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Can Chlamydia abortus be transmitted by embryo transfer in goats?
Theriogenology 86 (2016) 1482–1488
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Can Chlamydia abortus be transmitted by embryo transfer in goats? M. Oseikria a, J.L. Pellerin a, A. Rodolakis b, F. Vorimore c, K. Laroucau c, J.F. Bruyas a, C. Roux a, S. Michaud a, M. Larrat a, F. Fieni a, * a
LUNAM University, Oniris, Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering, Sanitary Security of Reproduction Biotechnology Unit, Nantes, France INRA, Animal Infectious Diseases and Public Health, Tours, France c ANSES, Animal Health Laboratory, Bacterial Zoonosis Unit, Maisons-Alfort, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2015 Received in revised form 10 May 2016 Accepted 11 May 2016
The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8–16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 107 Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at 20 C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: C. abortus Embryo transfer IETS Zona pellucida Caprine embryo PCR
1. Introduction
* Corresponding author. Tel.: þ33 2 40687711; fax: þ33 2 40687748. E-mail address: francis.fi[email protected] (F. Fieni). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.05.006
The classification of Chlamydiaceae has recently changed; there is now only one genus, Chlamydia, in the Chlamydiaceae family. Chlamydia abortus is one of 11 species in this genus [1].
M. Oseikria et al. / Theriogenology 86 (2016) 1482–1488
C. abortus is a gram-negative bacterium, which grows in the cytoplasm of eukaryotic cells; its unique life cycle includes a resistant infectious form, the elementary bodies (EBs), which alternate with a metabolically active noninfectious form, the reticulate bodies (RBs). The EB attach to the membrane of the host cell and promote endocytosis in a membrane limited vacuole called the inclusion, which does not fuse with lysosomes. The EB then transform into RB, which replicate by binary fission. After several divisions, the inclusion is filled with RB, which then transform back into infectious EB. These EB are released through host cell lysis or extrusion of the inclusion out of the host cell [2]. It is the causal agent of enzootic abortion of ewes [3] and is the most common infectious cause of abortion in many small ruminant-rearing countries [4,5], with the exception of Australia and New Zealand [6]. In addition to significant economic losses [7,8], C. abortus presents a zoonotic risk; exposure of pregnant women to infected sheep can lead to severe septicemia in the mother resulting in spontaneous abortion or stillbirth [9,10]. In sheep and goats, C. abortus infection typically causes abortion during the last two or 3 weeks of gestation or the birth of stillborn or weak lambs that die in the first days of life [8], although infected goats may abort at any time during pregnancy [11–13]. Other than reproductive failure, sheep and goats rarely display clinical signs of C. abortus infection, other than vulval discharge 2–3 days before abortion. In some cases, goats may shed C. abortus in vaginal fluids for up to 2 weeks before and after abortion; other signs, such as respiratory tract disease, polyarthritis, conjunctivitis, and retained placentas have been reported [11]. C. abortus can induce a persistent, subclinical infection in nonpregnant sheep and goats [3,14]. After abortion, ewes are considered to be immune to further lamb loss [3,15,16]. However, ewes can be chronically or persistently infected, continuing to shed organisms at estrus or at subsequent lambings [16–19]. It has also been reported that some lambs can be born healthy and survive infection, although they may go on to abort during their first pregnancy [15,20–22]. Sexual transmission of C. abortus is possible [9]; the bacteria have been found in fresh and cryopreserved semen, preputial washing fluid, in the male genital tracts of rams and bucks [23–26], and in the vaginal mucosa in sheep [17,27]. Experimental studies have shown that C. abortus can be excreted in semen of inoculated rams [28] and transmitted by experimentally infected semen to ewes [29]. Male fetuses can be contaminated in utero and adult males by mating with infected females [30]. These results reveal the main source of in utero infection and indicate a risk factor for the transmission of C. abortus during embryo transfer (ET). Embryo transfer is used, nationally and internationally, for the introduction, improvement, and preservation of livestock genetics. Embryos are generally considered to present a lower risk of infectious disease transmission than live animals, on the basis of the results of extensive research using zona pellucida (ZP)-intact, in vivo-produced,
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embryos [31]. To reduce the risk of pathogen transmission, the International Embryo Transfer Society (IETS) has established guidelines for bovine ET. Specific conditions, including donor sanitary status, rejection of embryos without an intact ZP, specific washing procedures (minimum of 10 washes in the culture medium so that each wash represents an 100-fold dilution of the previous wash, the use of a new sterile micropipette for each wash) are required to avoid pathogen transmission by ET [32]. In addition, treatment of the ZP with several enzymes, such as trypsin, has been shown to be effective for removal or inactivation of certain pathogenic agents [32], with no deleterious effects on embryonic development [33,34]. However, these protocols must be tested for each pathogen and for embryos from each different species [32]. To our knowledge, there are no reported studies into the interaction of caprine or bovine embryos and C. abortus. To investigate the risk of C. abortus transmission via caprine ET, our study aimed to determine whether C. abortus adheres to or infects ZP-intact early caprine embryos in vivo after in vitro infection. We also evaluated the efficacy of the IETS washing procedure recommended to decontaminate bovine embryos exposed to C. abortus in vitro. 2. Materials and methods 2.1. Production of embryos 2.1.1. Goats Fourteen healthy 3- to 6-year-old Saanen or Alpine goats from flocks in the Deux-Sèvres region of France were used as embryo donors. These goats were certified C. abortus free using ELISA for blood serum and conventional polymerase chain reaction (C-PCR) for vaginal swabs. 