154 NOTES phosphate. Cette solution ne doit pas etre preparee sortir la lamelle qu'on fixe 'a I'a!cool methylique extemporanement. durant cinq minutes. Les temps sont les suivants: Coloration des frottis La technique est ensuite la suivante: Elimination pendant cinq minutes avec la solution aqueuse de de l'alcool et coloration par la fuchsine basique en fuchsine basique. Lavage 'a l'eau. Coloration solution aqueuse a 0,25 % pendant trente minutes. pendant trente secondes avec la solution aqueuse de Lavage a l'eau. Coloration avec la solution de vert vert malachite 'a 0,8 %. Lavage a l'eau. Recoloration malachite a 0,8% pendant deux minutes. Lavage a au vert malachite pendant trente secondes. Lavage a l'eau. Nouvelle coloration au vert malachite pendant l'eau. Sechage. Examen. cinq minutes. Lavage a l'eau. Sechage de la lamelle Pour decolorer et recolorer les lames, la methode que l'on monte sur une lame porte-objet. est celle que nous avons decrite.a Pour la decoloration et la recoloration des lamelles, Elle consiste essentiellement en une decoloration les temps sont de 10 minutes pour l'acide citrique et 'a l'acide citrique 'a 0,5 % pendant deux minutes, puis de 10 minutes pour l'acide chlorhydrique. a l'acide chlorhydrique a 1 /O pendant une minute, L'examen des preparations colorees selon la suivie d'un lavage ai l'eau. methode decrite montre que l'age et la taille des Avant de recolorer les lames, on verifie que les inclusions intracytoplasmiques n'influencent pas corpuscules ont ete bien decolores en appliquant le leur reaction tinctoriale. Les inclusions apparaissent colorant de contraste de la premiere coloration. toutes colorees en rouge vif, coloration qui persiste Les lames sont ensuite decolorees de nouveau avant meme apres plusieurs mois. * d'appliquer la coloration voulue. * * Dans le cas des lamelles de cultures cellulaires, le Nous remercions MM. Ben Hamida, Hammami et tube est vide de son milieu de culture sans faire Khelil pour leur contribution technique.
Study of Serological Cross-reactions of Exo-erythrocytic Schizonts of Avian, Rodent and Primate Malaria Parasites by the Fluorescent Antibody Technique * by H. M. S. EL-NAHAL, Department of Parasitology, London School of Hygiene and Tropical Medicine, London, England
The fluorescent antibody (FA) technique provides Studies on the tissue phase of malaria parasites by a new tool for immunological studies on parasites the FA technique are apparently limited to only found in tissues. Carver & Goldman a used the FA 2 reports.c. d technique on frozen sections and sections fixed in The present work was undertaken in order to ethanol-acetic-acid and embedded in paraffin to study cross-reactions of exo-erythrocytic (EE) detect Toxoplasma gondii in brain tissues of infected schizonts of several species of mammalian and avian mice and rabbits. Shaw & Voller b used smears and malaria parasites by the indirect FA technique. sections of infected hamster's liver prepared by the freeze-substitution method for immunofluorescent Materials and methods studies on Leishmania. Strains of parasites. The following strains of plasmodia were used: Plasmodium berghei yoelii * This work received financial support from the World (17X strain); P. malariae (VS strain); P. cynomolgi Health Organization and was carried out during the author's bastianellii (the original strain); P. cynomolgi (langur tenure of a scholarship from the Government of the United Arab Republic. strain); P, gallinaceum (8B strain). a Carver, R. K. & Goldman, M. (1959) Amer. J. clin. Path., 32, 159. c Ingram, R. L. & Carver, R. K. (1963) Science, 139, 405. b Shaw, J. J. & Voller, A. (1964) Trans. roy. Soc. trop. Med. d Voller, A. & Taffs, L. F. (1963) Trans. roy. Soc. trop. Hyg., 58, 349. Med. Hyg., 57, 32. 2050B FLUORESCENT-ANTIBODY STUDIES OF EXO-ERYTHROCYTIC MALARIA SCHIZONTS 155
