Redefining piscine lactococcosis.

Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae. However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)-L. garvieae, L. petauri, and L. formosensis-which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis. Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention. IMPORTANCE: Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

Rapid identification of Salmonella serovars Enteritidis and Typhimurium using whole cell matrix assisted laser desorption ionization - Time of flight mass spectrometry (MALDI-TOF MS) coupled with multivariate analysis and artificial intelligence.

Salmonella is a common food-borne pathogen with Enteritidis and Typhimurium being among the most important serovars causing numerous outbreaks. A rapid method was investigated to identify these serovars using whole-cell MALDI-TOF MS coupled with multivariate analysis and artificial intelligence and 113 Salmonella strains, including 38 Enteritidis (SE), 38 Typhimurium (ST) and 37 strains from 32 other Salmonella serovars (SG). Datasets of ions (presence/absence) with high discriminative power were created using newly developed criteria and subject to multivariate analyses and eight artificial intelligence (AI) tools. Principal Component Analysis based on 55 or 88 selected ions separated SE, ST and SG without overlap on the first three principal components. Datasets were partitioned using five partitioning methods with 70% of samples for AI model training and 30% for validation. Of the eight AI models evaluated, high performance (HP) SVM and HP Neural were the top performers, identified three serovar groups 97% correctly on average (range 82%-100%) according to the validation results. Selection of serovar specific ions facilitated differentiation of serotypes using unsupervised model PCA and improved the accuracy of classification using AI significantly (p < 0.01). MALDI-TOF MS incorporated with advanced data processing and classification tools is a promising method to allow rapid identification of Salmonella serovars of concern in routine diagnostic laboratories.

AAV Vectors Pseudotyped with Capsids from Porcine and Bovine Species Mediate In Vitro and In Vivo Gene Delivery.

Adeno-associated virus (AAV) vectors are among the most widely used delivery vehicles for in vivo gene therapy as they mediate robust and sustained transgene expression with limited toxicity. However, a significant impediment to the broad clinical success of AAV-based therapies is the widespread presence of pre-existing humoral immunity to AAVs in the human population. This immunity arises from the circulation of non-pathogenic endemic human AAV serotypes. One possible solution is to use non-human AAV capsids to pseudotype transgene-containing AAV vector genomes of interest. Due to the low probability of human exposure to animal AAVs, pre-existing immunity to animal-derived AAV capsids should be low. Here, we characterize two novel AAV capsid sequences: one derived from porcine colon tissue and the other from a caprine adenovirus stock. Both AAV capsids proved to be effective transducers of HeLa and HEK293T cells in vitro. In vivo, both capsids were able to transduce the murine nose, lung, and liver after either intranasal or intraperitoneal administration. In addition, we demonstrate that the porcine AAV capsid likely arose from multiple recombination events involving human- and animal-derived AAV sequences. We hypothesize that recurrent recombination events with similar and distantly related AAV sequences represent an effective mechanism for enhancing the fitness of wildtype AAV populations.

Laboratory investigation of cases of fatal bacterial pneumonia in dairy cows.

Objective: Bacterial bronchopneumonia occurs in mature dairy cows but much of the information is extrapolated from knowledge of the disease in calves. The study was prompted by perceptions of an increasing occurrence and a paucity of information on fatal Mannheimia haemolytica pneumonia in dairy cows in Ontario. The study objectives were to describe the seasonality, main pathogens involved, and suggested predisposing factors for cases of fatal bacterial bronchopneumonia in mature dairy cows submitted for postmortem examination to a diagnostic laboratory, and to evaluate if the frequency of such submissions has increased over time. Animals: Mature dairy cows. Procedure: Retrospective study of cases submitted for postmortem examination to a diagnostic laboratory from 2007-2020 that were diagnosed as bacterial bronchopneumonia. Results: Most of the postmortem cases of bacterial bronchopneumonia in dairy cows were submitted from November to February (54% of cases). Mannheimia haemolytica was isolated from lung of 61/101 cases. Viruses were only identified in 8/55 cases tested. A minority (29/92) of bacterial isolates had in vitro resistance to antimicrobials used to treat pneumonia. Frequently suggested predisposing factors included recent introductions or movement of animals, recent or imminent calving, inclement weather, concurrent diseases, and poor ventilation in barns. Conclusion and clinical relevance: This study describes seasonal and annual trends, major pathogens, antimicrobial resistance profiles, and suggested predisposing factors in Ontario dairy cows submitted to a diagnostic laboratory for postmortem investigation of pneumonia and provides insights for understanding why outbreaks occur.

