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Azithromycin Inhibits Vertical Transmission of Toxoplasma gondii in Calomys callosus (Rodentia: Cricetidae)
Placenta 30 (2009) 884–890
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Azithromycin Inhibits Vertical Transmission of Toxoplasma gondii in Calomys callosus (Rodentia: Cricetidae) I.N. Costa a, M.B. Angeloni a, L.A. Santana a, B.F. Barbosa a, M.C.P. Silva a, A.A. Rodrigues a, C. Rostkowsa b, P.M. Magalha˜es c, J.D.O. Pena b, D.A.O. Silva b, J.R. Mineo b, E.A.V. Ferro a, * a
ˆndia, Av. Para ´ 1720, Bloco 2B, Uberla ˆndia 38405-320, MG, Brazil Laboratory of Histology and Embriology, Instituto de Cieˆncias Biome´dicas, Universidade Federal de Uberla ˆ ndia, Av. Para ´ , 1720, Bloco 4C, Uberla ˆ ndia 38405-320, MG, Brazil Laboratory of Immunoparasitology, Universidade Federal de Uberla c Laboratory of Agrotechnology, Universidade Estadual de Campinas, Rua Alexandre Cazelatto, 999, Vila Betel, Campinas 13081-970, SP, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 4 August 2009
Toxoplasma gondii infection during pregnancy may cause severe consequences to the embryo. Current toxoplasmosis treatment for pregnant women is based on the administration of spiramycin or a drug combination as sulphadiazine-pyrimethamine-folinic acid (SPFA) in cases of confirmed fetal infection. However, these drugs are few tolerated and present many disadvantages due to their toxic effects to the host. The aim of this study was to evaluate the effectiveness of different treatments on the vertical transmission of T. gondii, including azithromycin, Artemisia annua infusion, spiramycin and SPFA in Calomys callosus as model of congenital toxoplasmosis. C. callosus females were perorally infected with 20 cysts of T. gondii ME49 strain at the day that a vaginal plug was observed (1st day of pregnancy – dop). Treatment with azithromycin, A. annua infusion, and spiramycin started at the 4th dop, while the treatment with SPFA started at the 14th dop. Placenta and embryonic tissues were collected for morphological and immunohistochemical analyses, mouse bioassay and PCR from the 15th to 20th dop. No morphological changes were seen in the placenta and embryonic tissues from females treated with azithromycin, spiramycin and SPFA, but embryonic atrophy was observed in animals treated with A. annua infusion. Parasites were found in the placenta and fetal (brain and liver) tissues of animals treated with SPFA, A. annua infusion and spiramycin, although the number of parasites was lower than in nontreated animals. Parasites were also observed in the placenta of animals treated with azithromycin, but not in their embryos. Bioassay and PCR results confirmed the immunohistochemical data. Also, bradyzoite immunostaining was observed only in placental and fetal tissues of animals treated with SPFA. In conclusion, the treatment with azithromycin showed to be more effective, since it was capable to inhibit the vertical transmission of T. gondii in this model of congenital toxoplasmosis. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Calomys callosus Congenital toxoplasmosis Treatment Azithromycin
1. Introduction Human toxoplasmosis is a disease worldwide distributed and caused by the protozoan Toxoplasma gondii. In immunocompetent individuals the acute infection is asymptomatic and self-limited, persisting in a latent form as tissue cysts [1]. In immunocompromised hosts, however, it may cause retinochoroiditis and fatal encephalitis when not treated [2,3]. Similarly, T. gondii infection during pregnancy may cause severe consequences to the embryo [4]. T. gondii may cross the placental barrier, causing neurological damage to the fetus and miscarriages [5]. Current toxoplasmosis treatment for pregnant women is based on the administration of
* Corresponding author. Tel./fax: þ55 34 3218 2240. E-mail address: [email protected] (E.A.V. Ferro). 0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2009.08.002
spiramycin in order to decrease the risk of fetal transmission. This decreased risk is related to high concentrations of spiramycin in the placental tissue, whereas disadvantage of this drug may be explained by the limited effectiveness in the parasite clearance and low penetration in fetal tissues [6]. If the diagnosis reveals fetal infection, the drug combination as sulphadiazine-pyrimethamine is usually indicated [7]. Sulphadiazine is a dihydropteroate synthase (DHPS) inhibitor, while pyrimethamine inhibits the dihydrofolate reductase (DHFR), and both enzymes are fundamental for the biosynthesis of T. gondii pyrimidines [8]. Even though the standard treatment recommended for toxoplasmosis is the combination of these drugs, numerous toxicity problems have been described [9], in addition to the potential teratogenic effect of pyrimethamine in the first trimester of pregnancy [6]. Suppression of the bone marrow is one of the most adverse effects of this treatment, although it may be
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prevented by simultaneous administration of folinic acid [4,10]. Despite these drugs have strategic action by inhibiting essential enzymes for the T. gondii metabolism, they are few tolerated, especially when used for long periods of time, and they present many disadvantages due to their harmful toxic effects to the host. Thus, it is imperative the development of studies that allow the validation of new targets of drugs in this field [4,5,11]. Azithromycin is one of the new generation macrolides with numerous advantages, such as a high oral bioviability, a rapid cell absorption and wide distribution in the organism, and administration only once a day [5]. In addition, azithromycin presents better pharmacokinetics and greater tissue concentration than spiramycin, and has also shown a lower incidence of side effects, especially hepatotoxic effects [6]. Azithromycin mechanism of action is based on the inhibition of protein synthesis in both T. gondii tachyzoite and bradyzoite stages [6,12], but it may present limited effectiveness against T. gondii, requiring high drug concentration [6]. Artemisinin, an active component present in the herb Artemisia annua L., has been investigated as alternative treatment for toxoplasmosis [13]. The infusion of this herb has been used in the malaria treatment, a disease caused by another Apicomplexa protozoan, Plasmodium sp [14] and recently it was also investigated in experimental acquired toxoplasmosis [15]. Artemisinin and derivatives have been effective against T. gondii [16,17], but there are no studies currently that prove a possible effectiveness of artemisinin in the treatment of congenital toxoplasmosis. Calomys callosus is a rodent of the Cricetidae family found largely in Central Brazil. Previous studies have shown a high susceptibility of these animals to T. gondii, being considered suitable experimental models to study the dynamics of congenital toxoplasmosis [18–20]. It was demonstrated that when these animals are infected with the T. gondii ME49 strain, vertical transmission mainly occurs during the acute stage, likewise in humans [18,20]. However, there are no studies focusing conventional or alternative treatment for congenital toxoplasmosis in this animal model. The aim of this study was to evaluate the effectiveness of different treatments on the vertical transmission of T. gondii, including azithromycin, A. annua infusion, spiramycin and drug association as sulphadiazine-pyrimethamine-folinic acid (SPFA) in C. callosus as model of congenital toxoplasmosis.
