Identification of genetic markers of resistance to macrolide class antibiotics in Mannheimia haemolytica isolates from a Saskatchewan feedlot.

Mannheimia haemolytica is a major contributor to bovine respiratory disease (BRD), which causes substantial economic losses to the beef industry, and there is an urgent need for rapid and accurate diagnostic tests to provide evidence for treatment decisions and support antimicrobial stewardship. Diagnostic sequencing can provide information about antimicrobial resistance genes in M. haemolytica more rapidly than conventional diagnostics. Realizing the full potential of diagnostic sequencing requires a comprehensive understanding of the genetic markers of antimicrobial resistance. We identified genetic markers of resistance in M. haemolytica to macrolide class antibiotics commonly used for control of BRD. Genome sequences were determined for 99 M. haemolytica isolates with six different susceptibility phenotypes collected over 2 years from a feedlot in Saskatchewan, Canada. Known macrolide resistance genes estT, msr(E), and mph(E) were identified in most resistant isolates within predicted integrative and conjugative elements (ICEs). ICE sequences lacking antibiotic resistance genes were detected in 10 of 47 susceptible isolates. No resistance-associated polymorphisms were detected in ribosomal RNA genes, although previously unreported mutations in the L22 and L23 ribosomal proteins were identified in 12 and 27 resistant isolates, respectively. Pangenome analysis led to the identification of 79 genes associated with resistance to gamithromycin, of which 95% (75 of 79) had no functional annotation. Most of the observed phenotypic resistance was explained by previously identified antibiotic resistance genes, although resistance to the macrolides gamithromycin and tulathromycin was not explained in 39 of 47 isolates, demonstrating the need for continued surveillance for novel determinants of macrolide resistance.IMPORTANCEBovine respiratory disease is the costliest disease of beef cattle in North America and the most common reason for injectable antibiotic use in beef cattle. Metagenomic sequencing offers the potential to make economically significant reductions in turnaround time for diagnostic information for evidence-based selection of antibiotics for use in the feedlot. The success of diagnostic sequencing depends on a comprehensive catalog of antimicrobial resistance genes and other genome features associated with reduced susceptibility. We analyzed the genome sequences of isolates of Mannheimia haemolytica, a major bovine respiratory disease pathogen, and identified both previously known and novel genes associated with reduced susceptibility to macrolide class antimicrobials. These findings reinforce the need for ongoing surveillance for markers of antimicrobial resistance to support improved diagnostics and antimicrobial stewardship.

Tissue and cellular tropism of Eptesicus fuscus gammaherpesvirus in big brown bats, potential role of pulmonary intravascular macrophages.

Gammaherpesviruses (γHVs) are recognized as important pathogens in humans but their relationship with other animal hosts, especially wildlife species, is less well characterized. Our objectives were to examine natural Eptesicus fuscus gammaherpesvirus (EfHV) infections in their host, the big brown bat (Eptesicus fuscus), and determine whether infection is associated with disease. In tissue samples from 132 individual big brown bats, EfHV DNA was detected by polymerase chain reaction in 41 bats. Tissues from 59 of these cases, including 17 from bats with detectable EfHV genomes, were analyzed. An EfHV isolate was obtained from one of the cases, and electron micrographs and whole genome sequencing were used to confirm that this was a unique isolate of EfHV. Although several bats exhibited various lesions, we did not establish EfHV infection as a cause. Latent infection, defined as RNAScope probe binding to viral latency-associated nuclear antigen in the absence of viral envelope glycoprotein probe binding, was found within cells of the lymphoid tissues. These cells also had colocalization of the B-cell probe targeting CD20 mRNA. Probe binding for both latency-associated nuclear antigen and a viral glycoprotein was observed in individual cells dispersed throughout the alveolar capillaries of the lung, which had characteristics of pulmonary intravascular macrophages. Cells with a similar distribution in bat lungs expressed major histocompatibility class II, a marker for antigen presenting cells, and the existence of pulmonary intravascular macrophages in bats was confirmed with transmission electron microscopy. The importance of this cell type in γHVs infections warrants further investigation.

