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Immunohistochemical study of the cyst of Besnoitia besnoiti
Veterinary Parasitology 91 (2000) 1–6
Immunohistochemical study of the cyst of Besnoitia besnoiti M. Irigoien, E. Del Cacho∗ , M. Gallego, F. López-Bernad, J. Qu´ılez, C. Sánchez-Acedo Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain Received 20 December 1999; received in revised form 20 March 2000; accepted 6 April 2000
Abstract The present study has been undertaken in order to provide information on the molecular structure of the cysts of Besnoitia besnoiti. To that end, immunohistochemical techniques have been used to investigate the expression of several enzymes and proteins implicated in the cellular membrane permeability of bradyzoites. Paraffin and frozen sections, which were obtained from subcutaneous tissue samples taken from naturally infected cattle (coming from northeast Spain), were treated with a panel of antibodies. These were specific for Na+ , K+ -ATPase, alkaline phosphatase, calmodulin, S100 protein, heat shock proteins, hsp60, and hsp70. Positive-cysts for the said antibodies were found in 23.3% of the cows studied. Bradyzoites showed a positive immunoreaction in every positive cyst with respect to all these antibodies. In addition to the low percentage of positive animals, it is worth noting that positive and unstained cysts were observed in the same tissue section. These results suggest that bradyzoites may pass through both active and dormant metabolic phases. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Besnoitia besnoiti; Immunohistochemistry
1. Introduction Besnoitia besnoiti is a protozoan that forms cysts in the tissue of infected cattle and has considerable economic impact in certain regions of Europe. Cattle infection is thought to result mainly from the ingestion of sporulated oocysts from cat faeces (Peteshev et al., 1974), but biting flies, feeding on the skin lesions of cattle, have also been incriminated as another route of transmission through tachyzoites and bradyzoites (Pols, 1960). Although ∗ Corresponding author. Tel.: +34-76-761556; fax: +34-76-761612. E-mail address: [email protected] (E. Del Cacho)
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 2 6 0 - 0
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M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
a very significant amount of information is available on the morphological characteristics of B. besnoiti, comparatively little is known about the molecular structure of the subcutaneous cysts. Such knowledge is essential for an understanding of cyst development and survival. The present immnunohistochemical study has been undertaken in order to provide information on the enzymes and proteins implicated in the cellular membrane permeability of bradyzoites. The following proteins and enzymes were studied, (1) Na+ , K+ -ATPase, which is a plasma membrane enzyme, commonly known as the sodium and potassium pump, and is an active transport unit in all biological membranes, (2) Alkaline phosphatase, a group of nonspecific membrane-bound phosphomonoesterases that catalyze the hydrolysis and transphosphorylation of phosphate monoesters at optimum alkaline pH (Scheibe et al., 1991), (3) two calcium binding-proteins, calmodulin and S100 protein, which are believed to be involved in the regulation of intracellular calcium, and (4) two heat shock proteins (hsp), hsp60 and hsp70, a group of phylogenetically well-conserved proteins that may have pivotal roles in the adaptation to physiological stress. Immunohistochemical techniques were applied to tissue sections containing cysts of B. besnoiti in order to study the expression of hsp proteins (60 and 70) and calcium binding-proteins (calmodulin and S100), as well as the distribution of adenosine triphosphatase and alkaline phospatase. For that purpose, we used monoclonal antibodies against each of the above-mentioned proteins and enzymes.
