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Detection of exoerythrocytic stages of Plasmodium berghei in fixed liver tissue and cultured cells by an immunoperoxidase antibody technique
624 TRANSACTIONS OF THE ROYAL.SOCIETY OFTROPICAI. MEDICINEANDHYGIENE,VOL. 76, No. 5, 1982
Detection of exoerythrocytic stages of Plasmodium berghei in fixed liver tissue and cultured cells by an immunoperoxidase antibody technique M. R. HOLLINGDALE Biomedical
Research Institute,
121 I1 Parklawn
Summary
An immunoperoxidaseantibody (IPA) techniqueis describedfor the detectionof exoerythrocytic(EE) stagesof Plasnwdium bergheiin rat liver sectionsor cultured WI38 cells.EE stagesweredetectedin liver sectibns12hoursafter inoculation of sporozoites,but asearly asthree hours after the inoculationof WI38 ceils. By 42 hours in liver sections and 48 hours in WI38 cells, EE parasiteswereeasilyvisible by low powermicroscopy,and merozoiteswereclearlyseen by 72 hours within EE parasites in WI38 cells, or extracellularly on adjacent cells.
Introduction
The indirect immunofluorescent antibody (IFA) technique has been widely used for the detection of exoerythrocytic (EE) stages of malarial parasites in fixed -liver sections of sporozoite infected animals or man (INGRAM & CARVER. 1963: VOLLER & TAFFS. 1963; EL NAHAL, 1967; ‘WARD & CONRAN, 1968: KROTOSKI et al., 1973). EE stages have been iden-
tified as early is 48. hours f
and Methods
Slides for histological study were prepared from Carnoy’s fixed livers from 21-day-old rats (SpragueDawley) inoculated intravenously with 300,000 sporozoites of P. berghei and prepared 6, 12, 29 and 42 hours later. Slide cultures of human embryonic lung cells, W138, grown in medium NCTC-135 with 10% foetal bovine serum were inoculated with P. berahei (ANKA strain) sporozoites (HOLLINGDALE et al., 19811.Cultures were fixed with cold methanol at 3. 12,2& 48 and 72 hours after inoculation, rinsed thre; times in Dulbecco’s PBS, and reacted in the immunoperoxidase test.
AND P. LELAND Drive, Rockville, Maryland
20852, USA
Seraused were mouse anti-P. bwghei red blood cell (RBC) stage sera, prepared by infecting mice repeatedly with RBC stages and curing with chloroquine, and reactive with both RBC stagesand sporozoites; rabbit antiserum to mouse immunoglobulins conjugated with horseradish peroxidase (DAKO-immunoglobulins a/s). Immunoperoxidase antibody technique
Fixed liver sections were immersed in xylene to remove paraffin, and rehvdrated in Dulbecco’s PBS, pH 7*4.-Liver s&ions 0; fixed cells were reacted for 30 min at 22°C with mouse anti-RBC stage sera diluted 1:40 in PBS. Slides were rinsed wi& three washes of PBS for five min each and reacted for 30 min at 22°C with peroxidase conjugate diluted 1:80 in PBS. After three washes of five min each in PBS, slides were reacted with substrate consisting of 10 mg 3,3’ diaminobenzidine tetrahydrochloride in 20 ml Tris-HCl buffer (O.OSM,pH 7.6) containing 0.01% hydrogen peroxide. After three min, slides were rinsed with deionized water and examined by light microscopy, using a blue filter. Control slides were prepared using normal mouse serum or substrate alone. Results and Discussion
EE stages of P. berghei in liver sections fixed 12 hours aft& sporozoite-inoculation and reacted with anti-RBC serum were clearlv visible as dark brown bodies against unstained batkground cells (Fig. la), and measured 5 pm in diameter. By 42 hours, parasiteswere large (30 pm) and were easily detected at 100 x magnification, and at 900 X stained cytoplasmic structure and parasite and parasitophorous membranes could be seen (Fig. lb). Control sections using normal mouse serum or substrate only gave no reaction. EE trophozoites in cultured WI38 cells could be seen as early as three hours after sporozoite inoculation (Fig. 2a), measured 4 pm and were surrounded by a stained parasitophorous vacuole membrane. By 48 hours (Fig. 2b), EE parasites were largkr (29 pm) and easily enumerated at 100 x magnification. Often, parasite material adjacent to the host cell nucleus (perinuclear staining, Fig. 2b) was observed. Merozoites from mature EE parasites were produced in culture after 72 hours and were clearly stained bv the immunoDeroxidase techniaue (Fig. 3), either within the p&asitophorous va&ole & & extracellular merozoites on the surface of adjacent ceils. Because the preparations are permanent, prolonged observation of parasiteswas possible and more structure of the parasites could be seen than with IFA.
M.
R.
HOLLINGDALE
AND
P.
LELAND
625
Fig. 1. Immunoperoxidase antibody reaction of fixed rat liver sections at (a) 12 hours and (b) 42 hours after inoculation with 6’. berg&i sporozoites. Exwrythrocytic parasites appear darkly stained against non-reactive tissue. (x 900). Fig. 2. Immunoperoxidase antibody reaction of cultured WI38 cells at (a) 3 hours and (b) 48 hours after inoculation with P. berghi sporozoites. Exoerythrocytic parasites (EE) are clearly defined within a vacuole bounded by stained parasitophorous vacuole (PV) membrane. Perinuclear staining (PN) visible adjacent to nucleus (N) of host WI38 cell. (x 800).
EE parasites of P. berghi were not detected in liver sections with IPA earlier than 12 hours after inoculation. It is not clear whether six-hour liver sections lacked EE parasites, or whether their small size made
their detection difficult. However, that EE trophozoites were seen as early as three hours in cultured cells, suggests that application of IPA to cultured EE stages rather than liver sections may be more useful for
626
SEROLOGICAL
DETECTION
OF
berghei
P.
EE
STAGES
Acknowledgements
The authors thank Dr. R. S. Nussenzweig (Division of Microbiology, New York University, New York) for the histologic slides, and Dr. R. L. Beaudoin (Department of Immunoparasitology, Naval Medical Research Institute., Bethesda, Maryland, USA) for the anti-l’. berghn sera. Ths work was made possible with funds provided by Contract DSPE-C-0079from the U.S. Agency for International Development.
References Danforth, H. D., Orjib, A. U. & Nussenzweig, R. (1978). Immunofluorescent staining of exoerythrocytic schizonts
of Plasmodium berghei in fixed liver tissue with stagespec&z immune serum. Journal
of Parasitology,
64,
1123-1125. El Nahal, H. M. S. (1967). Study of serologic cross-reactions of exoerythrocytic schizonts of avian,rodentandprimate malaria parasites by the fluorescent antibodytechnique. Bulletin of the World Health Organization, 37, 154-158. Hollingdale, M. R., Leef, J. L., McCullough, M. & Beaudoin, R. L. (1981). In vitro cultivation of the exoerytbrocytic stage of Plasmodium berghei from sporozoites. Science, 213, 1021-1022. Ingram, R. L. & Carver, R. K. (1963). Malaria parasites: Fluorescent antibody technique for tissue stage study. Science, 139, 405-406. Krotoski, W. A., Jumper, J. R. & Collins, W. E. (1973). Demonstration of exoerythrocytic schizonts of simian plasmodia in fixed tissue by immunofluorescence. Amerf;;-I{2umal
Fig. 3. Immunoperoxidase antibodyreactionof culturedWI38 cells 72 hoursafter inoculationwith P. bmghei sporozoites.Merozoites (arrows)are within the exoerythrocyticparasiteand free on the surfaceof adjacentcells. (X 900).