Eptesipox virus-associated lesions in naturally infected big brown bats.

Bats have many unique qualities amongst mammals; one of particular importance is their reported tolerance to viruses without developing disease. Here, the authors present evidence to the contrary by describing and demonstrating viral nucleic acids within lesions from eptesipox virus (EfPV) infection in big brown bats. One hundred and thirty bats submitted for necropsy from Saskatchewan, Canada, between 2017 and 2021 were screened for EfPV by polymerase chain reaction (PCR); 2 had amplifiable poxvirus DNA. The lesions associated with infection were oral and pharyngeal ulcerations and joint swelling in 2/2 and 1/2 cases, respectively. These changes were nonspecific for poxvirus infection, although intracytoplasmic viral inclusion bodies within the epithelium, as observed in 2/2 bats, are diagnostic when present. Viral nucleic acids, detected by in situ hybridization (ISH), were observed in the epithelium adjacent to ulcerative lesions from both cases and within the joint proliferation of 1 case. A new isolate of EfPV was obtained from 1 case and its identity was confirmed with electron microscopy and whole genome sequencing. Juxtanuclear replication factories were observed in most cells; however, rare intranuclear virus particles were also observed. The significance of the presence of virus particles within the nucleus is uncertain. Whole genome assembly indicated that the nucleotide sequence of the genome of this EfPV isolate was 99.7% identical to a previous isolate from big brown bats in Washington, USA between 2009 and 2011. This work demonstrates that bats are not resistant to the development of disease with viral infections and raises questions about the dogma of poxvirus intracytoplasmic replication.

Improving the consistency of experimental swine dysentery inoculation strategies.

Swine dysentery (SD) caused by pathogenic Brachyspira spp. is an economic challenge for the swine industry. In research settings, experimental reproduction of swine dysentery typically relies on intragastric inoculation which has shown variable success. This project aimed to improve the consistency of the experimental inoculation protocol used for swine dysentery in our laboratory. Over six experiments, we evaluated the influence of group housing in inoculated pigs using a frozen-thawed broth culture of strongly hemolytic B. hyodysenteriae strain D19 (Trial A), compared the relative virulence of B. hyodysenteriae strains D19 and G44 (Trial B), compared inoculum volumes (50 mL vs 100 mL) for G44 and B. hampsonii 30446 (Trial C), and performed three independent trials evaluating intragastric inoculation using different oral inoculation methods: oral feed balls (Trial D), and oral syringe bolus of 100 mL (Trial E) or 300 mL (Trial F). Intragastric inoculation with a fresh broth culture of B. hyodysenteriae strain G44 resulted in a shorter incubation period and a higher proportionate duration of mucohemorrhagic diarrhea (MMHD) compared to D19. Intragastric inoculation with either 50 or 100 mL of B. hampsonii 30446 or B. hyodysenteriae (G44) were statistically equivalent. Oral inoculation with 100 mL or 300 mL also yielded similar results to intragastric inoculation but was more expensive due to the additional work and supplies associated with syringe training. Our future research will use intragastric inoculation with 100 mL of a fresh broth culture containing B. hyodysenteriae strain G44 as it yields a high incidence of mucohaemorrhagic diarrhea with a reasonable cost.

Culling of Urban Norway Rats and Carriage of Bartonella spp. Bacteria, Vancouver, British Columbia, Canada.

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.

Infection With a Novel Rickettsiella Species in Emperor Scorpions (Pandinus imperator).

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.

An optimized swine dysentery murine model to characterize shedding and clinical disease associated with "Brachyspira hampsonii" infection.

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.

Disseminated yeast (Order Saccharomycetales) infection in a Muscovy duckling (Cairina moschata).

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.

Infection disséminée à levures (OrdreSaccharomycetales) chez un caneton musqué(Cairina moschata). Un caneton musqué a été présenté pour la nécropsie en raison de mortalités se produisant à une ferme. Un examen microscopique a révélé des lésions inflammatoires multisystémiques par des organismes intralésionnels et intracytoplasmiques s'apparentant à des levures. Nous avons identifié ces agents comme des levures appartenant à l'ordre Saccharomycetales en nous fondant sur des données obtenues auprès de l'opéron ARN risbosomique.(Traduit par Isabelle Vallières).