Objectif: La bronchopneumonie bactérienne survient chez les vaches laitières matures, mais une grande partie de l'information est extrapolée à partir de la connaissance de la maladie chez les veaux. L'étude a été motivée par la perception d'une occurrence croissante et d'un manque d'information sur la pneumonie mortelle à Mannheimia haemolytica chez les vaches laitières en Ontario. Les objectifs de l'étude étaient de décrire la saisonnalité, les principaux agents pathogènes impliqués et les facteurs prédisposants suggérés pour les cas de bronchopneumonie bactérienne mortelle chez les vaches laitières matures soumises à un examen post-mortem à un laboratoire de diagnostic, et d'évaluer si la fréquence de telles soumissions a augmenté au fil du temps. Animaux: Vaches laitières matures. Procédure: Étude rétrospective des cas soumis pour examen post-mortem à un laboratoire de diagnostic, entre 2007 et 2020, qui ont été diagnostiqués comme une bronchopneumonie bactérienne. Résultats: La plupart des cas post-mortem de bronchopneumonie bactérienne chez les vaches laitières ont été soumis de novembre à février (54 % des cas). Mannheimia haemolytica a été isolée du poumon de 61/101 cas. Des virus n'ont été identifiés que dans 8/55 cas testés. Une minorité (29/92) d'isolats bactériens présentaient une résistance in vitro aux antimicrobiens utilisés pour traiter la pneumonie. Les facteurs prédisposants fréquemment suggérés comprenaient des introductions ou des déplacements récents d'animaux, un vêlage récent ou imminent, des conditions météorologiques défavorables, des maladies concomitantes et une mauvaise ventilation dans les étables. Conclusion et pertinence clinique: Cette étude décrit les tendances saisonnières et annuelles, les principaux agents pathogènes, les profils de résistance aux antimicrobiens et les facteurs prédisposants suggérés chez les vaches laitières de l'Ontario soumises à un laboratoire de diagnostic pour une enquête post-mortem sur la pneumonie et fournit des informations pour comprendre pourquoi les épidémies se produisent.(Traduit par Dr Serge Messier).

Observational study on antimicrobial resistance in Escherichia coli and Salmonella isolates from Ontario calf samples submitted to a diagnostic laboratory from 2007 to 2020.

The objectives of this study were to i) describe Escherichia coli and Salmonella isolates; ii) investigate the temporal trends in antimicrobial resistance (AMR) profiles; and iii) evaluate the impact of season and age on these AMR profiles from diagnostic and post-mortem samples in Ontario calves ≤ 2-months-old submitted from 2007 to 2020 to the Animal Health Laboratory in Guelph, Ontario, Canada. Antimicrobial susceptibility testing results were measured by the Kirby-Bauer disk diffusion method. A total of 1291 isolates with AMR profiles were obtained from calves, with E. coli (n = 434) and Salmonella (n = 378) being the most common bacteria characterized for AMR. For E. coli, 79% of isolates tested showed a positive result in F5/K99, whereas for Salmonella isolates, S. Typhimurium (33%) and S. Dublin (22%) were the 2 most common serotypes identified. Multivariable logistic regression models were built to evaluate AMR profiles for E. coli (n = 414) and Salmonella (n = 357) to each antimicrobial tested. Most E. coli isolates (91%) and Salmonella isolates (97%) were resistant to at least one of the antimicrobials tested. In general, E. coli and Salmonella had higher odds of resistance in calves aged ≥ 2 wk compared to 1-week-old calves, and little difference was seen in the level of resistance over the years observed or between seasons in most of the antimicrobials tested. Prospective research should investigate potential risk factors for the development of AMR in calves examples being antimicrobial use and farm management practices.

