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Protection against Toxoplasma gondii brain cyst formation in mice immunized with Toxoplasma gondii cytoskeleton proteins and Lactobacillus casei as adjuvant
Veterinary Parasitology 160 (2009) 311–315
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Protection against Toxoplasma gondii brain cyst formation in mice immunized with Toxoplasma gondii cytoskeleton proteins and Lactobacillus casei as adjuvant Federico Martı´nez-Go´mez a, Luis Francisco Garcı´a-Gonza´lez b, Ricardo Mondrago´n-Flores c, Carlos Ramo´n Bautista-Garfias d,* a
Departamento de Parasitologı´a ENCB-IPN, Mexico Centro Interdisciplinario de Ciencias de la Salud, unidad Santo Toma´s, Mexico Departamento de Bioquı´mica Cinvestav-IPN, Mexico d CENID-PAVET, INIFAP, Col. Progreso, 62550 Jiutepec, Morelos, Mexico b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 June 2008 Received in revised form 14 November 2008 Accepted 17 November 2008
The aim of the study was to evaluate the protection generated in mice against Toxoplasma gondii brain cyst burden by vaccination with T. gondii cytoskeleton proteins using Lactobacillus casei as adjuvant. One hundred and sixty-eight NIH mice were randomly allocated into eight groups of 21 mice each. Animals were immunized as follows: in group 1 with Toxoplasma lysate antigen (TLA) in Freund’s modified adjuvant, containing L. casei (FMA), in group 2 with Toxoplasma cytoskeleton proteins (TCPs) in FMA, in group 3 with FMA, in group 4 with phosphate buffered saline (PBS), in group 5 with L. casei dead by heath (Lc), in group 6 with Freund’s complete adjuvant (FCA), in group 7 with TLA in FCA, and in group 8 with TCP in FCA. Mean brain cyst burden (S.E.M.) was assessed in mice 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). The percentages of reduction in cyst burden per brain (P < 0.01) as compared with the group 4 (control: mean 3181 97.5) were 77.25% (724 98) in group 1, 88.02% (381 97.5) in group 2, 38.92% (1943 130.3) in group 3, 44.31% (1771.4 102) in group 5, 59.28% (1295.2 99.1) in group 7 and 55.69% (1409.5 89.9) in group 8. In order of importance, the best protection was obtained in groups 2, 1, 7, 8, 5 and 3. Noticeably the mice inoculated with L. casei alone showed a significant reduction in T. gondii brain cysts (P < 0.01), while those animals treated with FCA alone did not. Additionally, IgM anti-T. gondii antibody levels, as determined by ELISA 2 weeks after challenge, were highest in group 2 (P < 0.01) than in the other seven groups. Results suggest that T. gondii cytoskeleton proteins with L. casei as adjuvant constitute a good antitoxoplasmosis vaccine candidate. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Toxoplasma gondii cytoskeleton proteins Immunization Lactobacillus casei Adjuvant
1. Introduction Toxoplasma gondii is a protozoan parasite which infects many species of warm-blooded mammals worldwide,
* Corresponding author. Tel.: +52 777 3192848x120; fax: +52 777 3192848x129. E-mail addresses: [email protected], carlosramon.bautistagarfi[email protected] (C.R. Bautista-Garfias). 0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.11.017
including the man and most birds (Joiner and Dubremetz, 1993; Kim and Weiss, 2004) causing toxoplasmosis. In humans, clinical toxoplasmosis is generally limited either to immunocompromised individuals or to congenital disease resulting from an acute infection of the expectant mother (Black and Boothroyd, 2000; Dunn et al., 1999; Petersen et al., 2001). It has become one of the main opportunistic pathogens for AIDS patients causing toxoplasmic encephalitis (Luft and Remington, 1992; Richards et al., 1995). T. gondii is one of the most
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significant foodborne parasites because it is common, its acquisition can be devastating and it places an immense financial burden on society (Roberts et al., 1994; Lee, 2000). It has been pointed out that most apicomplexan parasites share a set of cytoskeletal structures essential for parasite survival and pathogenesis (Morrissette and Sibley, 2002). In this context, central to both invasion and proliferation is a group of structures at one end of the parasite, the apical complex. In T. gondii the apical complex is built around the conoid, an assembly of spirally arranged fibers that is actively motile during invasion. Also, in the conoid/apical complex of this parasite approximately 200 proteins, representing 70% of its cytoskeletal protein components, have been identified (Hu et al., 2006). On the other hand, it has been demonstrated that the inoculation of the lactic bacteria Lactobacillus casei in mice previous to infection with parasitic protozoa generates a protective response, such as the cases of Babesia microti (Bautista-Garfias et al., 2005), Plasmodium chabaudi (Martı´nez-Gomez et al., 2006) and Trypanosoma cruzi (Bautista-Garfias et al., 2008). In experiments of immunization against trichinellosis in mice, it also has been shown that L. casei functions as adjuvant when is inoculated with antigens of Trichinella spiralis larvae (Bautista-Garfias et al., 2004). Partial protection has been obtained in studies of vaccination against toxoplasmosis using parasite’s surface molecules or antigens from other organelles (Beguetto et al., 2005; Ismael et al., 2003; Leyva et al., 2001; Martin et al., 2004); however, the potential protective role of the parasite’s cytoskeleton is unknown. On the basis of the previous information, in the present study the protective role of T. gondii cytoskeleton proteins, using L. casei as adjuvant, was evaluated against brain cyst formation in mice. 2. Materials and methods 2.1. Animals Experiment was conducted using adult female NIH mice with a body weight of 22–25 g. Animals were housed in polysulphonate cages with clean saw-dust, fed on commercial mice pellets and allowed continual access to drinking water. They were maintained at the animal facility of the National School of the Biological Sciences of the IPN under regulated conditions of humidity and temperature. 2.2. T. gondii strains Two T. gondii strains were used: the virulent RH strain (Sabin, 1941) and the brain cyst forming Me49 strain (Suzuki et al., 1989). The virulent RH strain was utilized to obtain peritoneal tachyzoites from which antigen was prepared for immunization and for detection of anti-T. gondii antibodies by ELISA. The less virulent Me49 strain was used for the challenge of immunized mice.
