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TOXOPLASMOSIS: ABORTION IN SHEEP AND GOATS
 David Buxton

Courtesy : Festschrift - Dr. S. Ramachandran

Introduction
Clinical toxoplasmosis in sheep and goats is of economic importance in many countries. The disease is manifest as abortion and occurs when these animals suffer a primary infection with the protozoan parasite Toxoplasma gondii while pregnant (1). Toxoplasma gondii is an obligate intracellular parasite that occurs worldwide and can infect all warm-blooded animals, including man. It has a simple two-stage asexual life-cycle in all hosts, involving bradyzoites in tissue cysts and tachyzoites, as well as a sexual life cycle in cats, when toxoplasma oocysts are produced in the faeces. Oocysts ingested by susceptible pregnant sheep or goats, may trigger disease, the severity of which depends in part on the stage of gestation when infection is established. Diagnostic methods include macroscopic and microscopic placental and foetal pathology, serology and the use of the polymerase chain reaction (PCR). Control methods include management procedures to reduce exposure of susceptible sheep to oocysts but vaccination and the use of certain pharmaceutical preparations may be applicable in some countries. Toxoplasma may also cause serious human illness.

Clinical disease
Sporulated T.gondii oocysts, ingested by a susceptible pregnant sheep or goat, excyst and four days later tachyzoites can be found in the mesenteric lymph nodes, where they multiply (2) and are in turn released into the blood to cause a parasitaemia. This may last from the fifth until the twelfth day after infection (3,4,5), disseminating the parasite to many tissues. The cessation of the parasitaemia coincides with the onset of a protective immune response and infection then persists as bradyzoites within tissue cysts. However, in the pregnant ewe and doe infection may establish in the gravid uterus where maternal immunological responses are altered. During pregnancy the mammalian maternal immune system does not reject the semi-allogeneic foetus, which carries paternal antigens. Over the years, there have been attempts to explain this (6) and it is now clear that several mechanisms combine to permit pregnancy in outbred populations (7). One such appears to be the down-regulation of certain cytokines (interferon-gamma, tumour necrosis factor-alpha and interleukin-2) at the materno-foetal interface that are dangerous to the foetus (8). However, this phenomenon may leave the foetus and its placenta open to invasion by Toxoplasma. Toxoplasms initially parasitise the caruncular septa, the maternal tissues of the placentome, before invading the adjacent trophoblast cells of the foetal villi and from there the rest of the foetus (9). In the early stages of gestation the ability of the foetus to recognize and respond to the parasite is negligible but develops it progressively so that lambs and kids are born immunocompetent (10). 


Thus the outcome of infection early in gestation can result in foetal death and resorption/abortion while infection in the latter part of gestation may have no clinical effect, the offspring being born normal but infected and immune. In sheep typical clinical signs of toxoplasma abortion usually result following infection in mid gestation, with ewes producing stillborn and/or weak lambs often accompanied by a small, mummified foetus. Cotyledons on the accompanying placenta/s will also show lesions visible to the naked eye (1). Abortion and neonatal mortality in goats is essentially similar to that seen in sheep whether occurring naturally (11,12,13,14,15) or produced experimentally (16,17). During an acute infection in goats toxoplasms may be excreted in the milk (18,19) and be a possible source of human infection if drunk unpasteurised (19). Also, experimentally at least, toxoplasms may be present in goat semen for a variable time after infection (20) but the epidemiological significance of this, as in sheep (21), may be very slight. 

Pathology
Macroscopic changes: Characteristically the placental cotyledons appear bright to dark red and speckled with white foci of necrosis 2 to 3 mm in diameter which may be sparse or so numerous that they can become confluent while the intercotyledonary allanto-chorion appears normal (22,23). Visible changes in lambs and kids vary, the most obvious being the mummified foetus. Foetuses dying later in gestation are born in various stages of decomposition often with clear to bloody subcutaneous oedema and a variable amount of clear to bloodstained fluid in body cavities (22). However, while these latter changes indicate an intrauterine infection they are not specific to infection with Toxoplasma. 


