Isolation of atypical enteropathogenic Escherichia coli from chicken and chicken-derived products.

Atypical enteropathogenic Escherichia coli (EPEC) strains from chicken and chicken-derived products were isolated and characterised. The strains presented a wide variety of serotypes, some have been reported in other animal species (O2:H40, O5:H40) and in children with diarrhoea (O8:H-). Most of the strains carried intimin ß. The results indicate that chicken and chicken products are important sources of atypical EPEC strains that could be associated with human disease, and highlight the need to improve hygiene practices in chicken slaughtering and meat handling.

Distribution of additional virulence factors related to adhesion and toxicity in Shiga toxin-producing Escherichia coli isolated from raw products in Argentina.

UNLABELLED: A total of 73 Shiga toxin-producing Escherichia coli (STEC) isolates, belonging to 25 serotypes and isolated from raw products in Argentina, were examined for the occurrence of genes responsible for bacterial adhesions to intestine, ehaA (EHEC autotransporter), lpfAO113 (long polar fimbriae), sab (STEC autotransporter [AT] contributing to biofilm formation), ecpA (E. coli common pilus), hcpA (haemorrhagic coli pilus), elfA (E. coli laminin-binding fimbriae), sfpA (sorbitol-fermenting EHEC O157 fimbriae plasmid-encoded) and of the toxigenic gene cdt-V (cytolethal distending toxin). Our study showed different adhesin profiles that are not linked to one specific serotype and that all analysed isolates possess, besides stx genes, some adherence genes. Several of the isolates contained also multiple toxin genes. The results of the present work alert the presence of genes coding for additional adhesins and cdt-V toxin in LEE-negative STEC strains that occur in foods, and this traits could increase their pathogenic potential. SIGNIFICANCE AND IMPACT OF THE STUDY: Meat products are one of the main vehicles of Shiga toxin-producing E. coli, and the presence of genes coding for additional adhesins and toxins could increase their pathogenic potential. There is a need for a more detailed characterization of the strains in regard to these extra virulence factors.

Peritoneal carcinomatosis: imaging with 64-MDCT and 3T MRI with diffusion-weighted imaging.

Peritoneal carcinomatosis is usually associated with a poor overall survival rate. Recently, introduction of more aggressive surgical treatment and intraperitoneal chemotherapy appears to significantly increase the overall survival rate for these patients. A detailed preoperative assessment of peritoneal carcinomatosis could be very challenging in the field of imaging, but a new aggressive surgical approach requires an accurate preoperative assessment of the disease. Cross-sectional imaging using CT and MRI with diffusion-weighted imaging (DWI) sequences is important for appropriate management of patients with peritoneal carcinomatosis. Appreciation of the spectrum of diagnostic patterns and pitfalls as well as different sites of involvement of peritoneal carcinomatosis using CT and DWI is crucial for appropriate surgical treatment.

Multiplex PCR assay for the detection of five putative virulence genes encoded in verotoxigenic Escherichia coli plasmids.

The aim was to perform a pentavalent PCR assay for the detection of putative virulence genes encoded in VTEC plasmids, katP, espP, subA, stcE, and ehxA. The five-specific primer pairs used in the assay do not interfere with each other and generate amplification products of 914, 774, 556, 399, and 262 bp. It was selected at random 39 strains belonged to 20 serotypes in order to evaluate the multiplex in a wide variety of strains. The results of this study indicate that it is possible to perform simultaneous amplification and search for recognized plasmid-encoded virulence markers from different E. coli serotypes and apply this technique to the genetic characterization of E. coli strains isolated from reservoirs, foods or patients. This complementary technique is a useful tool to detect interstrain differences for epidemiological studies and to provide information that could be related to the risk of human infection.

Enteropathogenic Escherichia coli contamination at different stages of the chicken slaughtering process.

