423Vet. Res. 34 (2003) 423–433© INRA, EDP Sciences, 2003DOI: 10.1051/vetres:2003017

Original article

Experimental Coxiella burnetii infection in pregnant goats: excretion routes

Nathalie ARRICAU BOUVERY*, Armel SOURIAU, Patrick LECHOPIER, Annie RODOLAKIS

Pathologie Infectieuse et Immunologie, INRA, Tours-Nouzilly, 37380 Nouzilly, France

(Received 15 October 2002, accepted 21 March 2003)

Abstract – Q fever is a widespread zoonosis caused by Coxiella burnetii. Infected animals,shedding bacteria by different routes, constitute contamination sources for humans and theenvironment. To study Coxiella excretion, pregnant goats were inoculated by the subcutaneousroute in a site localized just in front of the shoulder at 90 days of gestation with 3 doses of bacteria(108, 106 or 104 I.D.). All the goats aborted whatever the dose used. Coxiella were found by PCRand immunofluorescence tests in all placentas and in several organs of at least one fetus per goat.At abortion, all the goats excreted bacteria in vaginal discharges up to 14 days and in milk samplesup to 52 days. A few goats excreted Coxiella in their feces before abortion, and all goats, excretedbacteria in their feces after abortion. Antibody titers against Coxiella increased from 21 days postinoculation to the end of the experiment. For a Q fever diagnostic, detection by PCR andimmunofluorescence tests of Coxiella in parturition products and vaginal secretions at abortionshould be preferred to serological tests.

Coxiella burnetii / goat / experimental infection / abortion / shedding

1. INTRODUCTION

Coxiella burnetii is an obligate intracel-lular bacterium responsible for Q fever, awidespread zoonosis [14]. Infection inman and animals has been worldwidereported [4, 5] with the exception of NewZealand [18]. Human infection could beacute (pneumonia, hepatitis, influenza-likesymptoms and headache), or chronic (endo-carditis, granulomatous hepatitis and abor-tion). In animals, C. burnetii usually causesreproductive disorders (abortion, retained

placenta, endometritis, infertility and lowbirth weight) that are responsible for impor-tant economic losses.

The source of human infection is oftenunidentified, although sheep and goats aremore frequently involved in the diseasecycle than other animal species. The mainroute of C. burnetii infection is by inhala-tion of contaminated aerosols containingthe microorganism shed from infectedanimals [1, 25, 26, 28, 30, 31, 34, 35].After becoming infected, female animalsshed large quantities of Coxiella into the

* Correspondence and reprintsTel.: (33) 2 47 42 76 34; fax: (33) 2 47 42 77 79; e-mail: Nathalie.Bouvery@tours.inra.fr

424 N. Arricau Bouvery et al.

environment during abortion or normaldelivery through birth fluids, placenta andfetal membranes [3, 6, 23, 24, 32]. Moreo-ver, following parturition, these infectedanimals excrete the bacteria via the urine,feces, vaginal discharges and milk for sev-eral months [10, 17, 19, 20]. Nevertheless,the C. burnetii shedding kinetics is not wellknown. Oral transmission is less common,but the consumption of contaminated rawmilk and dairy-products could be a sourceof infection [8, 12, 16]. Thus, most peoplecould be contaminated by C. burnetii, butstaffs working with ruminants appear to bethe most exposed [17]. An improvedknowledge of excretion routes of C. bur-netii by domestic ruminants is requiredto reduce the risk of human and animalcontaminations. Experimental models havealready been performed on sheep or cattle[7, 22, 34]. Some discrepancies have beenobtained between the results that may beexplained by the different route, the infec-tive dose and the C. burnetii strain used.No model is available yet to reproduceQ fever in goats. Moreover, very few datahave been reported about Coxiella excre-tion by naturally infected goats. However,Q fever abortions in goat herds are moreimportant than in sheep flocks since theycould rise up to 90% in females [23], andgoat cheese produced from raw milk iseaten in many countries. So, the setting upof an experimental infection of goats isnecessary to better understand the routesand duration of Coxiella excretion.