2.1.2. Synchronization and superovulation The donor goats were synchronized by inserting intravaginal sponges impregnated with 45 mg of fluorogestone acetate (Chronogest, Intervet, Angers, France) for 11 days, combined with the intramuscular injection of 125 mg of prostaglandin analog (Estrumate, Shering-Plough Veterinaire, France) 48 hours before sponge removal (Day 9). Superovulation was induced by intramuscular injection of porcine FSH (pFSH; Merial, University of Liege, Belgium) given twice daily at 12-hour intervals for 3 days (Days 9, 10, and 11). The total dose of pFSH was 16 Armor units per goat, administered in decreasing amounts (4–4, 2–2, and 2–2). A 132-mg dose of porcine LH was added to the pFSH preparation on the last two injections (66 mg per injection) [35]. 2.1.3. Fertilization The donor goats were mated by 3- to 6-year-old Saanen or Alpine bucks from the Deux-Sèvres region of France, 24–36 hours after sponge removal. These bucks of proven fertility were certified C abortus negative, by ELISA and PCR and regularly controlled. 2.1.4. Collection of embryos Embryos were collected by laparotomy 4 to 5 days after the onset of estrus. The goats were anesthetized with 5 mL of Zoletil 100 (Virbac, Nice, France) and were prepared for
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aseptic surgery. The uterus and ovaries were exposed via ventral midline laparotomy. The collection technique for collecting goat embryos was described by Lamara et al. [36]. In short, 40 mL of collection medium (EURO FLUSH, IMV, Aigle, France) were used to flush the contents of the oviducts into the uterine horns, where the embryos were recovered into a sterile bottle via a Foley catheter introduced at the greater curvature just above the bifurcation of the horn. Surgical closure was routine. The embryos were recovered from the collection medium and evaluated, and only those with an intact ZP were used. The collection media were centrifuged for 1 hour at 13,000 g and the pellets frozen at 20 C for PCR analysis. 2.2. Chlamydia abortus strain The C. abortus strain (AB7) used in this study was isolated from the placenta of an aborted sheep. The strain was grown on vitelline membranes from specific pathogenfree–embryonated chicken eggs. The bacterial load of the Chlamydia suspension used for the experiment was estimated at 108 bacteria/mL by immunofluorescence, aliquoted, and frozen at 80 C. To ensure purity, each aliquot used for in vitro contamination was diluted with 10-mL phosphate buffer saline then centrifuged twice for 15 minutes at 2000 g. The supernatant was recovered and centrifuged for 1 hour at 13,000 g. The pellet was diluted 1:10 in the exposure medium giving a final calculated concentration of 107 bacteria/mL. 2.3. Experimental design To study the susceptibility of early ZP-intact goat embryos produced in vivo against C. abortus infection in vitro, 52 ZP-intact embryos (8–16 cells), obtained from 14 donors for the three repetitions (4, 5, five goats per repetition respectively), were used in this experiment. The embryos were randomly divided into two groups (infected embryos and control noninfected embryos). The first group comprised 45 ZP-intact embryos (5, 15, and 25 in the first, second, and third repetitions, respectively) and was divided into nine batches of five embryos. These embryos were placed in 1-mL minimum essential medium (M2414, Sigma) supplemented with 10% fetal calf serum, 1% L-glutamine (2-mM final), 1% HEPES (0.01 M final), 2.5-mg/mL amphotericin B, with 4 107 C. abortus per milliliter of AB7 strain. After incubation for 18 hours at 37 C in an atmosphere of 5% CO2, the embryos were recovered. Embryo development and the integrity of the ZP were confirmed. The embryos were then washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution, according to the IETS guidelines [31]. A new sterile pipette was used for each successive wash; each wash corresponded to a dilution of 1:100 of the previous medium. In parallel, the second group of remaining (seven ZP-intact) embryos (2, 2, and 3 in the first, second, and third repetitions, respectively) were used as controls by being
subjected to similar procedures but without exposure to C. abortus. The embryo collection mediums of each donor goat and the 10 wash baths for each batch of embryos were collected separately and centrifuged for 1 hour at 13,000 g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at 20 C before examination for evidence of C. abortus using PCR. 2.4. Conventional PCR After thawing, DNA was extracted from the batches of embryos and the wash bath pellets using a “DNA Mini kit Qiagen-France” in accordance with the manufacturer’s instructions; samples were then stored at 20 C, awaiting PCR analysis. The detection of Chlamydia DNA was performed by amplifying a DNA fragment of 82 bp from the ompA gene (outer membrane protein), a single copy in the C. abortus genome, using the following primers: forward, CpaOMP1-F 50 -GCA ACT GAC ACT AAG TCG GCT ACA-30 and reverse, CpaOMP1-R 50 -ACA AGC ATG TTC AAT CGA TAA GAG A-30 , [37] (Eurofins MWG Operon, Ebersberg, Germany). For each sample, 10 mL of extracted DNA were added to 20 mL of amplification solution (mix-PCR) containing 6 mL of reaction buffer 5X Hot FIREPol Blend Master Mix Ready To Load with BSA and 7.5 MgCl2 (Solis BioDyne, Tartu, Estonia), 0.9 mL of each primer CpaOMP1-F and CpaOMP1-R (20 mM, Eurofins MWG Operon, Ebersberg, Germany), and 12.2 mL of diethylpyrocarbonate-treated water. Amplification was conducted in a thermal cycler (Mastercycler Eppendorf). After initial denaturation at 95 C for 15 minutes, the samples were subjected to a series of 45 cycles comprising: 30 seconds denaturation at 95 C, 1-minute hybridization at 60 C, and a 2-minutes elongation phase at 72 C. This was followed by a final elongation phase at 72 C for 10 minutes. Products were visualized by electrophoresis: 10 mL for each well of 2% agarose gel containing 50-mL ethidium bromide at 1 mg/mL in 1X TBE buffer (Tris-Borate-EDTA, SIGMA, Saint Quentin Fallavier, France). Two controls were performed: a positive control of C. abortus strain (AB7 11091) and a negative control (distilled water). Five microliters of smart-ladder (Solis BioDyne, Tartu, Estonia) were used as a molecular weight marker. This marker comprises 12 bands calibrated between 3000 and 100 bp. After 15 minutes of electrophoresis at 80 V and then 45 minutes at 110 V, the bands were visualized using transillumination with ultraviolet light (312 nm). Samples analyzed for Chlamydia DNA using PCR were considered positive when a band of 82 bp, corresponding to the positive control, was visualized on agarose gel electrophoresis under UV light. The sensitivity of this PCR method has been confirmed in our laboratory (SSBR, Oniris, France); it detects up to 20 bacteria per mL of bacterial suspension (Table 1). 2.5. Real-time PCR Real-time PCR was used to amplify a DNA fragment of 82 bp from the ompA gene, a single copy in the C. abortus genome, as described previously [37,38]. The detection threshold was determined using decimal serial dilutions of
