The intracellular bacterium Waddlia chondrophila, which belongs to the Chlamydiales order, was found to be associated with miscarriage in humans. There is little to no knowledge regarding the mode of infection, impact on the neonate and pathophysiology of this emerging bacterium. We have previously shown that W. chondrophila induces a systemic infection, organ pathology and elicits T helper type 1-associated humoral immunity in a murine model of genital infection. In the present study, we took advantage of this model of infection to evaluate the impact of this bacterium on the mouse pregnancy. We used two routes of inoculation, vaginal and intrauterine, to introduce infection before and after mating. Our results show that genital infection by W. chondrophila did not have any significant impact on gestation length and maternal weight gain, nor on the number of offspring and their weight. This observation indicates that the mouse model of infection is not suitable to study the effect of W. chondrophila on pregnancy and alternative models of infection, including in vitro ones, should be used. Moreover, an indirect immunopathological mechanism activated by this bacterium should be further explored

Ammerdorffer, A.

Baud, D.

Buffe, Y.

Greub, G.

Stojanov, M.

Vouga, M.

English

Serveur académique lausannois

ORIGINAL ARTICLEImpact of Waddlia chondrophila infection on pregnancy in the mouseD. Baud1, A. Ammerdorffer1,2, Y. Buffe1, M. Vouga1, G. Greub3 and M. Stojanov11) Materno-fetal and Obstetrics Research Unit, Department Woman-Mother-Child, Lausanne University Hospital, Switzerland, 2) Department of MedicalMicrobiology and Infection Control, Laboratory of Immunogenetics, Vrije Universiteit Amsterdam, the Netherlands and 3) Institute of Microbiology, LausanneUniversity Hospital and University of Lausanne, SwitzerlandAbstractThe intracellular bacterium Waddlia chondrophila, which belongs to the Chlamydiales order, was found to be associated with miscarriage inhumans. There is little to no knowledge regarding the mode of infection, impact on the neonate and pathophysiology of this emergingbacterium. We have previously shown that W. chondrophila induces a systemic infection, organ pathology and elicits T helper type 1-associated humoral immunity in a murine model of genital infection. In the present study, we took advantage of this model of infection toevaluate the impact of this bacterium on the mouse pregnancy. We used two routes of inoculation, vaginal and intrauterine, to introduceinfection before and after mating. Our results show that genital infection by W. chondrophila did not have any significant impact ongestation length and maternal weight gain, nor on the number of offspring and their weight. This observation indicates that the mousemodel of infection is not suitable to study the effect of W. chondrophila on pregnancy and alternative models of infection, including in vitroones, should be used. Moreover, an indirect immunopathological mechanism activated by this bacterium should be further explored.© 2019 The Author(s). Published by Elsevier Ltd.Keywords: Adverse pregnancy outcomes, chlamydiales, genital model, mouse, Waddlia chondrophilaOriginal Submission: 24 April 2019; Revised Submission: 14 November 2019; Accepted: 18 November 2019Article published online: 23 November 2019Corresponding authors: M. Stojanov, Department of Woman-Mother-Child, Lausanne University Hospital, Switzerland.Corresponding authors: D. Baud, Department of Woman-Mother-Child, Lausanne University Hospital, Switzerland.E-mails: david.baud@chuv.ch (D. Baud), milos.stojanov@chuv.ch (M. Stojanov)IntroductionWaddlia chondrophila is an obligate intracellular bacterium thathas previously been associated with miscarriage in humans andcattle [1,2]. Several studies performed in different geographicalregions showed that women with high antibody titres againstW. chondrophila were at a higher risk of miscarriage comparedwith control women [3,4]. Moreover, W. chondrophila wasidentified by quantitative PCR and immunohistochemistry in thegenital tract and placenta of women with miscarriages [5].Similar findings were identified in cattle [6]. Nevertheless, theThis is an open access artiexact mechanism of action of W. chondrophila that leads to thisincreased rate of miscarriage is still unknown.Wheelhouse et al. have recently tried to imitateW. chondrophila-associated abortion in a bovine model ofinfection using intravenous inoculation of this bacterium intogravid cows [7]. Although the study did not confirm the role ofW. chondrophila as an abortive agent, one animal showedpathogen-associated lesions in the placenta, suggesting atropism towards reproductive organs. To study the role ofW. chondrophila in miscarriages, we have previously developed agenital mouse-model of infection [8]. Intrauterine infection byW. chondrophila was able to induce a systemic infection andorgan pathology, while eliciting a T helper type 1-associatedhumoral immunity, typical of Chlamydia-like bacteria.Infection with W. chondrophila (later leading to persistentinfection) may occur much earlier than conception. Pregnancyis characterized by a relative deficit in T helper type 1 cell-mediated immunity and so may lead to the reactivation of aninduced persistent W. chondrophila infection, cell damage andmiscarriage [9]. An autoimmune response to an epitopeNew Microbe and New Infect 2020; 33: 100619© 2019 The Author(s). Published by Elsevier Ltdcle under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)https://doi.org/10.1016/j.nmni.2019.1006192 New Microbes and New Infections, Volume 33 Number C, --- 2020 NMNI(HSP60; 60-kDa heat-shock protein) shared by a fetal andchlamydial antigen is another hypothesis that may explain howW. chondrophila may be involved in abortion [9]. Finally,persistent infection may induce the secretion of pro-inflammatory cytokines, which have an abortigenic effect. Toinvestigate these hypotheses, an animal model of infection isneeded.With this study, we investigate whether pre- and post-mating infection with W. chondrophila induces adverse preg-nancy outcomes in mice.Materials and methodsWaddlia chondrophila inoculaWaddlia chondrophila was cultured and purified as describedpreviously [10]. In short, Acanthamoeba castellanii were used togrow W. chondrophila ATCC VR-1470 in 75-cm2 culture flasks(Corning, Corning, NY, USA) at 32°C in peptone–yeast–glucose medium. Cell cultures were harvested and filteredthrough a 5-μm filter (Merck Millipore, Darmstadt, Germany).The concentration of the culture was determined by quantita-tive PCR as previously described [11]. For the mock control,the same procedure was performed without W. chondrophila.MiceAll animal experiments were approved by the Office Vétérin-aire du Canton de Vaud, Lausanne, Switzerland (authorizations2090.0 and 2090.1) and performed according to our institu-tional guidelines for animal experiments.Female and male C57BL/6 mice (8–12 weeks old) wereobtained from Charles River Laboratories (L’Arbresle, France)and housed under pathogen-free conditions at the animal facilityof the University of Lausanne (UNIL, Epalinges, Switzerland).Upon mating, mice were housed in single-use filter-top cages,and water and food were provided ad libitum.One week before infection with W. chondrophila, all femalemice were treated subcutaneously 0.1 μg oestradiol (Sigma-Aldrich Chemie, Buchs, Switzerland) and 2 mg medrox-yprogesterone acetate (Depo-Provera, Pfizer, New York, NY,USA) to induce oestrus, during which mice are more suscep-tible to infections [12].Infection before pregnancy: intrauterine infectionprocedureInfection by the intrauterine route was performed as previouslydescribed [8]. Infections were performed in three separate ex-periments, for a total of 22 mice per group (W. chondrophila andmock controls). Infection was performed by injecting 100 μLmock control or W. chondrophila inoculum (~107 bacteria/μL).