Laboratório de Esquistossomose, Instituto de Pesquisa René Rachou-Fiocruz, Av. Augusto de Lima 1715, Belo Horizonte, MG, Brasil 2 - PDF

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424 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(4): , June 2011 Interaction between primary and secondary sporocysts of Schistosoma mansoni and the internal defence system of Biomphalaria resistant

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424 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(4): , June 2011 Interaction between primary and secondary sporocysts of Schistosoma mansoni and the internal defence system of Biomphalaria resistant and susceptible to the parasite Ana Carolina Alves de Mattos 1, Raquel Lopes Martins-Souza 2, John Robert Kusel 3, Paulo Marcos Zech Coelho 1 / + 1 Laboratório de Esquistossomose, Instituto de Pesquisa René Rachou-Fiocruz, Av. Augusto de Lima 1715, Belo Horizonte, MG, Brasil 2 Laboratório de Parasitologia, Universidade Federal de Alfenas, Alfenas, MG, Brasil 3 Division of Infection and Immunity, University of Glasgow, Scotland, UK The outcome of the interaction between Biomphalaria and Schistosoma mansoni depends on the response of the host internal defence system (IDS) and the escape mechanisms of the parasite. The aim of this study was to evaluate the responsiveness of the IDS (haemocytes and soluble haemolymph factors) of resistant and susceptible Biomphalaria tenagophila lineages and Biomphalaria glabrata lineages in the presence of in vitro-transformed primary sporocysts and secondary sporocysts obtained from infected B. glabrata. To do this, we assayed the cellular adhesion index (CAI), analysed viability/mortality, used fluorescent markers to evaluate the tegumental damage and transplanted secondary sporocysts. B. tenagophila Taim was more effective against primary and secondary sporocystes than the susceptible lineage and B. glabrata. Compared with secondary sporocysts exposed to B. tenagophila, primary sporocysts showed a higher CAI, a greater percentage of dead sporocysts and were labelled by lectin from Glycine max and Alexa-Fluor 488 fluorescent probes at a higher rate than the secondary sporocysts. However, the two B. tenagophila lineages showed no cercarial shedding after inoculation with secondary sporocysts. Our hypothesis that secondary sporocysts can escape the B. tenagophila IDS cannot be confirmed by the transplantation experiments. These data suggest that there are additional mechanisms involved in the lower susceptibilty of B. tenagophila to S. mansoni infection. Key words: Schistosoma mansoni - sporocysts - Biomphalaria tenagophila - internal defence system - escape mechanisms Planorbidae of the genus Biomphalaria are the intermediate hosts of the parasite Schistosoma mansoni. A high degree of specificity is an important characteristic of the mollusc-digenea interaction. According to the literature, a restricted number of Biomphalaria species and lineages are susceptible to a specific strain of S. mansoni (Basch 1976). Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea are the three species responsible for the transmission of schistosomiasis in Brazil (Paraense 1975). Many authors (Corrêa et al. 1979, Santos et al. 1979, Bezerra et al. 1997, 2003, Martins-Souza et al. 2003, Rosa et al. 2004, 2005, 2006, Barbosa et al. 2006, Coelho et al. 2008, Pereira et al. 2008) have described the existence of a geographic lineage of B. tenagophila (provided from Ecological Station of Taim, state of Rio Grande do Sul, Brazil) resistant to S. mansoni. This lineage represents an important population in which to study the defence mechanisms of these invertebrate hosts to S. mansoni infection. The compatibility between S. mansoni and Biomphalaria is established by genetic factors of the parasite and of the Financial support: CAPES, PRONEX-FAPEMIG, FIOCRUZ + Corresponding author: Received 18 November 2010 Accepted 16 February 2011 intermediate host (Adema & Loker 1997). Biomphalaria has an internal defence system (IDS) composed of cells (haemocytes) and