Backyard chickens - A cross-sectional survey of current and prospective backyard chicken owners in Ontario (2019-2021).

Objective: Chickens are a natural reservoir for zoonotic pathogens. Humans and pets may be at increased risk of illness due to contact with poultry, their eggs, and their environment. We aimed to identify any knowledge, attitude, or practice gaps among current and prospective backyard chicken owners. Animal: Backyard chickens. Procedure: Responses were collected through an anonymous online survey from December 2019 to March 2021. Respondents were asked questions regarding household demographics, previous, current, or future backyard chickens, primary reasons for having or wanting backyard chickens, and about animal handling practices and zoonotic disease awareness. Results: There were 279 respondents from Ontario (85.9%). Reported reasons for having or wanting backyard chickens included for eggs (94.0%), as pets (49.6%), and as a hobby (62.4%). Interestingly, 8.1% wanted chickens for their meat. Just over 1/5 (21.1%) of those with current or recent flocks allowed the birds to come into their house. Just over 7% incorrectly indicated rabies virus could be transmitted by backyard chickens. Conclusions: Reasons for having or wanting backyard chickens included food and companionship. Many owners reported allowing the chickens entry into their homes, highlighting increased opportunities for zoonotic pathogen transmission. There were misconceptions regarding pathogens transmissible by chickens.

Poulets urbains ­ Une enquête transversale auprès des propriétaires actuels et potentiels de poulets urbains en Ontario (2019-2021). Objectif: Les poulets sont un réservoir naturel pour des agents pathogènes zoonotiques. Les humains et les animaux de compagnie peuvent courir un risque accru de maladie en raison du contact avec la volaille, leurs oeufs et leur environnement. Notre objectif était d'identifier les lacunes en matière de connaissances, d'attitudes ou de pratiques chez les propriétaires actuels et potentiels de poulets urbains. Animal: Poulets urbains. Procédure: Les réponses ont été recueillies par le biais d'une enquête en ligne anonyme de décembre 2019 à mars 2021. Les répondants ont été interrogés sur la démographie des ménages, les poulets urbains précédents, actuels ou futurs, les principales raisons d'avoir ou de vouloir des poulets urbains, et sur les pratiques de manipulation des animaux et la sensibilisation aux maladies zoonotiques. Résultats: Il y avait 279 répondants de l'Ontario (85,9 %). Les raisons déclarées d'avoir ou de vouloir des poulets urbains incluaient pour les oeufs (94,0 %), comme animaux de compagnie (49,6 %) et comme passe-temps (62,4 %). Fait intéressant, 8,1 % voulaient des poulets pour leur viande. Un peu plus de 1/5 (21,1 %) de ceux qui ont des troupeaux actuels ou récents ont permis aux oiseaux d'entrer dans leur maison. Un peu plus de 7 % ont indiqué à tort que le virus de la rage pouvait être transmis par des poulets urbains. Conclusions: Les raisons d'avoir ou de vouloir des poulets urbains comprenaient la nourriture et la compagnie. De nombreux propriétaires ont déclaré autoriser l'entrée des poulets dans leurs maisons, soulignant les possibilités accrues de transmission d'agents pathogènes zoonotiques. Il y avait des idées fausses concernant les agents pathogènes transmissibles par les poulets.(Traduit par Dr Serge Messier).

Identification of appropriate reference genes for qPCR studies in Staphylococcus pseudintermedius and preliminary assessment of icaA gene expression in biofilm-embedded bacteria.

BACKGROUND: Quantitative PCR is rapidly becoming the standard method for analyzing gene expression in a wide variety of biological samples however it can suffer from significant error if stably expressed reference genes are not identified on which to base the analysis. Suitable reference genes for qPCR experiments on Staphylococcus pseudintermedius have yet to be identified. RESULTS: Three reference genes in S. pseudintermedius were identified and validated from a set of eight potential genes (proC, gyrB, rplD, rho, rpoA, ftsZ, recA, sodA). Two strains of S. pseudintermedius were used, and primer specificity and efficiency were confirmed and measured. Ranking of the genes with respect to expression stability revealed gyrB, rho and recA as the best reference genes. This combination was used to quantify expression of a single biofilm associated gene, icaA, in logarithmic, stationary and biofilm growth phases, revealing that expression was significantly upregulated in the biofilm growth phase in both strains. CONCLUSION: Three reference genes, gyrB, rho and recA, were identified and validated for use as reference genes for quantitative PCR experiments in S. pseudintermedius. Also, the biofilm associated gene icaA was shown to be significantly upregulated in biofilm samples, consistent with its role in biofilm production.

