Specific antigenic determinants on the membrane surface of Entamoeba histolytica that distinguish it from other Entamoeba species were demonstrated. Evidence for these antigenic determinants was obtained with a monoclonal antibody to E. histolytica which showed not only specificity but also sensitivity as demonstrated in enzyme linked immunosorbent assay. Immunofluorescence microscopy showed that the monoclonal antibody recognized an epitope present on the membrane surface of E. histolytica trophozoites. The epitope detected by the monoclonal antibody was present in three components of different molecular weight. These components may have a common precursor or may be the result of enzymatic degradation under the conditions tested.

In a 12-month longitudinal study, a cohort of Mexican HIV+/AIDS patients was checked several times for Entamoeba infection, with the parasites identified, as E. histolytica or E. dispar, using PCR. The polymorphic region of the parasites' chitinase genes was investigated by PCR, with the variation in amplicon sizes being used as a measure of the genetic variation among the isolates. The patients found infected with Entamoeba at the start of the study displayed varied patterns of infection clearance and re-infection. The analysis of the polymorphisms in the chitinase gene revealed seven polymorphic patterns in the E. histolytica isolates investigated and three in the E. dispar isolates. Many of the patients were each re-infected with Entamoeba at least once during the 12 months of follow-up.As seen in a previous study in Mexico, none of the E. histolytica-infected patients developed any clinical symptoms of invasive amoebiasis during the follow-up period. The results highlight the complexity of the host-parasite relationship in human amoebiasis.

We described an E. histolytica genotype detected in the drainage material of amoebic liver abscess from six Mexican patients. Five of theme experiences an ALA that needed drainage due to the risk of rupture and one developed a lung empyema consequence of the opening of the primary liver abscess to thoracic cavity. The material obtained through a sonography guide hepatic puncture was treated for DNA extraction. The DNA was used to amplify the polymorphic region of chitinase gene. The PCR products were sequenced, translated, aligned and compared with the already chitinase sequences reported in Gen Bank data base. The alignment of our sequence did not show identity with reported data so it was registered in the Gen Bank data base with the reference number EF 445962. Our results support the previous observations of the possible particular geographic distribution of E. histolytica and E. dispar strains all over the world and their high genetic variability.

The aim of the present work was to clone and express the crt gene of the E. histolytica parasite. We use the PCR technique to amplify the crt gene using specific oligonucleotides and the nuclear genomic DNA of E. histolytica. PCR products were cloned in pBluescript KS+. For the expression of CRT, the plasmids were sub-cloned in pProEx-HT-b. We obtained a 1200 bp PCR product that corresponds to the complete crt gene, and two small amplification products of 650 and 550 bp that encodes the C-terminal and N-terminal end of CRT, respectively. These fragments show an identity of 100 % with the gene EAL49855, which encodes for a probable CRT reported in 2005. The recombinant protein EhCRT was sequenced by tandem mass spectrometry (LC/ESI-MS/MS) and all peptides sequenced corresponded to CRT. The three recombinant peptides were characterized with three anti-CRT heterologous antibodies.

The encysting and excysting process in E histolytica is highly difficult to reproduce in culture, however, in the reptile pathogen E invadens, the process has been reproduced in a very reliable manner and used as a model. Some proteins, like chitinase and other proteins, are involved in both the encysting and excysting processes in intestinal protozoa parasites. Chitinase proteins are highly polymorphic between Entamoeba species and strains. In the present work, we characterized the Entamoeba invadens chitinase amino acids of a tandem repeat region, in order to study the polymorphism in the chitinase gene in this species. Our results show that the aa repeat region displays a single aa polymorphism (SAAP) - a different kind of polymorphism than that observed in the E histolytica and E dispar species. The repeat sequences in E invadens have seven segments, which in 10 isolates were located at the same position. In only three of these strains, the SAAP were observed in two of the repeated regions.

The objective of this work was to evaluate the frequency of Entamoeba histolytica/Entamoeba dispar intestinal infection in HIV+/AIDS subjects and their HIV− close relatives or sexual partners. Enteric parasites were investigated in stool samples by microscopic examination and E. histolytica and E. dispar were identified by PCR. We found by microscopic analysis in HIV+/AIDS group that the E. histolytica/E. dispar complex was present in 5.9% of the members, while in the HIV− group was 2.9%. With PCR we found that the E. histolytica prevalence was 25.3% in the HIV+/AIDS group and 18.5% in the HIV-group. The difference in the results obtained with the microscopic and PCR is due to the different sensibility of the procedures. Besides, we found patients who were infected with E. histolytica in both groups were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.