885
2.4. Experimental groups Ninety-six C. callosus virgin females aged 3 months, weighting 30 2 g, were mated with males and checked daily for the presence of a vaginal plug that was considered as the first day of pregnancy (dop). All pregnant females were divided into four groups of 24 animals according to each drug treatment, as follows: group I (azithromycin), group II (A. annua infusion), group III (spiramycin) and group IV (SPFA). Each group was then subdivided into four subgroups of six animals per group, being two subgroups perorally infected with 20 cysts of T. gondii ME 49 strain in the 1st dop, and treated with the respective drugs or PBS (control) as illustrated in Fig. 1. Animals of the group I received azithromycin (9 mg/24 h), animals of the group II received A. annua infusion (1.0 mg/8 h) and animals of the group III received spiramycin (0.15 mg/8 h), from the 4th dop until the euthanasia day. Animals of the group IV received a drug combination (SPFA) consisting of sulphadiazine (1.5 mg/ 12 h), pyrimethamine (0.025 mg/12 h), and folinic acid (0.0075 mg/24 h) from 14th dop until the euthanasia day. All drugs were administered by oral route and diluted in 0.5 ml of sterile PBS, except for the A. annua infusion that was prepared in water. The doses of spiramycin and SPFA herein used were adapted to C. callosus body weight based on the recommended doses for pregnant women [15,23], whereas the azithromycin dose was used as described previously [6]. The animals were euthanized from the 15th to 20th dop, when placenta and embryonic tissues were collected for morphological and immunohistochemical analyses, mouse bioassay and polymerase chain reaction (PCR) to detect the parasite. Blood samples were also collected in the 1st dop and when animals were euthanized (15th to 20th dop) to determine the levels of IgG antibodies to T. gondii by immunoenzymatic assays (ELISA). 2.5. Morphological and immunohistochemical analyses For morphological analyses, specimens were fixed in 10% formalin and 0.1 M phosphate buffer (pH 7.4), dehydrated and embedded in glycol-methacrylate resin. Sections with 2 mm were stained with 0.25% toluidine blue and examined in a photomicroscope (Reichert Jung Polyvar, Lab. Optica Robert Koch, Austria). For immunolocalization of the parasites, fixed specimens were dehydrated and embedded in paraffin. Sections with 4 mm were placed on glass slides and processed as previously described [18]. Samples were first incubated for 10 min at room temperature with 5% acid acetic to block endogenous alkaline phosphatase or alternatively, with 3% hydrogen peroxide to block endogenous peroxidase, and then
2. Materials and methods 2.1. Animals C. callosus (Canabrava strain) came from a resident colony housed at the Institute of Biomedical Sciences, Federal University of Uberlaˆndia, Brazil. The animals were kept under standard conditions on a 12-h light, 12-h dark cycle in a temperaturecontrolled room (25 2 C) with food and water ad libitum. All procedures were conducted according to institutional guidelines for animal ethics. 2.2. Parasites Cysts of T. gondii ME49 strain were obtained from brains of C. callosus infected orally 30–45 days earlier with 20 cysts as described elsewhere [20]. Brains were removed, washed in sterile 0.01 M phosphate-buffered saline (PBS, pH 7.2), homogenized and cysts were counted under light microscopy. 2.3. Artemisia annua L. infusion Artemisia annua L. seeds (Center of Chemical, Biological and Agricultural Researches, University of Campinas, Brazil) were sown in a greenhouse, the plants were harvested and the aerial parts were dried, ground and stored at low humidity [15]. A. annua L. infusion was prepared from the infusion of 2 g of dried herb in 1000 ml boiling distilled water. The mixture was briefly stirred, covered and left in rest for 10 min. Next, the mixture was filtered and cooled at room temperature [21,22]. A. annua L. infusion was stored as stock solution (2 mg/ml) at 4 C in the dark and showed a specific content of artemisinin of 4.906 mg/ml (0.2% of total) by HPLC [15].
Fig. 1. Schedule of the experimental design carried out with pregnant Calomys callosus females infected or not with Toxoplasma gondii ME49 strain and submitted to different types of treatment.
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with 2% normal goat serum or alternatively, bovine serum albumin (BSA) for 30 min at 37 C to block non-specific binding sites. Next, samples were incubated for 12 h at 4 C with hyperimmune rabbit anti-T. gondii or anti-BAG5 (bradyzoite antigen-5) sera and with biotinylated goat anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO, USA) for 30 min at 37 C. The reaction was amplified by using the streptavidinbiotinylated alkaline phosphatase complex (Biomeda, Foster City, CA, USA) and developed with fast red-naphtol (Sigma). Alternatively, the reaction was amplified with streptavidin-biotinylated peroxidase complex (DAKO Corporation, CA, USA) and developed with fast 3,30 -diaminobenzidine tablet sets (Sigma), when appropriate. Samples were counterstained with Mayer’s hematoxylin and examined under a light microscope (BX40, Olympus, Tokyo, Japan) and an image analysis software
(HLImageþþ97, Western Vision Software, San Diego, CA, USA). Analysis was done in three sections of each tissue, totalizing 60 images for each analyzed tissue. Results were expressed in immunostaining index (IS) by using the following formula: IS ¼ number of stained images/0.01872 total number of images, where 0.01872 represents the fixed area (mm2) of each image determined by the software.
2.6. Bioassay and PCR Detection of T. gondii was first evaluated by mouse bioassay as described elsewhere [20,24]. Placentas and embryonic tissues (liver and brain) were homogenized in PBS and separately inoculated intraperitoneally in Swiss mice, in duplicate. After
Fig. 2. Photomicrographs of placental and embryonic tissues from Calomys callosus females infected with Toxoplasma gondii ME49 strain at the 1st day of pregnancy (dop) and treated with azithromycin, Artemisia annua infusion, spiramycin, drug association (SPFA) or sterile PBS. (a) tachyzoites (arrowheads) in the placental junctional zone of females treated with SPFA at 19th dop; (b) tachyzoites (arrowheads) in the placental junctional zone from females treated with PBS at 19th dop; (c) parasites (arrow) in placental junctional zone from females treated with spiramycin at 19th dop. (d) Parasites (arrow) in the placental junctional zone from females treated with PBS at 18th dop; (e) parasites (arrow) in the placental junctional zone from females treated with A. annua infusion at 18th dop; (f) parasites are not observed in liver of embryos from females treated with azithromycin at 19th dop; (g) parasites (arrow) in the liver of embryos from females treated with SPFA at 19th dop. (a, b, g): Immunohistochemical staining using alkaline phosphatase and Fast red naphthol, counterstaining by Mayer’s hematoxylin;(c–f): Immunohistochemical staining using horseradish peroxidase and Fast DAB, counterstaining by Mayer´s hematoxylin. Bars: 35 mm (a, b, d, e and f); 70 mm (c); 10 mm (g).
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A conventional ELISA to detect IgG antibodies to T. gondii was carried out as previously described [20]. Plates were coated overnight at 4 C with T. gondii soluble antigen (10 mg/ml) in carbonate buffer 0.06 M (pH 9.6). Plates were washed with PBS plus 0.05% Tween 20 (PBS-T) and incubated with serum samples of C. callosus or Swiss mice diluted at 1:16 in PBST plus 5% skim milk (PBS-TM) for 1 h at 37 C. After washing, plates were incubated with peroxidase labeled rabbit anti-C. callosus IgG prepared according to Wilson and Nakane [27] diluted at 1:250 or peroxidase labeled goat anti-mouse IgG (Sigma) diluted 1:1000 in PBS-TM and incubated for 1 h at 37 C. After new washes, the reaction was developed with 0.03% hydrogen peroxide and 1 mg/ml 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 in 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, values of EI >1.2 were considered positive. 2.8. Statistical analyses Data were expressed as mean standard deviation (SD) and the differences between groups were determined using ANOVA and Bonferroni multiple comparison tests or Student t test, when appropriate (GraphPad Prism version 4.0; GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant when p < 0.05.
