Trypanosoma cruzi is a flagellate of the Kinetoplastida Order, Family Trypanosomatidae, characterized by the presence of one flagellum and a single mitochondrion in which is situated the Kinetoplast, a specialized DNA-containing organelle. The identification of this parasite by morphological and biological features does not offer difficulties and differential is only required for Trypanosoma rangeli, a non-pathogenic flagellate which infects humans in some areas of Central and South America and is transmitted by some of the same vectors that transmit T. cruzi.
T. cruzi is not a homogeneous population and is composed rather by a pool of strains which circulate in both the domestic and sylvatic cycles involving humans, vectors and animal reservoirs of the parasite. Isolation and study of T. cruzi populations from different origins demonstrated the presence of a large range of strains with distinct characteristics. This intriguingly intraspecific variation has been extensively investigated by biological characterization which includes morphology of blood forms, curves of parasitemia, virulence, pathogenicity and sensitivity to drugs. The antigenic make-up of different population has been studied by analysis of radioiodinated cell surface membrane components and band distribution in SDS-Page gel electrophoresis as well as monoclonal antibodies. Cross-reacting as well as strain specific antigens have been detected.
As strain traits may be influenced by environmental conditions or laboratory handling, new approaches have been recently introduced for T. cruzi characterization at the molecular level. Thus, lectins with different specificities for surface membrane carbohydrate residues were used for taxonomic purpose. Isoenzymes (different molecular forms of a same enzyme detected by eletrophoresis) are being extensively used to classify T. cruzi. Different zymodemes (a group of strains sharing the same isoenzyme pattern for a set of enzymes) have been detected in strains isolated from various hosts and geographical regions permitting to type the populations. Eletrophoresis of Kinetoplast DNA minicircles fragmented by restriction-endonucleases allowed the identification of different patterns or "schizodemes". When large numbers of T. cruzi strains are processed quantitative and qualitative differences in these patterns are observed. An extensive review is discussed eslewhere.
Kineplast DNA minicircles from T. cruzi display conserved and variable regions. Where molecular probes of the conserved DNA sequences are being used for the detection of the parasite in blood or vectors, probes of the highly variable regions of the minicircles are being used to group subpopulation of T. cruzi. The high variable DNA sequences can be amplified by PCR ("polymerase chain reaction") giving origion to probes to be used in the typing of a large number of T. cruzi strains.
The available diversified methods used to type or group T. cruzi strains may face drawbacks that interfere with the results. They are, for instance, the isolation of T.cruzi populations from hosts that harbor more than a single strain; the selection of populations during the maintenance of strains in laboratory conditions or during their amplification in experimental animals or in cultures; the mismanage and/misname of parasite populations in the laboratory. Nevertheless, some authors succeeded on grouping T. cruzi strains using a combination of parameters like parasite morphology, parasitemia and parasite tissue distribution. Moreover, isoenzyme studies have detected strain makers that in some circumstances permitted to trace back the flow of T. cruzi populations among the domestic and sylvatic infection cycles.
T. cruzi evolves during its cycle into different forms which are identified by the relative position of the Kinetoplast in relation to the cell nucleus and the flagellum emergence. In the trypomastigote stages the Kinetoplast is at the posterior end of the parasite and therefore of the nucleus; the flagellum emerges from the flagellar pocket which is located near by the Kinetoplast. In epimastigotes stages the Kinetoplast and the flagellar pocket are found in a position anterior to the nucleus. Finally, the amastigote stages are rounded stages displaying at eletron microscopy a short inconspicuous flagellum; these stages multiply intracellularly in the host cells.
As previously described T. cruzi has a single tubular mitochondrion which shares with the similar organelle from mammalian cells some features as the presence of DNA, cristae and a number of enzymes detected in its interior membrane. The Kinetoplast, a fibrous network of DNA which constitutes 20-25% of the total parasite DNA is located at the mitochondrion. Electron microscopy studies have shown that the K-DNA molecules are organized as associated minicircles and maxicircles. Although each Kinetoplast comprises 20.000-25.000 minicircles, the role played by the K-DNA has not been established. However, evidence from the number of base pairs and the coding capability of the mincircles suggest that they would only translate small proteins whose existence and importance have not been yet disclosed. Maxicircles, however, owing to their size and molecular weight are likely to code for enzymes which participate of the parasite metabolism. Whatever its role, K-DNA seems to be essential for the parasite viability. Drug-included "dyskinetoplastic" T. cruzi forms (developmental stages lacking the Kinetoplast) are unable to perform a normal cycle. There is evidence that in other trypanosome species in which "dyskinetoplastic" stages are regularly found in nature as Trypanosoma equiperdum, T. evansi, the extra-nuclear DNA is apparently distributed in the mitochondrion.
