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.
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.
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.
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.
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.
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.
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 A260/280 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.
► Commercial DNA/RNA extraction kits vary in yield, quality and purity of preparations. ► The RNeasy mini kit produced RNA samples of consistently high yield and quality. ► Ribotype or cell form did not impact the yield or quality of DNA preparations. ► Evidence of shearing in DNA preparations did not affect subsequent PCR.
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.
While Clostridium difficile has been extensively studied in acute care facilities (ACFs), there is limited information about long-term care facilities (LTCFs), despite the high occurrence of putative ...risk factors (e.g. age, antimicrobial use, healthcare system contact).
To evaluate C. difficile colonization in elderly patients and residents from ACF and its associated LTCF.
Stool swabs were collected from 884 LTCF and elderly (>65 years) hospital patients. Selective culture, PCR ribotyping and toxin gene characterization were performed.
C. difficile was isolated from 92/410 (22.4%) ACF and 89/474 (18.8%) LTCF samples. Ribotypes 027 (35%) and 020 (10.4%) predominated in the LTCF while ribotypes AI-82/1 (20.7%) and ribotype O (14.1%) predominated at the ACF (P=0.031). In the LTCF, C. difficile colonization was associated with a history of PPI use, and the interaction terms of male residents with prior medical leave of absence, and a prior history of C. difficile infection (CDI) combined with fluoroquinolone use. In the ACF, C. difficile colonization was associated with length of stay, feeding through a tube, antibiotic use, immunosuppressive therapy and VRE colonization, as well as the interaction terms for cephalosporin and fluoroquinolone use, prior CDI and cephalosporin use, and prior CDI and fluoroquinolone use.
C. difficile colonization by ACF and LTCF residents was common, despite a low apparent incidence of CDI. The association with PPI provides further evidence of the potential importance of this commonly used drug class in C. difficile colonization. Wide genetic diversity was present, highlighting the likelihood of multiple unidentified routes of C. difficile acquisition.
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