397 TRANSACTIONS
OF THE ROYAL
SOCIETY OF TROPICAL
MEDICINE
AND HYGIENE,
What happens when microfilariae
VOL. 70. Nos. 5/6.1976.
die ?
D. M. BRYCESON Diseases, 4 St. Pancras Way, London NW1 OPE
ANTHONY
Physician,
Hospital for Tropical
The first three papers in this symposium described the clinical pathology of onchocerciasis, particularly its ocular manifestations, with some precision; they showed how this varied geographically and suggested that some of the variation might be due to differences in pathogenicity between geographically distinct microfilariae. This fourth paper attempts to consider the evidence which suggests a role for the host or patient’s immune response in the pathology of onchocerciasis. There is general agreement that pathology is localized to sites where microfilariae die in the skin, eye and subcutaneous tissues. But this may not be the whole story. There is little information on the distribution of microfilariae in other tissues of the body-the kidney for example (NNOCHIRI, 1964) and no longitudinal study of morbidity and mortality has been presented hitherto. Little is known of the systemic effects of the disease, although the physical debility of severe chronic onchocerciasis is well documented. There is no information as to how, if at all, man is able naturally to kill microfilariae of Onchocerca voZvulus. That in most African infections microfilarial densities increase with age and eventually level off has been confirmed; in certain circumstances microfilarial densities may eventually decline. Cameroonian patients cured of onchocerciasis became reinfected and microfilariae reached pretreatment densities in 4 to 9 years (DUKE, 1968). In the occasional patient with localized hypertrophic lesions, however, and in the Arabian form of the diseases, sowda, microfilarial densities are low in the face of severe allergic pathology; it is uncertain whether this situation represents partial protection or simply a light infection. The humoral immune response to infection with 0. voIvulus has many components. Circulating antibodies can be detected by immunofluorescence and in enormous titres by indirect haemagglutination in every patient (personal observations), by complement fixation in lOO’/. (BOZICEVICH et al., 1947) and by_-_ gel nrecinitation _ in 60-O%(CAPRON, GENTI~INI & VERNES, 1968). Reaginic antibodies have been demonstrated by skin testing in 70-100 % (BOZICEVICH et al,. 1947, RIDLEY & STOTT, 1961). No correlation between pathology or protection and titre, biological activity or immunoglobulin class of antibody has ever been shown. Cell-mediated immunity, on the other hand, has been little studied in onchocerciasis and evidence for its efficacy or presence is scant. It has been suggested that onchocerciasis, like leprosy, presents a spectrum of disease, from high resistance as seen in Arabia, to low resistance as seen typically in West Africa, and that this resistance might be cell-mediated. Histology of skin lesions in sowda may show lymphocytic infiltration and occasional epithelioid cell granuloma formation. But there is no other published evidence of in vitro or in vivo cell-mediated immune responses in onchocerciasis. Lymphocytes from nine patients in Nigeria (BRYCESON, unpublished) were cultured in vitro in the presence of
antigens extracted from adult worms and microfilariae of 0. volvulus. Cultures were set up in 199 medium supplemented by 30% autologous serum or foetal calf serum with antigens at concentrations of 1.0 pg, 10 pg and 100 pg protein per ml and incubated for three to five days. No blast transformation was detected either by direct examination of stained cytocentrifuge preparations or by incorporation of 3H thymidine. Lymphocyte transformation in the presence of phytohaemagglutination was normal in all nine patients. These results suggest there is a specific defect or suppression of cell-mediated immunity in West African patients with onchocerciasis. The situation in patients with sowda has not been explored. The absence of any natural means of killing microfilariae has made it possible to study the pathological effects of drug-induced killing. The precise mode of action of diethylcarbamazine (d.e.c.) remains obscure. It has no microfilaricidal action in vitro (HAWKING, SEWELL & THURSTON, 1950), but in vivo it stimulates rapid mobilization and redistribution of microfilariae in the tissues and fluids of the body (BUCK, 1973 ; FUGLSANG & ANDERSON, 1974) and allows the host to kill them. Electron microscopy of skin biopsies taken before and after administration of