Peptides derived from phage display libraries as potential neutralizers of Shiga toxin-induced cytotoxicity in vitro and in vivo.

AIMS: To use the phage display technique to develop peptides with the capability to neutralize the cytotoxicity induced by Stx1 and Stx2 toxins produced by Shiga toxin-producing Escherichia coli (STEC). METHODS AND RESULTS: The phage display technique permitted the development of three peptides, named PC7-12, P12-26 and PC7-30, which bind to the globotriaosylceramide (Gb3) receptor for Shiga toxins produced by STEC. Moreover, these peptides were capable of competing efficiently with the Shiga toxins for binding to Gb3. The peptides described herein partially inhibited the Stx-induced cytotoxicity of cell-free filtrates of STEC O157 : H7 and purified Stx toxins in Vero cells. The inhibition of lethality induced by Stx toxins in mice indicated that peptide PC7-30 inhibited the lethality caused by Stx1 (2LD50) in mice. CONCLUSIONS: The phage display technique permitted the development of peptides that inhibited the cytotoxicity induced by Stx toxins in vitro. Peptide PC7-30 inhibited the lethality of Stx1 in vivo; this molecule would be a promising candidate for the development of therapeutic agents for STEC-related diseases in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of Gb3, the common receptor for Stx1 and Stx2, may contribute to the development of efficient neutralizers for both toxins, and our approach would be an interesting alternative for the development of therapeutic molecules for the treatment of diseases caused by STEC strains.

Detection of virulence genes in Escherichia coli strains isolated from diarrheic and healthy feces of dairy calves in Brazil

The aim of this work was to test 101 strains of E. coli for virulence factors associated with enterotoxigenic and enterohemorrhagic pathotypes of E. coli isolated from diarrheic and non-diarrheic calves. The virulence factors of E. coli Stx1 (Shiga toxin), Stx2, Ehly (Enterohemolysin), the eae gene, LT-II (heat-labile enterotoxin), STa (heat-stable toxin), and adhesins K99 and F41 were detected by PCR. Serogroups were determined by serological methods and Stx production was observed by biological assays in Vero cells. The frequency of the eae gene was higher in isolates from diarrheic calves (35/58, 60.3%) than in non-diarrheic calves (8/43, 18.6%; P 0.001). The gene for Stx1 occurred at high frequencies in the diarrheic strains (24/58, 41.3%) as well as in non-diarrheic (19/43, 44.2%) ones and all strains that were Stx positive by PCR showed cytotoxicity in Vero cells. Stx2 was found in ten strains, Ehly in eight strains, and LT-II in only two strains. Twenty-eight strains were negative for all of the PCR assays, including for F41 and K99 adhesins. The serogroups O7, O23, O4, O8, O153 and O156 were observed most frequently. Our results show that strains of E. coli isolated from cattle have similar virulence factors genes to strains isolated from cases of diseases in humans and may be a source of potentially pathogenic STEC for humans.

O objetivo deste trabalho foi detectar em 101 amostras de E. coli isoladas de bezerros com e sem diarreia, fatores de virulência associados aos patotipos de E. coli enterotoxigênica e enterohemorrágica. Os fatores de virulência de E. coli Stx1 (Shiga toxina), Stx2, Ehly (Enterohemolisina), o gene eae, LT-II (enterotoxina termo-lábil), STa (toxina termo-estável), e adesinas K99 e F41 foram detectados por PCR. Os sorogrupos foram determinados por métodos sorológicos e a produção de Stx foi observada através de ensaios biológicos em células Vero. A frequência de detecção do gene eae foi maior nos isolados de bezerros com diarreia (35/58, 60,3%) do que em bezerros saudáveis (8/43, 18,6%; P 0.001). O gene da toxina Stx1 foi detectado em alta frequência em amostras diarreicas (24/58, 40,3%), bem como em amostras não diarréicas (19/43, 44,2%) e todas as amostras positivas para toxina Stx em PCR mostraram citotoxicidade em células Vero. Stx2 foi encontrada em dez amostras, Ehly em oito amostras, e LT-II em duas amostras. Vinte e seis amostras foram negativas para todos os ensaios de PCR, incluindo para as adesinas F41 e K99. Os sorogrupos O7, O23, O4, O8, O153 e O156 foram detectados com maior frequência. O trabalho mostra que amostras de E. coli isoladas de bovinos apresentam fatores de virulência semelhantes à isolados de casos de doenças em humanos e possivelmente é uma fonte para STEC potencialmente patogênicas para humanos.