Enteric parasites of free-roaming, owned, and rural cats in prairie regions of Canada.

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.

Parasites entériques des chats errants, des chats avec propriétaire et des chats ruraux dans les régions des Prairies du Canada. Cette étude avait pour but de déterminer la prévalence, l'intensité et le potentiel zoonotique des parasites gastro-intestinaux chez les chats errants et les chats animaux de compagnie dans les régions urbaines de la Saskatchewan et une région rurale du Sud-Ouest de l'Alberta. Des échantillons de fèces ont été analysés à l'aide d'une méthode modifiée de flottaison au sucrose à double centrifugation afin de détecter les œufs d'helminthe et les ookystes coccidiens et une épreuve d'immunofluorescence a été réalisée pour détecter Giardia et Cryptosporidium. La prévalence des endoparasites était supérieure dans les échantillons provenant de chats ruraux de l'Alberta (41 % de 27) et de chats errants de la Saskatchewan (32 % de 161) que dans ceux de chats appartenant à des clients (6 % de 31). Les parasites identifiés à l'aide de techniques morphologiques et moléculaires incluaient Toxocara cati, Toxascaris leonina, des œufs de type Baylisascaris, Eucoleus aerophilus, Taenia taeniaeformis, Isospora spp., Cryptosporidium spp., et un génotype zoonotique A de Giardia duodenalis. Cette étude démontre les différences significatives dans la prévalence des endoparasites chez les populations félines et la valeur des techniques moléculaires dans les études basées sur les fèces afin d'identifier et de déterminer le potentiel zoonotique.(Traduit par Isabelle Vallières).

Confirmation that "Brachyspira hampsonii" clade I (Canadian strain 30599) causes mucohemorrhagic diarrhea and colitis in experimentally infected pigs.

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 108 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.

Using clotted, pelleted blood samples for direct molecular detection of Bartonella spp. in small mammal wildlife surveillance studies.

OBJECTIVE: Bartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals. RESULTS: DNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.

Environmental and sociodemographic factors associated with zoonotic pathogen occurrence in Norway rats (Rattus norvegicus) from Windsor, Ontario.

AIMS: Rat-associated zoonotic pathogen transmission at the human-wildlife interface is a public health concern in urban environments where Norway rats (Rattus norvegicus) thrive on abundant anthropogenic resources and live in close contact with humans and other animal species. To identify potential factors influencing zoonotic pathogen occurrence in rats, we investigated associations between environmental and sociodemographic factors and Leptospira interrogans and Bartonella spp. infections in rats from Windsor, Ontario, Canada, while controlling for the potential confounding effects of animal characteristics (i.e., sexual maturity and body condition). METHODS AND RESULTS: Between November 2018 and June 2021, 252 rats were submitted by collaborating pest control professionals. Kidney and spleen samples were collected for L. interrogans and Bartonella spp. PCR and sequencing, respectively. Of the rats tested by PCR, 12.7% (32/252) were positive for L. interrogans and 16.3% (37/227) were positive for Bartonella species. Associations between infection status and environmental and sociodemographic variables of interest were assessed via mixed multivariable logistic regression models with a random intercept for social group and fixed effects to control for sexual maturity and body condition in each model. The odds of L. interrogans infection were significantly higher in rats from areas with high building density (odds ratio [OR]: 3.76; 95% CI: 1.31-10.79; p = 0.014), high human population density (OR: 3.31; 95% CI: 1.20-9.11; p = 0.021), high proportion of buildings built in 1960 or before (OR: 11.21; 95% CI: 2.06-60.89; p = 0.005), and a moderate number of reports of uncollected garbage compared to a low number of reports (OR: 4.88; 95% CI: 1.01-23.63; p = 0.049). A negative association was observed between median household income and Bartonella spp. infection in rats (OR: 0.26; 95% CI: 0.08-0.89; p = 0.031). CONCLUSIONS: Due to the complexity of the ecology of rat-associated zoonoses, consideration of environmental and sociodemographic factors is of critical importance to better understand the nuances of host-pathogen systems and inform how urban rat surveillance and intervention efforts should be distributed within cities.