d.e.c. (GIBSON et al., 1976) shows that the subcuticular electrolucent layer of the microfilariae increases in thickness; perhaps this indicates increased cuticular permeability. At the same time, electron dense aggregates, which are consistent in appearance with immune complexes, appear on the surface of microfilariae, followed by degeneration of muscular components. Light microscopy shows accumulation of histiocytes and eosinophils around the degenerating microfilariae (HAWKING, 1952: CONNOR et al. 1970). Karla Schillhorn van Veen and I (unpublished) used direct and indirect immuno-fluorescence to study in vitro the binding of immunoglobulins in the sera of patients and uninfected controls to heat killed microfilariae. By this method antibodies in the IgG and IgE fractions of patients’ sera were shown to bind to dead, but not to live, microfilariae. Complement binding in vitro was not demonstrated but complement levels in the sera of four patients undergoing a reaction following treatment with d.e.c. fell sharply and persisted low for over 24 hours (BRYCESON& WARRELL, in preparation). These seemingly benign and beneficial events may have remarkably severe consequences for host as well as parasite. In Ethiopia, OOMEN (1969) reported the death of seven onchocerciasis patients after treatment with d.e.c. All seven were in poor physical condition and five of them had other diseases as well, which could have caused or contributed to death, but there was a remarkably constant pattern of events following d.e.c. administration in five of the patients, who became shocked and comatose and died in two days despite vigorous attempts at resuscitation, which included the use of corticosteroids. The sixth patient died suddenly and the seventh after coma of 13 days. It may possibly be relevant that these patients
398
WHATHAPPENS
WHENMICROFILARIAEDIE?
did not have an eosinophilia before treatment, nor did they develop clinical features of the Mazotti reaction to treatment. FUGLSANG & ANDERSON (1974) reported sudden but transient collapse and coma, associated with respiratory distress, in two patients after treatment with d.e.c., and the development of acute respiratory distress in four others. There seemed to be an association with the appearance of microfilariae in the sputum. Warrell and I (in preparation) studied in detail clinical, physiological and immunological features of the Mazotti reaction in nine patients in the Nigerian savanna. Clinical features observed were intensification of symptoms, local inflammation of skin and conjunctiva and generalized systemic disturbance including chills, restlessness, sneezing and coughing. Physiological changes included fever, tachypnoea and especially hypotension which was noted in five of nine patients and was profound in one, who collapsed after sitting up in bed and remained with a systolic pressure under 80 mm Hg for over three hours. During the reaction circulating eosinophils diminished profoundly in all cases and disappeared completely in two. There was a rough correlation between the onset of the clinical reaction and eosinophil disappearance suggesting that an adequate supply of tissue eosinophils may prevent clinical features of the Mazotti reaction and perhaps reduce the associated inflammatory effects. t A hypothetical and incomplete sequence of events during the reaction is shown on the figure. It is interesting that these physiologicallchanges can be controlled by
Mast
IgE antibod;H
Release histamine
cell
degranulation
"Death" of mf.
3
Local & systemic anaphylaxis
3
Inflammation - vasodilation
Q!L
INHIBITION
Physiological changes
EOSINOPHILS / PHAGOCYTOSIS
YL
Fever
& 3
eutr ophi 1 Enzyme hist iocy t e release haao cyto S IS emen ,-w--ef ed
Antigen antibod complexe
IgG antibodies
Possible pathology
of
3 m~d~~~~~s+
u
Release & recognition of antigene n L
corticosteroids but not by antihistamines. The phenomenon of microfilarial migration is, however, unaffected (FAZEN et al., 1976). Histological evidence of the damaging effects of microfilarial death have been reported by HAWKING (1952), CONNOR et al. (1970) and GIBSON et al. (1976). The migration of microfilariae into the epidermis is followed by the formation of intraepidermal cysts and abscess. In the dermis eosinophil and histocyte infiltrate around microfilariae is followed by the appearance of pools of mucin and by fibrinoid necrosis of collagen fibres. Although the precise relationship between these acute changes and the ultimate loss of collagen and elastin fibres and the proliferation of hyaline scar