2. Materials and methods 2.1. Source of parasite Thirty bovines with besnoitiosis, aged 3–7 years, and coming from different areas of Navarra and Huesca (northeast Spain) were used. All animals exhibited clinical signs of chronic besnoitiosis, i.e. alopecic dermatitis, scleroderma, hyperkeratosis and ocular cysts. B. besnoiti bradyzoites were identified by way of direct light microscopic examination in Giemsa-stained skin biopsy smears (Sannusi, 1991). Small pieces of subcutaneous tissue from naturally infected cattle were obtained from various affected areas (face, head, and dorsum from the neck to the pelvis), using local anesthesic infiltration and sterile operative procedures. Subcutaneous tissue samples were frozen in liquid nitrogen or fixed in Bouin’s solution for 90 min to obtain paraffin sections (5 m). Cryostat sections (5–7 m) were fixed in 100% acetone for 10 min. Serial sections were obtained from each of the tissue samples. 2.2. Immunohistochemical techniques Immunohistochemical techniques were applied to both frozen and paraffin sections in order to assess consistence of the results. After rehydration in phosphate-buffered saline (PBS) (pH 7.2) or deparaffination, sections were exposed to immunohistochemical staining by using an avidin–biotin complex technique (Vector Lab., Burlingame, CA). Endogenous peroxidase was inactivated with 1.7% hydrogen peroxide in 100% ethanol for 30 min. After washing in PBS (pH 7.2), the
M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
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slides were incubated in normal serum (blocking reagent) for 10 min, followed by incubation with the primary antibody at a 1:100 dilution for 1 h. As the primary antibody, we used the monoclonal antibodies that recognized the following proteins or enzymes: hsp70 (clone BRM-22, Sigma, St. Louis MO), hsp60 (clone LK2, Sigma), ATPase (clone 5F10, Sigma), alkaline phosphatase (clone AP-59, Sigma), calmodulin (mixture of clones 2D1, 1F11 and 6D4, Sigma), and S100 protein (whole molecule, Sigma). Each of these antibodies was applied to separate serial sections obtained from both the same cyst and from several different cysts. In addition, each of the sections of a series was stained with one of the six antibodies used. In this way, we could follow the antibody staining for all six antibodies in the same cyst. Sections were then incubated with a biotinylated anti-mouse or anti-rabbit serum (when primary antibody recognized S100 protein whole molecule) (Vector Lab.) for 30 min. An avidin–biotin peroxidase complex (Vector Lab.) was subsequently applied for 45 min. The peroxidase reaction was developed with diaminobenzidine (DAB) and hydrogen peroxide solution (20 mg DAB in 100 ml of 0.05 M Tris–HCl buffer, pH 7.6, containing 0.005% H2 O2 ) for 5 min. As negative controls, sections of all samples were incubated with normal serum instead of the primary antisera, with the remaining of the procedure being unaltered. The negative control sections were devoid of positive cells.
3. Results Each of the six antibodies used in the study recognized the molecule it was raised against in cysts of B. besnoiti. Thus, we found positive cysts for each of the following: calmodulin, S100 protein, hsp60, hsp70, ATPase, and alkaline phosphatase (Fig. 1 (1)–(4)). Positive cysts were observed in 7 out of the 30 cows studied (23.3%). In the samples taken from each of these seven cows, which totalled 250 cysts, only 17.67% were found to be positive. Attention should particularly be drawn to the fact that we observed positive and unstained cysts in the same tissue sections (Fig. 1 (1) and (2)). Bradyzoites showed a positive immunoreaction in every positive cyst with respect to all the antibodies mentioned above (Fig. 1 (1)–(4)). The remaining structures of the cyst were devoid of immunoreactivity, except for the host cell, which showed a strong positive reaction solely for the ATPase (Fig. 1 (3)). Therefore, in the positive cysts for ATPase, positivity for this enzyme was found in both the bradyzoites and in the host cell. The immunoreaction pattern of staining which was found for the antibodies used in the present study was equal in both frozen and paraffin sections.
4. Discussion In the present study, the host cell and bradyzoites within the Besnoitia cyst showed ATPase activity. The surface ATPase in bradyzoites may signify energy-dependent surface membrane processes, developed as a result of cellular differentiation or activation (Docampo and Moreno, 1999). Our results indicated that the expression of ATPase in the host cell took place at approximately the same time as the maturation process of the bradyzoites. This suggests that the host cell might play an important role in the development
4
M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
Fig. 1. (1) S100-positive bradyzoites within cutaneous cysts of Besnoitia besnoiti. Note the presence of a negative cyst (arrow). Bar=500 m. Paraffin section. (2) Detection of hsp70 in B. besnoiti cysts. Bradyzoites showing a positive immunoreaction (central cysts) and bradyzoites devoid of positivity for hsp70. Bar=250 m. Paraffin section. (3) ATPase-positive cyst. Note a strong positive immunoreaction in the host cell containing the cyst (arrowheads). Bradyzoites within the cyst were also positive for this enzyme. Bar=200 m. Paraffin section. (4) Phosphatase alkaline was found in bradyzoites within the positive cysts for this enzyme. Bar=250 m. Paraffin section.