© 2019 The Author(s). Published by Elsevier Ltd, NMNI, 33, 100619This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/liceTwo weeks after the introduction of the infection, two femaleswere placed in a cage housing a male mouse. This was chosenaccording to our previous experiments, which demonstrated amaximum immune response and organ pathology at 2 weekspost-infection [8]. Vaginal plug assessments were completed on adaily basis and once observed, female mice were housed inseparate cages to avoid cross-contamination. Blood samples (~50μL) were collected from the tail before infection, 2 days beforemating and at days 7 and 14 post-mating.Infection during the pregnancy: vaginal infectionprocedureIn total, six experiments were performed, including 26 mockcontrols and 26 W. chondrophila-infected mice. Mating wascarried out by placing two females with one male per cage.Vaginal plug assessments to identify sexual activity werecompleted daily. One week after the appearance of the vaginalplug, mice were infected vaginally with 20 μL mock orW. chondrophila inoculum (~5 × 107 bacteria/μL) via manualrestraint and infection using a pipette and sterile filter tip. Noanaesthesia was used for this procedure. fter infection, fe-males were housed separately to prevent cross-contamination. Blood samples (~50 μL) were collected fromthe tail before infection, 2 days before mating and at days 7and 14 post-mating.Microimmunofluorescence assayAll blood samples collected before and during the infection, aswell as at euthanasia, were used to isolate plasma usingMicrovette 500 μL Lithium-Heparin (Sarstedt, Numbrecht,Germany). Samples were centrifuged for 5 min at 2000 g andplasma was collected in a separate tube and stored at –20°Cuntil further processing. Microimmunofluorescence was usedto detect anti-W. chondrophila antibodies using a formalin-inactivated (0.3%) W. chondrophila strain ATCC VR-1470 asan antigen. Serum samples were used at dilutions of 1/32 and 1/64 in phosphate-buffered saline supplemented with 1% bovineserum albumin (Serva, Heildeberg, Germany). Goat anti-mouseIgG secondary antibodies conjugated to Alexa Fluor 488 dye(Thermo Fisher Scientific, Allschwil, Switzerland; diluted 1/1000) were used to detect the presence of IgG. Micro-immunofluorescence analyses were evaluated by two inde-pendent readers. Weighted Cohen’s κ coefficient wasdetermined for each experiment to judge the agreement of theresults and always ranked between ‘good’ and ‘very good’.Statistical analysisGRAPHPAD PRISM 7 software (GraphPad Software Inc., San Diego,CA, USA) was used to analyse and present data. The data areexpressed as mean (±standard error of the mean) unlessnses/by-nc-nd/4.0/).NMNI Baud et al. Waddlia chondrophila in murine pregnancy 3indicated otherwise. Differences between experimental groupswere tested using the Mann–Whitney U test, and a p value of<0.05 was considered statistically significant.ResultsVaginal versus intrauterine infection withW. chondrophilaWe have previously used the intrauterine route of infection toevaluate the pathogenicity of W. chondrophila. Although thisroute is useful for pre-mating infection, it is not compatible withinfection during gestation, as it would independently inducemiscarriages. For this reason, pregnant mice were infected viathe vaginal route.Schematic representations of the pre- and post-mating infec-tion procedures are depicted in Fig. 1(a). After co-habitation withmale mice, female mice were evaluated daily for the presence ofmating plugs. In our experimental scenario, the pregnancy rate ofvaginally infected mice was 92.3% (n = 52), with no significantdifference between mock andW. chondrophila-infected mice. Thepregnancy rate was lower for intrauterine-infected mice,reaching 70.5% (n = 44). The observed difference could beexplained by the difference in the invasiveness of the two routesof infection, as the catheter used for the intrauterine inoculationmight cause damage to the reproductive organs. This reasoning issupported by the fact that mice failing to become pregnant wereequally distributed between the mock-infected (n = 6) andW. chondrophila-infected (n = 7) groups.Impact of pre- and post-mating infection withW. chondrophila on pregnancySimilar serological results were seen at delivery for the twodifferent routes of inoculation (Fig. 2(a)). We avoided bloodFIG. 