Preparation of tissues. Heavy suspensions of Chicken immune sera. From chickens with blood- sporozoites of P. b. yoelii (from infected Anopheles induced chronic infections with P. gallinaceum and stephensi), P. malariae, P. c. bastianellii, P. cyno- P. juxtanucleare. molgi, langur strain (from infected An. labranchiae Fluorescent antibody test. Sections of tissues atroparvus), in cold 199 medium as descrited by prepared by the freeze-substitution method were Morgan et al.e were inoculated intravenously into a cut at 5t,u floated over 18 % sodium sulfate on micro- mouse, a chimpanzee and 2 rhesus monkeys, res- scope slides, left to dry in air and kept overnight pectively. A 1-week-old chicken was inoculated in a desiccator over calcium chloride before fluores- intraperitoneally with citrated chicken blood cent staining. After removal of the wax with xylol, infected with P. gallinaceum. the sections were treated with absolute ethanol and The liver of the mouse was removed 50 hours after then with aqueous ethanol of increasing dilution, sporozoite inoculation, while liver biopsies from the and washed with phosphate-buffered saline (PBS). chimpanzee and the 2 monkeys were made 13 days The sections were stained as usual by the indirect and 7 days after the infection, respectively. The FA technique. Sera were allowed to react for 1 hour chicken was killed 3 weeks after the blood-induced and the fluorescein-labelled antispecies globulins infection and its brain was removed. for 45 minutes at room temperature in a humid Small blocks of the livers and the chicken brain chamber. were prepared by the freeze-substitution method f The brain smears were first fixed in acetone at as modified by Voller & Taffs.d The blocks were - 200C for 5 minutes, then dried quickly in air, rapidly cooled to - 70°C in petroleum spirit, kept washed in PBS and stained as described. for 3 days in absolute ethanol at - 70°C, and Each serum was titrated in 2-fold serial dilutions embedded in a mixture of polyester wax (99 %) and starting at 1: 5 against the EE schizonts of the 5 cetyl alcohol (1 %). They were kept at -20°C until malaria parasites. used. A number of impression smears from the The test was controlled as follows. chicken brain were also made by squashing small pieces of tissue into thin smears between 2 slides. (1) Apart from the preparations stained using normal sera, a parallel series of slides were treated Sera. The test sera were obtained from rats, with the fluorescein-labelled antispecies globulins rabbits, man, monkeys and chickens. Sera from without any serum. normal animals and a man were used as contr6ls. (2) After the preparations were examined by the Rat immune sera. These sera were obtained from fluorescent microscope, they were restained with rats recovered from and hyperimmunized against Giemsa's stain to confirm two points: first that the the blood stages of P. berghei berghei, P. b. yoelii, fluorescent objects were EE schizonts and secondly P. vinckei and P. chabaudi.g that EE schizonts were present in preparations Rabbit immune sera. These sera were from rabbits which did not show fluorescence. The cover-slip immunized against the soluble antigens of the blood was removed carefully and the sections were fixed stages of the 4 rodent malaria parasites and P. in Carnoy's fluid for 20 minutes. They were washed cynomolgi bastianellii. The preparation of the soluble in methylated spirit, then in tap water and stained as antigens and the immunization of the rabbits were usual with Giemsa's stain. This method was found described in a previous paper.9 most satisfactory and gave good differentiation between the EE schizonts and the tissues (Fig. 1). Monkey immune sera. These sera were from monkeys with blood-induced chronic infections with Results P. cynomolgi bastianellii, P. c. ceylonensis, P. cyno- nmolgi (langur strain), P. inui and P. shortti. All the preparations treated with normal control sera and those treated with fluorescein-labelled Human immune sera. From patients with long- antispecies globulins without any serum were standing infections with P. malariae (VS strain) or negative throughout. P. falciparum. Sera immune to P. cynomolgi subspecies were found to give a positive reaction when used at a e Morgan, J. F., Morton, H. J. & Parker, R. C. (1950) Proc. Soc. exp. Biol. (N. Y.), 73, 1. dilution of 1: 20-1 : 40 but were negative at higher f Balfour, B. M. (1961) Immunology, 4, 206. concentrations, indicating the existence of a prozone. g El-Nahal, H. M. S. (1967) Bull. Wld Hlth Org., 36, 423. The table shows the reciprocal antibody levels 156 NOTES
