Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes

Veterinary Parasitology 185 (2012) 91–100

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Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes P.X. Marques a , J. O’ Donovan c , E.J. Williams a , J. Gutierrez a,f , S. Worrall a , M. McElroy b , A. Proctor a , C. Brady b , D. Sammin d , H. Bassett a , D. Buxton e , S. Maley e , B.K. Markey a , J.E. Nally a,∗ a

School of Agriculture, Food Science and Veterinary Medicine, Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland Central Veterinary Research Laboratory, Department of Agriculture and Food, Staccumny Lane, Backweston, Celbridge, Co. Kildare, Ireland c Regional Veterinary Laboratory, Department of Agriculture, Fisheries and Food, Coosan, Athlone, Co. Westmeath, Ireland d Regional Veterinary Laboratory, Department of Agriculture, Fisheries and Food, Kilkenny, Co. Kilkenny, Ireland e Moredun Research Institute, Edinburgh, Scotland, United Kingdom f Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Apdo. 257, 37071 Salamanca, Spain b

a r t i c l e

i n f o

Article history: Received 15 June 2011 Received in revised form 12 October 2011 Accepted 19 October 2011 Keywords: Ovine Amniotic and allantoic fluid Abortion Toxoplasma gondii

a b s t r a c t Toxoplasma gondii, an intracellular protozoan parasite, is one of the major causes of infectious abortion in sheep. To further understand the pathogenesis of toxoplasmosis, serum, amniotic and allantoic fluids and foetal stomach contents were collected from experimentally infected pregnant ewes to determine pathogen numbers and other markers of infection. Fifteen pregnant ewes (90 days of gestation) were each orally inoculated with 3000 sporulated oocysts of T. gondii. Serum samples were collected weekly following challenge. Amniotic and allantoic fluids and foetal stomach contents were collected at 21, 25, 28, 33 and 35 days post-infection. Characteristic placental lesions were detected in 1 of 4 challenged ewes at day 25, 3 of 4 challenged ewes at day 28 and in all challenged ewes at days 33 and 35 post-infection. T. gondii was detected only sporadically in amniotic and allantoic fluids before 35 days of infection, by real-time PCR, and only in ewes with placental lesions. At 35 days post-infection, high numbers of parasite were detected in both amniotic and allantoic fluids. An increase in the number of fluids from challenged animals with IgM and IgG was detected over time, except for IgG in allantoic fluid, which was detected in all samples from day 21 post-infection. IgG in amniotic and allantoic fluids was shown to be specific for T. gondii, and reacted with antigens with an apparent molecular mass of approximately 22 kDa and 30 kDa. Results suggest a maternal source of immunoglobulin in the allantoic fluid and a foetal source of immunoglobulin in the amniotic fluid early in infection but that both sources may contribute immunoglobulin to both fluids at a later stage. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Toxoplasma gondii, an intracellular protozoan parasite, is one of the major causes of abortion of ewes in the United

∗ Corresponding author. Tel.: +353 1 716 6182; fax: +353 1 716 6185. E-mail address: [email protected] (J.E. Nally). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.10.028

Kingdom (UK) (Mearns, 2007), and Ireland (DAFF, 2006, 2007, 2008, 2009). In the United Kingdom, from 2000 to 2006, 30% of ovine abortions submitted for analysis were positive for T. gondii (Mearns, 2007). In Ireland, in 2008, 20.6% of submitted abortions (20/97) were positive for T. gondii (DAFF, 2008). In a surveillance report from Scotland in 2009, it was reported that 27% of diagnosed ovine abortions were caused by T. gondii (SACVS, 2009).

