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Cryptosporidium parvum: oocysts purification using potassium bromide discontinuous gradient
Veterinary Parasitology 92 (2000) 223–226
Short communication
Cryptosporidium parvum: oocysts purification using potassium bromide discontinuous gradient Emilio Entrala a,∗ , Jose-Manuel Molina-Molina a,b , Maria-José Rosales-Lombardo a , Manuel Sánchez-Moreno a , Carmen Mascaró-Lazcano a b
a Departamento de Parasitolog´ıa, Facultad de Ciencias, Severo Ochoa s/n, Granada 18071, Spain Empresa Municipal de Abastecimiento y Saneamiento de Granada (EMASAGRA S.A.), Granada, Spain
Received 2 March 2000; accepted 14 June 2000
Abstract Cryptosporidium parvum oocysts were purified using a discontinuous potassium bromide density gradient, composed by three solutions of 6, 16 and 28% (w/v) KBr in Tris–EDTA buffer. Fecal samples containing oocysts were washed to diminish interfering lipids and applied to the gradient. After centrifugation, oocysts can be easily aspirated from a clear band, diluted and washed by centrifugation in phosphate buffer to remove residual KBr. This method allows the purification of large amounts of highly purified C. parvum oocysts, using low cost reagents and a standard table-top centrifuge. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cryptosporidium; Oocyst; Purification; Density gradient; Potassium bromide
The production of significant amounts of highly purified oocysts from feces of infected animals can be a very expensive and time-consuming task for labs involved in C. parvum research. Sucrose flotation, coupled with Percoll or cesium chloride gradients, are frequently used for oocyst purification for biochemical, molecular and in vitro culture studies (Upton, 1997), but have some inconveniences. They usually require samples previously concentrated over sucrose, and Percoll® and cesium chloride are expensive reagents. The aim of the present work is to develop a technique for the purification of high amounts of C. parvum oocysts, suitable for biochemical and molecular studies, using standard equipment and low cost reagents. ∗ Corresponding author. Tel.: +34-958-243263; fax: +34-958-243174. E-mail address: [email protected] (E. Entrala).
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 3 1 5 - 0
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E. Entrala et al. / Veterinary Parasitology 92 (2000) 223–226
Samples were collected from experimentally infected newborn goats (Ortega-Mora and Wright, 1994) or naturally infected newborn Holstein calves from local dairy farms. C. parvum infection was diagnosed using a modified acid-fast stain (Entrala et al., 1995). Positive samples were diluted with saline solution (NaCl 0.9%) and filtered to remove particulate material. The feces were concentrated by centrifugation (3000×g, 10 min., 4◦ C) and washed twice by centrifugation with saline solution under the same conditions. To improve oocyst yield and purity, prevention of lipid aggregation in the feces was performed by two different washing protocols: samples with low concentration of fat were repeatedly washed by centrifugation (3000×g, 10 min., 4◦ C) in Tris–EDTA buffer (Tris base 50 mM, EDTA 10 mM), until a clear supernatant was obtained. Samples with high fat content should be subjected to diethyl ether extraction (briefly, dilute fecal suspension 1:1.5 with diethyl ether, shake until a complete dispersion is obtained, centrifuge 1000×g for 10 min. at 4◦ C). The pellet obtained was washed twice by centrifugation in Tris–EDTA to remove residual ether. In both cases, the last pellet is resuspended in the minimum volume of Tris–EDTA buffer before being applied to the gradient. The gradient of potassium bromide (Sigma, P9881) consisted of three solutions of 6, 16 and 28% (w/v) KBr in Tris–EDTA buffer. From bottom to top, 7 ml of ice-cold 28, 16 and 6% KBr solutions were carefully layered into 25 ml clear plastic centrifuge tubes. Three millilitres of the concentrated fecal suspension was carefully layered on top of the gradient, and the tubes were centrifuged in a Beckman GS-15R centrifuge equipped with a swing-out S4180 rotor at 3000×g for 1 h at 4◦ C. After this, a white band over 16% KBr solution can be observed, containing C. parvum oocysts free of debris and bacteria (Fig. 1a). This band
Fig. 1. Centrifuge tubes showing a band (arrow) of C. parvum oocysts after KBr discontinuous gradient performed at (a) 3000×g, 1 h, 4◦ C; (b) 16,000×g, 1 h, 4◦ C.