Detection of virulence genes in escherichia coli strains isolated from diarrheic and healthy faces of dairy calves in Brazil / Detecção de genes de virulência em amostras de Escherichia coli isoladas de fezes de bezerros com a diarréia no Brasil

The aim of this work was to test 101 strains of E. coli for virulence factors associated with enterotoxigenic and enterohemorrhagic pathotypes of E. coli isolated from diarrheic and non-diarrheic calves. The virulence factors of E. coli Stx1 (Shiga toxin), Stx2, Ehly (Enterohemolysin), the eae gene, LT-II (heat-labile enterotoxin), STa (heat-stable toxin), and adhesins K99 and F41 were detected by PCR. Serogroups were determined by serological methods and Stx production was observed by biological assays in Vero cells. The frequency of the eae gene was higher in isolates from diarrheic calves (35/58, 60.3%) than in non-diarrheic calves (8/43, 18.6%; P < 0.001). The gene for Stx1 occurred at high frequencies in the diarrheic strains (24/58, 41.3%) as well as in non-diarrheic (19/43, 44.2%) ones and all strains that were Stx positive by PCR showed cytotoxicity in Vero cells. Stx2 was found in ten strains, Ehly in eight strains, and LT-II in only two strains. Twenty-eight strains were negative for all of the PCR assays, including for F41 and K99 adhesins. The serogroups O7, O23, O4, O8, O153 and O156 were observed most frequently. Our results show that strains of E. coli isolated from cattle have similar virulence factors genes to strains isolated from cases of diseases in humans and may be a source of potentially pathogenic STEC for humans.(AU)

O objetivo deste trabalho foi detectar em 101 amostras de E. coli isoladas de bezerros com e sem diarreia, fatores de virulência associados aos patotipos de E. coli enterotoxigênica e enterohemorrágica. Os fatores de virulência de E. coli Stx1 (Shiga toxina), Stx2, Ehly (Enterohemolisina), o gene eae, LT-II (enterotoxina termo-lábil), STa (toxina termo-estável), e adesinas K99 e F41 foram detectados por PCR. Os sorogrupos foram determinados por métodos sorológicos e a produção de Stx foi observada através de ensaios biológicos em células Vero. A frequência de detecção do gene eae foi maior nos isolados de bezerros com diarreia (35/58, 60,3%) do que em bezerros saudáveis (8/43, 18,6%; P < 0.001). O gene da toxina Stx1 foi detectado em alta frequência em amostras diarreicas (24/58, 40,3%), bem como em amostras não diarréicas (19/43, 44,2%) e todas as amostras positivas para toxina Stx em PCR mostraram citotoxicidade em células Vero. Stx2 foi encontrada em dez amostras, Ehly em oito amostras, e LT-II em duas amostras. Vinte e seis amostras foram negativas para todos os ensaios de PCR, incluindo para as adesinas F41 e K99. Os sorogrupos O7, O23, O4, O8, O153 e O156 foram detectados com maior frequência. O trabalho mostra que amostras de E. coli isoladas de bovinos apresentam fatores de virulência semelhantes à isolados de casos de doenças em humanos e possivelmente é uma fonte para STEC potencialmente patogênicas para humanos.(AU)