M. Oseikria et al. / Theriogenology 86 (2016) 1482–1488 Table 1 Verification of the sensitivity of conventional PCR to detect, for four repetitions, Chlamydia DNA in samples of Chlamydia abortus strain (AB7) at dilutions from one bacterium/mL to 50 bacteria/mL. Repetition
1
10
20
30
40
50
Negative control
1 2 3 4
þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
purified genomic DNA from C. abortus. DNase-RNase free water was used as a negative control. In short, the final 20 mL reaction mixture included 2 mL of sample DNA, 10 mL of 2 TaqMan universal master mix (Applied Biosystems, USA), 1 mL of a mixture of forward and reverse primers (0.6 mM, EurofinsMWG Operon, Ebersberg, Germany), and 2 mL of TaqMan probe (0.1 mM, Eurofins MWG Operon). Water was added to make up to a final volume of 20 mL. All real-time PCRs were performed in duplicate in an ABIPRISM Sequence Detection System 7300 (Applied Biosystems). The thermal cycling conditions were 50 C for 2 minutes and an initial cycle of heating at 95 C for 10 minutes (single denaturation step), followed by 45 cycles at 95 C for 15 seconds, and 60 C for 1 minute (annealing and extension). The cycle threshold value was calculated automatically. Data were analyzed with the corresponding software. The C. abortus titers in the samples were calculated in comparison with a standard curve obtained from a standard serial dilution of a titrated DNA. 3. Results After incubation for 18 hours with 4 107 C. abortus per milliliter for 18 hours, embryo development was confirmed as an increased number of embryos cells, and the ZP of all embryos were intact. Chlamydia DNA was detected by conventional PCR (Fig. 1) in all batches of five infected ZP-intact 8- to 16-cell
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embryos (9/9) after being washed 10 times (Table 2). Chlamydia DNA was also detected in all 10 wash baths for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively (Table 2). However, bacterial DNA was not detected in any of the 14 embryo collection mediums as in any of the embryos or wash baths from the control groups. After 10 wash cycles, all of the exposure baths and batches of embryos were tested using real-time PCR to quantify the final bacterial load (Fig. 2). The bacterial load in the exposure baths ranged from 2.2 to 5.0 107 bacteria/ mL. The bacterial load for embryos after the 10 wash baths ranged from 0.9 to 4.5 103 bacteria/mL (Table 3). 4. Discussion To confirm our results, we did three repetitions of the experiment. For each repetition, we had one control group of embryos without exposure to C. abortus to ensure there was no external (other than the experimental infection) contamination of the embryos. A strain of C. abortus strain (AB7) isolated from the placenta of an aborted sheep was used in this study. The pathogenic potential of Chlamydia is unlikely to be dependent on the species from which the strain was isolated. AB7 strains are a well-known strain for which in vitro production and biomolecular detection is well codified. AB7 strains colonized the placenta and fetus more strongly than POS, AB16, and LLG strains. Inoculation of the AB7 strain induced very high levels of IFN-g in nonpregnant mice [39]. The type of bacterial strain can also interfere in the connection between the ZP and the pathogenic agent, and it has recently been shown that certain strains of bovine viral diarrhea virus adhere more easily to the ZP of in vitro-produced bovine embryos [40]. The embryo is wrapped in a protective layer (ZP) until the blastocyst stage. The ZP is composed of three different glycoproteins, which create a mechanical barrier against viruses and bacteria [41–43]; the porosity of the matrix is apparently too small, allowing only the smallest of molecules to infiltrate the embryo from the external medium
Table 2 Detection of Chlamydia abortus in successive embryo washes and batches of five infected zona pellucida-intact 8- to 16-cell embryosa after 10 wash baths using conventional PCR.
Fig. 1. Example of conventional PCR amplification of Chlamydia-DNA. DNA from embryos or wash bath pellet was used to perform nested-PCR using specific sets of oligonucleotide primers to amplify 82 bp from the ompA gene. Following nested-PCR reactions, each PCR product was separated on 1.5% agarose gel and the bands visualized by staining with ethidium bromide. M ¼ Smart Ladder used as a molecular weight standard. Lanes 1–5: embryo samples (positive), lanes 6–10: wash bath pellet (lanes 6–8 and 10, positive; lane 9 negative). Cþ: positive tissue control (infected cell culture). C: negative control (distilled water). PCR, polymerase chain reaction.
Batches number
Last wash positive for C. abortus
Batches of embryos
1 2 3 4 5 6 7 8 9
9 10 10 10 10 10 8 10 10
þ þ þ þ þ þ þ þ þ
Abbreviation: PCR, polymerase chain reaction. a Exposed to 107 Chlamydia abortus for 18 h.
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Fig. 2. Typical real-time PCR results of Chlamydia-infected samples with our Taqman assay. (A) Ampliplots of the standard curve generated using serial 10-fold dilutions of the Chlamydia reference solution (see Materials and methods); a to e: duplicates obtained with bacterial amount ranging from 2 107 to 2 103 per mL; f: nontemplate control. (B) Standard curve corresponding to Fig. 2A data. The slope is 3.49 (near the theoretical 3.32 value) and the satisfactory R2 is 0.999. (C) Typical duplicate ampliplots generated using either the initial contaminating Chlamydia solution (a) or a 10th washing embryo solution postinfection (b); (c): nontemplate control solution. PCR, polymerase chain reaction.
[44]. Most of the pathogenic agents investigated to date are unable to penetrate the ZP of in vitro-produced embryos, with the exception of Leptospira [45,46]. Lamara et al. [36], reported that an intact ZP is a strong barrier that protects the caprine embryo from caprine arthritis encephalitis
Table 3 Quantification of Chlamydia abortus in embryo exposure baths and in batches of five infected zona pellucida-intact 8- to 16-cell embryos using real-time PCR (in bacteria/mL). Batches number
Exposure bath
1 2 3 4 5 6 7 8 9
2.2 3.5 3.5 3.5 5.0 5.0 5.0 5.0 5.0
107 107 107 107 107 107 107 107 107
Abbreviation: PCR, polymerase chain reaction.
