Further enzymic characters of Trypanosoma cruzi and their evaluation for strain identification

Starch-gel electrophoresis of 38 enzymes was attempted with extracts of Trypanosoma cruzi culture forms. 18 of the enzymes that gave discrete electrophoretic bands were selected for routine characterization of T. cruzi stocks; the enzymes were: aspartate aminotransferase (E.C. 2.6.1.1, ASAT); alanine aminotransferase (E.C.2.6.1.2, ALAT); phosphoglucomutase (E.C.2.7.5.1, PGM); glucosephosphate isomerase (E.C.5.3.1.9, GPI); malate dehydrogenase (oxaloacetate decarboxylating) (NADP⁺) (E.C.1.1.1.40, ME); glucose 6-phosphate dehydrogenase (E.C.1. 1.1.49, G6PD); malate dehydrogenase (E.C.1.1.1.37, MDH); aconitate hydratase (E.C.4.2.1.3, ACON); isocitrate dehydrogenase (NADP⁺) (E.C.1.1.1.42, ICD); alcohol dehydrogenase (NADP⁺) (E.C.1.1.1.2, ADH); lactate dehydrogenase (E.C.1.1.1.27, LDH); aminopeptidase (cytosol) (E.C.3.4.11.1, PEP); pyruvate kinase (E.C.2.7.1.40, PK); phosphoglycerate kinase (E.C.2.7.2.3, PGK); enolase (E.C.4.2.1.11, ENO); hexokinase (E.C.2.7.1.1, HK); mannosephosphate isomerase (E.C.5.3.1.8, MPI); and glutamate dehydrogenase (E.C. 1.4.1.2, GD). ADH (NADP⁺) in the genus Trypanosoma, and PGK, MPI and ENO, in T. cruzi, were apparently demonstrated for the first time.Between six and 18 enzymes were used to characterize more than 250 T. cruzi stocks, newly isolated from a wide range of sources in northern and central Brazil. All stocks were identified as belonging to T. cruzi zymodemes 1, 2 or 3, as originally defined—that is, by combination of electrophoretic patterns of ASAT, ALAT, PGM, GPI, ME and G6PD. The composite range of results with all enzymes confirmed the presence of three principal T. cruzi zymodemes, but some enzymic characters overlapped between zymodemes and others suggested subgroups within individual zymodemes. Seven (MDH, ACON, LDH, PK, PGK, ENO, HK) of the 18 enzymes did not distinguish the three zymodemes; five (ASAT, PGM, GPI, ICD, PEP) distinguished all three zymodemes; 10 (ASAT, ALAT, PGM, GPI, ME, G6PD, ICD, ADH, PEP, GD) distinguished zymodemes 1 and 2, of which seven plus MPI and eight plus MPI separated zymodemes 1 from 3 and 2 from 3 respectively. T. cruzi stocks were taken from a small area of the natural species distribution; the full range of enzymic characters within the species T. cruzi is expected to be far more complex.The epidemiological distribution of the zymodemes continued to accord with local transmission cycles and supported the hypothesis that distinct T. cruzi strains might be responsible for the enigmatic distribution of chronic Chagas's disease.Some of the difficulties in the empirical selection of new electrophoretic methods and the interpretation of results were presented, and the present and prospective significance of T. cruzi enzymic characters was discussed.Until the stability and genetic basis of T. cruzi enzymic characters are better understood it is recommended that isoenzymic profiles be confirmed routinely, both before and after stocks are used experimentally, as representative of a given zymodeme. A multiple biochemical approach to T. cruzi strain identification is recommended, using characters suitable for a numerical taxonomy.     

Leishmania in the Old World: 2. Heterogeneity among L. tropica zymodemes

Isoenzyme profiles of 27 stocks of Leishmania tropica from widely separated geographical areas were compared with those of reference strains of L. tropica and L. major using starch-gel electrophoresis of 13 enzymes (GPI, GD, ES, PGM, PEPD, NH, ASAT, ALAT, PK, MPI, 6PGD, SOD, MDH). 18 zymodemes were seen. L. tropica showed considerable intraspecific variation which did not correlate with its epidemiological uniformity. Isolates from cases of cutaneous and visceral leishmaniasis and leishmaniasis recidivans were identified as L. tropica. Only one isoenzyme band was held in common with the enzyme profile of the L. major reference strain thus supporting the status of L. tropica as a separate species.     