3. Results 3.1. Morphological and immunohistochemical analyses No morphological alteration was observed in placental tissues, but the presence of T. gondii parasites in all placental layers (basal decidua, junctional zone and labirint zone) was noted, although with smaller number of parasites in infected C. callosus females and treated with different drugs compared to untreated infected controls. However, embryos in atrophy process were observed in females treated with A. annua infusion (data not shown). Parasite immunostaining was detected in the junctional zone of C. callosus placenta, showing immunostained tachyzoites in the group treated with SPFA (Fig. 2a) in low intensity when compared to untreated controls (Fig. 2b and d). Similar pattern of immunostaining was observed in placental tissues from females treated with spiramycin (Fig. 2c) and A. annua infusion (Fig. 2e). Parasites were not observed in embryos from females treated with azithromycin (Fig. 2f). On the contrary, tachyzoites were frequently detected in the liver of embryos from females treated with SPFA (Fig. 2g), as well as in spiramycin and A. annua infusion groups (data not shown). Bradyzoite immunostaining was detected only in placental tissues from animals treated with SPFA and euthanized from the 15th to 19th dop, although it was scarcely and heterogeneously distributed in relation to tachyzoite immunostaining (data not shown). In infected females treated with azithromycin, there was a predominance of tachyzoites in the placental labyrinth zone from 15th to 19th dop, but no parasites were observed in the placenta at the 20th dop.
Tachyzoite immunostaining was observed in placental tissues from females in all treatment groups, however placentas from untreated controls showed significantly higher IS index than placental tissues from treated females. In addition, tachyzoite IS index in placental tissues from females treated with azithromycin was lower than that observed in placenta from females submitted to the other three treatments (Fig. 3a). Bradyzoite immunostaining was also detected in placental tissues from females of untreated controls, but no immunostaining was seen in the groups treated with azithromycin, A. annua infusion, and spiramycin, except for the SPFA group, which showed a significantly higher IS index (p < 0.05) (Fig. 3a). In embryonic tissues, the parasite IS index was lower when females were treated with SPFA, A. annua infusion and spiramycin as compared to the respective controls. In the embryonic tissues from females treated with azithromycin, no tachyzoite immunostaining was detected (Fig. 3b). In contrast, bradyzoite IS index was higher in embryonic tissues from females treated with SPFA in relation to the respective controls (p < 0.05) (Fig. 3b). Bradyzoite immunostaining was not detected in embryonic tissues from females treated with azithromycin, A. annua infusion and spiramycin (p < 0.05) (Fig. 3b). 3.3. Bioassay and PCR Mouse bioassay results showed that mice inoculated with placentas from infected females and treated with PBS showed seroconversion in the 15th day post-inoculation. On the other hand,
Control / Tachyzoites Treatment / Tachyzoites
a Parasite IS index (mm 2) in plac ental tiss ues
2.7. ELISA
3.2. Parasite immunostaining quantification
Control / Bradyzoites
20
Treatment / Bradyzoites
*
15
10
* 5 #
b
*
*
*
0
Parasite IS index (mm 2) in embryonic tiss ues
15, 30 and 45 days of inoculation, blood samples from mice were collected and sera obtained were analyzed for the detection of IgG antibodies to T. gondii in ELISA. The presence of T. gondii DNA was investigated by PCR as previously described [20,25]. Placentas and embryonic tissues previously treated with 100 mg/ml proteinase K were 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 was performed using the primers 50 TCTTCCCAGAGGTGGATTTC-30 (sense, nucleotides 151–171) and 50 CTCGACAATACGCTGCTTG-30 (antisense, nucleotides 682–663), amplifying a fragment of 531 bp of the B1 gene of T. gondii. 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 [26]. PCR products were analyzed in 1% agarose gel containing 0.5 mg/ml of ethidium bromide and visualized under UV illumination.
887
Azithromycin
&
*
*
A. annua infusion
* Spiramycin
SPFA
5 4
*
*
*
* 3
*
2 1 0
#
*
Azithromycin
* A. annua infusion
&
* Spiramycin
SPFA
Fig. 3. Tachyzoite and bradyzoite immunostaining index (IS) in placentas (a) and embryonic tissues (b) from Calomys callosus females infected with Toxoplasma gondii (ME49 strain) and treated with azithromycin, Artemisia annua infusion, spiramycin, drug association (SPFA) or sterile PBS (control). * Statistically significant differences for parasite IS index between control and all treatment groups. # Statistically significant differences for tachyzoite IS index between treatment with azithromycin and the other treatment groups. & Statistically significant differences for bradyzoite IS index between treatment with SPFA and the other treatment groups. Data are represented in mean SD of all values of independent dop (15th to 20th).
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mice inoculated with placental tissues from infected females and treated with SPFA demonstrated a delayed seroconversion, that is, in the 45th day post-inoculation. Similar results were observed in the group treated with azithromycin, even though it was observed seroconversion only in mice inoculated with placental tissues. No seroconversion was found in mice that were inoculated with placentas from non-infected C. callosus females. The presence of T. gondii DNA in placental tissues from infected females and treated with azithromycin, A. annua infusion, spiramycin, SPFA or PBS was also confirmed by PCR (Table 1 and Fig. 4). Bioassay data demonstrated no seroconversion in mice inoculated with liver and brain from embryos of infected females and treated with azithromycin, while those inoculated with embryonic tissues from females treated with SPFA had seroconversion in the 45th day post-inoculation (Table 1). No seroconversion was also found in mice that were inoculated with embryonic tissues from non-infected C. callosus females. The presence of T. gondii DNA was detected in embryonic tissues from females treated with A. annua infusion, spiramycin, SPFA or PBS, but not in the group treated with azithromycin (Table 1 and Fig. 4).
1
2
3
4
5
6
7
8
9
10
1000 bp 700 bp 500 bp
531 bp
Fig. 4. Representative PCR analysis of the Toxoplasma gondii 35-copy B1 gene in placentas and embryonic tissues collected from 15th to 20th days of pregnancy (dop) in Calomys callosus females infected or not with T. gondii (ME49 strain) and treated or not with azithromycin, Artemisia annua infusion, spiramycin or drug association (SPFA). (Lane 1) T. gondii RH strain tachyzoite DNA (positive control); (Lane 2) placenta and embryonic tissues from non-infected and non-treated C. callosus females (negative control); (Lane 3) placenta and (Lane 4) embryonic tissues from C. callosus females infected with T. gondii and treated with SPFA; (Lane 5) placenta and (Lane 6) embryonic tissues from C. callosus females infected with T. gondii and treated with azithromycin; (Lane 7) placenta and (Lane 8) embryonic tissues from C. callosus females infected with T. gondii and treated with A. annua infusion; (Lane 9) placenta and (Lane 10) embryonic tissues from C. callosus females infected with T. gondii and treated with spiramycin. Arrows on the left indicate the molecular marker in base pairs (bp) and the arrow on the right indicates the fragment of 531 bp of the B1 gene of T. gondii.