Batches of embryos 0.9 1.6 2.9 4.5 3.4 2.5 2.3 2.4 1.3
103 103 103 103 103 103 103 103 103
virus infection. The ZP, especially of in vivo-derived embryos, acts as a barrier for most diseases (enzootic bovine leucosis, foot and mouth disease, blue tongue virus, Brucella abortus) [47]. Because C. abortus-DNA was detected in all batches of ZP-intact–washed embryos, this study clearly demonstrates the ability of C. abortus to adhere to or penetrate ZP-intact embryos. This proves that there is a risk of C. abortus transmission after ET. Furthermore, the routine procedures proposed by the IETS are not effective for removing the bacterium from ZP-intact caprine embryos infected in vitro. This result supports previous studies in goats, reporting the binding of Coxiella burnetii to ZP-intact embryos and the inefficacy of routine IETS procedures for removing it from ZP-intact caprine embryos collected in vivo or produced in vitro after infection in vitro [48,49]. In a previous study, with bovine embryos exposed in vitro to Mycoplasma bovis, Mycoplasma bovigenitalium, Mycobacterium avium, after washing, bacteria were isolated from all batches of ZP-intact embryos [50,51]. Washing procedures and antibiotic treatment for the removal of bacterial agents that include Streptococcus agalactiae, Actinomyces pyogenes, Escherichia coli [52], M. avium ssp. paratuberculosis [51],
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Campylobacter fetus [53], and Mycoplasmas [54], from in vivo-derived bovine embryos have not been completely effective. Also, the treatment of in vivo-derived bovine embryos with trypsin in addition to the standard IETS washing was not fully effective for Mycoplasmas [54], and E. coli [52]. Embryos were experimentally exposed to 4 107 C. abortus per milliliter for 18 hours. This high concentration of inoculums with an 18-hour exposure period was chosen to maximize the chances of the bacteria coming into contact with the ZP. It should be noted that under natural conditions, the highest concentration of C. abortus was observed after abortion where they were detected in the fetal membranes and in the cotyledon at a concentration of about 107 Chlamydia per milligram of tissue [55,56]. Such bacterial levels found at abortion are associated with inflammation, which reduces the survival of the oocytes and/or embryos in the female genital tract. However, it appears that the adhesion of the bacteria to the ZP is dose related. In our study, the in vitro experimental conditions of infection are considerably different to natural infection conditions observed in the field. Real-time PCR analysis of embryos after washing identified an average of 2.4 103 Chlamydial/mL, i.e. (0.9–4.5 103 Chlamydia/mL), these concentrations are consistent with the experimental dose required to either elicit abortion [29] or the birth of weak lambs [27], after vaginal inoculation with C. abortus, or latent infection resulting in abortion at a pregnancy after intranasal administration of nonpregnant sheep with a low dose of C. abortus (5 103 inclusion forming units/mL) [13]. Similar conclusions were drawn from early research conducted by McEwen et al., who noted that the infectivity and pathological effects of a small dose was equal to or greater than the effects of a large dose of C. abortus when administered before mating [57]. This positive result does not necessarily mean that transferred in vivo-collected embryos will always transmit the infection. Real-time PCR used alone, without cell-culture assay, does not differentiate between DNA from “live” and “dead” C. abortus, so a positive result does not prove that the embryo carries an infectious pathogen. However, a negative result should indicate that embryos are free of pathogenic agents [58]. Finally, the interpretation of results obtained for C. abortus with in vivoderived caprine embryos recovered from synchronized and superovulated donor goats should not be extrapolated to another species or to in vitro-produced goat embryos, because of physiological and morphological differences between such embryos [59,60]. Our results report that C. abortus adheres to and/or penetrates the ZP of in vivo-derived caprine embryos after in vitro infection, and that the IETS washing protocol for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential mean of transmission of the bacterium during ET from infected donor goat to healthy recipients and/or their offspring. Nevertheless, the positive results of this in vitro study should be considered as worst case scenarios and should not be extrapolated to field situations. Further work is needed (i) to determine the location of the bacterium after in vitro infection and washing, (ii) to
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investigate whether the treatment of Chlamydia-infected embryos with trypsin or antibiotic in addition to the standard IETS washing would eliminate the bacterium from the ZP, and (iii) to assess the risks associated with ET transmission under normal circumstances (in vivo-in vivo experiment). References [1] Sachse K, Laroucau K, Riege K, Wehner S, Dilcher M, Creasy HH, et al. Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst Appl Microbiol 2014;37: 79–88. [2] Moulder JW. Interaction of Chlamydiae and host cells in vitro. Microbiol Rev 1991;55:143–90. [3] Aitken ID, Longbottom D. Chlamydial abortion. In: Diseases of sheep. Oxford: Blackwell Publishing Ltd; 2008. p. 105–12. [4] Aitken ID. Chlamydial abortion. In: Martin WB, Aitken ID, editors. Diseases of sheep. 3rd ed. Edinburgh: Blackwell; 2000. p. 81–6. [5] Longbottom D, Livingstone M. Vaccination against Chlamydial infections of man and animals. Vet J 2006;171:263–75. [6] Rodolakis A, Laroucau K. Chlamydiaceae and Chlamydial infections in sheep or goats. Vet Microbiol 2015;181:107–18. [7] Jimenez-Estrada JM, Escobedo-Guerra MR, Arteaga-Troncosa G, Lopez-Hurtado M, Haro-Cruz MJ, Oca-Jimenez RM, et al. Detection of Chlamydophila abortus in sheep (Ovis aries) in Mexico. Am J Anim Vet Sci 2008;3:91–5. [8] Rodolakis A. Chlamydiose et Fièvre Q, similitudes et différences entre ces deux zoonoses. Renc Rech Ruminants 2006;13:395–402. [9] Longbottom D, Coulter LJ. Animal chlamydioses and zoonotic implications. J Comp Pathol 2003;128:217–44. [10] Baud D, Regan L, Greub G. Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes. Curr Opin Infect Dis 2008;21:70–6. [11] Rodolakis A, Boullet C, Souriau A. Chlamydia psittaci experimental abortion in goats. Am J Vet Res 1984;45:2086–9. [12] Matthews J. Abortion. In: Diseases of the goat. Oxford: Blackwell Science; 1999. p. 22–36. [13] Longbottom D, Livingstone M, Maley S, van der Zon A, Rocchi M, Wilson K, et al. Intranasal infection with Chlamydia abortus induces dose-dependent latency and abortion in sheep. PLoS One 2013;8: e57950. [14] Rocchi MS, Wattegedera S, Meridiani I, Entrican G. Protective adaptive immunity to Chlamydophila abortus infection and control of ovine enzootic abortion (OEA). Vet Microbiol 2009;135:112–21. [15] Rodolakis A, Salinas J, Papp J. Recent advances on ovine chlamydial abortion. Vet Res 1998;29:275–88. [16] Livingstone M, Wheelhouse N, Maley SW, Longbottom D. Molecular detection of Chlamydophila abortus in post-abortion sheep at oestrus and subsequent lambing. Vet Microbiol 2009;135:134–41. [17] Papp JR, Shewen PE. Localization of chronic Chlamydia psittaci infection in the reproductive tract of sheep. J Infect Dis 1996;174: 1296–302. [18] Wilsmore AJ, Izzard KA, Wilsmore BC, Dagnall GJ. Breeding performance of sheep infected with Chlamydia psittaci (ovis) during their preceding pregnancy. Vet Rec 1990;126:40–1. [19] Papp JR, Shewen PE, Gartley CJ. Abortion and subsequent excretion of chlamydiae from the reproductive tract of sheep during estrus. Infect Immun 1994;62:3786–92. [20] Papp JR, Shewen PE. Chlamydia psittaci infection in sheep: a paradigm for human reproductive tract infection. J Reprod Immunol 1997;34:185–202. [21] Entrican G, Buxton D, Longbottom D. Chlamydial infection in sheep: immune control versus fetal pathology. J R Soc Med 2001;94:273–7. [22] Philips HL, Clarkson MJ. Investigation of pre-natal Chlamydophila abortus (Chlamydia psittaci) exposure of female lambs and the outcome of their first pregnancy. Vet J 2002;163:329–30. [23] Amin AS. Comparison of polymerase chain reaction and cell culture for the detection of Chlamydophila species in the semen of bulls, buffalo-bulls, and rams. Vet J 2003;166:86–92. [24] Gautam R, Purohit VD. Isolation of chlamydia psittaci from genitalia of healthy exotic and crossbred service rams. Indian J Anim Sci 2001;71. [25] Kauffold J, Henning K, Bachmann R, Hotzel H, Melzer F. The prevalence of chlamydiae of bulls from six bull studs in Germany. Anim Reprod Sci 2007;102:111–21.