Detection of virulence genes in escherichia coli strains isolated from diarrheic and healthy faces of dairy calves in Brazil / Detecção de genes de virulência em amostras de Escherichia coli isoladas de fezes de bezerros com a diarréia no Brasil

The aim of this work was to test 101 strains of E. coli for virulence factors associated with enterotoxigenic and enterohemorrhagic pathotypes of E. coli isolated from diarrheic and non-diarrheic calves. The virulence factors of E. coli Stx1 (Shiga toxin), Stx2, Ehly (Enterohemolysin), the eae gene, LT-II (heat-labile enterotoxin), STa (heat-stable toxin), and adhesins K99 and F41 were detected by PCR. Serogroups were determined by serological methods and Stx production was observed by biological assays in Vero cells. The frequency of the eae gene was higher in isolates from diarrheic calves (35/58, 60.3%) than in non-diarrheic calves (8/43, 18.6%; P < 0.001). The gene for Stx1 occurred at high frequencies in the diarrheic strains (24/58, 41.3%) as well as in non-diarrheic (19/43, 44.2%) ones and all strains that were Stx positive by PCR showed cytotoxicity in Vero cells. Stx2 was found in ten strains, Ehly in eight strains, and LT-II in only two strains. Twenty-eight strains were negative for all of the PCR assays, including for F41 and K99 adhesins. The serogroups O7, O23, O4, O8, O153 and O156 were observed most frequently. Our results show that strains of E. coli isolated from cattle have similar virulence factors genes to strains isolated from cases of diseases in humans and may be a source of potentially pathogenic STEC for humans.

O objetivo deste trabalho foi detectar em 101 amostras de E. coli isoladas de bezerros com e sem diarreia, fatores de virulência associados aos patotipos de E. coli enterotoxigênica e enterohemorrágica. Os fatores de virulência de E. coli Stx1 (Shiga toxina), Stx2, Ehly (Enterohemolisina), o gene eae, LT-II (enterotoxina termo-lábil), STa (toxina termo-estável), e adesinas K99 e F41 foram detectados por PCR. Os sorogrupos foram determinados por métodos sorológicos e a produção de Stx foi observada através de ensaios biológicos em células Vero. A frequência de detecção do gene eae foi maior nos isolados de bezerros com diarreia (35/58, 60,3%) do que em bezerros saudáveis (8/43, 18,6%; P < 0.001). O gene da toxina Stx1 foi detectado em alta frequência em amostras diarreicas (24/58, 40,3%), bem como em amostras não diarréicas (19/43, 44,2%) e todas as amostras positivas para toxina Stx em PCR mostraram citotoxicidade em células Vero. Stx2 foi encontrada em dez amostras, Ehly em oito amostras, e LT-II em duas amostras. Vinte e seis amostras foram negativas para todos os ensaios de PCR, incluindo para as adesinas F41 e K99. Os sorogrupos O7, O23, O4, O8, O153 e O156 foram detectados com maior frequência. O trabalho mostra que amostras de E. coli isoladas de bovinos apresentam fatores de virulência semelhantes à isolados de casos de doenças em humanos e possivelmente é uma fonte para STEC potencialmente patogênicas para humanos.

Putative new heat-stable cytotoxic and enterotoxic factors in culture supernatant of Escherichia coli isolated from drinking water

Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40 percent) among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60ºC and 100ºC for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2 percent) were positive for ST II (estB) and two (5 percent) positive for αHly (hlyA). Gene amplification of SLT (stx 1, stx 2), ST I (estA), LT (eltI, eltII), EAST1 (astA), EHly (enhly) and plasmid-encoded enterotoxin (pet) were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.(AU)

Putative new heat-stable cytotoxic and enterotoxic factors in culture supernatant of Escherichia coli isolated from drinking water

Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40 percent) among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60¨¬C and 100¨¬C for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2 percent) were positive for ST II (estB) and two (5 percent) positive for ¥áHly (hlyA). Gene amplification of SLT (stx 1, stx 2), ST I (estA), LT (eltI, eltII), EAST1 (astA), EHly (enhly) and plasmid-encoded enterotoxin (pet) were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.