2.3. Preparation of T. gondii antigens 2.3.1. Toxoplasma lysate antigen (TLA) ˜ o Osorio TLA was obtained in accordance with Castan and Sarracent Pe´rez (2002) with modifications. Briefly, tachyzoites of the virulent T. gondii RH strain were obtained from the peritoneal fluid of infected mice (48– 72 h of infection). The material was washed three times with phosphate buffered saline (PBS) (pH 7.2) at 500 g for 5 min, passed through 25- and 27-gauge needles, and passed through filter membranes of 5 mm pore size to remove debris and host cells. The parasites were resuspended PBS, sonicated (Vibra-cell, Sonics materials) on ice at 10,000 Hz, 7 cycles of 20 s each. Then the sonicated suspension was centrifuged in the cold (4 8C) at 7800 g during 30 min. The supernatant was recovered and a mixture of protease inhibitors (PMSF 5 mM, Pestatin A 1 mM, EDTA 1 mM) was added, then the T. gondii extract (TLA) was filtered through a 0.22 mm Millipore membrane, and protein content determined by the Bradford method (1976) using bovine serum albumin as standard. The TLA was stored in aliquots at 20 8C until use. 2.3.2. Toxoplasma cytoskeleton proteins (TCPs) As in the previous section, tachyzoites of the virulent T. gondii RH strain were obtained from the peritoneal fluid of infected mice (48–72 h of infection). The material was washed three times with PBS (pH 7.2) at 500 g for 5 min, passed through 25- and 27-gauge needles, and passed through filter membranes of 5 mm pore size (Millipore, IRE) to remove debris and host cells. The filtered material was washed twice in PBS and centrifuged at 500 g for 5 min and then adjusted in PBS at a concentration of 30– 40 106 tachyzoites/mL. The isolation of the cytoskeleton fraction was a carried out according with the method previously described (Patron et al., 2005). Briefly, the suspension was centrifuged at 800 g for 10 min, and then the pellet was resuspended in the cytoskeleton intracellular stabilizing medium PHEM (a mixture of 10 mM Hepes, 10 mM MgCl2, 10 mM EGTA, and 1 mM Cl2 EGTA) containing 50 mg/mL of Na-p-tosyl-L-lysine chloromethyl ketone (TPCK), and 17.4 mg/mL of phenylmethylsulphonyl fluoride (PMSF) as well as 0.1% Triton X-100 for 5 min; after that the cytoskeleton fraction was pelleted by ultracentrifugation at 150,000 g at 4 8C for 15 min and then the material was resuspended in PBS and kept at 4 8C until used. The cytoskeleton fraction was incubated in the solubilization buffer (2% b-mercaptoethanol, 2 mM Tris– HCl, and 1% SDS). Then, the material was run on 12% SDSPAGE gels in accordance with the method of Laemmli (1970). Afterwards, the gel was placed in 10 volumes of Coomassie Blue (Coomassie Brilliant Blue R 250) staining solution for 4 h, rocking gently to distribute the dye evenly over the gel. Destaining was monitored visually and adjusted accordingly. 2.3.3. Bacteria The strain ATCC 7469 of L. casei ssp rhamnosus (Bautista-Garfias et al., 1999) was grown in MRS broth at 37 8C during 18 h. The microorganisms were harvested and centrifuged at 5000 g during 10 min and then
F. Martı´nez-Go´mez et al. / Veterinary Parasitology 160 (2009) 311–315 Table 1 Experimental design. Group (n = 21)
1 2 3 4 5 6 7 8
Treatment/mouse (intraperitoneal) Day 0
Day 14
Day 28
TLA + FMA TCP + FMA FMA PBS Lc FCA TLA + FCA TCP + FCA
TLA + FIA TCP + FIA FIA PBS PBS PBS TLA + FIA TCP + FIA
TLA TCP PBS PBS PBS PBS TLA TCP
TLA: 20 mg of Toxoplasma gondii RH strain lysate antigen; FMA: Freund’s modified adjuvant; FIA: Freund’s incomplete adjuvant; TCP: 20 mg of T. gondii RH strain cytoskeleton proteins; PBS:100 mL of phosphate buffered saline; Lc: Lactobacillus casei 1 108 cfu dead by heath; FCA: Freund’s complete adjuvant.
washed several times in PBS (pH 7.2). After a viable count the L. casei was adjusted at a concentration of 109 bacteria/ mL, later these were killed by heath during 20 min at a boiling water bath (95 8C) and then they were lyophilized. 2.3.4. Freund’s modified adjuvant (FMA) To each millilitre of Freund’s incomplete adjuvant (Sigma, MO, USA) 1 mg of lyophilized dead L. casei were added to constitute the FMA. 2.4. Experimental design Immunization. Mice were randomly divided into eight groups of 21 mice per group. Each mouse was inoculated intraperitoneally (ip) as follows: in group 1 with of TLA in Freund’s modified adjuvant (FMA), in group 2 with TCP in FMA, in group 3 with FMA, in group 4 with PBS, in group 5 with L. casei dead by heath (Lc), in group 6 with Freund’s complete adjuvant (FCA) (Sigma, MO, USA), in group 7 with TLA in FCA, and in group 8 TCP in FCA (Table 1). 2.5. Parasitological and serological techniques 2.5.1. Challenge The brains of NIH mice with an infection of 8 weeks with the T. gondii Me49 strain were obtained, and then these were homogenized in 2 mL of PBS with glucose and using a glass homogenizer. Brain cysts were counted as follows: 5 mL of brain suspension was placed on a glass slide with an equal volume of Lugol’s iodine and mounted with a cover slip. The cysts, stained brown were counted at 40 magnification. Then the suspension was adjusted to the appropriated dose. Each mouse was infected orally 1 week after the 3rd booster immunization with 100 mL of the brain homogenate containing 20 cysts. 2.5.2. T. gondii brain cyst burden estimation 8 weeks alter challenge, all mice were humanitarianly sacrificed in accordance with the Mexican official norm NOM-062-ZOO-1999 (http://senasica2.bio.com.mx/default.asp?doc=743), and their brains were collected. Each brain was homogenized in 2 mL of PBS and Toxoplasma brain cysts were counted as indicated above.
