Discovery of Benzopyrrolizidines as Promising Antigiardiasic Agents.

Current treatments for giardiasis include drugs with undesirable side effects, which increase the levels of therapeutic desertion and promote drug resistance in the parasites. Herein, we describe the antigiardiasic evaluation on Giardia lamblia trophozoites of a structurally diverse collection of 74 molecules. Among these scaffolds, we discovered a benzopyrrolizidine derivative with higher antigiardiasic activity (IC50 = 11 µM) and lower cytotoxicity in human cell cultures (IC50 = 130 µM) than those displayed by the current gold-standard drugs (metronidazole and tinidazole). Furthermore, this compound produced morphologic modifications of trophozoites, with occasional loss of one of the nuclei, among other changes not observed with standard giardicidal drugs, suggesting that it might act through a novel mechanism of action.

Toxocara: Seroprevalence in Mexico.

In Mexico, toxocariasis, like some other parasitosis in humans, is not a disease of conventional surveillance or immediate notification. Seroprevalence studies are scarce, six dealing with paediatric populations and eight dealing with adults; the reports were only from four states in Mexico. There were 1596 children, and the seroprevalence was 13.8%. In the case of adults, there were 1827 subjects, and seroprevalence was 4.7%. There is a significant positive association between seroprevalence and the paediatric population P<0.0001 (OR, 3.285; 95% CI, 2.541-4.279). It is advisable to perform competitive ELISAs and add another diagnostic test, such as Western blot or the detection of circulating antigens to reduce diagnostic uncertainty. This neglected parasitosis can be confused with retinoblastoma. Therefore, there is a risk of ocular enucleation. It is necessary to sensitise the authorities of the Ministry of Health and decision-makers, to provide economic support for epidemiological surveillance of this zoonotic parasite.

Antigiardiasic activity of Cu(II) coordination compounds: Redox imbalance and membrane damage after a short exposure time.

Giardiasis is a widespread illness that affects inhabitants of underdeveloped countries, being children and seniors the highest risk population. The several adverse effects produced by current therapies besides its increasing ineffectiveness due to the appearance of resistant strains evidence the urgent need for new therapeutic approaches. We present the antigiardiasic effect of eight Cu(II) coordination compounds, which belong to the family Casiopeínas. Two of them, 4,7-diphenyl-1,10-phenanthroline(acetylacetonato)copper(II) nitrate (CasIII-Ha,36 µM) and 4,7-diphenyl-1,10-phenanthroline(glycinato)copper(II) nitrate (CasI-gly,36 µM) have shown the best antiproliferative effect in Giardia intestinalis trophozoite cultures, both with the higher lipophilic character of the series. The antiproliferative effect of these coordination compounds is attributable to its capacity to interact with the cellular membrane and to increase reactive oxygen species (ROS) concentration within the parasite since the first hours of exposure, (2-6 h). We found that these compounds mainly induced the cell death of trophozoites by apoptosis, contrary to metronidazole, which induces apoptosis and necrosis in the same ratio. The cytotoxic effects on lymphocytes and macrophages isolated from human peripheral blood allowed us to establish a selectivity index and in turn, identify and propose the best candidates to continue with the assays in animal models. The selected molecules do not include the most active compounds against trophozoites, instead of that, we propose the compounds 4',4'-dimethyl-2,2'-bipyridine(acetylacetonato)copper(II) nitrate (CasIII-ia,IC50 = 156 µM) and 4,7-dimethyl-1,10-phenanthroline(acetylacetonato) copper(II) nitrate (CasIII-Ea,IC50 = 125 µM), which possess an antiproliferative efficacy comparable with Metronidazole but also are those that produce the lowest effect on the viability of human lymphocytes and macrophages.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom.

BACKGROUND: Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. METHODS: The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. RESULTS: P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. CONCLUSION: These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

Background Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods: The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results: P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion: These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.(AU)

Disulfiram as a novel inactivator of Giardia lamblia triosephosphate isomerase with antigiardial potential.

Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM) has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222) by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug.

Toxocara canis: Analysis of the kinetics of antigen release and antibody production in an in vivo model for the detection of past or present infection.