studying the structure of young EE parasites. Little is known of the entry of sporozoites into susceptible cells, and its study in cultured cells using IPA may help elucidate the mechanism. Although the ultrastructure of EE parasitesin liver has been studied by electron microscopy (Review, see SINDEN, 1978) many aspectsof the cycle of development remain to be elucidated. In vitro cultured EE stagesprovide the opportunity for a more complete investigation. BecauseEE stagesare difficult to locate in cultured cells, immunoperoxidase technique may be a useful preliminary step for ultrastructural studies. As with liver sections, both surface and internal antigens are reactive in the immunoperoxidase technique in cultures fixed with methanol. The structure of red blood stageparasiteshas been studied using products of the diaminobenzidine reaction (LANGRETH, 1976), and studies are in progress to apply the immunoperoxidase technique to studies of the ultrastructure of cultured P. berghei EE parasites.
of Tropical
Medicine
and Hygiene,
22,
Krotoski, $. A., Collins, W. E., Broderson, J. R., Warren, M. & Krotoski, D. M. (1981). The 48-hour exoerythrocytic stage of Plasmodium .~nomolp’ bastianellii. fIT
Journal of Tropical Medicme and Hygiene, 30,
Langreth; S. G. (1976). Feeding mechanismsin extracellular Babesia microti and Plasmodium lophurae. Journal of Protozoology, 23, 215-223. Nakane, P. K. & Pierce, C. B. (1967). Enzyme-labelled antibodies for the light and electron microscopic localiza$;m3$ tissue antigens. Journal of Cell Biology, 33, Sells, P. G: & Burton, M. (1981). Identification of Leishmnia amastigotes and their antigens in formalin fixed tissue by immunoperoxidase staining. Transactions of the ~6~~~6%ciety
of Tropical Medicine and Hygiene, 75,
Sinden, R.‘E. (1978). Cell biology. In: Rodent Malaria. Killick-Kendrick, R. & Peters, W. (Editors). London: Academic Press, pp. 106-108. Voller, A. & Taffs, L. F. (1963). Fluorescent antibody staining of exo-erythrocytic stages of Plasmodium gallinaceum. Transactions of the Royal So&y Medicine and Hygiene, 57, 32-33.
of Tropical
Ward, I’. A. & Conran, P. B. (1968). Application of fluorescent antibody to exoerythrocytic stages of simian malaria. Journal of Parasitology, 54, 171-172.
,:: Accepted for publication
21st Januay,
1982.
Detection of exoerythrocytic stages of Plasmodium berghei in fixed liver tissue and cultured cells by an immunoperoxidase antibody technique M. R. HOLLINGDALE Biomedical
Research Institute,
121 I1 Parklawn
Summary
An immunoperoxidaseantibody (IPA) techniqueis describedfor the detectionof exoerythrocytic(EE) stagesof Plasnwdium bergheiin rat liver sectionsor cultured WI38 cells.EE stagesweredetectedin liver sectibns12hoursafter inoculation of sporozoites,but asearly asthree hours after the inoculationof WI38 ceils. By 42 hours in liver sections and 48 hours in WI38 cells, EE parasiteswereeasilyvisible by low powermicroscopy,and merozoiteswereclearlyseen by 72 hours within EE parasites in WI38 cells, or extracellularly on adjacent cells.
Introduction
The indirect immunofluorescent antibody (IFA) technique has been widely used for the detection of exoerythrocytic (EE) stages of malarial parasites in fixed -liver sections of sporozoite infected animals or man (INGRAM & CARVER. 1963: VOLLER & TAFFS. 1963; EL NAHAL, 1967; ‘WARD & CONRAN, 1968: KROTOSKI et al., 1973). EE stages have been iden-
tified as early is 48. hours f
and Methods
Slides for histological study were prepared from Carnoy’s fixed livers from 21-day-old rats (SpragueDawley) inoculated intravenously with 300,000 sporozoites of P. berghei and prepared 6, 12, 29 and 42 hours later. Slide cultures of human embryonic lung cells, W138, grown in medium NCTC-135 with 10% foetal bovine serum were inoculated with P. berahei (ANKA strain) sporozoites (HOLLINGDALE et al., 19811.Cultures were fixed with cold methanol at 3. 12,2& 48 and 72 hours after inoculation, rinsed thre; times in Dulbecco’s PBS, and reacted in the immunoperoxidase test.