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2.5.3. Serum samples Sera were collected from mice 7 days after the 3rd booster dose and on days 14, 21, and 28 post-infection by caudal vein bleeding. Sera were stored at 20 8C for the determination of antibodies. 2.5.4. Measurement of antibody responses TLA-specific IgM antibodies were detected and quantified by ELISA assay (Engvall and Perlmann, 1971) on serum samples from individual mice. Briefly, 96-well microtiter plates (Nunc) were coated overnight at 4 8C with TCP at a concentration of 50 mg/mL. After washing, the plates were blocked with 1% BSA in Tris-buffered saline for 2 h at room temperature and then were overlaid with 1:250 diluted sera. They were then detected with horseradish peroxidaseconjugated goat anti-mouse IgM (Sigma, MO, USA). Orthophenylenediamine-dihydrochloride (Sigma, MO, USA) was used as substrate. Optical densities (O.D.s) were measured with a microplate reader (MRX II, DYN-EX Technologies) at 450 nm. IgM anti-T. gondii antibody levels were expressed as group mean S.E.M. of individual O.D. values. 2.6. Statistical analysis The statistical significance of differences was determined from the means S.E.M. by analysis of variance (ANOVA) with software Paquete de disen˜os experimentales FAUANL. Version 2.5 (Olivares, 1994). When differences were found the Tukey test was used, and these were considered significant when P values were <0.01. 3. Results 3.1. T. gondii cytoskeleton proteins The TCP showed 19 components in 12% SDS-PAGE gels, which ranged between 11 and 133 kDa (data not shown). 3.2. T. gondii brain cyst burden The lowest brain cyst burdens (P < 0.01) were observed in mice immunized with TCP in FMA (381 97.5) and TLA in FMA (724 98) as compared with those obtained in animals of the control group (3181 89.32) and the other five groups. This protective response was even better (P < 0.01) than that showed in mice of groups vaccinated with TCP in FCA (1409.5 90) and TLA in FCA (1295.2 99.1). The brain cyst burden was significantly lower (P < 0.01) in mice treated with L. casei alone (1771.4 102) than that observed in control animals or in mice immunized with FCA (2971.4 85.4) (Table 2). 3.3. IgM antibody responses to T. gondii The levels of IgM anti-Toxoplasma antibodies were significantly higher (P < 0.01) at 14 days after challenge in sera from mice vaccinated with TCP mixed with FMA as compared with the values obtained in the sera from the animals of the other seven groups (Table 3). The IgM levels in groups TLA + FMA, TLA + FCA, and TCP + FCA were similar but higher (P < 0.01) than the values showed in groups FMA, PBS, Lc, and FCA (Table 3).
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Table 2 Mean cyst burden (S.E.M.) per brain in mice which received different treatments 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). Group
n
Treatment
Mean number of brain, cysts S.E.M.
P
Percentage reductiona
1 2 3 4 5 6 7 8
21 21 21 21 21 21 21 21
TLA + FMA TCP + FMA FMA PBS Lc FCA TLA + FCA TCP + FCA
723.8 98.0 381.0 97.5 1942.9 130.3 3181.0 89.32 1771.4 101.7 2971.4 85.4 1295.2 99.1 1409.5 89.9
D D B A BC A C C
77.25 88.02 38.92 – 44.31 6.59 59.28 55.69
TLA: Toxoplasma lysate antigen; FMA: Freund’s modified adjuvant; TCP: Toxoplasma cytoskeleton proteins; PBS: phosphate buffered saline; Lc: Lactobacillus casei dead; FCA: Freund’s complete adjuvant. P: different literals in the same column indicate significant difference (P < 0.01, Tukey test). a As compared with control group (PBS). Table 3 Mean (S.E.M.) anti-T. gondii antibody (IgM) levels in sera of mice which received different treatments, 2 weeks after challenge with T. gondii Me49 strain, as determined by ELISA. Group
n
Treatment
Mean IgM anti-T. gondii (O.D. at 450 nm)
P
1 2 3 4 5 6 7 8
7 7 7 7 7 7 7 7
TLA + FMA TCP + FMA FMA PBS Lc FCA TLA + FCA TCP + FCA
0.8228 0.037 1.4642 0.103 0.3585 0.106 0.1664 0.062 0.3228 0.024 0.270 0.014 0.6885 0.029 0.6328 0.037
B A C C C C B B
TLA: Toxoplasma lysate antigen; FMA: Freund’s modified adjuvant; TCP: Toxoplasma cytoskeleton proteins; PBS: phosphate buffered saline; Lc: Lactobacillus casei dead; FCA: Freund’s complete adjuvant. P: different literals in the same column indicate significant difference (P < 0.01, Tukey test).
