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Susceptibility to Vertical Transmission of Toxoplasma gondii is Temporally Dependent on the Preconceptional Infection in Calomys callosus
Placenta 28 (2007) 624e630
Susceptibility to Vertical Transmission of Toxoplasma gondii is Temporally Dependent on the Preconceptional Infection in Calomys callosus B.F. Barbosa a, D.A.O. Silva b, I.N. Costa a, J.D.O. Pena c, J.R. Mineo b, E.A.V. Ferro a,* a
Laboratory of Histology and Embriology, Universidade Federal de Uberlaˆndia, Av. Para´ 1720, Uberlaˆndia 38405-320, MG, Brazil b Laboratory of Immunoparasitology, Universidade Federal de Uberlaˆndia, Uberlaˆndia 38405-320, MG, Brazil c Laboratory of Molecular Biology, Institute of Biomedical Sciences, Universidade Federal de Uberlaˆndia, Uberlaˆndia 38405-320, MG, Brazil Accepted 31 October 2006
Abstract Toxoplasma gondii is an obligate intracellular parasite that causes a variety of clinical syndromes, but the infection is more severe in immunocompromised individuals and in cases of congenital toxoplasmosis. This study aimed to verify if the susceptibility to vertical transmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus. Twelve C. callosus females were infected with 20 cysts of T. gondii ME49 strain and divided into three groups of four animals that were mated after approximately 10 days (group 1), 30 days (group 2), and 50 days (group 3) of infection. The animals were sacrificed from the 17th to 20th day of pregnancy, when placentas and embryos were collected for morphological and immunohistochemical studies, mouse bioassay for evaluating seroconversion and PCR for detecting parasite DNA. Serum samples from C. callosus females and mice used in bioassay were analysed for the detection of IgG antibodies to T. gondii by ELISA. Detection of T. gondii was observed by mouse bioassay and PCR in placentas and embryos from C. callosus females infected around 10 days pre-conception. However, only placentas, but not embryos, from females infected around 30 and 50 days pre-conception showed positivity for parasite DNA and seroconversion by mouse bioassay. In conclusion, this study model shows that vertical transmission of T. gondii may take place when maternal infection occurs within one month before conception, thus demonstrating the time of preconceptional seroconversion that rule out a risk of congenital toxoplasmosis. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Toxoplasma gondii; Calomys callosus; Congenital toxoplasmosis; Preconceptional infection; Maternalefetal interface
1. Introduction Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii and is usually asymptomatic in immunocompetent subjects. However, when the infection occurs in immunocompromised patients or during pregnancy, it can lead to serious disorders as encephalitis or congenital toxoplasmosis, respectively [1]. The vertical transmission is not obligatory and occurs in 20e50% of maternal primary
* Corresponding author. Tel./fax: þ55 34 3218 2240. E-mail address: [email protected] (E.A.V. Ferro). 0143-4004/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2006.10.011
infection by T. gondii during pregnancy [2] with a more frequent involvement of the strains I and II in humans [3]. Maternal infection prior to conception normally excludes the risk of fetal infection and congenital toxoplasmosis, which usually follows T. gondii infection acquired in early pregnancy [1]. However, preconceptional infection can occasionally lead to implication for the fetus in immunodeficient women [4] and in immunologically competent women with clinical signs as cervical adenopathies [5,6]. Accordingly, two unusual cases of congenital toxoplasmosis were reported, one occurring after preconceptional infection with cervical adenopathies and the other occurring after maternal infection at the very end of pregnancy with maternal seronegativity at delivery [7]. In
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the infection acquired at the very end of pregnancy, with the mother still seronegative at delivery, the rate of transmission from mother to fetus is around 70% during this period [8]. Thus, the risk of congenital toxoplasmosis can be better evaluated when the maternal seroconversion and the time of primary infection is more accurately determined to lead to an efficient therapeutic strategy. If the infection is confirmed, anti-parasitic treatment during pregnancy may prevent fetal infection and serious damages, such as chorioretinitis or neurological defects [1]. Calomys callosus, a rodent of the family Cricetidae widely distributed in Central Brazil, has been reported as important natural reservoir for Trypanosoma cruzi [9,10], but also susceptible to several infectious diseases as schistosomosis and leishmaniasis [11]. Our previous studies have demonstrated the high susceptibility of C. callosus to T. gondii infection, when the presence of the parasite was detected in several tissues, particularly liver, spleen, lung and brain [12]. Also, this rodent was demonstrated to be a suitable experimental model to study the dynamics of congenital toxoplasmosis, due to the ability of a highly virulent strain of T. gondii (RH strain) to infect trophoblast cells during the early blastocysteendometrial relationship [13]. In another previous study, we demonstrated the vertical transmission of T. gondii in C. callosus acutely infected with the ME49 strain during pregnancy, but not in chronically infected animals. In addition, considering the sequence of events leading to the infection of the various organs, the placental trophoblast cells are infected later on during pregnancy, but were unable to completely stop the progression of T. gondii infection towards the fetal tissues [14]. Considering the possibility of vertical transmission of T. gondii when maternal seroconversion occurs a few weeks before conception and that so far this condition has not been kinetically studied in detail, the present study aimed to verify if the susceptibility to vertical transmission of T. gondii is temporally dependent on the preconceptional infection in C. callosus.
2. Materials and methods 2.1. Animals C. callosus of the Canabrava strain came from a resident colony housed at the Institute of Tropical Medicine of S~ao Paulo and were kindly provided by Dr Judith Kloetzel. The animals were kept under specific pathogen-free conditions on a 12-h light, 12-h dark cycle in a temperature-controlled room (25 2 C) with food and water ad libitum. All procedures were conducted according to institutional guidelines for animal ethics.
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2.3. Experimental groups Twelve C. callosus virgin females aged 2e3 months were perorally infected with 20 cysts of T. gondii ME49 strain and then divided into three groups of four animals that were mated with males after different times of infection as follows: group 1 (8e13 days after infection), group 2 (28e35 days after infection), and group 3 (42e57 days after infection). The presence of a vaginal plug was considered 1st day of pregnancy (dop). The animals were euthanised from the 17th to 20th dop, when placentas and embryos were collected for morphological and immunohistochemical assays, mouse bioassay and polymerase chain reaction (PCR) for the detection of Toxoplasma. Blood samples were collected at 1st dop and when animals were euthanised (17th to 20th dop) to determine the levels of IgG antibodies to T. gondii by immunoenzymatic assays (ELISA).