2. MATERIALS AND METHODS

2.1. Coxiella burnetii strain

The Coxiella burnetii strain CbC1, usedin the study, was originally isolated fromthe placenta of an aborted goat in a Frenchcaprine herd (Allier France). It was identi-fied as a Coxiella species by phenotypicand genotypic characterization. This strainwas isolated by intraperitoneal inoculationof 3 OF1 mice (8 weeks old) with 0.2 mL

of goat placenta homogenate. The micewere killed nine days post inoculation (p.i.)and their spleens were sampled and reinoc-ulated to specific pathogen free embryo-nated hen eggs. C. burnetii CbC1 in its 3rdpassage in the chicken embryo was aliq-uoted and frozen at –70 °C before use asthe challenge strain (109 infective mousedoses (I.D.)/mL). The infective mousedose was calculated by peritoneal inocula-tion of mice with decimal dilutions ofC. burnetii CbC1 and detection of Coxiellain the spleen by PCR.

2.2. Animals and experimental protocol

The animal experimentation was carriedout over a four-month period in a level 3biosecurity experimental building reservedfor trained staff only working in adequateconditions of protection. Nineteen one-year-old and healthy pregnant goats,obtained from Chlamydia and Coxiellaspecific serologically negative herds withno history of abortion, were randomlyallotted into three experimental groups.They were inoculated by the subcutaneousroute in a site localized just in front ofthe shoulder at 90 days of gestation, with108 (6 goats, 108 group), 106 (6 goats,106 group) or 104 I.D. of the CbC1 strain(7 goats, 104 group). The animals werekept in separate rooms in a security build-ing for about 6 weeks after delivery. Theanimals were observed daily for clinicalsigns. Rectal temperatures were measured,once a day, the day before and during8 days p.i. At the end of the study, the ani-mals were necropsied for a detection ofC. burnetii in different organs.

2.3. Diagnostic tests

2.3.1. Samples

Blood samples were collected for sero-logical tests at the time of inoculation andthen fortnightly. Fecal samples were col-lected directly from the rectum 24 days

Experimental infection of goats with Coxiella burnetii 425

after inoculation and then fortnightly. Pla-cental cotyledons were collected at parturi-tion. The spleen, liver, lung, stomach andperitoneal fluids were collected fromaborted fetuses, still-borns or kids dyingwithin 24 h after birth. Vaginal swabs werecollected with dry sterile cotton swabsfrom each animal after parturition, on the3 subsequent days and then every week.Milk samples were taken at the parturitionday and daily for five days after, and thenevery week. Prescapular, mammary andhead (submaxillary, parotid, retropharyn-geal) lymph nodes, liver, spleen and udderwere collected following necropsy of allinfected animals at the end of the experi-ment. The samples were kept at –80 °C forfurther PCR analysis to detect C. burnetii.

2.3.2. Serological tests

The sera were tested for the presence ofspecific antibodies for C. burnetii with anELISA assay (CHEKIT-Q-Fever enzymeimmuno-assay kit; Bommeli diagnostics,Switzerland) which was carried out as rec-ommended by the manufacturer, usingmicrotiter plates precoated with a mix ofpurified C. burnetii phase I + II antigens.Positive and negative controls wereincluded in each test. The sample OD wereexpressed as a percentage of the positivecontrol OD value which was considered tobe 100%. Goat sera were considered posi-tive if they had an OD percentage of 50%or more, doubtful if the OD percentage wasbetween 40 and 50%, and negative if theOD percentage was less than 40%.

2.3.3. C. burnetii DNA purificationfrom samples

Feces and vaginal swab samples wereprepared as previously described [10].Placental cotyledons, organs and lymphnodes from goats and fetus organs werehomogenized with a Stomacher (SewardMedical, London, UK) in sterile salinebuffer. C. burnetii DNA was extractedfrom 50 µL of stomach content, peritoneal

fluid, organ and lymph node homogenates,or from 100 µL of milk or feces with theQIAamp DNA mini kit (Qiagen), accord-ing to the manufacturer’s instructions.

2.3.4. PCR assay

PCR assays were performed as previ-ously described [9] with 2.5 µL of DNAextract. The PCR reaction was carried outin an automated DNA thermal cycler(UNO Thermobloc; Biometra); PCR prod-ucts (687 bp; 10 µL) were analyzed byelectrophoresis on 1% agarose gel, stainedwith ethidium bromide, visualized underan ultra-violet transilluminator and photo-graphed. When no DNA was amplified,1:10 and 1:100 DNA dilutions were madeand new PCR were performed. If the PCRwas positive at one of these dilutions, thesample was then considered positive.

2.3.5. Immunofluorescence assay

An indirect immunofluorescence (IFA)test was applied for C. burnetii detection insmears of placental cotyledon samples on3-well or 18-well microscope slides (PolyLabo), as previously described [10] usinga specific serum obtained from mice inoc-ulated with the Nine Mile C. burnetiistrain. The slides were examined with a40� objective (400� magnification) undera fluorescence microscope (Leitz Aristop-lan) for brilliant green staining of C. bur-netii microorganisms. The number of C.burnetii was estimated in different micro-scopic fields and scored as either noorganisms (–) or 10 or more C. burnetiispecific views per field (+). Positive Cox-iella and negative controls were includedin each test run.

2.4. Statistical analysis

The results were compared by theKruskal-Wallis test with StatXact 5 CYTELSoftware Corporation.

426 N. Arricau Bouvery et al.

3. RESULTS

3.1. Clinical response after challenge

A rise of rectal temperatures was onlyseen in the 108 group; for the two othergroups, as there was no control group, theirrectal temperatures were not different tothe ones before inoculation, except for twogoats (Fig. 1). This fever began on the3rd day after inoculation for all the goats ofthe 108 group and lasted 3 to 5 days. Anacute localized reaction also developedaround the site of inoculation after 4 daysp.i. for all the goats of this group, exceptfor goat No. 132. Only 2 goats of the106 group presented a rise in rectal temper-ature, which began on the 5th and 7th daysafter challenge and lasted 3 and 1 daysrespectively; these 2 goats also showed anacute localized reaction after 5 days p.i.The rectal temperatures of the 104 groupgoats were never over 39.5 °C until the8th day. No acute localized reaction wasvisible for the 104 group goats.

3.2. Antibody response

According to the threshold of positivitygiven by the manufacturer of the kit, allgoats became seropositive except the abortedgoat No. 132 (108 group) which had adoubtful serology throughout the experi-mentation. There was a seroconversion forall the infected goats which began around

21 days p.i. and gradually increased up tothe end of the experiment (Fig. 2), but it wasbelow the threshold of positivity of the kitthe first weeks after inoculation up to7 weeks for the majority of the goats of thethree groups. According to the threshold ofpositivity of the kit, the first antibodiesagainst phase I + II antigens of C. burnetiiwere detected 42 days after inoculation for5/7 of the 104 group, 3/6 of the 106 groupand 0/6 goats of the 108 group, anddetection of antibodies was only positivefor 3/7 goats (104 groups), 2/6 goats

Figure 1. Mean changes in body tem-perature after the infectious challengeof 6 goats of the 108 group (�), 6 goatsof the 106 group (�) and 7 goats of the104 group (�). Rectal temperatures(°C) were taken the day of pre-inocula-tion (–1), the day of inoculation (0) andthe following days (1 to 8).

Figure 2. ELISA serological responses follow-ing Coxiella burnetii infection. According tothe threshold of positivity given by the markerof the kit, the goats were considered to be posi-tive if they had an OD percentage of 50% ormore. The arrows represent the two abortionperiods. Goat 108 group (�); goat 106 group(�); goat 104 group (�).

Experimental infection of goats with Coxiella burnetii 427

(106 groups) and 1/6 goats (108 groups) atthe day of abortion, but all the aborted goatsbecame ELISA positive between 1 and36 days after parturition (between days 40and 82 p.i.).

3.3. Abortion syndrome and fetal contamination (Tab. I)

One goat (No. 132 of the 108 group)aborted 12 days after the inoculation; allthe other pregnant goats aborted betweendays 25 and 48 p.i., corresponding to days115 and 138 of gestation. Whatever thedose used, none of the kids survived morethan 24 h. There were two abortion out-breaks, the first around 29 days p.i. and thesecond around 43 days p.i., with no signif-icant difference between the duration ofpregnancies between the 3 groups.

The cotyledons of all the aborted goatswere positive for C. burnetii by immun-ofluorescence test and PCR. C. burnetiiwas detected in the majority of the abortedkids whatever the dose used. The most fre-quently contaminated samples were theliver (79% positive) and lungs (77% posi-tive). There was no significant differencebetween the 3 groups.