Étude observationnelle sur la résistance aux antimicrobiens d'isolats d'Escherichia coli et de Salmonella provenant d'échantillons de veaux de l'Ontario soumis à un laboratoire de diagnostic de 2007 à 2020. Les objectifs de cette étude étaient de i) décrire les isolats d'Escherichia coli et de Salmonella; ii) étudier les tendances temporelles des profils de résistance aux antimicrobiens (RAM); et iii) évaluer l'impact de la saison et de l'âge sur ces profils de RAM à partir d'échantillons diagnostiques et post-mortem de veaux de l'Ontario âgés de ≤ 2 mois soumis de 2007 à 2020 au Laboratoire de santé animale de Guelph, Ontario, Canada. Les résultats des tests de sensibilité aux antimicrobiens ont été mesurés par la méthode de diffusion en disque de Kirby-Bauer. Un total de 1291 isolats avec des profils de RAM ont été obtenus à partir de veaux, E. coli (n = 434) et Salmonella (n = 378) étant les bactéries les plus courantes caractérisées pour la RAM. Pour E. coli, 79 % des isolats testés ont montré un résultat positif en F5/K99, alors que pour les isolats de Salmonella, S. Typhimurium (33 %) et S. Dublin (22 %) étaient les deux sérotypes les plus fréquemment identifiés. Des modèles de régression logistique multivariable ont été construits pour évaluer les profils de RAM pour E. coli (n = 414) et Salmonella (n = 357) pour chaque antimicrobien testé. La plupart des isolats d'E. coli (91 %) et des isolats de Salmonella (97 %) étaient résistants à au moins un des antimicrobiens testés. En général, E. coli et Salmonella présentaient un risque de résistance plus élevé chez les veaux âgés de ≥ 2 semaines par rapport aux veaux âgés d'une semaine, et peu de différence a été observée dans le niveau de résistance au cours des années observées ou entre les saisons pour la plupart des antimicrobiens testés. La recherche prospective devrait étudier les facteurs de risque potentiels pour le développement de la RAM chez les veaux, par exemple l'utilisation d'antimicrobiens et les pratiques de gestion de la ferme.(Traduit par Dr Serge Messier).

Diagnostic and public health investigation of Mycobacterium tuberculosis infection in a dog in Ontario, Canada.

A 4-y-old, female mixed-breed dog was presented to the Ontario Veterinary College for further evaluation of multiple pulmonary and hepatic masses, intrathoracic lymphadenitis, and recent development of a pyogranulomatous pleural effusion. Along with other comprehensive tests, a thoracic lymph node biopsy was performed, and Mycobacterium tuberculosis complex infection was confirmed by real-time PCR. The dog's condition declined post-operatively, and euthanasia was elected. Postmortem examination confirmed severe granulomatous pneumonia, hepatitis, intrathoracic and intraabdominal lymphadenitis, omentitis, and nephritis. Line-probe assays performed on samples collected postmortem confirmed the species as M. tuberculosis. 24-loci MIRU-VNTR genotyping, spoligotyping, and whole-genome sequencing revealed relations to known human isolates, but no epidemiologic link to these cases was investigated. Given the concern for potential human exposure during this animal's disease course, a public health investigation was initiated; 45 individuals were tested for M. tuberculosis exposure, and no subsequent human infections related to this animal were identified. Our case highlights the need for more readily available, minimally invasive testing for the diagnosis of canine mycobacteriosis, and highlights the ability of canid species to act as potential contributors to the epidemiology of M. tuberculosis infections.

Spatio-Temporal Variation in the Prevalence of Major Mastitis Pathogens Isolated From Bovine Milk Samples Between 2008 and 2017 in Ontario, Canada.

An understanding of the spatio-temporal distribution of several groups of mastitis pathogens can help to inform programs for the successful control and management of mastitis. However, in the absence of an active surveillance program such information is not readily available. In this retrospective study we analyzed passive surveillance data from a diagnostic laboratory with an aim to describe the spatio-temporal trend of major mastitis pathogens between 2008 and 2017 in Ontario dairy cattle. Data for all milk culture samples submitted to the Animal Health Laboratory (AHL) at the University of Guelph between 2008 and 2017 was accessed. Descriptive analyses were conducted to identify the major pathogens and Chi-square goodness-of-fit tests were used to compare between multiple proportions. Likewise, univariable logistic regression analysis was performed to determine if there was a change in the probability of isolating the major mastitis pathogens depending on geography or time. Seasonality was assessed by calculating the seasonal relative risk (RR). Of a total of 85,979 milk samples examined, more than half of the samples (61.07%) showed no growth and the proportion of samples that showed no growth almost halved during the study period. Of the samples (36.21%, n = 31,133) that showed any growth, the major bacterial pathogens were Staphylococcus aureus (15.60%), Non-aureus Staphylococci (NAS) (5.04%), Corynebacterium spp. (2.96%), and Escherichia coli (2.00%). Of the NAS, the major species reported were Staphylococcus chromogenes (69.02%), Staphylococcus simulans (14.45%), Staphylococcus epidermidis (12.99%), and Staphylococcus hyicus (2.13%). A temporal change in the prevalence of contagious pathogens like S. aureus and Corynebacterium spp. was observed with an increasing odds of 1.06 and 1.62, respectively. Likewise, except for Trueperella pyogenes, the prevalence of all the major environmental mastitis pathogens increased during the study period. The isolation of most of the pathogens peaked in summer, except for S. aureus, T. pyogenes, and Streptococcus dysgalactiae which peaked in spring months. Interestingly, a regional pattern of isolation of some bacterial pathogens within Ontario was also observed. This study showed a marked spatio-temporal change in the prevalence of major mastitis pathogens and suggests that a regional and seasonal approach to mastitis control could be of value.

