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Molecular weight separation of very large DNA
Parasl?:ologyToday, vol. 3, no. Z 1987
60
Molecular Weight Separation of Very Large DNA H,J.S, Dawkins Gel electrophoresis has many applications in parasitology, especially for the separation of enzymes, immunoglobulins and DNA, but the ability to separate molecules based on size is usually restricted to within the upper and lower ranges of rnolecular weight. These limitations ore particularly evident in macramolecular DNA electrophoresis 1,2, although recent innovations in agarosegel electrophoresis have substantially reduced these boundaries and ore permitting the separation of very large DNA molecules and intact chromosomes of many organisms. In this article, Hugh Dawkins explains these techniques and their principle variants.
Conventional agarosegel electrophoresis separates native DNA molecules very effectivelyonthebasisofmolecularweight provided they are approximately in the range of 1-40 kilobase pairs (kb) which is of the order of 106-107 Da. Above the threshold size of approximately 30-40 kb, DNA molecules exhibit size-independent mobilities and co-migrate in an agarose gel matrixL2. The co-migration occurs because the molecules are larger than the pore size of the matrix 1,2and in order for the molecules to enter the gel they must orient longitudinally with respect to the gel pores. This orientation occurs when an electric field is applied across the gel. As the pores are not regular throughout the agarose gel matrix the forward motion of the molecule is serpentine in nature. With the beginning of serpentine motion, which has been termed "reptation "3,4,the capacity of the gel matrix to sieve the molecules is lost because the charge on the DNA and the friction exerted on the moving molecules are both proportional to size. Consequently, all large molecules in a constant electric field and a homogeneous gel matrix are subjected to the counterproductive forces of forward motion and friction. Any movement of large molecules is thus dependent on gel pore size and field strength and is not a function of molecular weight. In 1983, Schwartz and colleaguess addressed the problem of separating large DNA molecules based on data from viscoelastic techniques for DNA molecular weight determinations 3 and the understanding of the co-migration phenomenon of DNAL2. They theorized that by forcing large molecules periodically to change direction it would be possible to develop new ways of size-dependent DNA separation. In order to achieve the directional changes a complex electrode geometry was designed in conjunction with an electrical switching unit5,6. This enabled periodic application across the electrophoresis gel of two different electric fields at right anglesto each other in the horizon-
tal plane (Fig. I). The electrode array geometry was manipulated, as were the pulse times of the alternating fields, until large DNA was resolved on the basis of molecular weight 6. With the publication of their technique various modifications of their original apparatus have been made in many laboratories, with the result that very few laboratories have identical electrode geometries or switching units 7-12. Nevertheless, three main electrode formats have been adopted, shown schematically in Fig. I and 2.
PFGE and OFAGE
Although all units are a form of pulsed field-gradient gel electrophoresis (PFGE) units, I will reserve this term for the array depicted in Fig. I. Theothertwo electrode arrangements (Fig. 2) will be referred to here as orthogonal field alternation gel electrophoresis (OFAGE). This latter term was coined for the configuration depicted in Fig. 2b 12,13but examination of the electrode arrangement in Fig. 2a and b reveals the similarity of the two systems. The electrode geometry has a marked effect on the fields generated and therefore affects both the electrophoretic pattern of the DNA separation and the molecular weight separation range. In addition, separations are also markedly influenced by the pulse times employed and the agarose concentration of the gel. PFGE uses a single non-homogeneous field and a single homogeneous electric field in alternation to achieve separation6-9. Both OFAGE systems use two nonhomogeneous fields in alternation giving the net forward direction at a diagonal between the arrays6J2-15, While PFGE and OFAGE are effective, better resolution and separation as well as more linear migration patterns are attributed to OFAGE 6,13. All these factors are directly attributable to the array geometry. In a non-homogeneous field, the f~ld strength progressively weakens. A consequence of this is that the DNA at the front of a band experiences a weaker current than at the rear of the band thus sharpening and focusing the bands. This happens in only one direction in PFGE.The second advanttage, that of more linear migration patterns, is not simply one of aesthetic appearance but permits better lane-tolane comparisons and more samples per gel. These are both important considerations when the gels areto be used in hybridization studies as it simplifies the analysis and interpretation of the results. How does field alternation achieve separation? Alternation of the electric field at selected and controlled pulse times prevents DNA from establishing reptation. Consequently, a combination of the sieving capacity of the gel and the size related turning time of the DNA permits effective separation on the basis of molecular weight. To date much of the published work has been confined to yeast 6.9,13,14,
~) 1987, [Isevier Science Publishers B.V., Amsterdam 0169-4758/87/$0200
Parasitology Today, vol. 3, no. 2, 1987
61
