Mice Intragastric Infected with Insect and Blood Trypomastigotes of Trypanosoma cruzi IV: Differences and Similarities on the Evolution Profile and Response to Etiological Treatment.

PURPOSE: Our goal was to analyze the outcome of infection and response to benznidazole (BZ) treatment in mice intragastrically inoculated with trypomastigotes forms of Trypanosoma cruzi from different origins. METHODS: Twenty-four Swiss mice were divided in two groups and inoculated, by gavage, with 1 × 104 blood trypomastigotes (BT) or insect-derived metacyclic trypomastigotes (IT) of AM14 strain (T. cruzi IV). Half of the animals of each group were treated with BZ (TBZ), from 10 to 30th days after the inoculation, and the other constituted the untreated control groups (NT). After the etiological treatment, all mice were immunosuppressed with cyclophosphamide for three weeks. Parasitological and molecular parameters, infectivity, cumulative mortality, and reactivation post-immunosuppression rates were obtained. RESULTS: Animals inoculated with BT showed lower pre-patent period and early day of the maximum parasitemia, as well as a higher maximum peak of parasitemia than the IT animals. However, both, BT and IT animals, did not respond to BZ treatment (0.0% of cure). CONCLUSION: We conclude that the infective form influences in the outcome of infection, but not the response to the etiological treatment in mice intragastrically infected with the T. cruzi IV strain studied.

Essential oils from Syzygium aromaticum and Zingiber officinale, administered alone or in combination with benznidazole, reduce the parasite load in mice orally inoculated with Trypanosoma cruzi II.

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas disease (CD) or American trypanosomiasis, an important public health problem in Latin America. Benznidazole (BZ), a drug available for its treatment, has limited efficacy and significant side effects. Essential oils (EOs) have demonstrated trypanocidal activity and may constitute a therapeutic alternative. Our aim was to evaluate the efficacy of the EOs of clove (CEO - Syzygium aromaticum) and ginger (GEO - Zingiber officinale), administered alone and in combination with BZ, in Swiss mice infected with T. cruzi. METHODS: The animals were inoculated with 10,000 blood trypomastigotes of the Y strain of T. cruzi II by gavage and divided into four groups (n = 12 to 15): 1) untreated control (NT); 2) treated with BZ; 3) treated with CEO or GEO; and 4) treated with BZ + CEO or GEO. The treatments consisted of oral administration of 100 mg/kg/day, from the 5th day after parasite inoculation, for 20 consecutive days. All groups were submitted to fresh blood examination (FBE), blood culture (BC), conventional PCR (cPCR) and real-time PCR (qPCR), before and after immunosuppression with cyclophosphamide. RESULTS: Clove and ginger EOs, administered alone and in combination with BZ, promoted suppression of parasitemia (p < 0.0001), except for the animals treated with CEO alone, which presented a parasitemia curve similar to NT animals. However, there was a decrease in the BC positivity rate (p < 0.05) and parasite load (< 0.0001) in this group. Treatment with GEO alone, on the other hand, besides promoting a decrease in the BC positivity rate (p < 0.05) and parasite load (p < 0.01), this EO also resulted in a decrease in mortality rate (p < 0.05) of treated mice. CONCLUSIONS: Decreased parasite load, as detected by qPCR, was observed in all treatment groups (BZ, CEO, GEO and BZ + EOs), demonstrating benefits even in the absence of parasitological cure, thus opening perspectives for further studies.

Suprahyoid Muscle Activity in Patients with Chagasic Megaesophagus.

The objective of this investigation was to evaluate the activity of the suprahyoid musculature during swallowing and to correlate the findings with the degree of megaesophagus, oral and pharyngeal videofluoroscopy and esophageal manometry in patients with achalasia caused by Chagas' disease. Twenty-nine patients with positive serology for Trypanosoma cruzi and dysphagia (Chagas' disease group) and 29 individuals matched by sex and age (control group) participated in the study. Surface electromyography of the suprahyoid musculature and videofluoroscopy during swallowing of paste and liquid consistencies were performed. Canonical correlation analysis of the MANOVA test results showed that the Chagas' disease group had lower electromyographic activity when compared with controls. Overlapping circles of radiological findings were found for megaesophagus. The Spearman test showed a positive correlation between the electromyographic activity in the maximum voluntary isometric contraction and the time of pharyngeal transit for both liquid (p = 0.014) and paste (p = 0.047). The logistic regression test showed no association between electromyographic activity of the suprahyoid muscles and esophageal manometry results (p > 0.05). In conclusion, individuals with chagasic megaesophagus have reduced electromyographic activity of the suprahyoid muscles during swallowing, in addition to a greater recruitment of the suprahyoid musculature with increased pharyngeal transit time.