The size and shape of the Kinetoplast is variable in the different developmental stages. In trypomastigotes it displays a basket-like shape due to a peculiar arrangement of the DNA loops in several layers whereas in epi-and amastigotes is presented as a rod-like aspect.
The T. cruzi flagellum is connected to the basal body and emerges from a specialized invagination, the flagellar pocket which is apparently involved in the ingestion and uptake of nutrients of the external medium. The antique concept that the adhesion of the flagellum to the cell body was due to the existence of a virtual membrane has been abandoned but the mechanism of this connection is so far uncertain. Recent observations by electron microscopy and freeze fracture strongly suggest that particle clusters distributed at regular spaces or in a linear array observed in the flagellum and the body surface membrane of trypomastigotes may represent "rudimentary desmosomes" which participate in the adhension process.
Another characteristic of trypanosomatids in general is the presence of subpellicular microtubules which are organized as a cytoskeleton of the organism but also plays a role in other more complex functions such as the process of cellular differentiation, motility and tissue migration. Very little is known on the structure of T. cruzi cytoskeleton but recently actin filaments and a - and b - tubulins associated to the microtubules had been reported. Interestingly, the flagellar pocket is deprived of subpellicular microtubules but at this region are present pinocytotic vesicles which incorporate macromolecules and other substances from the external medium and the host cell cytoplasm. The cytostome, an invagination of the surface membrane, is another region in which incorporation of pinocytotic vesicles and larger particles occurs. In addition to these structures and organelles above described which are more specific to trypanosomatids, this group of protozoa shares with the eucariotic cells the endoplasmic reticulum, ribosomes and Golghi complex. The presence of peroxisomes, defined as membrane-bound cytoplasmatic organelles containing enzymes such as catalase and oxidases, have been reported on T. cruzi, however, their activity seems to be significantly lower than in mammalian cells, probably because of the lower concentration of the enzymes. A comprehensive review of the morphology and biology of T. cruzi, including organelle functions, has been published by De Souza.
Because, T. cruzi has to accomplish an intracellular cycle in the vertebrate host which implicates in an interaction with a number of different cells, and since a strong immune response is present in the infected host mostly induced by surface antigens, the plasma membrane of T. cruzi has been intensively investigated in the last years in many laboratories. The large number of already known surface macromolecular complexes, glycoproteins, polysaccharides and lipids, relevant for diagnosis, immuneprotection, immunopathology and other aspects will be reported elsewhere in this book. Surface membrane components of T. cruzi involved in cell recognition and interiorization will be discussed later in this chapter.
Differences in the T. cruzi blood trypomastigotes have been described by Chagas in 1909 in his classical paper on the discovery of Chagas disease. More recent investigations revealed the existence of different morphological patterns of blood forms which are correlated to biological characteristics. Studies carried out with the two "polar" T. cruzi strains Y and CL shows that infected animals display parasitemia in which predominate, respectively, slender and broad trypomastigotes. Moreover, striking differences are observed in the curves of parasitemia induced by both strains in experimental hosts as mice, dogs and rabbits. In experimentally infected mice the Y strain exhibits a tropism for cells from the mononuclear phagocytic system as demonstrated by the peculiar parasitism of macrophages from spleen, liver and bone marrow; in contrast, the CL strain induces a negligible parasitism of these cells. As both strains infect muscle cells, the concept of "macrophagic" and "non-macrophagic" strains has been suggested to characterize their tropism. In vitro experiments with mouse peritoneal resident macrophages confirmed the in vivo findings In addition, blood forms of the Y strain (but not CL strain) collected from infected mice at the acute phase are readily lysed by complement via membrane-bound specific immunoglobulins. The resistance of CL strain to this complement-mediated lysis strongly suggests that its blood forms are equipped with evasion mechanisms that are lacking in the Y population (review: Brener).