Enteropathogenic Escherichia coli is a foodborne pathogen that produces potentially fatal infant diarrhea, noticeably in developing countries. The aim of this study was to detect EPEC contamination by PCR at different stages of the chicken slaughtering process. We collected swabs from chicken cloacae and washed carcasses (external and visceral cavity) during the slaughtering process in 3 sampling occasions. Unwashed eviscerated carcasses were also sampled (at the visceral cavity) in the second and third sampling occasions. Enteropathogenic Escherichia coli was detected in 6 to 28% of cloacal samples, 39 and 56% of unwashed eviscerated carcasses, and 4 to 58% of washed carcasses. None of the samples were positive for bfpA, suggesting contamination with atypical EPEC. The detection of EPEC at different stages of the chicken slaughtering process showed that the proportion of contaminated samples remained or even increased during processing. In addition, the high proportion of contaminated carcasses during chicken processing represents a risk for the consumers and a challenge to improve procedures for those working in the sanitary control service.

ArgO145, a Stx2a prophage of a bovine O145:H- STEC strain, is closely related to phages of virulent human strains.

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H- strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145.

Molecular epidemiology of Shiga toxin-producing O113:H21 isolates from cattle and meat.

The serotype O113:H21 is considered one of the relevant non-O157 STEC serotypes associated with severe human infections. Due to the increased detection of O113 strains and their relationship with clinical cases, which emphasizes the importance of this serogroup as an emerging pathogen, our aim was to determine the characteristics of STEC O113:H21 strains circulating in bovine cattle and retail meat from Argentina. For this purpose, we determined the presence and combinations of various virulence genes (and their variants) related to adhesion and toxicity in a collection of 34 isolates. Their genetic relatedness using multiple-locus variable-number tandem repeat analysis (MLVA) was also studied. Subtyping of stx genes indicated that O113:H21 strains circulating in Argentina mainly present stx2a alone or together with stx2c or, less frequent, with stx2d , all of which are subtypes associated with human disease. We found plasmid markers, such as saa, ehxA and subA, in a higher proportion than previous studies, and five variants of saa, two of which were novel ones. In relation to MLVA subtyping, we detected a limited diversity among the isolates considering that several loci were not discriminative and, that in some farms, the same clone seemed to remain circulating throughout the year. The O113:H21 strains studied harbour several toxin and adhesion genes (saa, espP, fimCD, ehaA, iha, hcpA, elfA, lpfO113, ecpA, subA, cdt-V) and Stx subtypes associated with human disease. Results also highlighted that subtyping of stx and saa is useful to discriminate O113:H21 strains that share virulence genes. In conclusion, this study shows that a number of O113:H21 strains that occur in foods and bovines could be pathogenic for humans. This situation calls for further attention in the prevention and control of foodborne disease caused by these strains.

Focal adhesion kinase is involved in angiotensin II-mediated protein synthesis in cultured vascular smooth muscle cells.

The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal-regulated kinase (ERK) 1/2 and p70(S6K) pathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70(S6K) pathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II-dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II-induced ERK1/2 activation, but p70(S6K) activation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II-induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II-induced FAK phosphorylation and ERK1/2 activation, but not p70(S6K) phosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II-induced ERK1/2 activation, c-fos induction, and enhanced protein synthesis in vascular smooth muscle cells.

Cellular biomarkers for monitoring estuarine environments: transplanted versus native mussels.

In developed countries, estuarine environments are often subjected to chemical pollution, whose biological impact is profitably evaluated by the use of multi-biomarker approaches on sentinel species. In this paper, we investigate genotoxicity and lysosomal alterations in the Mediterranean mussel (Mytilus galloprovincialis), from the estuary of the River Cecina (Tuscany, Italy), selected as "pilot basin" within the Water Frame Directive (2000/60 European Community). Both native and 1 month transplanted mussels were used in order to compare these two approaches in terms of sensitiveness of specific biomarker responses. Genotoxic effects were evaluated as strand breaks, by single cell gel electrophoresis (or Comet assay), and as chromosomal alterations, by the micronucleus test in gill cells. Lysosomal alterations were assessed by the neutral red retention time (in haemocytes), lipofuscin accumulation and ultrastructure (in digestive cells). Heavy metal bioaccumulation was also analysed. Mussels from the River Cecina showed a general alteration of all the biomarkers investigated, accompanied by an elevation of tissue metal levels. However, some differences in specific responses occurred between transplanted and native mussels. Early biomarkers, such as those based on DNA and lysosomal membrane integrity, were induced at similar degree in native and transplanted mussels; while alterations resulting from cumulative events, as the increase of micronuclei frequency were much more elevated in native specimens (23.1+/-7.6) than in transplanted (9.3+/-4.7) and reference ones (5.8+/-5.2). Similarly, the comparison between lipofuscin accumulation and mean lysosomal diameter in impacted and control sites, gave significant differences exclusively with transplanted mussels. These results suggest that the parallel use of caged and native mussels in environmental biomonitoring can improve the characterization of the study area.