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is carefully aspirated using a Pasteur pipette, and the oocysts are diluted 1:3 with distilled water and centrifuged (3000×g, 10 min, 4◦ C). The pellet is resuspended and washed twice using phosphate saline buffer (PBS, pH 7.2) and centrifugation. Recovered oocysts are quantified using a hematocytometer. In some cases, higher centrifugal forces may be required to enhance the purity of the final suspension. Potassium bromide gradient can be centrifuged under the conditions described for cesium chloride gradient (Taghi-Kilani and Sekla, 1987), 16,000×g for 1 h at 4◦ C. Fig. 1b shows a gradient performed under this conditions, using a Beckman J2-21M centrifuge equipped with a JS-13 rotor. Using this gradient, it is possible to obtain 1×105 –2×108 oocysts per Sterilin tube in 3–4 h. The purity of the oocyst suspension is highly influenced by fecal fat content, so repeated washes in Tris–EDTA or ether extraction must be performed before applying to the gradient. Clumps of fat, bacteria and debris contaminate the oocyst band when poorly defatted suspensions are used. Recovery of oocysts is influenced by oocyst viability, since only water impermeable oocysts can be recovered using this hyperosmotic gradient. This enrichment or selective concentration of viable oocysts has been previously observed (Bukhari and Smith, 1995) when hyperosmotic sucrose density or zinc sulphate flotation techniques were used for oocyst concentration. In this way, viability of KBr purified C. parvum oocysts, determined using a fluorogenic vital dye uptake assay (Campbell et al., 1992), reached values close to 100% (99.6±0.4%; n=6), although the yield of oocysts varies from 30–90% (depending on initial oocyst viability). In summary, this technique allows the rapid and cost-efficient purification of C. parvum oocysts in a similar manner to that obtained using cesium chloride gradients, but has many advantages: good results can be achieved using a standard table-top centrifuge (suitable for 4000 rpm or 3000×g) and disposable tubes, the cost of potassium bromide is much lower than that of cesium chloride, and the band containing the oocysts is easily aspirated since a greater separation is achieved from contaminating bands. Axenic oocysts, suitable for in vitro culture, can be obtained using this method, but the centrifugal conditions used for cesium chloride gradient must be selected (16,000×g for 1 h at 4◦ C).
Acknowledgements This work was supported by CICYT AMB97-0982.0 project and Plan Propio de la Universidad de Granada.
References Bukhari, Z., Smith, H.V., 1995. Effect of three concentration techniques on viability of Cryptosporidium parvum oocysts recovered from bovine feces. J. Clin. Microbiol. 33, 2592–2595. Campbell, A.T., Robertson, L.J., Smith, H.V., 1992. Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes. Appl. Environ. Microbiol. 58, 3488–3493. Entrala, E., Rueda-Rubio, M.C., Janssen, D., Mascaró, C., 1995. Influence of hydrogen peroxide on acid-fast staining of Cryptosporidium parvum oocysts. Int. J. Parasitol. 25, 1473–1477.
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E. Entrala et al. / Veterinary Parasitology 92 (2000) 223–226
Ortega-Mora, L.M., Wright, S.E., 1994. Age-related resistance in ovine cryptosporidiosis: patterns of infection and humoral immune response. Infect. Immunol. 62, 5003–5009. Taghi-Kilani, R., Sekla, L., 1987. Purification of Cryptosporidium oocysts and sporozoites by cesium chloride and Percoll gradients. Am. J. Trop. Med. Hyg. 36, 505–508. Upton, S.J., 1997. In vitro cultivation. In: Fayer, R. (Ed.), Cryptosporidium and cryptosporidiosis. CRC Press Inc. pp. 181–207.