3.4. Excretion of C. burnetii

3.4.1. Fecal excretion

C. burnetii was detected in the feces ofall aborted goats, whatever the dose used(Fig. 3). The first detection happened25 days p.i. in fecal samples of 6 goats,which excreted bacteria before the abor-tion. Moreover fecal excretion was discon-tinuous for 4/9 goats of the 106 group and5/8 goats of the 104 group; negative resultswere obtained from some samples duringthe period of excretion. The analysis of theresults with the Kruskal-Wallis test did notshow a significant difference between thethree groups. The mean duration of excre-tion (comprising continuous and discontin-uous shedding) was 20 days.

3.4.2. Vaginal excretion

At the time of abortion and the 2 subse-quent days, C. burnetii organisms wereintensively shed in vaginal fluids of allaborted goats except for the goat No. 132of the 108 group which aborted 12 days p.i.(Fig. 4). This excretion decreased withtime and Coxiella was not detected 3 days

Table I. Results of the abortion syndrome of goats inoculated with Coxiella burnetii andbacteriological investigations in placental cotyledons and fetuses by PCR.

Challenge dose

108 106 104

Goatsnumber of goats 6 6 7duration of gestation (days)a 122 ± 13 130 ± 10 124 ± 7number of fetus/goat 1.7 ± 0.8 2.5 ± 0.8 2.6 ± 0.8Placentab (No. positive/total) 6/6 6/6 7/7Fetus (No. positive/total)Peritoneal content 3/7 9/15 14/16Stomach content 3/8 4/13 13/19Spleen 6/9 13/15 11/19Lung 7/9 14/15 12/19Liver 7/9 15/15 12/19Total (% positive) 62 75 67a Mean ± SD.b All placenta smears were also positive by immunofluorescence.

428 N. Arricau Bouvery et al.

after abortion for the 108 group, 10 daysfor the 104 group and 14 days for the106 group. The mean duration of excretionwas 2 days for the 108 group, 8 days for the106 group and 4.5 days for the 104 group.Analysis of these results with the Kruskal-Wallis test showed only significant differ-ence between the 108 group and the106 group (P = 0.0085).

3.4.3. Milk excretion

C. burnetii was always excreted in milkafter abortion and the following days,except for goat No. 132 (Fig. 5). This goatexcreted Coxiella in milk 27 and 34 daysafter abortion. During the first five days,

the number of goats excreting Coxiella inmilk was higher in the 106 and 104 groupsthan the 108 group; later, the number ofgoats which shed Coxiella in milk washigher and this excretion persisted more inthe 106 group than in the 108 and104 groups. Two goats (one of the 108

group and one of the 106 group) shed Cox-iella until the end of the experiment,52 days after their abortion. Four goats,(1 of the 106 group, and 3 of the 104 group)presented a discontinuous excretion. Thereis no statistical difference between the3 groups for the duration of milk excretion.The mean duration of excretion (compris-ing continuous and discontinuous shed-ding) was 24 days.

Figure 3. Assessment by PCR of the shedding of Coxiella burnetii in fecal samples the day ofchallenge and the following days. � positive PCR, � negative PCR.

Figure 4. Assessment by PCR of the shedding of C. burnetii in vaginal swabs the day of abortionand the following days. � positive PCR, � negative PCR.

Experimental infection of goats with Coxiella burnetii 429

3.5. Presence of Coxiella burnetii in organs of goats after necropsy

At the end of the experimentation, C.burnetii was detected in the udder of7 goats (2 of the 108 group including goatNo. 132, 2 of the 106 group and 3 of the104 group), and in the mammary lymphnodes of 1 goat of the 104 group (Tab. II).No Coxiella was detected in the spleen, theliver, and the prescapular or head lymphnodes.

4. DISCUSSION

To study Coxiella excretion in goats,3 doses of Coxiella were used in order toget goats with different clinical signs: onegroup with a majority of abortions andexcretions and another group with onlybacterial excretion. These doses were cho-sen because Brooks et al. [13] inoculatedpregnant ewes at about the 100th day ofgestation with 2.1 �� 105 plaque-formingunits (pfu) of phase I Nine Mile C. bur-netii, and showed that only one ewe fromsix had aborted. The subcutaneous route ofchallenge is artificial and does not repre-sent the normal routes of infection (inhala-tion of contaminated aerosols, and, rarely,oral route or intradermal inoculation byarthropods), but it is less dangerous than anaerosol challenge and it allowed us to get

inoculums under control to have reproduc-ible results. All the subcutaneous inocu-lated goats, with a C. burnetii CbC1 strainaborted whatever the dose used. So, fol-lowing this route of challenge, less viablebacteria of the Coxiella burnetii-CbC1strain were required to induce abortionwhen pregnant goats were inoculated at90 days of gestation. In this experimenta-tion the goats were inoculated earlier thanthose in the experimentation of Brookset al. [13], the counting viable bacteriawere made with ID rather than pfu and thechallenge strain CbC1 was isolated from agoat (same species in this experiment)