soluble haemolymph factors that are stimulated in the presence of parasites (van der Knaap & Loker 1990, Gliński & Jarosz 1997, Hahn et al. 2000, 2001). Parasite escape mechanisms that respond to the host IDS can assure the survival and adaptability of the parasite (Yoshino & Bayne 1983, Adema & Loker 1997). Previous studies have demonstrated that success of the infection depends on the parasite interfering with the mollusc IDS (Fryer & Bayne 1990). The parasite can disturb the IDS by producing proteases (Yoshino et al. 1993) and by the action of enzymes that neutralise reactive oxygen species (Connors & Yoshino 1990). In addition, it has been demonstrated that sporocysts release glycoproteins (excreted/secreted products) that bind to carbohydrate-binding receptors on the haemocyte cell surface to attenuate the initial cellular recognition of the parasite (Johnston & Yoshino 2001). These molecules can inhibit IDS mechanisms (Adema & Loker 1997) by suppressing the synthesis of many haemocyte molecules (Lodes et al. 1991) or by reducing haemocyte motility and phagocytic activity (Fryer & Bayne 1990, Lodes & Yoshino 1990). Other important parasite escape mechanisms include the use of molecular mimicry, likely through the production of sporocyst surface membrane molecules similar to those present on the host, and the ability of the parasite to absorb host antigens (Yoshino & Bayne 1983, Damian 1987, Salzet et al. 2000, Lehr et al. 2008, 2010, Peterson et al. 2009, van Die & Cummings online memorias.ioc.fiocruz.br B. tenagophila-sporocyst interaction Ana Carolina Alves de Mattos et al ). According to Salzet et al. (2000), the latter mechanism can prevent the recognition of the parasite by the host IDS. Furthermore, van Die and Cummings (2010) and Lehr et al. (2010) have suggested that glycans play a role in the parasite molecular mimicry process. Although the evolutionary advantages of this adaptive process for the parasite are well understood, it is not known how this process interferes with schistosomiasis (Salzet et al. 2000) or whether this mechanism could interfere with the snail s resistance mechanisms. To this end, the aim of this study was to evaluate the mechanisms of B. tenagophila Taim (resistant) and Cabo Frio (susceptible) IDS systems in the presence of primary sporocysts transformed in vitro and secondary sporocysts derived from infected snails. To accomplish this, we measured cellular adhesion, assayed sporocysts viability and mortality, evaluated the tegumental damage using fluorescent markers and transplanted secondary sporocysts. MATERIALS AND METHODS Snails and parasites - Snails B. tenagophila (Taim and Cabo Frio lineages) and B. glabrata (12-14 mm of diameter) and the S. mansoni LE strain were used in this work. The snails had been maintained for more than 30 years in the Mollusc Room of the René Rachou Research Center - Oswaldo Cruz Foundation. Production of primary and secondary sporocysts - Primary sporocysts were obtained from miracidia that were cultivated and transformed in vitro according to the technique described by Mattos et al. (2006) and Bahia et al. (2006). Briefly, livers of mice 50 days after infection with S. mansoni were macerated, sedimented and transferred to a glass flask containing 500 ml of water without chlorine. This flask was exposed to artificial light, and the miracidia were collected. The miracidia were transformed in Roswell Park Memorial Institute-1640 culture medium containing 5% foetal bovine serum (FBS) (Gibco Limited, Paisley, Scotland, UK) and 100 μg/ml penicillin/streptomycin antibiotics (Sigma) in biochemical oxygen demand (BOD) incubators at 26ºC for 24 h. The primary sporocysts were then washed with Chernin Balanced Saline Solution (CBSS) (ph 7.4; 100 mosm). The secondary sporocysts were obtained from B. glabrata infected with S. mansoni (Pereira et al. 1984). Briefly, the cephalopodal region from snails infected with 50 miracidia was dissected after 13 days of infection. This tissue, with the secondary sporocysts, was ground and fragments were transferred to