Aspartate aminotransferase in Leishmania is a broad-spectrum transaminase

The substrate specificity of aspartate aminotransferase (ASAT, E.C. 2.6.1.1.) from Leishmania was examined following observations of artefacts on gels stained for alanine aminotransferase (ALAT, E.C. 2.6.1.2.) after thin-layer starch-gel electrophoresis. Leishmanial ASAT acted on L-aspartate, L-alanine, L-tryptophan and L-tyrosine. Interpretation of ALAT zymograms must thus take into account the presence of interfering ASAT bands, and the need is emphasized for rigorous controls in isoenzyme electrophoresis.     

Leishmania in the Old World: 3. The distribution of L. aethiopica zymodemes

Isoenzyme profiles of 28 stocks of Leishmania aethiopica were compared with those of reference strains of L. aethiopica, L. tropica and L. major using starch-gel electrophoresis of 13 enzymes (GPI, GD, ES, PGM, PEPD, NH, ASAT, ALAT, PK, MPI, 6PGD, SOD, MDH). 13 zymodemes were seen. L. aethiopica showed some infraspecific variation. Stocks from Phlebotomus longipes and Procavia habessinica were indistinguishable from those from man. Stocks from cases of diffuse cutaneous and cutaneous leishmaniasis were indistinguishable. Only one enzyme pattern was held in common with the L. tropica reference strain enzyme profile, and none with the L. major reference strain. The status of L. aethiopica as a separate species is supported.     

Leishmaniasis in Brazil: XV. Biochemical distinction of Leishmania mexicana amazonensis,L. braziliensis braziliensis and L. braziliensis guyanensis—aetiological agents of cutaneous leishmaniasis in the Amazon Basin of Brazil

Enzymic profiles of the three known agents of human cutaneous leishmaniasis in the lower Amazon region are compared. Of 14 enzymes, 10 (ASAT, ALAT, GPI, G6PD, MDH, ACON, PEP, HK, MPI and ACP) differentiate Leishmania mexicana amazonensis from L. braziliensis braziliensis or L. braziliensis guyanensis: this supports their taxonomic status as distinct species. In contrast, only slight mobility differences of four enzymes (ASAT, ALAT, PGM, MPI) separate L. b. braziliensis and L. b. guyanensis, which are distinguished biochemically for the first time: this indicates that they are closely related.Four stocks of L. b. panamensis correspond with L. b. guyanensis on mobilities of 10 enzymes (ASAT, ALAT, PGM, GPI, G6PD, MDH, PK, HK, MPI, ACP), although these two subspecies are known to be separable by kinetoplast DNA buoyancies and the enzyme 6PGDH.The generation of practical, regional biochemical keys to the medically important leishmanias is discussed.     

Leishmania in the Old World: 1. The geographical and hostal distribution of L. major zymodemes

135 stocks of Leishmania major from man, reservoir hosts and sandflies were characterized using thin-layer starch-gel electrophoresis of 13 enzymes: MDH, 6PGD, GD, SOD, ASAT, ALAT, PK, PGM, ES, NH, PEPD, MPI, GPI. Homogeneity in this species was demonstrated by identical electrophoretic mobilities in nine enzymes. Polymorphism in four enzymes: 6PGD, GPI, PEPD, ES, gave six zymodemes among the collection. Stocks from sandflies and several species of burrowing rodents were indistinguishable from those from man in the same areas. Stocks of Leishmania from North-West India were identified as L. major. In some foci the distribution of zymodemes has some correlation with the presence of particular rodent reservoir hosts. The enzymic homogeneity of L. major throughout its geographical and host range appears to be correlated with the close association between L. major and sandflies of the subgenus Phlebotomus. The status of L. major as a distinct species is supported.     

Leishmania in the Old World: 4. The distribution of L. donovani sensu lato zymodemes

Isoenzyme profiles of 67 stocks of Leishmania donovani sensu lato from across the Old World were compared with those of reference strains of L. donovani sensu stricto, L. infantum, L. major, L. tropica and L. aethiopica using starch-gel electrophoresis of 13 enzymes (GPI, GD, ES, PGM, PEPD, NH, ASAT, ALAT, PK, MPI, 6PGD, SOD, MDH). 12 zymodemes were seen. Isolates from man, Canis familiaris, Vulpes vulpes, Rattus rattus, Arvicanthis sp. and Phlebotomus martini were examined. Several zymodemes comprised stocks from man and C. familiaris, two of which also included wild animal isolates. Isolates from cases of post-kala-azar dermal leishmaniasis and visceral leishmaniasis were indistinguishable. L. donovani s.l., including L. donovani s.s. and L. infantum, formed a coherent group with a striking degree of enzymic homogeneity. Only one enzyme pattern was held in common with the L. tropica and L. aethiopica reference strains and two with the L. major reference strain enzyme profile.     

