Effects of eight neuropsychiatric copy number variants on human brain structure.

Many copy number variants (CNVs) confer risk for the same range of neurodevelopmental symptoms and psychiatric conditions including autism and schizophrenia. Yet, to date neuroimaging studies have typically been carried out one mutation at a time, showing that CNVs have large effects on brain anatomy. Here, we aimed to characterize and quantify the distinct brain morphometry effects and latent dimensions across 8 neuropsychiatric CNVs. We analyzed T1-weighted MRI data from clinically and non-clinically ascertained CNV carriers (deletion/duplication) at the 1q21.1 (n = 39/28), 16p11.2 (n = 87/78), 22q11.2 (n = 75/30), and 15q11.2 (n = 72/76) loci as well as 1296 non-carriers (controls). Case-control contrasts of all examined genomic loci demonstrated effects on brain anatomy, with deletions and duplications showing mirror effects at the global and regional levels. Although CNVs mainly showed distinct brain patterns, principal component analysis (PCA) loaded subsets of CNVs on two latent brain dimensions, which explained 32 and 29% of the variance of the 8 Cohen's d maps. The cingulate gyrus, insula, supplementary motor cortex, and cerebellum were identified by PCA and multi-view pattern learning as top regions contributing to latent dimension shared across subsets of CNVs. The large proportion of distinct CNV effects on brain morphology may explain the small neuroimaging effect sizes reported in polygenic psychiatric conditions. Nevertheless, latent gene brain morphology dimensions will help subgroup the rapidly expanding landscape of neuropsychiatric variants and dissect the heterogeneity of idiopathic conditions.

Nicoletella semolina in the airways of healthy horses and horses with severe asthma.

BACKGROUND: Nicoletella semolina was identified in the airways of horses and its low prevalence could be because of its difficult differentiation from other Pasteurellaceae. OBJECTIVES: To develop a molecular method for the identification of N. semolina and to evaluate its prevalence in the mouth and the airways of healthy and severe asthmatic horses. ANIMALS: Six healthy and 6 severely asthmatic horses in phase I, 10 severely asthmatic horses in phase II, and 10 healthy horses in phase III. METHODS: Cohort (phases I and II) and cross-sectional (phase III) studies. Quantitative polymerase chain reaction primers targeting the sodA gene were optimized. N. semolina was quantified in oral and nasal washes and in bronchoalveolar lavage fluid (BALF; phase I, sampled twice), in nasal washes and BALF (phase II, sampled twice), and in nasal washes (phase III). RESULTS: N. semolina was found in the nose of 5, 10, and 9 horses in phases I, II, and III, respectively (first sampling for phases I and II). Six BALF from 5 different horses were positive for N. semolina in phase II. In phase I, there was no significant difference in the nasal loads of healthy horses (median (range): 2.04 × 104 copies/mL (0-2.44 × 105 )) and asthmatic horses in exacerbation (3.75 × 102 (0-4.84 × 106 ); Wilcoxon's rank sum test, P = .57). CONCLUSIONS AND CLINICAL IMPORTANCE: N. semolina is commonly found in the airways of horses. The potential pathogenicity of N. semolina remains to be elucidated, but the molecular technique we developed will facilitate future studies.

Persistence of a Clostridium perfringens Strain in a Broiler Chicken Farm over a Three-Year Period.

Clostridium perfringens, a commensal of the intestinal tract of many animal species, has been associated with necrotic enteritis (NE), an economically significant poultry disease. Clostridium perfringens is known to survive in the environment for extended periods of time through the formation of spores. These spores have the potential to be transmitted to subsequent flocks. Persistence of a single C. perfringens strain in a broiler chicken farm environment has, however, been poorly documented. The aim of this study was to compare multiple isolates of C. perfringens collected over time in a single farm with recurrent episodes of NE. Isolates were recovered from the intestines of chickens affected with NE (2014 and 2016 outbreaks) and from healthy chickens (2017), as well as from environmental samples (2016). PCR characterization of those isolates showed that all sampling groups contained netB-positive isolates except for the environmental samples. Moreover, results showed that all environmental isolates were positive for the cna adhesin whereas other groups had lower numbers of cna-positive isolates. Biofilm formation assays showed that most of the isolates were able to form biofilm. Pulsed-field gel electrophoresis analysis showed that one clone was present in every sampling group, with the exception of the 2014 outbreak. However, one clone found in the latter group was highly similar, having 94% similarity with the persistent C. perfringens clone. This study describes for the first time the persistence of a C. perfringens strain on a broiler chicken house over a 3-yr period.