but significant work has been conducted on Plasmodium 8,16. Leishmania7,17, trypanosomes ~,~8, Giardia ~9 and, more recently, human DNA 10,20.In the case of yeast and protozoa it has been possible to separate entire intact chromosomes 6-20. The application of infrequent-cutting restriction enzymes, or restriction enzymes that recognize AT sequences in GC rich organisms (and vice versa) has permitted the preparation cf very large restriction fragments for agarose gel separations I0,15,20. The potential applications of PFGE and OFAGE are far reaching. However, the complex geometry of the electrode arrays and the requirements for electronic switching units, efficient buffer recirculation and cooling systems in addition to a requirement of two power supply units for field alternation have restricted the spread of this technique. PFGE and OFAGE are usually performed at temperatures ranging from 4t 6°C, with high voltage and current output to achieve the separations. The heat generated by the high voltage and current has made cooling and recirculation of the buffer essential to maintain a constant temperature throughout the 2048 h over which these gels are run. Thus the establishment of OFAGE and PAGE requires a considerable physical and financial commitment by a laboratory. Field Inversion The limitations of PFGE and OFAGE have been overcome through a more recent innovation which resulted from experimentation with less complex electrode geometries 4. It was demonstrated that periodic inversion of a homogeneous electric field resulted in the effective separation of large molecular weight DNA molecules. The technique of field inversion gel electrophoresis (FIGE) uses conventional agarose electrophoresis apparatus in which the homogeneous field is periodically inverted with the net forward motion becoming a function of the ratio of the inversion periods. Increasing the time period of the field inversions at a given forward to reverse ratio results .n the same molecular weight separation that is seen in PFGE and OFAGE. The principle remains the same in that the molecules are being forced to change direction but in this instance in a single dimension. It could be viewed that all that has occurred is that a more obtuse angle (180 °) has been applied to the field and the field gradient has been exchanged for a gradient increase in the field inversion periods. Separation is achieved because with increasing molecular weight it is more difficult for
DNA to cease forward motion, become re-oriented sufficiently to enter the gel pores and begin to migrate in the reverse direction. Thus the DNA is, in essence, shuffled apart. FIGE has made large DNA separation available to all laboratories with standard electrophoresis equipment. The only requirement is for a switching unit capable of altering the variables of pulse time and ratio. Carle and colleagues4 used a programmed personal computer with an external switching unit to alter their electrophoretic parameters; our laboratory uses an integrated microprocessor controlled switching unit (FIGET, by Acronym Pty Ltd, Boronia, Victoria). Both the personal computer and FIGET allow precise control over the variables of pulse time gradient, ratio and the total time period of the electrophoresis run. The recent development of FIGE has meant that published work is very scarce. However, in the short period of time it has been available molecular weight separations of the order of 2000 kb have been achieved4. FIGE allows finer control over the electrophoretic variables than PFGE and OFAGE, and further manipulation of conditions should enhance its ability to separate large DNA. One area
in which additional control over the conditions is possible is in using vertical gel electrophoresis units because they allow measurement of the current actually passing through the gel. Vertical gel FIGE overcomes the problems of heating experience by PFGE, OFAGE and the original FIGE, because it uses low current which passes through gel only and permits very simple but effective temperature control of the system. In addition, vertical agarose gel electrophoresis systems allow multiple gels, and thus large numbers of samples, to be run simultaneously under similar defined conditions. Intact Chromosomal D N A All three systems discussed in this article have brought significant advances to the field of nucleic acid electrophoresis. One simple advantage that has arisen is the ability to prepare intact chromosomal DNA. This is possible because the organisms or cells are embedded and digested in agarose blocks which give great stability to the DNA and make its storage and handling very simple. The whole procedure is far simpler than conventional techniques for genomic DNA preparation, and over-
62
Parasitology Today, vol. 3, no. 2, 1987
comes may problems such as DNA strand shearing and the hazards of working with toxic chemicals. Restriction fragment length polymorphism analysis can also be made more accurate by using intact chromosomes rather than genomic DNA. The more fundamental advantages of large nucleic acid separations are that it enables electrophoretic karyotyping of many species 7,8,14,17.19 and also simplifies the identification of chromosomal rearrangements and deletions. Also the isolation of individual chromosomes for restriction digestion is now possible. In addition, linkage mapping by using overlapping cosmid clones to 'walk' along the large restriction fragments improves the efficiency of genomic mapping and simplifies the identification of repeat sequences1°.20. The separation of large DNA molecules also permits direct probing of DNA that has not been cut with restriction enzymes, and the separation of large insertion fragments containing more than one gene.
