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.

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.

Serologic and urinary PCR survey of leptospirosis in healthy cats and in cats with kidney disease.

BACKGROUND: Although there is serologic evidence of exposure of cats to Leptospira spp., clinical disease is rarely reported in cats. OBJECTIVE: To compare the seropositivity and urinary polymerase chain reaction (PCR) status for Leptospira spp. between healthy (H) cats and cats with kidney disease (KD), to investigate the serovars potentially involved, and to evaluate potential risk factors. ANIMALS: Two hundred and forty client-owned cats. METHODS: Cats were prospectively recruited and classified based on physical examination, complete blood count, serum biochemistry profile, and urinalysis (125 H and 115 KD cats). Leptospira spp. serology (titers ≥1 : 100 considered positive) and urinary PCR were performed in all cats. Data assessing risk factors, obtained from a questionnaire, were evaluated using logistic regression models. RESULTS: Seropositivity for Leptospira spp. was statistically different between groups: 7.2% (9/125) and 14.9% (17/114) in the H and KD, respectively (P = .05). The proportion of PCR-positive cats was not. The most common serovars detected serologically were Pomona (n = 16) and Bratislava (n = 8). Risk factors for seropositivity included outdoor and hunting lifestyles (P = .03 and P < .001, respectively), the presence of another cat in the household (P < .01), and the sampling period, with the greatest number of cases identified between June and August (P =.02). CONCLUSIONS: Seropositivity was significantly greater in KD cats, suggesting that the role of Leptospira spp. in KD in cats should be further investigated. The detection of urinary shedding of leptospires in several cats identifies a potential role in the transmission of the organism.

Image Signature: Highlighting Sparse Salient Regions.

We introduce a simple image descriptor referred to as the image signature. We show, within the theoretical framework of sparse signal mixing, that this quantity spatially approximates the foreground of an image. We experimentally investigate whether this approximate foreground overlaps with visually conspicuous image locations by developing a saliency algorithm based on the image signature. This saliency algorithm predicts human fixation points best among competitors on the Bruce and Tsotsos [1] benchmark data set and does so in much shorter running time. In a related experiment, we demonstrate with a change blindness data set that the distance between images induced by the image signature is closer to human perceptual distance than can be achieved using other saliency algorithms, pixel-wise, or GIST [2] descriptor methods.

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.

Use of a bacterial antimicrobial resistance gene microarray for the identification of resistant Staphylococcus aureus.

As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Québec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene.

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.

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.

Identification of an inducible bacteriophage in a virulent strain of Streptococcus suis serotype 2.

Streptococcus suis infection is considered to be a major problem in the swine industry worldwide. Most virulent Canadian isolates of S. suis serotype 2 do not produce the known virulence markers for this pathogen. PCR-based subtraction hybridization was adapted to isolate unique DNA sequences which were specific to virulent strains of S. suis isolated in Canada. Analysis of some subtracted DNA clones revealed significant homology with bacteriophages of gram-positive bacteria. An inducible phage (named Ss1) was observed in S. suis following the incubation of the virulent strain 89-999 with mitomycin C. Phage Ss1 has a long noncontractile tail and a small isometric nucleocapsid and is a member of the Siphoviridae family. Ss1 phage DNA appears to be present in most Canadian S. suis strains tested in this study, which were isolated from diseased pigs or had proven virulence in mouse or pig models. To our knowledge, this is the first report of the isolation of a phage in S. suis.

Binding to sulfatide and enterotoxicity of various Escherichia coli STb mutants.

Binding of the 48 amino acid polypeptide of the mature heat-stable Escherichia coli enterotoxin b (STb) to the functional receptor sulfatide (SFT) constitutes the first step in inducing secretory diarrhoea in the intestinal lumen of animals. The NMR structure of this toxin dictated the choice of amino acids for site-directed mutagenesis to delineate the binding site of STb to SFT. Amino acids facing the solvent either in the loop or the hydrophobic alpha-helix were selected. Seventeen site-specific mutants of STb toxin were produced and purified by high-pressure liquid chromatography. Enterotoxicity of the 17 mutants was determined using a rat loop assay and binding was evaluated using a microtitre plate binding assay. Both hydrophobic and electrostatic interactions are important for STb attachment. When mutations (F37K, I41S and M42S) were introduced into the hydrophobic alpha-helix to lessen hydrophobicity, binding activity and enterotoxicity decreased by more than sixfold. The loop defined by C21 and C36 also made specific contributions. Mutants generated at basic residues (K22, K23 and R29) within this region exhibited both reduced binding activities and reduced toxic activities. For all STb mutants constructed and analysed, when binding to SFT was reduced, a reduction in toxicity equivalent or greater was noted, indicating that binding to SFT is a step that precedes the toxic effect observed for STb toxin. Significantly, when the negatively charged D30 was substituted for either alanine or valine, the binding to SFT was about twice that of native STb, whereas the enterotoxicity was reduced by half.

Streptococcus suis serotypes characterized by analysis of chaperonin 60 gene sequences.