and survival of the cyst. The alkaline phosphatase of the eukaryotic and mammalian cells is involved in the regulation of the protein phosphorylation (Xu and Kantrowitz, 1991). Therefore, the alkaline phosphatase found in the cysts of B. besnoiti could play an important role in the regulation of the maturation process of the parasite, since phosphorylation of proteins is associated with cell differentiation and transformation (Scheibe et al., 1991). The role of calcium as a second messenger in the regulation of intracellular processes is now well established. Genes encoding calcium-binding proteins have been identified in various parasitic protozoa, including Plasmodium falciparum (Robson and Jennings, 1991), Toxoplasma gondii (Ng et al., 1995), Eimeria maxima and Eimeria tenella (Dunn et al., 1996). In addition, we have found positivity for S100 protein and calmodulin in the bradyzoites of B. besnoiti. Taking into account the different functions suggested by Del Cacho et al. (1998) and López-Bernad et al. (2000) for S100 protein in E. tenella and E. brunetti, we can speculate on its role in the bradyzoites of Besnoitia. Since calcium-binding proteins are important in microtubule assembly-disassembly and could regulate microtubule mediated motility, the positive immunoreaction for S100 protein that we have found in bradyzoites may be connected with the active movement that this parasite stage develops before host cell invasion occurs. In addition, there are indications that S100 protein and calmodulin are implicated in membrane permeability and are considered to be cell-growth
M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
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regulators, as they mediate the control of numerous enzymes modulated by calcium (Parsons et al., 1993). Heat shock proteins have been detected in several parasitic protozoa, including Trypanosoma, Leishmania, Toxoplasma, Plasmodium (Maresca and Carratù, 1992) and Eimeria (Dunn et al., 1995). In the present study, we have identified hsp70 and hsp60 in bradyzoites, which is indicative that hsp synthesis in B. besnoiti, as in other protozoan parasites, may be a pathway towards adaptation for survival in new environmental conditions during differentiation within the host. Furthermore, based on the presence of hsp within the bradyzoites of Toxoplasma (Weiss et al., 1998) and sporozoites of E. tenella (Dunn et al., 1995) when these have the ability to initiate infection, it is feasible that hsp in the bradyzoites of Besnoitia may play a role during infection. In this regard, evidence has been provided that hsp60 and hsp70 play important roles in infectivity and virulence in a number of protozoan parasites (Maresca and Carratù, 1992).The present results demonstrate that some cysts were devoid of positivity for all six antibodies employed, whereas other cysts showed the simultaneous expression of the molecules which are recognized by these six antibodies. The lack of immunoreactivity noted in the negative cysts indicated that these did not express the proteins which are recognized by the antibodies. This finding suggests that the negative-cysts were metabolically different from the positive-cysts, which expressed the molecules identified by the antibodies. Taken together, these results suggest that the bradyzoites pass through active and dormant metabolic phases. Phases of high metabolic activity might be related to the development and maturation process that the bradyzoites undergo before reaching infective capacity. However, the current results do not allow us to determine whether this maturation process of bradyzoites is a continuum or, by contrast, whether active and dormant phases are discontinuous. References Del Cacho, E., López-Bernad, F., Gallego, M., Qu´ılez, J., Sánchez-Acedo, C., 1998. Expression and localization of an S100 protein-like molecule in Eimeria tenella. J. Parasitol. 84, 1174–1177. Docampo, R., Moreno, S.N.J., 1999. Acidocalcisome: a novel Ca2+ storage compartment in trypanosomatids and apicomplexan parasites. Parasitol. Today 15, 443–448. Dunn, P.P.J., Billington, K., Bumstead, J.M., Tomley, F.M., 1995. Isolation and sequences of cDNA clones for cytosolic and organellar hsp70 species in Eimeria tenella ssp. Mol. Biochem. Parasitol. 70, 211–215. Dunn, P.P.J., Bumstead, J.M., Tomley, F.M., 1996. Sequence, expression and localization of calmodulin-domain protein kinases in Eimeria tenella and Eimeria maxima. Parasitology 113, 439–448. López-Bernad, F., del Cacho, E., Gallego, M., Qu´ılez, J., Sánchez-Acedo, C., 2000. Immunohistochemical study of S100-like protein in Eimeria brunetti and Eimeria acervulina.. Vet. Parasitol. 88, 1–6. Maresca, B., Carratù, L., 1992. The biology of the heat shock response in parasites. Parasitol. Today 8, 260–266. Ng, H.C., Singh, M., Jeyaseelan, K., 1995. Molecular cloning of a Snf1 type protein kinase gene from Toxoplasma gondii. Biochem. Mol. Biol. Int. 35, 155–165. Parsons, M., Valentine, M., Carter, V., 1993. Protein kinases in divergent eukaryotes: identification of protein kinase activities regulated during trypanosome development. Proc. Nat. Acad. Sci. USA 90, 2656–2660. Peteshev, V.M., Galuzo, I.G., Polomoshnov, A.P., 1974. Koshki-definitivnye khozyaeva besnoitii. Izv. Akad. Nauk Kaz. SSR. Ser. Biol. 1, 33–38. Pols, J.W., 1960. Studies on bovine besnoitiosis with special reference to the aetiology. Onderstepoort J. Vet. Res. 28, 265–357. Robson, K.J.H., Jennings, M.W., 1991. The structure of the calmodulin gene of Plasmodium falciparum. Mol. Biochem. Parasitol. 46, 36–43.
