Cloning and expression of the porcine attaching and effacing-associated (paa) gene of enteropathogenic Escherichia coli.

Porcine enteropathogenic Escherichia coli (PEPEC) produce an outer membrane protein (intimin) called Paa (porcine attaching and effacing-associated), which is involved in the pathogenesis of E. coli in piglets with diarrhea. The paa gene of a PEPEC strain isolated in Paraná, Brazil, was amplified by polymerase chain reaction, sequenced, and cloned into the pTrcHisTOPO2 vector. The deduced amino acid sequence encoded by the paa gene of PEPEC from Paraná, Brazil, showed 99% homology to the sequences from other PEPEC strains. In this study, the overexpression of recombinant Paa (rPaa) using alternative induction strategies was attempted. The auto-induction protocol showed excellent results for rPaa protein production with 0.4% (w/v) lactose. The rPaa protein is insoluble and was purified with Triton X-100 wash as a total antigen. This method produced a relatively high yield of rPaa. rPaa was recognized by serum from pigs immunized with the PEPEC strain. These results suggest that rPaa could be included in the development of a vaccine against swine colibacillosis.

Implications of extubation failure and prolonged mechanical ventilation in the postoperative period following elective intracranial surgery

Patients undergoing neurosurgery are predisposed to a variety of complications related to mechanical ventilation (MV). There is an increased incidence of extubation failure, pneumonia, and prolonged MV among such patients. The aim of the present study was to assess the influence of extubation failure and prolonged MV on the following variables: postoperative pulmonary complications (PPC), mortality, reoperation, tracheostomy, and duration of postoperative hospitalization following elective intra-cranial surgery. The study involved a prospective observational cohort of 317 patients submitted to elective intracranial surgery for tumors, aneurysms and arteriovenous malformation. Preoperative assessment was performed and patients were followed up for the determination of extubation failure and prolonged MV (>48 h) until discharge from the hospital or death. The occurrence of PPC, incidence of death, the need for reoperation and tracheostomy, and the length of hospitalization were assessed during the postoperative period. Twenty-six patients (8.2 percent) experienced extubation failure and 30 (9.5 percent) needed prolonged MV after surgery. Multivariate analysis showed that extubation failure was significant for the occurrence of death (OR = 8.05 [1.88; 34.36]), PPC (OR = 11.18 [2.27; 55.02]) and tracheostomy (OR = 7.8 [1.12; 55.07]). Prolonged MV was significant only for the occurrence of PPC (OR = 4.87 [1.3; 18.18]). Elective intracranial surgery patients who experienced extubation failure or required prolonged MV had a higher incidence of PPC, reoperation and tracheostomy and required a longer period of time in the ICU. Level of consciousness and extubation failure were associated with death and PPC. Patients who required prolonged MV had a higher incidence of extubation failure.

Implications of extubation failure and prolonged mechanical ventilation in the postoperative period following elective intracranial surgery.

Patients undergoing neurosurgery are predisposed to a variety of complications related to mechanical ventilation (MV). There is an increased incidence of extubation failure, pneumonia, and prolonged MV among such patients. The aim of the present study was to assess the influence of extubation failure and prolonged MV on the following variables: postoperative pulmonary complications (PPC), mortality, reoperation, tracheostomy, and duration of postoperative hospitalization following elective intra-cranial surgery. The study involved a prospective observational cohort of 317 patients submitted to elective intracranial surgery for tumors, aneurysms and arteriovenous malformation. Preoperative assessment was performed and patients were followed up for the determination of extubation failure and prolonged MV (>48 h) until discharge from the hospital or death. The occurrence of PPC, incidence of death, the need for reoperation and tracheostomy, and the length of hospitalization were assessed during the postoperative period. Twenty-six patients (8.2%) experienced extubation failure and 30 (9.5%) needed prolonged MV after surgery. Multivariate analysis showed that extubation failure was significant for the occurrence of death (OR = 8.05 [1.88; 34.36]), PPC (OR = 11.18 [2.27; 55.02]) and tracheostomy (OR = 7.8 [1.12; 55.07]). Prolonged MV was significant only for the occurrence of PPC (OR = 4.87 [1.3; 18.18]). Elective intracranial surgery patients who experienced extubation failure or required prolonged MV had a higher incidence of PPC, reoperation and tracheostomy and required a longer period of time in the ICU. Level of consciousness and extubation failure were associated with death and PPC. Patients who required prolonged MV had a higher incidence of extubation failure.

[Vital capacity reduction in postoperative of elective craniotomy]. / Disminución de la capacidad vital en el período postoperatorio de la craneotomía electiva.