tissue, changes which characterize onchocerciasis, is not yet established, the death of microfilariae seems to be the important trigger. If the patients’ response to dying microfilariae is essential to the pathology of onchocerciasis, to what extent might geographically based differences in patients’ responses or in antigenic composition of 0. volvuIus contribute to geographical differences in pathology? Using sera and onchocercal antigens prepared from adult worms and nodules taken from patients from the Cameroonian forest and savanna it was possible, by the use of a cross-absorbed indirect haemagglutination technique, to show that (see BRYCF.SON et al., 1976): (i) Onchocercal worms from forest and savanna were antigenically distinct, savanna worms possessing an antigen which forest worms lacked;
sequences following
f
L ocal inf lammation microabscesses ? collaen damage
3-
of immunologically mediated death of microfilariae in onchocerciasis
A. D. M. BRYCESON
(ii) Forest patients responded with the production of an antibody to this antigen, but savanna patients did not respond to it. The obvious anomaly in these findings is difficult to interpret, but perhaps another helminth, such as Ascnris or Lou Zoa, common in the forest but rare in the savanna, carries the missing forest antigen. Whatever the interpretation, this study lends weight to the burden of this symposium: that the host’s immune response underlies the pathogenesis of onchocerciasis, and that variations of this response contribute to geographical differences in the patterns of disease as seen in the forest and savanna belts of West Africa. Acknowledgements I am grateful to Dr. D. H. Connor and Dr. D. W. Gibson who kindly lent me illustrations from their recent papers. References Bozicevich, J., Donovan, A., Mazotti, L., Diaz, A., Francisco, A. & Padilla, E. (1947). Intradermal and complement fixation reactions elicited by various antigens in persons infected with Onchocerca volvulus. American Journal of Tropical Medicine and Hygiene, 27, 51-62. Buck A. A. (1973). Onchocerciusis. Geneva : World Health Organization. Bryceson, A. D. M., Schillhorn van Veen, K., Oduloju, A. J. & Duke, B. 0. L. (1976). Antigenic diversity among Onchocercu volvulus in Nigeria, and immunological differences between onchocerciasis in the savanna and forest of Cameroon. Clinical and Experimental Immunology, 24, 168-176. Capron, A., Gentilini, M. & Vernes, A. (1968). Le diagnostique immunologique des filarioses. Possibilitees nouvelles offertes par l’immuno-electrophorihe. Pathologie et Biologie, 16,1039-1045. Connor, D. H., Morrison, N. E., Kerdel-Vegas, F., Berkoff, H. A., Johnson, F., Tunnicliffe, R., Failing,
399
F. C., Hale, L. N. & Lindquist, K. (1970). Onchocercal dermatitis, lymphadenitis, and elephantiasis in the Ubangi Territory. Human Pathology, 1, 553-579. Duke, B. 0. L. (1968). Reinfections with Onchocercu volvulus in cured patients exposed to continuing transmission. Bulletin of the World Health Organization, 39, 307-309. Fazen, L. E., Anderson, R. I., Fazen, Lynn E. & Marroquin, H. F. (1976). Clinical and laboratory changes consequent to diethylcarbamazine in patients with onchocerciasis. American Journal of Tropical Medicine and Hygiene, 25, 250-256. Fuglsang. H. & Anderson. J. (1974). Microfilariae of &chGerca volvulus in blood and urine before, during, and after treatment with diethylcarbamazine. Journal of Helminthology, 48, 93-97. Fuglsang, H. & Anderson, J. (1974a). Collapse during treatment of onchocerciasis with diethylcafbamazine. Transactions of the Royal Societv of Trooical Medicine and Hygiene, 68, 72-73. . Gibson, D. W., Connor, D. H., Brown, L. H., Fuglsang, H., Anderson, J., Duke, B. 0. L. &Buck, A. A. (1976). Ultrastructure of microfilariae and host tissues before and after treatment with diethylcarbamazine (Hetrazan). American Journal of Tropical Medicine and Hygiene, 25, 74-87. Hawking, F. (1952). A histological study of onchocerciasis treated with Hetrazan. British Medical Journal, i, 992-994. Hawking, F., Sewell, P. & Thurston, J. P. (1950). The mode of action of Hetrazan on filarial worms. British Journal of Pharmacology, 5,217-238. Nnochiri, E. (1964). Observations on onchocercal lesions seen in autopsy specimens in Western Nigeria. Annals of Tropical Medicine and Parasitology, 58, 89-93. Oomen, A. P. (1969). Studies on onchocerciusis and elephantiusis in Ethiopia. Haarlem: De Erven F. Bohn N. V., p. 52. Ridley, D. S. & Stott, G. J. (1961). The skin test in filariasis using Setaria cervi. Journal of Tropical Medicine and Hygiene, 64,297-299.