Differential expression of proteins in genetically distinct Trypanosoma cruzi samples (TcI and TcII DTUs) isolated from chronic Chagas disease cardiac patients.

BACKGROUND: Trypanosoma cruzi, a hemoflagellate protozoan parasite and the etiological agent of Chagas disease (CD), exhibits great genetic and biological diversity. Infected individuals may present clinical manifestations with different levels of severity. Several hypotheses have been proposed to attempt to correlate the diversity of clinical signs and symptoms to the genetic variability of T. cruzi. This work aimed to investigate the differential expression of proteins from two distinct genetic groups of T. cruzi (discrete typing units TcI and TcII), isolated from chronically infected individuals displaying the cardiac form of CD. For this purpose, epimastigote forms of the two isolates were cultured in vitro and the cells recovered for protein extraction. Comparative two-dimensional (2D) gel electrophoreses were performed and differentially expressed spots selected for identification by mass spectrometry, followed by database searching and protein categorization. RESULTS: The 2D electrophoretic profiles revealed the complex composition of the T. cruzi extracted proteome. Protein spots were distributed along the entire pH and molecular mass ranges attesting for the integrity of the protein preparations. In total, 46 differentially expressed proteins were identified present in 40 distinct spots found in the comparative gel analyses. Of these, 16 displayed upregulation in the gel from TcI-typed parasites and 24 appeared overexpressed in the gel from TcII-typed parasites. Functional characterization of differentially expressed proteins revealed major alterations associated with stress response, lipid and amino acid metabolism in parasites of the TcII isolate, whilst those proteins upregulated in the TcI sample were primarily linked to central metabolic pathways. CONCLUSIONS: The comparative 2D-gel electrophoresis allowed detection of major differences in protein expression between two T. cruzi isolates, belonging to the TcI and TcII genotypes. Our findings suggest that patients displaying the cardiac form of the disease harbor parasites capable of exhibiting distinct proteomic profiles. This should be of relevance to disease prognosis and treatment.

Outcome of oral infection in mice inoculated with Trypanosoma cruzi IV of the Western Brazilian Amazon.

A new epidemiological view of American trypanosomiasis or Chagas disease has been formulated in recent decades. Oral transmission of the etiological agent of Chagas disease, Trypanosoma cruzi, has been the most common form of transmission. The T. cruzi discrete typing units TcI and TcIV have been involved in tens outbreaks of acute cases of Chagas disease in the Brazilian Amazon region. We investigated the intensity of infection in mice that were orally inoculated (OR group) with four strains of TcIV that were isolated from two outbreaks of acute Chagas disease that was orally acquired in the state of Amazonas, Brazil. We compared the OR group with mice that were intraperitoneally inoculated (IP group). Blood samples were analyzed by fresh blood examination, hemoculture, and conventional and qualitative real-time polymerase chain reaction (PCR). Samples of different tissues were analyzed by quantitative real-time PCR. The OR group exhibited a higher maximum peak of parasitemia, greater rates of positivity, and higher parasite loads in different tissues during acute infection compared with the IP group, indicating a greater intensity of orally acquired infection. Mice that were orally inoculated with TcIV strains that were obtained from two outbreaks of orally acquired Chagas disease in Amazonas, Brazil, exhibited a more intense course of infection compared with intraperitoneally inoculated mice, reflected by higher levels of parasitemia and parasite loads.

Spatial distribution and enteroparasite contamination in peridomiciliar soil and water in the Apucaraninha Indigenous Land, southern Brazil.

The prevalence and distribution of soil and water samples contaminated with enteroparasites of humans and animals with zoonotic potential (EHAZP) in Apucaraninha Indigenous Land (AIL), southern Brazil, was evaluated. An environmental survey was conducted to evaluate the presence of parasitic forms in peridomiciliary soil and associated variables. Soil samples were collected from 40/293 domiciles (10 domiciles per season), from November 2010 to June 2011, and evaluated by modified methods of Faust et al. and Lutz. Analyses of water from seven consumption sites were also performed. The overall prevalence of soil samples contaminated by EHAZP was 23.8 %. The most prevalent parasitic forms were cyst of Entamoeba spp. and eggs of Ascaris spp. The highest prevalence of contaminated soil samples was observed in winter (31 %). The probability map obtained with geostatistical analyses showed an average of 47 % soil contamination at a distance of approximately 140 m. The parasitological analysis of water did not detect Giardia spp. or Cryptosporidium spp. and showed that all collection points were within the standards of the Brazilian law. However, the microbiological analysis showed the presence of Escherichia coli in 6/7 sampled points. Despite the low level of contamination by EHAZP in peridomiciliar soil and the absence of pathogenic protozoa in water, the AIL soil and water (due to the presence of fecal coliforms) are potential sources of infection for the population, indicating the need for improvements in sanitation and water treatment, in addition periodic treatment of the population with antiparasitic.