.... s
All of these attributes are more readily achieved if lane-to-lane comparisons are accurate. FIGE has this property and because it uses conventional electrophoresis equipment also has the added advantage of allowing more meaningful inter-laboratory comparisons. The ease with which the FIGE technology can be adopted by a laboratory with conventional electrophoresis equipment makes it a very attractive technique. Hugh Dawkins ~sat the RegionalVeterinary Laboratory, PO Box 388, Benalla, 3672 Victoria, Australia References
I Fangman, W.L. (1978) Nucl, Acid. Res. 5, 563665 2 Lumpkin, O.]. and Zimm, B.H. (1982) Biopolymers I I, 2315-2316 3 Klotz, LC. and Zimm, B.H. (1972) Macromolecules 5, 471-48 I 4 Carle, G.F., Frank, M. and Olson, M.V, (1986) Science 232, 65-68 5 Schwartz, D.C. et al, ( 1983) Cold Spring Harbour
Symposium of Quantitative Biology 47, 189-195 6 Schwartz, D.C, and Cantor, C.R. (1984) Cell 37, 67-75 7 Spithill, T.W, and Samaras, N. (I 985) Nucl. Acid Res. 13.4155-4169 8 Kemp, D.J. et af (I 985) Nature 315,347-350 9 Snell, R,G. and Wilkins, R.J, (1986) Nud. Acid Res. 14,4401-4406 I 0 Hardy, D.A. et al. (1986) Nature 323,453-455 II Johnson, P.J. and Borst, P. (1986) Gene 43, 213-220 12 Smith, C.L and Cantor, C.R. (1986) Nature 319,701-702 13 Carle, G,F, and Olson, M,V. (1984) Nucl. Acids Res, 12, 5647-5664 14 Carle, G.F. and Olson, M.V. (1985) P. N. A S. 82, 3756-3760 15 Smith, C.L. et al. (I 986) in Genetic Engineering 8, pp 45-70 (Setlow, J.K, and Hollaender, A., eds) Plenum Press, New York 16 Van der Ploeg, L,H.T. et al. (I 985) Science 229, 658-66 I 17 Scholler, JK,. Reed, S.G, and Stuart, K, (1986) Mof, Biochem. Parasitol. 20, 279-293 18 Bernards, A. et af. (1986) Gene 42, 313-322 19 Upcroft, P., Boreham, P.F.L, and Upcroft, J. Appf. Env. Microbiol. (in press) 20 Smith, C.L and Cantor, C,R, (1986) Cold Spring Harbour Symposium (in press)
Wanted: Entrepreneurial Parasitologists M. Muller
At the recent World Water '86 conference in London, the Director of Water Supply and Urban Development in the Operations Policy Department of the World Bank chastised the assembled engineers for behaving like religious fanatics, convinced that their work is good and righteous but not willing (or able) to demonstrate this. There must be an opening here for an entrepreneurial parasitologist.
While the control of human parasites is frequently given as an objective of sanitation programmes the reality is that parasitic disease is only one component of the health problems dealt with by such programmes and, compared with other infections, of relatively minor importance. But this need not mean that parasitologists have nothing to offer the sanitation technicians. Just as the microbiologists provide the tools flor use in the control of water quality, so too could parasitologists make a contribution to the control and assessment of sanitation technologies.
Health Indicators In the field of water supply, indicators of efficacy are relatively easy to come by. Water quality is measurable by well-
established techniques. Water usage can also be evaluated relatively simply, by observation and questionnaire. In sanitation, however, it is difficult to gain information by interview or by observation. And it is difficult to assessthe efficacy of a sanitation technology whose object is extensive (prevention of the spread of pathogens to the environment) rather than intensive (prevention of transmission of pathogens to the consumer) as in the supply of drinking water. One approach has been to attempt direct assessment of the impact on community health of sanitation schemes. This has brought mixed results I -scarcely surprising given the many confounding variables which intervene. The response has been twofold; there are ongoing attempts to improve the impact studies, while the sceptics question the justification for them in the first place. There is a conceptual point which has not received enough emphasis. In water supply, while the provision of pure water does not necessarily lead to better health (because of the many behavioural changes that must accompany it) its purity is an important intermediate objective which is worthwhile to monitor. What is lacking in the case of sanitation is a similar intermediate, technical (non-behavioural) indicator. This is currently of particular importance because many agencies are
promoting the adoption of new on-site sanitation technologies for the developing countries without any clear idea of how to assess their relative merits or monitor their relative impact.
Parasitological Indicators The communities in which the new sanitation technologies are being adopted tend to be those in which the rate of parasite infection is high. These parasites, by the nature of their life cycle, would seem to offer useful indicators. Although other organisms can be used, Ascaris offers a particularly attractive indicator. Its life cycle, is effectively restricted to man. It is resistant to extreme conditions and persists in the environment. Its different soil stages can help to date the pollution event and indicate its subsequent pathways. Most importantly it is among the most widespread of parasitic infections.
Previous Work The idea of using parasitological indicators is not new. Groenen 2 reviewed the extensive literature and suggested a programme of sampling to detect total egg infestation and the proportion of each
~)1987. ElsevqerScience Publishers BV. Amsterdam 0169-475~/87/$02.00
60
Molecular Weight Separation of Very Large DNA H,J.S, Dawkins Gel electrophoresis has many applications in parasitology, especially for the separation of enzymes, immunoglobulins and DNA, but the ability to separate molecules based on size is usually restricted to within the upper and lower ranges of rnolecular weight. These limitations ore particularly evident in macramolecular DNA electrophoresis 1,2, although recent innovations in agarosegel electrophoresis have substantially reduced these boundaries and ore permitting the separation of very large DNA molecules and intact chromosomes of many organisms. In this article, Hugh Dawkins explains these techniques and their principle variants.
Conventional agarosegel electrophoresis separates native DNA molecules very effectivelyonthebasisofmolecularweight provided they are approximately in the range of 1-40 kilobase pairs (kb) which is of the order of 106-107 Da. Above the threshold size of approximately 30-40 kb, DNA molecules exhibit size-independent mobilities and co-migrate in an agarose gel matrixL2. The co-migration occurs because the molecules are larger than the pore size of the matrix 1,2and in order for the molecules to enter the gel they must orient longitudinally with respect to the gel pores. This orientation occurs when an electric field is applied across the gel. As the pores are not regular throughout the agarose gel matrix the forward motion of the molecule is serpentine in nature. With the beginning of serpentine motion, which has been termed "reptation "3,4,the capacity of the gel matrix to sieve the molecules is lost because the charge on the DNA and the friction exerted on the moving molecules are both proportional to size. Consequently, all large molecules in a constant electric field and a homogeneous gel matrix are subjected to the counterproductive forces of forward motion and friction. Any movement of large molecules is thus dependent on gel pore size and field strength and is not a function of molecular weight. In 1983, Schwartz and colleaguess addressed the problem of separating large DNA molecules based on data from viscoelastic techniques for DNA molecular weight determinations 3 and the understanding of the co-migration phenomenon of DNAL2. They theorized that by forcing large molecules periodically to change direction it would be possible to develop new ways of size-dependent DNA separation. In order to achieve the directional changes a complex electrode geometry was designed in conjunction with an electrical switching unit5,6. This enabled periodic application across the electrophoresis gel of two different electric fields at right anglesto each other in the horizon-
tal plane (Fig. I). The electrode array geometry was manipulated, as were the pulse times of the alternating fields, until large DNA was resolved on the basis of molecular weight 6. With the publication of their technique various modifications of their original apparatus have been made in many laboratories, with the result that very few laboratories have identical electrode geometries or switching units 7-12. Nevertheless, three main electrode formats have been adopted, shown schematically in Fig. I and 2.