OF THE ROYAL
SOCIETY OF TROPICAL
MEDICINE
AND HYGIENE,
What happens when microfilariae
VOL. 70. Nos. 5/6.1976.
die ?
D. M. BRYCESON Diseases, 4 St. Pancras Way, London NW1 OPE
ANTHONY
Physician,
Hospital for Tropical
The first three papers in this symposium described the clinical pathology of onchocerciasis, particularly its ocular manifestations, with some precision; they showed how this varied geographically and suggested that some of the variation might be due to differences in pathogenicity between geographically distinct microfilariae. This fourth paper attempts to consider the evidence which suggests a role for the host or patient’s immune response in the pathology of onchocerciasis. There is general agreement that pathology is localized to sites where microfilariae die in the skin, eye and subcutaneous tissues. But this may not be the whole story. There is little information on the distribution of microfilariae in other tissues of the body-the kidney for example (NNOCHIRI, 1964) and no longitudinal study of morbidity and mortality has been presented hitherto. Little is known of the systemic effects of the disease, although the physical debility of severe chronic onchocerciasis is well documented. There is no information as to how, if at all, man is able naturally to kill microfilariae of Onchocerca voZvulus. That in most African infections microfilarial densities increase with age and eventually level off has been confirmed; in certain circumstances microfilarial densities may eventually decline. Cameroonian patients cured of onchocerciasis became reinfected and microfilariae reached pretreatment densities in 4 to 9 years (DUKE, 1968). In the occasional patient with localized hypertrophic lesions, however, and in the Arabian form of the diseases, sowda, microfilarial densities are low in the face of severe allergic pathology; it is uncertain whether this situation represents partial protection or simply a light infection. The humoral immune response to infection with 0. voIvulus has many components. Circulating antibodies can be detected by immunofluorescence and in enormous titres by indirect haemagglutination in every patient (personal observations), by complement fixation in lOO’/. (BOZICEVICH et al., 1947) and by_-_ gel nrecinitation _ in 60-O%(CAPRON, GENTI~INI & VERNES, 1968). Reaginic antibodies have been demonstrated by skin testing in 70-100 % (BOZICEVICH et al,. 1947, RIDLEY & STOTT, 1961). No correlation between pathology or protection and titre, biological activity or immunoglobulin class of antibody has ever been shown. Cell-mediated immunity, on the other hand, has been little studied in onchocerciasis and evidence for its efficacy or presence is scant. It has been suggested that onchocerciasis, like leprosy, presents a spectrum of disease, from high resistance as seen in Arabia, to low resistance as seen typically in West Africa, and that this resistance might be cell-mediated. Histology of skin lesions in sowda may show lymphocytic infiltration and occasional epithelioid cell granuloma formation. But there is no other published evidence of in vitro or in vivo cell-mediated immune responses in onchocerciasis. Lymphocytes from nine patients in Nigeria (BRYCESON, unpublished) were cultured in vitro in the presence of
antigens extracted from adult worms and microfilariae of 0. volvulus. Cultures were set up in 199 medium supplemented by 30% autologous serum or foetal calf serum with antigens at concentrations of 1.0 pg, 10 pg and 100 pg protein per ml and incubated for three to five days. No blast transformation was detected either by direct examination of stained cytocentrifuge preparations or by incorporation of 3H thymidine. Lymphocyte transformation in the presence of phytohaemagglutination was normal in all nine patients. These results suggest there is a specific defect or suppression of cell-mediated immunity in West African patients with onchocerciasis. The situation in patients with sowda has not been explored. The absence of any natural means of killing microfilariae has made it possible to study the pathological effects of drug-induced killing. The precise mode of action of diethylcarbamazine (d.e.c.) remains obscure. It has no microfilaricidal action in vitro (HAWKING, SEWELL & THURSTON, 1950), but in vivo it stimulates rapid mobilization and redistribution of microfilariae in the tissues and fluids of the body (BUCK, 1973 ; FUGLSANG & ANDERSON, 1974) and allows the host to kill them. Electron microscopy of skin biopsies taken before and after administration of d.e.c. (GIBSON