RECIPROCAL ANTIBODY TITRES OF TEST SERA TITRATED AGAINST THE EXO-ERYTHROCYTIC SCHIZONTS OF P. BERGHEI YOELII, P. MALARIAE, P. CYNOMOLGI BASTIANELLII, P. CYNOMOLGI (LANGUR STRAIN) AND P. GALLINACEUM USING THE INDIRECT IMMUNOFLUORESCENT TECHNIQUE
Exo-erythrocytic schizonts P. Sera a P. b. P. P. c. cynomolgi P. yoelii malariae bastianellii (langur gallinaceum strain)
Rat sera immune to: P. b. berghei 20 <5 <5 <5 <5 P. b. yoelii 40 <5 <5 <5 <5 P. vinckei 20 <5 <5 <5 <5 P. chabaudi 20 <5 <5 <5 <5 Control <5 <5 <5 <5 <5
Rabbit sera immune to: P. b. berghei sa 80 <5 <5 <5 <5 P. b. yoelii sa 80 <5 <5 <5 <5 P. vinckei sa 40 <5 <5 <5 <5 P. chabaudi sa 80 <5 <5 <5 <5 P. c. bastianellii sa <5 <5 40 40 <5 Control <5 <5 <5 <5 <5
Monkey sera immune to: P. c. bastianellii <5 <5 40 20 P. c. ceylonensis <5 <5 20 20 <5 P. cynomolgi (langur strain) <5 <5 20 20 <5 P. inui <5 <5 <5 <5 <5 P. shortti <5 <5 <5 <5 <5 Control <5 <5 <5 <5 <5
Human sera immune to: P. malariae <5 40 <5 <5 <5 P. falciparum <5 <5 <5 <5 <5 Control <5 <5 <5 <5 <5
Chicken sera immune to: P. gallinaceum <5 <5 <5 <5 80(40b) P. juxtanucleare <5 <5 <5 <5 <5 Control <5 <5 <5 <5 <5
a sa = soluble antigen. b Value found with smear. (All other values are for sections.) FIG. 1 EXO-ERYTHROCYTIC SCHIZONT OF P. B. YOELII a
to.;
a The liver tissue was prepared by the freeze-substitution method. Sections were first stained by the indirect immunofluorescent technique and then restained with Giemsa's stain after fixation in Carnoy's fluid.
FIG. 2 FLUORESCENT IMMATURE EXO-ERYTHROCYTIC SCHIZONT OF P. MALARIAE a
E b ~ A4t I,> ¢ L-
a Serum used was 1: 5 anti-P. malariae obtained from a patient. Note the fluorescent cytoplasmic flocculi and the non-fluorescent nuclei. FIG. 3 FLUORESCENT EXO-ERYTHROCYTIC SCHIZONTS OF P. B. yoELlI a
Immature schizont: cytoplasmic flocculi highly Almost matuire schizont: non-fluorescent nuclei fluorescent with few non-fluorescent nuclei arranged in circles around highly fluorescent cytoplasm
Mature schizont: fluorescent flocculi replaced Mature schizont showing three non-fluorescent by evenly distributed fluorescent merozoites, areas (vacuoles) between the merozoites nuclei of which did not fluoresce
a Serum used obtained from rabbit immunized against soluble antigen of blood forms of homologous malaria parasites, 1 :5 dilution (cf. Landau, I. & Killick-Kendrick, R. (1966) Trans. roy. Soc. trop.Med. Hyg., 60, 633, Plate II, Fig. 1 and Fig. 4, and Plate I, Fig. 9). FIG. 4 FLUORESCENT EXO-ERYTHROCYTIC SCHIZONTS OF P. C. BASTIANELLII (A) AND P. CYNOMOLGI (LANGUR STRAIN) (B) a
5 ! _ -_ r r jNl __iE r r " W".iS _i___itil_-,___.-__|i_5__2Ei-l_1___jL__..n n|arjlws ;lIazs -____| * 4. |.Sn__Ssixt*is.a;St x_ . _l rw11 1_h1E#, zS_...... ,_w_9_ i_ ^'_> i i E -f __ f ^.S ___ _ _C__X_ _. s_____t.R_ S:
s' 5__MR's.t''tR w_Y ..S i i- .. _i __ ,_ wD-D'N -_s
_ 1' D',': S *' ,sr: _i;
a Note non-fluorescent vacuoles between fluorescent cytoplasmic flocculi. Serum S*-obtained from a monkey__=chronically infected with P. c. bastianellii,_._at 1 : 5 dilution. FIG. 5 FLUORESCENT EXO-ERYTHROCYTIC SCHIZONTS OF P. GALLINACEUM, IN SMEAR (A) AND SECTION (B) OF INFECTED CHICKEN'S BRAIN
A ~~~ ~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
a Serum 1: 5 anti-P. gallinaceum. Note schizont curving in capillary bifurcation in Fig.5A. FLUORESCENT-ANTIBODY STUDIES OF EXO-ERYTHROCYTIC MALARIA SCHIZONTS 157 of the sera. The results shown in this table may be phenomenon was most probably due to the fact that summarized as follows. as uninfected chickens become older, they become (1) P. b. yoelii EE schizonts cross-reacted with all less susceptible to exo-erythrocytic schizogony rather sera immune to the blood stages of the 4 rodent than to the effect of acquired immunity. Young malaria parasites, but not with sera immune to chicks were found to die after P. gallinaceum infec- primate or avian malaria parasites. tion from profuse exo-erythrocytic schizogony, (2) P. malariae EE schizonts reacted only with while old chickens similarly infected did not develop the serum immune to P. malariae, but not with other these forms in their brains although they developed sera immune to