Characterization of Leishmania spp. from Kuwait by isoenzyme electrophoresis

Leishmania stocks isolated from skin lesions of 26 patients in Kuwait were compared among themselves and with Leishmania stocks collected from other parts of the Old and New Worlds on the basis of their isoenzyme patterns for seven enzymes by means of thin layer starch gel electrophoresis. The enzymes examined were: alanine aminotransferase (ALAT) E.C.2.6.1.2; aspartate aminotransferase (ASAT) E.C.2.6.1.1; glucose-phosphate isomerase (GPI) E.C.5.3.1.9; glucose-6-phosphate dehydrogenase (G6PD) E.C.1.1.1.49; malate dehydrogenase (MDH) E.C.1.1.1.37; malic enzyme (ME) E.C.1.1.1.40 and phosphoglucomutase (PGM) E.C.2.7.5.1.The isoenzyme patterns obtained fell clearly into 11 groups for the stocks of Leishmania tested. The Kuwaiti stocks separated into six groups. The patterns obtained with 15 Kuwaiti stocks were identical with those obtained with Leishmania tropica major (identified on clinical and geographical characteristics); seven stocks were identical with L. tropica minor similarly identified; three stocks had isoenzyme patterns different from each other and from all other leishmanias examined in this study; and one stock gave isoenzyme patterns identical with those of an L. donovani isolated in the Sudan.The isoenzyme patterns were the same in three stocks classified as L. tropica minor, L. aethiopica and a Leishmania isolated in Baghdad which caused a visceral disease in rats.     

A genetic comparison between Brazilian and Bolivian zymodemes of Trypanosoma cruzi

The enzyme profiles of three major Brazilian Trypanosoma cruzi zymodemes (Brazilian Z1, Brazilian Z2, Brazilian Z3) and of two principal Bolivian zymodemes were compared. Relationships were assessed both intuitively and by calculating genetic distances. One of the Bolivian zymodemes, Bolivian Z1, was closely related to the Brazilian Z1. The second Bolivian zymodeme, Bolivian Z2, was related to Brazilian Z2 but differed from all the Brazilian zymodemes in the occurrence of typical heterozygous isozyme patterns in five out of 12 enzyme loci. Parental stocks and clones of Bolivian Z2 had the same putative heterozygous patterns. The evidence from enzyme profiles on the ploidy of T. cruzi and the possibility of recombination was considered. The presence of putative heterozygous patterns in Bolivian Z2 supported the hypothesis that T. cruzi is diploid. The definition of T. cruzi as a single polytypic species or as a species complex was considered to be dependent on the presence or absence of genetic recombination between or within the zymodemes, which has not been demonstrated in the ecotopes so far examined.     

Chagas's disease in the Amazon Basin: II. The distribution of Trypanosoma cruzi zymodemes 1 and 3 in Pará State,north Brazil

In Pará State, Brazil, 123 Trypanosoma cruzi stocks were isolated from 12 silvatic mammal species, five silvatic triatomine species and individuals with acute Chagas's disease. 100 T. Cruzi stocks were identified as zymodeme (Z) 1, 17 as Z3 and 6 as Z3 with Z1 ASAT character, but none were T. Cruzi Z2. Z1 was predominantly isolated from arboreal mammals, especially Didelphis marsupialis; Z3 was mainly found in terrestrial or burrowing mammals, particularly Dasypus novemcinctus and Monodelphis brevicaudata. It is not clear whether gene exchange occurs between the groups designated as zymodemes but the enzymic “distance” between T. Cruzi Z1, Z2 and Z3, their different geographical distributions, host associations and local transmission cycles support the view that these zymodemes represent taxonomic units of fundamental epidemiological significance. T. cruzi (Z1) was isolated for the first time from the silky anteater (Cyclopes didactylus).     

Leishmaniasis in Brazil. XXII: Characterization of Leishmania from man, dogs and the sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) isolated during an outbreak of visceral leishmaniasis in Santarém, Pará State

During epidemiological studies on an outbreak of visceral leishmaniasis in Santarém, Pará State, north Brazil, isolates of Leishmania from two children, three dogs and six naturally infected specimens of the sandfly Lutzomyia longipalpis were compared, biochemically, by starch-gel enzyme electrophoresis. They have proved to be indistinguishable from each other, and from a reference strain of Leishmania chagasi Cunha & Chagas, 1937 from a case of human visceral leishmaniasis from Bahia State, north-east Brazil, on their enzyme profiles for ASAT, ALAT, PGM, GPI, MDH and MPI. Lu. longipalpis is the principal, and possibly the only vector to man in the Amazon Region of Brazil.     

