Prevalence of Coxiella burnetii seropositivity and shedding in farm, pet and feral cats and associated risk factors in farm cats in Quebec, Canada.

Cats represent a potential source of Coxiella burnetii, the aetiological agent of Q fever in humans. The prevalence and risk factors of C. burnetii infection in farm, pet and feral cats were studied in Quebec, Canada, using a cross-sectional study. Serum samples were tested using a specific enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against C. burnetii, whereas rectal swabs were assayed using real-time quantitative polymerase chain reaction (qPCR) for the molecular detection of the bacteria. Potential risk factors for farm cats were investigated using clinical examinations, questionnaires and results from a concurrent study on C. burnetii farm status. A total of 184 cats were tested: 59 from ruminant farms, 73 pets and 52 feral cats. Among farm cats, 2/59 (3.4%) were ELISA-positive, 3/59 (5.1%) were ELISA-doubtful and 1/59 (1.7%) was qPCR-positive. All pets and feral cats were negative to C. burnetii ELISA and qPCR. Farm cat positivity was associated with a positive C. burnetii status on the ruminant farm (prevalence ratio = 7.6, P = 0.03). Our results suggest that although pet and feral cats do not seem to pose a great C. burnetii risk to public health, more active care should be taken when in contact with cats from ruminant farms.

A review of the current evidence of fruit phenolic compounds as potential antimicrobials against pathogenic bacteria.

Fruits are among the main natural sources of phenolic compounds (PC). These compounds exert important antioxidant properties primarily associated with the presence of hydroxyl groups in their molecular structure. Additionally, the antibacterial effects of fruit phenolic-rich extracts or individual PC commonly found in fruits have been an emerging research focus in recent years. This review discusses by first time the available literature regarding the inhibitory effects of fruit PC on pathogenic bacteria, including not only their direct effects on bacterial growth and survival, but also their effects on virulence factors and antibiotic resistance, as well as the possible mechanism underlying these inhibitory properties. The results of the retrieved studies show overall that the antibacterial effects of fruit PC vary with the target bacteria, type of PC and length of exposure to these compounds. The type of solvent and procedures used for extraction and fruit cultivar also seem to influence the antibacterial effects of phenolic-rich fruit extracts. Fruit PC have shown wide-spectrum antibacterial properties besides being effective antibiotic resistance modifying agents in pathogenic bacteria and these effects have shown to be associated with interruption of efflux pump expression/function. Furthermore, fruit PC can cause down regulation of a variety of genes associated with virulence features in pathogenic bacteria. Results of available studies indicate the depolarization and alteration of membrane fluidity as mechanisms underlying the inhibition of pathogenic bacteria by fruit PC. These data reveal fruit PC have potential antimicrobial properties, which should be rationally exploited in solutions to control pathogenic bacteria.

Is Escherichia coli urinary tract infection a zoonosis? Proof of direct link with production animals and meat.

Recently, it has been suggested that the Escherichia coli causing urinary tract infection (UTI) may come from meat and animals. The purpose was to investigate if a clonal link existed between E. coli from animals, meat and UTI patients. Twenty-two geographically and temporally matched B2 E. coli from UTI patients, community-dwelling humans, broiler chicken meat, pork, and broiler chicken, previously identified to exhibit eight virulence genotypes by microarray-detection of approximately 300 genes, were investigated for clonal relatedness by PFGE. Nine isolates were selected and tested for in vivo virulence in the mouse model of ascending UTI. UTI and community-dwelling human strains were closely clonally related to meat strains. Several human derived strains were also clonally interrelated. All nine isolates regardless of origin were virulent in the UTI model with positive urine, bladder and kidney cultures. Further, isolates with the same gene profile also yielded similar bacterial counts in urine, bladder and kidneys. This study showed a clonal link between E. coli from meat and humans, providing solid evidence that UTI is zoonosis. The close relationship between community-dwelling human and UTI isolates may indicate a point source spread, e.g. through contaminated meat.

Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry.

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential.

Development of a new integrated diagnostic test for identification and characterization of pathogens.

Animal diseases directly cause multi-million dollar losses world-wide. Therefore a rapid, highly specific, cost-effective diagnostic test for detecting a large set of bacterial virulence and antimicrobial resistance genes simultaneously is necessary. Hence, our group, the BCBG (Bacterial Chips Bacterial Genes) group, proposes developing a powerful molecular tool (DNA microarray) to detect a broad range of infectious agents, their endogenous main virulence factors and antibiotic resistance genes simultaneously. Effectively, a 70-mer oligonucleotide microarray capable of detecting the presence or absence of 169 Escherichia coli virulence genes or virulence marker genes as well as their variants, in addition to 30 principal antimicrobial resistance genes previously characterized in E. coli strains was developed by our group. This microarray was validated with a large collection of well characterized pathogenic and reference E. coli strains. Moreover, we are developing a new powerful clinical diagnostic microarray tool, to identify pathogenic bacteria of veterinary interest. The commercialization of this assay would allow same day diagnosis of infectious agents and their antibiotic resistance resulting in early treatment. In addition, this technology is also applicable to microbial quality control of food and water.