rodent, simian, human and bird a high level of parasitaemia. malaria parasites including the 2 simian quartan On the other hand, acquired immunity has a parasites, P. inui and P. shortti. powerful effect on erythrocytic stages in avian malaria. Huff & Coulston n noticed this difference (3) P. c. bastianellii and P. cynomolgi (langur in the action of acquired immunity on tissue and strain) EE schizonts cross-reacted with sera immune blood stages of P. but with gallinaceum and P. relictum and to P. cynomolgi subspecies not other sera believed that this difference was best explained on immune to mammalian and avian malaria species. the assumption that the EE schizonts and blood (4) P. gallinaceum EE schizonts reacted only with forms were antigenically different. This suggestion P. gallinaceum immune serum and not with P. was disproved by the present work and by similar juxtanucleare immune serum or sera immune to work by Ingram & Carver c on the tissue schizonts mammalian malaria parasites. of P. gallinaceum and P. cynomolgi and by Voller (5) The titres obtained were generally of a low & Taffs d on the EE schizonts of P. gallinaceum. level. The same sera were found to give much higher These authors stained the tissue stages of the antibody levels when titrated against the blood stages parasites by the immunofluorescent technique using of the homologous parasites. sera immune to the homologous blood stages. The fluorescent EE schizonts of the species In the present work the EE schizonts of P. b. examined are shown in Fig. 2- Fig. 5. yoelii, P. malariae, P. c. bastianellii, P. cynomolgi Discussion and conclusions (langur strain) and P. gallinaceum were stained by the indirect FA technique when treated with sera Acquired immunity is known to have no effect on immune to the homologous blood forms, indicating the tissue stages of malaria parasites, at least on a common or between Shortt and his antigen antigens the tissue those of the mammalian species.h-i schizonts and the corresponding erythrocytic stages. colleagues found EE schizonts of P. vivax in the liver This also shows that the FA technique detects anti- of an immune volunteer after a massive dose of the bodies rather than actual functional immunity. sporozoites of the parasite. Similarly, Garnham Most of the workers studied the cross-reactions & k found no difference in the Bray significant between malaria parasites in relation to their ery- number of EE schizonts of P. cynomolgi in the livers throcytic stages using the FA The of immune and non-immune monkeys when they technique.so-x were inoculated with an equal dose of sporozoites. However, Corradetti I suggested that acquired n Huff, C. G. & Coulston, F. (1946) J. infect. Dis., 78, 99. immunity might have an effect on the EE schizonts 0 Voller, A. (1962) Bull. Wid Hlth Org., 27, 283. P Voller, A. (1963) Studies on immunity in malaria, London of P. gallinaceum. He found that the tissue stages (Thesis). disappeared from the brains of chickens 6 weeks q Voller, A. (1965) Ann. Soc. belge Mid. trop., 45, 385. after infection. Garnham m showed that this r Tobie, J. E., Kuvin, S. F., Contacos, P. G., Coatney, G. R. & Evans C. B. (1962) Amer. J. trop. Med. Hyg., 11, 589. h Shortt, H. E., Garnham, P. C. C., Covell, G. & Shute, 8 Tobie, J. E., Kuvin, S. F., Contacos, P. G., Coatney P. G. (1948) Brit. med. J., 1, 547. G. R. & Evans, C. B. (1963) J. Amer. med. Ass., 184, 945. i Shortt, H. E. & Gamham, P. C. C. (1948) Trans. roy. t Diggs, C. L. & Sadun, E. H. (1965) Exp. Parasit., 16, 217. Soc. trop. Med. Hyg., 41, 785. u Collins, W. E., Jeffery, G. M., Guinn, E. & Skinner, J. C. i Shortt, H. E. (1951) Trans. roy. Soc. trop. Med. Hyg., (1966) Amer. J. trop. Med. Hyg., 15, 11. 45, 175. VCollins, W. E., Skinner, J. C. & Guinn, E. G. (1966) k Garnham, P. C. C. & Bray, R. S. (1956) Rev. bras. Malar., Amer. J. trop. Med. Hyg., 15, 483. 8, 151. WCollins, W. E., Skinner, J. C., Guinn, E., Dobrovolny, 1 Corradetti, A. (1955) Trans. roy. Soc. trop. Med. Hyg., C. G. & Jones, F. E. (1965) J. Parasit., 51, 81. 49, 311. x Voller, A., Garnham, P. C. C. & Targett, G. A. T. (1966) m Garnham, P. C. C. (1967) Bull. Soc. Path. exot., 59, 549. J. trop. Med. Hyg., 69, 121.