Worldwide, Toxocara canis is an important zoonotic nematode of public health concern. This soil-transmitted helminth causes visceral larva and ocular larva migrans in paratenic hosts. The detection of T. canis larva migrans is complicated because current immunological tests detect only IgG antibodies, which can cross-react with antigens from other parasites and cannot distinguish between the past and present infection. Analysis of antigen release and antibody production could help improve the detection of larva migrans. Here, we report the kinetics of antigen release, IgM and IgG production in an in vivo model for the detection of past or present infection. We used four groups of seven mice: two groups infected orally with 50 or 100 embryonated eggs, and the other two infected intraperitoneally with 50 or 100 live larvae. We obtained blood samples at 0, 3, 7, and 14days and, then, every two weeks until day 140. Sandwich ELISA and indirect ELISA were performed for antigen capture and the detection of immunoglobulins, respectively. Mice inoculated with larvae developed an immune response faster than those inoculated with eggs. In all groups, antigen capture was positive starting at 3days until 140days post-inoculation (dpi). Detection of immunoglobulins was at 14 or 28dpi in mice inoculated with larvae or eggs, respectively. Negative IgM values were detected at days 98 and 112. The samples remained positive for IgG until the last day of the experiment. Data suggest that in mice inoculated with T canis eggs, some larvae did not hatch, others died or never reached the bloodstream. Based on our model, we propose that there is early infection when only antigens are present, and active larva migrans when antigen and immunoglobulins are detected, implying an immune response of the host against the antigen. Our study offers a view into the parasite-host relationship and enables us to infer if there are live larvae. Additionally, these findings provide a foundation for the diagnosis and differentiation of recent infection and active larva migrans.

Piqueria trinervia as a source of metabolites against Giardia intestinalis.

CONTEXT: Piqueria trinervia Cav. (Asteraceae) is a plant species with a long history in traditional medicine to cure diarrhoea and other digestive disorders. OBJECTIVE: The study investigates the antigiardial activity of piquerol, trinervinol, red oil and two fractions (F1 and F2) from P. trinervia. MATERIALS AND METHODS: P. trinervia was collected in the Ajusco in Mexico City. Aerial parts were ground and mixed with water to obtain the extract, which was treated with dichloromethane to isolate piquerol and trinervinol (P & T). Remnants were the red oil, fractions 1 and 2 (RO, F1 & F2). Trophozoites of Giardia intestinalis were treated with P, T, RO, F1 and F2 at different concentrations (0.78-200 µg/mL) for 48 h. Antigiardial activity was measured using the methylene blue reduction, and the cytotoxicity assayed on human fibroblasts and Vero cells by reduction of tetrazolium salts. RESULTS: Trinervinol and piquerol showed antigiardial activity with an IC50 = 2.03 and 2.42 µg/mL, and IC90 = 13.03 and 8.74 µg/mL, respectively. The concentrations of trinervinol (CC50 = 590 µg/mL) and piquerol (CC50 = 501 µg/mL) were not cytotoxic to human fibroblasts. CONCLUSIONS: Compounds from P. trinervia showed antigiardial activity; to enhance this activity, piquerol and trinervinol can be chemically modified.

Giardia duodenalis genotypes among schoolchildren and their families and pets in urban and rural areas of Sinaloa, Mexico.

INTRODUCTION: Giardiasis is a human health concern worldwide, especially among schoolchildren. Giardia duodenalis genotypes A and B are infective to humans, but their zoonotic potential remains controversial. In Mexico, the most prevalent genotype is A, but B was also detected in southeastern Mexico. In Sinaloa state, northwestern Mexico, giardiasis is highly prevalent, but Giardia genotypes have been poorly studied. METHODOLOGY: This study aimed to investigate the distribution and clinical-epidemiological correlation of G. duodenalis genotypes in schoolchildren and their families and pets in urban and rural areas of Sinaloa state, Mexico. RESULTS: Among 395 schoolchildren (274 urban, 121 rural), 76 (49 urban, 27 rural) were infected with G. duodenalis. In total, 22 families (15 urban, 7 rural) of infected schoolchildren, consisting of 60 family members (41 urban, 19 rural) and 21 pet dogs (15 urban, 6 rural) were examined; 10 family members (5 urban, 5 rural) and 5 pet dogs (3 urban, 2 rural) of 10 families (6 urban, 4 rural) were infected. After PCR-RFLP analyses of vsp417 and gdh genes, genotype prevalence among infected urban schoolchildren was 79.5% AI, 12.8% AII, and 7.7% mixed AI+B. However, only AI genotype was found in family members and pets. In the rural area, only the AI genotype was detected. Genotypes were not correlated with clinical manifestations. CONCLUSIONS: This paper shows the presence of B genotype in northwestern Mexico for the first time. Detection of AI genotype in dogs suggested the possible role of dogs as the reservoir for human giardiasis in Sinaloa, Mexico.

