Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

INVITED REVIEW Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS   Semiramis GUIMARÃES(1), Maria Inês Leme SOGAYAR(1) & Marcello F. de FRANCO(2)     SUMMARY Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon. KEYWORDS: Giardia duodenalis; Review; Antigens; Isoenzymes; Proteases; Nucleic acids.     INTRODUCTION Giardia, a flagellate found in the small intestine of mammals, birds, reptiles and amphibians, was first observed in 1681 by LEEUWENHOEK in his own feces and later by LAMBL in 1859125. The genus Giardia was created by KUNSTLER (1882) when he observed a flagellate in the intestine of tadpoles of amphibian anurans71. Giardia goes through a simple life cycle during which the parasite occurs in two distintic morphological forms, i.e., a trophozoite and a cyst. The trophozoite is piriform, bilaterally symmetrical and measures approximately 10-12 µm in length by 5-7 µm in width. It presents four pairs of flagella and a ventral structure found only in the genus Giardia, denoted ventral disk or adhesive disk, which permits parasite adhesion to the intestinal mucosa of the host. Below the adhesive disk there are two parallel formations denoted median bodies which are found only in the genus Giardia and whose morphology has been used for species determination. With respect to internal organization, the trophozoites present two identical nuclei and no mitochondria. The Golgi complex was recently demonstrated in encysted trophozoites43,105 . The cyst is oval, 8-12 µm long and 7-10 µm wide, and has a 0.3-µm thick outer wall of a glycoprotein nature denoted cystic wall. Internally there are two or four nuclei, flagellar axonemes, ribosomes and fragments of the ventral disk. During the life cycle, the trophozoites adhering to the mucosa of the small intestine multiply by binary division and go through an encystation process. The cysts are excreted together with the feces of the host and may remain viable for several months in the environment. The cyst is the infecting form, which, after being ingested by the host, undergoes excystation in the duodenum and releases two trophozoites which divide by binary division, causing a new infection. Transmission occurs by fecal-oral contamination, with the host becoming infected by ingesting contaminated water and food, or from person to person through contaminated hands or through homosexual relations. Giardia and giardiasis have been extensively studied over the last 15 years but fundamental questions continue to be unanswered despite all efforts. The taxonomy of Giardia is still controversial and species determination has been performed considering the host of origin and some morphological characteristics. Apparently, the host of origin is not a valid criterion since DNA analysis shows that Giardia species from different hosts are identical, whereas isolates from the same host may be markedly different90. On the other hand, many investigators consider the morphology of the median bodies to be the only feature that can be used as a differential criteria12,85. Thus, the genus Giardia can be divided into three species: Giardia duodenalis which infects mammals, birds and reptiles; Giardia muris which infects rodents, birds and reptiles; Giardia agilis which infects amphibians38. In addition to these three species, two others detected in birds in electron microscopy studies have been proposed, i.e., Giardia psittaci32 and Giardia ardeae33, respectively detected in parrots and in blue herons. Giardia lamblia and Giardia intestinalis are used as synonyms of G. duodenalis, especially for Giardia isolates of human origin. According to the norms of zoologic nomenclature, the species G. duodenalis is correct and the use of G. lamblia and G. intestinalis only contributes to increasing the confusion in the scientific literature127. Therefore, in this review, we will use G. duodenalis for all human isolates. The development of axenic culture of Giardia, i.e., the establishment and growth of the parasite in culture media free from other organisms, has facilitated the study of the biology of the parasite3,84. Improved results with the axenic culture of the parasite were obtained after the development of a culture medium supplemented with bovine bile and cysteine66 and of techniques for in vitro excystation permitting the isolation, large-scale production and maintenance of the protozoa in the laboratory13, 14, 65, 78, 108. Most axenic isolates belong to the G. duodenalis group and are of human origin129 . A report of axenic culture of G. muris45 is questionable since this species appears to be refractory to axenization127. Giardiasis presents a cosmopolitan distribution, with 10 to 15% of the world population, on average, being infected. High prevalences ranging from 20 to 60% are commonly observed among children29,44,82,87, especially those living in institutions15, 26, 48, 67, 103 . The parasite can cause diarrhea associated with problems of malabsorption especially among children, leading to delayed growth and development. However, most infections are asymptomatic. This clinical variability has been attributed to factors related to the parasite (mechanical, proteolytic, immunologic), to the host (diet, intestinal motility, nutritional and immunologic status) and to host-parasite interactions (immune response and association with the intestinal microflora). The specific pathogenic mechanisms of Giardia, the factors responsible for the conversion of an asymptomatic infection to a symptomatic one and their impact on the host-parasite relationship still need investigation. In this review we shall present the most important studies on the characterization of G. duodenalis strains in which isoenzyme and protein patterns, protease activity and the antigenic and DNA characteristics of different isolates were investigated using biochemical, immunochemical and molecular techniques. These studies were conducted in order to expand the knowledge of this parasite and to detect differences between strains that might be associated with aspects of the host-parasite relationship and with the heterogeneity of clinical manifestations, immune response and effective treatment of human giardiasis.   ELECTROPHORETIC ANALYSIS OF PROTEINS Most studies of giardiasis have used sodium duodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) for the analysis of the pattern of proteins present in various types of Giardia strain preparations. The knowledge of the composition and complexity of (...truncated)