Molecular basis for porcine parvovirus detection in dead fetuses.

Reproductive failures are still common grounds for complaint by commercial swine producers. Porcine parvovirus (PPV) is associated with different clinical reproductive signs. The aim of the present study was to investigate PPV fetal infection at swine farms having ongoing reproductive performance problems. The presence of virus in fetal tissues was determined by nested-polymerase chain reaction assay directed to the conserved NS1 gene of PPV in aborted fetuses, mummies and stillborns. Fetuses show a high frequency of PPV infection (96.4%; n = 28). In 60.7% of the fetuses, PPV were detected in all tissue samples (lung, heart, thymus, kidney, and spleen). Viral infection differed among fetal tissues, with a higher frequency in the lung and heart (p < 0.05). Fetuses with up to 99 days of gestational age and from younger sows showed a higher frequency of PPV (p < 0.05). No significant difference in the presence of PPV was detected among the three clinical presentations. The results suggest that PPV remains an important pathogenic agent associated with porcine fetal death.

Molecular basis for porcine parvovirus detection in dead fetuses

Reproductive failures are still common grounds for complaint by commercial swine producers. Porcine parvovirus (PPV) is associated with different clinical reproductive signs. The aim of the present study was to investigate PPV fetal infection at swine farms having ongoing reproductive performance problems. The presence of virus in fetal tissues was determined by nested-polymerase chain reaction assay directed to the conserved NS1 gene of PPV in aborted fetuses, mummies and stillborns. Fetuses show a high frequency of PPV infection (96.4%; n = 28). In 60.7% of the fetuses, PPV were detected in all tissue samples (lung, heart, thymus, kidney, and spleen). Viral infection differed among fetal tissues, with a higher frequency in the lung and heart (p < 0.05). Fetuses with up to 99 days of gestational age and from younger sows showed a higher frequency of PPV (p < 0.05). No significant difference in the presence of PPV was detected among the three clinical presentations. The results suggest that PPV remains an important pathogenic agent associated with porcine fetal death.

A low molecular weight enterotoxic hemolysin from clinical Enterobacter cloacae.

Seven of 50 Enterobacter cloacae strains from clinical isolates produced small turbid zones of hemolysis in horse and sheep blood agar plates, and the culture supernatants were also positive for hemolytic activity. The hemolysin was partially purified from the culture supernatant of E. cloacae by ultrafiltration (PM-10 membrane) and extraction with acetone. Semipurified hemolysin was stable to heating (100 degrees C, 30 min) and was soluble in organic solvents (acetone, ethanol, and methanol). The toxin showed no loss of biological activity after treatment with trypsin and was stable to acid treatment at pH 2.0 but not at a pH greater than 7.0. In the rat intestinal loop assay, the hemolysin caused hemorrhagic fluid accumulation and severe histological alterations. These findings indicate that this hemolysin may be a putative virulence factor in E. cloacae infections.

Induction of apoptosis in HT29 human intestinal epithelial cells by the cytotoxic enterotoxin of Aeromonas hydrophila.

The cytotoxic enterotoxin produced by Aeromonas hydrophila is considered to be the main virulence factor in gastrointestinal infections mediated by this pathogen. In this study, we examined the morphological and apoptotic effects of this toxin on HT29 cells, using light and electron microscopy in situ, as well as agarose gel electrophoresis of cell DNA. Cells treated with the cytotoxic enterotoxin became round and lost their polarity as well as their adhesion to each other and to the substrate. Cytoplasmic blebbing and nuclear condensation also occurred. DNA fragmentation was detected by TUNEL labelling and agarose gel electrophoresis. These results show that the cytotoxic enterotoxin of A. hydrophila can induce apoptosis in human intestinal cells in culture.