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Seroprevalence in sheep increases with age, reflecting increased exposure to the parasite over time (Dubey and Beattie, 1988). IgM is more readily detected during the first month of infection, with IgG becoming the predominant immunoglobulin subclass during the second month (Blewett et al., 1982, 1983; McColgan et al., 1988). Production of IgM in foetal lymphoid tissues increases until 20 dpi (110 days gestation), but is short-lived and levels return to baseline levels by 30 dpi when it is replaced by an IgG response (Buxton and Finlayson, 1986). The importance of IgM during early acute virulent T. gondii infection has been demonstrated using mice that lack surface and secretory IgM but maintain normal B-cell functionality and isotype class switching (Couper et al., 2005). Specific IgM molecules bind to tachyzoites in vivo, limiting parasite systemic dissemination during early infection (Couper et al., 2005). Amniotic fluid has been used as a diagnostic fluid in humans for the detection of the parasite and reactive antibodies (Chabbert et al., 2004; Chumpitazi et al., 1995; Grover et al., 1990; Kasper et al., 2009; Nagy et al., 2006; Pelloux et al., 1996, 1998; Romand et al., 2004; Thalib et al., 2005). Few studies have examined antibody content of amniotic and allantoic fluids in sheep whereas foetal stomach contents, largely derived from amniotic fluid, are routinely used for diagnosis of abortion due to bacterial infections (Devenish et al., 2005; Guler et al., 2006; Leyla et al., 2003). Morris and Barron (1980) showed the presence of IgG in allantoic fluid from normal pregnant ewes and Omwandho et al. (1997) confirmed detection of IgG bound to normal placentas. These results suggest that antibody may be part of the immunological regulation of normal pregnancy (Omwandho et al., 1997). It has been suggested that IgG can cross the placental barrier to the allantoic sac (Morris and Barron, 1980; Omwandho et al., 1997), but not to the amnion (Morris and Barron, 1980). Several studies have shown that maternal antibodies do not cross the normal ruminant placenta (Dubey et al., 1987b; Griebel, 1998; Sammin et al., 2009). A possible exception is when placental lesions permit a confluence of circulation between dam and foetus (Poitras et al., 1986). In the present study, allantoic and amniotic fluids, foetal sera and foetal stomach contents from T. gondii infected pregnant ewes were examined for the presence of IgM and IgG. IgG antibodies were examined for specificity to T. gondii antigens. These results were correlated with maternal serology, the detection of T. gondii DNA in amniotic and allantoic fluids and the presence of lesions consistent with toxoplasmosis in placental tissues. This study contributes to our understanding of the pathogenesis of ovine toxoplasmosis and identifies a potential use for foetal stomach content in the diagnosis of congenital T. gondii infection in sheep.

a multidisciplinary study of the immunopathogenesis of toxoplasmosis. Fifteen pregnant ewes (90 days of gestation), seronegative for antibodies to T. gondii when tested with a commercial latex agglutination test (MAST ToxoreagentTM , Merseyside, United Kingdom) and an in-house ELISA test, were orally inoculated with 3000 sporulated oocysts of T. gondii M4 strain. In addition, ten seronegative pregnant ewes were inoculated orally with distilled water as negative controls. Blood samples were collected weekly from day 0 until time of euthanasia. Ewes were euthanized at 21 (2 controls and 2 infected), 25 (2 controls and 4 infected), 28 (2 controls and 4 infected), 33 (2 controls and 3 infected) and 35 (2 controls and 2 infected) days post-infection (dpi) with intravenous sodium barbital (Euthatal, Merial Animal Health Ltd., UK). Serum was collected from each foetus after it was exteriorised from the uterus. Amniotic and allantoic fluids and foetal stomach contents were collected from each foetus using 20 ml syringes with 18 gauge 1 in. needles (BD plastipak, USA).

2.2. Histopathological examination Five placentomes, selected at random, were collected for each foetus. The placentomes were sectioned and fixed in zinc salt fixative for 24 h and then processed routinely for histological examination. Placentomes displaying lesions consistent with toxoplasmosis were further examined by immunolabelling for T. gondii antigen. Briefly, placentome sections (5 ␮m thick) were cut from the zinc salt fixed, paraffin embedded blocks, mounted on positively charged glass slides (Superfrost® Plus, MenzelGlässer, Braunschweig, Germany), air dried and then incubated at 57 ◦ C for 1 h. Mounted sections were deparaffinized and immunohistochemical labelling was carried out on a Ventana® Benchmark LT (Syntec Scientific Ltd., Ireland) automated immunohistochemistry staining machine. Slides were incubated with a 1:100 working dilution of a polyclonal rabbit anti-T. gondii antibody (Thermo Scientific, Cheshire, UK) at 37 ◦ C for 32 min, and epitopebound primary antibody was immunolabelled using the Ventana® ultra-ViewTM DAB detection kit (Syntec Scientific Ltd., Dublin, Ireland). Sections were counterstained with Ventana haematoxylin and bluing reagent (Syntec Scientific Ltd., Dublin, Ireland). Following counterstaining sections were dehydrated through graded alcohols and xylene and then coverslipped. Sections were then examined by a veterinary pathologist for the presence of positively staining structures consistent with the morphology of T. gondii. Histopathological examination of foetal brain from ewes euthanized at 33 and 35 dpi were as previously described (O’Donovan et al., 2011).

2. Materials and methods 2.3. DNA extraction and real-time PCR 2.1. Experimental infections Experimental infections were carried out under license from the Department of Health and Children as part of

DNA was extracted from amniotic and allantoic fluids and processed for real-time PCR as previously described (Gutierrez et al., 2010).

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2.4. Detection and purification of immunoglobulin in the amniotic and allantoic fluids and foetal stomach contents Thirty microliters of each fluid were subjected to electrophoresis by 1D SDS-PAGE and detection of IgG and IgM was performed by immunoblotting as previously described (Marques et al., 2011). IgG was purified from 1 ml of each sample of amniotic (3 infected and 2 controls) and allantoic fluid (5 infected and 12 controls) and foetal stomach contents (5 infected and 13 controls) by column chromatography using Protein S affinity columns, NabTM Spin Kit (Pierce, Thermo Fisher Scientific Inc., UK), as per manufacturer’s instructions. Eluted IgG was dialysed using Slide-A-Lyzer dialysis cassettes (Pierce, Thermo Fisher Scientific Inc., UK) using PBS as exchanging buffer. Each eluted fraction was separated by SDS-PAGE for analysis by Sypro Ruby staining (Sigma Aldrich, Ireland) or immunoblotting for confirmation of purified IgG. 2.5. Immunofluorescence Vero cells (Green Monkey Kidney) were cultured in maintenance medium (Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5 ␮g/ml gentamycin and 5% heat-inactivated foetal bovine serum (FBS) (Invitrogen Corporation, USA), in a humidified incubator at 37 ◦ C and 5% CO2 . Vero cell concentration was adjusted to 3 × 105 /ml with IMDM medium supplemented with 5 ␮g/ml gentamicin and 2% FBS, 24 h before being infected with T. gondii, FR-OVI ARI 2007-14 strain (BRC Toxoplasma, Reims, France) at 1:10 (cell: parasite) ratio. At 96 h post-inoculation, cells were harvested using cell scrapers, and the number of parasites was counted in a Neubauer chamber. The specificity of purified IgG for T. gondii was confirmed by indirect immunofluorescence (IFA). In brief, Vero cells (5 × 104 ) grown in Trac culture bottles (Sterilin Limited, London, UK) for 24 h in 1 ml of maintenance medium (described above) were inoculated with 500 ␮l of tachyzoites (∼5 × 105 ). The Tracs were incubated at 37 ◦ C, 5% CO2 for 72 h. Cells were then washed with PBS, fixed with methanol for 20 min, washed again with PBS, blocked with blocking buffer (2% milk powder, 50 mM glycine) for 30 min and incubated overnight at room temperature with 25 ␮g/ml of purified IgG diluted in blocking buffer. Hyper-immune anti-T. gondii sheep serum (1:400) or PBS were used to provide positive and negative controls, respectively. Cells were washed again with PBS and incubated with donkey anti-sheep IgG FITC conjugate antibody (Sigma-Aldrich Ireland Ltd.) diluted 1:100 in blocking buffer, for 1–2 h at 37 ◦ C. Fluorescing tachyzoites were visualized using a Nikon Eclipse E400 Fluorescence Microscope (Nikon U.K. Ltd.). 2.6. Immunoblot One and two-dimensional (1D and 2D) immunoblotting of commercially produced tachyzoites (RH strain, Europa Bioproducts Ltd., United Kingdom) were performed using ewe sera and IgG purified from amniotic and allantoic and foetal stomach contents samples as previously described (Marques et al., 2010, 2011). For 2D immunoblotting,

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tachyzoites (100 ␮g) were pelleted by centrifugation at 10,000 × g for 10 min at room temperature, solubilised in rehydration buffer and separated by 2D gel electrophoresis using 7 cm Immobiline DryStrips (pH of 4–7) as previously described (Marques et al., 2010, 2011). Total proteins were visualized after staining with SYPRO Ruby protein gel stain (Sigma-Aldrich Inc., Ireland) under UV light in the BioSpectrum AC Imaging System (Ultra-Violet Products Ltd., UK). Alternatively, proteins were transferred to polyvinylidene difluoride (PVDF) membranes for one and two-dimensional (1D and 2D) immunoblotting as previously described (Marques et al., 2010, 2011). 3. Results 3.1. Detection of IgM and IgG in allantoic and amniotic fluids, and foetal stomach contents Allantoic and amniotic fluids, and foetal stomach contents obtained from T. gondii experimentally infected pregnant ewes contained significant amounts of IgM and IgG, as detected by immunoblotting (Fig. 1). IgM was detected at approximately 90 kDa (Fig. 1A), and IgG was detected at approximately 50 and 25 kDa, corresponding to heavy and light chains of sheep IgG, respectively (Fig. 1B). IgM was detected in 1 of 3 samples of allantoic fluids by 21 days post-infection (dpi) (Table 1). By 25 dpi, IgM was detected in 2 of 7 samples of allantoic fluid, 1 of 7 samples of amniotic fluid and 1 of 7 samples of foetal stomach contents. By 28 dpi, IgM was detected in 3/5 of the allantoic fluids, 4/5 of the amniotic fluids and 5/5 of the foetal stomach contents. At 33 and 35 dpi, IgM was detected in all three fluids in all infected ewes. IgM was not detected at any time points in samples from non-infected control animals. IgG was detected in allantoic and amniotic fluids and foetal stomach contents of both infected and non-infected animals (Table 2). Almost all allantoic fluids from infected ewes had IgG at all time points: 3/3, 7/7, 3/5, 2/4 and 2/2 at 21, 25, 28, 33 and 35 dpi, respectively. In the amniotic fluids, IgG was not detected at 21 and 33 dpi (0/3 and 0/4, respectively), but was detected in 6/7 samples at 25 dpi, in 4/5 samples at 28 dpi and in 2/2 samples at 35 dpi. In foetal stomach contents, IgG was detected in 1/3 samples at 21 dpi and 4/7 samples at 25 dpi IgG was detected in all samples of foetal stomach contents at 28, 33 and 35 dpi. 3.2. Detection of placental lesions and T. gondii in the amniotic and allantoic fluids At 25 dpi, placental lesions consistent with toxoplasmosis were detected in one of four ewes. At 28 dpi, placental lesions were present in three of four experimentally infected ewes (Table 3). At 33 and 35 dpi, lesions were detected in all placentas (Fig. 2). Brain lesions consistent with T. gondii infection were found in all foeti from ewes euthanized at 33 and 35 dpi (O’Donovan et al., 2011). Lesions were not detected in any placentomes from control ewes or challenged ewes sampled at 21 dpi. T. gondii antigen was demonstrated by IHC in at least one placentome from every infected ewe at 33 and 35 dpi and one ewe (1515) at 28 dpi. T. gondii was detected only sporadically in

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Fig. 1. Representative 1D immunoblot of allantoic and amniotic fluids, and foetal stomach contents to detect (A) IgM and (B) IgG. IgM (∼90 kDa) is indicated (arrow head). IgG heavy chains (∼50 kDa) (arrow) and light chains (asterisk) (∼25 kDa) are indicated. Molecular mass (kDa) standards are indicated on the left. Table 1 Numbers of allantoic fluid, amniotic fluid and foetal stomach contents positive for IgM. Days post infectiona

Day gestation

Number of samples

Allantoic fluid positive for IgM

Amniotic fluid positive for IgM

Foetal stomach contents positive for IgM

21 25 28 33 35

111 115 118 123 125

3 7 5 4 2

1 2 3 4 2

0 1 4 4 2

0 1 5 4 2

a

Post-infection.

amniotic and allantoic fluids before 35 days of infection, by real-time PCR. At 35 dpi, high numbers of parasite were detected in both fluids tested.

purified IgG ranged from 0.06 to 0.9 ␮g/␮l of amniotic, allantoic fluid or foetal stomach contents.

3.4. Specificity of purified IgG by immunofluorescence 3.3. Purification of IgG IgG was purified from allantoic and amniotic fluids, and foetal stomach contents from both experimentally infected pregnant ewes and non-infected control ewes that were positive for IgG. Purified IgG was confirmed by SYPRO ruby staining and immunoblot (Fig. 3). The concentration of

All IgG samples purified from allantoic and amniotic fluids and foetal stomach contents of infected ewes reacted specifically with tachyzoites of T. gondii cultured in vero cells (Fig. 4). IgG purified from non-infected control ewes (Table 2) was not reactive with T. gondii, except for two samples (data not shown). In one of these non-infected

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Table 2 Numbers of allantoic fluid, amniotic fluid and foetal stomach contents positive for IgG. Days post infectiona

21 25 28 33 35

Day gest b

111 115 118 123 125

Total a b c d e

Number of samples

Allantoic fluid positive for IgG

Amniotic fluid positive for IgG

Foetal stomach contents positive for IgG

Cc

Cc

Cc

Cc

Id

3 3 6 4 3

3 7 5 4 2

19

22

Id

2 2 3e 3 2 2

Id

Id

3 7 3 2 2

0 1 0 1 0

0 6 4 0 2

1 2 5 2 3

1 4 5 4 2

13

12

18

13

16

Post-infection. Days gestation. Fluids from negative control ewes. Fluids from infected ewes. 5/6 allantoic control samples were analysed.