4. Discussion Although the biology and physiology of T. gondii is widely investigated, the treatment for toxoplasmosis presents still limited efficacy due to their substantial side effects [5]. In the present study, we evaluated the effectiveness of azithromycin, A. annua infusion, spiramycin and SPFA on the vertical transmission of T. gondii in the C. callosus model that was already established to
Table 1 Summary of data obtained in assays to detect the presence of Toxoplasma gondii in placenta and embryonic tissues from Calomys callosus females in different treatment groups and days of pregnancy. Groups of animals*
Parasite detection Immunohistochemical assay
Mouse bioassay
Placenta
Embryonic tissues
Placenta
Embryonic tissues
PCR Placenta
Embryonic tissues
Azithromycin 15 dop 16 dop 17 dop 18 dop 19 dop 20 dop
þ þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ
A. annua infusion 15 dop 16 dop 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
þ þ þ þ þ þ
þ þ þ þ þ þ
Spiramycin 15 dop 16 dop 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
þ þ þ þ þ þ
þ þ þ þ þ þ
SPFA 15 dop 16 dop 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
þ þ þ þ þ þ
þ þ þ þ þ þ
*Animals were orally infected with 20 cysts of T. gondii (ME49 strain) at the 1st day of pregnancy (dop) and treated with azithromycin, Artemisia annua infusion, spiramycin or drug association as sulphadiazine plus pyrimethamine plus folinic acid (SPFA), as described in material and methods. Placenta and embryonic tissues were analyzed from the 15th to 20th dop. þ, presence of parasites; , absence of parasites.
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investigate congenital toxoplasmosis. No morphological alteration was observed in both placental and fetal tissues from females treated with azithromycin, spiramycin or SPFA. However, in females treated with A. annua infusion, embryos in atrophy process were observed. Similar embryotoxic effects were also demonstrated in a recent study of treatment with artemisinin in a malaria model, mainly during the first trimester of pregnancy [28]. Accordingly, toxicity of the artemisinin and its derivates have been investigated and showed that these compounds reach the embryonic erythroblast causing depletion of the red cells and consequent anemia, resulting in tissue damages and embryonic death [29–31]. However, a recent study demonstrated that A. annua infusion was effective to control systemic infection of C57BL/ 6 mice with a cystogenic T. gondii strain, due to its low toxicity and its inhibitory action directly against the parasite, resulting in a well tolerated therapeutic tool [15]. In the present study, immunohistochemical assays demonstrated that the kinetics of infection by T. gondii in different placental layers from females submitted to all treatments was the same observed in placentas from untreated females as described elsewhere [18]. Thus, the infection occurs first in giant trophoblast cells and in the labyrinth zone and later in spongiotrophoblasts. The labyrinth zone is directly bathed by the mother blood [18]. The embryonic infection was similar to that described in the literature and the most infected organ was the liver, which is one of the most damaged in congenital infections [32]. In all groups herein analyzed, bradyzoite immunostaining was observed in placental and fetal tissues only from females treated with SPFA. Interestingly, bradyzoite immunostaining in both placental and fetal tissues was higher than the control. Previous studies conducted in vitro demonstrated that the cellular stress of T. gondii may lead to early transformation from tachyzoites into bradyzoites and tissue cysts [33]. The early formation of cysts may be related to a strategy used by the parasite to evade the drug mechanisms of action. In addition, cysts are forms of parasite protection against external factors, such as the immune response [33,34]. However, in placental tissues from females treated with azithromycin, A. annua infusion or spiramycin, an increased number of bradyzoite immunostaining was not observed, indicating that possibly the treatments act by eliminating the parasite and preventing the chronification of the illness. Concerning azithromycin, a previous study demonstrated that this drug acts in tachyzoites and cysts of T. gondii, being effective also for bradyzoites [6]. However, so far there is limited information focusing the effectiveness of this drug in congenital toxoplasmosis. A recent report from our group demonstrated that in pregnant C. callosus treated with azithromycin, there was a reduction of T. gondii in the brains of mothers and no parasites were detected in eyes of fetuses, indicating that azithromycin may represent an alternative treatment for toxoplasmosis during pregnancy [35]. A significant decrease of parasite load in the group of females treated with SPFA was observed, even though this treatment was not able to avoid the vertical transmission of T. gondii. A previous study demonstrated that these drugs may reduce up to 70% the transmission of congenital toxoplasmosis due to their synergistic action and ability to across the placental barrier [36]. Similar to those results observed with SPFA in the present study, the data of immunohistochemistry, bioassay and PCR showed that the treatment with A. annua infusion or spiramycin were not able to hinder the fetal transmission of T. gondii. However, it was observed a significant decrease in parasite IS index in these treatment groups when compared with the respective controls, demonstrating a partial efficacy of these drugs against T. gondii. Concerning the mechanisms of action of both drugs, the mechanism for which the artemisinin inhibits the response of
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T. gondii involves the downregulation of the protein secretion of micronemes, which are calcium-dependent events [11]. Thus, artemisinin inhibits the calcium storage in the endoplasmatic reticulum of the parasite, and consequently, the secretion of proteins during the process of cellular invasion [11]. On the other hand, spiramycin, like other macrolides, has activity against T. gondii by inhibiting the parasite protein synthesis through the linking to the 50S subunit of the ribosome [37,38]. In this study, it was demonstrated that fetal infection occurred even when females were treated with spiramycin, showing that this drug was not capable to inhibit the maternal–fetal transmission. Similar results were found in previous studies showing that spiramycin reduces, but not avoids the transmission to the fetus [4,12,39]. In contrast, our data showed that in females treated with azithromycin, the infection was restricted to the placental tissues, with no evidence of fetal infection. A new ribosomal gene sequence was discovered through T. gondii DNA amplification by PCR and the ribosome codified with this new sequence of genes was shown to be more sensitive to the macrolide/lincosamide group of antibiotics. Therefore, it may be target for azithromycin action and other inhibitors of protein synthesis [40]. Azithromycin has been considered a powerful prophylactic agent even for highly virulent T. gondii strains [6]. Altogether, our results demonstrated that the treatment of pregnant C. callosus females with azithromycin was effective for inhibiting the vertical transmission of T. gondii, suggesting that it may be an alternative drug of choice for treatment of congenital infection since it is able to inhibit the fetal infection and offers new perspectives for the treatment of congenital toxoplasmosis.
Acknowledgements This work was supported by Brazilian Research Agencies (FAPEMIG, CNPq and CAPES).
