Introduction: Detection of pathogens associated with bovine respiratory disease (BRD) typically involves several laboratory tools, with results limited to a defined list of targets. This study adapted a previously reported method for metagenomic sequencing of nasal swabs to describe sequencing data from BRD associated viruses. Changes in virus composition were identified between arrival to a feedlot and 14 days on feed (DOF). These data were also assessed for the simultaneous characterization of bacteria and antimicrobial resistance genes (ARGs). Methods: Nasal swabs were obtained from fall-placed calves (FPC) and yearlings (YRL) from western Canadian commercial feedlots. Evidence of respiratory viruses were identified by sampling 380 animals during processing on arrival to the feedlot and again after 14 DOF using Nanopore metagenomic sequencing. Results: Twenty-one distinct viruses from 12 viral families were identified, with multiple viruses detected in most samples. In FPC arrival samples, the most common BRD associated viruses were bovine rhinitis B virus (BRBV; 46%), bovine coronavirus (BCoV; 32%), influenza D virus (IDV; 17%), bovine respiratory syncytial virus (BRSV; 8.5%), and bovine parainfluenza virus 3 (BPIV-3; 4.2%). The prevalences of bovine herpesvirus type 1 (BoHV-1; 2.7%), BPIV-3 (12%), BRSV (26%), and IDV (51%) were higher in 14 DOF samples compared to arrival samples (p < 0.05). Bovine viral diarrhea virus 1 (BVDV-1) and 2 (BVDV-2) were rarely detected at either time. The most prevalent viruses detected in YRL arrival samples were BRBV (42%), BRSV (39%), BPIV-3 (20%), IDV (16%), BCoV (12%), and BVDV-2 (7.5%). The prevalences of BRSV (60%), BPIV-3 (39%), and BVDV-2 (17%) were higher in 14 DOF samples than arrival samples (p < 0.05). BRSV (OR 7.0, 1.7-29) and BPIV-3 (OR 5.7, 1.5-21) were more likely to be detected in arrival samples from YRL than FPC (p = 0.01). In 14 DOF samples, BPIV-3 (OR 4.9, 1.3-19, p = 0.02) and BVDV-2 (OR 13, 2.0-83, p = 0.01) were identified more frequently in YRL than FPC. These data allowed the identification of respiratory bacteria and 33 ARGs in parallel with assessment of the viral components. The most prevalent bacteria detected in FPC at arrival were Mannheimia haemolytica (35%), Histophilus somni (35%) and Pasteurella multocida (23%). Detection of M. haemolytica increased at 14 DOF (p = 0.02), while P. multocida detection decreased (p = 0.03). At both arrival and 14 DOF in YRL, M. haemolytica was the most prevalent bacterium, followed by P. multocida and H. somni with no significant differences between arrival and 14 DOF samples. ARGs were detected more frequently in the 14 DOF samples than at arrival for both FPC (p = 0.03) and YRL (p = 0.01). The most commonly detected ARGs were associated with resistance to lincosamides and aminoglycosides; however, ARGs associated with other antimicrobials used in cattle including tetracyclines were also identified. Discussion: Changes in the prevalence of BRD associated viruses early in the feeding period reflect transmission and the potential risk of developing the disease. Frequent detection of BCoV, BRSV, and BPIV-3 in newly arrived feedlot cattle suggests the need for improved vaccination before shipping or limitations in existing commercial vaccine preparations.