4. Discussion 4.1. T. gondii cytoskeleton proteins Although only 19 components were observed in the CTP by 12% SD-PAGE of this study, the cytoskeleton of T. gondii is by far very complex. In this respect, it has been shown by using molecular tools that in the conoid/apical complex of T. gondii approximately 200 proteins, representing 70% of its cytoskeletal protein components have been identified (Hu et al., 2006). However, the CTP obtained from tachyzoites was excellent, when mixed with an adjuvant containing L. casei, for inducing protection against brain cyst formation in mice. 4.2. T. gondii brain cyst burden We obtained significant reductions in brain parasite burden after challenge with the T. gondii Me49 strain in NIH immunized with TCP (88%) and TLA (77.25%) and it is worth to note that good protection was generated by L. casei alone up to 1 month after intraperitoneal inoculation in mice. Other studies in which different T. gondii antigen preparations and adjuvants were used have shown similar percentages of protection (even lower to the reported by us) against T. gondii brain cyst formation in mice. In this respect, El-Malky et al. (2005) obtained a 64% reduction in
brain parasite burden (BPB) after challenge with the RRA (Beverly) strain of T. gondii in C57BL/6 mice vaccinated with TLA and CpG as adjuvant in comparison to non-CpG– TLA treated group. Swiss mice immunized with tachyzoites plus tissue cysts in liposomes or purified tachyzoite antigen in liposomes showed reductions in BPB of 85.85% and 85.4%, respectively after challenge with the P strain of T. gondii (Elsaid et al., 1999) as compared with control mice. Beuvillain et al. (2007) observed a significant reduction (59.75%) in BPB after challenge with the T. gondii 76K strain (30 cysts per mouse) in CBA/J mice immunized with exosomes secreted by the dendritic cell line SRDC pulsed in vitro with T. gondii-derived antigens. It has been pointed out that adjuvants play an important role in the efficacy of vaccines. In addition to increasing the strength and kinetics of an immune response, adjuvants also play a role in determining the type of immune response generated. In this context, it is known that IFNg plays a critical role in protection against T. gondii infection (Susuki et al., 1988; Suzuki et al., 1989b; Liu et al., 2006; LaRosa et al., 2008). It is also known that several species of lactobacilli, including L. casei, induce the production of protective IFNg (Bautista-Garfias et al., 1999; Castanheira et al., 2007). Although we did not measure the production of IFNg we believe that L. casei promoted the production of this cytokine. 4.3. IgM antibody responses to T. gondii Significant levels of IgM anti-T. gondii (P < 0.01) were observed in mice vaccinated with TCP and TLA mixed with Freund’s modified adjuvant or FCA as compared with mice which received FMA, PBS, Lc or FCA alone. In accordance with the results obtained we believe that the IgM anti-T. gondii antibodies induced, played a role in the whole protection observed. In this respect, it has been shown that T. gondii-specific IgM antibodies are important for limiting dissemination of parasite infection in BALB/c mice by preventing host cell invasion (Couper et al., 2005); however, the animals treated with FMA or L. casei alone showed significant protection (P < 0.01) as compared with mice treated with PBS (control group) or FCA. This observation is supported by the notion that the main protection against T. gondii is carried out by the cellular arm of the immune response (Liu et al., 2006).
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5. Conclusion The protective response against T. gondii cyst formation induced in NIH mice by L. casei dead alone (group 5) was similar to that generated by FMA (group 3) and better than that induced by FCA (group 6). The addition of L. casei to the incomplete Freund’s adjuvant used for the immunization of mice with TLA (group 1) or TCP (group 2) generated a better protection than that observed in mice immunized with these antigenic preparations mixed with FCA (groups 7 and 8); however, there were no differences between animals of the groups immunized with TLA or TCP and mixed with FMA. In this context, it is worth to note that the difference in the IgM anti-T. gondii antibody levels observed particularly between these two later groups (P < 0.01) might have been influenced by the antigen used in the ELISA. Taken together the results of the present study suggest that T. gondii cytoskeleton proteins using L. casei as adjuvant constitute a good anti-toxoplasmosis vaccine candidate. Acknowledgements To the Research Office of the National Polytechnic Institute (IPN), for financial support. To Dr. Pascal Rene Herion Scohy, Immunology Department of the Biomedical Research Institute from the National Autonomous University of Mexico (UNAM) for providing the T. gondii Me49 strain. References ˜a, M., Ixta, O., Martı´nez, F., Aguilar, B., Corte´s, Bautista-Garfias, C.R., Ordun A., 1999. Enhancement of resistance in mice treated with Lactobacillus casei: effect on Trichinella spiralis infection. Vet. Parasitol. 80, 251–260. Bautista-Garfias, C.R., Posadas Beltra´n, A., Ixta Rodrı´guez, O., 2004. Immunization of BALB/c mice with an antigen from Trichinella spiralis muscle larvae using Lactobacillus casei as adjuvant. Vet. Mex. 35, 359–368. Bautista-Garfias, C.R., Go´mez, M., Aguilar, B., Ixta, B.R., Martı´nez, O., Mosqueda, F.J., 2005. The treatment of mice with Lactobacillus casei induces protection against Babesia microti infection. Parasitol. Res. 97, 472–477. Bautista-Garfias, C.R., Torres-Alvarez, M.C., Martı´nez-Go´mez, F., 2008. The inoculation of Lactobacillus casei into mice induces a protective response against Trypanosoma cruzi (Ninoa strain). Vet. Mex. 39, 139–144. Beguetto, E., Nielsen, H.V., Del Porto, P., Buffolano, W., Guglietta, S., Felici, F., Petersen, E., Gargano, N., 2005. A combination of antigenic regions of Toxoplasma gondii microneme proteins induces protective immunity against oral infection with parasite cysts. J. Infect. Dis. 191, 637– 645. Beuvillain, C., Ruiz, S., Guiton, R., Bout, D., Dimier-Poisson, I., 2007. A vaccine based on exosomes secreted by dendritic cell line confers protection against T. gondii infection in syngeneic and allogeneic mice. Microbes Infect. 9, 1614–1622. Black, M.W., Boothroyd, J.C., 2000. Lytic cycle of Toxoplasma gondii. Microbiol. Mol. Biol. Rev. 64, 607–623. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of protein– dye binding. Anal. Biochem. 72, 248–254. Castanheira, L.G., de Andrade Castro, J.M., Martins-Filho, M.A., Nicoli, J.R., Vieira, L.Q., Crocco Alonso, L.C., 2007. Lactobacillus delbrueecki as potential skin adjuvant for induction of type 1 immune responses. Front. Biosci. 12, 1300–1307. ˜ o Osorio, J.C., Sarracent Pe´rez, J., 2002. Inmunizacio´n intranasal de Castan ratones con la proteı´na SAG2 de Toxoplasma gondii asociada con la toxina cole´rica. Rev. Cubana Invest. Biomed. 21, 35–45.