Ultrastructural study of enteropathogenic Escherichia coli O111ab:H2 infection in an infant with acute diarrhea.

Enteropathogenic Escherichia coli is the most important cause of acute diarrhea in developing countries, specially in infants under one year of age. Enteropathogenic Escherichia coli strains are able to induce profound cytoskeletal alterations in the enterocyte known as attaching and effacing lesions, associated with the formation of cuplike pedestals. We report an Enteropathogenic Escherichia coli O11ab:H2 strain isolated from an infant with acute diarrhea, on the eleventh day of disease, that caused attaching and effacing lesion and penetrated the enterocyte, as well as invaded the HeLa cell tissue culture in vitro and the rabbit ileal loop assay in vivo, in the ultrastructural study. This observation indicates that the severe lesions of the small bowel caused by an enteropathogenic Escherichia coli O111ab:H2 strain can occur even in the early stages of the infection.

Plasmid coding for drug resistance and invasion of epithelial cells in enteropathogenic Escherichia coli 0111:H.

Enteropathogenic Escherichia coli (EPEC) can adhere to, invade and multiply in human epithelial cells. To define the elements required for bacterial invasion, we isolated from an 0111:H- EPEC a 6.6 kb plasmid that is capable of conferring to an avirulent, non-adherent E. coli K12 strain (DK1) the capacity to invade epithelial cells. With this system a dissociation was possible between bacterial invasion and adherence to epithelial cells. Bacteria containing this plasmid synthesise a protein of 32 kDa (pl 4.93) which seemed to be required for cell invasion. The results provide a new basis for strategies to prevent EPEC infections.

Rotavirus excretion in naturally infected pigs with and without diarrhoea.

Seven hundred and fifty faecal samples from piglets ranging from 1 to 60 days old were studied for the presence of group A rotavirus by polyacrylamide gel electrophoresis (PAGE) and by enzyme immunoassay (EIA). From 451 diarrhoeic pigs, 117 (25.94%) were positive for rotavirus and only 45 (15.05%) of 299 pigs without diarrhoea excreted the virus (P < 0.005). When these animals were separated into four age groups with regard to the presence or absence of diarrhoea, it was observed that the excretion of rotavirus was associated with diarrhoea in piglets, both before and after weaning.

Bluetongue virus: production and study of viral antigen for serological diagnosis.

A soluble antigen, produced from the culture supernatant of VERO cells infected with bluetongue virus serotype 4 (BTV-S4) and concentrated by sequential ultrafiltration with membranes with cut-off values 10(3) and 25 x 10(3) NMWP, showed complete identity to standard antigens when compared by agar gel immunodiffusion (AGID) and SDS-PAGE profiles, revealing that the main protein component responsible for the AGID reaction has a molecular weight of about 60 kDa corresponding probably to the NS1 protein.

Teste de coaglutinaçäo para detecçäo de rotavirus / Coagglutination assay for detection of rotaviruses

Resumo: O teste de coaglutinaçäo foi utilizado para a detecçäo de rotavírus em fezes de origem humana e de suínos. Suspensäo de Staphylococcus aureos, produtor de proteina A, foi sensibilizada com uma diluiçäo seriada de antissoro anti-rotavirus do grupo A mostrando que quando foi utilizada a diluiçäo a 1:20, o teste foi capaz de detectar tanto antígeno SA11 como também o extrato fecal, ambos diluídos. Um total de 89 amostras de fezes absorvidas com S. aureus foram testadas por coaglutinaçäo e por um ensaio imunoenzimático. A análise estatística dos resultados obtidos mostrou uma concordância de 0,91 entre os dois testes o que levou-nos a conluir que a coaglutinaçäo é um método simples, rápido, sensível e pouco dispendioso para a detecçäo de rotavírus diretamente do material fecal. Além da utilizaçäo do soro diluído para a sensibilizaçäo de s> aureus ficou demonstrado também que, esta mistura, quando estocada a 4 graus C pode ser utilizada por até 6 meses após o seu preparo, sem implicar em resultados falso-positivos (au)

Comparison of indirect immunofluorescence with agar gel immunodiffusion for the diagnosis of bluetongue virus infection.