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[14] Ridder S, Van der Kooy F, Verpoorte R. Artemisia annua as a self-reliant treatment for malaria in developing countries. J Ethnopharmacol 2008;120: 302–14. [15] Oliveira TC, Silva DAO, Rostkowska C, Be´la SR, Ferro EAV, Magalha˜es PM, et al. Toxoplasma gondii: effects of Artemisia annua L. on susceptibility to infection in experimental models in vitro and in vivo. Exp Parasitol 2009; 122:233–41. [16] Hu K, Johnson J, Florens L, Fraunholz M, Suravajjala S, Dilullo C, et al. Cytoskeletal components of an invasion machine the apical complex of Toxoplasma gondii. PLoS Pathog 2006;2:13. [17] Sarciron ME, Saccharin C, Petavy AF, Peyron F. Effects of artesunate, dihydroartemisinin and an artesunate-dihydroartemisin combination against Toxoplasma gondii. Am Soc Trop Med Hyg 2000;62:73–6. [18] Ferro EAV, Silva DAO, Bevilacqua E, Mineo JR. Effect of Toxoplasma gondii infection kinetics on trophoblast cell population in Calomys callosus, a model of congenital toxoplasmosis. Infect Immun 2002;70:7089–94. [19] Oliveira JG, Silva NM, Santos AAD, Souza MA, Ferreira GLS, Mineo JR, et al. BeWo trophoblasts are unable to control replication of Toxoplasma gondii, even in the presence of exogeous IFN-g. Placenta 2006;27:691–8. [20] Barbosa BF, Silva DAO, Costa IN, Pena JD, Mineo JR, Ferro EAV. Susceptibility to vertical trasmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus. Placenta 2007;28:624–30. [21] Duarte MCT, Figueira GM, Pereira B, Magalha˜es PM, Delarmelina C. Atividade antimicrobiana de extratos hidroalcoo´licos de espe´cies da coleça˜o de plantas medicinais CPQBA/UNICAMP. Plantas 2005;14:11–33. [22] Rath K, Ta´xis K, Walz G, Gleiter CH, Li SM, Heid L. Pharmacokinetic study of artemisinin after oral intake of a traditional preparation of Artemisia annua L. (Annuam wormwood). Am J Trop Med Hyg 2004;7:128–32. [23] Remington JS, Mcleod R, Thulliez P, Desmonts G. Toxoplasmosis. In: Remington JS, Klein OJ, editors. Infectious diseases of the fetus and newborn infant. 5th ed. Philadelphia: Saunders; 2001. p. 205–356. [24] Freyre A, Falco`n 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;112:8–12. [25] Ferguson DJP. Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. Int J Parasitol 2004;34:347–60. [26] Silva NM, Rodrigues CV, Santoro MM, Reis LFL, Alvarez-Leite JL, Gazzinelli RT. Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and kynurenine formation during in vivo infection with Toxoplasma gondii:
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Azithromycin Inhibits Vertical Transmission of Toxoplasma gondii in Calomys callosus (Rodentia: Cricetidae) I.N. Costa a, M.B. Angeloni a, L.A. Santana a, B.F. Barbosa a, M.C.P. Silva a, A.A. Rodrigues a, C. Rostkowsa b, P.M. Magalha˜es c, J.D.O. Pena b, D.A.O. Silva b, J.R. Mineo b, E.A.V. Ferro a, * a
ˆndia, Av. Para ´ 1720, Bloco 2B, Uberla ˆndia 38405-320, MG, Brazil Laboratory of Histology and Embriology, Instituto de Cieˆncias Biome´dicas, Universidade Federal de Uberla ˆ ndia, Av. Para ´ , 1720, Bloco 4C, Uberla ˆ ndia 38405-320, MG, Brazil Laboratory of Immunoparasitology, Universidade Federal de Uberla c Laboratory of Agrotechnology, Universidade Estadual de Campinas, Rua Alexandre Cazelatto, 999, Vila Betel, Campinas 13081-970, SP, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 4 August 2009
Toxoplasma gondii infection during pregnancy may cause severe consequences to the embryo. Current toxoplasmosis treatment for pregnant women is based on the administration of spiramycin or a drug combination as sulphadiazine-pyrimethamine-folinic acid (SPFA) in cases of confirmed fetal infection. However, these drugs are few tolerated and present many disadvantages due to their toxic effects to the host. The aim of this study was to evaluate the effectiveness of different treatments on the vertical transmission of T. gondii, including azithromycin, Artemisia annua infusion, spiramycin and SPFA in Calomys callosus as model of congenital toxoplasmosis. C. callosus females were perorally infected with 20 cysts of T. gondii ME49 strain at the day that a vaginal plug was observed (1st day of pregnancy – dop). Treatment with azithromycin, A. annua infusion, and spiramycin started at the 4th dop, while the treatment with SPFA started at the 14th dop. Placenta and embryonic tissues were collected for morphological and immunohistochemical analyses, mouse bioassay and PCR from the 15th to 20th dop. No morphological changes were seen in the placenta and embryonic tissues from females treated with azithromycin, spiramycin and SPFA, but embryonic atrophy was observed in animals treated with A. annua infusion. Parasites were found in the placenta and fetal (brain and liver) tissues of animals treated with SPFA, A. annua infusion and spiramycin, although the number of parasites was lower than in nontreated animals. Parasites were also observed in the placenta of animals treated with azithromycin, but not in their embryos. Bioassay and PCR results confirmed the immunohistochemical data. Also, bradyzoite immunostaining was observed only in placental and fetal tissues of animals treated with SPFA. In conclusion, the treatment with azithromycin showed to be more effective, since it was capable to inhibit the vertical transmission of T. gondii in this model of congenital toxoplasmosis. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Calomys callosus Congenital toxoplasmosis Treatment Azithromycin
1. Introduction Human toxoplasmosis is a disease worldwide distributed and caused by the protozoan Toxoplasma gondii. In immunocompetent individuals the acute infection is asymptomatic and self-limited, persisting in a latent form as tissue cysts [1]. In immunocompromised hosts, however, it may cause retinochoroiditis and fatal encephalitis when not treated [2,3]. Similarly, T. gondii infection during pregnancy may cause severe consequences to the embryo [4]. T. gondii may cross the placental barrier, causing neurological damage to the fetus and miscarriages [5]. Current toxoplasmosis treatment for pregnant women is based on the administration of
* Corresponding author. Tel./fax: þ55 34 3218 2240. E-mail address: [email protected] (E.A.V. Ferro). 0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2009.08.002
spiramycin in order to decrease the risk of fetal transmission. This decreased risk is related to high concentrations of spiramycin in the placental tissue, whereas disadvantage of this drug may be explained by the limited effectiveness in the parasite clearance and low penetration in fetal tissues [6]. If the diagnosis reveals fetal infection, the drug combination as sulphadiazine-pyrimethamine is usually indicated [7]. Sulphadiazine is a dihydropteroate synthase (DHPS) inhibitor, while pyrimethamine inhibits the dihydrofolate reductase (DHFR), and both enzymes are fundamental for the biosynthesis of T. gondii pyrimidines [8]. Even though the standard treatment recommended for toxoplasmosis is the combination of these drugs, numerous toxicity problems have been described [9], in addition to the potential teratogenic effect of pyrimethamine in the first trimester of pregnancy [6]. Suppression of the bone marrow is one of the most adverse effects of this treatment, although it may be
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prevented by simultaneous administration of folinic acid [4,10]. Despite these drugs have strategic action by inhibiting essential enzymes for the T. gondii metabolism, they are few tolerated, especially when used for long periods of time, and they present many disadvantages due to their harmful toxic effects to the host. Thus, it is imperative the development of studies that allow the validation of new targets of drugs in this field [4,5,11]. Azithromycin is one of the new generation macrolides with numerous advantages, such as a high oral bioviability, a rapid cell absorption and wide distribution in the organism, and administration only once a day [5]. In addition, azithromycin presents better pharmacokinetics and greater tissue concentration than spiramycin, and has also shown a lower incidence of side effects, especially hepatotoxic effects [6]. Azithromycin mechanism of action is based on the inhibition of protein synthesis in both T. gondii tachyzoite and bradyzoite stages [6,12], but it may present limited effectiveness against T. gondii, requiring high drug concentration [6]. Artemisinin, an active component present in the herb Artemisia annua L., has been investigated as alternative treatment for toxoplasmosis [13]. The infusion of this herb has been used in the malaria treatment, a disease caused by another Apicomplexa protozoan, Plasmodium sp [14] and recently it was also investigated in experimental acquired toxoplasmosis [15]. Artemisinin and derivatives have been effective against T. gondii [16,17], but there are no studies currently that prove a possible effectiveness of artemisinin in the treatment of congenital toxoplasmosis. Calomys callosus is a rodent of the Cricetidae family found largely in Central Brazil. Previous studies have shown a high susceptibility of these animals to T. gondii, being considered suitable experimental models to study the dynamics of congenital toxoplasmosis [18–20]. It was demonstrated that when these animals are infected with the T. gondii ME49 strain, vertical transmission mainly occurs during the acute stage, likewise in humans [18,20]. However, there are no studies focusing conventional or alternative treatment for congenital toxoplasmosis in this animal model. The aim of this study was to evaluate the effectiveness of different treatments on the vertical transmission of T. gondii, including azithromycin, A. annua infusion, spiramycin and drug association as sulphadiazine-pyrimethamine-folinic acid (SPFA) in C. callosus as model of congenital toxoplasmosis.