a nylon mesh (200 µm) and maintained in contact with CBSS (ph 7.4) for 2 h in a water bath at 28ºC. Next, CBSS from below the mesh was centrifuged at 1,000 rpm for 2 min and the secondary sporocysts were counted under a microscope. Haemolymph collection and counting of haemocytes - Whole haemolymph was collected from B. tenagophila (Taim and Cabo Frio lineages) and B. glabrata. Each snail shell was cleaned with 70% alcohol and maintained overnight in antibiotic solution (100 μg/ml penicillin/ streptomycin and 4 mg amphotericin B). Total haemolymph (TH fraction) was collected by cardiac puncture using a 21G needle (Zelck & Becker 1990, Bezerra et al. 1997) and centrifuged at 1,000 rpm for 10 min at 4ºC. The supernatant was transferred to another tube [(S) soluble fraction only] and the pelleted haemocytes were resuspended in CBSS supplemented with 2% essential aminoacids (Sigma), 5% FBS and 2% antibiotics penicillin/streptomycin; haemocytes were resuspended in a CBSS volume equal to the haemolymph that was initially collected [(H) fraction of haemocytes only]. The haemocytes were then diluted 1/10 in CBSS solution containing 0.4% Trypan Blue and counted in a Neubauer s chamber (Martins-Souza et al. 2009). In the experiments, haemolymph from 15 snails was pooled and the post-separation fractions were called TH, H or S. Evaluation of the cellular adhesion index (CAI) and the viability/mortality of primary and secondary sporocysts after contact with B. tenagophila (Taim and Cabo Frio lineages) and B. glabrata IDS components - The experiments were performed in 24-well culture plates. Thirty microliters of S and TH fractions and 2 x 10 5 H fraction from B. tenagophila (Taim and Cabo Frio lineages) and B. glabrata were separately exposed to 20 primary or secondary sporocysts. CBSS was then added for a final volume of 300 µl/well. For measuring the CAI and for viability/mortality experiments, 10 wells were prepared for each lineage and each fraction; wells with only sporocysts were used as controls. The culture plates were maintained in BOD incubators at 26ºC. After 2 h incubation, 100 sporocysts from each lineage were analysed to determine their CAI using the protocol described by Castillo and Yoshino (2002). Sporocyst CAIs were scored as arbitrary values ranging from 1-4 according to the following parameters: CAI 1: no adherent haemocytes on the sporocyst surface; CAI 2: up to 10 adherent haemocytes; CAI 3: between haemocytes; CAI 4: more than 50 adherent haemocytes. The formula to calculate CAI was: CAI = Total binding value Number of sporocysts scored The total binding value was obtained by summing the individual values (1-4) of all the sporocysts scored from each lineage and haemolymph fraction. The following statistical analyses were used: an unpaired, twotailed Student t test/mann-whitney test (2-tailed) or the One-Way Analysis of Variance/Kruskal-Wallis and Dunn s Multiple Comparison test. Analyses were carried out using GraphPad Prism 4 software. Differences were considered significant if p Sporocyst viability was assessed 6 h post-incubation using an inverted microscope. The proportion of dead sporocysts was identified by 0.4% Trypan Blue staining. All the sporocysts present in each well were counted. The Chi-square test with Fisher s exact test (when necessary) was used to analyse the data. These tests were conducted using the Minitab 14 software. Evaluation of primary and secondary sporocyst tegumental damage after contact with B. tenagophila (Taim and Cabo Frio lineages) IDS components - Thirty microliters of fractions S, TH and 2 x 10 5 H fraction from the B. tenagophila Taim and Cabo Frio lineages were separately 426 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(4), June 2011 incubated with 20 primary or secondary sporocysts in 24- well culture plates. The volume of each well was topped up to 300 µl with CBSS after the distribution of IDS and sporocysts. The culture plates were maintained in BOD incubators at 26ºC. Wells containing only sporocysts were used as a control. Six experimental wells