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Can Chlamydia abortus be transmitted by embryo transfer in goats? M. Oseikria a, J.L. Pellerin a, A. Rodolakis b, F. Vorimore c, K. Laroucau c, J.F. Bruyas a, C. Roux a, S. Michaud a, M. Larrat a, F. Fieni a, * a
LUNAM University, Oniris, Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering, Sanitary Security of Reproduction Biotechnology Unit, Nantes, France INRA, Animal Infectious Diseases and Public Health, Tours, France c ANSES, Animal Health Laboratory, Bacterial Zoonosis Unit, Maisons-Alfort, France b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2015 Received in revised form 10 May 2016 Accepted 11 May 2016
The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8–16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 107 Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at 20 C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: C. abortus Embryo transfer IETS Zona pellucida Caprine embryo PCR
1. Introduction
* Corresponding author. Tel.: þ33 2 40687711; fax: þ33 2 40687748. E-mail address: francis.fi[email protected] (F. Fieni). 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.05.006
The classification of Chlamydiaceae has recently changed; there is now only one genus, Chlamydia, in the Chlamydiaceae family. Chlamydia abortus is one of 11 species in this genus [1].
M. Oseikria et al. / Theriogenology 86 (2016) 1482–1488
C. abortus is a gram-negative bacterium, which grows in the cytoplasm of eukaryotic cells; its unique life cycle includes a resistant infectious form, the elementary bodies (EBs), which alternate with a metabolically active noninfectious form, the reticulate bodies (RBs). The EB attach to the membrane of the host cell and promote endocytosis in a membrane limited vacuole called the inclusion, which does not fuse with lysosomes. The EB then transform into RB, which replicate by binary fission. After several divisions, the inclusion is filled with RB, which then transform back into infectious EB. These EB are released through host cell lysis or extrusion of the inclusion out of the host cell [2]. It is the causal agent of enzootic abortion of ewes [3] and is the most common infectious cause of abortion in many small ruminant-rearing countries [4,5], with the exception of Australia and New Zealand [6]. In addition to significant economic losses [7,8], C. abortus presents a zoonotic risk; exposure of pregnant women to infected sheep can lead to severe septicemia in the mother resulting in spontaneous abortion or stillbirth [9,10]. In sheep and goats, C. abortus infection typically causes abortion during the last two or 3 weeks of gestation or the birth of stillborn or weak lambs that die in the first days of life [8], although infected goats may abort at any time during pregnancy [11–13]. Other than reproductive failure, sheep and goats rarely display clinical signs of C. abortus infection, other than vulval discharge 2–3 days before abortion. In some cases, goats may shed C. abortus in vaginal fluids for up to 2 weeks before and after abortion; other signs, such as respiratory tract disease, polyarthritis, conjunctivitis, and retained placentas have been reported [11]. C. abortus can induce a persistent, subclinical infection in nonpregnant sheep and goats [3,14]. After abortion, ewes are considered to be immune to further lamb loss [3,15,16]. However, ewes can be chronically or persistently infected, continuing to shed organisms at estrus or at subsequent lambings [16–19]. It has also been reported that some lambs can be born healthy and survive infection, although they may go on to abort during their first pregnancy [15,20–22]. Sexual transmission of C. abortus is possible [9]; the bacteria have been found in fresh and cryopreserved semen, preputial washing fluid, in the male genital tracts of rams and bucks [23–26], and in the vaginal mucosa in sheep [17,27]. Experimental studies have shown that C. abortus can be excreted in semen of inoculated rams [28] and transmitted by experimentally infected semen to ewes [29]. Male fetuses can be contaminated in utero and adult males by mating with infected females [30]. These results reveal the main source of in utero infection and indicate a risk factor for the transmission of C. abortus during embryo transfer (ET). Embryo transfer is used, nationally and internationally, for the introduction, improvement, and preservation of livestock genetics. Embryos are generally considered to present a lower risk of infectious disease transmission than live animals, on the basis of the results of extensive research using zona pellucida (ZP)-intact, in vivo-produced,
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embryos [31]. To reduce the risk of pathogen transmission, the International Embryo Transfer Society (IETS) has established guidelines for bovine ET. Specific conditions, including donor sanitary status, rejection of embryos without an intact ZP, specific washing procedures (minimum of 10 washes in the culture medium so that each wash represents an 100-fold dilution of the previous wash, the use of a new sterile micropipette for each wash) are required to avoid pathogen transmission by ET [32]. In addition, treatment of the ZP with several enzymes, such as trypsin, has been shown to be effective for removal or inactivation of certain pathogenic agents [32], with no deleterious effects on embryonic development [33,34]. However, these protocols must be tested for each pathogen and for embryos from each different species [32]. To our knowledge, there are no reported studies into the interaction of caprine or bovine embryos and C. abortus. To investigate the risk of C. abortus transmission via caprine ET, our study aimed to determine whether C. abortus adheres to or infects ZP-intact early caprine embryos in vivo after in vitro infection. We also evaluated the efficacy of the IETS washing procedure recommended to decontaminate bovine embryos exposed to C. abortus in vitro. 2. Materials and methods 2.1. Production of embryos 2.1.1. Goats Fourteen healthy 3- to 6-year-old Saanen or Alpine goats from flocks in the Deux-Sèvres region of France were used as embryo donors. These goats were certified C. abortus free using ELISA for blood serum and conventional polymerase chain reaction (C-PCR) for vaginal swabs. 