Introduction:Very few studies have examined the diagnostic sensitivity and specificity of currently available laboratory tests for detecting respiratory pathogens in cattle, and even fewer have examined test performance on samples from animals before the onset of clinical disease. Methods:In this study, Bayesian latent class modeling (BLCM) was used to assess diagnostic test performance in the absence of a gold standard on nasal swabs collected from 19 western Canadian feedlots. Viruses associated with bovine respiratory disease (BRD) were identified using qPCR from a commercial diagnostic laboratory from 760 nasal swabs collected from fall-placed calves (FPC) and yearlings (YRL) at and shortly after feedlot arrival. Using BLCM, the qPCR results were compared to previously reported matching nanopore metagenomic sequencing data for these same samples. Based on BLCM, test sensitivities and specificities were estimated for the detection of bovine coronavirus (BCoV), bovine herpesvirus type 1 (BoHV-1), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and influenza D virus (IDV). Estimates informed by BLCM were not available for the detection of bovine viral diarrheal virus (BVDV) because qPCR did not detect this virus in any samples. Results:Diagnostic sensitivity of qPCR was higher than metagenomic sequencing for detecting BCoV (qPCR 0.90, 95% CrI 0.81-0.99; sequencing 0.35, 95% CrI 0.25-0.46) and BoHV-1 (qPCR 0.39, 95% CrI 0.19-0.99; sequencing 0.04, 95% CrI 0.01-0.15). However, the estimated diagnostic sensitivity of metagenomic sequencing was higher than qPCR for identifying BRSV (qPCR 0.32, 95% CrI 0.22-0.43; sequencing 0.60, 95% CrI 0.44-0.77). No significant difference among sensitivities was noted for the detection of BPIV-3 (qPCR 0.42, 95% CrI 0.21-0.66; sequencing 0.52, 95% CrI 0.19-0.87) and IDV (qPCR 0.65, 95% CrI 0.53-0.79; sequencing 0.60, 95% CrI 0.48-0.73). Diagnostic specificity was comparable for most viruses, except for BCoV, where metagenomic sequencing (BCoV 0.91, 95% CrI 0.88-0.95) outperformed qPCR (BCoV 0.59, 95% CrI 0.51-0.68). The specificity and sensitivity for detection of BRD-associated bacteria from the same metagenomic data were also similar to those estimated for culture and qPCR results for the same samples. Discussion:Estimated test sensitivities of both nanopore metagenomic sequencing and qPCR for the detection of BRD viruses of interest in nasal swab samples were moderate to very low for most viruses. While the tests varied in their ability to detect individual viruses, data from this study suggest nanopore metagenomic sequencing offers a potential alternative for diagnostic laboratories to identify three of six important BRD viruses as well as bacteria associated with BRD.

Glycogen and its breakdown products, maltose and malto-oligosaccharides, are important carbon sources for vaginal bacteria including Gardnerella species. MusEFGKI transport systems for maltose and malto-oligosaccharides have been identified in all Gardnerella species; however, unlike in other species, the Gardnerella swidsinskii operon encodes two substrate-binding proteins (SBPs) (MusE1345, MusE1346, ~60% amino acid identity). Two SBPs could allow binding of additional ligands, providing a competitive advantage to G. swidsinskii relative to other species with only one SBP. Our objectives were to determine if both genes are expressed in G. swidsinskii and compare the specificity and affinity of G. swidsinskii MusE SBPs for glycogen breakdown products. Gene expression analysis showed the presence of a polycistronic transcript spanning both SBP encoding genes; however, musE1346 transcripts were more abundant, likely due to the presence of an additional promoter identified in the intergenic region. No difference in the relative expression of either gene was observed in isolates grown in media supplemented with glycogen or maltotriose. Predicted structures of both SBPs were highly similar and characteristic of previously characterized maltose-binding proteins. Both proteins had a high affinity for maltose, maltotriose and maltotetraose (K d 10-6 to 10-7 M) and much lower affinities to maltopentaose and maltohexaose (K d 10-3 to 10-4 M). Our results demonstrate that the affinities of G. swidsinskii MusE SBPs for maltose and malto-oligosaccharides are similar under the same experimental conditions.