2.4. Morphological and immunohistochemical assays For conventional light microscopy, specimens were fixed by immersion in 10% formalin in 0.1 M phosphate buffer (pH 7.4), dehydrated, and embedded in methacrylate resin. Sections measuring 2 mm in thickness were stained with 0.25% toluidine blue and then examined in a photomicroscopy (Reichert-JungPolyvar, Lab. Optica Robert Koch, Austria). For immunolocalisation of the parasites, specimens fixed were dehydrated and embedded in paraffin. Sections measuring 4 mm in thickness were placed on glass slides and processed as previously described [14]. Briefly, samples were first incubated (10 min at room temperature) with 5% acid acetic to block endogenous alkaline phosphatase and then (30 min at 37 C) with 2% normal goat serum to block non-specific binding sites. Next, samples were incubated (12 h at 4 C) with rabbit anti-T. gondii serum and then (30 min at 37 C) with biotinylated goat anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO, USA). The reaction was amplified by using the ABC system (Biomeda, Foster City, CA, USA) and developed with fast red-naphthol (Sigma). Samples were counterstained with Mayer’s haematoxylin and examined in photomicroscopy.
2.5. Detection of T. gondii in placentas and embryo tissues Detection of T. gondii was first evaluated by mouse bioassay as described elsewhere [15], with minor modifications. Placentas and embryo tissues (liver and brain) were homogenised in PBS and separately inoculated in mice Swiss by intraperitoneal route, in duplicate. After 30e45 days of inoculation, blood samples from mice were collected and sera obtained were analysed for the detection of IgG antibodies to T. gondii in ELISA. The presence of T. gondii DNA was investigated by PCR as previously described [16]. Briefly, placentas and embryo tissues were treated with 100 mg/ ml proteinase K (Invitrogen Life Technologies, S~ao Paulo, Brazil) in buffer containing 10 mM TriseHCl, 0.1 M EDTA, 0.5% SDS, pH 8.0, at 50 C during 3 h. Samples were then submitted to phenol/chloroform/isoamyl alcohol (25:24:1, pH 8.0) extraction and DNA was precipitated from the aqueous phase by treatment with 2.5 volumes of cold ethanol. Qualitative PCR detecting the 35-copy B1 gene of T. gondii was performed using the primers 50 TCTTCCCAGAGGTGGATTTC-30 (sense, nucleotides 151e171) and 50 CTCGACAATACGCTGCTTG-30 (antisense, nucleotides 682e663) (Invitrogen Life Technologies, S~ao Paulo, Brazil), which should amplify a fragment of 531 bp. After initial incubation for 3 min at 95 C, samples were subjected to 38 cycles of denaturing at 94 C for 1 min, annealing for 1.2 min at 62 C, and extension for 2 min at 72 C [17]. PCR products were analysed by 1% agarose gel containing 0.5 mg/ml of ethidium bromide and visualised under UV illumination.
2.2. Parasites 2.6. ELISA and ELISA-avidity Cysts of T. gondii ME49 strain were obtained from brains of C. callosus infected 30e45 days earlier with 20 cysts via oral route. The brains were removed, washed in sterile 0.01 M phosphate-buffered saline (PBS) pH 7.2, and homogenised with a syringe and a 25 7 gauge needle. The brain preparations were further washed by centrifugation at 1000 g for 10 min in PBS and cysts were counted under light microscopy (10 magnification).
A conventional ELISA to detect IgG antibodies to T. gondii was carried out as previously described [14] with some modifications in order to confirm the pre-conceptional seroconversion as indicator of infection of C. callosus females. Polystyrene microtitre plates were coated overnight at 4 C with T. gondii soluble antigen at 10 mg/ml in carbonate buffer 0.06 M (pH 9.6). Plates
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were washed with PBS plus 0.05% Tween-20 (PBST) and incubated with serum samples of C. callosus diluted at 1:32 in PBST for 1 h at 37 C. After washing, plates were incubated with peroxidase labelled rabbit anti-C. callosus IgG, prepared according to Wilson and Nakane [18] diluted at 1:250 in PBST and incubated for 1 h at 37 C. After new washes, the reaction was developed with a substrate solution consisting of 0.03% hydrogen peroxide and 1 mg/ml of o-phenylenediamine (OPD) in 0.1 M citrate-phosphate buffer (pH 5.0). The reaction was stopped with 2 N H2SO4 and optical density (OD) was measured at 492 nm by using a plate reader (Titertek Multiskan Plus, Flow Laboratories, Geneva, Switzerland). Results were expressed in ELISA index (EI) as follows:
EI ¼ OD sample/cut-off, where cut-off was established as mean OD values of negative control sera plus three standard deviations. Based on screening tests performed with negative and positive control sera, EI > 1.2 values were considered positive results. To determine phases of infection (recent or chronic), an ELISA-avidity was conducted as described elsewhere [19] with some modifications. It was performed as above described for conventional ELISA, except for a modification in the serial washing step just after incubation with serum samples (diluted 1:32) in quadruplicate. Plates were washed once with PBST and half the wells were soaked for 10 min with 6 M urea in PBST. After three
Fig. 1. Photomicrographs of placentas and embryo tissues from C. callosus infected with ME49 strain of T. gondii in different preconceptional time periods and analysed from 17th to 20th days of pregnancy. Staining by toluidine blue shows the presence of parasites in (a) labyrinth cells (asterisk) at the transition region to the spongiotrophoblast area in placenta from animals of group 1 (8e13 days of preconceptional infection) and (b) decidua (asterisks) from animals of group 2 (28e35 days of preconceptional infection); absence of parasites in (c) trophoblastic cells (arrows) and spongiotrophoblast area (star) from animals of group 3 (42e57 days of preconceptional infection). Immunohistochemical staining using alkaline phosphatase and fast red naphthol showing the presence of parasites in (d) brain areas from embryos of group 1 (arrowhead); absence of parasites in (e) brain and (f) liver areas from embryos of groups 2 and 3, respectively. Counterstaining by Mayer’s haematoxylin. Bars: a,c,e,f: 35 mm; b,d: 14 mm.
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Immunohistochemical assay revealed the presence of T. gondii in the labyrinth zone cells of animals of groups 1 and 2 (data not shown). Only embryos from animals of group 1 were multi-infected including brain, liver, back, connective tissue and retro-abdominal region (Fig. 1d). In contrast, embryos from females of groups 2 and 3 did not show any evidence of parasites (Fig. 1e and f). Serological assays carried out at 1st dop showed that seroconversion was verified only in one animal with 13 days of infection of group 1 (Fig. 2a), presenting a low IgG avidity (IA ¼ 38%), while animals with 8e9 days of infection were seronegative to T. gondii (EI < 1.2). In contrast, all animals of group 1 showed high serum levels of antibodies to T. gondii with IgG avidity ranging from 40% to 62% (mean IA ¼ 51%), when analysed from 17th to 20th dop corresponding to 25e 27 days after infection (Fig. 2a). In group 2 (Fig. 2b), all animals were seropositive to T. gondii at both time periods analysed: 1st dop (28e35 days after infection) and from 17th to 20th dop (48e52 days after infection) showing mean IgG avidity indexes of 63% (range 58e66%) and 65% (range 54e80%), respectively. In the same way, all animals of group 3 showed high levels of antibodies to T. gondii at two time periods analysed: 1st dop (42e57 days after infection) and from 17th to 20th dop (58e 74 days after infection) showing mean IgG avidity indexes of 72% (range 61e91%) and 73% (range 71e78%), respectively (Fig. 2c). Mouse bioassay revealed seroconversion (EI > 1.2) in all mice that were inoculated with placentas and embryo tissues from C. callosus females of group 1 in all periods of time analysed (Fig. 3a), but only in mice inoculated with placentas
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3.2. Vertical transmission of toxoplasmosis is temporally dependent on the maternal pre-conceptional infection in C. callosus
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3.1. Placental labyrinth zone and basal decidua from C. callosus are the sites most often infected by T. gondii No morphological changes were observed in placentas of C. callosus infected with T. gondii, but parasites, when present, were noted in all three layers: basal decidua, junctional zone and labyrinth zone. In animals of group 1 (8e13 days of preconceptional infection) and group 2 (28e35 days of preconceptional infection), cells of the labyrinth zone and of basal decidua were the most often infected (Fig. 1a and b). No parasite was found in placentas from animals of group 3 (42e57 days of preconceptional infection) (Fig. 1c).
