Use of ferritin-labelled antibodies for differentiating Leishmania species and other Trypanosomatidae*

Bull. Org. nwnd. Santi 1973, 48, 289-297Bull. Wid Hlth Org.J Use of ferritin-labelled antibodies for differentiating Leishmania species and other Trypanosomatidae* V. M. SAF'JANOVA 1 & A. A. AVAKJAN2 A new immunomorphological test for differentiating Leishmania species, based on electron-microscopic revelation of the localization of their specific antigens, is described. The diagnostic value of treating the promastigotes of Leishmania and other Trypanosoma- tidae with ferritin-labelled antibodies was determined in cross experiments. The immuno- ferritin test reveals both the serogroup of a given species of Leishmania and specific anti- genic differences between different species. However, it fails to differentiate different strains of a given species of Leishmania, which attests to its species-specificity. The urgent problem of systematizing the taxonomy of the causative agents of leishmaniases in different parts of the world-Europe, Asia, Africa, and the Americas-as well as of the mucocutaneous leish- maniases of the New World is still unresolved. Elaboration ofa differential serodiagnostic method is of considerable theoretical importance in relation to the evolution and systematization of these micro- organisms, and is of practical importance in relation to many problems of epidemiology and epizo- otiology. Rational control of these widespread dis- eases with natural foci is impossible unless these problems are solved. Most workers (e.g., Hodukin, 1957; Garnham, 1962; Nicoli, 1963; Adler, 1964) believe that Leish- mania species can be differentiated only on the basis of their morphological features when stained by Giemsa's or other methods, the characteristics of their cultures in nutrient media, their biochemical characteristics, serological and immunological data, and signs manifested by the vertebrate and inverte- brate hosts of the parasites. These criteria have not all been equally well elabo- rated, nor has their taxonomic importance been ade- quately determined. This is particularly true of the morphological features of Leishmania, and-with the exception of L. enrietti-no differences in the * From the Gamaleja Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow D-98, USSR. 1 Senior Scientific Officer, Vector Laboratory, Depart- ment of Diseases Occurring in Natural Foci. 'Head, Department of the Morphology of Micro- organisms. structure of Leishmania species that might be of diagnostic value have yet been revealed by means of the optical microscope. The use of electron micro- scopy, however, has proved more successful. Studies conducted at our institute have shown that the distance between the centres of the cytoplasmic microtubules found in promastigotes in ultrathin sections directly under the osmiophilic layer of the cytoplasmic membrane (Manukjan, 1966) differs in various species of Leishmania, measuring about 3 500-4 200 nm in L. tropica major and about 5 800-6 700 nm in promastigotes obtained from Turkmenian reptiles (Manukjan & Saf'janova, 1968).3 Further data on the diagnostic value of this characteristic would be of great interest. Compara- tive studies of these formations in various genera of the Trypanosomatidae (Angelopoulos, 1969, 1970) have also revealed quantitative differences. The purpose of this study was to develop a new immunomorphological method of differentiating Leishmania species based on the treatment of their promastigotes with immunoferritin-labelled rabbit sera. The immunoferritin method was first employed by Singer (1959) to reveal the tobacco mosaic virus. Subsequently it found wide application in virology, bacteriology, and pathologic histology, and in proto- zoology it has been used to study Paramecium aurelia (Mott, 1963), Trypanosoma equiperdum (Boisson et al., 1969), and T. brucei (Vickerman & Luckins, "Also Avakjan, A. A., unpublished observations re- ported at the WHO Travelling Seminar on Leishmaniasis, Moscow, 1967. 3009 -289- 290 V. M. SAF JANOVA & A. A. AVAKJAN 1969). It should be noted, however, that until recently all studies employing the immunoferritin method were aimed at revealing the localization of specific antigens of normal or pathologically altered cells. The present study is, as far as we know, the first attempt to use the method for differentiating protozoa. The direct method, which-according to the literature-is more specific than the indirect method, was used. MATERIALS AND METHODS The following strains of Leishmania and Crithidia were used.' 1. Strain 60-L. tropica major Jak. Isolated by E. M. Belova in November 1965 in the Turkmenian SSR from a patient affected with acutely necrotizing (zoonotic) cutaneous leishmaniasis. It has now lost -its virulence. 2. Strain VI-L. tropica major Jak. Isolated by G. B. Gasan-zade in 1967 in Azerbaijan from a patient affected with acutely necrotizing (zoonotic) cutaneous leishmaniasis contracted in the Turk- menian SSR. Tests with golden hamsters have shown it to be highly virulent. 3. Strain X-L. tropica minor Jak. Isolated by C. B. Gasan-zade in 1968 in Baku (Azerbaijan) from a patient affected with the lipoid form of late ulcer- ating (anthroponotic) cutaneous leishmaniasis. Tests -with hamsters have shown it to be virulent. 4. Strain 7-L. adleri Heisch. Isolated by Heisch -in 1958 in Kenya from the lizard Latastia longi- -caudata revoili. Tests with hamsters have shown it -to be virulent. 