Microscopic changes: The most obvious histopathological changes are the necrotic foci, visible macroscopically in the cotyledons. They appear as large foci of coagulative necrosis, remarkably free of inflammatory cells, which may become mineralised with time. Rarely small numbers of intracellular and extracellular toxoplasms are visible, usually on the periphery of the necrotic lesions or in a villus, which is in the early stages of infection (9). In the foetal brain both primary and secondary lesions develop. Glial foci, typically surrounding a necrotic and sometimes mineralised centre, often associated with a mild lymphoid meningitis, represent a foetal immune response following direct damage by local parasite multiplication. Toxoplasms are only rarely found, usually at the periphery of the lesions. Focal leukomalacia, seen most commonly in the cerebral white matter cores, is also common and is possibly due to foetal anoxia in late gestation caused by advanced lesions in the placentome preventing sufficient oxygen transfer from mother to foetus (23). Immunohistochemistry can allow visualisation of intact toxoplasms as well as antigenic debris in histological sections of aborted materials, and is both a convenient and sensitive method. The ABC indirect immunoperoxidase method (Vector Laboratories, USA) and the peroxidase anti-peroxidase (PAP) technique (24) are equally good.

Diagnosis
Serology: Serology along with clinical and pathological observations is an important tool in the diagnosis of ovine and caprine toxoplasma abortion. The presence of specific antibodies in serum or tissue fluid from stillborn lambs or kids or in precolostral serum from live offspring indicates uterine infection. However, high toxoplasma antibody titres in sera taken from ewes and does within a few weeks of abortion or the production of stillborn lambs or kids can only suggest toxoplasmosis as titres remain relatively high for long periods after initial infection. Serology may also be used to indicate the degree of exposure to infection in a group of animals. The first method to be developed was the dye test (DT) of Sabin and Feldman (25) but it is expensive, time consuming and not without hazard as it requires live tachyzoites as antigen. The indirect immunofluorescent antibody test (IFAT) gives titres comparable with the DT but is safer as it uses killed tachyzoites (26) and the latex agglutination test (LAT) also performs well (26,27). The modified agglutination test (MAT) (28) has been shown to perform particularly well with goat sera (29) although for epidemiological studies the indirect haemagglutination test (IHA) (30), IFAT and LAT (31) are adequate. Both the IHA and LAT are easy to perform and the latter is available in kit form (Eiken Chemical Co., Japan) and neither test requires species-specific antisera or conjugates. The enzyme linked immunosorbent assay (ELISA) for T.gondii antibodies has been adapted for use in most domestic animals including sheep (32), goats. It can be used to distinguish IgM and IgG antibodies and as it is readily automated, it is suitable for handling large numbers of test sera. 


Polymerase chain reaction (PCR): Toxoplasma DNA may be identified in tissues with the PCR. Both the P30 and the B1 gene of T.gondii have been used as PCR targets for the detection of Toxoplasma in various clinical specimens collected from infected humans. Limited studies have also been carried out using ovine samples such as aborted placental material, brain and peritoneal fluid from aborted foetuses and blood, lymph nodes and lymph from artificially infected ewes (5,33). Detection of T.gondii DNA by amplification of the B1 gene is more sensitive than by amplification of the P30 gene. Currently with ovine and caprine material the technique is not used in routine diagnosis but the potential of the PCR to identify DNA in paraffin sections from histopathological tissue blocks may broaden its applicability (34).


Isolation: While the most direct and established method of demonstrating Toxoplasma infection in cases of abortion is to transmit the parasite from aborted material (foetal brain and placental cotyledons) to laboratory mice (35). It is slow and expensive. Toxoplasma may also be grown in tissue culture in virtually any mammalian cell line and while more rapid than mouse inoculation it is rarely used for routine diagnosis as it is expensive and test samples may frequently be heavily contaminated. A more rapid but less sensitive, method of isolation is the direct demonstration of T.gondii tissue cysts in smears stained with Giemsa following centrifugation of lamb brain homogenate on a discontinuous density gradient of 30% and 90% colloidal silica solution (36).
Life cycle: The asexual cycle involves the tachyzoite and the bradyzoite. Each crescent-shaped tachyzoite (about 5 mm by 1.5 mm) can actively penetrate a host cell, where it multiplies until the host cell ruptures to release more organisms to parasitize further cells. This process continues until the host develops immunity to the parasite when a persistent infection is established, extracellular organisms are thus eliminated, intracellular multiplication slows and tissue cysts develop (Fig.1). A small cyst contains only a few bradyzoites (the second stage of the asexual cycle) but a large one may contain thousands. Cysts are found most frequently in brain and skeletal muscle and represent the quiescent stage of the parasite within the host. Cysts eventually rupture to release the bradyzoites which then transform into tachyzoites, able to enter other cells to complete the asexual cycle (37).