Successful capture of Toxocara canis larva antigens from human serum samples.

BACKGROUND: Toxocara canis is a nematode that parasitizes dogs, while humans are paratenic hosts. When humans are infected the migrating larvae damage the liver, lungs and even the nervous system. Larva migrans diagnosis is based on immunological techniques; however, the commercial immunodiagnostic kits detect anti-T. canis antibodies which may cross-react with other parasites, mainly nematodes with extra-intestinal migration. Moreover, antibodies do not necessarily reflect an active infection; so detection and quantification of circulating antigens may provide appropriate and timely information for treatment, which prevents irreversible damage. Here we report the standardization of a monoclonal antibody based antigen capture ELISA to diagnose human toxocariasis without cross-reaction. METHODS: We developed anti-T. canis polyclonal antibodies in rabbits and a monoclonal antibody in mouse which did not cross-react with 15 antigens from several parasites. The sandwich ELISA standardization was performed using sera from mice experimentally infected. We tested the method using 29 positive and 58 negative human sera previously typified with a commercial kit, which detects antibodies. RESULTS: Only 5.0 µg/mL and 10 µg/mL polyclonal antibodies and monoclonal antibody, respectively, were needed in the sandwich ELISA standardization, detecting since 440 pg/mL larva antigens. Nine out of 29 antibody-positive sera were also positive for antigens and no false positive were found. Taking the antibody kit as the reference standard, the sensibility and specificity of the antigen test were 31% and 100%, respectively. CONCLUSIONS: With these tools we established a detection threshold as low as 440 pg/mL antigen. Monoclonal antibody is specific, and did not cross-react with antigens from other parasites. Detection of circulating antigens helps provide appropriate and timely treatment and prevents irreversible damage.

In vitro activity of 'Mexican Arnica' Heterotheca inuloides Cass natural products and some derivatives against Giardia intestinalis.

Giardiasis is a gastrointestinal disease that affects humans and other animals caused by parasitic protists of the genus Giardia. Giardia intestinalis (Syn. Giardia lamblia; Giardia duodenalis) infections can cause acute or chronic diarrhoea, dehydration, abdominal discomfort and weight loss. Metronidazole is the most widely used drug for treating giardiasis. Although effective, metronidazol has undesirable secondary effects. Plants used in traditional medicine as antidiarrhoeals or antiparasitics may represent alternative sources for new compounds to treat giardiasis. Heterotheca inuloides Cass. (Asteraceae/Compositae) plant is widely used in Mexican traditional medicine. The following secondary metabolites were isolated from H. inuloides flowers: 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-dihydroxy-3(4H)-isocadalen-4-one (3), 1R,4R-hydroxy-1,2,3,4-tetrahydrocadalen-15-oic acid (4), quercetin (5), quercetin-3,7,3'-trimethyl ether (6), quercetin-3,7,3',4'-tetramethyl ether (7) and eriodictyol-7,4'-dimethyl ether (8). The activity of these compounds against Giardia intestinalis trophozoites was assessed in vitro as was the activity of the semisynthetic compounds 7-acetoxy-3,4-dihydrocadalene (9), 7-benzoxy-3,4-dihydrocadalene (10), 7-acetoxycadalene (11), 7-benzoxycadalene (12), quercetin pentaacetate (13) and 7-hydroxycalamenene (14). Among these, 7-hydroxy-3,4-dihydrocadalene (1) and 7-hydroxycalamenene (14) were the most active, whereas the remaining compounds showed moderate or no activity. The G. intestinalis trophozoites exposed to compound 1 showed marked changes in cellular architecture along with ultrastructural disorganization. The aim of this study was to evaluate the giardicidal activity of selected H. inuloides metabolites and some semisynthetic derivatives using an in vitro experimental model of giardiasis.