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M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
Sannusi, A., 1991. A simple field diagnostic smear test for bovine besnoitiosis. Vet. Parasitol. 39, 185–188. Scheibe, R., Moller-Runge, I., Muller, W., 1991. Retinoic acid induces the expression of alkaline phosphatase in P19 teratocarcinoma cells. J. Biol. Chem. 266, 21300–21305. Xu, X., Kantrowitz, E., 1991. A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity. Biochemistry 30, 7789–7796. Weiss, L.M., Ma, Y.F., Takvorian, P.M., Tanowitz, H.B., Wittner, M., 1998. Bradyzoite development in Toxoplasma gondii and the hsp70 stress response. Infect. Immunity 66, 3295–3302.
Immunohistochemical study of the cyst of Besnoitia besnoiti M. Irigoien, E. Del Cacho∗ , M. Gallego, F. López-Bernad, J. Qu´ılez, C. Sánchez-Acedo Department of Animal Pathology, Faculty of Veterinary Sciences, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain Received 20 December 1999; received in revised form 20 March 2000; accepted 6 April 2000
Abstract The present study has been undertaken in order to provide information on the molecular structure of the cysts of Besnoitia besnoiti. To that end, immunohistochemical techniques have been used to investigate the expression of several enzymes and proteins implicated in the cellular membrane permeability of bradyzoites. Paraffin and frozen sections, which were obtained from subcutaneous tissue samples taken from naturally infected cattle (coming from northeast Spain), were treated with a panel of antibodies. These were specific for Na+ , K+ -ATPase, alkaline phosphatase, calmodulin, S100 protein, heat shock proteins, hsp60, and hsp70. Positive-cysts for the said antibodies were found in 23.3% of the cows studied. Bradyzoites showed a positive immunoreaction in every positive cyst with respect to all these antibodies. In addition to the low percentage of positive animals, it is worth noting that positive and unstained cysts were observed in the same tissue section. These results suggest that bradyzoites may pass through both active and dormant metabolic phases. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Besnoitia besnoiti; Immunohistochemistry
1. Introduction Besnoitia besnoiti is a protozoan that forms cysts in the tissue of infected cattle and has considerable economic impact in certain regions of Europe. Cattle infection is thought to result mainly from the ingestion of sporulated oocysts from cat faeces (Peteshev et al., 1974), but biting flies, feeding on the skin lesions of cattle, have also been incriminated as another route of transmission through tachyzoites and bradyzoites (Pols, 1960). Although ∗ Corresponding author. Tel.: +34-76-761556; fax: +34-76-761612. E-mail address: [email protected] (E. Del Cacho)
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 2 6 0 - 0
2
M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
a very significant amount of information is available on the morphological characteristics of B. besnoiti, comparatively little is known about the molecular structure of the subcutaneous cysts. Such knowledge is essential for an understanding of cyst development and survival. The present immnunohistochemical study has been undertaken in order to provide information on the enzymes and proteins implicated in the cellular membrane permeability of bradyzoites. The following proteins and enzymes were studied, (1) Na+ , K+ -ATPase, which is a plasma membrane enzyme, commonly known as the sodium and potassium pump, and is an active transport unit in all biological membranes, (2) Alkaline phosphatase, a group of nonspecific membrane-bound phosphomonoesterases that catalyze the hydrolysis and transphosphorylation of phosphate monoesters at optimum alkaline pH (Scheibe et al., 1991), (3) two calcium binding-proteins, calmodulin and S100 protein, which are believed to be involved in the regulation of intracellular calcium, and (4) two heat shock proteins (hsp), hsp60 and hsp70, a group of phylogenetically well-conserved proteins that may have pivotal roles in the adaptation to physiological stress. Immunohistochemical techniques were applied to tissue sections containing cysts of B. besnoiti in order to study the expression of hsp proteins (60 and 70) and calcium binding-proteins (calmodulin and S100), as well as the distribution of adenosine triphosphatase and alkaline phospatase. For that purpose, we used monoclonal antibodies against each of the above-mentioned proteins and enzymes.
