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.

Laboratory tools for the direct detection of bacterial respiratory infections and antimicrobial resistance: a scoping review.

Rapid laboratory tests are urgently required to inform antimicrobial use in food animals. Our objective was to synthesize knowledge on the direct application of long-read metagenomic sequencing to respiratory samples to detect bacterial pathogens and antimicrobial resistance genes (ARGs) compared to PCR, loop-mediated isothermal amplification, and recombinase polymerase amplification. Our scoping review protocol followed the Joanna Briggs Institute and PRISMA Scoping Review reporting guidelines. Included studies reported on the direct application of these methods to respiratory samples from animals or humans to detect bacterial pathogens ±ARGs and included turnaround time (TAT) and analytical sensitivity. We excluded studies not reporting these or that were focused exclusively on bioinformatics. We identified 5,636 unique articles from 5 databases. Two-reviewer screening excluded 3,964, 788, and 784 articles at 3 levels, leaving 100 articles (19 animal and 81 human), of which only 7 studied long-read sequencing (only 1 in animals). Thirty-two studies investigated ARGs (only one in animals). Reported TATs ranged from minutes to 2 d; steps did not always include sample collection to results, and analytical sensitivity varied by study. Our review reveals a knowledge gap in research for the direct detection of bacterial respiratory pathogens and ARGs in animals using long-read metagenomic sequencing. There is an opportunity to harness the rapid development in this space to detect multiple pathogens and ARGs on a single sequencing run. Long-read metagenomic sequencing tools show potential to address the urgent need for research into rapid tests to support antimicrobial stewardship in food animal production.

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.

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.

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.

Rapid and accurate taxonomic classification of cpn60 amplicon sequence variants.

The "universal target" region of the gene encoding the 60 kDa chaperonin protein (cpn60, also known as groEL or hsp60) is a proven sequence barcode for bacteria and a useful target for marker gene amplicon-based studies of complex microbial communities. To date, identification of cpn60 sequence variants from microbiome studies has been accomplished by alignment of queries to a reference database. Naïve Bayesian classifiers offer an alternative identification method that provides variable rank classification and shorter analysis times. We curated a set of cpn60 barcode sequences to train the RDP classifier and tested its performance on data from previous human microbiome studies. Results showed that sequences accounting for 79%, 86% and 92% of the observations (read counts) in saliva, vagina and infant stool microbiome data sets were classified to the species rank. We also trained the QIIME 2 q2-feature-classifier on cpn60 sequence data and demonstrated that it gives results consistent with the standalone RDP classifier. Successful implementation of a naïve Bayesian classifier for cpn60 sequences will facilitate future microbiome studies and open opportunities to integrate cpn60 amplicon sequence identification into existing analysis pipelines.

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.

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.

Birth Mode Does Not Determine the Presence of Shared Bacterial Strains between the Maternal Vaginal Microbiome and the Infant Stool Microbiome.

Dysbiosis of the neonatal gut microbiome during early life has been suggested as the missing link that may explain higher rates of certain diseases in caesarean section-delivered infants. Many studies report delivery mode-related dysbiosis in infants due to a lack of maternal vaginal microbiome exposure, prompting interventions to correct the neonatal gut microbiome by transferring these missing microbes after caesarean delivery. The maternal vaginal microbiome is among the first microbial exposures that many infants experience, yet little is known about the extent of direct transmission of maternal vaginal microbes. As part of the Maternal Microbiome Legacy Project, we aimed to determine if maternal vaginal bacteria are vertically transmitted to infants. We employed cpn60 microbiome profiling, culture-based screening, molecular strain typing, and whole-genome sequencing to determine whether identical maternal vaginal strains were present in infant stool microbiomes. We identified identical cpn60 sequence variants in both halves of maternal-infant dyads in 204 of 585 Canadian women and their newborn infants (38.9%). The same species of Bifidobacterium and Enterococcus were cultured from maternal and corresponding infant samples in 33 and 13 of these mother-infant dyads, respectively. Pulsed-field gel electrophoresis and whole-genome sequencing determined that near-identical strains were detected in these dyads irrespective of delivery mode, indicating an alternative source in cases of caesarean delivery. Overall, we demonstrated that vertical transmission of maternal vaginal microbiota is likely limited and that transmission from other maternal body sites, such as the gut and breast milk, may compensate for the lack of maternal vaginal microbiome exposure during caesarean delivery. IMPORTANCE The importance of the gut microbiome in human health and disease is widely recognized, and there has been a growing appreciation that alterations in gut microbiome composition during a "critical window" of development may impact health in later life. Attempts to correct gut microbiome dysbiosis related to birth mode are underpinned by the assumption that the lack of exposure to maternal vaginal microbes during caesarean delivery is responsible for dysbiosis. Here, we demonstrate that there is limited transmission of the maternal vaginal microbiome to the neonatal gut, even in cases of vaginal delivery. Furthermore, the presence of identical strains shared between mothers and infants in early life, even in cases of caesarean delivery, highlights compensatory microbial exposures and sources for the neonatal stool microbiome other than the maternal vagina.