PFGE and OFAGE
Although all units are a form of pulsed field-gradient gel electrophoresis (PFGE) units, I will reserve this term for the array depicted in Fig. I. Theothertwo electrode arrangements (Fig. 2) will be referred to here as orthogonal field alternation gel electrophoresis (OFAGE). This latter term was coined for the configuration depicted in Fig. 2b 12,13but examination of the electrode arrangement in Fig. 2a and b reveals the similarity of the two systems. The electrode geometry has a marked effect on the fields generated and therefore affects both the electrophoretic pattern of the DNA separation and the molecular weight separation range. In addition, separations are also markedly influenced by the pulse times employed and the agarose concentration of the gel. PFGE uses a single non-homogeneous field and a single homogeneous electric field in alternation to achieve separation6-9. Both OFAGE systems use two nonhomogeneous fields in alternation giving the net forward direction at a diagonal between the arrays6J2-15, While PFGE and OFAGE are effective, better resolution and separation as well as more linear migration patterns are attributed to OFAGE 6,13. All these factors are directly attributable to the array geometry. In a non-homogeneous field, the f~ld strength progressively weakens. A consequence of this is that the DNA at the front of a band experiences a weaker current than at the rear of the band thus sharpening and focusing the bands. This happens in only one direction in PFGE.The second advanttage, that of more linear migration patterns, is not simply one of aesthetic appearance but permits better lane-tolane comparisons and more samples per gel. These are both important considerations when the gels areto be used in hybridization studies as it simplifies the analysis and interpretation of the results. How does field alternation achieve separation? Alternation of the electric field at selected and controlled pulse times prevents DNA from establishing reptation. Consequently, a combination of the sieving capacity of the gel and the size related turning time of the DNA permits effective separation on the basis of molecular weight. To date much of the published work has been confined to yeast 6.9,13,14,
~) 1987, [Isevier Science Publishers B.V., Amsterdam 0169-4758/87/$0200
Parasitology Today, vol. 3, no. 2, 1987
61
but significant work has been conducted on Plasmodium 8,16. Leishmania7,17, trypanosomes ~,~8, Giardia ~9 and, more recently, human DNA 10,20.In the case of yeast and protozoa it has been possible to separate entire intact chromosomes 6-20. The application of infrequent-cutting restriction enzymes, or restriction enzymes that recognize AT sequences in GC rich organisms (and vice versa) has permitted the preparation cf very large restriction fragments for agarose gel separations I0,15,20. The potential applications of PFGE and OFAGE are far reaching. However, the complex geometry of the electrode arrays and the requirements for electronic switching units, efficient buffer recirculation and cooling systems in addition to a requirement of two power supply units for field alternation have restricted the spread of this technique. PFGE and OFAGE are usually performed at temperatures ranging from 4t 6°C, with high voltage and current output to achieve the separations. The heat generated by the high voltage and current has made cooling and recirculation of the buffer essential to maintain a constant temperature throughout the 2048 h over which these gels are run. Thus the establishment of OFAGE and PAGE requires a considerable physical and financial commitment by a laboratory. Field Inversion The limitations of PFGE and OFAGE have been overcome through a more recent innovation which resulted from experimentation with less complex electrode geometries 4. It was demonstrated that periodic inversion of a homogeneous electric field resulted in the effective separation of large molecular weight DNA molecules. The technique of field inversion gel electrophoresis (FIGE) uses conventional