Evaluation of commercial kits for extraction of DNA and RNA from Clostridium difficile.

Commercial nucleic acid extraction kits are a cost effective, efficient and convenient way to isolate DNA and RNA from bacteria. Despite the increasing importance of the gastrointestinal pathogen, Clostridium difficile, and the increased use of nucleic acids in its identification, characterization, and investigation of virulence factors, no standardized or recommended methods for nucleic acid isolation exist. Here, we sought to evaluate 4 commercial DNA extraction kits and 3 commercial RNA extraction kits assessing cost, labor intensity, purity, quantity and quality of nucleic acid preparations. The DNA extraction kits produced a range of concentrations (20.9-546 ng/ml) and A(260/280) ratios (1.92-2.11). All kits were suitable for DNA extraction with the exception of the Roche MagNA pure LC DNA isolation kit III which produced DNA of high yield but with substantial shearing, but that did not affect downstream PCR amplifications. For RNA extraction, the Qiagen RNeasy mini kit stood out producing preparations of consistently higher concentrations and higher RNA integrity numbers (RIN). The Roche MagNA pure LC RNA isolation kit produced preparations that could not be properly assigned RINs due to a failure to remove small RNAs which were interpreted as degradation. Good DNA and RNA yield are critical but methods are often overlooked. This study highlights the potential for critical variation between established commercial systems and the need for assessment of any extraction methods that are used.

Binary toxin locus analysis in Clostridium difficile.

The objective of this study was to compare full binary toxin loci (CDTloc) sequences from a collection of Clostridium difficile isolates in an effort to further understand the regulation of the binary toxin (CdtAB) and its putative regulator (CdtR). Sequences from different ribotypes and toxinotypes were analysed phylogenetically and for polymorphisms, non-sense mutations, promoter features and signal sequences. Expression of cdtA, which was also representative of cdtB expression, was measured by quantitative PCR (qPCR). Several consensus promoter features and various polymorphisms were identified including a non-sense mutation identified in a ribotype 078 cdtR gene that is predicted to result in a severely truncated protein. Despite this mutation, cdtA expression was still detected by qPCR. Dendrograms based on total sequences indicated that isolates belonging to the same ribotype shared the greatest similarity within the binary toxin locus. Although cdtR is thought to be involved in regulation of cdtA expression, a cdtR non-sense mutation did not inhibit expression of cdtA, suggesting that either the truncated protein is functional or another regulator of the binary toxin exists.

Clostridium difficile in seafood and fish.

The objective of this study was to determine the prevalence of Clostridium difficile contamination in retail seafood and fish from Canadian grocery stores. C. difficile was found in 4.8% (5/119) of the samples. This study, combined with studies of other food sources, suggests that widespread contamination of food is common.

Identification of cold-temperature-regulated genes in Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS). It causes disease primarily in fresh water-reared salmonids, but other fish species can also be affected. A diverse array of clinical conditions is associated with BCWD, including tail rot (peduncle disease), necrotic myositis, and cephalic osteochondritis. Degradation of connective and muscular tissues by extracellular proteases is common to all of these presentations. There are no effective vaccines to prevent BCWD or RTFS, and antibiotics are often used to prevent and control disease. To identify virulence factors that might permit development of an efficacious vaccine, cDNA suppression subtractive hybridization (SSH) was used to identify cold-regulated genes in a virulent strain of F. psychrophilum. Genes predicted to encode a two-component system sensor histidine kinase (LytS), an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein, and four housekeeping genes were upregulated at 8°C versus the level of expression at 20°C. Because no F. psychrophilum gene was known to be suitable as an internal standard in reverse transcription-quantitative real-time PCR (RT-qPCR) experiments, the expression stability of nine commonly used reference genes was evaluated at 8°C and 20°C. Expression of the 16S rRNA was equivalent at both temperatures, and this gene was used in RT-qPCR experiments to verify the SSH findings. With the exception of the ATCC 49513 strain, similar patterns of gene expression were obtained with 11 other representative strains of F. psychrophilum.

Prevalence of Clostridium difficile in retail pork.