Figure 5. Assessment by PCR of the shedding of C. burnetii in milk the day of abortion and thefollowing days. � positive PCR, � negative PCR.

Table II. PCR detection of C. burnetii fromorgans and mammary lymph nodes of the goatafter necropsy.

Challenge dose

108 106 104

Time between abortion and euthanasia (days)a

88 ± 7 83 ± 0 96 ± 0

PCR results (No. positive/total goats)

Udder 2/6 2/6 3/7

Mammary lymph nodes 0/6 0/6 1/7

Spleen 0/6 0/6 0/7

Liver 0/6 0/6 0/7

a Mean ± SD.

430 N. Arricau Bouvery et al.

rather than the Nine Mile strain isolatedfrom a tick; this could explain the differ-ences between the abortion rates. Any doseeffect on the abortion of the goats andplacental and fetal contaminations wasobserved here. The lower dose of Coxiellaburnetii inoculated by this route seems tobe sufficient to colonize placenta andfetuses, and to induce abortions. However,post-inoculation clinical signs showed adose dependant response. Increase in tem-perature associated with an acute localizedreaction was higher for the 108 group com-pared to the 104 group. This could becaused by the antigenic crowd which was10 000 times higher in the 108 group thanin the 104 group. The onset of fever thatoccurred on days 5–7 after inoculation for2–3 days has also been observed afterintra-venous inoculation of pregnant eweswith 107 ID 50 of C. burnetii [22].

One goat, No. 132 of the 108 group wasthe only goat of the experiment whichaborted 12 days after inoculation. Thisearly abortion should be due to the strongtemperature elevation after the inoculationof Coxiella rather than a massive infectionby the bacteria. Indeed this goat presentedthe highest temperature elevation (average41.3 °C for 3 days) among the goats of the108 group and Coxiella were only detectedby PCR in placental cotyledons but not invaginal discharges or in fetal organs. Cox-iella were found by IFA and PCR in theplacenta of all goats and in 95% of thefetuses. Placental and fetal contaminationshave also been reported in naturallyinfected goats [23, 27, 33]. The presence ofCoxiella in the stomach content of fetusesindicates that the bacteria contaminateamniotic liquid; so the placenta, which cancontain up to 109 organisms/g of tissue,and the amniotic liquid are importantsources of contamination of animals andthe environment.

Coxiella were detected only in the udderand mammary lymph nodes when euthana-sia occurred between 34 and 83 days afterabortion. Contamination of the mammary

lymph nodes and udder can cause chronicinfection in goats, which could shed Cox-iella in milk for a long period and perhapsduring successive lactating periods. Here,the goats were not milked after abortion butCoxiella excretion in residual mammarysecretions was still found 52 days later, atthe end of the observation. These goatswould perhaps shed bacteria during severalmonths after this last sample. Indeed insome naturally infected cows, animalscould continue to shed the Coxiella in theirmilk during successive lactating periods[12]. During this experimentation, 6 nonpregnant goats were inoculated with theCoxiella CbC1 strain (data not shown). Theday of euthanasia at 82 days p.i., C. burnetiiwas found in the liver of a goat inoculatedwith 108 I.D. As it has been shown in themouse, in which Coxiella were detected inthe liver 2 years after inoculation [29], Cox-iella was able to colonize the liver and couldinduce chronic infection in the goat. Thepresence of Coxiella in the liver could beresponsible for fecal shedding by goats.Fecal excretion was displayed in pregnantand non-pregnant goats, before and afterabortion, so it appears to be an importantroute of contamination of the environmentby the spread of contaminated dung on thefields.