were used for each lineage and each haemolymph fraction. After 3 h and 5 h of incubation, the sporocysts were labelled for 30 min with fluorescents probes Hoechst (a hydrophilic DNA-specific probe that binds with DNA only after damage to the cell membrane), lectin from Glycine max (soybean, specific for N-acetylgalactosamine) and Alexa-Fluor 488 phalloidin (specific for actin filaments). The parasites were then washed three times with CBSS and analysed with a fluorescence microscope (K-Zeiss). The filters used were as follows: red for lectin from Glycine max TRITC (excitation/maximal emission 543/571 nm), green for Alexa-Fluor 488 (excitation/maximal emission 499/520 nm) and blue for Hoechst (excitation/maximal emission 352/455 nm). Photographs were taken with a Canon EOS Digital Rebel XT camera. The colour histogram tool of the ImageJ 1.43 software was used to calculate mean fluorescence intensity/area for each image obtained in the experiments. Statistical analyses were performed using the Mann Whitney test (2-tailed) in the GraphPad Prism 4 software. Differences were considered significant if p Evaluating the development of secondary sporocysts from B. glabrata infected after sporocyst inoculations in B. tenagophila (Taim and Cabo Frio) and B. glabrata - To determine whether the IDS from B. tenagophila Taim can recognise the secondary sporocysts obtained from B. glabrata, we inoculated B. tenagophila with parasites originally recovered from B. glabrata. Thirty secondary sporocysts obtained from infected B. glabrata were inoculated into 65 B. tenagophila Taim and B. glabrata and into 35 B. tenagophila de Cabo Frio using a modified protocol described by Jourdane and Theron (1980). Briefly, snails were anaesthetised with sodium pentobarbital solution (0.4 mg/ml) for at least 6 h (Martins-Souza et al. 2001). A sample of secondary sporocysts was stained with Trypan Blue and the unstained parasites were counted. Next, the secondary sporocysts were inoculated (maximum 30 µl) into the cephalopodal area using a syringe and 30G needle. After inoculation for 14, 21, 27 and 35 days, the snails were exposed individually to artificial light for 1 h to verify the shedding of cercariae. RESULTS CAI of primary and secondary sporocysts - The CAI values are shown in Table I. The haemocytes from all Biomphalaria species were able to adhere to the surface of primary and secondary sporocysts. However, IDS of B. glabrata (mean CAI - TH: 1.89 ± 0.31, H: 1.87 ± 0.35) showed significantly lower CAI values compared to IDS of B. tenagophila Taim (mean CAI - TH: 2.13 ± 0.25, H: 2.02 ± 0.22) and Cabo Frio (mean CAI - TH: 2.32 ± 0.3, H: 1.98 ± 0.31) (p 0.03). Similarly, B. tenagophila Taim cell binding to secondary sporocysts (mean CAI - TABLE I Mean cellular adhesion index (CAI) values of Biomphalaria tenagophila (Taim and Cabo Frio) and Biomphalaria glabrata cells binding of primary and secondary sporocysts of Schistosoma mansoni Groups of IDS Primary sporocysts CAI ± SD Secondary sporocysts CAI ± SD B. tenagophila Taim TH 2.13 ± 0.25 b 2.03 ± 0.31 b H 2.02 ± 0.22 b 2.27 ± 0.30 a,b B. tenagophila Cabo Frio TH 2.32 ± 0.3 b,c 2.03 ± 0.23 b H 1.98 ± ± 0.12 b B. glabrata TH 1.89 ± ± 0.41 H 1.87 ± 0.35 c 1.41 ± 0.2 a: statistical difference between CAI of primary or secondary sporocysts exposed to B. tenagophila Taim and Cabo Frio internal defence system (IDS) [total hemolymph (TH) or fraction of hemocytes only (H)] and CAI of primary or secondary sporocysts exposed to Cabo Frio IDS (TH or H) p 0,03; b: statistical difference between CAI of primary or secondary sporocysts exposed to Taim IDS (TH or H) and CAI of primary or secondary sporocysts exposed to B. glabrata IDS (TH or H) p 0,03; c: statistical difference betwen CAI of primary and secondary sporocysts exposed to IDS from the same species or lineage (p 0,02). H: 2.27 ± 0.30) was significantly higher (p 0.03) than to the secondary sporocysts exposed to Cabo Frio (mean CAI - H: 1.99 ± 0.12) and B. glabrata (mean CAI - H: 1.41 ± 0.2). Moreover, there were significantly (p 0.02) more cells