EI
IgG avidity index (IA %)
additional washes, plates were incubated with peroxidase conjugate and revealed as above described. Results were expressed as ELISA index (EI) and the index of avidity (IA) was calculated by using the following formula: IA (%) ¼ [(EI samples with urea)/(EI samples without urea)] 100. Low avidity was arbitrarily considered for IA < 40%, intermediate for IA between 40% and 70%, and high avidity for IA > 70%, based on previous reports [14].
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Days after infection/pregnancy Fig. 2. Levels of IgG antibodies to T. gondii expressed in ELISA index (EI) and IgG avidity index (IA) expressed in percentage (%) in serum samples of C. callosus infected with ME49 strain of T. gondii prior conception. (a) animals of group 1 with 8e13 days of preconceptional infection, (b) animals of group 2 with 28e35 days of preconceptional infection and (c) animals of group 3 with 42e57 days of preconceptional infection were analysed at 1st and from 17th to 20th days of pregnancy.
from females of group 2 (Fig. 3b). In animals of group 3, seroconversion was verified only in mice inoculated with placentas from 17th and 18th dop (Fig. 3c). T. gondii DNA was detected in placentas and embryo tissues from all females of group 1 from 17th to 20th dop. However, T. gondii DNA was detected in placentas, but not in embryos, from all females of group 2, for all periods of time analysed. Concerning group 3, only placentas showed parasite DNA at 17th and 18th dop (Fig. 4). Table 1 summarises the results obtained for all assays to determine the presence of T. gondii in placenta and embryo tissues from C. callosus females, as well as the maternal seroconversion in different groups of animals infected before conception. 4. Discussion Toxoplasmosis is routinely diagnosed by using serologic tests for the detection of specific antibodies [3]. In the last
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Fig. 4. Representative PCR analysis of the T. gondii 35-copy B1 gene in placentas and embryo tissues collected from 17th to 20th days of pregnancy in C. callosus females infected with T. gondii ME49 strain around 10, 30 or 50 days prior conception. (1) 500 bp DNA ladder (Invitrogen); (2) T. gondii (RH strain) tachyzoite DNA (positive control); (3) placenta and (4) embryo tissues from non-infected C. callosus females (negative controls); (5) placenta and (6) embryo tissues from C. callosus females infected with T. gondii ME49 strain around 10 days prior conception (group 1); (7) placenta and (8) embryo tissues from C. callosus females infected with T. gondii ME49 strain around 30 days prior conception (group 2); (9) placenta and (10) embryo tissues from C. callosus females infected with T. gondii ME49 strain around 50 days prior conception (group 3). Numbers on the left indicate the molecular markers in base pairs (bp).
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Days of pregnancy Fig. 3. Levels of IgG antibodies to T. gondii expressed in ELISA index (EI) in serum samples of mice in bioassay. Mice were inoculated with placentas or embryo tissues collected from 17th to 20th days of pregnancy in C. callosus infected with T. gondii ME49 strain around 10 days (a), 30 days (b) or 50 days (c) prior to conception. Data are represented as mean SEM.
decades, specific IgG antibody avidity tests have been shown to be very useful for diagnosing recent infections and characterising the time of acquisition of Toxoplasma infection [19]. The risk of congenital toxoplasmosis is raised when maternal seroconversion is confirmed and the time of acquisition of infection is determined [8,20]. In the present study, recent or chronic phases of the infection were determined based on the results of serological assays by ELISA and ELISA avidity
that were performed at 1st dop and from 17th to 20th dop. Animals of group 1 (8e13 days of preconceptional infection) were either seronegative or exhibited a profile of low IgG avidity (IA ¼ 38%) at 1st dop, thus characterising a recent infection. IgG avidity showed an increasing profile (IA ¼ 51%) when analysed from 17th to 20th dop (25e27 days after infection), indicating an affinity maturation of IgG synthesis and reflecting a possible transient phase of infection. Animals from both group 2 and group 3 showed high levels of antibodies to T. gondii at time periods analysed with increasing IgG avidity indexes (IA ¼ 63% and IA ¼ 72%, respectively), showing characteristic profiles that lead to a chronic infection. In humans, T. gondii-specific IgG avidity may be broadly varied among individuals, but the presence of high avidity antibodies essentially rules out infection acquired in the last 3e5 months, whereas low avidity antibodies can persist beyond 3 months of infection [21]. In the present study, we evaluated the risk of congenital toxoplasmosis in C. callosus in three intervals of preconceptional infection with the ME49 strain of T. gondii. Vertical transmission was confirmed by morphological and immunohistochemical assays, as well as by mouse bioassay and PCR in females infected around 10 days (group 1) prior conception, but not around 30 and 50 days preconception (groups 2 and 3), respectively indicating that congenital toxoplasmosis does not take place when maternal infection occurs up to about one month before conception in this experimental model. As seen in other rodents, the labyrinth zone is the greatest area of the placenta from C. callosus and it is considered the
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Table 1 Summarised data obtained in all assays to determine the presence of Toxoplasma gondii in placenta and embryo tissues from Calomys callosus females and the maternal seroconversion in different groups of animals infected before conception Groups of animalsa
Parasite detection Immunohistochemical staining
Mouse bioassay
PCR
Maternal seroconversionb
Placenta
Embryo tissues
Placenta
Embryo tissues
Placenta
Embryo tissues
ELISA index (EI)
ELISA avidity (IA %)
Group 1 17 dop 18 dop 19 dop 20 dop
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
2.9 2.7 3.0 2.6
49.8 39.4 53.7 61.9
Group 2 17 dop 18 dop 19 dop 20 dop
þ þ þ þ
þ þ þ þ
þ þ þ þ
3.7 3.7 4.1 3.6
80.7 62.7 54.3 61.4
Group 3 17 dop 18 dop 19 dop 20 dop
þ þ
þ þ
3.1 3.2 3.5 3.6
71.2 71.0 71.8 77.8
a Animals were infected with T. gondii prior to conception at different time periods: group 1 (8e13 days of preconceptional infection), group 2 (28e35 days of preconceptional infection) and group 3 (42e57 days of preconceptional infection). Then, animals were euthanised and analysed at 17, 18, 19 and 20 days of pregnancy (dop). þ, presence of parasite; , absence of parasite. b Maternal seroconversion was determined by measuring levels of IgG antibodies to T. gondii in ELISA and expressed in ELISA index (EI), and values of EI > 1.2 were considered positive. IgG avidity was also determined by ELISA-avidity and expressed in percentage of avidity index (IA %). Low avidity was arbitrarily considered for IA < 40%, intermediate for IA between 40% and 70%, and high avidity for IA > 70%.