2. Materials and methods 2.1. Source of parasite Thirty bovines with besnoitiosis, aged 3–7 years, and coming from different areas of Navarra and Huesca (northeast Spain) were used. All animals exhibited clinical signs of chronic besnoitiosis, i.e. alopecic dermatitis, scleroderma, hyperkeratosis and ocular cysts. B. besnoiti bradyzoites were identified by way of direct light microscopic examination in Giemsa-stained skin biopsy smears (Sannusi, 1991). Small pieces of subcutaneous tissue from naturally infected cattle were obtained from various affected areas (face, head, and dorsum from the neck to the pelvis), using local anesthesic infiltration and sterile operative procedures. Subcutaneous tissue samples were frozen in liquid nitrogen or fixed in Bouin’s solution for 90 min to obtain paraffin sections (5 m). Cryostat sections (5–7 m) were fixed in 100% acetone for 10 min. Serial sections were obtained from each of the tissue samples. 2.2. Immunohistochemical techniques Immunohistochemical techniques were applied to both frozen and paraffin sections in order to assess consistence of the results. After rehydration in phosphate-buffered saline (PBS) (pH 7.2) or deparaffination, sections were exposed to immunohistochemical staining by using an avidin–biotin complex technique (Vector Lab., Burlingame, CA). Endogenous peroxidase was inactivated with 1.7% hydrogen peroxide in 100% ethanol for 30 min. After washing in PBS (pH 7.2), the
M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
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slides were incubated in normal serum (blocking reagent) for 10 min, followed by incubation with the primary antibody at a 1:100 dilution for 1 h. As the primary antibody, we used the monoclonal antibodies that recognized the following proteins or enzymes: hsp70 (clone BRM-22, Sigma, St. Louis MO), hsp60 (clone LK2, Sigma), ATPase (clone 5F10, Sigma), alkaline phosphatase (clone AP-59, Sigma), calmodulin (mixture of clones 2D1, 1F11 and 6D4, Sigma), and S100 protein (whole molecule, Sigma). Each of these antibodies was applied to separate serial sections obtained from both the same cyst and from several different cysts. In addition, each of the sections of a series was stained with one of the six antibodies used. In this way, we could follow the antibody staining for all six antibodies in the same cyst. Sections were then incubated with a biotinylated anti-mouse or anti-rabbit serum (when primary antibody recognized S100 protein whole molecule) (Vector Lab.) for 30 min. An avidin–biotin peroxidase complex (Vector Lab.) was subsequently applied for 45 min. The peroxidase reaction was developed with diaminobenzidine (DAB) and hydrogen peroxide solution (20 mg DAB in 100 ml of 0.05 M Tris–HCl buffer, pH 7.6, containing 0.005% H2 O2 ) for 5 min. As negative controls, sections of all samples were incubated with normal serum instead of the primary antisera, with the remaining of the procedure being unaltered. The negative control sections were devoid of positive cells.