The purpose of this study was to determine whether HIV-1 infected patients in our community were more susceptible to Entamoeba histolytica and Entamoeba dispar infection than non-HIV-infected individuals. The prevalence and frequency of invasive amebiasis was determined in 203 HIV+/AIDS subjects and 140 close relatives or sexual partners, all of whom were HIV-. Anti-E. histolytica antibodies (IgG, IgA) were assessed as indicators of E. histolytica invasive infection. Polymerase chain reaction (PCR) was used for the characterization of the Entamoeba species. The prevalence estimated with PCR data showed that E. histolytica infection was more common in the HIV+/AIDS group (25.32%), than in HIV- contacts (18.46%). E. histolytica + E. dispar infection was more frequent in HIV+/AIDS patients (13.3%), than in HIV- contacts (0.7%). E. histolytica and/or E. dispar infection was highly prevalent in HIV+/AIDS patients (34.1%) without evidence of recent or current invasive disease. Contacts of HIV+/AIDS patients who were infected with E. histolytica were asymptomatic cyst passers. Our results suggest that E. histolytica strains prevalent in the studied community appear to be of low pathogenic potential.

The frequency of Entamoeba histolytica and Entamoeba dispar infection was analyzed in a rural community in the state of Morelos, Mexico, through PCR technique by using specie specific primer. The E. histolytica specie was detected in 33 of 290 analyzed stool samples (11.4%), E. dispar specie was observed in 21 samples (7.2%) and both species of Entamoeba were detected in seven samples (2.4%). So a higher E. histolytica than E. dispar frequency infection was detected (13.8 versus 9.6%). Even though in our design we did not considered the follow-up of included individuals, the absence of invasive amebiasis cases in the studied population during our stay in town was unexpected.

The frequency of Entamoeba histolytica and Entamoeba dispar infection was analyzed in a rural community in the state of Morelos, Mexico, using polymerase chain reaction (PCR). Sociodemographic variables as risk factors for the infection were assessed. Results highlighted the number of individuals with intestinal parasites (43.1%) in the community, indicating extensive fecalism. A high frequency of E. histolytica asymptomatic infection, higher than E. dispar infection (13.8% versus 9.6%), was detected by PCR. Anti-amebic antibody levels (IgG) in serum and saliva (IgA) samples were not associated with E. histolytica intestinal infection. These findings suggest a predominant distribution of E. histolytica strains of low invasive potential in this community.

The intra-species polymorphism of E. histolytica and E. dispar species in endemic area is an important tool for geographic distribution and spread mechanism studies. Since E. dispar and E. histolytica shears ecological niche, cell cycle, and transmission mechanism for human host, we studied the intra-specie variation and distribution of E. dispar strains obtained from cyst passers, in two neighbor rural communities in Morelos Mexico. We analyzed the polymorphic region of the quitinase protein gene in isolates of E. dispar. In 45 isolates from one community we identified 12 different CHI patterns while in 15 isolates from the other community we identified 5 different patterns. However both communities share 4 patterns. This finding suggests the presence of strains with different geographic mobility.

Proteins of 45 and 29 kDa of Salmonella enterica serovar Typhi (S. 14 typhi) that previously showed to be immunodominant for cellular immune responses of individuals with typhoid fever were isolated. Immunization of BALB/c mice by subcutaneous route with these proteins confer-red 92% protection to mice challenged by intraperitoneal route with 100 DL50 of serovar Typhi. Immunization with the 45 kDa protein induced a strong antibody response (IgG1 and IgG2a). Both proteins induced a strong proliferative response of spleen cells from immunized mice.

Entamoeba histolytica is responsible for human invasive amebiasis. This disease has an endemic behavior in specific geographic areas throughout the world, and is commonly associated with population with low socioeconomic and educational conditions (1). It is in these endemic areas where the evaluation of prevalence of infection and disease through the detection of antiameba antibodies in serum and secretions is particularly valuable. Although there is an increasing number of new strategies to make available better antibody and antigen detection tests (2), the complexity of data analysis obtained in epidemiologic trials in highly endemic areas justifies the search for new antigens of E. histolytica with biological or molecular characteristics that may optimize antibody detection tests for use in field studies. In the present article, we describe the purification of three proteases of E. histolytica HM-1:IMSS that previously showed to be highly recognized (91.4%) by serum antibodies from patients with amebic dysentery (3), and in more than 95% of amebic liver abscess patients. Preliminary data suggest that antibody levels to these proteins decrease significantly after 6 months of treatment, which makes them attractive for the detection of new cases of invasive amebic disease.