Figure 1. Life cycle of Toxoplasma gondi


The sexual cycle may take place in all felids and is initiated when a non-immune individual ingests food contaminated by oocysts or containing tachyzoites or tissue cysts. In the case of the latter the cyst wall is dissolved by proteolytic enzymes in the stomach and small intestine and the released bradyzoites penetrate the epithelial cells of the small intestine. While the parasite spreads to brain and muscles where tissue cysts will develop (asexual cycle), simultaneously toxoplasms also undergo gametogeny (sexual cycle) in enteroepithelial cells. Here, in the small intestine (most commonly the ileum) gametocytes develop, over 3-15 days after infection. Microgametes form and are released to penetrate mature macrogametes triggering the formation of an oocyst wall around each fertilized gamete (Fig.1). The oocysts (10 x 12 mm in diameter), each almost filled by the sporont, are then discharged into the intestinal lumen to pass out in the faeces. Sporulation occurs within 1-5 days (depending on aeration and temperature) to produce two ellipsoidal sporocysts, each containing four sporozoites within each oocyst (37). Thus during the 4-12 days after ingesting tissue cysts the cat is capable of shedding millions of oocysts in its faeces, after which it will not normally excrete the parasite again. Cats may also become infected by ingesting oocysts or tachyzoites but in this case they tend to produce oocysts after 19 or 20 days, for only a day or two and in relatively small numbers and even then about half of these animals will not excrete oocysts (37). Thus the most significant amplification of parasite numbers in cats occurs following ingestion of tissue cysts in muscle and brain from persistently infected animals. 


While most environmental contamination by oocysts is due to domestic and feral cats other felids such as lions (38), tigers (39), cougars (40), jaguars, ocelots (41) and leopards (42) may play a significant role in countries where they have a significant presence. Felids may also be a source of toxoplasma infection in zoological collections. Toxoplasma oocysts may remain viable for many months and so infected cat faeces contaminating farm feeds (43), bedding, pasture (44) and drinking water can create potent, long-lasting sources of infection for herbivores (45,46). 


Animals persistently infected with T.gondii are important sources of infection for cats (47,48). Mice (49,50,51,52), but probably not rats (53) are particularly important because they can pass the parasite in utero without causing overt clinical disease or foetopathy. In this way a reservoir of T.gondii tissue cyst infection for cats can exist in a population of mice for generations. 

Control
A degree of control is possible in sheep and goats with an understanding of the life cycle of T.gondii. In addition a vaccine is available in some European countries for use in sheep and pharmaceutical preparations may be used in some circumstances.


Flock and herd management: During pregnancy a flock/herd, in which the majority are seronegative to T.gondii, could be at risk if allowed access to an environment contaminated by cat faeces and so, where possible, food and water should be kept free from soiling by cats. Other measures to reduce environmental contamination by oocysts should be aimed at reducing the number of cats capable of shedding oocysts. These could include selective culling of aged and diseased cats and attempts to control future breeding. The maintenance of a small healthy population of mature cats will reduce oocyst excretion as well as help control rodents. 


Vaccination: Natural infection with T.gondii stimulates protective immunity in both sheep and goats (54) but to date inactivated vaccines have not been effective (55,56,57). In 1988, the Ministry of Agriculture and Fisheries (New Zealand) launched a live attenuated toxoplasma vaccine for the control of ovine toxoplasmosis (58,59) and subsequently, after further study (60,61), it was marketed in the U.K. and Eire (Toxovax, Intervet U.K.) as a tissue-culture-grown vaccine. The vaccine, which consists of live S48 tachyzoites, is now also available in France, Spain and Portugal. Protective immunity in sheep given the live vaccine is largely cell mediated, (62) with CD4+ and CD8+ T cells (63) and interferon gamma (64,65) playing an important role in suppressing T.gondii parasitaemia in pregnant ewes (66) thereby protecting the developing foetus from exposure to infection. As the vaccine has a shelf life of only seven to 10 days, and is capable of infecting people, it must be handled with care, strictly according to the manufacturer’s recommendations. 
As with sheep, the majority of goats previously exposed to infection with T.gondii develop a protective immunity to the parasite so that they are protected against subsequent challenge during pregnancy (67), although repeat abortions have been recorded (68). Toxovax is not licensed for use in goats. Immunity induced in goats by experimental infection with the related coccidian parasite Hammondia hammondi has been shown to offer some cross-protection against challenge with T.gondii both in non pregnant (69) and pregnant animals (70,71) but this avenue of research has not been pursued. 