Genetic variation in angiotensin-converting enzyme does not prevent development of cardiac hypertrophy or upregulation of angiotensin II in response to aortocaval fistula.

BACKGROUND: Experimental and clinical evidence suggests that angiotensin II may be an important mediator of cardiac hypertrophy in response to hemodynamic stress. We investigated the effect of genetic variation in angiotensin-converting enzyme (ACE) on the development of cardiac hypertrophy and left ventricular (LV) dysfunction in response to volume overload. METHODS AND RESULTS: Male heterozygous ACE knockout (1/0) and wild-type (1/1) mice were studied 4 weeks after the creation of an aortocaval fistula (ACF). The LV weight/body weight ratio increased 74% in ACF versus sham-operated control mice but did not differ between genotypes. Echocardiographic circumferential stress versus rate-corrected velocity of circumferential shortening curves demonstrated depressed LV function in ACF versus sham-operated mice but no difference between genotypes. LV ACE activity was higher in 1/1 versus 1/0 mice and in ACF versus sham-operated mice, and it increased significantly more in the 1/1 versus the 1/0 mice after ACF (P<0.001 for effect of genotype, ACF/sham operation, and interaction term). LV angiotensin II was higher in ACF versus sham-operated mice but did not differ between genotypes, despite 3-fold higher LV ACE activity in ACF 1/1 versus ACF 1/0 mice. CONCLUSIONS: ACE underexpression does not prevent cardiac hypertrophy or LV dysfunction in response to volume overload. LV angiotensin II is unaffected by ACE genotype, both at baseline and after volume overload, indicating that the heart can maintain angiotensin II levels across a broad range of genetic ACE variation under both physiological and pathophysiological conditions.

Regulation of sodium-hydrogen exchange in vascular smooth muscle.

The Na+/H+ exchanger in vascular smooth muscle cells represents a major mechanism for sodium influx and is also one of the principal mechanisms responsible for the regulation of intracellular pH (pHi). In this review, the relationship between pHi and vascular smooth muscle cell growth, the regulation of Na+/H+ exchange by vasoactive agents and growth factors, and the second messenger pathways that may be involved in activation of Na+/H+ exchange have been discussed. The exchanger appears to be important in vascular smooth muscle cell growth, based on results that (1) Na+/H+ exchange is stimulated by hypertrophic and hyperplastic agonists, (2) vascular smooth muscle cell proliferation is induced by cytoplasmic alkalinisation in the absence of mitogens, (3) vascular smooth muscle cell proliferation is dependent on extracellular sodium, and (4) inhibitors of Na+/H+ exchange block cell growth. Several pathways appear capable of activating the exchanger in vascular smooth muscle cells as there is evidence for both calcium and protein kinase C dependent and independent pathways. We speculate that the calcium and protein kinase C dependent pathways may play a role in the contractile response of differentiated vascular smooth muscle cells in the vessel wall, while the calcium and protein kinase C independent pathways may be involved in the proliferative response observed after arterial injury and in tissue culture.

Does the crystal habit modulate the genotoxic potential of silica particles? A cytogenetic evaluation in human and murine cell lines.

Crystalline silica inhaled from occupational sources has been classified by IARC as carcinogenic to humans; in contrast, for amorphous silica, epidemiological and experimental evidence remains insufficient. The genotoxicity of crystalline silica is still debated because of the inconsistency of experimental results ("variability of silica hazard"), often related to the features of the particle surfaces. We have assessed the role of crystal habit in the genotoxicity of silica powders. Pure quartz (crystalline) and vitreous silica (amorphous), sharing the same surface features, were used in an in vitro study with human pulmonary epithelial (A549) and murine macrophage (RAW264.7) cell lines, representative of occupational and environmental exposures. Genotoxicity was evaluated by the comet and micronucleus assays, and cytotoxicity by the trypan blue method. Cells were treated with silica powders for 4 and 24h. Quartz but not vitreous silica caused cell death and DNA damage in RAW264.7 cells. A549 cells were relatively resistant to both powders. Our results support the view that crystal habit per se plays a pivotal role in modulating the biological responses to silica particles.