Amebiasis is caused by the protozoan Entamoeba histolytica. This disease, or the asymptomatic infection, is associated with poor socioeconomic conditions, malnutrition, and poor hygiene behavior, all common in developing countries. There is evidence of the existence of two species of quadrinucleated amebas in the human population ( E. histolytica and Entamoeba dispar ) with different pathogenic capabilities, making necessary the reevaluation of the actual frequency of infection and disease, because is a tendency to overestimate it in endemic areas where other intestinal infections causative of dysentery or bloody diarrhea are found. The opposite is frequent in nonendemic areas, where the presence of amebas is overlooked in stool microscopic examination. In the present work, we studied the prevalence of E. histolytica or E. dispar infection in the rural community of Coahuixtla, State of Morelos, Mexico. The selection of this community was made on the basis of the reported rate of new cases of intestinal amebiasis or amebic liver abscess in the state of Morelos (1). E. histolytica and E. dispar identification was performed through the molecular analysis of the cysts isolated directly from stool samples.

Formalin fixation is the most common storage, transportation and preservation method for stool samples. However, fixation dramatically reduces our ability to extract from stool samples DNA that is a suitable template for polymerase chain reaction (PCR)-based diagnostic tests. In this study we evaluated the effects of formalin concentration and of the time stored in fixative on the success of PCR amplification. We found that the deleterious effects of formalin are both time and concentration dependent and may result from fragmentation of fixed DNA during its purification.

The sera of cohorts of newborn infants and their mothers, characterized as cyst passers of Entamoeba with nonpathogenic zymodemes (E. dispar) and seropositive for amoebic antigens, were analyzed. Both cohorts were followed for a period of 12 months by microscopic examination of feces and determination of serum anti-amoebic antibody titers using the indirect hemagglutination assay. Control groups (noncyst passer mothers and their infants) were included and followed. To characterize antigens involved in the induction of IgG and IgA antibody responses, Western blots of serum from all participants were tested and immunoplots of the frequency of antigenic recognition were constructed. Results of clinical follow-up and microscopic examination of feces showed that during the 12-month period none of the cyst passer mothers had episodes of diarrhea attributable to E. histolytica invasion; five of 21 children of cyst passer mothers became infected during the study, five of five infected children developed serum antiamebic antibodies (titers 1:64-1:128); none of the cohort of children from cyst passer mothers had diarrhea due to E. histolytica. Western blot analysis showed that there are antigenic fractions that induce serum antibodies of the IgG and IgA classes against E. dispar very early in the host-parasite relationship. Our results suggest that mechanisms of antibody induction different from intestinal invasion may be operating in amebic infection. Intestinal absorption of antigen, systemic reflection of secretory antibody response, and priming of newborns by maternal anti-idiotypic antibody transfer are discussed.

Pigeon breeder's disease (PBD), a form of hypersensitivity pneumonitis caused by repeated inhalation of antigens of pigeon origin, is characterized by a diffuse inflammation of the lower respiratory tract. Although a variety of immunologic and nonimmunologic mechanisms have been described in the development of the disease, the pathogenesis is still far from clear. In this study we analyzed the T-lymphocyte proliferative response to a variety of avian antigens with use of peripheral blood mononuclear cells from 11 patients who had PBD and 10 healthy volunteers. We used a new method based on avian antigen-bearing nitrocellulose particles derived from Western blots to study the T-cell proliferative response to 15 antigenic fractions obtained from pigeon serum. With this technique, complex mixtures of antigens can be fractionated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and used for T-cell proliferation assays with selected antigenic determinants. A wide variety of responses were observed, and there were no reproducible patterns of reaction within either group. Nine of 10 healthy subjects responded to some soluble fractions. However, patients with PBD displayed the strongest response and responded to a significantly greater number of antigenic fractions. Fraction 2, representing a 220 kd molecular weight protein, was the only immunodominant antigen when both groups were compared; it was recognized by 73% of the patients with PBD and by only 20% of control subjects (p < 0.03). These findings show that T lymphocytes of patients with PBD recognize a wide range of bird proteins, which induce marked T-cell proliferation.

The proliferative responses of peripheral blood mononuclear cells of 10 subjects that had typhoid fever, and healthy volunteers without history of typhoid fever or immunization against disease, were analysed with antigen fractions from two protein extracts of Salmonella typhi. Fractions from each extract were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) electrophoresis, transferred to nitrocellulose filters by electroblotting and processed to obtain antigen‐bearing nitrocellulose particles for use in lymphocyte cultures. Although the individual proliferative responses were heterogeneous we identified two main immunogenic regions of 29–32 × 103 MW and 45–56 × 103 MW for both extracts. Even though there was no one particular antigenic fraction capable of stimulating lymphocytes from all individuals with a previous history of typhoid fever, the combination of three fractions 29–32, 41–45, 63–71 × 103 MW could be stimulatory for cells of 90% of these individuals. Also, four subjects that did not respond to unfractionated antigens gave proliferative responses to several fractions of the same extract. We have identified the main immunogenic fractions of S. typhi that might play a role during typhoid infection and postinfection immunity, and merit further purification and characterization.