2.1.2. Synchronization and superovulation The donor goats were synchronized by inserting intravaginal sponges impregnated with 45 mg of fluorogestone acetate (Chronogest, Intervet, Angers, France) for 11 days, combined with the intramuscular injection of 125 mg of prostaglandin analog (Estrumate, Shering-Plough Veterinaire, France) 48 hours before sponge removal (Day 9). Superovulation was induced by intramuscular injection of porcine FSH (pFSH; Merial, University of Liege, Belgium) given twice daily at 12-hour intervals for 3 days (Days 9, 10, and 11). The total dose of pFSH was 16 Armor units per goat, administered in decreasing amounts (4–4, 2–2, and 2–2). A 132-mg dose of porcine LH was added to the pFSH preparation on the last two injections (66 mg per injection) [35]. 2.1.3. Fertilization The donor goats were mated by 3- to 6-year-old Saanen or Alpine bucks from the Deux-Sèvres region of France, 24–36 hours after sponge removal. These bucks of proven fertility were certified C abortus negative, by ELISA and PCR and regularly controlled. 2.1.4. Collection of embryos Embryos were collected by laparotomy 4 to 5 days after the onset of estrus. The goats were anesthetized with 5 mL of Zoletil 100 (Virbac, Nice, France) and were prepared for
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aseptic surgery. The uterus and ovaries were exposed via ventral midline laparotomy. The collection technique for collecting goat embryos was described by Lamara et al. [36]. In short, 40 mL of collection medium (EURO FLUSH, IMV, Aigle, France) were used to flush the contents of the oviducts into the uterine horns, where the embryos were recovered into a sterile bottle via a Foley catheter introduced at the greater curvature just above the bifurcation of the horn. Surgical closure was routine. The embryos were recovered from the collection medium and evaluated, and only those with an intact ZP were used. The collection media were centrifuged for 1 hour at 13,000 g and the pellets frozen at 20 C for PCR analysis. 2.2. Chlamydia abortus strain The C. abortus strain (AB7) used in this study was isolated from the placenta of an aborted sheep. The strain was grown on vitelline membranes from specific pathogenfree–embryonated chicken eggs. The bacterial load of the Chlamydia suspension used for the experiment was estimated at 108 bacteria/mL by immunofluorescence, aliquoted, and frozen at 80 C. To ensure purity, each aliquot used for in vitro contamination was diluted with 10-mL phosphate buffer saline then centrifuged twice for 15 minutes at 2000 g. The supernatant was recovered and centrifuged for 1 hour at 13,000 g. The pellet was diluted 1:10 in the exposure medium giving a final calculated concentration of 107 bacteria/mL. 2.3. Experimental design To study the susceptibility of early ZP-intact goat embryos produced in vivo against C. abortus infection in vitro, 52 ZP-intact embryos (8–16 cells), obtained from 14 donors for the three repetitions (4, 5, five goats per repetition respectively), were used in this experiment. The embryos were randomly divided into two groups (infected embryos and control noninfected embryos). The first group comprised 45 ZP-intact embryos (5, 15, and 25 in the first, second, and third repetitions, respectively) and was divided into nine batches of five embryos. These embryos were placed in 1-mL minimum essential medium (M2414, Sigma) supplemented with 10% fetal calf serum, 1% L-glutamine (2-mM final), 1% HEPES (0.01 M final), 2.5-mg/mL amphotericin B, with 4 107 C. abortus per milliliter of AB7 strain. After incubation for 18 hours at 37 C in an atmosphere of 5% CO2, the embryos were recovered. Embryo development and the integrity of the ZP were confirmed. The embryos were then washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution, according to the IETS guidelines [31]. A new sterile pipette was used for each successive wash; each wash corresponded to a dilution of 1:100 of the previous medium. In parallel, the second group of remaining (seven ZP-intact) embryos (2, 2, and 3 in the first, second, and third repetitions, respectively) were used as controls by being
subjected to similar procedures but without exposure to C. abortus. The embryo collection mediums of each donor goat and the 10 wash baths for each batch of embryos were collected separately and centrifuged for 1 hour at 13,000 g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at 20 C before examination for evidence of C. abortus using PCR. 2.4. Conventional PCR After thawing, DNA was extracted from the batches of embryos and the wash bath pellets using a “DNA Mini kit Qiagen-France” in accordance with the manufacturer’s instructions; samples were then stored at 20 C, awaiting PCR analysis. The detection of Chlamydia DNA was performed by amplifying a DNA fragment of 82 bp from the ompA gene (outer membrane protein), a single copy in the C. abortus genome, using the following primers: forward, CpaOMP1-F 50 -GCA ACT GAC ACT AAG TCG GCT ACA-30 and reverse, CpaOMP1-R 50 -ACA AGC ATG TTC AAT CGA TAA GAG A-30 , [37] (Eurofins MWG Operon, Ebersberg, Germany). For each sample, 10 mL of extracted DNA were added to 20 mL of amplification solution (mix-PCR) containing 6 mL of reaction buffer 5X Hot FIREPol Blend Master Mix Ready To Load with BSA and 7.5 MgCl2 (Solis BioDyne, Tartu, Estonia), 0.9 mL of each primer CpaOMP1-F and CpaOMP1-R (20 mM, Eurofins MWG Operon, Ebersberg, Germany), and 12.2 mL of diethylpyrocarbonate-treated water. Amplification was conducted in a thermal cycler (Mastercycler Eppendorf). After initial denaturation at 95 C for 15 minutes, the samples were subjected to a series of 45 cycles comprising: 30 seconds denaturation at 95 C, 1-minute hybridization at 60 C, and a 2-minutes elongation phase at 72 C. This was followed by a final elongation phase at 72 C for 10 minutes. Products were visualized by electrophoresis: 10 mL for each well of 2% agarose gel containing 50-mL ethidium bromide at 1 mg/mL in 1X TBE buffer (Tris-Borate-EDTA, SIGMA, Saint Quentin Fallavier, France). Two controls were performed: a positive control of C. abortus strain (AB7 11091) and a negative control (distilled water). Five microliters of smart-ladder (Solis BioDyne, Tartu, Estonia) were used as a molecular weight marker. This marker comprises 12 bands calibrated between 3000 and 100 bp. After 15 minutes of electrophoresis at 80 V and then 45 minutes at 110 V, the bands were visualized using transillumination with ultraviolet light (312 nm). Samples analyzed for Chlamydia DNA using PCR were considered positive when a band of 82 bp, corresponding to the positive control, was visualized on agarose gel electrophoresis under UV light. The sensitivity of this PCR method has been confirmed in our laboratory (SSBR, Oniris, France); it detects up to 20 bacteria per mL of bacterial suspension (Table 1). 2.5. Real-time PCR Real-time PCR was used to amplify a DNA fragment of 82 bp from the ompA gene, a single copy in the C. abortus genome, as described previously [37,38]. The detection threshold was determined using decimal serial dilutions of