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Protection against Toxoplasma gondii brain cyst formation in mice immunized with Toxoplasma gondii cytoskeleton proteins and Lactobacillus casei as adjuvant Federico Martı´nez-Go´mez a, Luis Francisco Garcı´a-Gonza´lez b, Ricardo Mondrago´n-Flores c, Carlos Ramo´n Bautista-Garfias d,* a
Departamento de Parasitologı´a ENCB-IPN, Mexico Centro Interdisciplinario de Ciencias de la Salud, unidad Santo Toma´s, Mexico Departamento de Bioquı´mica Cinvestav-IPN, Mexico d CENID-PAVET, INIFAP, Col. Progreso, 62550 Jiutepec, Morelos, Mexico b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 June 2008 Received in revised form 14 November 2008 Accepted 17 November 2008
The aim of the study was to evaluate the protection generated in mice against Toxoplasma gondii brain cyst burden by vaccination with T. gondii cytoskeleton proteins using Lactobacillus casei as adjuvant. One hundred and sixty-eight NIH mice were randomly allocated into eight groups of 21 mice each. Animals were immunized as follows: in group 1 with Toxoplasma lysate antigen (TLA) in Freund’s modified adjuvant, containing L. casei (FMA), in group 2 with Toxoplasma cytoskeleton proteins (TCPs) in FMA, in group 3 with FMA, in group 4 with phosphate buffered saline (PBS), in group 5 with L. casei dead by heath (Lc), in group 6 with Freund’s complete adjuvant (FCA), in group 7 with TLA in FCA, and in group 8 with TCP in FCA. Mean brain cyst burden (S.E.M.) was assessed in mice 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). The percentages of reduction in cyst burden per brain (P < 0.01) as compared with the group 4 (control: mean 3181 97.5) were 77.25% (724 98) in group 1, 88.02% (381 97.5) in group 2, 38.92% (1943 130.3) in group 3, 44.31% (1771.4 102) in group 5, 59.28% (1295.2 99.1) in group 7 and 55.69% (1409.5 89.9) in group 8. In order of importance, the best protection was obtained in groups 2, 1, 7, 8, 5 and 3. Noticeably the mice inoculated with L. casei alone showed a significant reduction in T. gondii brain cysts (P < 0.01), while those animals treated with FCA alone did not. Additionally, IgM anti-T. gondii antibody levels, as determined by ELISA 2 weeks after challenge, were highest in group 2 (P < 0.01) than in the other seven groups. Results suggest that T. gondii cytoskeleton proteins with L. casei as adjuvant constitute a good antitoxoplasmosis vaccine candidate. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Toxoplasma gondii cytoskeleton proteins Immunization Lactobacillus casei Adjuvant
1. Introduction Toxoplasma gondii is a protozoan parasite which infects many species of warm-blooded mammals worldwide,
* Corresponding author. Tel.: +52 777 3192848x120; fax: +52 777 3192848x129. E-mail addresses: [email protected], carlosramon.bautistagarfi[email protected] (C.R. Bautista-Garfias). 0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.11.017
including the man and most birds (Joiner and Dubremetz, 1993; Kim and Weiss, 2004) causing toxoplasmosis. In humans, clinical toxoplasmosis is generally limited either to immunocompromised individuals or to congenital disease resulting from an acute infection of the expectant mother (Black and Boothroyd, 2000; Dunn et al., 1999; Petersen et al., 2001). It has become one of the main opportunistic pathogens for AIDS patients causing toxoplasmic encephalitis (Luft and Remington, 1992; Richards et al., 1995). T. gondii is one of the most
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significant foodborne parasites because it is common, its acquisition can be devastating and it places an immense financial burden on society (Roberts et al., 1994; Lee, 2000). It has been pointed out that most apicomplexan parasites share a set of cytoskeletal structures essential for parasite survival and pathogenesis (Morrissette and Sibley, 2002). In this context, central to both invasion and proliferation is a group of structures at one end of the parasite, the apical complex. In T. gondii the apical complex is built around the conoid, an assembly of spirally arranged fibers that is actively motile during invasion. Also, in the conoid/apical complex of this parasite approximately 200 proteins, representing 70% of its cytoskeletal protein components, have been identified (Hu et al., 2006). On the other hand, it has been demonstrated that the inoculation of the lactic bacteria Lactobacillus casei in mice previous to infection with parasitic protozoa generates a protective response, such as the cases of Babesia microti (Bautista-Garfias et al., 2005), Plasmodium chabaudi (Martı´nez-Gomez et al., 2006) and Trypanosoma cruzi (Bautista-Garfias et al., 2008). In experiments of immunization against trichinellosis in mice, it also has been shown that L. casei functions as adjuvant when is inoculated with antigens of Trichinella spiralis larvae (Bautista-Garfias et al., 2004). Partial protection has been obtained in studies of vaccination against toxoplasmosis using parasite’s surface molecules or antigens from other organelles (Beguetto et al., 2005; Ismael et al., 2003; Leyva et al., 2001; Martin et al., 2004); however, the potential protective role of the parasite’s cytoskeleton is unknown. On the basis of the previous information, in the present study the protective role of T. gondii cytoskeleton proteins, using L. casei as adjuvant, was evaluated against brain cyst formation in mice. 2. Materials and methods 2.1. Animals Experiment was conducted using adult female NIH mice with a body weight of 22–25 g. Animals were housed in polysulphonate cages with clean saw-dust, fed on commercial mice pellets and allowed continual access to drinking water. They were maintained at the animal facility of the National School of the Biological Sciences of the IPN under regulated conditions of humidity and temperature. 2.2. T. gondii strains Two T. gondii strains were used: the virulent RH strain (Sabin, 1941) and the brain cyst forming Me49 strain (Suzuki et al., 1989). The virulent RH strain was utilized to obtain peritoneal tachyzoites from which antigen was prepared for immunization and for detection of anti-T. gondii antibodies by ELISA. The less virulent Me49 strain was used for the challenge of immunized mice.