Genome Sequence Analysis of Clostridium chauvoei Strains of European Origin and Evaluation of Typing Options for Outbreak Investigations.

Black quarter caused by Clostridium (C.) chauvoei is an important bacterial disease that affects cattle and sheep with high mortality. A comparative genomics analysis of 64 C. chauvoei strains, most of European origin and a few of non-European and unknown origin, was performed. The pangenome analysis showed limited new gene acquisition for the species. The accessory genome involved prophages and genomic islands, with variations in gene composition observed in a few strains. This limited accessory genome may indicate that the species replicates only in the host or that an active CRISPR/Cas system provides immunity to foreign genetic elements. All strains contained a CRISPR type I-B system and it was confirmed that the unique spacer sequences therein can be used to differentiate strains. Homologous recombination events, which may have contributed to the evolution of this pathogen, were less frequent compared to other related species from the genus. Pangenome single nucleotide polymorphism (SNP) based phylogeny and clustering indicate diverse clusters related to geographical origin. Interestingly the identified SNPs were mostly non-synonymous. The study demonstrates the possibility of the existence of polymorphic populations in one host, based on strain variability observed for strains from the same animal and strains from different animals of one outbreak. The study also demonstrates that new outbreak strains are mostly related to earlier outbreak strains from the same farm/region. This indicates the last common ancestor strain from one farm can be crucial to understand the genetic changes and epidemiology occurring at farm level. Known virulence factors for the species were highly conserved among the strains. Genetic elements involved in Nicotinamide adenine dinucleotide (NAD) precursor synthesis (via nadA, nadB, and nadC metabolic pathway) which are known as potential anti-virulence loci are completely absent in C. chauvoei compared to the partial inactivation in C. septicum. A novel core-genome MLST based typing method was compared to sequence typing based on CRISPR spacers to evaluate the usefulness of the methods for outbreak investigations.

Revisiting Ophidiomycosis (Snake Fungal Disease) After a Decade of Targeted Research.

Emerging infectious diseases (EIDs) are typically characterized by novelty (recent detection) and by increasing incidence, distribution, and/or pathogenicity. Ophidiomycosis, also called snake fungal disease, is caused by the fungus Ophidiomyces ophidiicola (formerly "ophiodiicola"). Ophidiomycosis has been characterized as an EID and as a potential threat to populations of Nearctic snakes, sparking over a decade of targeted research. However, the severity of this threat is unclear. We reviewed the available literature to quantify incidence and effects of ophidiomycosis in Nearctic snakes, and to evaluate whether the evidence supports the ongoing characterization of ophidiomycosis as an EID. Data from Canada remain scarce, so we supplemented the literature review with surveys for O. ophidiicola in the Canadian Great Lakes region. Peer-reviewed reports of clinical signs consistent with ophidiomycosis in free-ranging, Nearctic snakes date back to at least 1998, and retrospective molecular testing of samples extend the earliest confirmed record to 1986. Diagnostic criteria varied among publications (n = 33), confounding quantitative comparisons. Ophidiomycosis was diagnosed or suspected in 36/121 captive snakes and was fatal in over half of cases (66.7%). This result may implicate captivity-related stress as a risk factor for mortality from ophidiomycosis, but could also reflect reporting bias (i.e., infections are more likely to be detected in captive snakes, and severe cases are more likely to be reported). In contrast, ophidiomycosis was diagnosed or suspected in 441/2,384 free-ranging snakes, with mortality observed in 43 (9.8 %). Ophidiomycosis was only speculatively linked to population declines, and we found no evidence that the prevalence of the pathogen or disease increased over the past decade of targeted research. Supplemental surveys and molecular (qPCR) testing in Ontario, Canada detected O. ophidiicola on 76 of 657 free-ranging snakes sampled across ~136,000 km2. The pathogen was detected at most sites despite limited and haphazard sampling. No large-scale mortality was observed. Current evidence supports previous suggestions that the pathogen is a widespread, previously unrecognized endemic, rather than a novel pathogen. Ophidiomycosis may not pose an imminent threat to Nearctic snakes, but further research should investigate potential sublethal effects of ophidiomycosis such as altered reproductive success that could impact population growth, and explore whether shifting environmental conditions may alter host susceptibility.