Clostridium difficile was isolated from 1.8% (7/393) of retail pork samples obtained from 4 Canadian provinces. Five ribotypes and 3 toxinotypes were identified. Three isolates were indistinguishable from the international outbreak strain ribotype 027 and were toxinotype III. Although the implications for food safety practices remain elusive, the frequency of toxigenic isolates and isolates indistinguishable from known human pathogenic strains suggests contaminated pork may be a source of C. difficile in humans.

Evaluation of candidate reference genes in Clostridium difficile for gene expression normalization.

Quantitative real-time polymerase chain reaction (qPCR) is a sensitive, efficient and reproducible technique for studying gene expression. Identification of stably expressed reference genes is required to avoid bias in these studies yet mostly unvalidated reference genes are used in studying gene expression in Clostridium difficile. Here, we sought to identify a set of stable reference genes used to normalize C. difficile expression data comparing exponential versus stationary phases of growth. Eight candidate reference genes (rpoA, rrs, gyrA, gluD, adk, rpsJ, tpi, and rho) were assessed in 3 C. difficile genotypes (ribotypes 027, 078, and 001). The primers were analyzed for efficiency and the 8 genes were ranked according to their stability. Overall, the genes rrs, adk, and rpsJ ranked among the most stable. Identification of the most stable genes was, however, strain dependent and suggests that selection of reference genes in a heterogeneous species, such as C. difficile, requires multiple genes to be assessed to confirm their stability within the strains being studied.

Translational control of the antibiotic inducibility of the PA5471 gene required for mexXY multidrug efflux gene expression in Pseudomonas aeruginosa.

The PA5471 gene required for induction of the MexXY multidrug efflux system in response to ribosome-targeting antimicrobials was itself shown to be inducible by ribosome-targeting antimicrobials (Y. Morita, M. L. Sobel, and K. Poole, J. Bacteriol. 188:1847-1855, 2006). Using a lacZ transcriptional reporter, drug inducibility of PA5471 was shown to require the entirety of the 367-bp PA5472-PA5471 intergenic region. A constitutive promoter activity was, however, localized to the first 75 bp of this region, within which a single PA5471 transcription initiation site was mapped. That 3' sequences of the intergenic region blocked PA5471 expression and made it antibiotic dependent was suggestive of an attenuation mechanism of control. A 13-amino-acid leader peptide (LP)-encoding open reading frame preceded by a Shine-Dalgarno sequence was identified ca. 250 bp upstream of the PA5471 coding sequence, and its expression and translation were confirmed using a lacZ translational reporter. Alteration of the initiation codon (M1T) or introduction of translational stop signals at codons 3 (Q3Am) and 8 (C8Op) of this LP sequence (PA5471.1) yielded high-level constitutive expression of PA5471, suggesting that interference with LP translation was linked to PA5471 gene expression. Consistent with this, a Q3K mutation in the LP sequence maintained the drug inducibility of PA5471 expression. Introduction of the LP Q3Am mutation into the chromosome of Pseudomonas aeruginosa yielded stronger expression of PA5471 than did antibiotic (chloramphenicol) exposure of wild-type P. aeruginosa, in agreement with lacZ transcriptional fusion data. Still, the Q3Am mutation yielded modest expression of mexXY, less than that seen for antibiotic-treated wild-type P. aeruginosa. These data suggest that PA5471 is not sufficient for MexXY recruitment in response to antibiotic exposure and that additional antibiotic-dependent effects are needed.

Phenotypic and genotypic analysis of Flavobacterium psychrophilum isolates from Ontario salmonids with bacterial coldwater disease.