2.3. Preparation of T. gondii antigens 2.3.1. Toxoplasma lysate antigen (TLA) ˜ o Osorio TLA was obtained in accordance with Castan and Sarracent Pe´rez (2002) with modifications. Briefly, tachyzoites of the virulent T. gondii RH strain were obtained from the peritoneal fluid of infected mice (48– 72 h of infection). The material was washed three times with phosphate buffered saline (PBS) (pH 7.2) at 500 g for 5 min, passed through 25- and 27-gauge needles, and passed through filter membranes of 5 mm pore size to remove debris and host cells. The parasites were resuspended PBS, sonicated (Vibra-cell, Sonics materials) on ice at 10,000 Hz, 7 cycles of 20 s each. Then the sonicated suspension was centrifuged in the cold (4 8C) at 7800 g during 30 min. The supernatant was recovered and a mixture of protease inhibitors (PMSF 5 mM, Pestatin A 1 mM, EDTA 1 mM) was added, then the T. gondii extract (TLA) was filtered through a 0.22 mm Millipore membrane, and protein content determined by the Bradford method (1976) using bovine serum albumin as standard. The TLA was stored in aliquots at 20 8C until use. 2.3.2. Toxoplasma cytoskeleton proteins (TCPs) As in the previous section, tachyzoites of the virulent T. gondii RH strain were obtained from the peritoneal fluid of infected mice (48–72 h of infection). The material was washed three times with PBS (pH 7.2) at 500 g for 5 min, passed through 25- and 27-gauge needles, and passed through filter membranes of 5 mm pore size (Millipore, IRE) to remove debris and host cells. The filtered material was washed twice in PBS and centrifuged at 500 g for 5 min and then adjusted in PBS at a concentration of 30– 40 106 tachyzoites/mL. The isolation of the cytoskeleton fraction was a carried out according with the method previously described (Patron et al., 2005). Briefly, the suspension was centrifuged at 800 g for 10 min, and then the pellet was resuspended in the cytoskeleton intracellular stabilizing medium PHEM (a mixture of 10 mM Hepes, 10 mM MgCl2, 10 mM EGTA, and 1 mM Cl2 EGTA) containing 50 mg/mL of Na-p-tosyl-L-lysine chloromethyl ketone (TPCK), and 17.4 mg/mL of phenylmethylsulphonyl fluoride (PMSF) as well as 0.1% Triton X-100 for 5 min; after that the cytoskeleton fraction was pelleted by ultracentrifugation at 150,000 g at 4 8C for 15 min and then the material was resuspended in PBS and kept at 4 8C until used. The cytoskeleton fraction was incubated in the solubilization buffer (2% b-mercaptoethanol, 2 mM Tris– HCl, and 1% SDS). Then, the material was run on 12% SDSPAGE gels in accordance with the method of Laemmli (1970). Afterwards, the gel was placed in 10 volumes of Coomassie Blue (Coomassie Brilliant Blue R 250) staining solution for 4 h, rocking gently to distribute the dye evenly over the gel. Destaining was monitored visually and adjusted accordingly. 2.3.3. Bacteria The strain ATCC 7469 of L. casei ssp rhamnosus (Bautista-Garfias et al., 1999) was grown in MRS broth at 37 8C during 18 h. The microorganisms were harvested and centrifuged at 5000 g during 10 min and then
F. Martı´nez-Go´mez et al. / Veterinary Parasitology 160 (2009) 311–315 Table 1 Experimental design. Group (n = 21)
1 2 3 4 5 6 7 8
Treatment/mouse (intraperitoneal) Day 0
Day 14
Day 28
TLA + FMA TCP + FMA FMA PBS Lc FCA TLA + FCA TCP + FCA
TLA + FIA TCP + FIA FIA PBS PBS PBS TLA + FIA TCP + FIA
TLA TCP PBS PBS PBS PBS TLA TCP
TLA: 20 mg of Toxoplasma gondii RH strain lysate antigen; FMA: Freund’s modified adjuvant; FIA: Freund’s incomplete adjuvant; TCP: 20 mg of T. gondii RH strain cytoskeleton proteins; PBS:100 mL of phosphate buffered saline; Lc: Lactobacillus casei 1 108 cfu dead by heath; FCA: Freund’s complete adjuvant.
washed several times in PBS (pH 7.2). After a viable count the L. casei was adjusted at a concentration of 109 bacteria/ mL, later these were killed by heath during 20 min at a boiling water bath (95 8C) and then they were lyophilized. 2.3.4. Freund’s modified adjuvant (FMA) To each millilitre of Freund’s incomplete adjuvant (Sigma, MO, USA) 1 mg of lyophilized dead L. casei were added to constitute the FMA. 2.4. Experimental design Immunization. Mice were randomly divided into eight groups of 21 mice per group. Each mouse was inoculated intraperitoneally (ip) as follows: in group 1 with of TLA in Freund’s modified adjuvant (FMA), in group 2 with TCP in FMA, in group 3 with FMA, in group 4 with PBS, in group 5 with L. casei dead by heath (Lc), in group 6 with Freund’s complete adjuvant (FCA) (Sigma, MO, USA), in group 7 with TLA in FCA, and in group 8 TCP in FCA (Table 1). 2.5. Parasitological and serological techniques 2.5.1. Challenge The brains of NIH mice with an infection of 8 weeks with the T. gondii Me49 strain were obtained, and then these were homogenized in 2 mL of PBS with glucose and using a glass homogenizer. Brain cysts were counted as follows: 5 mL of brain suspension was placed on a glass slide with an equal volume of Lugol’s iodine and mounted with a cover slip. The cysts, stained brown were counted at 40 magnification. Then the suspension was adjusted to the appropriated dose. Each mouse was infected orally 1 week after the 3rd booster immunization with 100 mL of the brain homogenate containing 20 cysts. 2.5.2. T. gondii brain cyst burden estimation 8 weeks alter challenge, all mice were humanitarianly sacrificed in accordance with the Mexican official norm NOM-062-ZOO-1999 (http://senasica2.bio.com.mx/default.asp?doc=743), and their brains were collected. Each brain was homogenized in 2 mL of PBS and Toxoplasma brain cysts were counted as indicated above.