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2.4. Experimental groups Ninety-six C. callosus virgin females aged 3 months, weighting 30 2 g, were mated with males and checked daily for the presence of a vaginal plug that was considered as the first day of pregnancy (dop). All pregnant females were divided into four groups of 24 animals according to each drug treatment, as follows: group I (azithromycin), group II (A. annua infusion), group III (spiramycin) and group IV (SPFA). Each group was then subdivided into four subgroups of six animals per group, being two subgroups perorally infected with 20 cysts of T. gondii ME 49 strain in the 1st dop, and treated with the respective drugs or PBS (control) as illustrated in Fig. 1. Animals of the group I received azithromycin (9 mg/24 h), animals of the group II received A. annua infusion (1.0 mg/8 h) and animals of the group III received spiramycin (0.15 mg/8 h), from the 4th dop until the euthanasia day. Animals of the group IV received a drug combination (SPFA) consisting of sulphadiazine (1.5 mg/ 12 h), pyrimethamine (0.025 mg/12 h), and folinic acid (0.0075 mg/24 h) from 14th dop until the euthanasia day. All drugs were administered by oral route and diluted in 0.5 ml of sterile PBS, except for the A. annua infusion that was prepared in water. The doses of spiramycin and SPFA herein used were adapted to C. callosus body weight based on the recommended doses for pregnant women [15,23], whereas the azithromycin dose was used as described previously [6]. The animals were euthanized from the 15th to 20th dop, when placenta and embryonic tissues were collected for morphological and immunohistochemical analyses, mouse bioassay and polymerase chain reaction (PCR) to detect the parasite. Blood samples were also collected in the 1st dop and when animals were euthanized (15th to 20th dop) to determine the levels of IgG antibodies to T. gondii by immunoenzymatic assays (ELISA). 2.5. Morphological and immunohistochemical analyses For morphological analyses, specimens were fixed in 10% formalin and 0.1 M phosphate buffer (pH 7.4), dehydrated and embedded in glycol-methacrylate resin. Sections with 2 mm were stained with 0.25% toluidine blue and examined in a photomicroscope (Reichert Jung Polyvar, Lab. Optica Robert Koch, Austria). For immunolocalization of the parasites, fixed specimens were dehydrated and embedded in paraffin. Sections with 4 mm were placed on glass slides and processed as previously described [18]. Samples were first incubated for 10 min at room temperature with 5% acid acetic to block endogenous alkaline phosphatase or alternatively, with 3% hydrogen peroxide to block endogenous peroxidase, and then
2. Materials and methods 2.1. Animals C. callosus (Canabrava strain) came from a resident colony housed at the Institute of Biomedical Sciences, Federal University of Uberlaˆndia, Brazil. The animals were kept under standard conditions on a 12-h light, 12-h dark cycle in a temperaturecontrolled room (25 2 C) with food and water ad libitum. All procedures were conducted according to institutional guidelines for animal ethics. 2.2. Parasites Cysts of T. gondii ME49 strain were obtained from brains of C. callosus infected orally 30–45 days earlier with 20 cysts as described elsewhere [20]. Brains were removed, washed in sterile 0.01 M phosphate-buffered saline (PBS, pH 7.2), homogenized and cysts were counted under light microscopy. 2.3. Artemisia annua L. infusion Artemisia annua L. seeds (Center of Chemical, Biological and Agricultural Researches, University of Campinas, Brazil) were sown in a greenhouse, the plants were harvested and the aerial parts were dried, ground and stored at low humidity [15]. A. annua L. infusion was prepared from the infusion of 2 g of dried herb in 1000 ml boiling distilled water. The mixture was briefly stirred, covered and left in rest for 10 min. Next, the mixture was filtered and cooled at room temperature [21,22]. A. annua L. infusion was stored as stock solution (2 mg/ml) at 4 C in the dark and showed a specific content of artemisinin of 4.906 mg/ml (0.2% of total) by HPLC [15].
Fig. 1. Schedule of the experimental design carried out with pregnant Calomys callosus females infected or not with Toxoplasma gondii ME49 strain and submitted to different types of treatment.
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with 2% normal goat serum or alternatively, bovine serum albumin (BSA) for 30 min at 37 C to block non-specific binding sites. Next, samples were incubated for 12 h at 4 C with hyperimmune rabbit anti-T. gondii or anti-BAG5 (bradyzoite antigen-5) sera and with biotinylated goat anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO, USA) for 30 min at 37 C. The reaction was amplified by using the streptavidinbiotinylated alkaline phosphatase complex (Biomeda, Foster City, CA, USA) and developed with fast red-naphtol (Sigma). Alternatively, the reaction was amplified with streptavidin-biotinylated peroxidase complex (DAKO Corporation, CA, USA) and developed with fast 3,30 -diaminobenzidine tablet sets (Sigma), when appropriate. Samples were counterstained with Mayer’s hematoxylin and examined under a light microscope (BX40, Olympus, Tokyo, Japan) and an image analysis software
(HLImageþþ97, Western Vision Software, San Diego, CA, USA). Analysis was done in three sections of each tissue, totalizing 60 images for each analyzed tissue. Results were expressed in immunostaining index (IS) by using the following formula: IS ¼ number of stained images/0.01872 total number of images, where 0.01872 represents the fixed area (mm2) of each image determined by the software.
2.6. Bioassay and PCR Detection of T. gondii was first evaluated by mouse bioassay as described elsewhere [20,24]. Placentas and embryonic tissues (liver and brain) were homogenized in PBS and separately inoculated intraperitoneally in Swiss mice, in duplicate. After
Fig. 2. Photomicrographs of placental and embryonic tissues from Calomys callosus females infected with Toxoplasma gondii ME49 strain at the 1st day of pregnancy (dop) and treated with azithromycin, Artemisia annua infusion, spiramycin, drug association (SPFA) or sterile PBS. (a) tachyzoites (arrowheads) in the placental junctional zone of females treated with SPFA at 19th dop; (b) tachyzoites (arrowheads) in the placental junctional zone from females treated with PBS at 19th dop; (c) parasites (arrow) in placental junctional zone from females treated with spiramycin at 19th dop. (d) Parasites (arrow) in the placental junctional zone from females treated with PBS at 18th dop; (e) parasites (arrow) in the placental junctional zone from females treated with A. annua infusion at 18th dop; (f) parasites are not observed in liver of embryos from females treated with azithromycin at 19th dop; (g) parasites (arrow) in the liver of embryos from females treated with SPFA at 19th dop. (a, b, g): Immunohistochemical staining using alkaline phosphatase and Fast red naphthol, counterstaining by Mayer’s hematoxylin;(c–f): Immunohistochemical staining using horseradish peroxidase and Fast DAB, counterstaining by Mayer´s hematoxylin. Bars: 35 mm (a, b, d, e and f); 70 mm (c); 10 mm (g).