Effects of tumor promoters on the expression of a tumor-related multigenic set in human cells.

We previously found that a minor subfraction of the human genomic DNA, corresponding to 2500-3000 nonrepetitive sequences of 3 kilobases each and designated as tumor-activated DNA (TaDNA) was transcriptionally active in Burkitt's lymphoma cells and almost inactive in normal lymphocytes growing in vitro following integration of the Epstein-Barr virus genome. Furthermore all the neoplastic cells in culture or primary neoplasms (leukemias, sarcomas, carcinomas) studied contained transcripts from most of the TaDNA sequences found in malignant lymphoblasts whereas normal cells growing in vitro contained only a few TaDNA transcripts. It is shown in the present study that treatments of the myeloid leukemia HL60 cells with various inducers of cell differentiation (dimethyl sulfoxide, retinoic acid, mezerein, 12-O-tetradecanoylphorbol-13-acetate, teleocidin) caused a dose-dependent reduction of the level of TaDNA transcripts, correlated with the diminution of c-myc transcripts. The 12-O-tetradecanoylphorbol-13-acetate treatment had this same effect on Burkitt's lymphoma cells (Raji or Namalwa) but the opposite effect on normal cells (Epstein-Barr virus-immortalized lymphocytes or fetal fibroblasts) where it enhanced the formation of Ta-DNA transcripts up to the levels found in untreated malignant cells. These data suggest two conclusions (a) TaDNA corresponds to a multigenic set which seems to be involved in modulation of the malignant phenotype and (b) depending on the origin of the cells, agents like 12-O-tetradecanoylphorbol-13-acetate may operate either as tumor promoters or as differentiation inducers through the control of TaDNA expression.

Differential expression of protooncogenes related to transformation and cancer progression in rat myoblasts.

We previously derived, from a nonmalignant clonal line of rat myogenic cells (L6 alpha 1), two sublines which have lost the capacity to differentiate, the M4 cell of low malignancy and the RMS4 cell of high malignancy. In the present study it is shown that 14 of 15 protooncogenes analyzed exhibit detectable levels of transcripts during L6 alpha 1 cell proliferation. When L6 alpha 1 cells from myotubes, the levels of c-abl, c-myb, and c-Ha-ras transcripts remain unchanged, the level of c-N-ras RNA is augmented, the level of c-erbB RNA is markedly reduced, and all other c-onc transcripts (c-erbA, c-sis, c-src, c-fes, c-fms, c-fos, c-myc, c-Ki-ras, and the putative tyrosine kinase transcript of the c-fgr gene) become hardly, if at all, detectable. Surprisingly, when the three cell types are growing at similar rates only, one protooncogene (c-mos) is not expressed at detectable levels in L6 alpha 1, two others (c-fos, c-erbA) are not expressed in M4 or in RMS4, and three additional ones (c-erbB, c-sis, c-src) are expressed in M4 but not in RMS4. Moreover the level of c-fes RNAs is markedly lower in RMS4 than in M4 or L6 alpha 1. By contrast, the level of two c-Ki-ras 5.4- and 2.2-kilobase transcripts is lower in M4 and L6 alpha 1 than in RMS4, and the latter contains another abundant c-Ki-ras 3.8-kilobase transcript which is hardly detectable in M4 and not at all in L6 alpha 1. These data suggest an activation of the c-Ki-ras gene in the malignant myoblasts and some relationship between the progression of malignancy and inactivation of certain other c-onc genes.

Changes in gene expression following neoplastic transformation of rat myogenic cells.

Two malignant sublines, M4 and RMS4 , were previously derived from the recloned L6 line of rat myogenic cells. Comparative studies in tissue culture and inoculation into suckling rats indicated that M4 cells and RMS4 cells may be considered as low-malignant and high-malignant cells, respectively, while L6 cells are not malignant. In the present work, we used extracts from L6 cells, M4 cells, and RMS4 cells collected during the period of exponential growth, to compare their polyadenylic acid-containing messenger RNA (mRNA) populations and the corresponding cell-free translation products. Analysis of the hybridization kinetics between radioactive complementary DNA and homologous or heterologous cellular RNAs indicated that L6 cells contained about 28,000 distinct polyadenylic acid-containing mRNA sequences of 1.8 kilobases each, of which 2,000 to 2,500 and 4,000 to 5,000 were missing (or at least were very infrequent) in M4 cells and RMS4 cells, respectively. Using a minor fraction of the RMS4 cell complementary DNAs, partially purified through repeated complementary DNA-RNA hybridization cycles, it was further shown that RMS4 cells contained at least 700 to 800 distinct mRNA species, mainly belonging to the class of low abundance, which appeared to be absent in L6 cells. Most of these mRNA species were also found with a lower frequency in M4 cells. Bidimensional analysis of the cell-free translation products directed by polyadenylic acid-containing mRNA revealed some remarkable differences, in particular the synthesis in a RMS4 cell extract of at least three major polypeptides, possibly related either to the neoplastic process itself or to the stage of malignant transformation.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

Rat myogenic c-mos cDNA: cloning sequence analysis and regulation during muscle development.