After abortion, all goats excreted Cox-iella either in their vaginal discharges ormilk and feces. Such excretion wasobserved on ewes and dairy cows in natu-rally infected flocks [10, 11, 21] but excre-tion only in colostrums was observed inewes inoculated with the Nine Mile strainof Coxiella [13]. There was no relationbetween the moment of abortion and theduration of excretion, but the goats, whichshed bacteria in vaginal discharges for thelongest period (6 to 14 days), also shedbacteria in mammary secretions for a longtime, between 24 and 52 days after abor-tion, but fecal excretion was very differentbetween the goats. Coxiella excretion inmilk and feces was discontinuous for sev-eral goats. In fact the number of Coxiellawas low in some samples of these goats

Experimental infection of goats with Coxiella burnetii 431

and the negative responses could be due tothe limit of detection by PCR (103 bacteria/mL). Discontinuous excretion has alsobeen reported for naturally infected cowswhere Coxiella were detected in the milkby induction of specific antibody produc-tion in mice or guinea pigs, following i.p.inoculation of milk samples [15]. No sta-tistical differences of fecal and milk shed-ding were observed between the threegroups and important differences wereobserved between goats among a samegroup. This lack of differences between thegroups could be explained by the high sen-sibility of the goat to the Coxiella infectionthus hiding the dose effect. In contrast, thevaginal excretion duration was shorter forthe goats of the 108 group than for the106 group. With regards to all the otherresults (duration of gestation, milk andfecal shedding…) this observation is diffi-cult to explain but these results should beconfirmed by further experiments withmore animals per group and a study of thisexcretion in relationship with the localimmune response.

Antibody responses of goats from the108 group began after those of the 2 othergroups and then were comparable to thoseof the goats from the other groups. We donot know if the high antigenic mass cap-tured the majority of the antibodies or if thehigh temperature elevation significantlyreduced the number of Coxiella in theorganism, and so delayed the immunologi-cal response. According to the threshold ofpositivity given by the manufacturer of thekit, 67% of the goats had a negative ELISAtest when they aborted and excreted Cox-iella. These results are in accordance withthe results of a study by Berri et al. [11],who carried out the relationships betweenthe shedding of Coxiella burnetii and sero-logical responses of 34 sheep and reportedthat the ELISA failed to detect half of theewes shedding C. burnetii through the vag-inal tract. Other studies have also shownthat seronegative cows could shed C. bur-netii in their milk [2, 12]. However, in ourstudy, the rate of antibody against Coxiella

increased from 21 days post inoculation tothe end of the experiment, but the valueswere under the threshold of positivity ofthe kit the first weeks p.i.. Further experi-ments could be made to adjust the thresh-old of positivity of this test. At 80 days p.i.,91% of inoculated goats had antibodiesagainst Coxiella, so it appears that serolog-ical tests can be used for the diagnosis ofinfected flocks. However, this test with theactual threshold of positivity cannot beused to identify the individual shedder thatcould represent a risk for public health.Indeed, Coxiella are very resistant to adrastic environment and infectious parti-cles can be found in cheese made with rawmilk or in dung and compost containingcontaminated feces. Detection of Coxiellain feces or milk by PCR, for example,appears to give more accurate results andsuch a diagnostic technique should limitthese risks of contamination. When anabortion arises in a flock, detection of Cox-iella in the placenta, the fetuses’ organsand vaginal secretions within 2 days afterparturition by PCR and immunofluores-cence tests should be privileged. Two sero-logical tests can be made: one on the day ofabortion and the other 3 weeks later toobserve an elevation of antibodies againstCoxiella.

This experimental infection shows thatwe can induce abortions and bacterialshedding due to Q fever in a goat model.Moreover, the 104 Coxiella I.D. and thePCR technique can be used for the study ofvaccines against Q fever or antibiotic treat-ments and that even the rate of abortion ishigher than that observed in natural flocks.

ACKNOWLEDGEMENTS

The authors are grateful to P. Bernardet,R. Delaunay, D. Gauthier and D. Musset forexcellent technical assistance in the mainte-nance of the animals, and Agnès Moutoussamyand Karine Ladenise for laboratory technicalassistance. We also appreciate the assistancerendered by Serge Bernard for statistical

432 N. Arricau Bouvery et al.

analysis. We wish to thank Laurence Guilloteauand Patricia Berthon for her help in writingthe manuscript. This work was supported byDGAL (grant S98/34) and INRA.

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