3. Results Each of the six antibodies used in the study recognized the molecule it was raised against in cysts of B. besnoiti. Thus, we found positive cysts for each of the following: calmodulin, S100 protein, hsp60, hsp70, ATPase, and alkaline phosphatase (Fig. 1 (1)–(4)). Positive cysts were observed in 7 out of the 30 cows studied (23.3%). In the samples taken from each of these seven cows, which totalled 250 cysts, only 17.67% were found to be positive. Attention should particularly be drawn to the fact that we observed positive and unstained cysts in the same tissue sections (Fig. 1 (1) and (2)). Bradyzoites showed a positive immunoreaction in every positive cyst with respect to all the antibodies mentioned above (Fig. 1 (1)–(4)). The remaining structures of the cyst were devoid of immunoreactivity, except for the host cell, which showed a strong positive reaction solely for the ATPase (Fig. 1 (3)). Therefore, in the positive cysts for ATPase, positivity for this enzyme was found in both the bradyzoites and in the host cell. The immunoreaction pattern of staining which was found for the antibodies used in the present study was equal in both frozen and paraffin sections.
4. Discussion In the present study, the host cell and bradyzoites within the Besnoitia cyst showed ATPase activity. The surface ATPase in bradyzoites may signify energy-dependent surface membrane processes, developed as a result of cellular differentiation or activation (Docampo and Moreno, 1999). Our results indicated that the expression of ATPase in the host cell took place at approximately the same time as the maturation process of the bradyzoites. This suggests that the host cell might play an important role in the development
4
M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
Fig. 1. (1) S100-positive bradyzoites within cutaneous cysts of Besnoitia besnoiti. Note the presence of a negative cyst (arrow). Bar=500 m. Paraffin section. (2) Detection of hsp70 in B. besnoiti cysts. Bradyzoites showing a positive immunoreaction (central cysts) and bradyzoites devoid of positivity for hsp70. Bar=250 m. Paraffin section. (3) ATPase-positive cyst. Note a strong positive immunoreaction in the host cell containing the cyst (arrowheads). Bradyzoites within the cyst were also positive for this enzyme. Bar=200 m. Paraffin section. (4) Phosphatase alkaline was found in bradyzoites within the positive cysts for this enzyme. Bar=250 m. Paraffin section.
and survival of the cyst. The alkaline phosphatase of the eukaryotic and mammalian cells is involved in the regulation of the protein phosphorylation (Xu and Kantrowitz, 1991). Therefore, the alkaline phosphatase found in the cysts of B. besnoiti could play an important role in the regulation of the maturation process of the parasite, since phosphorylation of proteins is associated with cell differentiation and transformation (Scheibe et al., 1991). The role of calcium as a second messenger in the regulation of intracellular processes is now well established. Genes encoding calcium-binding proteins have been identified in various parasitic protozoa, including Plasmodium falciparum (Robson and Jennings, 1991), Toxoplasma gondii (Ng et al., 1995), Eimeria maxima and Eimeria tenella (Dunn et al., 1996). In addition, we have found positivity for S100 protein and calmodulin in the bradyzoites of B. besnoiti. Taking into account the different functions suggested by Del Cacho et al. (1998) and López-Bernad et al. (2000) for S100 protein in E. tenella and E. brunetti, we can speculate on its role in the bradyzoites of Besnoitia. Since calcium-binding proteins are important in microtubule assembly-disassembly and could regulate microtubule mediated motility, the positive immunoreaction for S100 protein that we have found in bradyzoites may be connected with the active movement that this parasite stage develops before host cell invasion occurs. In addition, there are indications that S100 protein and calmodulin are implicated in membrane permeability and are considered to be cell-growth
M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
5