main region of nutrient exchanges between maternal and fetal blood. Three layers of trophoblast cells plus the fetal endothelium constitute this placental barrier that becomes more and more slender with the advance of the pregnancy. Therefore, once infected by pathogens there is no difficulty for the pathogens to reach the fetal tissues [22]. Our previous studies have demonstrated that C. callosus is a useful experimental model for congenital toxoplasmosis, since vertical transmission was observed in females infected with ME49 strain of T. gondii in acute phase, but not in chronic phase of the infection [14]. It has been demonstrated that maternal T. gondii infection in C. callosus before conception normally excludes the risk of fetal transmission [14]. However, the present study showed that this might not be true when maternal infection occurs a few weeks before conception, as demonstrated by results obtained by mouse bioassay and PCR. It was demonstrated that the T. gondii replication is restricted in the placental microenvironment, no crossing the placenta barrier and, thus, preventing the vertical transmission of the parasite to the embryo, when infection occurred 30 or 50 days before conception. Thus, the time of maternal seroconversion that rule out a probable risk of congenital toxoplasmosis in C. callosus is around 30 days preconception. It is worth noting that these results agree with our previous studies demonstrating that congenital infection did not occur in C. callosus females infected with T. gondii 100 days before conception [14]. As already reported in humans, further studies should be conducted in the C. callosus model to verify the possibility of materno-fetal transmission of T. gondii when maternal infection occurs at the very end of pregnancy, even when signs of seroconversion are absent at delivery [7,23].
A recent study has demonstrated that maternal infection with a variety of T. gondii strains occurring 2 months before conception in SpragueeDawley rats may act as a type of immunisation that prevents the vertical transmission of this parasite [15]. Accordingly, our findings demonstrated that as early as 1 month preconceptional infection can guarantee fetal protection in immunocompentent C. callosus. In conclusion, the results presented herein strengthen the role of C. callosus as a useful congenital toxoplasmosis model, since the vertical transmission occurs in the acute phase of infection, including unusual cases of preconceptional infection, as previously reported in human congenital toxoplasmosis [7,6]. In addition, the time of acquisition of preconceptional seroconversion that rules out a probable risk of congenital toxoplasmosis could be established in this experimental model. Acknowledgements This work was supported by Brazilian Research Agencies (FAPEMIG, CNPq and CAPES). References [1] Remington JS, McLeod R, Thulliez P, Desmonts G. Toxoplasmosis. In: Remington JS, Klein OJ, editors. Infectious disease of the fetus and newborn infant. 5th edn. Philadelphia: WB Saunders 2001. p. 205e346. [2] Jones JL, Lopez A, Wilson M, Schulkin J, Gibbs R. Congenital toxoplasmosis: a review. Obstetric Gynecol Surv 2001;56:296e305. [3] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004;363:1965e76.
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[4] Desmonts G, Couvreur J, Thulliez P. Congenital toxoplasmosis: five cases with mother-to-child transmission of pre-pregnancy infection. Press Med 1990;19:1445e9. [5] Villena I, Chemla C, Quereux C, Dupouy D, Leroux B, Foudrinier F, et al. Prenatal diagnosis of congenital toxoplasmosis transmitted by an immunocompetent woman infected before conception. Prenat Diagn 1998;18:1079e81. [6] Vogel N, Kirisits M, Michael E, Bach H, Hostetter M, Boyer K, et al. Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception. Clin Infect Dis 1996;23:1055e 60. [7] Chemla C, Villena I, Aubert D, Hornoy P, Dupouy D, Leroux B, et al. Preconceptional seroconversion and maternal seronegativity at delivery do not rule out the risk of congenital toxoplasmosis. Clin Diagn Lab Immunol 2002;9:489e90. [8] Dunn D, Wallon M, Peyron F, Petersen E, Peckham C, Gilbert R. Mother-to-child transmission of toxoplasmosis: risk estimates for clinical counselling. Lancet 1999;353:1829e33. [9] Borges MM, De Andrade SG, Pilatti CG, Do Prado Junior JC, Kloetzel JK. Macrophage activation and histopathological findings in Calomys callosus and Swiss mice infected with several strains of Trypanosoma cruzi. Mem Inst Oswaldo Cruz 1992;87:493e502. [10] Mello DA, Valin E, Teixeira ML. Various aspects of the behavior of wild strains of Trypanosoma cruzi in mice and Calomys callosus (Rodentia). Rev Sau´de Pu´blica 1979;13:314e25. [11] Mello DA. Note on breeding of Calomys expulsus, Lund, 1841 (Rodentia, Cricetidae) under laboratory condition. Rev Bras Pesq Med Biol 1977;10:107. [12] Favoreto Jr S, Ferro EAV, Clemente D, Silva DAO, Mineo JR. Experimental infection of Calomys callosus (Rodentia, Cricetidae) by Toxoplasma gondii. Mem Inst Oswaldo Cruz 1998;93:103e7. [13] Ferro EAV, Bevilacqua E, Favoreto Jr S, Silva DAO, Mortara RA, Mineo JR. Calomys callosus (Rodentia: Cricetidae) trophoblast cells as host cells to Toxoplasma gondii in early pregnancy. Parasitol Res 1999;85:647e54. [14] Ferro EAV, Silva DAO, Bevilacqua E, Mineo JR. Effect of Toxoplasma gondii infection kinetics on trophoblast cell population in Calomys
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[21] [22]
[23]
callosus, a model of congenital toxoplasmosis. Infect Immun 2002;70:7089e94. Freyre A, Falco´n J, Me´ndez J, Rodriguez A, Correa L, Gonzalez M. Toxoplasma gondii:partial cross-protection among several strains of the parasite against congenital transmission in a rat model. Exp Parasitol 2006;112:8e12. Grigg ME, Boothroyd JC. Rapid identification of virulent type 1 strains of the protozoan pathogen Toxoplasma gondii by PCR-restriction fragment length polymorphism analysis at the B1 gene. J Clin Microbiol 200;39:398e400. Silva NM, Rodrigues CV, Santoro MM, Reis LFL, Alvarez-Leite JI, Gazzinelli RT. Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and kynurenine formation during in vivo infection with Toxoplasma gondii: induction by endogenous gamma interferon and requirement of interferon regulatory factor 1. Infect Immun 2002;70:859e68. Wilson MB, Nakane PK. Antibody conjugated to horseradish peroxidase. In: Knapp W, Holubar K, Wick G, editors. Immunofluorescence and related staining technique. Amsterdam: Elsevier North Holland Biomedical; 1978. p. 215e24. Cozon GJN, Ferrandiz J, Nebhi H, Wallon M, Peyron F. Estimation of the avidity immunoglobulin G for routine diagnosis of chronic Toxoplasma gondii infection in pregnant woman. Eur J Clin Microbiol Infect Dis 1998;17:32e6. Foulon W, Villena I, Stray-Pedersen B, Decoster A, Lappalainen M, Pinon JM, et al. Treatment of toxoplasmosis during pregnancy: a multicenter study of impact on fetal transmission and children’s sequelae at age 1 year. Am J Obstet Gynecol 1999;180:410e5. Montoya JG. Laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. J Infect Dis 2002;185(Suppl. 1):S73e82. Ferro EAV. Cine´tica da infecc¸~ao congeˆnita de ce´lulas trofobla´sticas por Toxoplasma gondii na placenta de Calomys callosus 2000; Thesis. S~ao Paulo: Universidade de S~ao Paulo. 167pp. Wirden M, Botterel F, Romand S, Ithier G, Boure´e P. Congenital toxoplasmosis following primary maternal infection acquired during late pregnancy: relevance of serological screening after delivery. J Gynecol Biol Reprod 1999;28:566e7.
