Detection and counting of Cryptosporidium parvum in HCT-8 cells by flowcytometry.

The objective of the present study was to evaluate flowcytometry analysis (FCA) as a tool for rapidly and objectively estimating the percentage of cells infected with Cryptosporidium parvum in an in vitro model. We compared the results to those obtained with immunofluorescence assay (IFA) and evaluated the intra-assay variability of both assays and the inter-assay variability of IFA. Human ileocecal adenocarcinoma cells (HCT-8) were infected with different doses of excysted oocysts. After 24 hours, cells were analysed by FCA and by IFA using a monoclonal antibody that recognises a C. parvum antigenic protein and a lectin that binds with glycoproteins present in the parasitophorous vacuoles. The coefficient of variability in terms of the percentage of infected cells was lower for FCA (i.e., 13-14 %) than for IFA (i.e., 27-38 % when performed by a single operator and 19-22 % when performed by three operators), suggesting that FCA is more accurate, in that it is not subject to operator expertise. FCA also has the advantage of allowing the entire culture to be examined, thus avoiding problems with heterogeneity among microscopic fields. In light of these results, this method could also be used to test new anti-Cryptosporidium drugs

Proteomics Analysis and Protein Expression during Sporozoite Excystation of Cryptosporidium parvum (Coccidia, Apicomplexa)

Cryptosporidiosis, caused by coccidian parasites of the genus Cryptosporidium, is a major cause of human gastrointestinal infections and poses a significant health risk especially to immunocompromised patients. Despite intensive efforts for more than 20 years, there is currently no effective drug treatment against these protozoa. This study examined the zoonotic species Cryptosporidium parvum at two important stages of its life cycle: the non-excysted (transmissive) and excysted (infective) forms. To increase our understanding of the molecular basis of sporozoite excystation, LC-MS/MS coupling with a stable isotope N-terminal labeling strategy using iTRAQ (TM) reagents was used on soluble fractions of both non-excysted and excysted sporozoites, i.e. sporozoites both inside and outside oocysts were examined. Sporozoites are the infective stage that penetrates small intestinal enterocytes. Also to increase our knowledge of the C. parvum proteome, shotgun sequencing was performed on insoluble fractions from both non-excysted and excysted sporozoites. In total 303 C. parvum proteins were identified, 56 of which, hitherto described as being only hypothetical proteins, are expressed in both excysted and non-excysted sporozoites. Importantly we demonstrated that the expression of 26 proteins increases significantly during excystation. These excystation-induced proteins included ribosomal proteins, metabolic enzymes, and heat shock proteins. Interestingly three Apicomplexa-specific proteins and five Cryptosporidium-specific proteins augmented in excysted invasive sporozoites. These eight proteins represent promising targets for developing vaccines or chemotherapies that could block parasite entry into host cells

Prevalence and genetic characterization of Dientamoeba fragilis in asymptomatic children attending daycare centers

In order to provide additional data on the prevalence and genetic diversity of Dientamoeba fragilis in human populations, we conducted a study in children from low-income communities in Sao Paulo State, Brazil. Fecal samples from daycare center attendees up to 6 years old (n=156) and staff members (n=18) were submitted to PCR and sequencing of D. fragilis as well as to microscopic examination for the presence of other intestinal parasites. All children assessed were asymptomatic and 10.3% (16/156) were positive for D. fragilis. No worker was found to be positive. An association between Dientamoeba and coinfection with other intestinal parasites was observed. Concerning the genetic diversity, 14 and only two isolates were genotype 1 and genotype 2, respectively. Our findings outline interesting aspects: (1) asymptomatic children as carriers of Dientamoeba in communities in which environmental conditions ensure parasite transmission and, (2) association between Dientamoeba infection in young children and coinfection with other enteric parasites, reinforcing its transmission via the fecal–oral route

Molecular Characterization Of Intestinal Protozoa In Two Poor Communities In The State Of São Paulo, Brazil.

Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.810

Molecular characterization of intestinal protozoa in two poor communities in the state of Sao Paulo, Brazil

Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tiete in the State of Sao Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis8COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2011/52100-

Detection and counting of Cryptosporidium parvum

The objective of the present study was to evaluate flowcytometry analysis (FCA) as a tool for rapidly and objectively estimating the percentage of cells infected with Cryptosporidium parvum in an in vitro model. We compared the results to those obtained with immunofluorescence assay (IFA) and evaluated the intra-assay variability of both assays and the inter-assay variability of IFA. Human ileocecal adenocarcinoma cells (HCT-8) were infected with different doses of excysted oocysts. After 24 hours, cells were analysed by FCA and by IFA using a monoclonal antibody that recognises a C. parvum antigenic protein and a lectin that binds with glycoproteins present in the parasitophorous vacuoles. The coefficient of variability in terms of the percentage of infected cells was lower for FCA (i.e., 13-14 %) than for IFA (i.e., 27-38 % when performed by a single operator and 19-22 % when performed by three operators), suggesting that FCA is more accurate, in that it is not subject to operator expertise. FCA also has the advantage of allowing the entire culture to be examined, thus avoiding problems with heterogeneity among microscopic fields. In light of these results, this method could also be used to test new anti-Cryptosporidium drugs

Cryptosporidium parvum at Different Developmental Stages Modulates Host Cell Apoptosis In Vitro

We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected with Cryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show that C. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression

Multiple-Antibiotic Resistance Mediated by Structurally Related IncL/M Plasmids Carrying an Extended-Spectrum β-Lactamase Gene and a Class 1 Integron

A conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella enterica serotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extended-spectrum beta-lactamase gene, bla(SHV-5), was identified 3.5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb bla(SHV-5)–In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expanded-spectrum cephalosporins and aminoglycosides

Antibiotic Resistance Conferred by a Conjugative Plasmid and a Class I Integron in Vibrio cholerae O1 El Tor Strains Isolated in Albania and Italy

Multidrug-resistant Vibrio cholerae O1 El Tor strains isolated during the 1994 outbreak of cholera in Albania and Italy were characterized for the molecular basis of antibiotic resistance. All strains were found to be resistant to tetracycline, streptomycin, spectinomycin, trimethoprim, sulfathiazole, and the vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylteridine). Resistance genes were self-transferable by a conjugative plasmid of about 60 MDa, with the exception of spectinomycin resistance, which was conferred by the aadA1 gene cassette located in the bacterial chromosome within a class 1 integron. The resistance to trimethoprim and O/129 was conferred by the dfrA1 gene, which was present on the plasmid. Although the dfrA1 gene is known to be borne on an integron cassette, class 1, 2, or 3 intI genes were not detected as part of the plasmid DNA from the strains studied