M. Póvoa

Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Leishmania hertigi

30 Brazilian stocks of Leishmania mexicana amazonensis and 13 stocks of subspecies of Leishmania hertigi were characterized by starch-gel electrophoresis, using 18 enzymes selected from a total of 36 investigated. L. m. amazonensis was separable from subspecies of L. hertigi by enzymic profiles of 11 enzymes. The L. m. amazonensis stocks, which were from a wide range of hosts in a large geographical area, were enzymically extremely homogeneous, and could only be subdivided on two enzymes; sub-groups did not relate to each other or to any differences in epidemiological characters, including the clinical form of the human disease. 12 stocks regarded as L. hertigi deanei, that were isolated from Coendou prehensilis prehensilis and Coendou sp. in Pará State, Brazil, were separable into two sub-groups by three enzymes. A single stock of L. hertigi hertigi from Panama was separable from both enzymic sub-groups of L. h. deanei, in each case by three enzymes. The significance of these and other characters of diversity is discussed, together with the use of enzymes for the identification of the leishmaniae.

Chagas' disease in the Amazon Basin IV: Host records of Trypanosoma cruzi zymodemes in the States of Amazonas and Rondonia, Brazil

Of 151 Trypanosoma cruzi stocks from 117 different individual hosts collected in the states of Amazonas and Rondonia, 147 from 113 hosts were identified as zymodeme 1 (Z1). These included T. cruzi stocks from three marsupial species, two rodent species and three triatomine species although most were from the common opossum, Didelphis marsupialis. One T. cruzi stock from Rhodnius robustus was identified as Z1 with a Z3 PGM character, one from Sciurus sp. as Z3 and two from Monodelphis brevicaudata and Panstrongylus geniculatus as Z3 with a Z1 ASAT character. The ways in which stocks were isolated and grown up in vitro did not influence isozyme profile. These results support earlier evidence from Pará State that Z2 is absent from the Amazon basin and that the distribution of T. cruzi zymodemes in this region is quite different to that in endemic areas on the south of the continent.

A comparison of electrophoretic methods for isoenzyme characterization of trypanosomatids. I: Standard stocks of Trypanosoma cruzi zymodemes from northeast Brazil

Abstract An investigation of the relative merits of cellulose acetate electophoresis (CAE) and starch-gel electrophoresis (SGE) was made for 18 enzymes of T. cruzi using standard stocks of zymodemes Z1, Z2 and Z3. The 18 enzymes were those shown previously to be the most suited to routine screening of T. cruzi on starch-gel, namely, 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.G.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); mannose phosphate isomerase (E.C.5.3.1.8. MPI); and glutamate dehydrogenase (E.C.1.4.1.2. GD). Of these MDH and PEP failed to give satisfactory patterns on CAE. The cellulose acetate zymograms of the other 16 enzymes were as good as, and in some cases better than, those of starch. Increased CAE resolution for ME and G6PD enabled all three zymodemes to be distinguished. Single CAE bands replaced double SGE bands in some cases, and vice versa, without affecting the zymodeme classification. It was concluded that CAE and SGE were both suitable for isoenzyme characterization and were complementary to each other. CAE characterization of T. cruzi was recommended for use in field work and simple laboratories because of its simplicity, transportability, low maintenance requirements and low capital expenditure. Isoelectric focusing (IEF) of ASAT, ALAT, GPI and PGM on Ampholine PAG plates gave poor results, in our hands, and was considered impracticable for routine characterization of T. cruzi.

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, G5PD, 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.

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.

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.

Anion-exchange separation for neotropical trypanosomes: a preliminary trial and a description ofTrypanosoma devei from the tamarinSaguinus midas niger

Anion-exchange separation trials using DEAE-cellulose columns were performed with blood from two single species of marsupial and edentate, three species of rodent and single species of carnivore, primate, cayman and lizard.Trypanosoma cruzi was isolated fromDidelphis marsupialis, Dasypus novemcinctus andCoendou sp.T. (Megatrypanum) devei was isolated from the tamarinSaguinus midas niger and the mensural characters of the organism were redescribed. Anion-exchange separation was considered to be a valuable procedure for the taxonomist searching for new or little-known trypanosomes.