Maternal vaginal microbiome composition does not affect development of the infant gut microbiome in early life.

Birth mode has been implicated as a major factor influencing neonatal gut microbiome development, and it has been assumed that lack of exposure to the maternal vaginal microbiome is responsible for gut dysbiosis among caesarean-delivered infants. Consequently, practices to correct dysbiotic gut microbiomes, such as vaginal seeding, have arisen while the effect of the maternal vaginal microbiome on that of the infant gut remains unknown. We conducted a longitudinal, prospective cohort study of 621 Canadian pregnant women and their newborn infants and collected pre-delivery maternal vaginal swabs and infant stool samples at 10-days and 3-months of life. Using cpn60-based amplicon sequencing, we defined vaginal and stool microbiome profiles and evaluated the effect of maternal vaginal microbiome composition and various clinical variables on the development of the infant stool microbiome. Infant stool microbiomes showed significant differences in composition by delivery mode at 10-days postpartum; however, this effect could not be explained by maternal vaginal microbiome composition and was vastly reduced by 3 months. Vaginal microbiome clusters were distributed across infant stool clusters in proportion to their frequency in the overall maternal population, indicating independence of the two communities. Intrapartum antibiotic administration was identified as a confounder of infant stool microbiome differences and was associated with lower abundances of Escherichia coli, Bacteroides vulgatus, Bifidobacterium longum and Parabacteroides distasonis. Our findings demonstrate that maternal vaginal microbiome composition at delivery does not affect infant stool microbiome composition and development, suggesting that practices to amend infant stool microbiome composition focus factors other than maternal vaginal microbes.

Gardnerella Revisited: Species Heterogeneity, Virulence Factors, Mucosal Immune Responses, and Contributions to Bacterial Vaginosis.

Gardnerella species are associated with bacterial vaginosis (BV) and have been investigated as etiological agents of the condition. Nonetheless, the isolation of this taxon from healthy individuals has raised important questions regarding its etiological role. Recently, using advanced molecular approaches, the Gardnerella genus was expanded to include several different species that exhibit differences in virulence potential. Understanding the significance of these different species with respect to mucosal immunity and the pathogenesis and complications of BV could be crucial to solving the BV enigma. Here, we review key findings regarding the unique genetic and phenotypic diversity within this genus, virulence factors, and effects on mucosal immunity as they stand. We also comment on the relevance of these findings to the proposed role of Gardnerella in BV pathogenesis and in reproductive health and identify key gaps in knowledge that should be explored in the future.

Transport and Utilization of Glycogen Breakdown Products by Gardnerella spp. from the Human Vaginal Microbiome.