Susceptibility to Vertical Transmission of Toxoplasma gondii is Temporally Dependent on the Preconceptional Infection in Calomys callosus B.F. Barbosa a, D.A.O. Silva b, I.N. Costa a, J.D.O. Pena c, J.R. Mineo b, E.A.V. Ferro a,* a
Laboratory of Histology and Embriology, Universidade Federal de Uberlaˆndia, Av. Para´ 1720, Uberlaˆndia 38405-320, MG, Brazil b Laboratory of Immunoparasitology, Universidade Federal de Uberlaˆndia, Uberlaˆndia 38405-320, MG, Brazil c Laboratory of Molecular Biology, Institute of Biomedical Sciences, Universidade Federal de Uberlaˆndia, Uberlaˆndia 38405-320, MG, Brazil Accepted 31 October 2006
Abstract Toxoplasma gondii is an obligate intracellular parasite that causes a variety of clinical syndromes, but the infection is more severe in immunocompromised individuals and in cases of congenital toxoplasmosis. This study aimed to verify if the susceptibility to vertical transmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus. Twelve C. callosus females were infected with 20 cysts of T. gondii ME49 strain and divided into three groups of four animals that were mated after approximately 10 days (group 1), 30 days (group 2), and 50 days (group 3) of infection. The animals were sacrificed from the 17th to 20th day of pregnancy, when placentas and embryos were collected for morphological and immunohistochemical studies, mouse bioassay for evaluating seroconversion and PCR for detecting parasite DNA. Serum samples from C. callosus females and mice used in bioassay were analysed for the detection of IgG antibodies to T. gondii by ELISA. Detection of T. gondii was observed by mouse bioassay and PCR in placentas and embryos from C. callosus females infected around 10 days pre-conception. However, only placentas, but not embryos, from females infected around 30 and 50 days pre-conception showed positivity for parasite DNA and seroconversion by mouse bioassay. In conclusion, this study model shows that vertical transmission of T. gondii may take place when maternal infection occurs within one month before conception, thus demonstrating the time of preconceptional seroconversion that rule out a risk of congenital toxoplasmosis. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Toxoplasma gondii; Calomys callosus; Congenital toxoplasmosis; Preconceptional infection; Maternalefetal interface
1. Introduction Toxoplasmosis is an infection caused by the protozoan parasite Toxoplasma gondii and is usually asymptomatic in immunocompetent subjects. However, when the infection occurs in immunocompromised patients or during pregnancy, it can lead to serious disorders as encephalitis or congenital toxoplasmosis, respectively [1]. The vertical transmission is not obligatory and occurs in 20e50% of maternal primary
* Corresponding author. Tel./fax: þ55 34 3218 2240. E-mail address: [email protected] (E.A.V. Ferro). 0143-4004/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2006.10.011
infection by T. gondii during pregnancy [2] with a more frequent involvement of the strains I and II in humans [3]. Maternal infection prior to conception normally excludes the risk of fetal infection and congenital toxoplasmosis, which usually follows T. gondii infection acquired in early pregnancy [1]. However, preconceptional infection can occasionally lead to implication for the fetus in immunodeficient women [4] and in immunologically competent women with clinical signs as cervical adenopathies [5,6]. Accordingly, two unusual cases of congenital toxoplasmosis were reported, one occurring after preconceptional infection with cervical adenopathies and the other occurring after maternal infection at the very end of pregnancy with maternal seronegativity at delivery [7]. In
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the infection acquired at the very end of pregnancy, with the mother still seronegative at delivery, the rate of transmission from mother to fetus is around 70% during this period [8]. Thus, the risk of congenital toxoplasmosis can be better evaluated when the maternal seroconversion and the time of primary infection is more accurately determined to lead to an efficient therapeutic strategy. If the infection is confirmed, anti-parasitic treatment during pregnancy may prevent fetal infection and serious damages, such as chorioretinitis or neurological defects [1]. Calomys callosus, a rodent of the family Cricetidae widely distributed in Central Brazil, has been reported as important natural reservoir for Trypanosoma cruzi [9,10], but also susceptible to several infectious diseases as schistosomosis and leishmaniasis [11]. Our previous studies have demonstrated the high susceptibility of C. callosus to T. gondii infection, when the presence of the parasite was detected in several tissues, particularly liver, spleen, lung and brain [12]. Also, this rodent was demonstrated to be a suitable experimental model to study the dynamics of congenital toxoplasmosis, due to the ability of a highly virulent strain of T. gondii (RH strain) to infect trophoblast cells during the early blastocysteendometrial relationship [13]. In another previous study, we demonstrated the vertical transmission of T. gondii in C. callosus acutely infected with the ME49 strain during pregnancy, but not in chronically infected animals. In addition, considering the sequence of events leading to the infection of the various organs, the placental trophoblast cells are infected later on during pregnancy, but were unable to completely stop the progression of T. gondii infection towards the fetal tissues [14]. Considering the possibility of vertical transmission of T. gondii when maternal seroconversion occurs a few weeks before conception and that so far this condition has not been kinetically studied in detail, the present study aimed to verify if the susceptibility to vertical transmission of T. gondii is temporally dependent on the preconceptional infection in C. callosus.
2. Materials and methods 2.1. Animals C. callosus of the Canabrava strain came from a resident colony housed at the Institute of Tropical Medicine of S~ao Paulo and were kindly provided by Dr Judith Kloetzel. The animals were kept under specific pathogen-free conditions on a 12-h light, 12-h dark cycle in a temperature-controlled room (25 2 C) with food and water ad libitum. All procedures were conducted according to institutional guidelines for animal ethics.