We have isolated and sequenced a cDNA clone, homologous to the rat c-mos gene, from a cDNA library of rat skeletal muscles. The 3220 nucleotide cDNA clone codes for a protein of 339 amino acids (37.4 kDa). Both the nucleotide sequence and the deduced amino acid sequence show 60-90% overall homology to Xenopus, chicken, mouse and human mos. By Northern blot analysis, we detected two c-mos transcripts, one major of about 3.6 Kb long, and one minor of about 1.7 Kb long. These are differently regulated during the development of cardiac and skeletal muscles. By Western blot with two antibodies directed against two different portions of the mos protein, we observed in rat muscle two polypeptides of 43 kDa, and 75 kDa respectively.

Oligomerization of Escherichia coli enterotoxin b through its C-terminal hydrophobic alpha-helix.

Using a chemical cross-linker and gel electrophoresis or a dot blot overlay assay, we studied protein-protein interaction of STb toxin, a 48-residue amphiphilic polypeptide causing intestinal disorders. For the first time, we report on the oligomerization property of STb. This enterotoxin forms hexamers and heptamers in a temperature-independent fashion in presence or absence of its receptor (sulfatide) anchored in a 50-nm liposome or as a free molecule. Full STb structure integrity is necessary for its oligomerization as this process is not observed under reducing conditions in the presence of beta-mercaptoethanol. STb treatment with tetramethylurea (TMU) and different detergents prevented oligomerization. Site-directed mutagenesis decreasing overall STb hydrophobicity in the hydrophobic alpha-helix resulted in the incapacity to form oligomers. Taken together, these data suggest that the C-terminal hydrophobic alpha-helix corresponds to the domain of STb-STb inter-binding where hydrophobic interaction is involved.

The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction.

It was shown with the use of specific probes that mild micrococcal nuclease digestion released from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A+) RNAs, however larger released fragments include some transcribed sequences, while the nuclease resistant chromatin is considerably impoverished in coding sites. These evidences are the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5-2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.

Optimization of microbial DNA extraction and purification from raw wastewater samples for downstream pathogen detection by microarrays.

Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.

Cloning and characterization of a dextranase gene (dexS) from Streptococcus suis.

A gene (dexS) coding for a Streptococcus suis capsular type 2 dextranase (DexS) was detected in a recombinant gene library constructed in phage lambda ZapII, and its nucleotide sequence was determined. Sequence comparison showed that the dexS gene product had significant similarities with enzymes which hydrolyze glucose polymers. Moreover, conserved amino acids that are suggested to be part of the active site of the glucosidases are also found in DexS. The dexS gene, adjacent to the gene encoding a S. suis IgG-binding protein, encoded a protein of approximately 62 kDa which exhibited DexS activity.

Early effects of triidothyronine on the complexity of rat heart messenger RNAs.

Triiodothyronine (T3), injected daily into rats, induces heart hypertrophy. In a recent work we have shown that a single T3 injection stimulates RNA synthesis and modifies the translational products of myocardial mRNAs in reticulocyte lysates, quantitatively and qualitatively. In this work we show that T3 induces small but significant changes in mRNA size distribution after 4 h and much more important changes after 18 h. It also modifies the size distribution of their poly(A) tails. We studied the early effects of this compound on mRNA complexity, using the nucleic acid hybridization technique with DNA complementary to poly(A)+ RNAs. T3, 4 h after injection, suppresses approximately 15% of the sequences, mostly among rare sequences, and increases the frequency of the abundant sequences and of the sequences of intermediary abundance. A large part of this effect disappears 18 h after the injection. It may be concluded that T3 presents an early effect on gene expression, involving changes either at the gene level or and at some post transcriptional level.

Effects of global regulatory proteins and environmental conditions on fimbrial gene expression of F165(1) and F165(2) produced by Escherichia coli causing septicaemia in pigs.