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A conventional ELISA to detect IgG antibodies to T. gondii was carried out as previously described [20]. Plates were coated overnight at 4 C with T. gondii soluble antigen (10 mg/ml) in carbonate buffer 0.06 M (pH 9.6). Plates were washed with PBS plus 0.05% Tween 20 (PBS-T) and incubated with serum samples of C. callosus or Swiss mice diluted at 1:16 in PBST plus 5% skim milk (PBS-TM) for 1 h at 37 C. After washing, plates were incubated with peroxidase labeled rabbit anti-C. callosus IgG prepared according to Wilson and Nakane [27] diluted at 1:250 or peroxidase labeled goat anti-mouse IgG (Sigma) diluted 1:1000 in PBS-TM and incubated for 1 h at 37 C. After new washes, the reaction was developed with 0.03% hydrogen peroxide and 1 mg/ml 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 in 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, values of EI >1.2 were considered positive. 2.8. Statistical analyses Data were expressed as mean standard deviation (SD) and the differences between groups were determined using ANOVA and Bonferroni multiple comparison tests or Student t test, when appropriate (GraphPad Prism version 4.0; GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant when p < 0.05.
3. Results 3.1. Morphological and immunohistochemical analyses No morphological alteration was observed in placental tissues, but the presence of T. gondii parasites in all placental layers (basal decidua, junctional zone and labirint zone) was noted, although with smaller number of parasites in infected C. callosus females and treated with different drugs compared to untreated infected controls. However, embryos in atrophy process were observed in females treated with A. annua infusion (data not shown). Parasite immunostaining was detected in the junctional zone of C. callosus placenta, showing immunostained tachyzoites in the group treated with SPFA (Fig. 2a) in low intensity when compared to untreated controls (Fig. 2b and d). Similar pattern of immunostaining was observed in placental tissues from females treated with spiramycin (Fig. 2c) and A. annua infusion (Fig. 2e). Parasites were not observed in embryos from females treated with azithromycin (Fig. 2f). On the contrary, tachyzoites were frequently detected in the liver of embryos from females treated with SPFA (Fig. 2g), as well as in spiramycin and A. annua infusion groups (data not shown). Bradyzoite immunostaining was detected only in placental tissues from animals treated with SPFA and euthanized from the 15th to 19th dop, although it was scarcely and heterogeneously distributed in relation to tachyzoite immunostaining (data not shown). In infected females treated with azithromycin, there was a predominance of tachyzoites in the placental labyrinth zone from 15th to 19th dop, but no parasites were observed in the placenta at the 20th dop.
Tachyzoite immunostaining was observed in placental tissues from females in all treatment groups, however placentas from untreated controls showed significantly higher IS index than placental tissues from treated females. In addition, tachyzoite IS index in placental tissues from females treated with azithromycin was lower than that observed in placenta from females submitted to the other three treatments (Fig. 3a). Bradyzoite immunostaining was also detected in placental tissues from females of untreated controls, but no immunostaining was seen in the groups treated with azithromycin, A. annua infusion, and spiramycin, except for the SPFA group, which showed a significantly higher IS index (p < 0.05) (Fig. 3a). In embryonic tissues, the parasite IS index was lower when females were treated with SPFA, A. annua infusion and spiramycin as compared to the respective controls. In the embryonic tissues from females treated with azithromycin, no tachyzoite immunostaining was detected (Fig. 3b). In contrast, bradyzoite IS index was higher in embryonic tissues from females treated with SPFA in relation to the respective controls (p < 0.05) (Fig. 3b). Bradyzoite immunostaining was not detected in embryonic tissues from females treated with azithromycin, A. annua infusion and spiramycin (p < 0.05) (Fig. 3b). 3.3. Bioassay and PCR Mouse bioassay results showed that mice inoculated with placentas from infected females and treated with PBS showed seroconversion in the 15th day post-inoculation. On the other hand,
Control / Tachyzoites Treatment / Tachyzoites
a Parasite IS index (mm 2) in plac ental tiss ues
2.7. ELISA
3.2. Parasite immunostaining quantification
Control / Bradyzoites
20
Treatment / Bradyzoites
*
15
10
* 5 #
b
*
*
*
0
Parasite IS index (mm 2) in embryonic tiss ues
15, 30 and 45 days of inoculation, blood samples from mice were collected and sera obtained were analyzed for the detection of IgG antibodies to T. gondii in ELISA. The presence of T. gondii DNA was investigated by PCR as previously described [20,25]. Placentas and embryonic tissues previously treated with 100 mg/ml proteinase K were 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 was performed using the primers 50 TCTTCCCAGAGGTGGATTTC-30 (sense, nucleotides 151–171) and 50 CTCGACAATACGCTGCTTG-30 (antisense, nucleotides 682–663), amplifying a fragment of 531 bp of the B1 gene of T. gondii. 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 [26]. PCR products were analyzed in 1% agarose gel containing 0.5 mg/ml of ethidium bromide and visualized under UV illumination.
887
Azithromycin
&
*
*
A. annua infusion
* Spiramycin
SPFA
5 4
*
*
*
* 3
*
2 1 0
#
*
Azithromycin
* A. annua infusion
&
* Spiramycin
SPFA
Fig. 3. Tachyzoite and bradyzoite immunostaining index (IS) in placentas (a) and embryonic tissues (b) from Calomys callosus females infected with Toxoplasma gondii (ME49 strain) and treated with azithromycin, Artemisia annua infusion, spiramycin, drug association (SPFA) or sterile PBS (control). * Statistically significant differences for parasite IS index between control and all treatment groups. # Statistically significant differences for tachyzoite IS index between treatment with azithromycin and the other treatment groups. & Statistically significant differences for bradyzoite IS index between treatment with SPFA and the other treatment groups. Data are represented in mean SD of all values of independent dop (15th to 20th).
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mice inoculated with placental tissues from infected females and treated with SPFA demonstrated a delayed seroconversion, that is, in the 45th day post-inoculation. Similar results were observed in the group treated with azithromycin, even though it was observed seroconversion only in mice inoculated with placental tissues. No seroconversion was found in mice that were inoculated with placentas from non-infected C. callosus females. The presence of T. gondii DNA in placental tissues from infected females and treated with azithromycin, A. annua infusion, spiramycin, SPFA or PBS was also confirmed by PCR (Table 1 and Fig. 4). Bioassay data demonstrated no seroconversion in mice inoculated with liver and brain from embryos of infected females and treated with azithromycin, while those inoculated with embryonic tissues from females treated with SPFA had seroconversion in the 45th day post-inoculation (Table 1). No seroconversion was also found in mice that were inoculated with embryonic tissues from non-infected C. callosus females. The presence of T. gondii DNA was detected in embryonic tissues from females treated with A. annua infusion, spiramycin, SPFA or PBS, but not in the group treated with azithromycin (Table 1 and Fig. 4).