Factors associated with Salmonella enterica and Escherichia coli during downtime in commercial broiler chicken barns in Ontario.

Salmonella enterica and Escherichia coli are bacteria of concern to veterinary public health and poultry health. Our research aimed to determine the factors associated with S. enterica and E. coli in commercial broiler chicken barns during the rest period between flocks to identify the best methods of sanitation for bacterial load reduction. This involved collecting samples from September 2015 to July 2016 from the floors of 36 barns before sanitation (baseline) and at 2 time intervals after sanitation, followed by microbiological and molecular analysis. A priori variables of interest included sanitation procedure (dry cleaning, wet cleaning, disinfection), sampling point (baseline, 2 d after sanitation, 6 d after sanitation), and flooring type (concrete, wood). The odds of detecting S. enterica were higher on wooden floors that were wet-cleaned than on concrete floors that were dry-cleaned, lower in the winter and spring than in the fall, and lower when samples were collected 2 d and 6 d after sanitation than at baseline. For E. coli, the concentration was higher on wooden floors than on concrete floors and in the summer than in the fall, and it was lower in postsanitation samples from disinfected barns than in presanitation samples from dry-cleaned barns and in the winter than in the fall. Among E. coli isolates, factors associated with the presence of qacEΔ1, a gene associated with resistance to quaternary ammonium compounds, included sanitation procedure, flooring type, cycle length, and the number of times per yr the barn is disinfected. Our findings highlight the importance of cleaning after litter removal, although the sanitation procedure chosen might differ depending on which pathogen is present and causing disease issues; dry cleaning appears to be preferable for S. enterica control, especially in barns with wooden floors, whereas disinfection appears to be preferable for E. coli reduction.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Antimicrobial resistance in fecal Escherichia coli and Salmonella enterica isolates: a two-year prospective study of small poultry flocks in Ontario, Canada.

BACKGROUND: Although keeping small poultry flocks is increasingly popular in Ontario, information on the antimicrobial susceptibility of enteric bacteria of such flocks is lacking. The current study was conducted on small poultry flocks in Ontario between October 2015 and September 2017, and samples were submitted on a voluntary basis to Ontario's Animal Health Laboratory. From each submission, a pooled cecal sample was obtained from all the birds of the same species from the same flock and tested for the presence of two common enteric pathogens, E. coli and Salmonella. Three different isolates from each E. coli-positive sample and one isolate from each Salmonella-positive sample were selected and tested for susceptibility to 14 antimicrobials using a broth microdilution technique. RESULTS: A total of 433 fecal E. coli isolates (358 chicken, 27 turkey, 24 duck, and 24 game bird) and 5 Salmonella isolates (3 chicken, 1 turkey, and 1 duck) were recovered. One hundred and sixty-seven chicken, 5 turkey, 14 duck, and 15 game bird E. coli isolates were pan-susceptible. For E. coli, a moderate to high proportion of isolates were resistant to tetracycline (43% chicken, 81% turkey, 42% duck, and 38% game bird isolates), streptomycin (29% chicken, 37% turkey, and 33% game bird isolates), sulfonamides (17% chicken, 37% turkey, and 21% duck isolates), and ampicillin (16% chicken and 41% turkey isolates). Multidrug resistance was found in 37% of turkey, 20% of chicken, 13% of duck, and 8% of game bird E. coli isolates. Salmonella isolates were most frequently resistant to streptomycin, tetracycline, and sulfonamides. Resistance to cephalosporins, carbapenems, macrolides, and quinolones was infrequent in both E. coli and Salmonella isolates. Cluster and correlation analyses identified streptomycin-tetracycline-sulfisoxazole-trimethoprim-sulfamethoxazole as the most common resistance pattern in chicken E. coli isolates. Turkey E. coli isolates compared to all the other poultry species had higher odds of resistance to tetracycline and ampicillin, and a higher multidrug resistance rate. CONCLUSIONS: Escherichia coli isolates were frequently resistant to antimicrobials commonly used to treat poultry bacterial infections, which highlights the necessity of judicious antimicrobial use to limit the emergence of multidrug resistant bacteria.