Biological behaviour in mice of Trypanosoma cruzi isolates from Amazonas and Paraná, Brazil.

The biological behaviour of 23 Trypanosoma cruzi isolates in Swiss mice was compared. Nineteen isolates were obtained from patients in the acute phase of Chagas disease (13), sylvatic reservoir hosts (Didelphis marsupialis) (3), and triatomine bugs (Rhodnius robustus) (3) from four regions of the State of Amazonas (AM). Four isolates were obtained from chronic chagasic patients in the State of Paraná (PR): three autochthones, and one allochthone from the State of Minas Gerais. Only one isolate was unable to infect the mice. The AM and PR isolates showed the largest number of significant differences from each other. The former had lower mean values in the pre-patent (5.4 days) and patent (4.6 days) periods (PP), with the parasitaemia (Pmax) reaching a peak of 9.9×10(4) blood trypomastigotes (BT)/mL of blood by the 7th day following inoculation. The AM isolates also had higher positivity to fresh-blood examination (FBE) (84.1%) compared to haemoculture (HC) (58.7%) and polymerase chain reaction (PCR) (33.3%), in addition to higher mortality (2.9%). The PR isolates had higher values for PP (18.5 days) and Pmax (99.9×10(4)BT/mL) as well as higher positivity to FBE (87.2%), HC (100%), and PCR (83.3%). The correlations between the biological behaviour of the T. cruzi isolates and the clinical and epidemiological characteristics of Chagas disease are discussed.

Preinfection aerobic treadmill training improves resistance against Trypanosoma cruzi infection in mice.

Exercise performed before infections has been linked to improvement of the immune response against infections. The purpose of this study was to evaluate the influence of preinfection moderate-intensity treadmill training on acute Trypanosoma cruzi infection in mice. Ninety-nine female BALB/c mice were divided into 4 groups, as follows: training + infection (T+I) (n = 41); no training + infection (NT+I) (n = 38); training + no infection (T+NI) (n = 10); and no training + no infection (NT+NI) (n = 10). The exercise program for trained groups was carried out on a motorized treadmill for 8 weeks. Infected groups were inoculated with the Y strain of T. cruzi. Infectivity, prepatent period, patent period, parasitemia peak, mortality, survival time, weight, food intake, tumor necrosis factor-alpha serum levels, and peritoneal macrophage hydrogen peroxide production were evaluated. We found that preinfection training induced statistically significant reductions in parasitemia peak (p < 0.03) and weight loss (p < 0.04). However, no statistically significant differences were found for the other parameters evaluated when trained and nontrained infected groups were compared. We conclude that preinfection aerobic training induces some improvement in the immune response to T. cruzi infection in female BALB/c mice.

Usefulness of the polymerase chain reaction for monitoring cure of mice infected with different Trypanosoma cruzi clonal genotypes following treatment with benznidazole.

The capacity of the polymerase chain reaction (PCR) to detect the DNA of Trypanosoma cruzi was evaluated in 90 blood samples from BALB/c mice infected with T. cruzi cloned stocks of genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II), and treated with benznidazole. The results from the fresh blood examination, hemoculture, and ELISA allowed to group the treated animals into: cured (TC), dissociated (DIS) and non-cured (NC). The PCR detected T. cruzi DNA in 50.9%, 58.3% and 100.0% of the samples from TC, DIS and NC mice, respectively. These DNA possibly derives from live T. cruzi or from recently lysed parasites, suggests that these animals are in fact not cured. The difference between the PCR results and results obtained using other techniques was statistically significant and independent of the parasite genotype. The PCR described has therefore potential to be used in cure control of treated patients.

Infectivity for mice of Trypanosoma cruzi I and II strains isolated from different hosts.

In this paper, the infectivity for mice of Trypanosoma cruzi I and II strains isolated from sylvatic animals, triatomines, and humans is determined using fresh blood examination, hemoculture, culture of macerated organs, and polymerase chain reaction (PCR). Six strains were considered to have low infectivity (9.1-18.2%), five medium (27.3-45.4%), and one high (100.0%). Infectivity of T. cruzi strains isolated from sylvatic animals was significantly higher than that of strains isolated from humans and triatomines (p=0.0141). No significant difference was observed between the infectivity of T. cruzi I and II strains. The parasite was detected by fresh blood examination in one strain, by hemoculture and culture of macerated organs in four strains, and by PCR in all strains. We conclude that the infectivity is related to the host from which the strains were isolated, but the infectivity is not related to the genetic group of the parasite. We also conclude that the majority of the strains studied have low and medium infectivity for mice, and that PCR is an important tool to detect T. cruzi in strains with this biological characteristic.