AIMS: To assess the vital capacity (VC), tidal volume, minute volume and respiratory rate during the first four postoperative days of elective craniotomy and how they are correlated with smoking, associated diseases and respiratory symptoms. PATIENTS AND METHODS: Ninety-four patients were initially evaluated for elective craniotomy and they were included in this study only if they presented normal consciousness level and spontaneous breathing at the first postoperative. The preoperative and postoperative evaluations comprised physical examination and ventilometry up to the fourth postoperative. The repeated measures analysis of variance was used to the ventilation measurements. The significance level adopted for all the statistical tests was p = 0.05. RESULTS: Sixty-two patients were included in this study. There was a 20% fall in the VC from the first to the third postoperative (p = 0.001). Patients with systemic arterial hypertension presented in the preoperative period a lower mean VC (2.59 L) than the patients without (3.28 L) (p = 0.045). Smokers presented a lower mean VC (2.65 and 1.95 L) than the nonsmokers (3.13 and 2.43 L), both in the preoperative and in the postoperative, but with no statistic significance (p = 0.090). CONCLUSION: After elective craniotomy, there is a significant decrease in VC immediately after surgery, improving gradually thereafter; there was no difference in VC between the smoking and nonsmoking patients in the pre- and postoperative as well.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil.

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7.

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 microg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Toxoplasma gondii: evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice.

The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n=11) received 12.5 microg of each recombinant protein plus 0.5 microg of cholera toxin, group 2 (G2, n=11) received phosphate buffer saline (PBS) plus 0.5 microg of cholera toxin, and group 3 (G3, n=11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRA7. Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P<0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P>0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T. gondii.

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Atypical phenotypic characteristics of klebsiella pneumoniae isolates from an outbreak in a neonatal intensive care unit in Brazil

Extended-spectrum beta-Lactamase-producing (ESBL) Klebsiella sp.isolates from an outbreak in a Neonatal Intensive Care Unit (NICU) at a teaching hospital in Londrina, Paraná State, Brazil, presented atypical phenotypic characteristics that hampered their identification and the distinction between Klebsiella and Enterobacter species. Ten isolates were identified as K. pneumoniae due to negative reactions for motility and inducible beta-lactamase test (ESBL and AmpC) despite being positive for ornithyne descarboxilase. These isolates were genotyped by ribotyping and polymerase chain reaction (PCR) with repetitive extragenic palindromic sequences (REP). Ribotyping by means of an automated instrument and EcoRI and Pvu II as restriction enzymes resulted indetection of K. pneumoniae subspecie pneumoniae RIBO1 222-36-S-5 ribotype. Typing by REP-PCR showed that the 17 isolates from the outbreak were highly similar, belonging to one cluster with 100 percent of similarity, and that they presented more than 70 percent of similarity with K. pneumoniae ATCC 13883 and ATCC 10031, and 25 percent of similarity with E. aerogenes CDC 1680. In conclusion, the isolates of the outbreak were identified as Klebsiella pneumoniae, despite presenting ornithyne descarboxilase enzyme, which is an atypical characteristic of this Klebsiella species.

Isolados de Klebsiella sp. produtora de beta-lactamase de espectro estendido (ESBL), responsável por um surto na Unidade Neonatal de Terapia Intensiva (UNTI) do Hospital Universitário de Londrina, Paraná, Brasil apresentaram características fenotípicas atípicas que dificultaram sua identificação e a diferenciação entre as espécies Klebsiella pneumoniae e Enterobacter aerogenes. Dez isolados foram identificados como K. pneumoniae devido às reações negativas para motilidade e produção de enzimas beta-lactamases (ESBL e AmpC). Embora apresentassem teste positivo para ornitina descarboxilase. Estes isolados foram genotipados por ribotipagem e por reação em cadeia da polimerase (PCR) com oligonucleotídeos para "repetitive extragenic palindromic sequences" (REP). A ribotipagem com as enzimas de restrição EcoRI e Pvu II detectou o ribotipo de K. pneumoniae subespécie pneumoniae RIBO1 222-36-S-5. A técnica de REP-PCR mostrou que os isolados do surto foram similares, pertencentes a um grupo com 100 por cento de similaridade, e apresentaram mais de 70 por cento de similaridade com amostras padrão de K. pneumoniae (ATCC 13883 e 10031), e 25 por cento de similaridade com E. aerogenes CDC 1680. Concluindo, os isolados do surto da NICU mostraram se geneticamente relacionados e foram identificados como Klebsiella pneumoniae, embora apresentassem ornitina descarboxilase, característica atípica para esta espécie de Klebsiella.

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil.

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paraná, Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO® vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinantclone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post- immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.

Phylogenetic analysis of Anaplasma marginale strains from Paraná State, Brazil, using the msp1alpha and msp4 genes.