1. Sera from 190 cows and from 72 sheep were examined to compare the results obtained with the agar gel immunodiffusion (AGID) and indirect immunofluorescence (IIF) tests for the diagnosis of bluetongue (BT) disease. 2. In the AGID test, 96 of 190 (50.5%) cattle serum samples and 38 of 72 (52.7%) sheep serum samples were positive, for a total of 134 out of 262 (51.1%) sera. In the IIF test, 98 of 190 (51.6%) cattle serum samples and 39 of 72 (54.2%) sheep serum samples were positive, for a total of 137 out of 262 (52.3%) sera. 3. The fluorescence of the IIF test presented a granular cytoplasmic aspect, which in some cells was observed only on the cell membranes. 4. Statistical analysis of the data showed close agreement between the two techniques, regardless of the kind of sera examined. The IIF test showed high sensitivity (93.8% and 92.1%), specificity (91.4% and 88.2%) and positive (91.8% and 89.7%) and negative (93.48% and 90.9%) predictive values for cattle serum and sheep serum, respectively. 5. The results obtained with IIF were comparable to those obtained with the AGID test, indicating that both techniques can be used routinely in epidemiologic studies of BT. However, the IIF offers the additional advantages that it can be used for antibody quantification and for the detection of viral antigens in BT-infected cell lines.

Comparison of indirect immunofluorescencee with agar gel immunodiffusion for the diagnosis of bluetongue virus infection

Sera from 190 cows and from 72 sheep were examined to compare the results obtained with the agar gel immundiffusion (AGIP) and indirect immunofluorescence (IIF) tests for the diagnosis of bluetongue (BT) disease. In the AGIP test, 96 of 190 (50.5%) cattle serum samples and 38 of 72 (52.7%) sheep serum samples were positive, for a total of 134 out of 262 (51.1%) sera. In the IIF test, 98 of 190 (51.6%) cattle serum samples and 39 of 72 (54.2%) sheep serum samples were positive, for a total of 137 out of 262 (52.3%) sera. The fluorescence of the IIF test presented a granular cytoplasmic aspect, which in some cells was observed only on the cell membranes. Statistical analysis of the data showed close agreement between the two techniques, regardless of the kind of sera examined. The IIF test showed high sensitivity (93.8% and 92.1%), specificity (91.4% and 88.2%) and positive (91.8% and 89.7%) and negative (93.48% and 90.9%) predictive values for cattle serum and sheep serum, respectively. The results obtained with obtained with IIF were comparable to those obtained with the AGIP test, indicating that both techniques can be used routinely in epidemiologic studies of BT. However, the IIF offers the additional advantages that it can be used for antibody quantification and for the detection of viral antigens in BT-infected cell lines

Different lethal effects by enzyme-generated triplet indole-3-aldehyde in different Escherichia coli strains.

Strains of Escherichia coli which lack 4-thiouridine (S4U) exhibit a higher survival rate than their wild-type parents which contain S4U after treatment with enzyme-generated triplet indole-3-aldehyde. In a similar manner to results obtained with monochromatic 334 nm UV light, the survival is related to single-strand breakage of DNA in E. coli containing the pBR 322 plasmid. The effects of the excited states generated by an enzymatic system suggest that S4U is an important chromophore in the lethal effects observed. The results also suggest that the energy transferred from triplet indole-3-aldehyde to S4U may also be passed from S4U of t-RNA to DNA, possibly through a singlet oxygen intermediate generated by excited S4U, resulting in a decrease in the survival rate of E. coli containing S4U. These results emphasize the importance of excited states in biological systems.