agarose electrophoresis apparatus in which the homogeneous field is periodically inverted with the net forward motion becoming a function of the ratio of the inversion periods. Increasing the time period of the field inversions at a given forward to reverse ratio results .n the same molecular weight separation that is seen in PFGE and OFAGE. The principle remains the same in that the molecules are being forced to change direction but in this instance in a single dimension. It could be viewed that all that has occurred is that a more obtuse angle (180 °) has been applied to the field and the field gradient has been exchanged for a gradient increase in the field inversion periods. Separation is achieved because with increasing molecular weight it is more difficult for
DNA to cease forward motion, become re-oriented sufficiently to enter the gel pores and begin to migrate in the reverse direction. Thus the DNA is, in essence, shuffled apart. FIGE has made large DNA separation available to all laboratories with standard electrophoresis equipment. The only requirement is for a switching unit capable of altering the variables of pulse time and ratio. Carle and colleagues4 used a programmed personal computer with an external switching unit to alter their electrophoretic parameters; our laboratory uses an integrated microprocessor controlled switching unit (FIGET, by Acronym Pty Ltd, Boronia, Victoria). Both the personal computer and FIGET allow precise control over the variables of pulse time gradient, ratio and the total time period of the electrophoresis run. The recent development of FIGE has meant that published work is very scarce. However, in the short period of time it has been available molecular weight separations of the order of 2000 kb have been achieved4. FIGE allows finer control over the electrophoretic variables than PFGE and OFAGE, and further manipulation of conditions should enhance its ability to separate large DNA. One area
in which additional control over the conditions is possible is in using vertical gel electrophoresis units because they allow measurement of the current actually passing through the gel. Vertical gel FIGE overcomes the problems of heating experience by PFGE, OFAGE and the original FIGE, because it uses low current which passes through gel only and permits very simple but effective temperature control of the system. In addition, vertical agarose gel electrophoresis systems allow multiple gels, and thus large numbers of samples, to be run simultaneously under similar defined conditions. Intact Chromosomal D N A All three systems discussed in this article have brought significant advances to the field of nucleic acid electrophoresis. One simple advantage that has arisen is the ability to prepare intact chromosomal DNA. This is possible because the organisms or cells are embedded and digested in agarose blocks which give great stability to the DNA and make its storage and handling very simple. The whole procedure is far simpler than conventional techniques for genomic DNA preparation, and over-
62
Parasitology Today, vol. 3, no. 2, 1987
comes may problems such as DNA strand shearing and the hazards of working with toxic chemicals. Restriction fragment length polymorphism analysis can also be made more accurate by using intact chromosomes rather than genomic DNA. The more fundamental advantages of large nucleic acid separations are that it enables electrophoretic karyotyping of many species 7,8,14,17.19 and also simplifies the identification of chromosomal rearrangements and deletions. Also the isolation of individual chromosomes for restriction digestion is now possible. In addition, linkage mapping by using overlapping cosmid clones to 'walk' along the large restriction fragments improves the efficiency of genomic mapping and simplifies the identification of repeat sequences1°.20. The separation of large DNA molecules also permits direct probing of DNA that has not been cut with restriction enzymes, and the separation of large insertion fragments containing more than one gene.