Pharmaceuticals: Chemoprophylaxis with monensin given in the feed at the rate of 15 mg/animal/day during pregnancy, can significantly suppress a toxoplasma infection in sheep (32) as can the anticoccidial drug decoquinate fed daily at 2 mg/kg body weight (72) and both work best if they are already being fed to susceptible ewes at the time they encounter infection rather than after infection is established. 

Human infection
Toxoplasma can pose a serious threat to the unborn child if the mother becomes infected for the first time while pregnant (73). The rate of congenital infection varies from one region or country to another but is usually between 1 and 6 per 1000 pregnancies. Other people at risk of developing clinical illness include those who are immunosuppressed, such as organ transplant patients, victims of AIDS, people suffering from certain types of cancer and those undergoing certain forms of cancer therapy. The very young and very old may also be more susceptible. On occasions people with no apparent immune deficiency may develop an illness characterised by general malaise, fever and lymphadenopathy. 


Sources of infection: Most human infection appears to result either from exposure to an environment contaminated with toxoplasma oocysts or from ingestion of raw or lightly cooked meat containing toxoplasma tissue cysts (74). Sheep, goats and pigs once infected may remain so for life with bradyzoites in tissue cysts in brain and muscle. There is less risk of tissue cysts being present in cattle and deer. Free range poultry meat may also be infected. Cooking of red meat sufficient to induce a colour change to brown would be expected to kill the parasite while freezing and thawing will significantly reduce the viability of toxoplasma bradyzoites, if not kill them altogether (74). Those handling raw meat should take care to wash their hands afterwards. Sporulated oocysts may remain infectious for a long period so that vegetable plots and flower beds in which cats have defecated can present a risk of infection to the vulnerable. It is a sensible precaution to wash all vegetables whether they are to be cooked or not before they are eaten. Childrens’ sand pits may also be a source of infection if soiled by cats and so should be kept covered when not in use. On the farm, cats may also soil hay, bedding and bulk grain stores and so pose a threat of infection to farm staff as well as stock. Water contaminated with oocysts may also cause human infection (75).

Conclusions
Toxoplasmosis in sheep is important in its own right but is also a good model for studies of human toxoplasmosis as the latter more closely resembles the infection in sheep than it does the more extensively researched infection in mice (62). The very success of Toxoplasma as an intracellular parasite is that it would appear to be able to infect any nucleated cell-type in any warm-blooded vertebrate. This very ubiquity and the relative ease with which a live infection (and hence live vaccine) induces immunity is in indirect proportion to the difficulty scientists have had in developing a non-infectious vaccine. Success will require a detailed knowledge both of the parasite and the host response to it, including how tachyzoites bind to and subsequently invade cells (76,77) and the role of the secretory organelles in establishing the tachyzoite in its parasitophorous vacuole where it multiplies in the cell (76,78). In addition, as Toxoplasma naturally infects animals orally, research should not ignore the potential role of mucosal immunity. In support of this, experiments have shown that the purified major surface tachyzoite protein SAG1 (30kDa/P30 antigen) in association with “cholera toxin” presented to mice by the nasal route can very substantially reduce subsequent development of T.gondii tissue cysts in brain (79). Other avenues being explored include genetic immunization (80). Success with these initiatives will lead to an improved vaccine to prevent human and animal toxoplasmosis (81). 



References

  1. Buxton, D. (2000). Toxoplasmosis and neosporosis. In Diseases of sheep. 3rd edition. p. 86. Eds. W.B. Martin and I.D. Aitken. Blackwell Science, Oxford.