controls, IgG purified from allantoic fluids from both foeti of the same ewe were reactive, and in another case, only IgG purified from foetal stomach contents was reactive. 3.5. Specificity of purified IgG by immunoblot Total protein staining following SDS-PAGE analysis of a tachyzoite protein preparation showed a large number of proteins with molecular masses between 20 and 220 kDa (Fig. 5A). The more abundant proteins have molecular masses above 25 kDa. Reactive antigens between 20 and 80 kDa were detected with serum from experimentally infected ewe 1490 collected at 29 dpi (Fig. 5C). However,

immunoblotting of tachyzoites with serum from the same ewe collected at day 0 also reacted with antigenic proteins (Fig. 5B). Immunoblotting with sera from non-infected control ewes showed antigenic reactivity similar to day 0 of infected ewes. Immunoblotting of tachyzoites with purified IgG (allantoic fluid from ewe 1490) further confirmed its specificity for antigens of T. gondii, particularly antigens with an apparent molecular mass of approximately 22 kDa (Fig. 5D). Less reactive antigens were also detected at molecular mass between 25 and 120 kDa. An immunoblot was probed with IgG purified from a non-infected control ewe, negative by immunofluorescence (IF) (Fig. 5E) and a second one with IgG purified from a non-infected

Table 3 Results of histopathological examination of placentomes, detection of T. gondii by real time PCR, and detection of IgM and IgG in amniotic and allantoic fluids, and foetal stomach contents from T. gondii-infected ewes. Days post infection

Foetus ID

Placental lesionsa

Real time PCRb

21

1486A 1486B 1503A

Negative Negative Negative

0 0 0

0 0 0

25

1497A 1497B 1499A 1499B 1504A 1519A 1519B

Positive Negative Negative Negative Negative Negative Negative

31 0 0 0 0 0 0

28

1481A 1488A 1495A 1495B 1515A

Negative Positive Positive Negative Positive

33

1490A 1501A 1501B 1645A

35

1498A 1508A

Amniotic fluid

Allantoic fluid

Foetal stomach contents

IgM

IgG

IgM

IgG

IgM

IgG

− − −

− − −

+ − −

+ + +

− − −

− − +

0 0 0 0 0 0 0

+ − − − − − −

+ + + + + + +

+ + − − − − −

+ + + + + + +

+ − − − − − −

+ − + + − − +

0 0 0 0 0

0 0 0 0 57

+ + + + +

+ + − + +

+ + + − −

+ + + + −

+ + + + +

+ + + + +

Positive Positive Positive Positive

21 0 0 0

68 0 0 0

+ + + +

− − − −

+ + + +

− − + +

+ + + +

+ + + +

Positive Positive

84 241

730 11,776

+ +

+ +

+ +

+ +

+ +

+ +

Amniotic

a b

Allantoic

Hispathological examination of randomly selected placentomes for each foetus. Results obtained by real time PCR, expressed as average copy numbers detected per ml.

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and an isoelectric point (pI) between 4 and 5, circled in Fig. 6C, and at an apparent molecular mass of approximately 22 kDa and pI between 4 and 7, squared in Fig. 6C. Reactive antigens of more than 30 kDa and a pI between 4 and 5, were also detected, but most were also reactive with serum taken from the infected ewe at day 0 (Fig. 6B). Approximately 30 antigens were identified as reactive with purified IgG (allantoic fluid from ewe 1490) following 2D immunoblotting, Fig. 6D. The most reactive antigens had an apparent molecular mass of approximately 22 kDa, squared box in Fig. 6C and D. The highly reactive 30 and 22 kDa proteins were not detected with IgG purified from allantoic fluid of non-infected control ewes, which were negative by IF (Fig. 6E) nor was it detected with non-infected control ewes that were positive by IF (Fig. 6F). Fig. 2. Toxoplasma gondii placentitis. Photomicrograph of a necrotic focus (L) in an ovine placentome 35 days after the ewe was challenged orally with sporulated T. gondii oocysts. Normal placentome tissue, consisting of interdigitating maternal caruncular septa (m) and foetal cotyledonary villi (v), is present on the right. Intralesional T. gondii tachyzoites (t) are visible in the main image and inset (H&E).

control ewe, positive by IF (Fig. 5F). Several antigens were detected using the IgG purified from the control ewe which was positive by IF; however it did not react with the immunoreactive ∼22 kDa antigen which was detected with IgG purified from an infected ewe. Serum from foeti did not react with any antigens (data not shown). The proteome of T. gondii tachyzoites was examined by 2D gel electrophoresis (Fig. 6A). More than 100 spots were detected over a pH range of 4–7 with molecular masses between 20 kDa and 220 kDa. The most abundant proteins observed had a pI between 4.5 and 6 and a molecular mass between 25 and 220 kDa, in agreement with the 1D SDS-PAGE results (Fig. 6A). Immunoblotting of 2D gels was performed with sera from infected ewe 1490, 29 days postinfection (Fig. 6C). Highly reactive antigens were detected with an apparent molecular mass of approximately 30 kDa