AND P. LELAND Drive, Rockville, Maryland
20852, USA
Seraused were mouse anti-P. bwghei red blood cell (RBC) stage sera, prepared by infecting mice repeatedly with RBC stages and curing with chloroquine, and reactive with both RBC stagesand sporozoites; rabbit antiserum to mouse immunoglobulins conjugated with horseradish peroxidase (DAKO-immunoglobulins a/s). Immunoperoxidase antibody technique
Fixed liver sections were immersed in xylene to remove paraffin, and rehvdrated in Dulbecco’s PBS, pH 7*4.-Liver s&ions 0; fixed cells were reacted for 30 min at 22°C with mouse anti-RBC stage sera diluted 1:40 in PBS. Slides were rinsed wi& three washes of PBS for five min each and reacted for 30 min at 22°C with peroxidase conjugate diluted 1:80 in PBS. After three washes of five min each in PBS, slides were reacted with substrate consisting of 10 mg 3,3’ diaminobenzidine tetrahydrochloride in 20 ml Tris-HCl buffer (O.OSM,pH 7.6) containing 0.01% hydrogen peroxide. After three min, slides were rinsed with deionized water and examined by light microscopy, using a blue filter. Control slides were prepared using normal mouse serum or substrate alone. Results and Discussion
EE stages of P. berghei in liver sections fixed 12 hours aft& sporozoite-inoculation and reacted with anti-RBC serum were clearlv visible as dark brown bodies against unstained batkground cells (Fig. la), and measured 5 pm in diameter. By 42 hours, parasiteswere large (30 pm) and were easily detected at 100 x magnification, and at 900 X stained cytoplasmic structure and parasite and parasitophorous membranes could be seen (Fig. lb). Control sections using normal mouse serum or substrate only gave no reaction. EE trophozoites in cultured WI38 cells could be seen as early as three hours after sporozoite inoculation (Fig. 2a), measured 4 pm and were surrounded by a stained parasitophorous vacuole membrane. By 48 hours (Fig. 2b), EE parasites were largkr (29 pm) and easily enumerated at 100 x magnification. Often, parasite material adjacent to the host cell nucleus (perinuclear staining, Fig. 2b) was observed. Merozoites from mature EE parasites were produced in culture after 72 hours and were clearly stained bv the immunoDeroxidase techniaue (Fig. 3), either within the p&asitophorous va&ole & & extracellular merozoites on the surface of adjacent ceils. Because the preparations are permanent, prolonged observation of parasiteswas possible and more structure of the parasites could be seen than with IFA.
M.
R.
HOLLINGDALE
AND
P.
LELAND
625
Fig. 1. Immunoperoxidase antibody reaction of fixed rat liver sections at (a) 12 hours and (b) 42 hours after inoculation with 6’. berg&i sporozoites. Exwrythrocytic parasites appear darkly stained against non-reactive tissue. (x 900). Fig. 2. Immunoperoxidase antibody reaction of cultured WI38 cells at (a) 3 hours and (b) 48 hours after inoculation with P. berghi sporozoites. Exoerythrocytic parasites (EE) are clearly defined within a vacuole bounded by stained parasitophorous vacuole (PV) membrane. Perinuclear staining (PN) visible adjacent to nucleus (N) of host WI38 cell. (x 800).
EE parasites of P. berghi were not detected in liver sections with IPA earlier than 12 hours after inoculation. It is not clear whether six-hour liver sections lacked EE parasites, or whether their small size made
their detection difficult. However, that EE trophozoites were seen as early as three hours in cultured cells, suggests that application of IPA to cultured EE stages rather than liver sections may be more useful for
626
SEROLOGICAL
DETECTION
OF
berghei
P.
EE
STAGES
Acknowledgements
The authors thank Dr. R. S. Nussenzweig (Division of Microbiology, New York University, New York) for the histologic slides, and Dr. R. L. Beaudoin (Department of Immunoparasitology, Naval Medical Research Institute., Bethesda, Maryland, USA) for the anti-l’. berghn sera. Ths work was made possible with funds provided by Contract DSPE-C-0079from the U.S. Agency for International Development.