Multiple Gardnerella species frequently cooccur in vaginal microbiomes, and several factors, including competition for nutrients such as glycogen could determine their population structure. Although Gardnerella spp. can hydrolyze glycogen to produce glucose, maltose, maltotriose, and maltotetraose, how these sugars are transported and utilized for growth is unknown. We determined the distribution of genes encoding transporter proteins associated with the uptake of glucose, maltose, and malto-oligosaccharides and maltodextrins among Gardnerella species. A total of five different ABC transporters were identified in Gardnerella spp. of which MusEFGK2I and MalXFGK were conserved across all 15 Gardnerella isolates. RafEFGK and TMSP (trehalose, maltose, sucrose, and palatinose) operons were specific to G. vaginalis while the MalEFG transporter was identified in G. leopoldii only. Although no glucose specific sugar-symporters were identified, putative "glucose/galactose porters" and components of a phosphotransferase system were identified. In laboratory experiments, all Gardnerella isolates grew more in the presence of glucose, maltose, maltotriose, and maltotetraose compared to unsupplemented media. In addition, most isolates (10/15) showed significantly more growth on maltotetraose compared to glucose (Kruskal Wallis, P < 0.05) suggesting their preference for longer chain malto-oligosaccharides. Our findings show that although putative MusEFGK2I and MalXFGK transporters are found in all Gardnerella spp., some species-specific transporters are also present. Observed distribution of genes encoding transporter systems was consistent with laboratory observations that Gardnerella spp. grow better on longer chain malto-oligosaccharides. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community. Gardnerella produces enzymes to digest glycogen, an important nutrient source for vaginal bacteria, but little is known about the mechanisms in Gardnerella for uptake of the products of this digestion, or whether Gardnerella use some or all of the products. Our results indicate that Gardnerella may have evolved to preferentially use a subset of the glycogen breakdown products, which would help them reduce direct competition with some other bacteria in the vagina.

Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in Gardnerella spp. from the Vaginal Microbiome.

Gardnerella spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake, a process that depends on the combined action of glycogen-degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules, and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as "public goods" that can digest glycogen into maltose, maltotriose, and maltotetraose that can be used by the vaginal microbiota. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community, but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.

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.

Changes in the phenotypic susceptibility of Mannheimia haemolytica isolates to macrolide antimicrobials during the early feeding period following metaphylactic tulathromycin use in western Canadian feedlot calves.

Cattle at high-risk for bovine respiratory disease on entry to western Canadian feedlots are often treated metaphylactically with antimicrobials from the macrolide class. High levels of resistance to macrolides have been reported in Mannheimia haemolytica isolates from clinical samples, but it is less clear whether this trend extends to the broader feedlot population. The objective was to describe near-term [< 40 days on feed (DOF)] changes in the recovery and susceptibility of M. haemolytica isolates from healthy feedlot calves after metaphylactic exposure to tulathromycin. Eight cohorts of 100 calves (n = 800) were sampled via deep nasopharyngeal swab at entry processing (i.e., before metaphylaxis, at 1 DOF) and again at 13 DOF. Ten calves from each cohort (n = 80) were randomly sampled a third time at 36 DOF. Recovery of M. haemolytica isolates across all cohorts increased over the study period, from 33% (95% CI: 26.5 to 40.2%) at 1 DOF to 75% (95% CI: 71.4 to 78.3%) at 36 DOF. A significant shift in the minimum inhibitory concentration (MIC) distribution of tulathromycin from 1 DOF (MIC90 ≤ 8 µg/mL) to 13 DOF (MIC90 > 64 µg/mL) was observed. A subset of 36 isolates from 13 DOF screened for macrolide resistance genes via multiplex polymerase chain reaction all harbored the msrE and mphE genes. Recovery of M. haemolytica at 13 and 36 DOF did not decline in response to metaphylactic use of tulathromycin; conversely, we inferred the potential for rapid inter-pen spread of a macrolide-resistant clone by 13 DOF in 6 of 8 pens under selective pressure from antimicrobial use.