Anaplasma marginale is an obligate intraerythrocytic rickettsial pathogen (order, Rickettsiales: family, Anaplasmataceae) that causes bovine anaplasmosis. This disease is widely distributed in tropical and sub-tropical regions of the world and causes important economic losses to cattle production. Major surface protein (MSP)1a (msp1alpha gene) is one of the six MSPs identified on A. marginale from cattle, whose sequence and size vary according to the number of tandem 28- to 29-amino acid repeats. This study characterized the msp1alpha and msp4 genes obtained from three distinct Brazilian herds from the State of Paraná. Three strains of the msp1alpha and one strain of the msp4 gene were sequenced. The strains evaluated revealed PCR products of different size, representing three, five and six internal repeats. Sequence analyses confirmed the number of tandem sequence copies and revealed a high degree of sequence identity with strains from other Brazilian States, as well as strains from the USA, Europe and Israel. The msp1alpha DNA and amino acid sequences from A. marginale and DNA sequences of msp4 strains did not reveal distinct phylogeographical segregation. However, the amino acid sequences of msp4 demonstrated definite phylogeographical relationship. These results suggest that the amino acid sequences of msp4 should be used for phylogenetic identification of A. marginale strains and may be an important tool for the epidemiology and control of anaplasmosis. Additionally, the close similarity of the Paraná strains of A. marginale with strains from USA, Europe and Asia may reflect the introduction of these genes during the development of the Brazilian bovine herd.

Immunization of bovines using a DNA vaccine (pcDNA3.1/MSP1b) prepared from the Jaboticabal strain of Anaplasma marginale.

Anaplasma is a tick-borne ehrlichial pathogen of cattle that causes the disease, anaplasmosis. In the present study, a total of 11 Anaplasma marginale seronegative calves were assigned into two groups: one immunized (G1, n = 6) and one nonimmunized-control (G2, n = 5). Six calves were immunized by using a DNA vaccine containing the gene of a major surface protein, MSP1b, encoded by the plasmid identified as pcDNA3.1/MSP1b. Calves received three intramuscular inoculations of 100 microg of pcDNA3.1/MSP1b at a 20-day interval. The control group received buffer phosphate at the same schedule as the experimental group. The immune response elicited by immunization with pcDNA3.1/MSP1b was evaluated in mice and calves. Twenty days following initial immunization, specific serum antibody from four BALB/c mice bound MSP1b in immunoblots. Sixty days after the last immunization, all calves were challenged with cryopreserved A. marginale at a dose of 10(4) parasites/mL/animal by intravenous injection. Results of packed cell volume (PCV) and detection of infected erythrocytes in all experimental groups revealed that the decrease of PCV and detection of infected erythrocytes occurred at 28 to 42 days after challenge. Mean temperature values did not increase over 39.85 degrees C. Antibodies developed by immunized bovines from G2 were detected 14 days after challenge. MSP1b was characterized during the immunization period and MSP2 was the most predominant polypeptide at the challenge period. DNA of A. marginale was detected in all groups just after challenge by nested PCR assay. It can be concluded that all immunized bovines were partially protected against homologous challenge.

Early membrane exposure of phosphatidylserine followed by late necrosis in murine macrophages induced by Candida albicans from an HIV-infected individual.

The hypothesis that Candida albicans isolate (CR1) from an HIV-infected individual induced apoptosis of macrophages was examined by optical microscopy, binding of annexin V-FITC and analyses of DNA degradation (TUNEL tests and agarose gel electrophoresis). Resident murine peritoneal macrophages co-incubated for 5-15 min with C. albicans CR1 bound annexin V, whereas macrophages incubated with either heat-inactivated strain CR1, C. albicans 577 (isolated from a patient with mucocutaneous candidiasis) or C. albicans FCF14 (a mutant that did not produce proteases and phospholipases) did not bind annexin for up to 2 h of observation. However, macrophages exposed to C. albicans CR1 did not present the pattern of DNA degradation typical of apoptosis. Macrophages became increasingly permeable to propidium iodide from 30 min to 2 h after their exposure to C. albicans CR1. Most of the phagocytosed C. albicans CR1 yeast cells switched to germ-tubes inside the macrophages after incubation for 1-2 h. These results show that macrophages exposed to C. albicans CR1 presented early signs of apoptosis but progressed to necrosis, and suggest that Candida strains that readily switch to germ-tubes inside those apoptotic cells might have a competitive advantage in vivo because released germ-tubes resist further attack by macrophages.

Antigenic characterization of Anaplasma marginale isolates from different regions of Brazil.