BACKGROUND:The tick-borne spirochete Borrelia burgdorferi causes Lyme borreliosis in North America. In nature, rodent reservoir hosts are often infected with multiple strains of B. burgdorferi, which induce strain-specific antibody responses that are mediated by outer surface protein C (OspC). During pregnancy and lactation, female rodents infected with B. burgdorferi will transfer a finite amount of strain-specific maternal antibodies to their offspring. The purpose of our study was to investigate whether infection with multiple strains of B. burgdorferi in female mice reduces the per strain level of maternal anti-OspC IgG antibodies in their offspring. METHODS:The study used two B. burgdorferi strains, A and I, which carried ospC types A and I, respectively. Female C3H/HeJ mice were assigned to four different maternal infection treatments: (1) infected with strain A alone, (2) infected with strain I alone, (3) co-infected with both strains, and (4) uninfected. Female mice (dams) from all treatment groups were mated with uninfected males and allowed to produce offspring. The maternal anti-OspC IgG response was determined in the dams at 4 weeks post-infection and in the offspring at ≤2, 4, and 9 weeks of age. RESULTS:Infected dams transferred maternal anti-B. burgdorferi IgG antibodies to their offspring. The maternal anti-OspC IgG antibody titers waned over time and were specific for the OspC antigen of the infecting strain with limited cross-reactivity between strains. The absorbance of the maternal anti-OspC IgG antibodies in the offspring aged ≤2, 4, and 9 weeks were 83.6%, 46.9%, and 16.2% of the absorbance in the dams at 4 weeks post-infection, respectively. Compared to dams infected with single strains, co-infection in dams reduced the maternal anti-OspC IgG response in the offspring by 22.2% for strain A (p = 0.001) and 4.5% for strain I (p = 0.538). Our study suggests that co-infection with multiple strains can reduce the quantity and/or quality of maternal anti-OspC IgG antibodies per B. burgdorferi strain that are transferred from dams to their offspring. A future study will investigate whether co-infection in the dams reduces the protective efficacy of the maternal antibody response in the offspring against an infectious tick bite challenge.

IntroductionThe risk to humans and animals from antimicrobial resistance (AMR) has increased the emphasis on antimicrobial stewardship in food animal agriculture. Current stewardship recommendations include increasing diagnostic laboratory testing to inform antimicrobial use for bovine respiratory disease (BRD) management in beef feedlot production, yet the performance of newer molecular and sequencing-based diagnostic tests in commercial settings remains poorly characterized.MethodsUsing nasopharyngeal swabs collected from commercial feedlot calves as part of Canadian surveillance, this study evaluated diagnostic laboratory testing approaches for detecting key bacterial BRD pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasmopsis bovis) and associated AMR genes. Bayesian latent class models (BLCMs) were applied to compare traditional culture and antimicrobial susceptibility testing (AST) or qPCR with long-read metagenomic sequencing and recombinase polymerase amplification (RPA). Differences in detection of target bacteria and phenotypic or genotypic AMR were assessed across the early feeding period and between age cohorts.ResultsThis represents the first large-scale field evaluation of a recently developed, long-read metagenomic sequencing protocol implemented by a commercial laboratory for detecting BRD bacteria and AMR in respiratory samples (n = 760) collected by private veterinarians from western Canadian beef feedlots. Detection patterns for BRD bacteria and AMR using culture/AST and metagenomics were often similar between fall-placed calves and yearlings, but with differences from RPA. Detection of BRD bacteria had low sensitivity (< 65% for most organisms/tests), but higher specificity (>90% for all organisms/tests). Detection of macrolide and tetracycline resistance had low but variable sensitivity, with higher estimates for AST compared to metagenomics and RPA, and higher but variable specificity (>90% for most resistance outcomes/tests). Despite not using any targeted enrichment, metagenomic sequencing detected M. bovis although with a sensitivity lower than qPCR or RPA. Estimates of predictive value were most informative across the largest range of prevalence for AST, followed by metagenomics and then RPA.DiscussionThis work demonstrates the potential for large scale implementation of long-read metagenomic sequencing to support antimicrobial stewardship and AMR surveillance for feedlot cattle. The estimates of clinical diagnostic performance and predictive values provide evidence-based guidance for three different laboratory tests for BRD management.