Short communication: Detection of antibiotic resistance, mecA, and virulence genes in coagulase-negative Staphylococcus spp. from buffalo milk and the milking environment.

The aim of this study was to determinate whether coagulase-negative staphylococci (CNS) from buffalo milk or the milking environment possess virulence factors that are associated with intramammary infections or antimicrobial resistance. Milk samples (n = 320) from 80 lactating buffalo were evaluated for clinical and subclinical mastitis by physical examination, the strip cup test, California Mastitis Test (CMT), and somatic cell count (SCC) over a 4-mo period. In addition, swabs were obtained from the hands of consenting milkers (16), liners (64), and from the mouths (15) and nostrils (15) of buffalo calves. No clinical cases of mastitis were observed; however, CMT together with SCC results indicated that 8 animals had subclinical mastitis. Eighty-four CNS isolates were identified by MALDI-TOF MS and cydB real-time PCR (qPCR) and then evaluated by qPCR for presence of the eta, etb, sea, sec, cna, seb, sei, seq, sem, seg, see, and tst toxin genes, adhesion- and biofilm-associated genes (eno, ebps, fib, fnbA, coa), and the methicillin resistance gene (mecA). Resistance to antibiotics commonly used for mastitis treatment in Brazil was determined using the Kirby-Bauer test. Two strains were positive for the see and eta toxin genes; and mecA (1), eno (27), ebps (10), fnbA (10), and coa (5) genes were also detected. A notable number of isolates were resistant to erythromycin (30), penicillin (26), and cotrimoxazole (18); importantly, 10 vancomycin-resistant isolates were also detected. A smaller number of isolates were resistant to rifampicin (8), oxacillin (7), clindamycin (5), cefepime (4), tetracycline (3), ciprofloxacin (2), and chloramphenicol (1), and none were resistant to gentamicin or ciprofloxacin. Isolates with resistance to 2 (13 isolates), 3 (3), 4 (3), 5 (1), and 6 (1) antibiotics were detected. Taken together, our findings suggest that CNS isolates may not be a significant cause of clinical or even subclinical mastitis in buffaloes, but they may be a reservoir of virulence and antibiotic resistance genes.

Resistance to antimicrobials used at hatchery level among clinical Escherichia coli isolates from Ontario broilers.

Antimicrobials are used for prophylactic purposes in some flocks because chicks are susceptible to pathogenic bacterial infection at the hatchery stage. The objectives of this study were to assess temporal changes in resistance to ceftiofur, gentamicin, and spectinomycin in Escherichia coli clinical isolates from Ontario broiler chickens between 2008 and 2015, to determine whether stage of production, year and season are predictors of resistance, and whether published data on antimicrobial use help to interpret the temporal patterns in resistance. Logistic regression revealed that stage of production, year, and season were significant predictors of resistance to all 3 antimicrobials. Resistance to ceftiofur fluctuated over time, with a trend towards decreasing resistance between 2013 to 2015; resistance to gentamicin and spectinomycin increased over the study period, with significantly high resistance clusters identified from 2013 to 2015. Comparisons with published hatchery-level antimicrobial use data suggest that these trends may reflect changes in use of ceftiofur and spectinomycin in Ontario.

Résistance aux antimicrobiens utilisés dans les couvoirs chez des isolats d' Escherichia coli provenant de poulets à griller de l'Ontario. Les antimicrobiens sont utilisés à des fins préventives dans certains troupeaux car les oiseaux sont susceptibles à des infections causées par des bactéries pathogènes lorsqu'ils sont au couvoir. Les objectifs de la présente étude étaient d'évaluer les changements temporaux dans la résistance au ceftiofur, à la gentamicine, et à la spectinomycine chez des isolats cliniques d'Escherichia coli provenant de poulets à griller de l'Ontario entre 2008 et 2015, afin de déterminer si le stade de production, l'année et la saison sont des prédicteurs de résistance, et si les données publiées sur l'utilisation des antimicrobiens aide à interpréter les patrons temporaux de résistance. Une régression logistique a révélé que le stade de production, l'année, et la saison étaient des prédicteurs significatifs de résistance pour les trois antibiotiques. La résistance au ceftiofur fluctuait dans le temps, avec une tendance vers une diminution de résistance entre 2013 et 2015; la résistance à la gentamicine et à la spectinomycine a augmenté durant la période d'étude, avec des agrégats de résistance élevée significative entre 2013 et 2015. Des comparaisons avec les données publiées sur l'utilisation des antimicrobiens dans les couvoirs suggèrent que ces tendances pourraient refléter des changements dans l'utilisation du ceftiofur et de la spectinomycine en Ontario.(Traduit par Dr Serge Messier).

Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli isolated from small poultry flocks in Ontario, Canada: A two-year surveillance study.

Antimicrobial resistance in Campylobacter, common in poultry, is a global public health issue. The emergence and spread of antimicrobial resistant Campylobacter has been linked to the use of antimicrobials in food animals. Small poultry flocks are becoming increasingly popular not only as a source of food but also as pets, yet not all small flock owners are aware of proper antimicrobial use practices and safe food handling protocols. This trend could contribute to antimicrobial resistance. In order to determine the prevalence of antimicrobial resistance in Campylobacter in small poultry flocks, we analyzed data from birds that had been submitted to a diagnostic laboratory in Ontario between October 2015 and September 2017. A pooled cecal sample was obtained from each submission and cultured for Campylobacter jejuni and Campylobacter coli. Three isolates were recovered from each positive sample and tested for susceptibility to nine antimicrobials using a broth microdilution method. Overall, 176 isolates were recovered (141 chicken, 21 turkey, 6 duck, and 8 game bird). A high frequency of resistance to tetracycline was observed in the C. jejuni isolates from chickens (77%) and turkeys (100%), and in the C. coli isolates from turkeys (50%) and game birds (40%). Campylobacter jejuni isolates had higher odds of resistance to tetracycline (OR = 3.54, P ≤ 0.01) compared to C. coli isolates. Overall, there was a low frequency of resistance to quinolones and a very low frequency of resistance to macrolides. Multidrug resistance was uncommon. The high prevalence of tetracycline resistance emphasizes the importance of prudent antimicrobial use in small flocks. Although low, the presence of resistance to macrolides and quinolones, which are used to treat campylobacteriosis in humans, highlights the need for proper food safety and infection control practices by small flock owners to prevent exposure to antimicrobial resistant Campylobacter.

Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Multi-Laboratory Validation of a Loop-Mediated Isothermal Amplification Method for Screening Salmonella in Animal Food.

Loop-mediated isothermal amplification (LAMP) has gained wide popularity in the detection of Salmonella in foods owing to its simplicity, rapidity, and robustness. This multi-laboratory validation (MLV) study aimed to validate a Salmonella LAMP-based method against the United States Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method in a representative animal food matrix (dry dog food). Fourteen independent collaborators from seven laboratories in the United States and Canada participated in the study. Each collaborator received two sets of 24 blind-coded dry dog food samples (eight uninoculated; eight inoculated at a low level, 0.65 MPN/25 g; and eight inoculated at a high level, 3.01 MPN/25 g) and initiated the testing on the same day. The MLV study used an unpaired design where different test portions were analyzed by the LAMP and BAM methods using different preenrichment protocols (buffered peptone water for LAMP and lactose broth for BAM). All LAMP samples were confirmed by culture using the BAM method. BAM samples were also tested by LAMP following lactose broth preenrichment (paired samples). Statistical analysis was carried out by the probability of detection (POD) per AOAC guidelines and by a random intercept logistic regression model. Overall, no significant differences in POD between the Salmonella LAMP and BAM methods were observed with either unpaired or paired samples, indicating the methods were comparable. LAMP testing following preenrichment in buffered peptone water or lactose broth also resulted in insignificant POD differences (P > 0.05). The MLV study strongly supports the utility and applicability of this rapid and reliable LAMP method in routine regulatory screening of Salmonella in animal food.

Targeting discriminatory SNPs in Salmonella enterica serovar Heidelberg genomes using RNase H2-dependent PCR.

We report a novel RNase H2-dependent PCR (rhPCR) genotyping assay for a small number of discriminatory single-nucleotide polymorphisms (SNPs) that identify lineages and sub-lineages of the highly clonal pathogen Salmonella Heidelberg (SH). Standard PCR primers targeting numerous SNP locations were initially designed in silico, modified to be RNase H2-compatible, and then optimized by laboratory testing. Optimization often required repeated cycling through variations in primer design, assay conditions, reagent concentrations and selection of alternative SNP targets. The final rhPCR assay uses 28 independent rhPCR reactions to target 14 DNA bases that can distinguish 15 possible lineages and sub-lineages of SH. On evaluation, the assay correctly identified the 12 lineages and sub-lineages represented in a panel of 75 diverse SH strains. Non-specific amplicons were observed in 160 (15.2%) of the 1050 reactions, but due to their low intensity did not compromise assay performance. Furthermore, in silico analysis of 500 closed genomes from 103 Salmonella serovars and laboratory rhPCR testing of five prevalent Salmonella serovars including SH indicated the assay can identify Salmonella isolates as SH, since only SH isolates generated amplicons from all 14 target SNPs. The genotyping results can be fully correlated with whole genome sequencing (WGS) data in silico. This fast and economical assay, which can identify SH isolates and classify them into related or unrelated lineages and sub-lineages, has potential applications in outbreak identification, source attribution and microbial source tracking.