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome. BCWD has a considerable economic impact on aquaculture operations in Ontario, Canada, and our limited understanding of the population structure and epidemiology of F. psychrophilum isolates is an impediment to the development of improved management strategies. Seventy-five 16S rRNA gene and gyr polymerase chain reaction positive isolates of F. psychrophilum that had been collected over a 16-year period from farmed salmonids with tail rot, necrotic myositis, and osteochondrosis were characterized morphologically, biochemically, and genotypically. Although the isolates were homogeneous by preliminary biochemical and phenotypic characterization, two distinct biovars were found by API ZYM testing. As well, four restriction pattern types were detected by 16S rRNA polymerase chain reaction -- restriction fragment length polymorphism analysis and there was a significant (P < 0.001) correlation between biovar I and digestion with MaeIII and between biovar II and digestion with MnlI or no site (P < 0.05). Further heterogenity was detected by sequence analysis of a 194 bp stem loop 3 region of rRNA. Nine sequence types were identified; 40/46 biovar I isolates were sequence type "a", while 21/32 biovar II isolates belonged to either sequence type "c" or "d". More than one biovar and genotype was identified among the strains recovered from separate fish sampled from three groups of rainbow trout (Oncorhynchus mykiss) experiencing BCWD mortality events. No association was found between genotype or biovar and type of disease. Taken together, these data suggest that F. psychrophilum from Ontario can be grouped into two major lineages based on biovar and 16S rRNA polymorphisms, and although three major strain types were most frequently isolated in this study, it appears that the population of F. psychrophilum with pathogenic potential is quite heterogeneous.

Prevalence of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella multocida, and Streptococcus suis in representative Ontario swine herds.

Tonsillar and nasal swabs were collected from weanling pigs in 50 representative Ontario swine herds and tested for the presence of 5 important bacterial upper respiratory tract pathogens. All but 1 herd (2%) tested positive for Streptococcus suis by polymerase chain reaction (PCR); 48% of herds were S. suis serovar 2, 1/2 positive. In all but 2 herds there was evidence of Haemophilus parasuis infection. In contrast, toxigenic strains of Pasteurella multocida were detected by a P. multocida--enzyme-linked immunosorbant assay (PMT-ELISA) in only one herd. Seventy-eight percent of the herds were diagnosed positive for Actinobacillus pleuropneumoniae by apxIV PCR. Sera from finishing pigs on the same farms were also collected and tested by ELISA for the presence of A. pleuropneumoniae antibodies. Seventy percent of the herds tested had evidence of antibodies to A. pleuropneumoniae including serovars 1-9-11 (2%), 2 (4%), 3-6-8-15 (15%), 5 (6%), 4-7 (26%), and 12 (17%). This likely represents a shift from previous years when infection with A. pleuropneumoniae serovars 1, 5, and 7 predominated. At least 16% and possibly as many as 94% of the herds tested were Actinobacillus suis positive; only 3 of the 50 herds were both A. pleuropneumoniae and A. suis negative as judged by the absence of a positive PCR test for apxII. Taken together, these data suggest that over the past 10 years, there has been a shift in the presence of pathogenic bacteria carried by healthy Ontario swine with the virtual elimination of toxigenic strains of P. multocida and a move to less virulent A. pleuropneumoniae serovars. As well, there appears to be an increase in prevalence of S. suis serovar 2, 1/2, but this may be a reflection of the use of a more sensitive detection method.

Differential expression of Haemophilus parasuis genes in response to iron restriction and cerebrospinal fluid.

Haemophilus parasuis is an important opportunistic pathogen in swine of high health status, but to date no proven virulence factors have been described. As virulence factors are known to be regulated during disease, the objective of this study was to identify genes of a virulent serovar 5 strain with altered expression after iron restriction or in the presence of porcine cerebrospinal fluid (CSF), conditions that reflect in vivo growth conditions. Using differential-display reverse-transcriptase-mediated polymerase chain reaction, we found that homologues of genes encoding fructose bisphosphate aldolase (fba), adenylosuccinate synthetase (purA), 2',3'-cyclic nucleotide phosphodiesterase (cpdB), lipoprotein signal peptidase (lspA), pyrophosphate reductase (lytB), superoxide dismutase (sodC), tyrosyl t-RNA synthetase (tyrS), cysteine synthetase (cysK), an unknown protein, and a homologue of a hydrolase of the haloacid dehydrogenase superfamily were upregulated in response to iron restriction. In addition, the purA, cpdB, lspA, lytB, and sodC homologues, cDNAs homologous with a Na+/alanine symporter, fatty acid ligase (fadD), diadenosine tetraphosphatase (apaH), and an unknown protein were upregulated in response to CSF. In screening for the presence of these differentially expressed genes to assess their usefulness as diagnostic markers of high virulence potential, we detected homologues of all of these genes in all of the reference strains of the 15 established serovars. The hydrolase homologue, however, was expressed only in representative H. parasuis strains associated with a high virulence potential, suggesting that this enzyme may play a role in pathogenesis.