M. Oseikria et al. / Theriogenology 86 (2016) 1482–1488 Table 1 Verification of the sensitivity of conventional PCR to detect, for four repetitions, Chlamydia DNA in samples of Chlamydia abortus strain (AB7) at dilutions from one bacterium/mL to 50 bacteria/mL. Repetition
1
10
20
30
40
50
Negative control
1 2 3 4
þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
purified genomic DNA from C. abortus. DNase-RNase free water was used as a negative control. In short, the final 20 mL reaction mixture included 2 mL of sample DNA, 10 mL of 2 TaqMan universal master mix (Applied Biosystems, USA), 1 mL of a mixture of forward and reverse primers (0.6 mM, EurofinsMWG Operon, Ebersberg, Germany), and 2 mL of TaqMan probe (0.1 mM, Eurofins MWG Operon). Water was added to make up to a final volume of 20 mL. All real-time PCRs were performed in duplicate in an ABIPRISM Sequence Detection System 7300 (Applied Biosystems). The thermal cycling conditions were 50 C for 2 minutes and an initial cycle of heating at 95 C for 10 minutes (single denaturation step), followed by 45 cycles at 95 C for 15 seconds, and 60 C for 1 minute (annealing and extension). The cycle threshold value was calculated automatically. Data were analyzed with the corresponding software. The C. abortus titers in the samples were calculated in comparison with a standard curve obtained from a standard serial dilution of a titrated DNA. 3. Results After incubation for 18 hours with 4 107 C. abortus per milliliter for 18 hours, embryo development was confirmed as an increased number of embryos cells, and the ZP of all embryos were intact. Chlamydia DNA was detected by conventional PCR (Fig. 1) in all batches of five infected ZP-intact 8- to 16-cell
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embryos (9/9) after being washed 10 times (Table 2). Chlamydia DNA was also detected in all 10 wash baths for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively (Table 2). However, bacterial DNA was not detected in any of the 14 embryo collection mediums as in any of the embryos or wash baths from the control groups. After 10 wash cycles, all of the exposure baths and batches of embryos were tested using real-time PCR to quantify the final bacterial load (Fig. 2). The bacterial load in the exposure baths ranged from 2.2 to 5.0 107 bacteria/ mL. The bacterial load for embryos after the 10 wash baths ranged from 0.9 to 4.5 103 bacteria/mL (Table 3). 4. Discussion To confirm our results, we did three repetitions of the experiment. For each repetition, we had one control group of embryos without exposure to C. abortus to ensure there was no external (other than the experimental infection) contamination of the embryos. A strain of C. abortus strain (AB7) isolated from the placenta of an aborted sheep was used in this study. The pathogenic potential of Chlamydia is unlikely to be dependent on the species from which the strain was isolated. AB7 strains are a well-known strain for which in vitro production and biomolecular detection is well codified. AB7 strains colonized the placenta and fetus more strongly than POS, AB16, and LLG strains. Inoculation of the AB7 strain induced very high levels of IFN-g in nonpregnant mice [39]. The type of bacterial strain can also interfere in the connection between the ZP and the pathogenic agent, and it has recently been shown that certain strains of bovine viral diarrhea virus adhere more easily to the ZP of in vitro-produced bovine embryos [40]. The embryo is wrapped in a protective layer (ZP) until the blastocyst stage. The ZP is composed of three different glycoproteins, which create a mechanical barrier against viruses and bacteria [41–43]; the porosity of the matrix is apparently too small, allowing only the smallest of molecules to infiltrate the embryo from the external medium
Table 2 Detection of Chlamydia abortus in successive embryo washes and batches of five infected zona pellucida-intact 8- to 16-cell embryosa after 10 wash baths using conventional PCR.
Fig. 1. Example of conventional PCR amplification of Chlamydia-DNA. DNA from embryos or wash bath pellet was used to perform nested-PCR using specific sets of oligonucleotide primers to amplify 82 bp from the ompA gene. Following nested-PCR reactions, each PCR product was separated on 1.5% agarose gel and the bands visualized by staining with ethidium bromide. M ¼ Smart Ladder used as a molecular weight standard. Lanes 1–5: embryo samples (positive), lanes 6–10: wash bath pellet (lanes 6–8 and 10, positive; lane 9 negative). Cþ: positive tissue control (infected cell culture). C: negative control (distilled water). PCR, polymerase chain reaction.
Batches number
Last wash positive for C. abortus
Batches of embryos
1 2 3 4 5 6 7 8 9
9 10 10 10 10 10 8 10 10
þ þ þ þ þ þ þ þ þ
Abbreviation: PCR, polymerase chain reaction. a Exposed to 107 Chlamydia abortus for 18 h.
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Fig. 2. Typical real-time PCR results of Chlamydia-infected samples with our Taqman assay. (A) Ampliplots of the standard curve generated using serial 10-fold dilutions of the Chlamydia reference solution (see Materials and methods); a to e: duplicates obtained with bacterial amount ranging from 2 107 to 2 103 per mL; f: nontemplate control. (B) Standard curve corresponding to Fig. 2A data. The slope is 3.49 (near the theoretical 3.32 value) and the satisfactory R2 is 0.999. (C) Typical duplicate ampliplots generated using either the initial contaminating Chlamydia solution (a) or a 10th washing embryo solution postinfection (b); (c): nontemplate control solution. PCR, polymerase chain reaction.
[44]. Most of the pathogenic agents investigated to date are unable to penetrate the ZP of in vitro-produced embryos, with the exception of Leptospira [45,46]. Lamara et al. [36], reported that an intact ZP is a strong barrier that protects the caprine embryo from caprine arthritis encephalitis
Table 3 Quantification of Chlamydia abortus in embryo exposure baths and in batches of five infected zona pellucida-intact 8- to 16-cell embryos using real-time PCR (in bacteria/mL). Batches number
Exposure bath
1 2 3 4 5 6 7 8 9
2.2 3.5 3.5 3.5 5.0 5.0 5.0 5.0 5.0
107 107 107 107 107 107 107 107 107
Abbreviation: PCR, polymerase chain reaction.