  2. Dubey, J.P. (1984). Int. Goat Sheep Res., 2: 93.

  3. Dubey, J.P. and Sharma, S.P. (1980). J. Parasit., 66: 111.

  4. Reid, H.W. et al. (1982). J. Comp. Path., 92: 181.

  5. Wastling, J.M. et al. (1993). J. Med. Microbiol., 38: 360.

  6. Medawar, P.B. (1953). Symp. Soc. Exp. Biol., 7: 320.

  7. Chaouat, G. (1999). Foetal-maternal immunological relationships. Encyclopaedia of Life Sciences Online [www.els.net] article A 516. 

  8. Ragupathy, R. (1997). Immunology Today, 18: 478.

  9. Buxton, D. and Finlayson, J. (1986). J. Comp. Path., 96: 319.

  10. Salami, M. Al. et al. (1985). Haemopoiesis and the development of immunological reactivity in the sheep foetus. In Immunology of Sheep. p. 19. Eds. B. Morris and M. Miyasaka. Basle, Editiones (Roche).

  11. Munday, B.L. and Mason, R.W. (1979). Aust. vet. J., 55: 485.

  12. Chhabra, M.B. and Gautam, O.P. (1984). Caprine abortion and neonatal mortality associated with toxoplasmosis in India. In Les maladies de la chèvre, p. 719. Niort (France) Les Colloques de l’INRA, No. 28. 

  13. Dubey, J.P. (1981). J. Am. vet. Med. Ass., 178: 661.

  14. Dubey, J.P. et al. (1981). Am. J. vet. Res., 42: 1624.

  15. Nurse, G.H. and Lenghaus, C. (1986). Aust. vet. J., 63: 27.

  16. Dubey, J.P. et al. (1980). Am. J. vet. Res., 41: 1072.

  17. Dubey, J.P. (1988). Am. J. vet. Res., 49: 905.

  18. Dubey, J.P. (1980). J. Am. vet. Med. Ass., 177: 1203.

  19. Skinner, L.J. et al. (1990). Scand. J. Infect. Dis., 22: 359.

  20. Dubey, J.P. and Sharma, S.P. (1980). Am. J. vet. Res., 41: 794.

  21. Blewett, D.A. et al. (1982). Vet. Rec., 111: 73.

  22. Hartley, W.J. and Kater, J.C. (1963). Res. vet. Sci., 4: 326.

  23. Beverley, J.K. et al. (1971). Vet. Rec., 88: 124.

  24. Buxton D. et al. (1982). Res. vet. Sci., 32: 170.

  25. Uggla, A. et al. (1987). Am. J. vet. Res., 48: 348.

  26. Sabin, A.B. and Feldman, H.A. (1948). Science, 108: 660.

  27. Maley, S.W. et al. (1997). Vet. Rec., 140: 558.

  28. Trees, A.J. et al. (1989). Res. vet. Sci., 46: 67. 

  29. Desmonts, G. and Remington, J.S. (1980). J. Clin. Microbiol., 11: 562.

  30. Dubey, J.P. et al. (1985). Am. J. vet. Res., 46: 1137.

  31. Patton, S. et al. (1990). J. Parasit., 76: 74.

  32. Opel, U. et al. (1991). Vet. Parasit, 40: 181.

  33. Buxton, D. et al. (1988). J. Comp. Path., 98: 225.

  34. Owen, M.R. et al. (1998). Vet. Rec., 142: 445.

  35. Ellis, J.T. (1997). Neospora caninum: prospects for diagnosis and control using molecular methods. In Control of coccidiosis into the next millenium. p. 80. Proceedings of the VII International Coccidiosis Conference and European Union COST 820 Workshop. Sept. 1-5. Oxford.

  36. Fleck, D.G. and Kwantes, W. (1980). The Laboratory Diagnosis of Toxoplasmosis. Public Health Laboratory Service, London. Monograph series 13.

  37. Blewett, D.A. et al. (1983). Vet. Rec., 112: 98.

  38. Dubey, J.P. and Beattie, C. P. (1988). Toxoplasmosis of Man and Animals. CRC Press, Boca Raton.

  39. Ocholi, R.A. et al. (1989). Vet. Rec., 124: 515.

  40. Dorny, P and Fransen, J. (1989). Vet. Rec., 125: 647.

  41. Aramini, J.J. et al. (1998). J. Parasit., 84: 438.

  42. Jewell, M.L. et al. (1972). Am. J. trop. Med. Hyg., 21: 512.

  43. Lukesova, D. and Literak, I (1998). Vet. Parasit., 74: 1.

  44. Plant, J.W. et al. (1974). Aust. vet. J., 50: 19.

  45. Faull, W.B. et al. (1986). Vet. Rec., 119: 491. 

  46. Blewett, D.A. (1983). Br. vet. J., 139: 537. 

  47. Blewett, D.A. and Watson, W.A. (1983). Br. vet. J., 139: 546.

  48. Jackson, M.H. and Hutchison, W.M. (1989). The prevalence and source of Toxoplasma infection in the environment. In Advances in Parasitology, vol. 28, p. 55. Eds. J.R. Baker and R. Muller. Academic Press, London.