5. Strain Ag-Promastigotes of reptiles. Isolated -by V. N. V'jukov in 1964 in Turkmenia from the steppe agama (Agama sanguinolenta). It is non- -pathogenic to mammals. 6. Crithidia oncopelti Noguchi & Tilden from the -bug Oncopeltus fasciatus. The degree of antigenic relationship between the :strains of Leishmaniae was first studied in cross serological experiments by the modified test of Adler {(Saf'janova, 1966, 1967, 1970; Saf'janova & Aliev, 1971). It was established that they belong to two dif- ferent serogroups, those obtained from mammals 4strains 60, VI, and X) belonging to one group and -those from reptiles (strains 7 and Ag) to the other. 1 Obtained from the Vector Laboratory of the Gamaleja Institute. Both strains of L. t. major (60 and VI) were found to be serologically identical. Each is in antigenic rela- tionship of the second degree with L. t. minor (strain X), of the third degree with L. adleri (strain 7), and in the sixth degree with the strain of promasti- gotes isolated from the steppe agama (strain Ag).2 Correspondingly, Leishmania strains 7 and Ag (from reptiles), which are in relationship of the second degree with one another, are in relationship of the third and fourth degree, respectively, with Leishmania strains from mammals. For each strain of Leishmnania and for C. oncopelti we prepared, by the usual methods (Adler, 1963), rabbit antisera labelled with ferritin. The fractions of immunoglobulin isolated from each antiserum were labelled by the modified Singer method (Singer & Schick, 1961). The conjugate of immunoglobulin and ferritin was 2,4-diisocyanatotoluene. All the strains were examined by the electron microscope on the 7th to the 10th day of their culti- vation in a two-phase medium. To obtain a sediment, the liquid part of the medium from 10 test tubes containing about 1 million promastigotes per cm3 was centrifuged for 15 min at 300 rev/min. The sediment was fixed with glutaraldehyde and, after it had been washed and diluted with phosphate buffer, labelled antiserum was added to it. The antiserum was conjugated with the antigen contained in the sediment for 1 h at room temperature, after which the sediment was washed from the serum with phos- phate buffer. This was followed by additional fixa- tion with a buffered solution of osmium(VIII) oxide (10 g/litre) and then by dehydration in ethanol; butyl and methyl methacrylates (4: 1) were then added and the mixture was placed in gelatin capsules. Promastigotes of each strain were treated with a homologous antiserum and several heterologous antisera. Each experiment was performed three times, ten pouring blocks being prepared each time. The sections were cut with an LKB 4801 A ultratome and were stained with lead citrate. A JEM-60 electron microscope, with an accelerating voltage of 80 kV, was used. RESULTS The electron micrographs (Fig. 1-7) show the results of treating promastigotes of the different strains of Leishmania and C. oncopelti with ferritin- labelled homologous and heterologous antisera. ' According to the arbitrary gradation of Saf'janova (1970). Fig. 1. C. oncopelti, strain SO, treated with ferritin-labelled homologous antiserum. Numerous particles of ferritin are seen on the external surface of the cell. Along the cytoplasmic micro- tubules a concentration of ferritin granules is noted. Fig. 2. Promastigotes of reptiles, strain Ag, treated with ferritin-labelled homologous antiserum. An accumulation of ferritin granules is seen on the external surface of the cell. : *L [*-1vtsfi*t X P.*M Fig. 3. L. t. major, strain 60, treated with ferritin-labelled homologous antiserum. Granules of ferritin are seen on the external surface of the cytoplasmic membrane of the cell. -4' r ;I. r.f "As .Ikl jr.. Fig. 4. L. t. major, strain L. t. major, strain VI. 60, treated with ferritin-labelled antiserum against Fig. 5. L. t. minor, strain X, treated with ferritin-labelled heterologous antiserum against L. t. major, strain 60. Negative results: granules of ferritin are not observed on the external surface of the cell. Fig. 6. Promastigotes of reptiles, strain Ag, treated with ferritin-labelled hetero- logous antiserum against C. oncopelti, strain SO. Negative results. _ Fig. 7. L. t. major, strain 60, treated with ferritin-labelled heterologous anti- serum against C. on- copelti, strain SO. Negative results DIEFFERENTIATION OF LEISHMANIA SPP. Table 1. Results of treating representatives of ferritin-labelled rabbit antisera the family Trypanosomatidae with Labelled antisera Serogroup Strain L. t. L. t. L. t. L. Promasti- C. major major minor adlern gotes of onco- 60 VI X 7 the agama pe/ti Leishmania L. t. major 60 + + - - - - of mammals L.t.majorVI + + - - - - L. t. minor X - - + Leishmania L. adleri 7 - - - + of reptiles promastigotes of the agama - - - - + C. oncopelti C. oncopelti - - - - - + Promastigotes treated with homologous antisera (Fig. 1-3) showed numerous particles of ferritin on the external surface of the cytoplasmic membrane, indicating the settling of specific antibodies on it. No penetration of ferritin into the internal structures of the cell (the cytoplasmic membrane being undam- aged) was observed in any case. Sections through the body of C. oncopelti treated with homologous antiserum (Fig. 1) showed a con- centration of ferritin granules along the cytoplasmic microtubules located directly under the cytoplasmic membrane, which in some measure apparently reflects the localization of specific