Batches of embryos 0.9 1.6 2.9 4.5 3.4 2.5 2.3 2.4 1.3
103 103 103 103 103 103 103 103 103
virus infection. The ZP, especially of in vivo-derived embryos, acts as a barrier for most diseases (enzootic bovine leucosis, foot and mouth disease, blue tongue virus, Brucella abortus) [47]. Because C. abortus-DNA was detected in all batches of ZP-intact–washed embryos, this study clearly demonstrates the ability of C. abortus to adhere to or penetrate ZP-intact embryos. This proves that there is a risk of C. abortus transmission after ET. Furthermore, the routine procedures proposed by the IETS are not effective for removing the bacterium from ZP-intact caprine embryos infected in vitro. This result supports previous studies in goats, reporting the binding of Coxiella burnetii to ZP-intact embryos and the inefficacy of routine IETS procedures for removing it from ZP-intact caprine embryos collected in vivo or produced in vitro after infection in vitro [48,49]. In a previous study, with bovine embryos exposed in vitro to Mycoplasma bovis, Mycoplasma bovigenitalium, Mycobacterium avium, after washing, bacteria were isolated from all batches of ZP-intact embryos [50,51]. Washing procedures and antibiotic treatment for the removal of bacterial agents that include Streptococcus agalactiae, Actinomyces pyogenes, Escherichia coli [52], M. avium ssp. paratuberculosis [51],
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Campylobacter fetus [53], and Mycoplasmas [54], from in vivo-derived bovine embryos have not been completely effective. Also, the treatment of in vivo-derived bovine embryos with trypsin in addition to the standard IETS washing was not fully effective for Mycoplasmas [54], and E. coli [52]. Embryos were experimentally exposed to 4 107 C. abortus per milliliter for 18 hours. This high concentration of inoculums with an 18-hour exposure period was chosen to maximize the chances of the bacteria coming into contact with the ZP. It should be noted that under natural conditions, the highest concentration of C. abortus was observed after abortion where they were detected in the fetal membranes and in the cotyledon at a concentration of about 107 Chlamydia per milligram of tissue [55,56]. Such bacterial levels found at abortion are associated with inflammation, which reduces the survival of the oocytes and/or embryos in the female genital tract. However, it appears that the adhesion of the bacteria to the ZP is dose related. In our study, the in vitro experimental conditions of infection are considerably different to natural infection conditions observed in the field. Real-time PCR analysis of embryos after washing identified an average of 2.4 103 Chlamydial/mL, i.e. (0.9–4.5 103 Chlamydia/mL), these concentrations are consistent with the experimental dose required to either elicit abortion [29] or the birth of weak lambs [27], after vaginal inoculation with C. abortus, or latent infection resulting in abortion at a pregnancy after intranasal administration of nonpregnant sheep with a low dose of C. abortus (5 103 inclusion forming units/mL) [13]. Similar conclusions were drawn from early research conducted by McEwen et al., who noted that the infectivity and pathological effects of a small dose was equal to or greater than the effects of a large dose of C. abortus when administered before mating [57]. This positive result does not necessarily mean that transferred in vivo-collected embryos will always transmit the infection. Real-time PCR used alone, without cell-culture assay, does not differentiate between DNA from “live” and “dead” C. abortus, so a positive result does not prove that the embryo carries an infectious pathogen. However, a negative result should indicate that embryos are free of pathogenic agents [58]. Finally, the interpretation of results obtained for C. abortus with in vivoderived caprine embryos recovered from synchronized and superovulated donor goats should not be extrapolated to another species or to in vitro-produced goat embryos, because of physiological and morphological differences between such embryos [59,60]. Our results report that C. abortus adheres to and/or penetrates the ZP of in vivo-derived caprine embryos after in vitro infection, and that the IETS washing protocol for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential mean of transmission of the bacterium during ET from infected donor goat to healthy recipients and/or their offspring. Nevertheless, the positive results of this in vitro study should be considered as worst case scenarios and should not be extrapolated to field situations. Further work is needed (i) to determine the location of the bacterium after in vitro infection and washing, (ii) to
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investigate whether the treatment of Chlamydia-infected embryos with trypsin or antibiotic in addition to the standard IETS washing would eliminate the bacterium from the ZP, and (iii) to assess the risks associated with ET transmission under normal circumstances (in vivo-in vivo experiment). References [1] Sachse K, Laroucau K, Riege K, Wehner S, Dilcher M, Creasy HH, et al. Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst Appl Microbiol 2014;37: 79–88. [2] Moulder JW. Interaction of Chlamydiae and host cells in vitro. Microbiol Rev 1991;55:143–90. [3] Aitken ID, Longbottom D. Chlamydial abortion. In: Diseases of sheep. Oxford: Blackwell Publishing Ltd; 2008. p. 105–12. [4] Aitken ID. Chlamydial abortion. In: Martin WB, Aitken ID, editors. Diseases of sheep. 3rd ed. Edinburgh: Blackwell; 2000. p. 81–6. [5] Longbottom D, Livingstone M. Vaccination against Chlamydial infections of man and animals. Vet J 2006;171:263–75. [6] Rodolakis A, Laroucau K. Chlamydiaceae and Chlamydial infections in sheep or goats. Vet Microbiol 2015;181:107–18. [7] Jimenez-Estrada JM, Escobedo-Guerra MR, Arteaga-Troncosa G, Lopez-Hurtado M, Haro-Cruz MJ, Oca-Jimenez RM, et al. Detection of Chlamydophila abortus in sheep (Ovis aries) in Mexico. Am J Anim Vet Sci 2008;3:91–5. [8] Rodolakis A. Chlamydiose et Fièvre Q, similitudes et différences entre ces deux zoonoses. Renc Rech Ruminants 2006;13:395–402. [9] Longbottom D, Coulter LJ. Animal chlamydioses and zoonotic implications. J Comp Pathol 2003;128:217–44. [10] Baud D, Regan L, Greub G. Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes. Curr Opin Infect Dis 2008;21:70–6. [11] Rodolakis A, Boullet C, Souriau A. Chlamydia psittaci experimental abortion in goats. Am J Vet Res 1984;45:2086–9. [12] Matthews J. Abortion. In: Diseases of the goat. Oxford: Blackwell Science; 1999. p. 22–36. [13] Longbottom D, Livingstone M, Maley S, van der Zon A, Rocchi M, Wilson K, et al. Intranasal infection with Chlamydia abortus induces dose-dependent latency and abortion in sheep. PLoS One 2013;8: e57950. [14] Rocchi MS, Wattegedera S, Meridiani I, Entrican G. Protective adaptive immunity to Chlamydophila abortus infection and control of ovine enzootic abortion (OEA). Vet Microbiol 2009;135:112–21. [15] Rodolakis A, Salinas J, Papp J. Recent advances on ovine chlamydial abortion. Vet Res 1998;29:275–88. [16] Livingstone M, Wheelhouse N, Maley SW, Longbottom D. Molecular detection of Chlamydophila abortus in post-abortion sheep at oestrus and subsequent lambing. Vet Microbiol 2009;135:134–41. [17] Papp JR, Shewen PE. Localization of chronic Chlamydia psittaci infection in the reproductive tract of sheep. J Infect Dis 1996;174: 1296–302. [18] Wilsmore AJ, Izzard KA, Wilsmore BC, Dagnall GJ. Breeding performance of sheep infected with Chlamydia psittaci (ovis) during their preceding pregnancy. Vet Rec 1990;126:40–1. [19] Papp JR, Shewen PE, Gartley CJ. Abortion and subsequent excretion of chlamydiae from the reproductive tract of sheep during estrus. Infect Immun 1994;62:3786–92. [20] Papp JR, Shewen PE. Chlamydia psittaci infection in sheep: a paradigm for human reproductive tract infection. J Reprod Immunol 1997;34:185–202. [21] Entrican G, Buxton D, Longbottom D. Chlamydial infection in sheep: immune control versus fetal pathology. J R Soc Med 2001;94:273–7. [22] Philips HL, Clarkson MJ. Investigation of pre-natal Chlamydophila abortus (Chlamydia psittaci) exposure of female lambs and the outcome of their first pregnancy. Vet J 2002;163:329–30. [23] Amin AS. Comparison of polymerase chain reaction and cell culture for the detection of Chlamydophila species in the semen of bulls, buffalo-bulls, and rams. Vet J 2003;166:86–92. [24] Gautam R, Purohit VD. Isolation of chlamydia psittaci from genitalia of healthy exotic and crossbred service rams. Indian J Anim Sci 2001;71. [25] Kauffold J, Henning K, Bachmann R, Hotzel H, Melzer F. The prevalence of chlamydiae of bulls from six bull studs in Germany. Anim Reprod Sci 2007;102:111–21.
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