  49. Peach, W. et al. (1989). Ann. trop. Med. Parasit., 83: 173. 

  50. Eichenwald, H. (1948). Am. J. Dis. Child, 76: 307.

  51. Beverley, J.K.A. (1959). Nature, 183: 1348. 

  52. De Roever-Bonnet, H. (1969). Trop. Geogr. Med., 21: 443.

  53. Owen, M.R., and Trees, A.J. (1998). Parasitology, 116: 299.

  54. Dubey, J.P. et al. (1997). Parasitology, 115: 9.

  55. McColgan, C. et al. (1988). Vet. Rec., 123: 467. 

  56. Beverley, J.K. et al. (1971). Br. vet. J., 127: 529.

  57. Wilkins, M.F. et al. (1987). N.Z. vet. J., 35: 31.

  58. Buxton D. et al. (1989). Br. vet. J., 145: 451.

  59. O’Connell, E. et al. (1988). N.Z. vet. J., 36: 1.

  60. Wilkins, M.F. et al. (1988). N.Z. vet. J., 36: 86.

  61. Buxton, D. et al. (1991). Vet. Rec., 129: 89.

  62. Buxton, D. et al. (1993). Vet. Rec., 133: 310.

  63. Innes, E.A. and Wastling, J.M. (1995). Parasitol Today, 11: 268.

  64. Innes, E.A. et al. (1995). Parasite Immunol., 17: 151.

  65. Oura, C.A.L. et al. (1993). Parasite Immunol., 15: 535.

  66. Innes, E.A. et al. (1995). J. Comp. Path., 113: 89.

  67. Buxton, D. et al. (1994). J. Comp. Path., 111: 231. 

  68. Obendorf, D.L. et al. (1990). Aust. vet. J., 67: 233.

  69. Dubey, J.P. (1982). J. Am. vet. med. Ass., 180: 1220.

  70. Dubey, J.P. (1981). Am. J. vet. Res., 42: 2068.

  71. Dubey, J.P. (1981). Am. J. vet. Res., 42: 2155.

  72. Munday, B.L. and Dubey, J.P. (1988). Aust. vet. J., 65: 150.

  73. Buxton, D. et al. (1996). Vet. Rec., 138: 434.

  74. Hayde, M. and Pollak, A. (2000). Clinical picture. Neonatal signs and symptoms. In Congenital Toxoplasmosis. Chapter 3.1, p. 151. Eds. P. Ambroise-Thomas and E. Petersen. Springer-Verlag, Paris.

  75. Hall, S. et al. (2001). Epidemiology. In Toxoplasmosis, a comprehensive clinical guide. Chapter 3. Eds. D.H.M. Joynson and T.G. Wreghitt. Cambridge University Press, Cambridge. 

  76. Bowie, W.R. et al. (1997). Lancet, 350: 173.

  77. Joiner, K.A. and Dubremetz, J.F. (1993). Infection and Immunity, 61: 1169.

  78. Kasper, L.H. and Mineo, J.R. (1994). Parasitol Today, 10: 184.

  79. Cesbron-Delauw, M.F. (1994). Parasitol Today, 10: 293.

  80. Debard, N. et al. (1996). Infection and Immunity, 64: 2158.

  81. Nielsen, H.V. et al. (2000). Strategies for development of vaccines against Toxoplasma gondii. In Congenital Toxoplasmosis. p. 313. Eds. P. Ambroise-Thomas and E. Petersen, Springer-Verlag, Paris.

Authors Corresponding address: 

Dr. David Buxton
Moredun Research Institute, Pentlands Science Park, Bush Loan, Edinburgh EH22 0PZ, Scotland, U.K.

The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s).