adhering to the surface of primary sporocysts than of secondary sporocysts when they were exposed to B. tenagophila Cabo Frio (mean CAI - TH: 2.32 ± 0.3) and B. glabrata (mean CAI - H: 1.87 ± 0.35). There were no statistically significant differences between the different fractions of IDS (TH and H) used in the experiments. Viability/mortality of primary and secondary sporocysts - The results of the viability experiments are summarised in Table II. B. tenagophila Taim IDS resulted in significantly higher primary sporocysts mortality (TH = 47.5%, H = 58%, S = 56%) compared to IDS from B. tenagophila Cabo Frio (TH = 23%, H = 32%, S = 24.5%), B. glabrata (TH = 5%, H = 18%, S = 6%) and sporocysts control (20%) (p 0.03). However, the proportion of dead secondary sporocysts exposed to B. tenagophila Taim (TH = 7%, H = 8.5%, S = 6.5%) was significantly higher (p 0.03) only when compared to B. glabrata (TH = 3%, H = 3%, S = 2%). On the other hand, we observed that the secondary sporocysts died at a significantly lower rate (p 0.01) than the primary sporocysts when exposed to B. tenagophila Taim and Cabo Frio but not to B. glabrata. Presence of tegumental damage in primary and secondary sporocysts after contact with mollusc IDS (HT, H and S fractions) - No differences were observed in the labelling of sporocysts analysed 3 h and 5 h after incubation with different fractions of haemolymph TH B. tenagophila-sporocyst interaction Ana Carolina Alves de Mattos et al. 427 from each lineage. Tegumental damage was shown by Hoechst staining to be similar for all experimental conditions. However, primary sporocysts exposed to B. tenagophila Cabo Frio (mean fluorescence intensity/ area: 2.8 ± 1.6) were significantly more labelled (p = 0.03) than secondary sporocysts (mean fluorescence intensity/ area: 0.21 ± 0.05) (Fig. 1). Alexa-Fluor 488 was better in indicating the differences in the extent of tegument damage between the various experiments. The primary sporocysts exposed to Taim (mean fluorescence intensity/area: 2.5 ± 0.8) presented significantly more (p = 0.04) tegumental damage than both the sporocysts exposed to Cabo Frio (mean fluorescence intensity/area: 1.1 ± 0.84) and control primary sporocysts (mean fluorescence intensity/area: 1.1 ± 0.9). Moreover, the primary sporocysts exposed to Taim were labelled at a significantly higher rate (p = 0.04) than the secondary sporocysts (mean fluorescence intensity/area: 0.38 ± 0.2) (Fig. 2). A similar result was observed using lectin from Glycine max. Primary sporocysts exposed to Taim (mean fluorescence intensity/area: 2.76 ± 0.8) showed significantly higher (p 0.03) labelling than the sporocysts exposed to Cabo Frio (mean fluorescence intensity/area: 0.47 ± 0.43) and sporocysts control (mean fluorescence intensity/area: 0.7 ± 0.6) and higher labelling than secondary sporocysts exposed Taim (mean fluorescence intensity/ area: 0.12 ± 0.06) (Fig. 3). The labelling intensities for all experimental conditions are shown in the Figs 4-6. Development of secondary sporocysts isolated from B. glabrata and inoculated into B. tenagophila (Taim and Cabo Frio) and B. glabrata - Sixty-five B. tenagophila Taim were inoculated with secondary sporocysts provided from infected B. glabrata. Thirty days after inoculation, only seven snails were alive (11%) and none were shedding cercariae. Of the 35 B. tenagophila Cabo Frio similarly inoculated with B. glabrata-derived secondary sporocysts, 11 snails were alive at the end of the TABLE II Proportion of dead primary and secondary sporocysts of Schistosoma mansoni after exposure of parasite to the internal defence systems (IDS) of Biomphalaria tenagophila Taim (resistant), Cabo Frio (susceptible) and Biomphalaria glabrata Groups of IDS Dead primary sporocysts (%) Dead secondary sporocysts (%) B. tenagophila Taim TH 47.5 a,b,c,d 7 b H 58 a,b,c, d 8.5 b S 56 a,b,c,d 6.5 b B. tenagophila Cabo Frio TH 23 b,d 9 c H 32 d 5 S 24.5 b,d 5.5 B. glabrata TH 5 c 3 H 18 d 3 S 6 c 2 Control a: statistical differ
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