Short communication
Cryptosporidium parvum: oocysts purification using potassium bromide discontinuous gradient Emilio Entrala a,∗ , Jose-Manuel Molina-Molina a,b , Maria-José Rosales-Lombardo a , Manuel Sánchez-Moreno a , Carmen Mascaró-Lazcano a b
a Departamento de Parasitolog´ıa, Facultad de Ciencias, Severo Ochoa s/n, Granada 18071, Spain Empresa Municipal de Abastecimiento y Saneamiento de Granada (EMASAGRA S.A.), Granada, Spain
Received 2 March 2000; accepted 14 June 2000
Abstract Cryptosporidium parvum oocysts were purified using a discontinuous potassium bromide density gradient, composed by three solutions of 6, 16 and 28% (w/v) KBr in Tris–EDTA buffer. Fecal samples containing oocysts were washed to diminish interfering lipids and applied to the gradient. After centrifugation, oocysts can be easily aspirated from a clear band, diluted and washed by centrifugation in phosphate buffer to remove residual KBr. This method allows the purification of large amounts of highly purified C. parvum oocysts, using low cost reagents and a standard table-top centrifuge. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cryptosporidium; Oocyst; Purification; Density gradient; Potassium bromide
The production of significant amounts of highly purified oocysts from feces of infected animals can be a very expensive and time-consuming task for labs involved in C. parvum research. Sucrose flotation, coupled with Percoll or cesium chloride gradients, are frequently used for oocyst purification for biochemical, molecular and in vitro culture studies (Upton, 1997), but have some inconveniences. They usually require samples previously concentrated over sucrose, and Percoll® and cesium chloride are expensive reagents. The aim of the present work is to develop a technique for the purification of high amounts of C. parvum oocysts, suitable for biochemical and molecular studies, using standard equipment and low cost reagents. ∗ Corresponding author. Tel.: +34-958-243263; fax: +34-958-243174. E-mail address: [email protected] (E. Entrala).
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 3 1 5 - 0
224
E. Entrala et al. / Veterinary Parasitology 92 (2000) 223–226
Samples were collected from experimentally infected newborn goats (Ortega-Mora and Wright, 1994) or naturally infected newborn Holstein calves from local dairy farms. C. parvum infection was diagnosed using a modified acid-fast stain (Entrala et al., 1995). Positive samples were diluted with saline solution (NaCl 0.9%) and filtered to remove particulate material. The feces were concentrated by centrifugation (3000×g, 10 min., 4◦ C) and washed twice by centrifugation with saline solution under the same conditions. To improve oocyst yield and purity, prevention of lipid aggregation in the feces was performed by two different washing protocols: samples with low concentration of fat were repeatedly washed by centrifugation (3000×g, 10 min., 4◦ C) in Tris–EDTA buffer (Tris base 50 mM, EDTA 10 mM), until a clear supernatant was obtained. Samples with high fat content should be subjected to diethyl ether extraction (briefly, dilute fecal suspension 1:1.5 with diethyl ether, shake until a complete dispersion is obtained, centrifuge 1000×g for 10 min. at 4◦ C). The pellet obtained was washed twice by centrifugation in Tris–EDTA to remove residual ether. In both cases, the last pellet is resuspended in the minimum volume of Tris–EDTA buffer before being applied to the gradient. The gradient of potassium bromide (Sigma, P9881) consisted of three solutions of 6, 16 and 28% (w/v) KBr in Tris–EDTA buffer. From bottom to top, 7 ml of ice-cold 28, 16 and 6% KBr solutions were carefully layered into 25 ml clear plastic centrifuge tubes. Three millilitres of the concentrated fecal suspension was carefully layered on top of the gradient, and the tubes were centrifuged in a Beckman GS-15R centrifuge equipped with a swing-out S4180 rotor at 3000×g for 1 h at 4◦ C. After this, a white band over 16% KBr solution can be observed, containing C. parvum oocysts free of debris and bacteria (Fig. 1a). This band
Fig. 1. Centrifuge tubes showing a band (arrow) of C. parvum oocysts after KBr discontinuous gradient performed at (a) 3000×g, 1 h, 4◦ C; (b) 16,000×g, 1 h, 4◦ C.