et al., 1976) shows that the subcuticular electrolucent layer of the microfilariae increases in thickness; perhaps this indicates increased cuticular permeability. At the same time, electron dense aggregates, which are consistent in appearance with immune complexes, appear on the surface of microfilariae, followed by degeneration of muscular components. Light microscopy shows accumulation of histiocytes and eosinophils around the degenerating microfilariae (HAWKING, 1952: CONNOR et al. 1970). Karla Schillhorn van Veen and I (unpublished) used direct and indirect immuno-fluorescence to study in vitro the binding of immunoglobulins in the sera of patients and uninfected controls to heat killed microfilariae. By this method antibodies in the IgG and IgE fractions of patients’ sera were shown to bind to dead, but not to live, microfilariae. Complement binding in vitro was not demonstrated but complement levels in the sera of four patients undergoing a reaction following treatment with d.e.c. fell sharply and persisted low for over 24 hours (BRYCESON& WARRELL, in preparation). These seemingly benign and beneficial events may have remarkably severe consequences for host as well as parasite. In Ethiopia, OOMEN (1969) reported the death of seven onchocerciasis patients after treatment with d.e.c. All seven were in poor physical condition and five of them had other diseases as well, which could have caused or contributed to death, but there was a remarkably constant pattern of events following d.e.c. administration in five of the patients, who became shocked and comatose and died in two days despite vigorous attempts at resuscitation, which included the use of corticosteroids. The sixth patient died suddenly and the seventh after coma of 13 days. It may possibly be relevant that these patients
398
WHATHAPPENS
WHENMICROFILARIAEDIE?
did not have an eosinophilia before treatment, nor did they develop clinical features of the Mazotti reaction to treatment. FUGLSANG & ANDERSON (1974) reported sudden but transient collapse and coma, associated with respiratory distress, in two patients after treatment with d.e.c., and the development of acute respiratory distress in four others. There seemed to be an association with the appearance of microfilariae in the sputum. Warrell and I (in preparation) studied in detail clinical, physiological and immunological features of the Mazotti reaction in nine patients in the Nigerian savanna. Clinical features observed were intensification of symptoms, local inflammation of skin and conjunctiva and generalized systemic disturbance including chills, restlessness, sneezing and coughing. Physiological changes included fever, tachypnoea and especially hypotension which was noted in five of nine patients and was profound in one, who collapsed after sitting up in bed and remained with a systolic pressure under 80 mm Hg for over three hours. During the reaction circulating eosinophils diminished profoundly in all cases and disappeared completely in two. There was a rough correlation between the onset of the clinical reaction and eosinophil disappearance suggesting that an adequate supply of tissue eosinophils may prevent clinical features of the Mazotti reaction and perhaps reduce the associated inflammatory effects. t A hypothetical and incomplete sequence of events during the reaction is shown on the figure. It is interesting that these physiologicallchanges can be controlled by
Mast
IgE antibod;H
Release histamine
cell
degranulation
"Death" of mf.
3
Local & systemic anaphylaxis
3
Inflammation - vasodilation
Q!L
INHIBITION
Physiological changes
EOSINOPHILS / PHAGOCYTOSIS
YL
Fever
& 3
eutr ophi 1 Enzyme hist iocy t e release haao cyto S IS emen ,-w--ef ed
Antigen antibod complexe
IgG antibodies
Possible pathology
of
3 m~d~~~~~s+
u
Release & recognition of antigene n L
corticosteroids but not by antihistamines. The phenomenon of microfilarial migration is, however, unaffected (FAZEN et al., 1976). Histological evidence of the damaging effects of microfilarial death have been reported by HAWKING (1952), CONNOR et al. (1970) and GIBSON et al. (1976). The migration of microfilariae into the epidermis is followed by the formation of intraepidermal cysts and abscess. In the dermis eosinophil and histocyte infiltrate around microfilariae is followed by the appearance of pools of mucin and by fibrinoid necrosis of collagen fibres. Although the precise relationship between these acute changes and the ultimate loss of collagen and elastin fibres and the proliferation of hyaline scar tissue, changes