Escherichia coli O115:F165 strains are associated with septicaemia in young pigs and possess at least two types of fimbriae. F165(1) fimbriae belong to the P fimbrial family and F165(2) fimbriae belong to the S fimbrial family. Regulatory regions of foo (F165(1)) and fot (F165(2)) fimbrial gene clusters from wild-type strain 4787 were sequenced and characterised. Expression of F165(1) and F165(2) fimbrial genes was analysed by using lacZ and/or luxAB as reporter genes under the control of the native fimbrial promoters. Differential expression of fimbrial genes was observed. Global regulatory mechanisms such as catabolite repression, leucine-responsive regulatory protein (Lrp), methylation and DNA supercoiling were demonstrated to influence foo and fot expression. foo and fot expression was optimal at 37 degrees C and under aerobic conditions. Expression of foo was higher on minimal medium, whereas fot expression was higher on complex Luria-Bertani medium. This could reflect an in vivo differential expression.

Studies on a murine model for evaluation of virulence of Streptococcus suis capsular type 2 isolates.

Five different parameters, time of incubation of the culture, type of culture medium, inoculum, strain of inbred mice, and age of mice, were tested using the LD50 technique to standardize a murine model for the evaluation of the virulence of Streptococcus suis capsular type 2 isolates. A model using 28 day-old mice belonging to CF1 strain appeared to give the best results. The inoculum size was the parameter most influencing the 50% lethal dose obtained with mice. Inoculation with 1-ml volume of a bacterial suspension instead of 0.1 or 0.5 ml decreased the LD50. The standardized model was used to evaluate the virulence of some isolates of known pathogenicity for pigs. The minimum lethal dose was used in the model and it appeared that the virulence of Streptococcus suis capsular type 2 isolates can be measured from highly virulent to totally avirulent.

Cloning and characterization of the eae gene from a dog attaching and effacing Escherichia coli strain 4221.

We have cloned and determined the nucleotide sequence of the eae gene from a dog attaching and effacing (A/E) Escherichia coli (DEPEC) strain 4221. When comparing the predicted amino acid sequence of the eaeDEPEC to that of the Eae proteins from enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli O157:H7 (EHEC), Citrobacter freundii biotype 4280, and a swine A/E E. coli strain O45 (PEPEC), the overall sequence identity was 84, 81, 83 and 83%, respectively, with the greatest divergence at the C-terminal end, the putative receptor-binding portion. Interestingly, the DEPEC Eae shares the greatest identity at the C-terminal region with the Citrobacter freundii Eae protein. We have constructed and purified a maltose-binding fusion protein (MBP) containing the product of the entire eae gene of the DEPEC strain 4221. Binding of MBP-EaeDEPEC fusion protein to HEp-2 cells was demonstrated by immunofluorescence microscopy. In addition, the Eae protein of DEPEC (4221) demonstrated a strong serological relationship with that of EPEC (E2348/69) as observed using a polyclonal antiserum against MBP-EaeDEPEC fusion protein.

Cloning and characterization of the esp region from a dog attaching and effacing Escherichia coli strain 4221 and detection of EspB protein-binding to HEp-2 cells.

The espA, espB and espD genes from enteropathogenic Escherichia coli were previously shown to be essential for triggering the signal transduction in infected host cells. We have cloned and determined the nucleotide sequences of the espA, espB and espD homologues from an E. coli strain (4221) isolated from a dog which manifested the attaching and effacing lesions in the small intestine. This strain is designated as a dog enteropathogenic E. coli. When comparing predicted amino acid sequences to those of the corresponding proteins from enteropathogenic E. coli O127, enterohemorrhagic E. coli serotype O26, enterohemorrhagic E. coli O157 and rabbit enteropathogenic E. coli, the EspADEPEC protein showed the same level of similarity (75% identity) with EspA of enteropathogenic E. coli O127 and rabbit enteropathogenic E. coli. The EspBDEPEC protein showed the highest similarity with the EspB of enteropathogenic E. coli O127 (99% identity). The EspDDEPEC protein showed 88% identity with the EspDEPEC. We constructed and purified a maltose-binding fusion protein containing the product of the entire espBDEPEC gene of the dog enteropathogenic E. coli strain 4221. Purified maltose-binding protein-EspBDEPEC fusion protein was shown to bind efficiently to HEp-2 cells in a localized fashion as shown by immunofluorescence microscopy. In addition, when the dog enteropathogenic E. coli strain 4221 was grown in tissue culture medium (DMEM) supplemented with serum, a secreted 36-kDa protein was identified by immunoblot analysis using a polyclonal antiserum against the maltose-binding protein-EspBDEPEC fusion protein.

Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs.

We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA (E. coli attaching and effacing) gene. In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E. coli isolates was examined. The majority (20/22) of attaching and effacing positive, eaeA+ E. coli O45 isolates, but none of ten attaching and effacing negative, eaeA- or eaeA+ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E. coli O45 and the EaeA protein (intimin) of human enteropathogenic E. coli and enterohemorrhagic E. coli. In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E. coli strains was observed. Our results indicate an association between expression of the EaeA protein and attaching and effacing activity among O45 E. coli isolates. The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E. coli.