MLVA typing of Mycoplasma hyopneumoniae bacterins and field strains.

Because of the lack of information about both the genetic characteristics of Mycoplasma hyopneumoniae commercial vaccines and their relationship with field strains, the authors attempted to identify genetic subtypes of some M hyopneumoniae bacterins, and to compare them with M. hyopneumoniae field strains. Six commercial M hyopneumoniae bacterins and 28 bronchoalveolar lavages from pigs at slaughter from three herds were analysed by Multiple-Locus Variable number tandem repeat Analysis (MLVA) on p146R1, p146R3, H4, H5 and p95 loci. The results obtained showed the presence of more than one M hyopneumoniae genotype in some pigs and also in one of the bacterins analysed. It is also worth noting that MLVA typing allowed the distinction among circulating field strains and also when comparing them with vaccine strains, which, knowing the relatedness among them, could be useful in the research of the efficacy of the vaccines.

Genomic and chromosomal damage in the marine mussel Mytilus galloprovincialis: Effects of the combined exposure to titanium dioxide nanoparticles and cadmium chloride.

Titanium dioxide nanoparticles (TiO2-NPs) continuously released into waters, may cause harmful effects to marine organisms and their potential interaction with conventional toxic contaminants represents a growing concern for biota. We investigated the genotoxic potential of nanosized titanium dioxide (n-TiO2) (100 µg L(-1)) alone and in combination with CdCl2 (100 µg L(-1)) in Mytilus galloprovincialis after 4 days of in vivo exposure. RAPD-PCR technique and Micronucleus test were used to study genotoxicity. The results showed genome template stability (GTS) being markedly reduced after single exposure to n-TiO2 and CdCl2. Otherwise, co-exposure resulted in a milder reduction of GTS. Exposure to n-TiO2 was responsible for a significant increase of micronucleated cell frequency in gill tissue, while no chromosomal damage was observed after CdCl2 exposure as well as after combined exposure to both substances.

n-TiO2 and CdCl2 co-exposure to titanium dioxide nanoparticles and cadmium: Genomic, DNA and chromosomal damage evaluation in the marine fish European sea bass (Dicentrarchus labrax).

Due to the large production and growing use of titanium dioxide nanoparticles (n-TiO2), their release in the marine environment and their potential interaction with existing toxic contaminants represent a growing concern for biota. Different end-points of genotoxicity were investigated in the European sea bass Dicentrarchus labrax exposed to n-TiO2 (1mgL(-1)) either alone and combined with CdCl2 (0.1mgL(-1)) for 7 days. DNA primary damage (comet assay), apoptotic cells (diffusion assay), occurrence of micronuclei and nuclear abnormalities (cytome assay) were assessed in peripheral erythrocytes and genomic stability (random amplified polymorphism DNA-PCR, RAPD assay) in muscle tissue. Results showed that genome template stability was reduced after CdCl2 and n-TiO2 exposure. Exposure to n-TiO2 alone was responsible for chromosomal alteration but ineffective in terms of DNA damage; while the opposite was observed in CdCl2 exposed specimens. Co-exposure apparently prevents the chromosomal damage and leads to a partial recovery of the genome template stability.

Effects of the anti-calmodulin drugs calmidazolium and trifluoperazine on 45Ca transport in plasmalemmal vesicles from gastric smooth muscle.