antigens. The treatment of leptomonads of different strains of Leishmania and of C. oncopelti with labelled heterologous antisera (Fig. 5-7) yielded negative results. In the electron micrographs the cell mem- branes are seen to be clearly outlined and entirely free of ferritin particles, indicating that no specific antibodies had settled on them. Positive results were obtained (see Table 1) in all the experiments with homologous antisera and in the cross experiments with two different strains of L. tropica major (Fig. 4). In contrast, all the cross experiments with different species and subspecies of Trypanosomatidae (of the same serogroup as well as of different serogroups) invariably yielded negative results. DISCUSSION Our results warrant the conclusion that the immunoferritin test identifies both the serogroup and the species of Leishmania spp. It fails, however, to reveal differences between individual strains within the same species. Unlike the modified Adler test (Saf'janova & Aliev, 1971), the immunoferritin test does not enable the degree of serological relationship among different species and strains of Leishmania to be determined. In cross experiments different species and subspecies belonging to the same serogroup (e.g., L. t. major and L. t. minor) yield a clearly negative result, which is entirely analogous to that obtained in experiments with representatives of different serogroups of Leish- mania (e.g., L. t. major) and even with representa- tives of different genera of the family Trypanosoma- tidae (e.g., L. t. major and C. oncopelti). It is possible that the test is not equally specific for the different representatives of the Trypanoso- matidae. Thus in the experiments of Vickerman & Luckins (1969) on the same strain, T. brucei yielded a negative result in the cross immunoferritin test, indicating a heterology of their surface antigens. However, such a result could in some measure have been expected. By means of an agglutination test it was previously established that the antigen compo- sition of this parasite changes during its life cycle in line with the change in the surface layer of its cytoplasmic membrane (Vickerman & Luckins, 1969). The antigenic properties of Leishmania are appar- ently much more stable. Serological studies on more than 30 strains of different species of Leishmania (isolated from man, rodents, reptiles, and sandflies) conducted at this institute by means of a modified Adler test have shown their antigenic properties to be relatively invariable both in protracted cultivation 3 295 296 V. M. SAF'JANOVA & A. A. AVAKJAN in vitro and in passage through golden hamsters (Saf'janova, 1966, 1970; Saf'janova & V'jukov, 1967; Aliev, 1970, 1971). A firm conclusion on the specificity ofthe immuno- ferritin test for Leishmania must await the accumula- tion of more results. However, consideration of the relative stability of the antigenic properties of Leish- mania warrants the conclusion that the cross-reaction between strains of L. t. major revealed in our experi- ments and the complete absence of cross-reactions with all the heterologous strains indicate the pos- sibility of using the test for differentiating and iden- tifying Leishmania species. The serological and mor- phological criteria obtained by means of the immuno- ferritin method appear to be valuable in the overall approach to this problem. ACKNOWLEDGEMENTS This study was financially assisted by a grant from the World Health Organization. The authors thank graduate students B. A. Koselev and N. Losaberidze for their assistance. RtSUME EMPLOI D'ANTICORPS MARQUES A LA FERRITINE POUR DIFFERENCIER DES ESPECES DE LEISHMANIA ET D'AUTRES TRYPANOSOMATIDAE Afin d'6tudier comparativement l'ultrastructure et les affinites antigeniques d'esp6ces appartenant a la famille des trypanosomatides, on a eu recours a une methode de traitement par des immunserums de lapins marqu6s a la ferritine. On a utilise comme mat6riel des promastigotes de plusieurs especes de Leishmania (L. tropica major (2 souches), L. tropica minor, L. adleri), une souche de promastigotes isolee chez des reptiles (souche Ag) et une souche de Crithidia oncopelti. Tous les flagelles ont fait l'objet d'une serie d'6preuves, chaque souche 6tant traitee par un antiserum marque homologue et par plusieurs antiserums h6t6rologues. La repartition des particules de ferritine a 6te 6tudi6e par microscopie electronique sur des coupes ultraminces. On a obtenu des resultats positifs dans toutes les 6preuves utilisant des antiserums homologues ainsi que dans les epreuves croisees portant sur les deux souches de L. t. major. L'examen des coupes a r6v6l6 nettement la localisation des antigenes par suite de leur interaction avec les anticorps marques. On notait une concentration des particules de ferritine sur la surface externe de la membrane cytoplasmique et le long des microtubules du cytoplasme. Le traitement des souches par des serums marques heterologues a donne des r6sultats negatifs. Ces donnees permettent d'attribuer a l'epreuve de l'immunoferritine le pouvoir d'identifier le serogroupe et l'espece de Leishmania spp., mais non celui de diff6rencier les diverses souches d'une meme espEce. REFERENCES Adler, S. (1963) Israel J. exp. Med., 2, 31-34 Adler, S. (1964) Advanc. Parasit., 2, 35-96 Aliev, P. I. (1970) Med. Parazit. (Mosk.), 39, 293-299 Aliev, -. I. (1971) Med. Parazit. (Mosk.), 40, 191-197 Angelopoulos, E. 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