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is carefully aspirated using a Pasteur pipette, and the oocysts are diluted 1:3 with distilled water and centrifuged (3000×g, 10 min, 4◦ C). The pellet is resuspended and washed twice using phosphate saline buffer (PBS, pH 7.2) and centrifugation. Recovered oocysts are quantified using a hematocytometer. In some cases, higher centrifugal forces may be required to enhance the purity of the final suspension. Potassium bromide gradient can be centrifuged under the conditions described for cesium chloride gradient (Taghi-Kilani and Sekla, 1987), 16,000×g for 1 h at 4◦ C. Fig. 1b shows a gradient performed under this conditions, using a Beckman J2-21M centrifuge equipped with a JS-13 rotor. Using this gradient, it is possible to obtain 1×105 –2×108 oocysts per Sterilin tube in 3–4 h. The purity of the oocyst suspension is highly influenced by fecal fat content, so repeated washes in Tris–EDTA or ether extraction must be performed before applying to the gradient. Clumps of fat, bacteria and debris contaminate the oocyst band when poorly defatted suspensions are used. Recovery of oocysts is influenced by oocyst viability, since only water impermeable oocysts can be recovered using this hyperosmotic gradient. This enrichment or selective concentration of viable oocysts has been previously observed (Bukhari and Smith, 1995) when hyperosmotic sucrose density or zinc sulphate flotation techniques were used for oocyst concentration. In this way, viability of KBr purified C. parvum oocysts, determined using a fluorogenic vital dye uptake assay (Campbell et al., 1992), reached values close to 100% (99.6±0.4%; n=6), although the yield of oocysts varies from 30–90% (depending on initial oocyst viability). In summary, this technique allows the rapid and cost-efficient purification of C. parvum oocysts in a similar manner to that obtained using cesium chloride gradients, but has many advantages: good results can be achieved using a standard table-top centrifuge (suitable for 4000 rpm or 3000×g) and disposable tubes, the cost of potassium bromide is much lower than that of cesium chloride, and the band containing the oocysts is easily aspirated since a greater separation is achieved from contaminating bands. Axenic oocysts, suitable for in vitro culture, can be obtained using this method, but the centrifugal conditions used for cesium chloride gradient must be selected (16,000×g for 1 h at 4◦ C).
Acknowledgements This work was supported by CICYT AMB97-0982.0 project and Plan Propio de la Universidad de Granada.
References Bukhari, Z., Smith, H.V., 1995. Effect of three concentration techniques on viability of Cryptosporidium parvum oocysts recovered from bovine feces. J. Clin. Microbiol. 33, 2592–2595. Campbell, A.T., Robertson, L.J., Smith, H.V., 1992. Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes. Appl. Environ. Microbiol. 58, 3488–3493. Entrala, E., Rueda-Rubio, M.C., Janssen, D., Mascaró, C., 1995. Influence of hydrogen peroxide on acid-fast staining of Cryptosporidium parvum oocysts. Int. J. Parasitol. 25, 1473–1477.
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E. Entrala et al. / Veterinary Parasitology 92 (2000) 223–226
Ortega-Mora, L.M., Wright, S.E., 1994. Age-related resistance in ovine cryptosporidiosis: patterns of infection and humoral immune response. Infect. Immunol. 62, 5003–5009. Taghi-Kilani, R., Sekla, L., 1987. Purification of Cryptosporidium oocysts and sporozoites by cesium chloride and Percoll gradients. Am. J. Trop. Med. Hyg. 36, 505–508. Upton, S.J., 1997. In vitro cultivation. In: Fayer, R. (Ed.), Cryptosporidium and cryptosporidiosis. CRC Press Inc. pp. 181–207.