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2.5.3. Serum samples Sera were collected from mice 7 days after the 3rd booster dose and on days 14, 21, and 28 post-infection by caudal vein bleeding. Sera were stored at 20 8C for the determination of antibodies. 2.5.4. Measurement of antibody responses TLA-specific IgM antibodies were detected and quantified by ELISA assay (Engvall and Perlmann, 1971) on serum samples from individual mice. Briefly, 96-well microtiter plates (Nunc) were coated overnight at 4 8C with TCP at a concentration of 50 mg/mL. After washing, the plates were blocked with 1% BSA in Tris-buffered saline for 2 h at room temperature and then were overlaid with 1:250 diluted sera. They were then detected with horseradish peroxidaseconjugated goat anti-mouse IgM (Sigma, MO, USA). Orthophenylenediamine-dihydrochloride (Sigma, MO, USA) was used as substrate. Optical densities (O.D.s) were measured with a microplate reader (MRX II, DYN-EX Technologies) at 450 nm. IgM anti-T. gondii antibody levels were expressed as group mean S.E.M. of individual O.D. values. 2.6. Statistical analysis The statistical significance of differences was determined from the means S.E.M. by analysis of variance (ANOVA) with software Paquete de disen˜os experimentales FAUANL. Version 2.5 (Olivares, 1994). When differences were found the Tukey test was used, and these were considered significant when P values were <0.01. 3. Results 3.1. T. gondii cytoskeleton proteins The TCP showed 19 components in 12% SDS-PAGE gels, which ranged between 11 and 133 kDa (data not shown). 3.2. T. gondii brain cyst burden The lowest brain cyst burdens (P < 0.01) were observed in mice immunized with TCP in FMA (381 97.5) and TLA in FMA (724 98) as compared with those obtained in animals of the control group (3181 89.32) and the other five groups. This protective response was even better (P < 0.01) than that showed in mice of groups vaccinated with TCP in FCA (1409.5 90) and TLA in FCA (1295.2 99.1). The brain cyst burden was significantly lower (P < 0.01) in mice treated with L. casei alone (1771.4 102) than that observed in control animals or in mice immunized with FCA (2971.4 85.4) (Table 2). 3.3. IgM antibody responses to T. gondii The levels of IgM anti-Toxoplasma antibodies were significantly higher (P < 0.01) at 14 days after challenge in sera from mice vaccinated with TCP mixed with FMA as compared with the values obtained in the sera from the animals of the other seven groups (Table 3). The IgM levels in groups TLA + FMA, TLA + FCA, and TCP + FCA were similar but higher (P < 0.01) than the values showed in groups FMA, PBS, Lc, and FCA (Table 3).
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Table 2 Mean cyst burden (S.E.M.) per brain in mice which received different treatments 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). Group
n
Treatment
Mean number of brain, cysts S.E.M.
P
Percentage reductiona
1 2 3 4 5 6 7 8
21 21 21 21 21 21 21 21
TLA + FMA TCP + FMA FMA PBS Lc FCA TLA + FCA TCP + FCA
723.8 98.0 381.0 97.5 1942.9 130.3 3181.0 89.32 1771.4 101.7 2971.4 85.4 1295.2 99.1 1409.5 89.9
D D B A BC A C C
77.25 88.02 38.92 – 44.31 6.59 59.28 55.69
TLA: Toxoplasma lysate antigen; FMA: Freund’s modified adjuvant; TCP: Toxoplasma cytoskeleton proteins; PBS: phosphate buffered saline; Lc: Lactobacillus casei dead; FCA: Freund’s complete adjuvant. P: different literals in the same column indicate significant difference (P < 0.01, Tukey test). a As compared with control group (PBS). Table 3 Mean (S.E.M.) anti-T. gondii antibody (IgM) levels in sera of mice which received different treatments, 2 weeks after challenge with T. gondii Me49 strain, as determined by ELISA. Group
n
Treatment
Mean IgM anti-T. gondii (O.D. at 450 nm)
P
1 2 3 4 5 6 7 8
7 7 7 7 7 7 7 7
TLA + FMA TCP + FMA FMA PBS Lc FCA TLA + FCA TCP + FCA
0.8228 0.037 1.4642 0.103 0.3585 0.106 0.1664 0.062 0.3228 0.024 0.270 0.014 0.6885 0.029 0.6328 0.037
B A C C C C B B
TLA: Toxoplasma lysate antigen; FMA: Freund’s modified adjuvant; TCP: Toxoplasma cytoskeleton proteins; PBS: phosphate buffered saline; Lc: Lactobacillus casei dead; FCA: Freund’s complete adjuvant. P: different literals in the same column indicate significant difference (P < 0.01, Tukey test).