Antigenic characterization of A. marginale isolates has contributed to identifying the presence of common and restricts epitopes of major surface proteins (MSPs). The data may improve vaccine development to protect against A. marginale isolates from different regions. Brazilian A. marginale isolates were characterized antigenically by Western blot with monoclonal antibodies (MAbs) against MSPs and rabbit anti-MSP-4 from Florida strain. Six A. marginale isolates from MS, MG (AUFV1), SP, PR-L1, PR-HV, RS and Florida strain were tested with ANA22B1 to MSP-1a, AMR36A6 to MSP-1b, ANAF19E2 to MSP-2, AMG75C1 and AMG76B2 to MSP-3 and ANAF16C1 to MSP-5. ANA22B1 recognized MSP-1a epitope in all A. marginale isolates, and reacted with polypeptides of different size ranging 46-105kDa. MSP2 was not detected in MS and SP isolates by ANAF19E2, and only PR-L1 and MG (AUFV1) isolates reacted with MAbs which recognize MSP3 epitope. MSP4 and MSP5 were detected in all A. marginale isolates analyzed. The results revealed conservation of MSP-1a and MSP-5 epitopes among all Brazilian isolates, and showed antigenic variability to MSP-1b, MSP-2 and MSP-3 proteins, agreeing with recent data about the genetic diversity found in the polimorphic multigene family responsible for these proteins.

Genetic variability of avian Escherichia coli strains evaluated by enterobacterial repetitive intergenic consensus and repetitive extragenic palindromic polymerase chain reaction.

In this study, we tested the capability of enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) to detect genetic diversity among Escherichia coli strains isolated from chickens bearing clinical signs of colibacillosis and compared the genotypes so obtained with the O:H serotypes and virulence of those strains. The DNAs from 50 avian E. coli strains and from E. coli ATCC 25922 were used to amplify ERIC and REP sequences. DNA from avian strains produced from 8 to 17 bands by ERIC-PCR and from 6 to 20 bands by REP-PCR; E. coli ATCC produced 11 bands by both methods. ERIC and REP-PCR showed good discriminating power, and the dendograms based on the different patterns revealed extensive genetic diversity among the avian strains. Those strains were allocated into four major clonal clusters, each one with 60% of similarity by ERIC and REP-PCR, and those clusters corresponded to strains with different degrees of pathogenicity. However, 56% of the pathogenic strains (28/50) belonged to two out of three major clonal clusters, and 86% of the nonpathogenic strains tended to group in one cluster and one subgroup. The 32 serotypes detected were distributed in all clusters, and within a serogroup, different DNA fingerprints were observed; however, strains with same serotypes tended to form clusters with similarity coefficients greater than 80%. These results suggest that no specific serotype and genotype is responsible for colibacillosis and that REP and ERIC-PCR are reproducible techniques that can improve the studies needed to clarify the pathways to the pathogenesis of colibacillosis.

Use of repetitive DNA elements to define genetic relationships among Anaplasma marginale isolates.

Anaplasma marginale genomic DNA was tested for the presence of repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC)-like sequences in order to evaluate the genetic diversity of multiple A. marginale isolates. A. marginale isolates were obtained from cattle of six different states of Brazil, from the US and an Anaplasma centrale strain was obtained from Uruguay. Patterns obtained from A. marginale isolates varied from 14 to 17 fragments by REP-polymerase chain reaction (PCR) and 6 to 14 fragments by ERIC-PCR. All A. marginale isolates presented a 0.75-kb fragment by REP and two common fragments (0.38 and 1.0 kb) by ERIC-PCR. These two fragments were not detectable in A. centrale. Both methods produced similar patterns (80%) among A. marginale isolates obtained from the same region, although some isolates within regions shared less similarity. Isolates from Parana and Pernambuco, were differentiated by these methods. The study demonstrates the presence of ERIC and REP-like elements in A. marginale isolates and shows that A. marginale isolates and strains can be differentiated by these methods.

Prevalence of HEp-2 cell-adherent Escherichia coli and characterisation of enteroaggregative E. coli and chain-like adherent E. coli isolated from children with and without diarrhoea, in Londrina, Brazil.

A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells. Localised adherence was found only in isolates from cases. Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls. The AA isolates were tested for gene sequences associated with enteroaggregative E. coli (EAEC). Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively. The aggA gene was not found, although 7.9% were positive for aggC. The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively. The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.

Apoptosis of murine peritoneal macrophages induced by an avian pathogenic strain of Escherichia coli.

The mechanisms used by avian strains of Escherichia coli to invade the respiratory epithelia, leading to septicemia in poultry, are not well-established. In this work, we show that resident murine peritoneal macrophages infected in vitro with an avian strain of E. coli underwent apoptosis 4 h after infection (55.6% of apoptosis in infected cells versus 3.5% in non-infected cells). Heat-inactivated bacteria did not induce apoptosis and the inhibition of phagocytosis by pretreatment of cells with cytochalasin D reduced the number of apoptotic cells from 55.6 to 13.9% (P<0.05), showing that the bacteria must be intracellularly located and viable to induce apoptosis. Therefore, these data suggest that induction of macrophage apoptosis may be a pathogenic mechanism employed by avian E. coli to circumvent the host defences and invade the respiratory epithelia.