Use of an Iron Superoxide Dismutase Excreted by Trypanosoma Cruzi in the Diagnosis of Chagas Disease: Seroprevalence in Rural Zones of the State of Queretaro, Mexico

Abstract

Four iron superoxide dismutase (SODI, SODII, SODIII, and SODIV) activities with pI values of 6.9, 6.8, 5.25, and 3.8, respectively, were isolated from epimastigote forms of the Maracay strain of Trypanosoma cruzi cultivated at 28 degrees C in Grace's medium supplemented with 10% heat-inactivated fetal bovine serum. The activity of SODe (pI 3.8), which coincides with that of SODIV, was detected in Grace's medium without serum in which T. cruzi epimastigotes were cultivated for 24 hours at 28 degrees C. SODe, which was excreted into the medium by the parasite, was immunogenic and antibodies to SODe were detected in serum to a dilution of 1:2,500 by Western blot. The role of SODe is related to the establishment of the parasite within the host, and its high immunogenicity and specificity make it a useful molecular marker in diagnosing infection with this parasite. To validate a Western blot result using SODe as a antigen fraction, 1,029 sera of individuals from 11 municipalities in the state of Queretaro, Mexico were analyzed. Sampling was done randomly and results were compared with those for the same sera with three conventional serologic methods: an enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination (IHA), and an indirect immunofluorescence assay (IFA) to detect antibodies to T. cruzi SODe. Samples that were positive by these three techniques were also positive by the Western blot method. The seroprevalence values for SODe (8.16% by ELISA and Western blot) in Queretaro were considerably higher than those reported in regions of Mexico considered to be endemic for Chagas disease. These results support the use of SODe in the serodiagnosis of Chagas disease.

Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

“…Each peak was further purified separately by filtration chromatography, giving rise to a total of four SOD activities [called SOD I, II, III and IV, according to the nomenclature used by Villagrán et al (2005)]. This is consistent with the results revealed by IEF since the P85 fraction showed four bands of SOD activity.…”

“…The SOD activity identification is possible only under the native conditions of the protein; thus, we have detected four different isoenzymes by IEF 3-9 and native electrophoresis. According to Villagrán et al (2005), we have found different pI: 6.9, 6.8, 5.2 and 3.8 for SOD I, SOD II, SOD III and SOD IV, respectively. The pIs ob- Fig.…”

“…To date, the presence of an iron-containing SOD activity has been reported in numerous trypanosomatids, including T. cruzi , Temperton et al 1997, Villagrán et al 2005, Trypanosoma brucei (Dufernez et al 2006, Wilkinson et al 2006, different species of the genus Leishmania (Leishmania aethiopica, Genetu et al 2006, Leishmania chagasi, Paramchuk et al 1997, and Leishmania donovani, Bogdan et al 1990, Crithidia fasciculata (Le Trant et al 1983) and Phytomonas spp. (plant trypanosomatid) (Marín et al 2004), playing an important role in the survival and growth of the parasites.…”

“…In a previous study, we detected four SOD activities in epimatigote forms of T. cruzi, for which different isoelectric points (pI) are known (Villagrán et al 2005), but these have been neither purified nor biochemically characterized. Thus, this is the first study to focus on the purification and biochemical characterization of SODs in T. cruzi.…”

Identification of excreted iron superoxide dismutase for the diagnosis of Phtytomonas

“…Extracellular SODs (EcCu/ZnSODs) have been evidenced in invertebrates, in several species of nematodes, such as, Brugia pahangi (Tang et al 1994), Onchocerca volvulus (James et al 1994), and Caenorhabditis elegans (Fujii et al 1998) and the crustacean Callinectes sapidus (Brouwer et al 2003). Recently, Villagrán et al (2005) have shown the presence of a iron SODe in T. cruzi. Also, in mammals, several researchers have studied extracellular SOD (EC-SOD), which is the major extracellular antioxidant enzyme and plays a critical role in the pathogenesis of a variety of pulmonary, neurological, and cardiovascular diseases (Suliman et al 2001, Fukai et al 2002.…”