Intraepithelial giardia intestinalis: a case report and literature review.

The giardiasis is a neglected parasitic disease. The WHO has estimated more than 280 million of human infections each year; however, intraepithelial giardiasis is a rare entity, there are only 5 reports showing invasive giardiasis. A pediatric female patient with chronic abdominal pain, diarrhea, or pasty stools, without fever, was seen in the Gastroenterology and Nutrition Service. The stool studies were negative for pathogens and lactose hydrogen breath test was positive. The presumptive clinical diagnosis was giardiasis and the patient was empirically treated with nitazoxanide. But, the patient persisted with abdominal pain and pasty stools. Endoscopy was indicated to search for Helicobacter and Giardia. Guardian and patient gave written informed consent. Hematological profile was normal. The endoscopy was performed under general anesthesia and the biopsies and duodenal aspirate were obtained. The microscopic analyses of duodenal fluid showed Giardia trophozoites. Electron microscopic analysis was negative for Helicobacter pylori, but Giardia trophozoites with a typical crescent shape within the tissue were found. The patient was treated with tinidazole, subsequent tests showed that lactose absorption was normal, stool examinations were negative for Giardia and abdominal pain had stopped. This case suggest that intraepithelial giardiasis could be a common entity but unseen because the giardiasis diagnosis is usually made on fecal samples. Future studies are necessary to determine the role of intraepithelial trophozoites in giardiasis pathogenic mechanisms.

Prevalence and risk factors associated with Toxocara canis infection in children.

The objective of this study was to determine seroprevalence and identify risk factors associated with Toxocara canis infection. A clinical and epidemiological questionnaire and body mass index were used to assess the risk factors associated with human toxocariasis in 108 children with an age range of 2-16 years. Antibodies against Toxocara canis were detected using an ELISA test kit. Chi-square analysis and odds ratio (OR) were used to identify risk factors associated with Toxocara canis seropositivity. The prevalence of antibodies against Toxocara canis was greater (P = 0.02) in males than females (28.84% and 16.07%, resp.). Chi-square analysis and odds ratio revealed just one variable with P < 0.05, and OR > 1.0 was associated with seropositivity: the possession of dogs under one year old (OR = 1.78). Although not significant, the OR values suggest that other factors may be epidemiologically important for Toxocara presence such as not washing hands before meals, malnutrition, obesity, and use of public parks. Children in the age group >12 and <16 years old had higher seroprevalence to Toxocara canis (17.59%) than the >2 and <11 years old age group (4.62%). Toxocariosis infection needs to be prevented by pet deworming and hygienic measures after contact with dogs.

[Microsporidia in pediatric patients with leukemia or limphoma]. / Microsporidiosis en pacientes pediátricos con leucemia o linfoma.

INTRODUCTION: Microsporidia are intracellular micro-organisms, characterized by mature spores with chitin walls and by one extrusive polar tube through which they pour their sporoplasm to the host cells. In immunocompromised patients, Enterocytozoon bieneusi and Encephalitozoon intestinalis produce diarrhea and systemic dissemination. In Mexico there is not information about microsporidia in children with cancer. OBJECTIVE: The aim of this pilot study was to investigate the presence of microsporidia species in pediatric patients with leukemia or lymphoma. MATERIAL AND METHODS: We obtained fecal samples from thirteen patients. The samples were processed to detect microsporidia by both modified Ziehl-Neelsen and clacofluor white stains, DNA was isolated to amplify rRNA specific sequences, to identify E. bieneusi, E. intestinalis, E. cuniculi and E. hellem by DNA polymerase chain reaction (PCR). Other parasites and pathogenic bacteria were also tested. RESULTS: Based on morphologic traits 7/13 samples were found positives to microsporidia and 6/10 by PCR. Was identified E. bieneusi in three patients with leukemia and one with lymphoma, another two children with leukemia were infected with E. intestinalis. Almost all children were high-risk patients and in phase of re-induction, consolidation or with many chemotherapy treatments. All the patients with microspiridia did not present diarrhea at the moment of the sampling; however, in two children with diarrhea it was found Cyclospora cayetanensis. Also we obtained feces from five patients' mothers and microsporidia spores were identified by stain in all of them and by PCR it was diagnosed the species in three of them. CONCLUSION: It was demonstrated that the feces of patients with leukemia or lymphoma had microsporidia, therefore is necessary to know the prevalence of these microorganisms and to analyze their impact in evolution of cancer patients.