4. Discussion 4.1. T. gondii cytoskeleton proteins Although only 19 components were observed in the CTP by 12% SD-PAGE of this study, the cytoskeleton of T. gondii is by far very complex. In this respect, it has been shown by using molecular tools that in the conoid/apical complex of T. gondii approximately 200 proteins, representing 70% of its cytoskeletal protein components have been identified (Hu et al., 2006). However, the CTP obtained from tachyzoites was excellent, when mixed with an adjuvant containing L. casei, for inducing protection against brain cyst formation in mice. 4.2. T. gondii brain cyst burden We obtained significant reductions in brain parasite burden after challenge with the T. gondii Me49 strain in NIH immunized with TCP (88%) and TLA (77.25%) and it is worth to note that good protection was generated by L. casei alone up to 1 month after intraperitoneal inoculation in mice. Other studies in which different T. gondii antigen preparations and adjuvants were used have shown similar percentages of protection (even lower to the reported by us) against T. gondii brain cyst formation in mice. In this respect, El-Malky et al. (2005) obtained a 64% reduction in
brain parasite burden (BPB) after challenge with the RRA (Beverly) strain of T. gondii in C57BL/6 mice vaccinated with TLA and CpG as adjuvant in comparison to non-CpG– TLA treated group. Swiss mice immunized with tachyzoites plus tissue cysts in liposomes or purified tachyzoite antigen in liposomes showed reductions in BPB of 85.85% and 85.4%, respectively after challenge with the P strain of T. gondii (Elsaid et al., 1999) as compared with control mice. Beuvillain et al. (2007) observed a significant reduction (59.75%) in BPB after challenge with the T. gondii 76K strain (30 cysts per mouse) in CBA/J mice immunized with exosomes secreted by the dendritic cell line SRDC pulsed in vitro with T. gondii-derived antigens. It has been pointed out that adjuvants play an important role in the efficacy of vaccines. In addition to increasing the strength and kinetics of an immune response, adjuvants also play a role in determining the type of immune response generated. In this context, it is known that IFNg plays a critical role in protection against T. gondii infection (Susuki et al., 1988; Suzuki et al., 1989b; Liu et al., 2006; LaRosa et al., 2008). It is also known that several species of lactobacilli, including L. casei, induce the production of protective IFNg (Bautista-Garfias et al., 1999; Castanheira et al., 2007). Although we did not measure the production of IFNg we believe that L. casei promoted the production of this cytokine. 4.3. IgM antibody responses to T. gondii Significant levels of IgM anti-T. gondii (P < 0.01) were observed in mice vaccinated with TCP and TLA mixed with Freund’s modified adjuvant or FCA as compared with mice which received FMA, PBS, Lc or FCA alone. In accordance with the results obtained we believe that the IgM anti-T. gondii antibodies induced, played a role in the whole protection observed. In this respect, it has been shown that T. gondii-specific IgM antibodies are important for limiting dissemination of parasite infection in BALB/c mice by preventing host cell invasion (Couper et al., 2005); however, the animals treated with FMA or L. casei alone showed significant protection (P < 0.01) as compared with mice treated with PBS (control group) or FCA. This observation is supported by the notion that the main protection against T. gondii is carried out by the cellular arm of the immune response (Liu et al., 2006).
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5. Conclusion The protective response against T. gondii cyst formation induced in NIH mice by L. casei dead alone (group 5) was similar to that generated by FMA (group 3) and better than that induced by FCA (group 6). The addition of L. casei to the incomplete Freund’s adjuvant used for the immunization of mice with TLA (group 1) or TCP (group 2) generated a better protection than that observed in mice immunized with these antigenic preparations mixed with FCA (groups 7 and 8); however, there were no differences between animals of the groups immunized with TLA or TCP and mixed with FMA. In this context, it is worth to note that the difference in the IgM anti-T. gondii antibody levels observed particularly between these two later groups (P < 0.01) might have been influenced by the antigen used in the ELISA. Taken together the results of the present study suggest that T. gondii cytoskeleton proteins using L. casei as adjuvant constitute a good anti-toxoplasmosis vaccine candidate. Acknowledgements To the Research Office of the National Polytechnic Institute (IPN), for financial support. To Dr. Pascal Rene Herion Scohy, Immunology Department of the Biomedical Research Institute from the National Autonomous University of Mexico (UNAM) for providing the T. gondii Me49 strain. References ˜a, M., Ixta, O., Martı´nez, F., Aguilar, B., Corte´s, Bautista-Garfias, C.R., Ordun A., 1999. Enhancement of resistance in mice treated with Lactobacillus casei: effect on Trichinella spiralis infection. Vet. Parasitol. 80, 251–260. Bautista-Garfias, C.R., Posadas Beltra´n, A., Ixta Rodrı´guez, O., 2004. Immunization of BALB/c mice with an antigen from Trichinella spiralis muscle larvae using Lactobacillus casei as adjuvant. Vet. Mex. 35, 359–368. Bautista-Garfias, C.R., Go´mez, M., Aguilar, B., Ixta, B.R., Martı´nez, O., Mosqueda, F.J., 2005. The treatment of mice with Lactobacillus casei induces protection against Babesia microti infection. Parasitol. Res. 97, 472–477. Bautista-Garfias, C.R., Torres-Alvarez, M.C., Martı´nez-Go´mez, F., 2008. The inoculation of Lactobacillus casei into mice induces a protective response against Trypanosoma cruzi (Ninoa strain). Vet. Mex. 39, 139–144. Beguetto, E., Nielsen, H.V., Del Porto, P., Buffolano, W., Guglietta, S., Felici, F., Petersen, E., Gargano, N., 2005. A combination of antigenic regions of Toxoplasma gondii microneme proteins induces protective immunity against oral infection with parasite cysts. J. Infect. Dis. 191, 637– 645. Beuvillain, C., Ruiz, S., Guiton, R., Bout, D., Dimier-Poisson, I., 2007. A vaccine based on exosomes secreted by dendritic cell line confers protection against T. gondii infection in syngeneic and allogeneic mice. Microbes Infect. 9, 1614–1622. Black, M.W., Boothroyd, J.C., 2000. Lytic cycle of Toxoplasma gondii. Microbiol. Mol. Biol. Rev. 64, 607–623. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantitites of protein utilizing the principle of protein– dye binding. Anal. Biochem. 72, 248–254. Castanheira, L.G., de Andrade Castro, J.M., Martins-Filho, M.A., Nicoli, J.R., Vieira, L.Q., Crocco Alonso, L.C., 2007. Lactobacillus delbrueecki as potential skin adjuvant for induction of type 1 immune responses. Front. Biosci. 12, 1300–1307. ˜ o Osorio, J.C., Sarracent Pe´rez, J., 2002. Inmunizacio´n intranasal de Castan ratones con la proteı´na SAG2 de Toxoplasma gondii asociada con la toxina cole´rica. Rev. Cubana Invest. Biomed. 21, 35–45.
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