Changements dans la sensibilité phénotypique des isolats de Mannheimia haemolytica aux a ntibiotiques de la classe des macrolides au début de la période d'alimentation après l'utilisation m étaphylactique de tulathromycine chez les veaux des parcs d'engraissement de l'Ouest canadien. Les bovins à risque élevé de maladies respiratoires bovines à leur entrée dans les parcs d'engraissement de l'Ouest canadien sont souvent traités métaphylactiquement avec des antimicrobiens de la classe des macrolides. Des taux élevés de résistance aux macrolides ont été signalés chez les isolats de Mannheimia haemolytica provenant d'échantillons cliniques, mais il est moins clair si cette tendance s'étend à la population plus large des parcs d'engraissement. L'objectif était de décrire les changements à court terme [< 40 jours d'alimentation (DOF)] dans la récupération et la sensibilité des isolats de M. haemolytica provenant de veaux sains en parc d'engraissement après une exposition métaphylactique à la tulathromycine. Huit cohortes de 100 veaux (n = 800) ont été échantillonnées via un prélèvement nasopharyngé profond lors du traitement d'entrée (i.e., avant la métaphylaxie, à 1 DOF) et à nouveau à 13 DOF. Dix veaux de chaque cohorte (n = 80) ont été échantillonnés au hasard une troisième fois à 36 DOF. La récupération des isolats de M. haemolytica dans toutes les cohortes a augmenté au cours de la période d'étude, passant de 33 % (IC 95 % : 26,5 à 40,2 %) à 1 DOF à 75 % (IC 95 % : 71,4 à 78,3 %) à 36 DOF. Un changement significatif dans la distribution de la concentration minimale inhibitrice (MIC) de la tulathromycine de 1 DOF (MIC90 ≤ 8 µg/mL) à 13 DOF (MIC90 > 64 µg/mL) a été observé. Un sous-ensemble de 36 isolats de 13 DOF criblés pour les gènes de résistance aux macrolides via une réaction d'amplification en chaîne par polymérase multiplex hébergeaient tous les gènes msrE et mphE. L'isolement de M. haemolytica à 13 et 36 DOF n'a pas diminué en réponse à l'utilisation métaphylactique de la tulathromycine; à l'inverse, nous avons suggéré le potentiel de propagation rapide entre les enclos d'un clone résistant aux macrolides par 13 DOF dans 6 des 8 enclos sous la pression sélective de l'utilisation d'antimicrobiens.(Traduit par Dr Serge Messier).

Experimental natural transmission (seeder pig) models for reproduction of swine dysentery.

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.

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.

Vaginal microbiota associated with oncogenic HPV in a cohort of HPV-vaccinated women living with HIV.

BACKGROUND: Women living with HIV (WLWH) experience higher rates of human papillomavirus (HPV) infection and cervical cancer than women without HIV. Changes in the vaginal microbiome have been implicated in HPV-related disease processes such as persistence of high-risk HPV infection but this has not been well defined in a population living with HIV. METHODS: Four hundred and 20 girls and WLWH, age ≥9, across 14 clinical sites in Canada were enrolled to receive three doses of quadrivalent HPV vaccine for assessment of vaccine immunogenicity. Blood, cervical cytology, and cervico-vaginal swabs were collected. Cervico-vaginal samples were tested for HPV DNA and underwent microbiota sequencing. RESULTS: Principal component analysis (PCA) and hierarchical clustering generated community state types (CSTs). Relationships between taxa and CSTs with HPV infection were examined using mixed-effects logistic regressions, Poisson regressions, or generalized linear mixed-effects models, as appropriate. Three hundred and fifty-six cervico-vaginal microbiota samples from 172 women were sequenced. Human papillomavirus DNA was detected in 211 (59%) samples; 110 (31%) contained oncogenic HPV. Sixty-five samples (18%) were taken concurrently with incident oncogenic HPV infection and 56 (16%) were collected from women with concurrent persistent oncogenic HPV infection. CONCLUSIONS: No significant associations between taxa, CST, or microbial diversity and HPV-related outcomes were found. However, we observed weak associations between a dysbiotic microbiome and specific species, including Gardnerella, Porphyromonas, and Prevotella species, with incident HPV infection.

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.

Phylogenetic analysis of porcine circovirus 3 circulating in Canadian pigs.

INTRODUCTION: Porcine circovirus 3 (PCV3) has been detected in pigs worldwide and associated with several clinical signs. METHODS: To investigate the genetic diversity of PCV3 strains circulating in Canada, 44 PCV3 positive samples from Saskatchewan (2/44), Manitoba (2/44), Quebec (4/44), Alberta (11/44) and Ontario (25/44) submitted to diagnostic laboratories in Canada between 2019 and 2021 were sequenced and analyzed. RESULTS: Phylogenetic analysis of capsid genes showed that all of the 44 Canadian strains classified into PCV3a and segregated into seven lineages with common amino acid changes observed at A24V, R27K, N56D, T77S, Q98R, L150I (F) and R168K positions. CONCLUSION: Future studies are required to determine whether the polymorphisms in capsid proteins, as revealed in this study, could be associated with differences in the pathogenicity or antigenicity of PCV3 strains. This is the first phylogenetic analysis of PCV3 strains among different provinces in Canada.