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2.3. Experimental groups Twelve C. callosus virgin females aged 2e3 months were perorally infected with 20 cysts of T. gondii ME49 strain and then divided into three groups of four animals that were mated with males after different times of infection as follows: group 1 (8e13 days after infection), group 2 (28e35 days after infection), and group 3 (42e57 days after infection). The presence of a vaginal plug was considered 1st day of pregnancy (dop). The animals were euthanised from the 17th to 20th dop, when placentas and embryos were collected for morphological and immunohistochemical assays, mouse bioassay and polymerase chain reaction (PCR) for the detection of Toxoplasma. Blood samples were collected at 1st dop and when animals were euthanised (17th to 20th dop) to determine the levels of IgG antibodies to T. gondii by immunoenzymatic assays (ELISA).
2.4. Morphological and immunohistochemical assays For conventional light microscopy, specimens were fixed by immersion in 10% formalin in 0.1 M phosphate buffer (pH 7.4), dehydrated, and embedded in methacrylate resin. Sections measuring 2 mm in thickness were stained with 0.25% toluidine blue and then examined in a photomicroscopy (Reichert-JungPolyvar, Lab. Optica Robert Koch, Austria). For immunolocalisation of the parasites, specimens fixed were dehydrated and embedded in paraffin. Sections measuring 4 mm in thickness were placed on glass slides and processed as previously described [14]. Briefly, samples were first incubated (10 min at room temperature) with 5% acid acetic to block endogenous alkaline phosphatase and then (30 min at 37 C) with 2% normal goat serum to block non-specific binding sites. Next, samples were incubated (12 h at 4 C) with rabbit anti-T. gondii serum and then (30 min at 37 C) with biotinylated goat anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO, USA). The reaction was amplified by using the ABC system (Biomeda, Foster City, CA, USA) and developed with fast red-naphthol (Sigma). Samples were counterstained with Mayer’s haematoxylin and examined in photomicroscopy.
2.5. Detection of T. gondii in placentas and embryo tissues Detection of T. gondii was first evaluated by mouse bioassay as described elsewhere [15], with minor modifications. Placentas and embryo tissues (liver and brain) were homogenised in PBS and separately inoculated in mice Swiss by intraperitoneal route, in duplicate. After 30e45 days of inoculation, blood samples from mice were collected and sera obtained were analysed for the detection of IgG antibodies to T. gondii in ELISA. The presence of T. gondii DNA was investigated by PCR as previously described [16]. Briefly, placentas and embryo tissues were treated with 100 mg/ ml proteinase K (Invitrogen Life Technologies, S~ao Paulo, Brazil) in buffer containing 10 mM TriseHCl, 0.1 M EDTA, 0.5% SDS, pH 8.0, at 50 C during 3 h. Samples were then submitted to phenol/chloroform/isoamyl alcohol (25:24:1, pH 8.0) extraction and DNA was precipitated from the aqueous phase by treatment with 2.5 volumes of cold ethanol. Qualitative PCR detecting the 35-copy B1 gene of T. gondii was performed using the primers 50 TCTTCCCAGAGGTGGATTTC-30 (sense, nucleotides 151e171) and 50 CTCGACAATACGCTGCTTG-30 (antisense, nucleotides 682e663) (Invitrogen Life Technologies, S~ao Paulo, Brazil), which should amplify a fragment of 531 bp. After initial incubation for 3 min at 95 C, samples were subjected to 38 cycles of denaturing at 94 C for 1 min, annealing for 1.2 min at 62 C, and extension for 2 min at 72 C [17]. PCR products were analysed by 1% agarose gel containing 0.5 mg/ml of ethidium bromide and visualised under UV illumination.
2.2. Parasites 2.6. ELISA and ELISA-avidity Cysts of T. gondii ME49 strain were obtained from brains of C. callosus infected 30e45 days earlier with 20 cysts via oral route. The brains were removed, washed in sterile 0.01 M phosphate-buffered saline (PBS) pH 7.2, and homogenised with a syringe and a 25 7 gauge needle. The brain preparations were further washed by centrifugation at 1000 g for 10 min in PBS and cysts were counted under light microscopy (10 magnification).
A conventional ELISA to detect IgG antibodies to T. gondii was carried out as previously described [14] with some modifications in order to confirm the pre-conceptional seroconversion as indicator of infection of C. callosus females. Polystyrene microtitre plates were coated overnight at 4 C with T. gondii soluble antigen at 10 mg/ml in carbonate buffer 0.06 M (pH 9.6). Plates
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were washed with PBS plus 0.05% Tween-20 (PBST) and incubated with serum samples of C. callosus diluted at 1:32 in PBST for 1 h at 37 C. After washing, plates were incubated with peroxidase labelled rabbit anti-C. callosus IgG, prepared according to Wilson and Nakane [18] diluted at 1:250 in PBST and incubated for 1 h at 37 C. After new washes, the reaction was developed with a substrate solution consisting of 0.03% hydrogen peroxide and 1 mg/ml of o-phenylenediamine (OPD) in 0.1 M citrate-phosphate buffer (pH 5.0). The reaction was stopped with 2 N H2SO4 and optical density (OD) was measured at 492 nm by using a plate reader (Titertek Multiskan Plus, Flow Laboratories, Geneva, Switzerland). Results were expressed in ELISA index (EI) as follows:
EI ¼ OD sample/cut-off, where cut-off was established as mean OD values of negative control sera plus three standard deviations. Based on screening tests performed with negative and positive control sera, EI > 1.2 values were considered positive results. To determine phases of infection (recent or chronic), an ELISA-avidity was conducted as described elsewhere [19] with some modifications. It was performed as above described for conventional ELISA, except for a modification in the serial washing step just after incubation with serum samples (diluted 1:32) in quadruplicate. Plates were washed once with PBST and half the wells were soaked for 10 min with 6 M urea in PBST. After three
Fig. 1. Photomicrographs of placentas and embryo tissues from C. callosus infected with ME49 strain of T. gondii in different preconceptional time periods and analysed from 17th to 20th days of pregnancy. Staining by toluidine blue shows the presence of parasites in (a) labyrinth cells (asterisk) at the transition region to the spongiotrophoblast area in placenta from animals of group 1 (8e13 days of preconceptional infection) and (b) decidua (asterisks) from animals of group 2 (28e35 days of preconceptional infection); absence of parasites in (c) trophoblastic cells (arrows) and spongiotrophoblast area (star) from animals of group 3 (42e57 days of preconceptional infection). Immunohistochemical staining using alkaline phosphatase and fast red naphthol showing the presence of parasites in (d) brain areas from embryos of group 1 (arrowhead); absence of parasites in (e) brain and (f) liver areas from embryos of groups 2 and 3, respectively. Counterstaining by Mayer’s haematoxylin. Bars: a,c,e,f: 35 mm; b,d: 14 mm.