The anti-calmodulin drugs calmidazolium (CMZ) and trifluoperazine (TFP) were shown to have a number of effects on 45Ca transport by plasmalemmal vesicles from gastric smooth muscle. Although these compounds produced the expected dose-dependent inhibition of the plasmalemmal ATP-dependent Ca2+ transport system, they also evoked a Ca2+ release comparable to that observed in the presence of the Ca2+ ionophore, ionomycin. This increased transmembrane Ca2+ flux was so large that it accounted for much of the apparent decrease in 45Ca uptake produced by these agents. Thus, direct effects of CMZ and TFP on ATP-dependent 45Ca uptake could only be reliably assessed for brief (less than or equal to 30 seconds) drug exposures. The explanation for the observed effects of CMZ and TFP on membrane Ca2+ permeability is unclear. The increased transmembrane Ca2+ flux may reflect nonspecific effects on membrane permeability or it may reflect a specific interaction of the anticalmodulin drugs with a Ca2+ release channel or with the Ca2+ transport ATPase. In any case, these results suggest the need for caution in the design and interpretation of studies using both CMZ and TFP as anticalmodulin agents.

Na(+)-H+ exchanger expression in vascular smooth muscle of spontaneously hypertensive and Wistar-Kyoto rats.

The Na(+)-H+ exchanger has important modulatory effects on vascular smooth muscle cell proliferation and contractility. Increased Na(+)-H+ exchange activity is a general property of many tissues, including mesenteric artery and cultured vascular smooth muscle cells, in the spontaneously hypertensive rat (SHR). In the present work, we investigated whether alterations in the steady-state levels of specific Na(+)-H+ exchanger mRNA isoforms (NHE-1 through NHE-4) are associated with the observed increases in exchanger activity. Poly(A+) mRNA prepared from 12-week-old hypertensive SHR and normotensive Wistar-Kyoto (WKY) aorta, kidney, and intestine was hybridized to cDNAs specific for each NHE isoform. By Northern blot analysis, NHE-1 was detected in all tissues as well as cultured vascular smooth muscle cells and was not regulated differently in SHR compared with WKY tissues. There was no expression of NHE-2, NHE-3, or NHE-4 in SHR and WKY aortas or in cultured vascular smooth muscle cells from SHR and WKY aortas. Stimulation of NHE-1 mRNA expression by growth factors was similar in cultured SHR and WKY vascular smooth muscle cells. We conclude that the previously observed increase in exchanger activity in blood vessels and cultured vascular smooth muscle cells of the SHR is not caused by induction of the NHE-2, NHE-3, and NHE-4 isoforms or by alterations in steady-state NHE-1 mRNA expression. These findings suggest that posttranslational regulation of the Na(+)-H+ exchanger is responsible for increased activity in the SHR.

A 90-kD Na(+)-H+ exchanger kinase has increased activity in spontaneously hypertensive rat vascular smooth muscle cells.

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.

Integument ultrastructure of Oestrus ovis (L.) (Diptera:Oestridae) larvae: host immune response to various cuticular components.

The nasal bot fly, Oestrus ovis, was investigated to establish which specific cuticular component is most immunogenic to infested sheep and how larval cuticle attains a protective role, if any, against the host immune system. To accomplish these goals, larval cuticle was extracted by a variety of agents and tested against immune sera from infested sheep and experimentally immunized rabbits. The cuticle substructure remaining after extraction was examined to localize various immunogenic components. O. ovis larval integument comprises an inner cellular layer, the epidermis, and an overlying cuticle layer. In 3rd instar larvae, the cuticle comprises 2 additional layers: the procuticle with numerous pore canals and the epicuticle which includes the wax canals. Three additional layers, altogether comprising the cuticulin layer, are present external to the epicuticle. The epicuticle is completed by apposition of an amorphous electrondense material extending for up to 1 micron in thickness. When fixed with ruthenium red, cuticle becomes heavily stained all along the epicuticular surface in larvae of all developmental stages. However, in 3rd instar larvae, ruthenium red deposits are restricted to the cuticulin layer alone. By gel electrophoresis, 3rd instar larval cuticle is shown to contain a number of polypeptides ranging in molecular weight from 180 to 4.5 kDa. The number and relative concentration of low molecular weight polypeptides was shown to vary in relation to the extraction media employed. Cuticular fragments examined after extraction exhibit an altered ultrastructure. When tested by immunoblotting, the cuticular polypeptides most reactive against sheep antisera are in the range of 180-56 kDa. A similar reaction was also detected with sera from rabbits infested experimentally with O. ovis larvae. Results are interpreted in relation to differential polypeptide distribution within the larval cuticle and to accessibility of the host immune system.