1. Time course of vaginal andintrauterine infection models. Com-parison of the two models ofWaddliachondrophila inoculation before mat-ing (intrauterine infection) and aftermating (vaginal infection). Time-points at which blood samples werecollected are indicated by thearrows.This is an open access articsampling during gestation to minimize the stress on the preg-nant mice. All mock-infected mice had a negative serology.Exposure to W. chondrophila did not affect the duration of thegestation compared with mock controls for both routes ofinfection (Fig. 2(b)). There was a significant difference in lengthof gestation between the two routes of infection, which couldbe again explained by the increased invasiveness of the intra-uterine infection procedure, as mentioned above. The weightgain of the pregnant mice was similar for both mock-infectedand infected mice infected via the intrauterine route(Fig. 2(c)). Moreover, infection by W. chondrophila did not affectthe average number of pups per pregnant mouse (Fig. 2(d)), northe average weight of pups from the same litter (Fig. 2(d)),regardless of the route of infection.DiscussionWaddlia chondrophila has been linked to adverse pregnancyoutcomes in humans and bovines [2,6]. To study the abortiverole of this bacterium, we have previously developed a femalemouse model of genital infection in which intrauterine inocu-lation of the bacterium caused a strong immune response anddisseminated infection [8]. The present study aimed at evalu-ating the impact of W. chondrophila infection on reproductivesuccess using the above-mentioned murine model of infection.We used the intrauterine route of inoculation to study theimpact of infection before mating and the vaginal route to studythe influence of post-mating infection. The latter was used toavoid the potential higher risk of abortion induced by thetechnical aspects of intrauterine inoculation in already pregnantmice. The intrauterine route of infection was used beforemating to increase the number of bacteria reaching the uterinecavity, which we previously demonstrated to induce a© 2019 The Author(s). Published by Elsevier Ltd, NMNI, 33, 100619le under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).FIG. 2. Impact of Waddlia chon-drophila infection on mouse preg-nancy. (a) Serological analysis ofinfected female mice during the study(refer to Fig. 1 for the time-points).(b, c) Impact of W. chondrophila onlength of gestation and maternalweight gain, respectively. (d, e)Impact of W. chondrophila on thenumber of pups and their weight,respectively.4 New Microbes and New Infections, Volume 33 Number C, --- 2020 NMNIsignificant immune response and local, as well as distant pa-thology 2 weeks post-infection [8].Our results indicate that in our murine model of genitalinfection, W. chondrophila does not result in adverse pregnancyoutcomes despite eliciting an immune response. Both routes ofinoculation produced a strong immune response that peakedbetween days 14 and 21 post-infection. The infection did notimpact any of the outcomes presented in the study, includingthe litter size and weight of the pups, duration of the gestationand maternal weight gain.The fact that W. chondrophila was isolated from human andbovine samples, but never in rodents, may indicate that the© 2019 The Author(s). Published by Elsevier Ltd, NMNI, 33, 100619This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licemouse model of infection is not suitable to study the impact ofW. chondrophila on gestation. There are some similarities withChlamydia trachomatis, which also does not induce pathologiesin a mouse model of genital infection. When inoculated vaginallyor into the uterus, the bacterium is quickly cleared by themouse immune system [13]. Conversely, this infection is closelyrelated to Chlamydia muridarum, the murine counterpart ofC. trachomatis, which causes similar obstetric issues asC. trachomatis in humans [14].Recently, a bovine model of infection has been used toaddress this issue [7]. Although the infection did not produce asignificant increase in miscarriage, in one heifer (out of nine),nses/by-nc-nd/4.0/).NMNI Baud et al. Waddlia chondrophila in murine pregnancy 5W. chondrophila was successfully re-isolated from the diseasedplacenta. Although this suggests that there may be a tropismtowards the reproductive tract, it also indicates that the bac-terium is not systematically