In vitro activity of the F-6 fraction of oregano against Giardia intestinalis.

Giardiosis is a neglected parasitic disease that produces diarrhoea and different degrees of malabsorption in humans and animals. Its treatment is based on derivatives of 5-nitroimidazoles, benzimidazoles, nitrofuranes, acridine and nitrotiazoles. These drugs produce undesirable secondary effects, ranging from a metallic taste in the mouth to genetic damage and the selection of resistant strains; therefore, it is necessary to develop new therapeutic alternatives. We demonstrated that a 2-h treatment with 2·87 µg ml(-1) of fraction 6 of Lippia graveolens (F-6) was sufficient to kill half of an experimental Giardia intestinalis (Syn. G. duodenalis, G. lamblia) population, based on the reduction of MTT-tetrazolium salt levels. F-6 breaks the nuclear envelope and injures the ventral suckling disc. The major compounds of F-6 were characterized as naringenin, thymol, pinocembrin and traces of compounds not yet identified. The results suggest that Lippia is a potential source to obtain compounds with anti-Giardia activity. This knowledge is an important starting point to develop new anti-giardial drugs. Future studies will be required to establish the efficacy of F-6 in vivo using an animal model.

Trypanosoma cruzi: seroprevalence detected in the blood bank of the Instituto Nacional de Pediatría, Mexico City, in the period 2004 through 2009.

BACKGROUND: The second most common mode of Trypanosoma cruzi or Chagas disease transmission is via therapeutic blood transfusion. In Mexico, control of T. cruzi is still in its initial phase; in fact, there are only 14 studies published covering 10 states on T. cruzi seroprevalence in donated blood in Mexico. Here we present the results of 5 years of trypanosomiasis screening in the blood bank of the Instituto Nacional de Pediatría. STUDY DESIGN AND METHODS: Samples from all blood donated in the period from 2004 to 2009 were analyzed. We screened for T. cruzi using an enzyme-linked immunosorbent assay technique. Seropositive samples were then processed using the polymerase chain reaction (PCR) to detect a nuclear gene segment. RESULTS: A total of 37,333 samples were analyzed and a 0.17% (64 samples) T. cruzi seroprevalence was found. Donors were mostly from Mexico State and Mexico City, which is considered nonendemic for T. cruzi area. Of 64 seropositive samples, only two tested positive by PCR (3.12%), which amplified a 189-bp product from nuclear gene from the parasite. CONCLUSION: Although the seroprevalence of T. cruzi infection was low, this surveillance program prevented the infection of more than 100 children because each unit of blood provides 2.6 to 3.5 blood products. The majority of the donors were from Mexico State and Mexico City, which is a nonendemic area. The serodetection of T. cruzi in this region is evidence that is necessary to increase our understanding of its distribution in the Mexico City and surrounding places.

Prevalencia de Giardia intestinalis y predominio de genotipos zoonóticos en ovinos y bovinos de traspatio de cinco estados de la República Mexicana / Prevalence of Giardia intestinalis and zoonotic genotype predominance in small scale sheep and cattle farms in five states of the Mexican Republic

The aim of this paper was to discover the prevalence and assemblages of Giardia intestinalis, harbored in sheep and cows on familiar farms from five states of the Mexican Republic. Stool samples from 265 sheep and 174 cows were analyzed by centrifugation and flotation in zinc sulfate to search for cysts and ova. The samples with Giardia cysts were processed in a Sheather solution in order to isolate them. Afterwards, cultures were established in TYI-S-33, each one of which was the Giardia DNA source. The DNA was obtained and used as a template to amplify a fragment of the glutamate dehydrogenase (gdh) enzyme. The 430 bp amplicons were restricted with Nla IV and Rsa I in order to identify the restriction fragments length polymorphisms (RFLP's) patterns. From the cyst analysis, Giardia cysts in nine cows (5.1%) and 30 sheep (11.3%) were found. Then 10 axenic cultures (5 from sheep and 5 from cows) were set up. From the RFLP's pattern it was found that one cow had assemblage (AI), another two had a mixture of assemblages (AI + BIII) and the other two had (E + BIII). In sheep, it was found that two sheep had assemblage (AI) and the other three had a mixture of assemblages (AI + BIII). This is the first report in which zoonotic assemblages (A-I and BIII) predominance in ruminants from five states of Mexico have been demonstrated. Therefore, it is necessary to carry out further studies aimed at discovering other Giardia genotypes and transmission patterns between animals and humans in Mexico.