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Immunohistochemical assay revealed the presence of T. gondii in the labyrinth zone cells of animals of groups 1 and 2 (data not shown). Only embryos from animals of group 1 were multi-infected including brain, liver, back, connective tissue and retro-abdominal region (Fig. 1d). In contrast, embryos from females of groups 2 and 3 did not show any evidence of parasites (Fig. 1e and f). Serological assays carried out at 1st dop showed that seroconversion was verified only in one animal with 13 days of infection of group 1 (Fig. 2a), presenting a low IgG avidity (IA ¼ 38%), while animals with 8e9 days of infection were seronegative to T. gondii (EI < 1.2). In contrast, all animals of group 1 showed high serum levels of antibodies to T. gondii with IgG avidity ranging from 40% to 62% (mean IA ¼ 51%), when analysed from 17th to 20th dop corresponding to 25e 27 days after infection (Fig. 2a). In group 2 (Fig. 2b), all animals were seropositive to T. gondii at both time periods analysed: 1st dop (28e35 days after infection) and from 17th to 20th dop (48e52 days after infection) showing mean IgG avidity indexes of 63% (range 58e66%) and 65% (range 54e80%), respectively. In the same way, all animals of group 3 showed high levels of antibodies to T. gondii at two time periods analysed: 1st dop (42e57 days after infection) and from 17th to 20th dop (58e 74 days after infection) showing mean IgG avidity indexes of 72% (range 61e91%) and 73% (range 71e78%), respectively (Fig. 2c). Mouse bioassay revealed seroconversion (EI > 1.2) in all mice that were inoculated with placentas and embryo tissues from C. callosus females of group 1 in all periods of time analysed (Fig. 3a), but only in mice inoculated with placentas
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IA
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3.1. Placental labyrinth zone and basal decidua from C. callosus are the sites most often infected by T. gondii No morphological changes were observed in placentas of C. callosus infected with T. gondii, but parasites, when present, were noted in all three layers: basal decidua, junctional zone and labyrinth zone. In animals of group 1 (8e13 days of preconceptional infection) and group 2 (28e35 days of preconceptional infection), cells of the labyrinth zone and of basal decidua were the most often infected (Fig. 1a and b). No parasite was found in placentas from animals of group 3 (42e57 days of preconceptional infection) (Fig. 1c).
EI
IgG avidity index (IA %)
additional washes, plates were incubated with peroxidase conjugate and revealed as above described. Results were expressed as ELISA index (EI) and the index of avidity (IA) was calculated by using the following formula: IA (%) ¼ [(EI samples with urea)/(EI samples without urea)] 100. Low avidity was arbitrarily considered for IA < 40%, intermediate for IA between 40% and 70%, and high avidity for IA > 70%, based on previous reports [14].
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Days after infection/pregnancy Fig. 2. Levels of IgG antibodies to T. gondii expressed in ELISA index (EI) and IgG avidity index (IA) expressed in percentage (%) in serum samples of C. callosus infected with ME49 strain of T. gondii prior conception. (a) animals of group 1 with 8e13 days of preconceptional infection, (b) animals of group 2 with 28e35 days of preconceptional infection and (c) animals of group 3 with 42e57 days of preconceptional infection were analysed at 1st and from 17th to 20th days of pregnancy.
from females of group 2 (Fig. 3b). In animals of group 3, seroconversion was verified only in mice inoculated with placentas from 17th and 18th dop (Fig. 3c). T. gondii DNA was detected in placentas and embryo tissues from all females of group 1 from 17th to 20th dop. However, T. gondii DNA was detected in placentas, but not in embryos, from all females of group 2, for all periods of time analysed. Concerning group 3, only placentas showed parasite DNA at 17th and 18th dop (Fig. 4). Table 1 summarises the results obtained for all assays to determine the presence of T. gondii in placenta and embryo tissues from C. callosus females, as well as the maternal seroconversion in different groups of animals infected before conception. 4. Discussion Toxoplasmosis is routinely diagnosed by using serologic tests for the detection of specific antibodies [3]. In the last
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Fig. 4. Representative PCR analysis of the T. gondii 35-copy B1 gene in placentas and embryo tissues collected from 17th to 20th days of pregnancy in C. callosus females infected with T. gondii ME49 strain around 10, 30 or 50 days prior conception. (1) 500 bp DNA ladder (Invitrogen); (2) T. gondii (RH strain) tachyzoite DNA (positive control); (3) placenta and (4) embryo tissues from non-infected C. callosus females (negative controls); (5) placenta and (6) embryo tissues from C. callosus females infected with T. gondii ME49 strain around 10 days prior conception (group 1); (7) placenta and (8) embryo tissues from C. callosus females infected with T. gondii ME49 strain around 30 days prior conception (group 2); (9) placenta and (10) embryo tissues from C. callosus females infected with T. gondii ME49 strain around 50 days prior conception (group 3). Numbers on the left indicate the molecular markers in base pairs (bp).
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Days of pregnancy Fig. 3. Levels of IgG antibodies to T. gondii expressed in ELISA index (EI) in serum samples of mice in bioassay. Mice were inoculated with placentas or embryo tissues collected from 17th to 20th days of pregnancy in C. callosus infected with T. gondii ME49 strain around 10 days (a), 30 days (b) or 50 days (c) prior to conception. Data are represented as mean SEM.
decades, specific IgG antibody avidity tests have been shown to be very useful for diagnosing recent infections and characterising the time of acquisition of Toxoplasma infection [19]. The risk of congenital toxoplasmosis is raised when maternal seroconversion is confirmed and the time of acquisition of infection is determined [8,20]. In the present study, recent or chronic phases of the infection were determined based on the results of serological assays by ELISA and ELISA avidity
that were performed at 1st dop and from 17th to 20th dop. Animals of group 1 (8e13 days of preconceptional infection) were either seronegative or exhibited a profile of low IgG avidity (IA ¼ 38%) at 1st dop, thus characterising a recent infection. IgG avidity showed an increasing profile (IA ¼ 51%) when analysed from 17th to 20th dop (25e27 days after infection), indicating an affinity maturation of IgG synthesis and reflecting a possible transient phase of infection. Animals from both group 2 and group 3 showed high levels of antibodies to T. gondii at time periods analysed with increasing IgG avidity indexes (IA ¼ 63% and IA ¼ 72%, respectively), showing characteristic profiles that lead to a chronic infection. In humans, T. gondii-specific IgG avidity may be broadly varied among individuals, but the presence of high avidity antibodies essentially rules out infection acquired in the last 3e5 months, whereas low avidity antibodies can persist beyond 3 months of infection [21]. In the present study, we evaluated the risk of congenital toxoplasmosis in C. callosus in three intervals of preconceptional infection with the ME49 strain of T. gondii. Vertical transmission was confirmed by morphological and immunohistochemical assays, as well as by mouse bioassay and PCR in females infected around 10 days (group 1) prior conception, but not around 30 and 50 days preconception (groups 2 and 3), respectively indicating that congenital toxoplasmosis does not take place when maternal infection occurs up to about one month before conception in this experimental model. As seen in other rodents, the labyrinth zone is the greatest area of the placenta from C. callosus and it is considered the
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Table 1 Summarised data obtained in all assays to determine the presence of Toxoplasma gondii in placenta and embryo tissues from Calomys callosus females and the maternal seroconversion in different groups of animals infected before conception Groups of animalsa
Parasite detection Immunohistochemical staining
Mouse bioassay
PCR
Maternal seroconversionb
Placenta
Embryo tissues
Placenta
Embryo tissues
Placenta
Embryo tissues
ELISA index (EI)
ELISA avidity (IA %)
Group 1 17 dop 18 dop 19 dop 20 dop
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
2.9 2.7 3.0 2.6
49.8 39.4 53.7 61.9
Group 2 17 dop 18 dop 19 dop 20 dop
þ þ þ þ
þ þ þ þ
þ þ þ þ
3.7 3.7 4.1 3.6
80.7 62.7 54.3 61.4
Group 3 17 dop 18 dop 19 dop 20 dop
þ þ
þ þ
3.1 3.2 3.5 3.6
71.2 71.0 71.8 77.8
a Animals were infected with T. gondii prior to conception at different time periods: group 1 (8e13 days of preconceptional infection), group 2 (28e35 days of preconceptional infection) and group 3 (42e57 days of preconceptional infection). Then, animals were euthanised and analysed at 17, 18, 19 and 20 days of pregnancy (dop). þ, presence of parasite; , absence of parasite. b Maternal seroconversion was determined by measuring levels of IgG antibodies to T. gondii in ELISA and expressed in ELISA index (EI), and values of EI > 1.2 were considered positive. IgG avidity was also determined by ELISA-avidity and expressed in percentage of avidity index (IA %). Low avidity was arbitrarily considered for IA < 40%, intermediate for IA between 40% and 70%, and high avidity for IA > 70%.