Streptococcus suis is an important pathogen of swine which occasionally infects humans as well. There are 35 serotypes known for this organism, and it would be desirable to develop rapid methods methods to identify and differentiate the strains of this species. To that effect, partial chaperonin 60 gene sequences were determined for the 35 serotype reference strains of S. suis. Analysis of a pairwise distance matrix showed that the distances ranged from 0 to 0.275 when values were calculated by the maximum-likelihood method. For five of the strains the distances from serotype 1 were greater than 0.1, and for two of these strains the distances were were more than 0.25, suggesting that they belong to a different species. Most of the nucleotide differences were silent; alignment of protein sequences showed that there were only 11 distinct sequences for the 35 strains under study. The chaperonin 60 gene phylogenetic tree was similar to the previously published tree based on 16S rRNA sequences, and it was also observed that strains with identical chaperonin 60 gene sequences tended to have identical 16S rRNA sequences. The chaperonin 60 gene sequences provided a higher level of discrimination between serotypes than the 16S RNA sequences provided and could form the basis for a diagnostic protocol.

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.

Specific detection by PCR of Streptococcus agalactiae in milk.

The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk.

Atypical Actinobacillus pleuropneumoniae isolates that share antigenic determinants with both serotypes 1 and 7.

In the present study, the characterization of 3 atypical isolates of Actinobacillus pleuropneumoniae is presented. Two isolates (1B and 27E) showed positive reactions in coagglutination, immunodiffusion, and indirect hemagglutination tests for serotypes 1 and 7, whereas the third isolate (26B) reacted with antisera to serotypes 1, 4, and 7. These atypical isolates of A. pleuropneumoniae possessed a capsular polysaccharide (CPS) antigenically related to serotype 1 as well as an O-chain lipopolysaccharide antigenically related to serotype 7 or to serotypes 4 and 7, as shown by the use of monoclonal antibodies. Results of toxin profile and virulence assays for mice and pigs showed them to be more related to A. pleuropneumoniae serotype 7 field isolates. All 3 isolates induced antibodies mainly against serotype 7/4 O-long-chain lipopolysaccharide (LC-LPS) and, to a lesser extent, to the CPS of serotype 1, in experimentally infected pigs. Diagnostic laboratories that use a LC-LPS-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of A. pleuropneumoniae infection in swine would probably diagnose herds infected with these atypical isolates as being infected by A. pleuropneumoniae serotypes 7 or 4, whereas those that use a CPS-based ELISA would probably consider them as infected by A. pleuropneumoniae serotype 1.

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.

Isolation and characterization of a capsule-deficient mutant of Actinobacillus pleuropneumoniae serotype 1.

The capsular polysaccharides (CPS) play a major role in pathogenicity of Actinobacillus pleuroIpneumoniae, the causative agent of porcine pleuropneumonia. The purpose of the present study was to isolate a mutant in CPS biosynthesis by using a mini-Tn 10 transposon mutagenesis system and evaluate its adherence to host cells. One mutant apparently did not possess CPS as it did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 capsular antigen. Absence of capsule was confirmed by flow cytometry and also by transmission electron microscopy after polycationic ferritin labelling. The site of insertion of the mini-Tn 10 was determined and found to be in the cpxC gene. Its gene product, CpxC, is a protein involved in polysaccharide transport across the cytoplasmic membrane during CPS biosynthesis. Use of piglet tracheal frozen sections indicated that the CPS mutant adhered significantly (P=0.0001) more than the parent strain. The non-capsular mutant was less virulent in pigs compared to the parent strain and showed no mortality in experimentally infected pigs. The CPS mutant was however resistant to pig serum. This CPS mutant is the first A. pleuropneumoniae mutant in a CPS transport gene. It is also the first time that adherence of a CPS mutant of A. pleuropneumoniae is evaluated. Our observations indicate that capsular polysaccharides of A. pleuropneumoniae serotype 1 are not involved in adherence to piglet tracheal frozen sections but rather mask, at least in part, the adhesive functions.

Presence of the LEE (locus of enterocyte effacement) in pig attaching and effacing Escherichia coli and characterization of eae, espA, espB and espD genes of PEPEC (pig EPEC) strain 1390.

In the present study, attaching and effacing Escherichia coli (AEEC) O45 isolates from post-weaning pigs with diarrhoea were examined for the presence of the LEE (locus of enterocyte effacement) using various DNA probes derived from the LEE of human enteropathogenic E. coli (EPEC) strain E2348/69. The LEE fragment was conserved among the eae -positive pig isolates. The attaching and effacing activity of PEPEC (pig EPEC) O45 isolates is highly correlated with the presence of the LEE. Nevertheless, for some PEPEC isolates, the insertion site of the LEE is different or has diverged during evolution. The presence of the LEE fragment in PEPEC isolates provides further evidence that the LEE region is conserved among AEEC of different animal origins. In addition, the nucleotide sequence of the region containing the eae gene and esp genes of a pig AEEC isolate, strain 1390, was determined. Among examined Eae proteins, Eae of strain 1390 showed the highest similarity with Eae belonging to the beta intimin group such as the Eae of rabbit AEEC. Moreover, all pig strains that produced attaching and effacing lesions in piglets and pig ileal explants belonged to the beta intimin group. The deduced amino acid sequences of the EspA, EspB and EspD proteins of strain 1390 showed particularly strong homology to those of AEEC strains presenting a beta intimin allele. Thus, pig AEEC possess the LEE sequences, and for the strain 1390, sequences of the eae and esp regions are related to those of other AEEC, in particular, strains presenting a beta intimin allele, such as the rabbit AEEC.