References Danforth, H. D., Orjib, A. U. & Nussenzweig, R. (1978). Immunofluorescent staining of exoerythrocytic schizonts
of Plasmodium berghei in fixed liver tissue with stagespec&z immune serum. Journal
of Parasitology,
64,
1123-1125. El Nahal, H. M. S. (1967). Study of serologic cross-reactions of exoerythrocytic schizonts of avian,rodentandprimate malaria parasites by the fluorescent antibodytechnique. Bulletin of the World Health Organization, 37, 154-158. Hollingdale, M. R., Leef, J. L., McCullough, M. & Beaudoin, R. L. (1981). In vitro cultivation of the exoerytbrocytic stage of Plasmodium berghei from sporozoites. Science, 213, 1021-1022. Ingram, R. L. & Carver, R. K. (1963). Malaria parasites: Fluorescent antibody technique for tissue stage study. Science, 139, 405-406. Krotoski, W. A., Jumper, J. R. & Collins, W. E. (1973). Demonstration of exoerythrocytic schizonts of simian plasmodia in fixed tissue by immunofluorescence. Amerf;;-I{2umal
Fig. 3. Immunoperoxidase antibodyreactionof culturedWI38 cells 72 hoursafter inoculationwith P. bmghei sporozoites.Merozoites (arrows)are within the exoerythrocyticparasiteand free on the surfaceof adjacentcells. (X 900).
studying the structure of young EE parasites. Little is known of the entry of sporozoites into susceptible cells, and its study in cultured cells using IPA may help elucidate the mechanism. Although the ultrastructure of EE parasitesin liver has been studied by electron microscopy (Review, see SINDEN, 1978) many aspectsof the cycle of development remain to be elucidated. In vitro cultured EE stagesprovide the opportunity for a more complete investigation. BecauseEE stagesare difficult to locate in cultured cells, immunoperoxidase technique may be a useful preliminary step for ultrastructural studies. As with liver sections, both surface and internal antigens are reactive in the immunoperoxidase technique in cultures fixed with methanol. The structure of red blood stageparasiteshas been studied using products of the diaminobenzidine reaction (LANGRETH, 1976), and studies are in progress to apply the immunoperoxidase technique to studies of the ultrastructure of cultured P. berghei EE parasites.
of Tropical
Medicine
and Hygiene,
22,
Krotoski, $. A., Collins, W. E., Broderson, J. R., Warren, M. & Krotoski, D. M. (1981). The 48-hour exoerythrocytic stage of Plasmodium .~nomolp’ bastianellii. fIT
Journal of Tropical Medicme and Hygiene, 30,
Langreth; S. G. (1976). Feeding mechanismsin extracellular Babesia microti and Plasmodium lophurae. Journal of Protozoology, 23, 215-223. Nakane, P. K. & Pierce, C. B. (1967). Enzyme-labelled antibodies for the light and electron microscopic localiza$;m3$ tissue antigens. Journal of Cell Biology, 33, Sells, P. G: & Burton, M. (1981). Identification of Leishmnia amastigotes and their antigens in formalin fixed tissue by immunoperoxidase staining. Transactions of the ~6~~~6%ciety
of Tropical Medicine and Hygiene, 75,
Sinden, R.‘E. (1978). Cell biology. In: Rodent Malaria. Killick-Kendrick, R. & Peters, W. (Editors). London: Academic Press, pp. 106-108. Voller, A. & Taffs, L. F. (1963). Fluorescent antibody staining of exo-erythrocytic stages of Plasmodium gallinaceum. Transactions of the Royal So&y Medicine and Hygiene, 57, 32-33.
of Tropical
Ward, I’. A. & Conran, P. B. (1968). Application of fluorescent antibody to exoerythrocytic stages of simian malaria. Journal of Parasitology, 54, 171-172.
,:: Accepted for publication
21st Januay,
1982.