Con el fin de determinar la frecuencia y genotipos de Giardia intestinalis en ovinos y bovinos de traspatio de algunos estados de la República Mexicana, en este trabajo se colectaron heces de 265 ovinos y 174 bovinos, para la búsqueda de Giardia mediante coproparasitoscópicos (CPS) de concentración flotación. De las muestras fecales que resultaron positivas se obtuvieron los quistes por el método de Sheather. Los quistes se desenquistaron in vitro y los trofozoítos se mantuvieron en cultivo TYI-S-33 axénico. El ADN de los trofozoítos se obtuvo mediante extracciones fenólicas y se amplificó un segmento de ≈ 430 pb del gen de la enzima glutamato deshidrogenasa (gdh) por medio de la reacción en cadena de la ADN polimerasa (PCR), el producto se restringió con las enzimas Nla IV y Rsa I y se obtuvieron los polimorfismos de los fragmentos de restricción (RFLP). En los CPS se encontró a Giardia en nueve bovinos (5.1%) y 30 ovinos (11.3%). Se establecieron 10 cultivos axénicos (5 de bovinos y 5 de ovinos). En un bovino se encontró el genotipo (AI), dos tuvieron mezcla de los genotipos (AI + BIII) y los otros dos fueron (E + BIII). Un ovino fue genotipo (AI) y tres tuvieron mezcla de los genotipos (AI + BIII). Éste es el primer informe que presenta predominio de genotipos zoonóticos (AI y BIII) en ovinos y bovinos de México. Es necesario investigar los genotipos de Giardia y patrones de transmisión entre animales y humanos en México.

A simplified method for hatching and isolating Toxocara canis larvae to facilitate excretory-secretory antigen collection in vitro.

Human toxocariasis causes several dangerous syndromes that can involve the viscera, vision and central nervous system. Diagnosing toxocariasis requires the identification of antibodies against Toxocara canis or Toxocara cati excretions and secretions (ES). To obtain ES it is necessary to collect a large number of larvae. However, since the earliest work describing the culture of Toxocara larvae, few advances in the method have been made. It has been suggested that carbon dioxide triggers molecular mechanisms that enable nematode hatching. A similar hypothesis has been made regarding Giardia excystation. To test the hypothesis we used the Giardia excystation HBSS method to hatch embryonated T. canis eggs. We found that the HBSS method was more effective than the original De Savigny method. Our results suggest that both parasites require stimulation in an acidic environment, and the abrupt change to a basic milieu in duodenum. This physiological adaptation is successful to exploit the intestinal habitat.

Goblet cells: are they an unspecific barrier against Giardia intestinalis or a gate?

Giardiosis is one of the major intestinal parasitic diseases of human beings as well as wild and domesticated animals. Several protective mechanisms against infection have been described. However, specific information about relationship between giardiosis and the increased proliferation of goblet cells (GC) in patients infected with Giardia intestinalis (Syn. G. duodenalis, G. lamblia) is scarce. In this work, we compare and quantify the number of GC, and have inferred their metabolic state in the small intestine of dogs parasitized with Giardia intestinalis compared to dogs without parasites. Small intestine segments were processed using routine methods for histology and electron microscopy; areas and cells were screened with an Axiovision Ver. 4.0 system. Data were analyzed by ANOVA and comparison of averages. Parasitized dogs showed higher GC numbers than nonparasitized ones. Averages were: 20+/-0.81 GC/25 microm(2) with independent mucin granules and 11+/-1.53 GC/25 microm(2) that were expelling mucus, compared to 11+/-0.94 GC/25 microm(2) and 1+/-0.27 GC/25 microm(2), respectively, in nonparasitized dogs (Tukey, p<0.001). The increases in GC number seem to be an unspecific defensive mechanism against Giardia trophozoites. However, we found some evidence supporting that GC hyperplasia could be a prejudicial to epithelial barrier that gives rise to gates allowing for Giardia-tissue invasion.