which characterize onchocerciasis, is not yet established, the death of microfilariae seems to be the important trigger. If the patients’ response to dying microfilariae is essential to the pathology of onchocerciasis, to what extent might geographically based differences in patients’ responses or in antigenic composition of 0. volvuIus contribute to geographical differences in pathology? Using sera and onchocercal antigens prepared from adult worms and nodules taken from patients from the Cameroonian forest and savanna it was possible, by the use of a cross-absorbed indirect haemagglutination technique, to show that (see BRYCF.SON et al., 1976): (i) Onchocercal worms from forest and savanna were antigenically distinct, savanna worms possessing an antigen which forest worms lacked;
sequences following
f
L ocal inf lammation microabscesses ? collaen damage
3-
of immunologically mediated death of microfilariae in onchocerciasis
A. D. M. BRYCESON
(ii) Forest patients responded with the production of an antibody to this antigen, but savanna patients did not respond to it. The obvious anomaly in these findings is difficult to interpret, but perhaps another helminth, such as Ascnris or Lou Zoa, common in the forest but rare in the savanna, carries the missing forest antigen. Whatever the interpretation, this study lends weight to the burden of this symposium: that the host’s immune response underlies the pathogenesis of onchocerciasis, and that variations of this response contribute to geographical differences in the patterns of disease as seen in the forest and savanna belts of West Africa. Acknowledgements I am grateful to Dr. D. H. Connor and Dr. D. W. Gibson who kindly lent me illustrations from their recent papers. References Bozicevich, J., Donovan, A., Mazotti, L., Diaz, A., Francisco, A. & Padilla, E. (1947). Intradermal and complement fixation reactions elicited by various antigens in persons infected with Onchocerca volvulus. American Journal of Tropical Medicine and Hygiene, 27, 51-62. Buck A. A. (1973). Onchocerciusis. Geneva : World Health Organization. Bryceson, A. D. M., Schillhorn van Veen, K., Oduloju, A. J. & Duke, B. 0. L. (1976). Antigenic diversity among Onchocercu volvulus in Nigeria, and immunological differences between onchocerciasis in the savanna and forest of Cameroon. Clinical and Experimental Immunology, 24, 168-176. Capron, A., Gentilini, M. & Vernes, A. (1968). Le diagnostique immunologique des filarioses. Possibilitees nouvelles offertes par l’immuno-electrophorihe. Pathologie et Biologie, 16,1039-1045. Connor, D. H., Morrison, N. E., Kerdel-Vegas, F., Berkoff, H. A., Johnson, F., Tunnicliffe, R., Failing,
399
F. C., Hale, L. N. & Lindquist, K. (1970). Onchocercal dermatitis, lymphadenitis, and elephantiasis in the Ubangi Territory. Human Pathology, 1, 553-579. Duke, B. 0. L. (1968). Reinfections with Onchocercu volvulus in cured patients exposed to continuing transmission. Bulletin of the World Health Organization, 39, 307-309. Fazen, L. E., Anderson, R. I., Fazen, Lynn E. & Marroquin, H. F. (1976). Clinical and laboratory changes consequent to diethylcarbamazine in patients with onchocerciasis. American Journal of Tropical Medicine and Hygiene, 25, 250-256. Fuglsang. H. & Anderson. J. (1974). Microfilariae of &chGerca volvulus in blood and urine before, during, and after treatment with diethylcarbamazine. Journal of Helminthology, 48, 93-97. Fuglsang, H. & Anderson, J. (1974a). Collapse during treatment of onchocerciasis with diethylcafbamazine. Transactions of the Royal Societv of Trooical Medicine and Hygiene, 68, 72-73. . Gibson, D. W., Connor, D. H., Brown, L. H., Fuglsang, H., Anderson, J., Duke, B. 0. L. &Buck, A. A. (1976). Ultrastructure of microfilariae and host tissues before and after treatment with diethylcarbamazine (Hetrazan). American Journal of Tropical Medicine and Hygiene, 25, 74-87. Hawking, F. (1952). A histological study of onchocerciasis treated with Hetrazan. British Medical Journal, i, 992-994. Hawking, F., Sewell, P. & Thurston, J. P. (1950). The mode of action of Hetrazan on filarial worms. British Journal of Pharmacology, 5,217-238. Nnochiri, E. (1964). Observations on onchocercal lesions seen in autopsy specimens in Western Nigeria. Annals of Tropical Medicine and Parasitology, 58, 89-93. Oomen, A. P. (1969). Studies on onchocerciusis and elephantiusis in Ethiopia. Haarlem: De Erven F. Bohn N. V., p. 52. Ridley, D. S. & Stott, G. J. (1961). The skin test in filariasis using Setaria cervi. Journal of Tropical Medicine and Hygiene, 64,297-299.