main region of nutrient exchanges between maternal and fetal blood. Three layers of trophoblast cells plus the fetal endothelium constitute this placental barrier that becomes more and more slender with the advance of the pregnancy. Therefore, once infected by pathogens there is no difficulty for the pathogens to reach the fetal tissues [22]. Our previous studies have demonstrated that C. callosus is a useful experimental model for congenital toxoplasmosis, since vertical transmission was observed in females infected with ME49 strain of T. gondii in acute phase, but not in chronic phase of the infection [14]. It has been demonstrated that maternal T. gondii infection in C. callosus before conception normally excludes the risk of fetal transmission [14]. However, the present study showed that this might not be true when maternal infection occurs a few weeks before conception, as demonstrated by results obtained by mouse bioassay and PCR. It was demonstrated that the T. gondii replication is restricted in the placental microenvironment, no crossing the placenta barrier and, thus, preventing the vertical transmission of the parasite to the embryo, when infection occurred 30 or 50 days before conception. Thus, the time of maternal seroconversion that rule out a probable risk of congenital toxoplasmosis in C. callosus is around 30 days preconception. It is worth noting that these results agree with our previous studies demonstrating that congenital infection did not occur in C. callosus females infected with T. gondii 100 days before conception [14]. As already reported in humans, further studies should be conducted in the C. callosus model to verify the possibility of materno-fetal transmission of T. gondii when maternal infection occurs at the very end of pregnancy, even when signs of seroconversion are absent at delivery [7,23].
A recent study has demonstrated that maternal infection with a variety of T. gondii strains occurring 2 months before conception in SpragueeDawley rats may act as a type of immunisation that prevents the vertical transmission of this parasite [15]. Accordingly, our findings demonstrated that as early as 1 month preconceptional infection can guarantee fetal protection in immunocompentent C. callosus. In conclusion, the results presented herein strengthen the role of C. callosus as a useful congenital toxoplasmosis model, since the vertical transmission occurs in the acute phase of infection, including unusual cases of preconceptional infection, as previously reported in human congenital toxoplasmosis [7,6]. In addition, the time of acquisition of preconceptional seroconversion that rules out a probable risk of congenital toxoplasmosis could be established in this experimental model. Acknowledgements This work was supported by Brazilian Research Agencies (FAPEMIG, CNPq and CAPES). References [1] Remington JS, McLeod R, Thulliez P, Desmonts G. Toxoplasmosis. In: Remington JS, Klein OJ, editors. Infectious disease of the fetus and newborn infant. 5th edn. Philadelphia: WB Saunders 2001. p. 205e346. [2] Jones JL, Lopez A, Wilson M, Schulkin J, Gibbs R. Congenital toxoplasmosis: a review. Obstetric Gynecol Surv 2001;56:296e305. [3] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004;363:1965e76.
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[4] Desmonts G, Couvreur J, Thulliez P. Congenital toxoplasmosis: five cases with mother-to-child transmission of pre-pregnancy infection. Press Med 1990;19:1445e9. [5] Villena I, Chemla C, Quereux C, Dupouy D, Leroux B, Foudrinier F, et al. Prenatal diagnosis of congenital toxoplasmosis transmitted by an immunocompetent woman infected before conception. Prenat Diagn 1998;18:1079e81. [6] Vogel N, Kirisits M, Michael E, Bach H, Hostetter M, Boyer K, et al. Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception. Clin Infect Dis 1996;23:1055e 60. [7] Chemla C, Villena I, Aubert D, Hornoy P, Dupouy D, Leroux B, et al. Preconceptional seroconversion and maternal seronegativity at delivery do not rule out the risk of congenital toxoplasmosis. Clin Diagn Lab Immunol 2002;9:489e90. [8] Dunn D, Wallon M, Peyron F, Petersen E, Peckham C, Gilbert R. Mother-to-child transmission of toxoplasmosis: risk estimates for clinical counselling. Lancet 1999;353:1829e33. [9] Borges MM, De Andrade SG, Pilatti CG, Do Prado Junior JC, Kloetzel JK. Macrophage activation and histopathological findings in Calomys callosus and Swiss mice infected with several strains of Trypanosoma cruzi. Mem Inst Oswaldo Cruz 1992;87:493e502. [10] Mello DA, Valin E, Teixeira ML. Various aspects of the behavior of wild strains of Trypanosoma cruzi in mice and Calomys callosus (Rodentia). Rev Sau´de Pu´blica 1979;13:314e25. [11] Mello DA. Note on breeding of Calomys expulsus, Lund, 1841 (Rodentia, Cricetidae) under laboratory condition. Rev Bras Pesq Med Biol 1977;10:107. [12] Favoreto Jr S, Ferro EAV, Clemente D, Silva DAO, Mineo JR. Experimental infection of Calomys callosus (Rodentia, Cricetidae) by Toxoplasma gondii. Mem Inst Oswaldo Cruz 1998;93:103e7. [13] Ferro EAV, Bevilacqua E, Favoreto Jr S, Silva DAO, Mortara RA, Mineo JR. Calomys callosus (Rodentia: Cricetidae) trophoblast cells as host cells to Toxoplasma gondii in early pregnancy. Parasitol Res 1999;85:647e54. [14] Ferro EAV, Silva DAO, Bevilacqua E, Mineo JR. Effect of Toxoplasma gondii infection kinetics on trophoblast cell population in Calomys
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