regulators, as they mediate the control of numerous enzymes modulated by calcium (Parsons et al., 1993). Heat shock proteins have been detected in several parasitic protozoa, including Trypanosoma, Leishmania, Toxoplasma, Plasmodium (Maresca and Carratù, 1992) and Eimeria (Dunn et al., 1995). In the present study, we have identified hsp70 and hsp60 in bradyzoites, which is indicative that hsp synthesis in B. besnoiti, as in other protozoan parasites, may be a pathway towards adaptation for survival in new environmental conditions during differentiation within the host. Furthermore, based on the presence of hsp within the bradyzoites of Toxoplasma (Weiss et al., 1998) and sporozoites of E. tenella (Dunn et al., 1995) when these have the ability to initiate infection, it is feasible that hsp in the bradyzoites of Besnoitia may play a role during infection. In this regard, evidence has been provided that hsp60 and hsp70 play important roles in infectivity and virulence in a number of protozoan parasites (Maresca and Carratù, 1992).The present results demonstrate that some cysts were devoid of positivity for all six antibodies employed, whereas other cysts showed the simultaneous expression of the molecules which are recognized by these six antibodies. The lack of immunoreactivity noted in the negative cysts indicated that these did not express the proteins which are recognized by the antibodies. This finding suggests that the negative-cysts were metabolically different from the positive-cysts, which expressed the molecules identified by the antibodies. Taken together, these results suggest that the bradyzoites pass through active and dormant metabolic phases. Phases of high metabolic activity might be related to the development and maturation process that the bradyzoites undergo before reaching infective capacity. However, the current results do not allow us to determine whether this maturation process of bradyzoites is a continuum or, by contrast, whether active and dormant phases are discontinuous. References Del Cacho, E., López-Bernad, F., Gallego, M., Qu´ılez, J., Sánchez-Acedo, C., 1998. Expression and localization of an S100 protein-like molecule in Eimeria tenella. J. Parasitol. 84, 1174–1177. Docampo, R., Moreno, S.N.J., 1999. Acidocalcisome: a novel Ca2+ storage compartment in trypanosomatids and apicomplexan parasites. Parasitol. Today 15, 443–448. Dunn, P.P.J., Billington, K., Bumstead, J.M., Tomley, F.M., 1995. Isolation and sequences of cDNA clones for cytosolic and organellar hsp70 species in Eimeria tenella ssp. Mol. Biochem. Parasitol. 70, 211–215. Dunn, P.P.J., Bumstead, J.M., Tomley, F.M., 1996. Sequence, expression and localization of calmodulin-domain protein kinases in Eimeria tenella and Eimeria maxima. Parasitology 113, 439–448. López-Bernad, F., del Cacho, E., Gallego, M., Qu´ılez, J., Sánchez-Acedo, C., 2000. Immunohistochemical study of S100-like protein in Eimeria brunetti and Eimeria acervulina.. Vet. Parasitol. 88, 1–6. Maresca, B., Carratù, L., 1992. The biology of the heat shock response in parasites. Parasitol. Today 8, 260–266. Ng, H.C., Singh, M., Jeyaseelan, K., 1995. Molecular cloning of a Snf1 type protein kinase gene from Toxoplasma gondii. Biochem. Mol. Biol. Int. 35, 155–165. Parsons, M., Valentine, M., Carter, V., 1993. Protein kinases in divergent eukaryotes: identification of protein kinase activities regulated during trypanosome development. Proc. Nat. Acad. Sci. USA 90, 2656–2660. Peteshev, V.M., Galuzo, I.G., Polomoshnov, A.P., 1974. Koshki-definitivnye khozyaeva besnoitii. Izv. Akad. Nauk Kaz. SSR. Ser. Biol. 1, 33–38. Pols, J.W., 1960. Studies on bovine besnoitiosis with special reference to the aetiology. Onderstepoort J. Vet. Res. 28, 265–357. Robson, K.J.H., Jennings, M.W., 1991. The structure of the calmodulin gene of Plasmodium falciparum. Mol. Biochem. Parasitol. 46, 36–43.
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M. Irigoien et al. / Veterinary Parasitology 91 (2000) 1–6
Sannusi, A., 1991. A simple field diagnostic smear test for bovine besnoitiosis. Vet. Parasitol. 39, 185–188. Scheibe, R., Moller-Runge, I., Muller, W., 1991. Retinoic acid induces the expression of alkaline phosphatase in P19 teratocarcinoma cells. J. Biol. Chem. 266, 21300–21305. Xu, X., Kantrowitz, E., 1991. A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity. Biochemistry 30, 7789–7796. Weiss, L.M., Ma, Y.F., Takvorian, P.M., Tanowitz, H.B., Wittner, M., 1998. Bradyzoite development in Toxoplasma gondii and the hsp70 stress response. Infect. Immunity 66, 3295–3302.