.... s
All of these attributes are more readily achieved if lane-to-lane comparisons are accurate. FIGE has this property and because it uses conventional electrophoresis equipment also has the added advantage of allowing more meaningful inter-laboratory comparisons. The ease with which the FIGE technology can be adopted by a laboratory with conventional electrophoresis equipment makes it a very attractive technique. Hugh Dawkins ~sat the RegionalVeterinary Laboratory, PO Box 388, Benalla, 3672 Victoria, Australia References
I Fangman, W.L. (1978) Nucl, Acid. Res. 5, 563665 2 Lumpkin, O.]. and Zimm, B.H. (1982) Biopolymers I I, 2315-2316 3 Klotz, LC. and Zimm, B.H. (1972) Macromolecules 5, 471-48 I 4 Carle, G.F., Frank, M. and Olson, M.V, (1986) Science 232, 65-68 5 Schwartz, D.C. et al, ( 1983) Cold Spring Harbour
Symposium of Quantitative Biology 47, 189-195 6 Schwartz, D.C, and Cantor, C.R. (1984) Cell 37, 67-75 7 Spithill, T.W, and Samaras, N. (I 985) Nucl. Acid Res. 13.4155-4169 8 Kemp, D.J. et af (I 985) Nature 315,347-350 9 Snell, R,G. and Wilkins, R.J, (1986) Nud. Acid Res. 14,4401-4406 I 0 Hardy, D.A. et al. (1986) Nature 323,453-455 II Johnson, P.J. and Borst, P. (1986) Gene 43, 213-220 12 Smith, C.L and Cantor, C.R. (1986) Nature 319,701-702 13 Carle, G,F, and Olson, M,V. (1984) Nucl. Acids Res, 12, 5647-5664 14 Carle, G.F. and Olson, M.V. (1985) P. N. A S. 82, 3756-3760 15 Smith, C.L. et al. (I 986) in Genetic Engineering 8, pp 45-70 (Setlow, J.K, and Hollaender, A., eds) Plenum Press, New York 16 Van der Ploeg, L,H.T. et al. (I 985) Science 229, 658-66 I 17 Scholler, JK,. Reed, S.G, and Stuart, K, (1986) Mof, Biochem. Parasitol. 20, 279-293 18 Bernards, A. et af. (1986) Gene 42, 313-322 19 Upcroft, P., Boreham, P.F.L, and Upcroft, J. Appf. Env. Microbiol. (in press) 20 Smith, C.L and Cantor, C,R, (1986) Cold Spring Harbour Symposium (in press)
Wanted: Entrepreneurial Parasitologists M. Muller
At the recent World Water '86 conference in London, the Director of Water Supply and Urban Development in the Operations Policy Department of the World Bank chastised the assembled engineers for behaving like religious fanatics, convinced that their work is good and righteous but not willing (or able) to demonstrate this. There must be an opening here for an entrepreneurial parasitologist.
While the control of human parasites is frequently given as an objective of sanitation programmes the reality is that parasitic disease is only one component of the health problems dealt with by such programmes and, compared with other infections, of relatively minor importance. But this need not mean that parasitologists have nothing to offer the sanitation technicians. Just as the microbiologists provide the tools flor use in the control of water quality, so too could parasitologists make a contribution to the control and assessment of sanitation technologies.
Health Indicators In the field of water supply, indicators of efficacy are relatively easy to come by. Water quality is measurable by well-
established techniques. Water usage can also be evaluated relatively simply, by observation and questionnaire. In sanitation, however, it is difficult to gain information by interview or by observation. And it is difficult to assessthe efficacy of a sanitation technology whose object is extensive (prevention of the spread of pathogens to the environment) rather than intensive (prevention of transmission of pathogens to the consumer) as in the supply of drinking water. One approach has been to attempt direct assessment of the impact on community health of sanitation schemes. This has brought mixed results I -scarcely surprising given the many confounding variables which intervene. The response has been twofold; there are ongoing attempts to improve the impact studies, while the sceptics question the justification for them in the first place. There is a conceptual point which has not received enough emphasis. In water supply, while the provision of pure water does not necessarily lead to better health (because of the many behavioural changes that must accompany it) its purity is an important intermediate objective which is worthwhile to monitor. What is lacking in the case of sanitation is a similar intermediate, technical (non-behavioural) indicator. This is currently of particular importance because many agencies are
promoting the adoption of new on-site sanitation technologies for the developing countries without any clear idea of how to assess their relative merits or monitor their relative impact.
Parasitological Indicators The communities in which the new sanitation technologies are being adopted tend to be those in which the rate of parasite infection is high. These parasites, by the nature of their life cycle, would seem to offer useful indicators. Although other organisms can be used, Ascaris offers a particularly attractive indicator. Its life cycle, is effectively restricted to man. It is resistant to extreme conditions and persists in the environment. Its different soil stages can help to date the pollution event and indicate its subsequent pathways. Most importantly it is among the most widespread of parasitic infections.
Previous Work The idea of using parasitological indicators is not new. Groenen 2 reviewed the extensive literature and suggested a programme of sampling to detect total egg infestation and the proportion of each
~)1987. ElsevqerScience Publishers BV. Amsterdam 0169-475~/87/$02.00