A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks.

Necrotic enteritis (NE) is a major problem in antibiotic-free (ABF) chicken flocks and specific strains of Clostridium perfringens are known to induce NE. The objective of this study was to develop a chicken intestinal ligated loop model in order to compare the virulence of various C. perfringens strains recovered from consecutive ABF flocks with and without NE. Intestinal loops were surgically prepared in 10 anaesthetized specific-pathogen-free chickens and alternately inoculated with C. perfringens isolates or brain heart infusion (BHI) media. Histological lesion scoring was performed for each loop. All strains from NE-affected flocks induced histological lesions compatible with NE whereas inoculation of loops with a commensal C. perfringens strain or BHI did not. Among inoculated strains, CP0994 (netB-positive and cpb2-positive) and CP-2003-1256 (netB-positive) demonstrated mean histological lesion scores significantly higher (P < 0.01) than those obtained with a commensal strain or BHI whereas strain CP1073 (netB-negative and cpb2-positive) induced intestinal lesions without significantly higher scores. In loops where villi were colonized by Gram-positive rods, significantly higher (P < 0.01) mean histological lesion scores were observed. This result supports the hypothesis that colonization of the intestinal mucosa by C. perfringens is a critical step in the pathogenesis of NE. Finally, we demonstrated the importance of controlling virulent C. perfringens strains in ABF chicken flocks as a highly virulent strain can be present in consecutive flocks with NE and possibly affect multiple flocks.

Tolerance of Clostridium perfringens biofilms to disinfectants commonly used in the food industry.

Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxemia in animal species. Recently, it was shown to form mono-species biofilms, a structured community of bacterial cells enclosed in a self-produced extracellular matrix. Biofilms have been associated with tolerance to antibiotics, disinfectants, and physical and environmental stresses. Very little is known about the tolerance of C. perfringens biofilm toward disinfectants. In the present study, susceptibilities of C. perfringens biofilms to five types of commonly used disinfectants on farms and in food processing environments were analysed. In this paper, we show that C. perfringens mono-species biofilms can protect the bacterial cells from the action of potassium monopersulfate, quaternary ammonium chloride, hydrogen peroxide and glutaraldehyde solutions. However, sodium hypochlorite solution was shown to be effective on C. perfringens biofilms. Our investigation of dual-species biofilms of C. perfringens with the addition of Staphylococcus aureus or Escherichia coli demonstrated that overall, the mono-species biofilm of C. perfringens was more tolerant to all disinfectants than the dual-species biofilms. For the anaerobic grown biofilms, the mono-species biofilm of C. perfringens was more tolerant to sodium hypochlorite and quaternary ammonium chloride than the dual-species biofilms of C. perfringens with S. aureus or E. coli. This study demonstrates that C. perfringens biofilm is an effective protection mechanism to disinfectants commonly used on farms and in food processing environments.

Comparative transcriptomic analysis of Clostridium perfringens biofilms and planktonic cells.

Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxaemias in animal species. Recently, C. perfringens was shown to form biofilms, a structured community of bacterial cells enclosed in a self-produced extracellular matrix. However, very little is known on the subject and no information is available on gene expression in C. perfringens biofilms. To gain insights into the differences between free-living C. perfringens cells and those in biofilms, we used RNA sequencing. In total, 25.7% of genes showed differential expression in the two growth modes; about 12.8% of genes were up-regulated and about 12.9% were down-regulated in biofilms. We show that 772 genes were significantly differentially expressed between biofilms and planktonic cells from the supernatant of biofilms. Genes that were down-regulated in biofilm cells, relative to planktonic cells, included those involved in virulence, energy production, amino acid, nucleotide and carbohydrate metabolism, and in translation and ribosomal structure. Genes up-regulated in biofilm cells were mainly involved in amino acid and carbohydrate metabolism, transcription, inorganic ion metabolism and in defence mechanisms. This study provides new insights into the transcriptomic response of C. perfringens during biofilm formation.

Biofilm formation of Clostridium perfringens and its exposure to low-dose antimicrobials.

Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxemia in animal species. Very little is known on the biofilm of C. perfringens and its exposure to subminimal inhibitory concentrations of antimicrobials. This study was undertaken to address these issues. Most of the C. perfringens human and animal isolates tested in this study were able to form biofilm (230/277). Porcine clinical isolates formed significantly more biofilm than the porcine commensal isolates. A subgroup of clinical and commensal C. perfringens isolates was randomly selected for further characterization. Biofilm was found to protect C. perfringens bacterial cells from exposure to high concentrations of tested antimicrobials. Exposure to low doses of some of these antimicrobials tended to lead to a diminution of the biofilm formed. However, a few isolates showed an increase in biofilm formation when exposed to low doses of tylosin, bacitracin, virginiamycin, and monensin. Six isolates were randomly selected for biofilm analysis using scanning laser confocal microscopy. Of those, four produced more biofilm in presence of low doses of bacitracin whereas biofilms formed without bacitracin were thinner and less elevated. An increase in the area occupied by bacteria in the biofilm following exposure to low doses of bacitracin was also observed in the majority of isolates. Morphology examination revealed flat biofilms with the exception of one isolate that demonstrated a mushroom-like biofilm. Matrix composition analysis showed the presence of proteins, beta-1,4 linked polysaccharides and extracellular DNA, but no poly-beta-1,6-N-acetyl-D-glucosamine. This study brings new information on the biofilm produced by C. perfringens and its exposure to low doses of antimicrobials.

Genetic diversity of Mycoplasma hyopneumoniae isolates of abattoir pigs.

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, is present in swine herds worldwide. However, there is little information on strains infecting herds in Canada. A total of 160 swine lungs with lesions suggestive of enzootic pneumonia originating from 48 different farms were recovered from two slaughterhouses and submitted for gross pathology. The pneumonic lesion scores ranged from 2% to 84%. Eighty nine percent of the lungs (143/160) were positive for M. hyopneumoniae by real-time PCR whereas 10% (16/160) and 8.8% (14/160) were positive by PCR for M. hyorhinis and M. flocculare, respectively. By culture, only 6% of the samples were positive for M. hyopneumoniae (10/160). Among the selected M. hyopneumoniae-positive lungs (n=25), 9 lungs were co-infected with M. hyorhinis, 9 lungs with PCV2, 2 lungs with PRRSV, 12 lungs with S. suis and 10 lungs with P. multocida. MLVA and PCR-RFLP clustering of M. hyopneumoniae revealed that analyzed strains were distributed among three and five clusters respectively, regardless of severity of lesions, indicating that no cluster is associated with virulence. However, strains missing a specific MLVA locus showed significantly less severe lesions and lower numbers of bacteria. MLVA and PCR-RFLP analyses also showed a high diversity among field isolates of M. hyopneumoniae with a greater homogeneity within the same herd. Almost half of the field isolates presented less than 55% homology with selected vaccine and reference strains.

Characterization of hospital-associated lineages of ampicillin-resistant Enterococcus faecium from clinical cases in dogs and humans.

Ampicillin-resistant Enterococcus faecium (ARE) has rapidly emerged worldwide and is one of the most important nosocomial pathogens. However, very few reports are available on ARE isolates from canine clinical cases. The objective of this study was to characterize ARE strains of canine clinical origin from a veterinary teaching hospital in Canada and to compare them with human strains. Ten ARE strains from dogs and humans were characterized by multilocus sequence typing (MLST), pulsed field gel electrophoresis (PFGE), antibiotic susceptibility and biofilm activities, presence of rep-families, CRISPR-cas and putative virulence genes. All ARE strains (n = 10) were resistant to ciprofloxacin and lincomycin. Resistances to tetracycline (n = 6), macrolides (n = 6), and to high concentrations of gentamicin, kanamycin and streptomycin (n = 5) were also observed. Canine ARE isolates were found to be susceptible to vancomycin whereas resistance to this antibiotic was observed in human strains. Ampicillin resistance was linked to PBP5 showing mutations at 25 amino acid positions. Fluoroquinolone resistance was attributable to ParC, GyrA, and GyrB mutations. Data demonstrated that all canine ARE were acm (collagen binding protein)-positive and that most harbored the efaAfm gene, encoding for a cell wall adhesin. Biofilm formation was observed in two human strains but not in canine strains. Two to five rep-families were observed per strain but no CRISPR sequences were found. A total of six STs (1, 18, 65, 202, 205, and 803) were found with one belonging to a new ST (ST803). These STs were identical or closely related to human hospital-associated lineages. This report describes for the first time the characterization of canine ARE hospital-associated strains in Canada and also supports the importance of prudent antibiotic use in veterinary medicine to avoid zoonotic spread of canine ARE.

Characterization of genes encoding for acquired bacitracin resistance in Clostridium perfringens.

Phenotypic bacitracin resistance has been reported in Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC(90) (>256 µg/ml) was identical for both turkey and chicken isolates; whereas MIC(50) was higher in turkey isolates (6 µg/ml) than in chicken isolates (3 µg/ml). Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 µg/ml) and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens.