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We evaluated the feasibility of cffDNA extraction from the maternal blood samples regarding the threshold concentrations required for fetal sexing in pregnant cattle by PCR. In four trials, we 1) compared the extraction efficiency of seven methods using freshly harvested plasma/blood of cows carrying male fetii (150-240 d gestation) bovine amelogenin (bAML) and Y-specific gene sequences, 2) identified the minimum amounts of spiked cffDNA needed for a PCR for fetal sexing, 3) determined the most optimal protocol among three commercial kits for cffDNA extraction from neat and spiked plasma samples (181-240 d gestation) for PCR detection of Y-specific sequence and 4) tested Y-specific sequence PCR on pregnant cows at different stages of gestation (60-150 versus 151-240 d pregnant). In these experiments, blood samples from unbred dairy heifers (Canadian Holstein, n = 10), pregnant dairy cows (Canadian Holstein, 60-240 d gestation, n = 25 with male fetii), and aborting beef cows (Angus cross, n = 5, 100-150 d pregnant) were used for DNA extraction, spiking, and PCR. Extracted DNA from the blood samples of unbred heifers (n = 5) and bull calves (n = 5) served as controls in all trials. In the first trial, DNeasy Blood and Tissue, Qiagen DSP Virus, and NucleoMag cfDNA isolation kits were relatively successful among seven methods to isolate cffDNA from freshly harvested maternal plasma/blood of pregnant cows. In trial 2, using serial dilutions of cffDNA from male fetii spiked in cow plasma samples, a positive and unambiguous detection by PCR targeting Y-specific sequence and bAML gene was observed when spiked cffDNA concentration in plasma was31.3 pg/ml and2 ng/ml, respectively. In the third trial, the DNeasy Blood and Tissue kit was most successful in extracting cffDNA spiked at the minimum concentrations in maternal plasma and subsequent PCR for Y-specific sequence. In our fourth trial, more cows in the second half (9/10) of gestation showed a positive Y-specific PCR outcome than those in the first half (3/9, Fischer's exact test; P 0.05, 90%; CI: 55.5-99.75 vs 33%; CI: 7.5-70.1). In conclusion, we observed variability between different DNA extraction methodologies and stages of gestation results in the PCR for prenatal sexing. Thus, the current PCR methodologies are unreliable for detecting cffDNA in pregnant cows. Additionally, ≥10 (≥31.3 pg/ml of cffDNA) and ≥648 (≥2 ng/ml of cffDNA) copies of the whole fetal genome in bovine maternal plasma are needed for Y-specific PCR and bAML PCR, respectively.

We investigated the effects of culling on Bartonella spp. bacteria carriage among urban rats in Canada. We found that the odds of Bartonella spp. carriage increased across city blocks except those in which culling occurred. Removing rats may have prevented an increase in Bartonella spp. prevalence, potentially lowering human health risks.

Background Inflammatory bowel disease (IBD) is common in women of childbearing years, and active IBD during pregnancy is associated with increased rates of preterm delivery and low-birth-weight newborns. Changes in the vaginal microbiome have been associated with preterm delivery. We aimed to determine the taxonomic composition of the vaginal microbiota at 3 time points during pregnancy in a population of women with IBD. Methods Participants were recruited from the patient registry of the Preconception and Pregnancy IBD Clinic at Royal University Hospital in Saskatoon, Canada. Self-collected vaginal swabs were obtained from patients at each trimester. Microbiota profiles were created by cpn60 amplicon sequencing. Results We characterized the vaginal microbiota of 32 pregnant participants with IBD (33 pregnancies) during each trimester. A total of 32 of 33 pregnancies resulted in a live birth with 43.8% (n = 14 of 32, 2 missing) by caesarean section; 2 of 32 were preterm. Microbiota compositions corresponded to previously described community state types, with most participants having microbiota dominated by Lactobacillus crispatus. In 25 of 29 participants in which samples were available for more than 1 time point, there was no change in the community state type over time. Prevalence of Mollicutes (Mycoplasma and/or Ureaplasma) was significantly higher in pregnant participants with IBD than in a previously profiled cohort of 172 pregnant women without IBD who delivered at term. Conclusions The vaginal microbiome of participants with IBD was stable throughout pregnancy. Prevalence of Mollicutes, which has been associated with preterm delivery, warrants further study in this patient group.

Background Bartonella are intracellular bacteria that are transmitted via animal scratches, bites and hematophagous arthropods. Rodents and their associated fleas play a key role in the maintenance of Bartonella worldwide, with > 22 species identified in rodent hosts. No studies have addressed the occurrence and diversity of Bartonella species and vectors for small mammals in Arctic and Subarctic ecosystems, which are increasingly impacted by invasive species and climate change. Methods In this study, we characterized the diversity of rodent fleas using conventional PCR targeting the mitochondrial cytochrome c oxidase II gene (COII) and Bartonella species in rodents and shrews (n = 505) from northern Canada using conventional PCR targeting the ITS (intergenic transcribed spacer) region and gltA (citrate synthase) gene. Metagenomic sequencing of a portion of the gltA gene was completed on a subset of 42 rodents and four rodent flea pools. Results Year, total summer precipitation the year prior to sampling, average minimum spring temperature and small mammal species were significant factors in predicting Bartonella positivity. Occurrence based on the ITS region was more than double that of the gltA gene and was 34% (n = 349) in northern red-backed voles, 35% (n = 20) in meadow voles, 37% (n = 68) in deer mice and 31% (n = 59) in shrews. Six species of Bartonella were identified with the ITS region, including B. grahamii, B. elizabethae, B. washoensis, Candidatus B. rudakovii, B. doshiae, B. vinsonii subsp. berkhoffii and subsp. arupensis. In addition, 47% (n = 49/105) of ITS amplicons had Bartonella species (B. heixiaziensis) was detected during metagenomic sequencing of the gltA gene in 6/11 rodents that had ITS sequences with B. grahamii and B. heixiaziensis). Conclusion Our study calls attention to the usefulness of a combined approach to determine the occurrence and diversity of Bartonella communities in hosts and vectors. Graphical Abstract

Rickettsiella infection was diagnosed in 4 adult emperor scorpions ( Pandinus imperator) from 2 different collections over a 3-year period. One case had a 2-day history of weakness, failure to lift the tail, or respond to stimulation, with rapid progression to death. The other 3 cases were found dead. There were no gross lesions, but histologically the hemolymphatic vasculature and sinuses, presumed hematopoietic organ, heart, midgut and midgut diverticula, nerves, and skeletal muscle were infiltrated with phagocytic and granular hemocytes with necrosis. Phagocytic hemocytes contained abundant intracellular microorganisms that were Fite’s acid-fast-positive, Macchiavello-positive, variably gram-positive or gram-negative, and Grocott’s methenamine silver-negative. By transmission electron microscopy, hemocytes contained numerous phagocytic vacuoles with small dense bacterial forms (mean 0.603 × 0.163 μm) interspersed with large bacterial forms (mean 1.265 × 0.505 μm) and few intermediary forms with electron-dense nucleoids and membrane-bound crystalline arrays (average 4.72 μm). Transmission electron microscopy findings were consistent with bacteria of the family Coxiellaceae. Based on sequencing the 16S ribosomal RNA gene, the identity was confirmed as Rickettsiella, and phylogenetic analysis of protein-coding genes gidA, rspA, and sucB genes suggested the emperor scorpion pathogen as a new species. This study identifies a novel Rickettsiella causing infection in emperor scorpions and characterizes the unique pathological findings of this disease. We suggest this organism be provisionally named Rickettsiella scorpionisepticum.

The aetiology and pathogenesis of bovine respiratory disease (BRD) are complex and involve the interplay of infectious agents, management and environmental factors. Previous studies of BRD focused on ante‐mortem samples from the upper respiratory tract and identified several unconventional viruses. The lung, however, is the primary location where significant BRD lesions are usually found and is a common post‐mortem diagnostic specimen. In this study, results of high‐throughput virome sequencing, bacterial culture, targeted real‐time PCR and histological examination of 130 bovine pneumonic lungs from western Canadian cattle were combined to explore associations of microorganisms with different types of pneumonia. Fibrinous bronchopneumonia (FBP) was the predominant type of pneumonia (46.2%, 60/130) and was associated with the detection of Mannheimia haemolytica. Detection of Histophilus somni and Pasteurella multocida was associated with suppurative bronchopneumonia (SBP) and concurrent bronchopneumonia and bronchointerstitial pneumonia (BP&BIP), respectively. Sixteen viruses were identified, of which bovine parvovirus 2 (BPV2) was the most prevalent (11.5%, 15/130) followed by ungulate tetraparvovirus 1 (UTPV1, 8.5%, 11/130) and bovine respiratory syncytial virus (BRSV, 8.5%, 11/130). None of these viruses, however, were significantly associated with a particular type of pneumonia. Unconventional viruses such as influenza D virus (IDV) and bovine rhinitis B virus (BRBV) were detected, although sparsely, consistent with our previous findings in upper respiratory tract samples. Taken together, our results show that while virus detection in post‐mortem lung samples is of relatively little diagnostic value, the strong associations of H. somni and M. haemolytica with SBP and FBP, respectively, indicate that histopathology can be useful in differentiating bacterial aetiologies.

Bovine respiratory disease (BRD) causes considerable economic losses in North America. The pathogenesis involves interactions between bacteria, viruses, environment and management factors. Primary viral infection can increase the risk of secondary fatal bacterial infection. The objective of this study was to use metagenomic sequencing to characterize the respiratory viromes of paired nasal swabs and tracheal washes from western Canadian feedlot cattle, with or without BRD. A total of 116 cattle (116 nasal swabs and 116 tracheal washes) were analysed. The presence of influenza D virus (IDV), bovine rhinitis A virus (BRAV), bovine rhinitis B virus (BRBV), bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) was associated with BRD. Agreement between identification of viruses in nasal swabs and tracheal washes was generally weak, indicating that sampling location may affect detection of infection. This study reported several viruses for the first time in Canada and provides a basis for further studies investigating candidate viruses important to the prevention of BRD.

Swine dysentery is causally associated with Brachyspira hampsonii and B. hyodysenteriae infection. Given the importance of transmission models in understanding re-emergent diseases and developing control strategies such as vaccines, the objective of this experiment was to evaluate two experimental natural transmission (seeder pig) models in grower pigs, each with 24 animals. Seeder pigs were intragastrically inoculated using broth cultures of either B. hampsonii strain 30446 (genomovar II) or B. hyodysenteriae strain G44. In trial 1, three seeder pigs were placed into two pens containing nine susceptible contact pigs creating a 1:3 seeder:contact ratio. This was sufficient to achieve natural B. hampsonii infection of 13/18 (72%) contact pigs, however, the incidence of mucoid or mucohemorrhagic diarrhea (MMHD) in contact pigs differed significantly between pens (4/9 versus 9/9; P = 0.03). In trial 2, eight seeder pigs inoculated intragastrically with B. hampsonii did not develop MMHD but when re-inoculated with B. hyodysenteriae 14 days later, all developed mucohemorrhagic diarrhea within 13 days of re-inoculation. Two seeder pigs were placed into each of 4 contact pens each containing 4 pigs. This 1:2 seeder:contact ratio resulted in natural infection of 14/16 (87%) contact pigs with incubation period ranging from 9–15 days. There were no significant differences among pens in incubation period, duration, clinical period or severity of diarrhea. These trials demonstrated that a 1:2 seeder:contact ratio with groups of six grower pigs per pen sustained natural transmission of B. hyodysenteriae G44 with greater consistency in the incidence of MMHD among pens compared to a B. hampsonii 30446 transmission model using 1:3 seeder:contact ratio in pens of 12. Understanding why B. hampsonii intragastric inoculation failed in one experiment warrants additional research.

This protocol can be applied to determine the composition of a microbial community. The cpn60 gene (also known as groEL, hsp60) is present in almost all bacteria and a 552-558 bp region of the gene has been established as a barcode for species level identification of bacteria. The primer cocktail used in this protocol amplifies cpn60 barcode sequences from bacteria with a wide range of G+C content. Some species of Mycoplasma lack the cpn60 gene and therefore this method is not recommended to detect Mycoplasma. DNA sequences generated from this method could be compared to cpnDB, a public database of cpn60 sequences, for identification. Library preparation involves cpn60 amplicon generation, PCR clean-up, index PCR, index PCR clean-up, library quantification, normalization, pooling, library denaturation and loading. Time taken to complete depends on the number of samples included. If using 96 samples, the procedure takes 8 hours but there are several stages where the samples could be stored and continued the next day. Specific instructions are provided for the Illumina MiSeq platform, but the protocol could easily be adapted for other sequencing platforms.

Urban Norway rats (Rattus norvegicus) are a reservoir for Bartonella spp. - a genus of zoonotic bacteria transmitted by hematophagous vectors, particularly fleas. Rats and fleas may be infected with more than one Bartonella species; however, mixed infections may be difficult to detect using culture and/or mono-locus PCR. We set out to characterize Bartonella spp. using gltA PCR and Sanger sequencing on blood (n = 480) and Nosopsyllus fasciatus flea pools (n = 200) obtained from a population of urban Norways rats from Vancouver, Canada. However, when contamination of a subset of flea pools necessitated the use of a second target (ssrA) and the results of gltA and ssrA were discordant, a metagenomic approach was used to better characterize the Bartonella spp. present in these samples and our objective transitioned to comparing data obtained via metagenomics to those from PCR/sequencing. Among the Bartonella spp.-positive rats (n = 95), 52 (55.3%), and 41 (43.6%) had Sanger sequences consistent with Bartonella tribocorum and Bartonella vinsonii, respectively. One rat had a mixed infection. All sequences from Bartonella spp.-positive flea pools (n = 85), were consistent with B. tribocorum, and re-analysis of 34 bloods of varying Bartonella spp. infection status (based gltA PCR and sequencing) using ssrA PCR showed that the assay was capable of identifying B. tribocorum but not B. vinsonii. Metagenomics analysis of a subset of PCR-positive blood samples (n = 70) and flea pools (n = 24) revealed that both B. tribocorum and B. vinsonii were circulating widely in the study population with 31/70 (44.3%) rats and 5/24 (2.1%) flea pools infected with both species. B. vinsonii, however, made up a smaller relative proportion of the reads for samples with mixed infections, which may be why it was generally not detected by genus-specific PCR and Sanger sequencing. Further analysis of 16S-23S ITS sequences amplified from a subset of samples identified the B. vinsonii strain as B. vinsonii subsp. berkhoffii type II. This demonstrates the value of a metagenomic approach for better characterizing the ecology and health risks associated with this bacterium, particularly given that the less dominant species, B. vinsonii is associated with greater pathogenicity in people.

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What&rsquo, s In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

Swine dysentery is traditionally associated with Brachyspira hyodysenteriae, but the re-emergence of Brachyspira-associated disease in North America associated with a novel causative species, B. hampsonii, is now a concern for swine producers. The pathogenesis of Brachyspira-associated disease is not completely understood, and it is not known whether mixed infections of Brachyspira spp. are important in disease development. Deep sequencing of partial sequences of the nox gene amplified with genus-specific primers was used to detect Brachyspira spp. in 55 fecal samples from clinical cases of mucohaemorrhagic diarrhea in pigs from Western Canada that had been identified as positive for one or more Brachyspira species using established diagnostic tests. Synthetic mixtures of Brachyspira genomic DNA were included in the study to define detection limits for the technique and identify biases in detection of different species. Multiple species were detected in all clinical cases for which sufficient nox sequence data were generated (n = 47), indicating that mixed species Brachyspira infections are common, although in most cases, one species accounted for at least half of the sequences identified. In all cases, the species detected in the original diagnostic investigation of each case was also detected by nox sequencing. Results from synthetic communities indicated that the method was highly reproducible, but also indicated potential PCR bias against B. hampsonii genomovar I. Deep sequencing of the nox gene target is a suitable method for simultaneous detection of multiple Brachyspira species in clinical case material that may offer advantages over current, more targeted diagnostic approaches for investigating the significance of mixed infections in disease development.

A novel Brachyspira emerged in 2009 and has since become a production-limiting pathogen of pigs in North America. The name “ Brachyspira hampsonii” has been proposed for this novel taxon. “ Brachyspira hampsonii” is divided into two phylogenetically distinct clades based on the sequence of the NADH-oxidase ( nox) gene, although the clinical disease associated with clades I and II is indistinguishable and phenotypic characteristics that discriminate the clades have not been determined. The objectives of the current study were to enhance the description of the provisional species “ B. hampsonii” with biochemical profiles and morphometric data from isolates affecting Canadian swine and to investigate potentially diagnostically informative characteristics for this emerging pathogen. Biochemical profiles of isolates from different commercial swine barns in Western Canada showed that biochemical profiles were insufficient to distinguish “ B. hampsonii” clades I and II from each other or from other pathogenic Brachyspira. Hippurate hydrolysis, previously reported as uniformly negative in “ B. hampsonii,” was variable among Canadian isolates. Spirochete dimensions and flagella numbers for “ B. hampsonii” overlapped with other Brachyspira species. Taken together, these results indicate that nox gene sequencing remains a preferred method for identification and discrimination of “ B. hampsonii” from other pig-associated Brachyspira spp.

Mucohaemorrhagic diarrhea associated with Brachyspira hampsonii infection has emerged as a production-limiting disease in western Canada. This pathogen was first described in North America in 2010, and reports of its detection occurred concurrently in western Canada and the United States. Since that time, Brachyspira hampsonii has been detected in Europe, both in pigs and in waterfowl. The origin of B. hampsonii and the timing and reasons for its emergence are unknown. We conducted a retrospective study of historic, archived cases of porcine colitis to determine when B. hampsonii was first evident in western Canada. A total of 206 samples from 114 cases submitted from 57 different farms or productions systems in 1984 and 1999-2009 were screened using real-time PCR assays targeting B. hampsonii genomovars I and II, and Brachyspira hyodysenteriae (the traditional agent of swine dysentery). In most cases, positive real-time PCR results were confirmed by amplification and sequencing of additional gene targets. A total of 9, 7 and 5 samples tested positive for B. hampsonii (I), B. hampsonii (II) or B. hyodysenteriae respectively. The results of this study push the date of first appearance of B. hampsonii in pigs in western Canada back to 2002 (B. hampsonii (I)) and 2006 (B. hampsonii (II)), which is up to 7 years before the new species were first identified in fresh samples.

It is currently unclear how Toxoplasma gondii (Nicolle and Manceaux, 1908) persists in arctic tundra ecosystems in the absence of felid definitive hosts. To investigate potential transmission routes of T. gondii in a terrestrial arctic food web, we collected samples from two migratory herbivores, Ross’s Geese (Chen rossi (Cassin, 1861)) and Lesser Snow Geese (Chen caerulescens (L., 1758)), and from two resident herbivores, Nearctic brown lemmings (Lemmus trimucronatus (Richardson, 1825)) and collared lemmings (Dicrostonyx groenlandicus (Traill, 1823)), trapped at Karrak Lake, Nunavut, Canada. Antibodies were detected in 76 of 234 (32.4%) serum samples from Ross’s Geese and 66 of 233 (28.3%) serum samples from Lesser Snow Geese. We did not detect T. gondii antibodies in filter-paper eluate tested from thoracic fluid samples collected from 84 lemmings. We did not detect T. gondii DNA in brain tissue from these lemmings. Although a small sample size, our findings suggest that lemmings in this terrestrial arctic ecosystem are not exposed to, or infected with, the parasite. This suggests that oocysts are not introduced into the terrestrial arctic ecosystem at Karrak Lake via freshwater runoff from temperate regions. This study demonstrated that live adult arctic-nesting geese are exposed to T. gondii and therefore migratory herbivorous hosts are potential sources of T. gondii infection for predators in terrestrial arctic ecosystems.

Brachyspira hampsonii causes dysentery-like disease in infected pigs. Serial passage of a virulent swine isolate (P13) one-hundred times in laboratory culture medium was conducted to produce an attenuated strain, and to identify genomic determinants of virulence through comparison of genome sequences of the original and passaged strains. The resulting strain, P113, did not differ from P13 in terms of diagnostic biochemical characteristics but had an enhanced growth rate in culture, indicating laboratory adaptation. Whole genome sequencing of P113 revealed several single-nucleotide changes including a T to C transition that results in an R to G amino acid change in a putative mannose-1-phosphate guanylytransferase that is implicated in production of lipo-oligosaccharide. P113 was partially attenuated in a mouse model of infection, indicated by significantly fewer observations of abnormal feces in mice infected with P113 relative to P13. No differences were detected in bacterial shedding in feces, demonstrating that the ability of the organism to colonize mice was not affected. Passage through a mouse did not further alter the virulence of P113. Results of this study provide insight into genomic determinants of virulence in B. hampsonii and a live attenuated vaccine candidate.

Background The development of a mouse model as an in vivo pathogenicity screening tool for Brachyspira spp. has advanced the study of these economically important pathogens in recent years. However, none of the murine models published to date have been used to characterize the clinical signs of disease in mice, instead focusing on pathology following oral inoculation with various Brachyspira spp. The experiments described herein explore modifications of published models to characterize faecal consistency, faecal shedding and pathology in mice challenged with “Brachyspira hampsonii” clade II (Bhamp). Methods and results In Experiment 1, 24 CF-1 mice were randomly allocated to one of three inoculation groups: sham (Ctrl), Bhamp, or B. hyodysenteriae (Bhyo; positive control). Half of each group was fed normal mouse chow (RMH) while the other received a low-zinc diet (TD85420). In Experiment 2, eight CF-1 mice and nine C3H/HeN mice were divided into Ctrl or Bhamp inoculation groups, and all fed TD85420. In Experiment 1, mice fed TD85420 demonstrated more severe mucoid faeces (P = 0.001; Kruskal Wallis) and faecal shedding for a significantly greater number of days (P = 0.005; Kruskal Wallis). Mean faecal scores of Bhamp inoculated mice trended higher than Ctrl (P = 0.06; Wilcoxon rank-sum) as did those of Bhyo mice (P = 0.0; Wilcoxon rank-sum). In Experiment 2, mean faecal scores of inoculated CF-1 mice were significantly greater than in C3H mice (P = 0.049; Kruskal Wallis) but no group differences in faecal shedding were observed. In both experiments, mice clustered based on the severity of colonic and caecal histopathology but high lesion scores were not always concurrent with high fecal scores. Conclusion In our laboratory, CF-1 mice and the lower-zinc TD85420 diet provide a superior murine challenge model of “Brachyspira hampsonii” clade II. Electronic supplementary material The online version of this article (doi:10.1186/s12917-017-1166-5) contains supplementary material, which is available to authorized users.

Mucohemorrhagic diarrhea in pigs caused by Brachyspira spp. has a global distribution, and an economic impact on affected farms due to poor performance of animals. Demonstrations that "Brachyspira hampsonii" is pathogenic have been achieved using in vivo animal models, but a critical knowledge gap exists regarding the pathogenic mechanisms employed by Brachyspira. Here, we used in vitro organ culture of porcine colon to investigate interactions between "B. hampsonii" and explants during the first 12 h of contact. Explants were either inoculated with "B. hampsonii" or sterile culture broth. Responses to infection were evaluated by optical microscopy and quantitative PCR. Significantly greater numbers of necrotic crypt cells and thicker catarrhal exudate were observed on infected explants compared to controls. Spirochaetes were observed in the mucus layer, in contact with necrotic exfoliated cells, in crypts and the lamina propria. Statistical differences were observed in mRNA levels between inoculated and control explants for IL-1α, TNF-α and ZO-1 using a Bayesian analysis, but not observed using the ΔΔCq method. These results provide a demonstration of a porcine colon explant model for investigating interactions of Brachyspira with its host and show that initial effects on the host are observed within the first 12 h of contact.

A Muscovy duckling was presented for necropsy due to ongoing mortalities on a farm. Microscopic examination revealed multisystemic inflammatory lesions with intralesional and intracytoplasmic yeast-like organisms. We identified these agents as yeast belonging to the Order Saccharomycetales based on sequence data obtained from the ribosomal RNA operon.

The objective of this study was to determine prevalence, intensity, and zoonotic potential of gastrointestinal parasites in free-roaming and pet cats in urban areas of Saskatchewan (SK) and a rural region in southwestern Alberta (AB). Fecal samples were analyzed using a modified double centrifugation sucrose flotation to detect helminth eggs and coccidian oocysts, and an immunofluorescence assay to detect Giardia and Cryptosporidium. Endoparasite prevalence was higher in samples from rural AB cats (41% of 27) and free-roaming SK cats (32% of 161) than client-owned SK cats (6% of 31). Parasites identified using morphological and molecular techniques included Toxocara cati, Toxascaris leonina, Baylisascaris-type eggs, Eucoleus aerophilus, Taenia taeniaeformis, Isospora spp., Cryptosporidium spp., and zoonotic genotype A of Giardia duodenalis. This study demonstrates significant differences in endoparasite prevalence in feline populations, and the value of molecular techniques in fecal-based surveys to identify and determine parasite zoonotic potential.

Brachyspira is associated with diarrhea and colitis in pigs, and control of these pathogens is complicated by their complex ecology. Identification of wildlife reservoirs of Brachyspira requires the discrimination of colonized animals and those simply contaminated through environmental exposure. Lesser snow geese (Chen caerulescens caerulescens) were sampled in the Canadian arctic during the summer of 2011, and cloacal swabs were cultured on selective media. Brachyspira isolates were obtained from 15/170 (8.8 %) samples, and 12/15 isolates were similar to isolates previously recovered from pigs, including “Brachyspira hampsonii”, a recently characterized species associated with dysentery-like disease in pigs in North America. A pilot inoculation study with one strongly β-hemolytic B. hampsonii isolate resulted in fecal shedding of the isolate by inoculated pigs for up to 14 days post-inoculation, but no severe clinical disease. Results of this study indicate that lesser snow geese can be colonized by Brachyspira strains that can also colonize pigs. Millions of lesser snow geese (C. caerulescens caerulescens) travel through the major pork-producing areas of Canada and the USA during their annual migration, making them a potential factor in the continental distribution of these bacteria.

BackgroundMucohaemorrhagic diarrhea caused by Brachyspira hyodysenteriae, swine dysentery, is a severe production limiting disease of swine. Recently, pigs in western Canada with clinical signs indistinguishable from swine dysentery were observed. Despite the presence of spirochetes on fecal smears, recognized Brachyspira spp. including B. hyodysenteriae could not be identified. A phylogenetically distinct Brachyspira, called "B. hampsonii" strain 30446, however was isolated. The purpose of this study was to experimentally reproduce mucohaemorrhagic colitis and characterize strain 30446 shedding following inoculation.Methods and findingsEighteen 13-week-old pigs were randomly assigned to inoculation (n = 12) or control (n = 6) groups in each of two trials. In trial 1, pigs were inoculated with a tissue homogenate collected from clinically affected field cases. In trial 2, pigs were inoculated with a pure broth culture of strain 30446. In both trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls, all of which remained healthy. In animals with mucohaemorrhagic diarrhea, significantly more spirochetes were observed on Gram stained fecal smears, and higher numbers of strain 30446 genome equivalents were detected by quantitative PCR (qPCR). Strain 30446 was cultured from colon and/or feces of all affected but no control animals at necropsy.Conclusions"Brachyspira hampsonii" strain 30446 causes mucohaemorrhagic diarrhea in pigs following a 4-9 day incubation period. Fecal shedding was detectable by day 4 post inoculation, and rarely preceded the onset of mucoid or haemorrhagic diarrhea by more than 2 days. Culture and 30446-specific qPCR are reliable methods of detection of this organism in feces and tissues of diarrheic pigs. The emergence of a novel Brachyspira spp., such as "B. hampsonii", creates diagnostic challenges including higher risk of false negative diagnostic tests. We therefore recommend diagnostic laboratories routinely use Brachyspira culture, nox-based and species-specific PCR, and DNA sequencing to diagnose Brachyspira-associated colitis in pigs.

The discovery of the parasite Toxocara vitulorum in bison calves in the province of Manitoba, Canada is discussed. This parasite is more commonly found in the small intestines of bovid calves living in tropical and subtropical regions of the world. This is the first time that Toxocara vitulorum has been reported from hosts in Canada.

To the Editor: Carbapenem antimicrobial drugs are the line of defense against multidrug-resistant gram-negative bacterial infections. The global emergence of carbapenemase-producing organisms is a public health emergency because these enzymes confer resistance to nearly all β-lactam drugs and are often associated with multidrug or pandrug resistance (1). Alarmingly, reports of carbapenemase-producing organisms from environmental and animal sources, including food animals, are increasing (1). Recently, clinical isolates of Salmonella enterica serotype Kentucky that produce VIM-2 and OXA-48 were reportedly isolated from patients in France with a travel history to Africa and the Middle East, suggesting foodborne transmission of carbapenemase producers (2). To the best of our knowledge, before this report no foodborne carbapenemase-producing organisms had been identified in Canada and the United States, although the scope of antimicrobial drug resistance surveillance programs is limited to major agricultural products (poultry, beef, and pork) (3,4). In our modern, ethnically diverse societies, niche-market meat products, including imported foods, are becoming increasingly common. Worldwide dissemination of the Klebsiella pneumoniae, VIM, OXA, and New Delhi metallo-β-lactamase type carbapenemases among humans has been facilitated by intercontinental passenger travel, but the role of the global food trade in this dissemination has not been investigated (5,6). We describe a carbapenemase-producing organism isolated from a squid purchased from the seafood section of a food store. Among other items, the squid was purchased from a Chinese grocery store in Saskatoon, Canada, in January 2014 as part of a drug-resistance surveillance pilot study. Although no country-of-origin labeling was available for inspection, the store owner reported that, according to the distributor, this squid originated in South Korea. An organism with 95.5% sequence identity to Pseudomonas fluorescens was isolated on Mueller-Hinton agar with 2 μg/mL meropenem and identified by partial sequencing of the cpn60 gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KJ606641","term_id":"645930543","term_text":"KJ606641"}}KJ606641). Although the organism was not extensively resistant, it was resistant to all β-lactam drugs tested including ertapenem (Table). PCR amplification and sequencing confirmed that this organism contained VIM-2 carbapenemase (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KJ625238","term_id":"645930547","term_text":"KJ625238"}}KJ625238). Table Antimicrobial drug susceptibility of a VIM-2 producing Pseudomonas fluorescens–like organism isolated from food (squid), Saskatoon, Canada, January 2014 The presence of carbapenemase-producing organisms in the food supply is alarming. Although this organism may not be a pathogen, its contribution to the resistome and the potential for lateral gene transfer to clinically relevant bacteria is certainly a cause for concern. This finding indicates that the risk for exposure to carbapenemases extends beyond persons with particular travel histories, previous antimicrobial drug use, or hospitalization and into the general public. There is an urgent need for expanded resistance surveillance for carbapenemase-producing organisms and their resistance plasmids in food products that are not captured under current programs.

Background: “Brachyspira hampsonii”, discovered in North America in 2010 associated with dysentery-like illness, is an economically relevant swine pathogen resulting in decreased feed efficiency and increased morbidity, mortality and medication usage. “B. hampsonii” clade II strain 30446 has been shown to be causally associated with mucohemorrhagic diarrhea and colitis. Our objectives were to determine if “Brachyspira hampsonii” clade I strain 30599 is pathogenic to pigs, and to evaluate the relative diagnostic performance of three ante mortem sampling methodologies (direct PCR on feces, PCR on rectal GenoTube Livestock swabs, Brachyspira culture from rectal swabs). Five-week old pigs were intragastrically inoculated thrice with 10 8 genomic equivalents "B. hampsonii" (n= 12), or served as sham controls (n= 6). Feces were sampled and consistency assessed daily. Necropsies were performed 24 h after peak clinical signs. Results: One pig died due to unrelated illness. Nine of 11 inoculated pigs, but no controls, developed mucoid or mucohemorrhagic diarrhea (MHD). Characteristic lesions of swine dysentery were observed in large intestine. “B. hampsonii” strain 30599 DNA was detected by qPCR in feces of all inoculated pigs for up to 6 days prior to the onset of MHD. The organism was isolated from the feces and colons of pigs demonstrating MHD, but not from controls. B. intermedia was isolated from inoculated pigs without MHD, and from 5 of 6 controls. Conclusions: We conclude that “Brachyspira hampsonii” clade I strain 30599 is pathogenic and causes mucohemorrhagic diarrhea and colitis in susceptible pigs. Moreover, the three sampling methodologies performed similarly. GenoTube Livestock, a forensic swab designed to preserve DNA during shipping is a useful tool especially in settings where timely transport of diagnostic samples is challenging.

Background Brachyspira associated diarrhea is a re-emerging concern for Canadian swine producers. To identify critical control points for reducing the impact of Brachyspira on production, improved diagnostic tools and a better understanding of the on-farm epidemiology of these pathogens are required. A cross-sectional study was conducted for the detection of Brachyspira on a commercial, two-site, farrow-to-finish pork production unit in Saskatchewan, Canada with a clinical history of mucohaemorrhagic colitis associated with “B. hampsonii”. Results Rectal swabs from pigs at all production stages were collected over 13 weeks (n = 866). Two swabs were collected per pig for culture and Gram stain, and for PCR. Ninety-one culture positive samples were detected, with the highest prevalence of Brachyspira shedding in grower pigs (21%). No Brachyspira were detected in pre-weaned piglets. PCR and Gram stain of rectal swabs detected fewer positive samples than culture. The most prevalent species detected was B. murdochii; other species detected included B. pilosicoli, B. innocens, and “Brachyspira hampsonii”. Phylogenetic analysis revealed that several of the isolates, including some strongly beta-haemolytic isolates, might represent novel taxa. Conclusions Our results indicate that apparently healthy pigs can be colonized with diverse Brachyspira species, including some potential pathogens, and that frequency of shedding peaks in the grower stage. Difference in the detection rates of Brachyspira amongst culture, Gram stain or PCR on rectal swabs have implications for choice of detection methods and surveillance approaches that may be most effective in Brachyspira control strategies.