4. Discussion In this study it was found that IgM and IgG are present in allantoic and amniotic fluids and foetal stomach contents of T. gondii infected pregnant ewes. Purified IgG was specific for tachyzoites of T. gondii as demonstrated by immunofluorescence, and specific reactive antigens were characterized by 2D gel electrophoresis and immunoblotting. IgG, but not IgM, was also detected in allantoic and amniotic fluids and foetal stomach contents of noninfected control animals. This IgG was not specific for T. gondii by immunofluorescence except in the case of two ewes. Analysis by 2D immunoblotting showed that IgG purified from these two ewes reacted with different antigens than IgG purified from infected ewes. An increase in the number of fluids from challenged animals with IgM and IgG was detected over time, except for IgG in allantoic fluid. Two-dimensional immunoblots allowed the detection of two groups of antigens, one with ∼22 kDa and an isoelectric point (pI) between 4 and 7, and the other of ∼30 kDa and pI between 4 and 5, when sera and IgG purified from T. gondii infected ewe allantoic fluid were used, compared to non-infected controls.

Fig. 3. Purification of IgG. Lane 1, total sample, lane 2, flow through from column after sample application, and lane 3, eluted IgG, as detected by (A) total protein stain and (B) immunoblotting with anti-sheep IgG. IgG heavy chains (∼50 kDa) (arrows) and light chains (asterisk) (∼25 kDa) are indicated. M, molecular mass (kDa) standards are indicated.

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Fig. 4. Indirect immunofluorescence staining of vero cells infected with T. gondii (original magnification 1000×). (A) Negative control, (B) positive control, (C) and D) IgG (25 ␮g/ml) purified from allantoic fluid of non-infected and infected ewes, respectively, (E) and (F) IgG (25 ␮g/ml) purified from amniotic fluid of non-infected and infected ewes, respectively, (G) and (H) IgG (25 ␮g/ml) purified from foetal stomach content of a foetus of non-infected and infected ewes, respectively.

Sera taken at day 0 were reactive with antigens of T. gondii, as was IgG that was purified from the allantoic fluid of a non-infected control ewe that was reactive with T. gondii antigens by immunofluorescence; however, they did not react with antigens of 22 and 30 kDa which were detected using IgG purified from experimentally infected ewes. Reasons for reactivity of sera at day 0 include the possibility that antibodies against alternative pathogens may cross-react with antigens of T. gondii (Lunde and Jacobs, 1965; Riahi et al., 1998; Suggs et al., 1968). Alternatively, the antibodies in the sera may be “natural” antibodies to T. gondii as demonstrated in sera

from non-infected human adults and infants older than 3 months (Potasman et al., 1986b). Evidence of the presence of these antibodies has been shown by fluorescence and in electron microscopy studies (Dzbenski et al., 1976; Sulzer et al., 1971). Such antibodies react with several antigens including a 30 kDa antigen (Potasman et al., 1986b). After infection the intensity of staining of this and other antigens increased remarkably (Potasman et al., 1986a). When foetal sera was used to probe 1D gel immunoblots, no reactive antigens were detected. This is in contrast with the study by Dubey et al. (1987a) in which IgG antibodies against T. gondii were detected by MAT

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Figure 5. 1D SDS-PAGE of tachyzoites from Toxoplasma gondii. (A) Total protein staining of tachyzoites, (B) and (C) immunoblot with sera collected from an infected ewe at day 0 and 29 dpi, respectively, (D)–(F) immunoblot with IgG purified from allantoic fluid (100 ␮g) from an infected ewe, from a non-infected control ewe that was positive for IgG which was negative by immunofluorescence, and from a non-infected control that was positive for IgG which was positive by immunofluorescence. Molecular mass (kDa) standards are indicated in the left.