Leishmaniasis in Brazil: XVII. Enzymic characterization of a Leishmania from the armadillo,Dasypus novemcinctus (Edentata), from Pará State

A comparison of enzyme profiles, by starch-gel electrophoresis, has distinguished a Leishmania of armadillos (Dasypus novemcinctus), from Pará State, north Brazil, from Leishmania braziliensis braziliensis, L. braziliensis guyanensis, L. mexicana amazonensis, L. donovani sensu lato (from Bahia, Brazil), and L. hertigi deanei. The parasite was separated from L. b. braziliensis and L. b. guyanensis by 8 of the 14 enzymes used (ASAT, ALAT, PGM, GPI, G6PD, PEP, MPI and GD), although differences in the mobility of some of the enzymes were small. At least 9 of the enzymes separated the organism from L. m. amazonensis, L. donovani s.l., and L. h. deanei.     

Leishmaniasis in Brazil: XVIII. Further evidence incriminating the fox Cerdocyon thous (L) as a reservoir of Amazonian visceral leishmaniasis

Major endemic areas of visceral leishmaniasis in Brazil are located in the drier, poorly forested regions, principally in the northeastern States such as Ceará and Bahia. Cases of the human disease in the Amazon Region are rare, very sporadic, and seldom present opportunities for epidemiological study. Following the report of a fatal case near Salvaterra, the Island of Marajó, Pará State, a preliminary investigation has resulted in the isolation of a parasite regarded as Leishmania donovani chagasi from the viscera and skin of an apparently healthy fox, Cerdocyon thous, captured in the same locality. This represents the third recorded isolation of the parasite from this species of fox in the Amazon Region. The inapparent nature of the infections supports the suggestion that this canid may represent the primitive natural host of L. d. chagasi. C. thous is commonly associated with forested or wooded areas, and enzymic profiles for the enzymes ASAT, ALAT, PGM, GPI, MDH, MPI, G6PD, PEP and ACP failed to distinguish an isolate of L. d. chagasi from this animal in Pará from others obtained from cases of human visceral leishmaniasis in the neighbouring States of Maranhão, Ceará and Bahia. This suggests that the major, present-day endemics may have originated from a primary silvatic enzootic.     

The identification by isoenzyme patterns of two distinct strain-groups of Trypanosoma cruzi, circulating independently in a rural area of Brazil.

Culture forms of 17 Trypanosoma cruzi stocks¹, primarily isolated from a rural area of endemic Chagas' disease at São Felipe, Bahia, Brazil, were compared by the electrophoretic patterns of six enzymes: aspartate aminotransferase, alanine aminotransferase, glucose-6-phosphate dehydrogenase, malate dehydrogenase (decarboxylating) (NADP⁺), glucosephosphate isomerase and phosphoglucomutase. Two markedly distinct combinations of isoenzyme patterns were seen, justifying the arrangement of the 17 stocks into two strain-groups, each of which was enzymically homogeneous. One combination was characteristic of the 11 domestic stocks of T. cruzi derived from both human infections and domiciliated animals; the second was characteristic of the six sylvatic stocks derived from opossums and a sylvatic triatomine species. The enzyme patterns were independent of the original host and the type of culture medium used. Distinction of the two strain-groups accords with epidemiological evidence that the domestic and sylvatic transmission cycles in São Felipe do not overlap. It is suggested that the diverse enzyme characters of the two strain-groups circulating in São Felipe reflect diverse origins; the domestic form of T. cruzi probably invaded the area from the south of Brazil with the domestic triatomine vector, Panstrongylus megistus.     

The presence in Bolivia of two distinct zymodemes of Trypanosoma cruzi, circulating sympatrically in a domestic transmission cycle

The enzyme profiles of 109 Bolivian stocks of Trypanosoma cruzi were determined by cellulose acetate electrophoresis using the four enzymes: malate dehydrogenase (oxaloacetate decarboxylating) (NADP+) (E.C.1.1.1.40, ME), phosphogluconate dehydrogenase (E.C.1.1.1.44, 6PGDH), phosphoglucomutase (E.C.2.7.5.1, PGM) and glucosephosphate isomerase (E.C.5.3.1.9, GPI). As previously, two principal zymodemes were found sympatrically. Both were isolated from man, one appeared to be more frequent at high altitude, the other appeared to be more frequent at low altitude. These results agree with our previous hypothesis on the genetics of T. cruzi (diploidy, lack of actual sexuality).     