Evaluating Virulence-Associated Genes and Antimicrobial Resistance of Avian Pathogenic Escherichia coli Isolates from Broiler and Broiler Breeder Chickens in Ontario, Canada.

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis in poultry, an economically important disease worldwide. In Ontario, Canada, early and late systemic bacterial infections due to APEC were the most commonly reported diseases in broiler chickens. In 2016, Ontario poultry veterinarians submitted samples from 331 cases of broiler and broiler breeder chickens with a high suspicion of colibacillosis to the Animal Health Laboratory (Guelph, Ontario, Canada) for bacterial culture. Escherichia coli isolates from those samples were tested with multiplex PCR to detect the presence of 13 virulence-associated genes. The most common genes identified were sitA (detected in 93% of isolates), iss (88%), iroN (85%), iutA (79%), ompT (77%), and etsB (67%). In 94% of isolates, at least three virulence-associated genes were detected. Antimicrobial susceptibility testing of isolates by using the disk diffusion method revealed high frequencies of resistance to tetracycline (57% of isolates), gentamicin (50%), spectinomycin (46%), and ampicillin (44%). Relatively fewer isolates were resistant to trimethoprim-sulfamethoxazole (18%), ceftiofur (15%), kanamycin (11%), and apramycin (3%). A high proportion (46%) of the isolates were multidrug resistant (≥3 antimicrobial classes). On the basis of multivariable, mixed effects logistic regression models, statistically significant associations ( P ≤ 0.05) were identified between the following: ampicillin resistance and the presence of kpsII (odds ratio [OR] = 1.88), tsh (OR = 0.46), and ireA (OR = 0.32); ceftiofur resistance and etsB (OR = 2.98) and kpsII (OR = 2.61); gentamicin resistance and ompT (OR = 3.89) and sitA (OR = 3.54); kanamycin resistance and papC (OR = 50.10); spectinomycin resistance and ireA (OR = 2.50) and iutA (OR = 3.15); trimethoprim-sulfamethoxazole resistance and ompT (OR = 0.14) and tsh (OR = 0.31); and tetracycline resistance and cvaC (OR = 2.12), eitA (OR = 2.15), and papC (OR = 8.27). On the basis of a multivariable, mixed effects Poisson regression model, the number of antimicrobials to which an isolate was resistant increased with the presence of eitA (risk ratio [RR] = 1.37), iroN (RR = 1.24), papC (RR = 1.34), and sitA (RR = 1.77) and decreased with the presence of tsh (RR = 0.79). On the basis of bivariable logistic regression models, age group and time of sample collection were not significantly associated with resistance to individual antimicrobials, the presence of multidrug resistance, or the presence of virulence-associated genes. Our results provide information on antimicrobial resistance and virulence gene patterns currently present on Ontario broiler chicken and broiler breeder farms that can be used as a benchmark from which to measure changes.

An epidemiological study of Streptococcus suis serotypes of pigs in Ontario determined by a multiplex polymerase chain reaction.

The purpose of the study was to identify the serotypes of Streptococcus suis from tonsil swabs in clinically ill and healthy pigs in Ontario using a multiplex polymerase chain reaction (PCR) method. Although 22 different serotypes were identified, most isolates were S. suis-like bacteria or untypable.

Étude épidémiologique des sérotypes Streptococcus suisdes porcs en Ontario déterminés par amplification en chaîne par polymérase multiplexe. Le but de cette étude consistait à identifier les sérotypes de Streptococcus suis provenant d'écouvillons des amygdales chez des porcs cliniquement malades et en santé en Ontario en utilisant une méthode multiplexe d'amplification en chaîne par polymérase (PCR). Même si 22 sérotypes différents ont été identifiés, la plupart des isolats étaient des bactéries de type S. suis ou non typables.(Traduit par Isabelle Vallières).