Canadian Public Health Laboratory Network position statement: Non-culture based diagnostics for gastroenteritis and implications for public health investigations.

As clinical laboratories transition to using culture-independent detection test (CIDT) panels for cases of acute gastroenteritis, culture of clinical specimens is becoming less common. The reduction in bacterial cultures available for public health activities is expected to hinder surveillance and outbreak response by public health laboratories at the local, provincial, national and international levels. These recommendations are intended to serve as guidelines for the implementation of CIDT panels in frontline laboratories in Canada. The United States of America has already seen a significant reduction in culture of stool specimens despite the Association of Public Health Laboratories recommendation to perform reflex culture on positive CIDT specimens. Priority public health organisms addressed in these Canadian guidelines include Shiga toxin-producing Escherichia coli, Shigella and Salmonella and, under regional circumstances, other organisms such as Campylobacter jejuni/coli and Yersinia enterocolitica. These recommendations suggest active engagement between primary diagnostic laboratories and provincial public health laboratories to determine the workflow and protocols for reflex or parallel culture. Consequently, notifiable disease definitions will also need modification, with consultation of all stakeholders. Stakeholders need to work together to enhance recovery of bacterial isolates with best practices used for stool transport and storage.

Usefulness of High-Quality Core Genome Single-Nucleotide Variant Analysis for Subtyping the Highly Clonal and the Most Prevalent Salmonella enterica Serovar Heidelberg Clone in the Context of Outbreak Investigations.

Salmonella enterica serovar Heidelberg is the second most frequently occurring serovar in Quebec and the third-most prevalent in Canada. Given that conventional pulsed-field gel electrophoresis (PFGE) subtyping for common Salmonella serovars, such as S. Heidelberg, yields identical subtypes for the majority of isolates recovered, public health laboratories are desperate for new subtyping tools to resolve highly clonal S. Heidelberg strains involved in outbreak events. As PFGE was unable to discriminate isolates from three epidemiologically distinct outbreaks in Quebec, this study was conducted to evaluate whole-genome sequencing (WGS) and phylogenetic analysis as an alternative to conventional subtyping tools. Genomes of 46 isolates from 3 Quebec outbreaks (2012, 2013, and 2014) supported by strong epidemiological evidence were sequenced and analyzed using a high-quality core genome single-nucleotide variant (hqSNV) bioinformatics approach (SNV phylogenomics [SNVphyl] pipeline). Outbreaks were indistinguishable by conventional PFGE subtyping, exhibiting the same PFGE pattern (SHEXAI.0001/SHEBNI.0001). Phylogenetic analysis based on hqSNVs extracted from WGS separated the outbreak isolates into three distinct groups, 100% concordant with the epidemiological data. The minimum and maximum number of hqSNVs between isolates from the same outbreak was 0 and 4, respectively, while >59 hqSNVs were measured between 2 previously indistinguishable outbreaks having the same PFGE and phage type, thus corroborating their distinction as separate unrelated outbreaks. This study demonstrates that despite the previously reported high clonality of this serovar, the WGS-based hqSNV approach is a superior typing method, capable of resolving events that were previously indistinguishable using classic subtyping tools.

Legionella pneumophila monoclonal antibody subgroups and DNA sequence types isolated in Canada between 1981 and 2009: Laboratory Component of National Surveillance.

Legionella pneumophila (Lp) is a significant cause of nosocomial, community-acquired, and travel-associated pneumonia in industrialized regions. Legionellosis has been a nationally notifiable disease in Canada since 1986, with an average of 75 cases reported annually; however, only the most severe, and often fatal, cases are reported or investigated. Here, epidemiological relationships, types, and distribution of Lp referrals to the Canadian national reference center were studied. Lp strains from different years, sources, and geographic locations were subtyped using a sequence-based typing (SBT) scheme and by the 'Joly' and/or 'Dresden' monoclonal antibody panels. Included were 128 epidemiologically unrelated clinical and 86 unrelated environmental strains. Sixty-four (index of diversity [IOD] = 0.964) and 45 (IOD = 0.888) sequence types (STs) were observed among clinical and environmental sources, respectively. Serogroup (sg) 1 was represented by 60.2% (77/128) and 52.3% (45/86) of clinical and environmental strains, respectively, and 63.6% (49/77) and 15.6% (7/45) of those were mAb2-positive, respectively. Serogroup 1, ST1 accounted for 14.1% (18/128) and 30.2% (26/86) of unrelated clinical and environmental isolates, respectively. This database will serve as a basis for Canadian epidemiological surveillance efforts and is linked to global surveillance initiatives curated by the European Working Group for Legionella Infections (EWGLI) network.

Unbiased cocaine conditioned place preferences (CPP) obscures conditioned locomotion, and nimodipine blockade of cocaine CPP is due to conditioned place aversions.

The effect of nimodipine (0, 0.1, 1.0 and 10 mg/kg, SC), a dihydropyridine L-type Ca2+ channel antagonist, on the establishment of cocaine-(10 mg/kg IP) conditioned place preferences (CPP) was investigated. Nimodipine produced conditioned place aversions (CPA) on its own; reductions in cocaine CPP are apparently due to this CPA. There is a high negative correlation between time spent in the CS+ compartment and the difference in locomotion rates between the CS+ and the non-drug (CS-) compartments, independent of drug effects. This relationship is responsible for an increased rate of locomotion observed in the CS- compartment in cocaine-conditioned rats. Analysis of covariance indicated that cocaine CPP occurred independently of cocaine's effects on locomotion. Furthermore, cocaine produces an increase in the rate of locomotion in the CS+ compartment when time spent in this compartment is equated with time spent in the CS- compartment. This suggests that cocaine's effects on CPP and "conditioned" locomotion are due to separate mechanisms of action. On the other hand, nimodipine-induced place aversions and locomotor rates are not independent of each other, indicating a common mechanism of action, or that one is a consequence of the other. It is concluded that place preferences and place aversions can sometimes be secondary to compartment-specific locomotor changes, and locomotion effects can be confounded by differential times spent in each compartment. The relationships between these two behaviours must be controlled for before conclusions of CPP or CPA can be drawn in drug conditioning studies.

Classically conditioned motor effects do not occur with cocaine in an unbiased conditioned place preferences procedure.

Classical conditioning and behavioural sensitisation of motor activity induced with cocaine (10mg/kg, i.p.) were examined using an unbiased two-compartment conditioned place preference (CPP) procedure. Habituation of the rats to the testing environment prior to training was varied (i.e. either the rats were habituated to the environment for three 30min sessions or they were not) in order to examine a possible influence of latent inhibition on conditioned locomotion or behavioural sensitisation. Furthermore, rats were either trained with an explicit CS+ (cocaine-paired compartment) and CS- (vehicle-paired compartment), or else they were trained with no barrier between the compartments (effectively a single-compartment procedure with no explicit CS-) in order to examine a possible influence of stimulus change (training rats while confined to one compartment, but testing with no barrier between compartments). On a drug-free test day with free access to both compartments, rats previously exposed to cocaine in one compartment (CS+) and vehicle in the second compartment (CS-) spent more time in the CS+ compartment (conditioned place preference). However, under no circumstance was the rate of motor activity higher in the CS+ compartment than in the CS- compartment, as would be expected if cocaine-induced motor activity was classically conditioned to contextual cues. Whether or not increased activity extinguished with repeated drug-free exposures to previously drug-paired contexts depended on habituation experience. In addition, both habituation and current access to compartments (free or restricted) determined the presence of post-extinction sensitisation to a challenge dose of cocaine (7.5mg/kg). Classical conditioning and non-associative sensitisation, independently or together, cannot account for this pattern of results.

Conditioned place preferences, conditioned locomotion, and behavioral sensitization occur in rats treated with diethylpropion.

Diethylpropion is a centrally acting appetite-suppressing drug thought to act primarily through catecholamine pathways in the brain. In the present study, four doses of diethylpropion (0, 10, 20, and 40 mg/kg, intraperitoneally) were administered to rats to examine the hypothesis that the drug has psychomotor stimulant properties such as the ability to induce conditioned behaviors and behavioural sensitization. The rats were administered drug and then vehicle on alternating days, and confined to a "drug" or vehicle-paired side of a two-compartment box for 16 pairings. Only the 10-mg/kg dose of diethylpropion increased spontaneous locomotor activity in comparison to vehicle; the 20- and 40-mg/kg doses significantly decreased spontaneous locomotion. All doses of diethylpropion decreased spontaneous rearing, and the 20-and 40-mg/kg doses produced significantly less rearing than the 10-mg/kg one. At the 10-mg/kg dose, conditioned place preferences, conditioned locomotion, and conditioned rearing were observed. The 40-mg/kg dose produced conditioned rearing and conditioned defecation. In response to a 5-mg/kg challenge injection of diethylpropion, behavioural sensitization in locomotion and rearing occurred in rats that had previously received any one of the three doses of diethylpropion. Over 36 days, decreased weight gain was observed only in the 20- and 40-mg/kg groups. The rats were killed 48 h after the last drug injection, and whole brain was analyzed for levels of the catecholamines, homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT (not a catecholamine), and 5-hydroxyindoleacetic acid (5-HIAA) by HPLC with electrochemical detection.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of nimodipine and/or haloperidol on the expression of conditioned locomotion and sensitization to cocaine in rats.

The development of classical conditioning of cocaine's locomotor effects can be dissociated from the development of sensitization to cocaine by co-administration of haloperidol, a dopamine D2-like receptor antagonist, and nimodipine, an L-type calcium channel antagonist. The effects of these agents on the expression of conditioning and sensitization are described in the present report. Rats were given injections of vehicle or cocaine (10 mg/kg, IP) for 10 days before placement in a specific context in which locomotor activity was recorded. Neither haloperidol (0.05 mg/kg, IP) nor nimodipine (10 mg/kg, SC) influenced the expression of classical conditioning of cocaine's locomotor effects to the situational context on a subsequent cocaine-free test. Combined treatment of rats with both drugs did block classical conditioning with cocaine. Nimodipine, but not haloperidol, blocked the expression of behavioural sensitization to cocaine after a cocaine challenge. It is concluded that the expression of cocaine-induced classical conditioning can be pharmacologically dissociated from the expression of behavioural sensitization to cocaine. Furthermore, the effects of nimodipine and haloperidol on the expression of conditioning and sensitization are different from their effects on the development of these phenomena.

Nimodipine and haloperidol attenuate behavioural sensitization to cocaine but only nimodipine blocks the establishment of conditioned locomotion induced by cocaine.

The classical conditioning of the behavioural effects of cocaine has been shown to contribute to behavioural sensitization. In the present experiments, it was demonstrated that the effects of cocaine in rats can be conditioned to contextual stimuli. Furthermore, sensitization to cocaine's locomotor effects were demonstrated, and shown to be context specific. Nimodipine (10 mg/kg, SC), an L-type dihydropyridine Ca2+ channel antagonist, appeared to completely block the establishment of conditioning of cocaine's effects, but only partially blocked sensitization to cocaine. Haloperidol (0.05 mg/kg, IP), a relatively specific D2 dopamine receptor antagonist, attenuated behavioral sensitization but had no influence on the establishment of the conditioned component of cocaine. These results indicate that the sensitization to, and the development of classical conditioning of, cocaine's behavioural effects can be pharmacologically dissociated, but that a non-associative process involved in sensitization is normally overridden by conditioning factors.