1t 158 NOTES
blood stages of malaria parasites were found to fall cross-reacted with sera immune to the 4 rodent antigenically into 4 groups. Strong cross-reactions malaria parasites, indicating a common antigen occur between members of each group but not bet- (probably a basic antigen) between P. b. yoelii tissue ween members of different groups. These groups are: schizonts and the erythrocytic stages of rodent (1) the primate malaria parasites; (2) the rodent parasites. malaria parasites; (3) the avian malaria parasites of EE schizonts of P. gallinaceum fall into a further the subgenus Haemamoeba; (4) the avian malaria separate group and show no cross-reaction with P. parasites of the subgenus Novyella. juxtanucleare immune serum or sera immune to the From the results obtained in the present work it mammalian species. seems that the EE schizonts of some primate The low titres obtained might indicate that the malaria parasites are more species-specific than the test detected a " basic antigen " shared between EE blood forms. Thus while the blood stages of P. schizonts and their corresponding blood stages. vivax, P. malariae, P. falciparum, P. ovale, P. cynomolgi subspecies, P. gonderi, P. knowlesi, * * P. inui, P. shortti, P. coatneyi, P. fieldi and P. brasilianum cross-react, the EE schizonts of P. I wish to thank Professor P. C. C. Garnham, under P. bastianellii whose supervision this work was done, for his guidance, malariae do not. Similarly, cynomolgi invaluable help and constant encouragement. and P. cynomolgi (langur strain) EE schizonts My sincere thanks are due to Dr R. S. Bray, Dr J. R. reacted only with sera immune to P. cynomolgi Baker and Dr M. Wery for all their help and advice. subspecies. I am grateful to Mr R. Killick-Kendrick, Mr P. E. On the other hand, EE schizonts of P. b. yoelii Nesbitt, Mr T. Oxbrow, Mr M. Guy and Mr G. Gill for behaved like their blood forms." These EE schizonts their extremely helpful technical assistance.
Some Cardiomyopathies of Animals: a Review indicating Possible Analogies with Human Diseases
by ISOBEL W. JENNINGS, Veterinary Pathologist, Department ofPathology, University of Cambridge, Cambridge, England
The range of cardiomyopathies in animals is wide munities, on locally grown food supplies, so that a and includes most of the conditions known to affect knowledge of mineral deficiencies or excesses in lccal the human myocardium. The purpose of this review soil is indispensable for the understanding of indige- is to draw attention to some ofthe naturally occurring nous disease. As an example, one might quote the oc- conditions in animals which, by analogy, may prove currence of selenium-deficient soils in Scandinavia, useful in the study of cardiomyopathies in man. New Zealand and in parts of the United States of America. Animals reared in these areas are subject Nutritional myopathy to nutritional myopathy, which is preventable by the It is perhaps in the study of nutrition that compara- addition of selenium to the foodstuff. Muth (1963) tive cardiology has most to contribute, in particular describes the natural history of selenium-responsive to the preventive aspect of cardiomyopathy. Much " white muscle " disease in animals in the USA. importance is placed by the veterinarian on animal Cardiac lesions are commonly seen in calves and nutrition, particularly at the early state of organo- lambs. In the latter, they usually occur as well- genesis, and during the all-important perinatal weeks defined subendocardial plaques simulating white -when dietary deficiencies may cauEe irreparable enamel. Microscopically, affected fibrils show classic- damage in the developing organs. Farm animals may al Zenker's degeneration with progressive calcifica- often be dependent, as are many rural human com- tion. Muth also lists useful references on nutritional 2050c