Isolation and characterization of Brachyspira spp. including "Brachyspira hampsonii" from lesser snow geese (Chen caerulescens caerulescens) in the Canadian Arctic.

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 ß-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.

Fecal shedding of Brachyspira spp. on a farrow-to-finish swine farm with a clinical history of "Brachyspira hampsonii"-associated colitis.

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.

Evaluating cpn60 for high-resolution profiling of the mammalian skin microbiome and detection of phylosymbiosis.

Despite being the most widely used phylogenetic marker for amplicon-based profiling of microbial communities, limited phylogenetic resolution of the 16S rRNA gene limits its use for studies of host-microbe co-evolution. In contrast, the cpn60 gene is a universal phylogenetic marker with greater sequence variation capable of species-level resolution. This research compared mammalian skin microbial profiles generated from cpn60 and 16S rRNA gene sequencing approaches, testing for patterns of phylosymbiosis that suggest co-evolutionary host-microbe associations. An ~560 bp fragment of the cpn60 gene was amplified with universal primers and subjected to high-throughput sequencing. Taxonomic classification of cpn60 sequences was completed using a naïve-Bayesian QIIME2 classifier created for this project, trained with an NCBI-supplemented curated cpn60 database (cpnDB_nr). The cpn60 dataset was then compared to published 16S rRNA gene amplicon data. Beta diversity comparisons of microbial community profiles generated with cpn60 and 16S rRNA gene amplicons were not significantly different, based on Procrustes analysis of Bray-Curtis and UniFrac distances. Despite similar relationships among skin microbial profiles, improved phylogenetic resolution provided by the cpn60 gene sequencing permitted observations of phylosymbiosis between microbial community profiles and their mammalian hosts that were not previously observed with 16S rRNA gene profiles. Subsequent investigation of Staphylococcaceae taxa using the cpn60 gene showed increased phylogenetic resolution compared the 16S rRNA gene profiles, revealing potential co-evolutionary host-microbe associations. Overall, our results demonstrate that 16S rRNA and cpn60 marker genes generate comparable microbial community composition patterns while cpn60 better facilitates analyses, such as phylosymbiosis, that require increased phylogenetic resolution.

Complete genome sequence of Mannheimia bovis isolated from a healthy feedlot calf in Saskatchewan, Canada.

A lack of whole genome sequences for Mannheimia spp. other than Mannheimia haemolytica complicates their identification. Here, we present the genome sequence of Mannheimia bovis 39324.S-11, isolated from a healthy calf on a feedlot in Saskatchewan, Canada, and compare it to ZY190616T, which is currently the only other isolate of M. bovis for which sequence is publicly available.

Toxocara vitulorum in a bison (Bison bison) herd from western Canada.

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.

Identifying the minimum concentrations of cell-free fetal DNA in maternal blood required for bovine fetal sexing using PCR.

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 was >31.3 pg/ml and >2 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.

Combining deep sequencing and conventional molecular approaches reveals broad diversity and distribution of fleas and Bartonella in rodents and shrews from Arctic and Subarctic ecosystems.

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 < 97% identity to sequences in GenBank, possibly due to a limited reference library or previously unreported species. An additional Bartonella species (B. heixiaziensis) was detected during metagenomic sequencing of the gltA gene in 6/11 rodents that had ITS sequences with < 97% identity in GenBank, highlighting that a limited reference library for the ITS marker likely accounted for low sequence similarity in our specimens. In addition, one flea pool from a northern red-backed vole contained multiple species (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.

Composition and Stability of the Vaginal Microbiota of Pregnant Women With Inflammatory Bowel Disease.

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.

Composition of the vaginal microbiota was stable throughout pregnancy. Prevalence of Mollicutes was significantly higher in individuals with inflammatory bowel disease than in a previously profiled cohort of 172 pregnant women without inflammatory bowel disease who delivered at term.