associated with adverse pregnancyoutcomes.In conclusion, infection with W. chondrophila was not asso-ciated with an increased rate of miscarriage in our mousemodel of genital infection. Given the strong serological link inhumans and the genital tropism of this bacterium, futureresearch should focus more on immunopathological mecha-nisms as direct infection by W. chondrophila might not be themain cause of adverse pregnancy outcomes.Conflicts of interestThe authors have no conflicts of interest to declare.Author contributionsMS, AA and DB conceived the experimental design. AA, YB,MV and MS performed the experiments. MS, AA, YB, MV andDB interpreted the data. MS and DB prepared the figures andwrote the manuscript. GG reviewed the manuscript. All au-thors read and approved the final manuscript.FundingThis work was supported by the Department of Obstetrics andGynaecology, Lausanne University Hospital, Switzerland and bythe Swiss National Science Foundation grant numbers 310030-156169/1, 320030-169853/1 and 320030-169853/2 attributedto David Baud. David Baud is also supported by the FondationLeenaards through the Bourse pour la relève académique, bythe Fondation Divesa and by the Loterie Romande. No conflictsof interest to declare.This is an open access articReferences[1] Ammerdorffer A, Stojanov M, Greub G, Baud D. Chlamydia trachomatisand chlamydia-like bacteria: new enemies of human pregnancies. CurrOpin Infect Dis 2017;30:289–96.[2] Baud D, Greub G. Intracellular bacteria and adverse pregnancy out-comes. Clin Microbiol Infect 2011;17:1312–22.[3] Baud D, Thomas V, Arafa A, Regan L, Greub G.Waddlia chondrophila, apotential agent of human fetal death. Emerg Infect Dis 2007;13:1239.[4] Hornung S, Thuong BC, Gyger J, Kebbi-Beghdadi C, Vasilevsky S,Greub G, et al. Role of Chlamydia trachomatis and emerging Chlamydia-related bacteria in ectopic pregnancy in Vietnam. Epidemiol Infect2015;143:2635–8.[5] Baud D, Goy G, Osterheld M-C, Croxatto A, Borel N, Vial Y, et al.Role of Waddlia chondrophila placental infection in miscarriage. EmergInfect Dis 2014;20:460–4.[6] Dilbeck-Robertson P, McAllister MM, Bradway D, Evermann JF.Results of a new serologic test suggest an association of Waddliachondrophila with bovine abortion. J Vet Diagn Invest 2003;15:568–9.[7] Wheelhouse N, Flockhart A, Aitchison K, Livingstone M, Finlayson J,Flachon V, et al. Experimental challenge of pregnant cattle with theputative abortifacient Waddlia chondrophila. Sci Rep 2016;6.[8] Vasilevsky S, Gyger J, Piersigilli A, Pilloux L, Greub G, Stojanov M, et al.Waddlia chondrophila induces systemic infection, organ pathology, andelicits Th1-associated humoral immunity in a murine model of genitalinfection. Front Cell Infect Microbiol 2015;5:76.[9] Baud D, Regan L, Greub G. Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes. Curr Opin Infect Dis2008;21:70–6.[10] Baud D, Vulliemoz N, Ammerdorffer A, Gyger J, Greub G,Castella V, et al. Waddlia chondrophila, a Chlamydia-related bacterium,has a negative impact on human spermatozoa. Hum Reprod2017;1–8.[11] Goy G, Croxatto A, Posfay-Barbe KM, Gervaix A, Greub G. Devel-opment of a real-time PCR for the specific detection of Waddliachondrophila in clinical samples. Eur J Clin Microbiol Infect Dis 2009;28:1483–6.[12] Kaushic C, Ashkar AA, Reid LA, Rosenthal KL. Progesterone increasessusceptibility and decreases immune responses to genital herpesinfection. J Virol 2003;77:4558–65.[13] Sturdevant GL, Caldwell HD. Innate immunity is sufficient for theclearance of Chlamydia trachomatis from the female mouse genital tract.Pathogens Dis 2014;72:70–3.[14] Gondek DC, Olive AJ, Stary G, Starnbach MN. CD4+ T cells arenecessary and sufficient to confer protection against Chlamydia tra-chomatis infection in the murine upper genital tract. J Immunol2012;189:2441–9.© 2019 The Author(s). Published by Elsevier Ltd, NMNI, 33, 100619le under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Impact of Waddlia chondrophila infection on pregnancy in the mouse.

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Impact of Waddlia chondrophila infection on pregnancy in the mouse.

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