1
2
3
4
5
6
7
8
9
10
1000 bp 700 bp 500 bp
531 bp
Fig. 4. Representative PCR analysis of the Toxoplasma gondii 35-copy B1 gene in placentas and embryonic tissues collected from 15th to 20th days of pregnancy (dop) in Calomys callosus females infected or not with T. gondii (ME49 strain) and treated or not with azithromycin, Artemisia annua infusion, spiramycin or drug association (SPFA). (Lane 1) T. gondii RH strain tachyzoite DNA (positive control); (Lane 2) placenta and embryonic tissues from non-infected and non-treated C. callosus females (negative control); (Lane 3) placenta and (Lane 4) embryonic tissues from C. callosus females infected with T. gondii and treated with SPFA; (Lane 5) placenta and (Lane 6) embryonic tissues from C. callosus females infected with T. gondii and treated with azithromycin; (Lane 7) placenta and (Lane 8) embryonic tissues from C. callosus females infected with T. gondii and treated with A. annua infusion; (Lane 9) placenta and (Lane 10) embryonic tissues from C. callosus females infected with T. gondii and treated with spiramycin. Arrows on the left indicate the molecular marker in base pairs (bp) and the arrow on the right indicates the fragment of 531 bp of the B1 gene of T. gondii.
4. Discussion Although the biology and physiology of T. gondii is widely investigated, the treatment for toxoplasmosis presents still limited efficacy due to their substantial side effects [5]. In the present study, we evaluated the effectiveness of azithromycin, A. annua infusion, spiramycin and SPFA on the vertical transmission of T. gondii in the C. callosus model that was already established to
Table 1 Summary of data obtained in assays to detect the presence of Toxoplasma gondii in placenta and embryonic tissues from Calomys callosus females in different treatment groups and days of pregnancy. Groups of animals*
Parasite detection Immunohistochemical assay
Mouse bioassay
Placenta
Embryonic tissues
Placenta
Embryonic tissues
PCR Placenta
Embryonic tissues
Azithromycin 15 dop 16 dop 17 dop 18 dop 19 dop 20 dop
þ þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ
A. annua infusion 15 dop 16 dop 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
þ þ þ þ þ þ
þ þ þ þ þ þ
Spiramycin 15 dop 16 dop 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
þ þ þ þ þ þ
þ þ þ þ þ þ
SPFA 15 dop 16 dop 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
þ þ þ þ þ þ
þ þ þ þ þ þ
*Animals were orally infected with 20 cysts of T. gondii (ME49 strain) at the 1st day of pregnancy (dop) and treated with azithromycin, Artemisia annua infusion, spiramycin or drug association as sulphadiazine plus pyrimethamine plus folinic acid (SPFA), as described in material and methods. Placenta and embryonic tissues were analyzed from the 15th to 20th dop. þ, presence of parasites; , absence of parasites.
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investigate congenital toxoplasmosis. No morphological alteration was observed in both placental and fetal tissues from females treated with azithromycin, spiramycin or SPFA. However, in females treated with A. annua infusion, embryos in atrophy process were observed. Similar embryotoxic effects were also demonstrated in a recent study of treatment with artemisinin in a malaria model, mainly during the first trimester of pregnancy [28]. Accordingly, toxicity of the artemisinin and its derivates have been investigated and showed that these compounds reach the embryonic erythroblast causing depletion of the red cells and consequent anemia, resulting in tissue damages and embryonic death [29–31]. However, a recent study demonstrated that A. annua infusion was effective to control systemic infection of C57BL/ 6 mice with a cystogenic T. gondii strain, due to its low toxicity and its inhibitory action directly against the parasite, resulting in a well tolerated therapeutic tool [15]. In the present study, immunohistochemical assays demonstrated that the kinetics of infection by T. gondii in different placental layers from females submitted to all treatments was the same observed in placentas from untreated females as described elsewhere [18]. Thus, the infection occurs first in giant trophoblast cells and in the labyrinth zone and later in spongiotrophoblasts. The labyrinth zone is directly bathed by the mother blood [18]. The embryonic infection was similar to that described in the literature and the most infected organ was the liver, which is one of the most damaged in congenital infections [32]. In all groups herein analyzed, bradyzoite immunostaining was observed in placental and fetal tissues only from females treated with SPFA. Interestingly, bradyzoite immunostaining in both placental and fetal tissues was higher than the control. Previous studies conducted in vitro demonstrated that the cellular stress of T. gondii may lead to early transformation from tachyzoites into bradyzoites and tissue cysts [33]. The early formation of cysts may be related to a strategy used by the parasite to evade the drug mechanisms of action. In addition, cysts are forms of parasite protection against external factors, such as the immune response [33,34]. However, in placental tissues from females treated with azithromycin, A. annua infusion or spiramycin, an increased number of bradyzoite immunostaining was not observed, indicating that possibly the treatments act by eliminating the parasite and preventing the chronification of the illness. Concerning azithromycin, a previous study demonstrated that this drug acts in tachyzoites and cysts of T. gondii, being effective also for bradyzoites [6]. However, so far there is limited information focusing the effectiveness of this drug in congenital toxoplasmosis. A recent report from our group demonstrated that in pregnant C. callosus treated with azithromycin, there was a reduction of T. gondii in the brains of mothers and no parasites were detected in eyes of fetuses, indicating that azithromycin may represent an alternative treatment for toxoplasmosis during pregnancy [35]. A significant decrease of parasite load in the group of females treated with SPFA was observed, even though this treatment was not able to avoid the vertical transmission of T. gondii. A previous study demonstrated that these drugs may reduce up to 70% the transmission of congenital toxoplasmosis due to their synergistic action and ability to across the placental barrier [36]. Similar to those results observed with SPFA in the present study, the data of immunohistochemistry, bioassay and PCR showed that the treatment with A. annua infusion or spiramycin were not able to hinder the fetal transmission of T. gondii. However, it was observed a significant decrease in parasite IS index in these treatment groups when compared with the respective controls, demonstrating a partial efficacy of these drugs against T. gondii. Concerning the mechanisms of action of both drugs, the mechanism for which the artemisinin inhibits the response of
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T. gondii involves the downregulation of the protein secretion of micronemes, which are calcium-dependent events [11]. Thus, artemisinin inhibits the calcium storage in the endoplasmatic reticulum of the parasite, and consequently, the secretion of proteins during the process of cellular invasion [11]. On the other hand, spiramycin, like other macrolides, has activity against T. gondii by inhibiting the parasite protein synthesis through the linking to the 50S subunit of the ribosome [37,38]. In this study, it was demonstrated that fetal infection occurred even when females were treated with spiramycin, showing that this drug was not capable to inhibit the maternal–fetal transmission. Similar results were found in previous studies showing that spiramycin reduces, but not avoids the transmission to the fetus [4,12,39]. In contrast, our data showed that in females treated with azithromycin, the infection was restricted to the placental tissues, with no evidence of fetal infection. A new ribosomal gene sequence was discovered through T. gondii DNA amplification by PCR and the ribosome codified with this new sequence of genes was shown to be more sensitive to the macrolide/lincosamide group of antibiotics. Therefore, it may be target for azithromycin action and other inhibitors of protein synthesis [40]. Azithromycin has been considered a powerful prophylactic agent even for highly virulent T. gondii strains [6]. Altogether, our results demonstrated that the treatment of pregnant C. callosus females with azithromycin was effective for inhibiting the vertical transmission of T. gondii, suggesting that it may be an alternative drug of choice for treatment of congenital infection since it is able to inhibit the fetal infection and offers new perspectives for the treatment of congenital toxoplasmosis.
Acknowledgements This work was supported by Brazilian Research Agencies (FAPEMIG, CNPq and CAPES).
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