(modified agglutination test) 28 days post infection. Other studies also detected reactive IgG in foetal sera (Buxton and Finlayson, 1986; Hedstrom et al., 1989; Hunter et al., 1982; Munday and Corbould, 1975), but discordant results may be due to a multiplicity of factors; placental lesions were observed 10–15 days earlier post challenge, which would suggest that foetal infection occurred earlier than our study (Buxton and Finlayson, 1986). Additionally, a different challenge infection was used; bradyzoites in tissue cysts compared to sporulated oocysts, and although IgM and IgG were in circulation in infected foeti at 20 dpi, T. gondii-specific IgM and IgG was only detected at 30 dpi, which is near the end time point of our study. In the present study, several reactive antigenic proteins were detected by 2D immunoblotting with molecular

masses between 28 and 32 kDa, including the 30 kDa antigen that was only detected after infection. Some of these proteins may correspond to the major surface antigen (designated gp30 or SAG 1) which is highly immunogenic (Charles et al., 2007). This protein has been implicated in the stimulation of both humoral and cellular responses in mice (Khan et al., 1988; McLeod et al., 1991). Ma et al. (2009) identified 4 isoforms of this protein with pI between 5.4 and 6.0. In a recent study in sheep, vaccination with plasmid DNA containing the gene sequence for SAG1 did not stimulate IFN-␥ production, but a humoral response was apparent (Li et al., 2010). The antigenic protein observed at 22 kDa may correspond to the surface antigen protein 2 (SAG2) that was used by Lunden et al. to immunize sheep (Lunden, 1995) and mice (Lunden et al.,

Fig. 6. 2D SDS-PAGE of tachyzoites from Toxoplasma gondii. (A) Total protein staining of tachyzoites, (B) and (C) immunoblot with sera collected from an infected ewe at day 0 and 29 dpi, respectively, (D)–(F) immunoblot with IgG purified from allantoic fluid (100 ␮g) from infected ewe 1490 at 29 days post-infection, from a non-infected control ewe that was positive for IgG which was negative by immunofluorescence, and from a non-infected control that was positive for IgG which was positive by immunofluorescence. Molecular mass (kDa) standards are indicated in the left.

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1997). It has also been used for serological diagnosis in humans (Aubert et al., 2000; Parmley et al., 1992) and cats (Huang et al., 2004). The other prominent antigens likely represent a number of proteins previously identified by Ma et al. (2009) and Zhang et al. (2011). An additional important question raised in this study is the origins of the immunoglobulin found in allantoic and amniotic fluids and foetal stomach contents. Their origin can be hypothesized based on the following chain of events during infection; when T. gondii tachyzoites reach the ovine pregnant uterus, they infect the placenta where they multiply. T. gondii tachyzoites are disseminated to the foetus haematogenously, even in the absence of visible placental lesions. At that point, normally the DNA of the parasite can be detected in the foetal organs, mainly in the brain and aqueous humour (Gutierrez et al., 2010). High levels of IgM were detected in the foetal stomach contents. High levels of IgM were also detected in amniotic fluid, which may suggest that this immunoglobulin originated from the foetus. IgG was first detected in foetal stomach contents and then in the amniotic fluid, which suggests that this immunoglobulin is also derived from the foetus. Logan et al. (1981) suggested that the immunoglobulins detected in bovine abomasal fluids may be due to a local production in response to antigenic stimulation of the orogastrointestinal tract. This local production may explain the presence of IgG in the foetal stomach contents in the absence of IgG in the foetal sera in the present study. Examination of the allantoic fluid suggests that the IgM and IgG detected in this fluid had a maternal origin, since almost all allantoic fluids had immunoglobulins even when they were not detected in foetal stomach contents. Omwandho et al. (1997) proposed that normal immunological regulation of the ovine pregnancy may involve the production of maternal antibodies directed to specific antigens in the placenta that have to be transported across the placental barrier by some, as yet unidentified, mechanism. The same mechanism might also be used to transport T. gondii-specific antibodies across the placental barrier. The results of the present study and in studies by Morris and Barron (1980) and by Omwandho et al. (1997), appear to contradict the generally accepted fact that maternal antibodies do not cross the placenta, even when severely damaged (Dubey et al., 1987b; Griebel, 1998; Sammin et al., 2009). However, studies from Poitras et al. (1986) and Gabriel et al. (2005) have shown that antibody transfer is observed in association with placental lesions which produced confluence of maternal and foetal circulations. This work supports the potential use of foetal stomach contents as a diagnostic fluid for T. gondii. In the clinical or laboratory investigation of ovine abortion outbreaks, amniotic fluid is not readily available, but abomasal fluid (representing ingested amniotic fluid) is routinely sampled for culture of abortifacient microorganisms (Logan et al., 1981). In humans, amniotic fluid has been used as a diagnostic sample for serology (Chumpitazi et al., 1995) and parasite detection by polymerase chain reaction (Chabbert et al., 2004; Grover et al., 1990; Kasper et al., 2009; Nagy et al., 2006; Pelloux et al., 1996, 1998; Romand et al., 2004; Thalib et al., 2005). In the present study, specific antibody

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detection rather than parasite detection produced more consistent results. This is the first study trying to examine and correlate events in terms of tissue infection and humoral response in ewe, foetus, amniotic and allantoic fluids, in an attempt to improve our understanding of the host–pathogen interactions of T. gondii in pregnant infected ewes. Acknowledgements This work was funded by grant number RSF06 394 from the Research Stimulus Fund, Department of Agriculture, Fisheries and Food. JEN is funded by grant number 05/YI2/B696 President of Ireland Young Researcher Award from Science Foundation Ireland. References Aubert, D., Maine, G.T., Villena, I., Hunt, J.C., Howard, L., Sheu, M., Brojanac, S., Chovan, L.E., Nowlan, S.F., Pinon, J.M., 2000. Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay. J. Clin. Microbiol. 38, 1144–1150. Blewett, D.A., Bryson, C.E., Miller, J.K., 1983. Studies of antibody titres in experimentally induced ovine toxoplasmosis. Res. Vet. Sci. 34, 163–166. Blewett, D.A., Miller, J.K., Buxton, D., 1982. Response of immune and susceptible ewes to infection with Toxoplasma gondii. Vet. Rec. 111, 175–178. Buxton, D., Finlayson, J., 1986. Experimental infection of pregnant sheep with Toxoplasma gondii: pathological and immunological observations on the placenta and foetus. J. Comp. Pathol. 96, 319–333. Chabbert, E., Lachaud, L., Crobu, L., Bastien, P., 2004. Comparison of two widely used PCR primer systems for detection of Toxoplasma in amniotic fluid, blood, and tissues. J. Clin. Microbiol. 42, 1719–1722. Charles, E., Callegan, M.C., Blader, I.J., 2007. The SAG1 Toxoplasma gondii surface protein is not required for acute ocular toxoplasmosis in mice. Infect. Immun. 75, 2079–2083. Chumpitazi, B.F., Boussaid, A., Pelloux, H., Racinet, C., Bost, M., Goullier-Fleuret, A., 1995. Diagnosis of congenital toxoplasmosis by immunoblotting and relationship with other methods. J. Clin. Microbiol. 33, 1479–1485. Couper, K.N., Roberts, C.W., Brombacher, F., Alexander, J., Johnson, L.L., 2005. Toxoplasma gondii-specific immunoglobulin M limits parasite dissemination by preventing host cell invasion. Infect. Immun. 73, 8060–8068. DAFF (Ed.), 2006. Regional Veterinary Laboratories Surveillance Report 2006. Department of Agriculture, Fisheries and Food, Dublin, 16 pp. DAFF (Ed.), 2007. Regional Veterinary Laboratories Surveillance Report 2007. Department of Agriculture, Fisheries and Food, 24 pp. DAFF (Ed.), 2008. Regional Veterinary Laboratories Surveillance Report 2008. Department of Agriculture, Fisheries and Food, 30 pp. DAFF (Ed.), 2009. Regional Veterinary Laboratories Surveillance Report 2009. Department of Agriculture, Fisheries and Food, 21 pp. Devenish, J., Brooks, B., Perry, K., Milnes, D., Burke, T., McCabe, D., Duff, S., Lutze-Wallace, C.L., 2005. Validation of a monoclonal antibodybased capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus. Clin. Diagn. Lab. Immunol. 12, 1261–1268. Dubey, J.P., Beattie, C.P., 1988. Toxoplasmosis of Animals and Man. CRC Press, Boca Raton, FL, 220 pp. Dubey, J.P., Emond, J.P., Desmonts, G., Anderson, W.R., 1987a. Serodiagnosis of postnatally and prenatally induced toxoplasmosis in sheep. Am. J. Vet. Res. 48, 1239–1243. Dubey, J.P., Hughes, H.P., Lillehoj, H.S., Gamble, H.R., Munday, B.L., 1987b. Placental transfer of specific antibodies during ovine congenital toxoplasmosis. Am. J. Vet. Res. 48, 474–476. Dzbenski, T.H., Michalak, T., Plonka, W.S., 1976. Electron microscopic and radioisotopic studies on cap formation in Toxoplasma gondii. Infect. Immun. 14, 1196–1201. Gabriel, S., Geldhof, P., Phiri, I.K., Cornillie, P., Goddeeris, B.M., Vercruysse, J., 2005. Placental transfer of immunoglobulins in cattle infected with Schistosoma mattheei. Vet. Immunol. Immunopathol. 104, 265–272. Griebel, P.J., 1998. Sheep immunology and goat peculiarities. In: Pastoret, P.-P., Griebel, P., Bazin, H., Govaerts, A. (Eds.), Handbook of Vertebrate Immunology. Academic Press, London, pp. 485–554.

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