocytic in¢ltra-tion was observed and numerousTcells were detectedin the interplacentomal areas of the pregnant uterus.Several mechanisms may impair the entry of lympho-cytes into the maternal placenta.The production of in-hibitory cytokines such as IL-10 (Bennett et al., 1999),the induction of activatedT-cell apoptosis mediated byFas ligand (Mor et al.,1998), or the depletion of trypto-phane (Entrican, 2001) inhibit the maternal immuneresponse against allogenic fetal tissues. These inhibi-tory mechanisms lead to an immunologically privi-leged area suitable for the multiplication ofintracellular pathogens.A strong immune responsewas observed in the inter-

placentomal areas of the pregnant uterus from day 40p.i. In these areas, the in¢ltration of Tcells and neutro-philsmayhave prevented C. burnetiidissemination fromplacentomes to other maternal organs. Although noantigen was detected immunohistochemically, the po-sitive results obtained by PCR analysis suggest thatsome bacteria reach these areas, probably through theuterine lumen, to induce a strong local immune re-sponse.The mammary glands showed major immuno-histopathological changes and C. burnetii wasdemonstrated in milk in all goats after abortion. Mas-titiswas detected in onlyone animal, possiblydue to thedi¡use character of the lymphocytic in¢ltration, whichmay have lessened the likelihood of detection at themoment of the sampling. Other maternal organsshowed no obvious lesions; the presence of C. burnetii inthe liver and lung was con¢rmed, however, by PCRanalysis. It would seem that the maternal immune re-sponse e¡ectively controlled bacterial multiplication.In contrast, lesions in the liver and spleen were de-scribed in an experimental murine model of C. burnetiiinfection (Baumg�rtner et al.,1993).In the present study, lesionswere observed in some of

the aborted fetuses, but neither lesions nor C. burnetiiantigens were detected in fetuses from goats killed dur-ing gestation. Lesions, when seen, consisted of vasculi-tis of allantoic vessels in the chorionic mesenchyme,and the in¢ltration of mononuclear cells (character-ized asTcells). In addition, mild-to-moderate perivas-cular hepatitis, with neutrophils and lymphocytessurrounding the vessels, was observed in some of the

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J. Sanchez et al.114

aborted foetuses. In studies on abortions in naturallyinfected animals (Van Moll et al., 1993), the ¢ndingswere similar to those of the present study, with di¡erentdegrees of pathological change but no C. burnetii immu-nolabelling. On the other hand, the early detection of C.burnetii DNA in fetal tissue demonstrated the simulta-neous colonization of this tissue and the placentomes.The number of fetal PCR-positive samples increasedup to the moment of the abortion, without the produc-tion of obvious histopathological lesions. It is possiblethat the early immune response detected in fetal chor-ion and liver controlled bacterial multiplication. Earlyfetal immune response to placental pathogens has beenreported in other infections in ruminants (Buxton et al.,1990; Andrianarivo et al., 2001). Recent reports (Andohet al., 2003; Stein et al., 2005) suggest that, in the absenceof a host response, C. burnetii spreads widely to di¡erentorgans; this may indicate a relatively e⁄cient fetal im-mune response in ourmodel of infection.The search forpossible persistent infection in this experiment gave ne-gative results, neither lesions nor bacterial DNA beingdetected in the animals maintained until day 120 p.a.This suggests that the speci¢c immune response in-duced by C. burnetii multiplication was su⁄cient toeliminate the infection.In conclusion, C. burnetii-induced abortion in goats

was associated with massive bacterial multiplicationin the trophoblast cells in the weeks immediately pre-ceding abortion. Except for a few di¡erences, the abor-tions displayed many chronological and pathologicalsimilarities to those induced by other intracellularpathogens in ruminants. It is therefore necessary toseek a speci¢c diagnosis in such cases. Immunohisto-chemical techniques, which make use of ¢xedmaterial,are free from risk to the operator; they are not, how-ever, su⁄ciently sensitive to detect antigen in samplesfrom maternal or fetal organs other than placenta.PCR assays would appear to be more sensitive.

Acknowledgments

C.M.Mart|¤ nez was the recipient of a predoctoral grantfromMinisterio Ciencia yTecnolog|¤ a.

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Received, November 7th, 2005

Accepted, June 10th, 2006

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