Influence of geographical factors in the distribution of pathogenic zymodemes of Entamoeba histolytica: identification of zymodeme XIV in India

Stocks of Entamoeba histolytica isolated and maintained in a variety of culture media, both axenic and polyxenic, were compared with each other and with strains previously characterized. The present stocks were isolated from subjects living in various cities in India. Using the enzyme patterns of: E C 5319 glucose phosphate isomerase (GPI); E C 11140 L-malate: NADP+ oxidoreductase (oxaloacetate decarboxylating) (ME): E C 2751 phosphoglucomutase (PGM); and E C 2711 hexakinase (HK), developed after electrophoresis, three zymodemes were identified in 54 individual isolations of E. histolytica. Most importantly only one zymodeme associated with pathogenicity occurred, identified 28 times, among the collection, one of which was from a liver abscess.     

The isolation and isoenzyme characterization of Leishmania braziliensis subsp. from patients with cutaneous leishmaniasis acquired in Belize

Leishmanial organisms were cultivated from cutaneous lesions of British military personnel returning from Belize. Isoenzyme profiles of the freshly isolated organisms and 'marker' strains of New World Leishmania spp. were compared using 10 enzymes (ALAT, ASAT, ME, GPI, MPI, PGM, SOD, 6-PGDH, G-6-PDH and MDH), by starch gel electrophoresis. 19 of the 22 new isolates from Belize were isoenzymically indistinguishable from Leishmania braziliensis braziliensis (10 out of 10 enzymes) and clearly differentiated from L. b. guyanensis and L. b. panamensis (different in 6 out of 10 enzymes) and from L. mexicana mexicana and L. m. amazonensis (9 out of 10 enzymes). Two isolates closely resembled L. m. mexicana and one could not be positively identified. This is the first report of autochthonous human leishmaniasis caused by L. braziliensis group organisms as far north as latitude 16 degrees N.     

Delimitation of Trypanosoma cruzi zymodemes by numerical taxonomy

Based on the absence or presence of 24 isoenzyme characters, Brazilian Trypanosoma cruzi stocks can be separated by methods of numerical taxonomy into three principal, distinct groups; these groups correspond to the three T. cruzi zymodemes previously reported from north Brazil. A distinction is drawn between taxonomy, which may be of zoological interest only, and identification of medically important groups according to one or a few distinguishing characters.     

Enzyme electrophoresis in characterizing the causative organism of Gambian trypanosomiasis

Trypanosoma (Trypanozoon) brucei includes three morphologically identical subspecies which are poorly defined by clinical behaviour; T. b. brucei does not infect man, whereas T. b. rhodesiense causes an acute, and T. b. gambiense a chronic, disease. Thirty-three isolates of the complex, each of which had previously been identified on clinical or other criteria, were compared by the electrophoretic patterns of two trypanosomal enzymes, alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT). One particular ALAT pattern clearly segregated a group of human pathogens of which all except one were labelled T. b. gambiense. The exception was labelled T. b. rhodesiense, and in addition three putative T. b. gambiense isolates did not have this pattern; it is suggested that only one presents a serious anomaly. The T. b. gambiense group could also be subdivided by three ASAT patterns which coincided with known groupings based on serological criteria.     

Epidemiological aspects of three Trypanosoma cruzi zymodemes in Bahia State,Brazil

Culture forms of 104 stocks of Trypanosoma cruzi isolated in different regions of the State of Bahia were compared by electrophoresis of six enzymes. The three distinct combinations of isoenzyme patterns seen were designated Z1, Z2 and Z3. In an area of endemic Chagas's disease in eastern Bahia, T. cruzi Z1 was associated with sylvatic mammals and sylvatic triatomines, whereas T. cruzi Z2 was associated with a separate domestic cycle of transmission. T. cruzi Z1 was also found in sylvatic triatomines from other parts of the State. In contrast, in an area of the São Francisco Valley region of western Bahia, both T. cruzi Z1 and Z2 were isolated from man, domestic animals, and peridomestic rats. T. cruzi Z3 was isolated from an armadillo and from Panstrongylus geniculatus, a triatomine commonly found in armadillo burrows.Both T. cruzi Z1 and Z2 appeared to be pathogenic in man: T. cruzi Z1 was isolated from patients with acute Chagas's disease and from a single patient with chronic cardiac manifestations. T. cruzi Z2 was isolated from some asymptomatic individuals but was also associated with acute disease and chronic cardiac and digestive syndromes.