Mannheimia haemolytica is an opportunistic bacterial pathogen associated with the economically costly bovine respiratory disease. Two genotypes have been described, of which genotype 2 is more strongly associated with disease. Several previous studies have investigated the genomic differences between the genotypes and/or the major serotypes (1, 2 and 6) of M. haemolytica , however we still lack a clear basis for the greater disease association of genotype 2 (serotypes 1 and 6) and demonstrations of phenotypic differences are scarce. This work builds upon previous investigations to identify genes that differentiate the two genotypes with a particular focus on genes that may play a role in virulence and fitness in the respiratory tract microbiome. We identified 422 genotype differentiating genes in a collection of 208 M. haemolytica genomes (62 genotype 1, 146 genotype 2). Genotype differentiating genes including genotype-associated variants of a TonB-dependent siderophore receptor homolog, transferrin binding protein B, leukotoxin A, and IgA1 proteases. We also identified a genotype 1 associated lytic transglycosylase, and a genotype 2 specific highly immunogenic outer membrane lipoprotein. These results expand our knowledge of what differentiates the genotypes of M. haemolytica and provides information that can be utilized to inform diagnostic strategies. The results also highlight the relative lack of information regarding M. haemolytica from sheep and goats and geographic regions other than Canada and the United States. ### Competing Interest Statement The authors have declared no competing interest.

Mannheimia haemolytica is an opportunistic bacterial pathogen associated with the economically costly bovine respiratory disease. Two genotypes have been described, of which genotype 2 is more strongly associated with disease. Several previous studies have investigated the genomic differences between the genotypes and/or the major serotypes (1, 2 and 6) of M. haemolytica, however we still lack a clear basis for the greater disease association of genotype 2 (serotypes 1 and 6) and demonstrations of phenotypic differences are scarce. This work builds upon previous investigations to identify genes that differentiate the two genotypes with a particular focus on genes that may play a role in virulence and fitness in the respiratory tract microbiome. We identified 422 genotype differentiating genes in a collection of 206 unique M. haemolytica genomes (61 genotype 1, 145 genotype 2). Genotype differentiating genes included genotype-associated variants of a TonB-dependent siderophore receptor homolog, transferrin binding protein B, leukotoxin A, and IgA1 proteases. We also identified a genotype 1 associated lytic transglycosylase, and a genotype 2 specific highly immunogenic outer membrane lipoprotein. Genotype 2 genomes were significantly larger in size and contained more predicted protein coding genes than genotype 1 genomes. These results expand our knowledge of what differentiates the genotypes 1 and 2 of M. haemolytica and provides information that can be used as the basis for laboratory investigations of corresponding phenotypic differences.

ABSTRACT Gardnerella spp. significantly influence female reproductive health and are indicators of bacterial vaginosis, a common gynecological disorder. Lack of genetic tools for Gardnerella spp. is a hindrance to fully understanding their role in the vaginal microbiome, and no naturally occurring plasmids have yet been identified in these organisms. The aim of this study was to transform Gardnerella vaginalis and characterize transformants carrying Bifidobacterium-E. coli shuttle vector pKO403-lacZ′-Sp. G. vaginalis ATCC 49145 was selected for protocol development based on its high growth rate, lack of restriction activity, and susceptibility to spectinomycin. Low efficiency (~102 cfu/µg of plasmid DNA) but reproducible transformation was achieved. The expression of the spectinomycin resistance gene and the β-galactosidase gene of pKO403-lacZ′-Sp in G. vaginalis ATCC 49145 resulted in an increase in spectinomycin tolerance from 2 µg/mL (MIC) to >512 µg/mL, and an appreciable increase in β-galactosidase activity compared with the wild type. Plasmid copy number was determined to be ~3 per genome copy. Plasmid was lost rapidly in the absence of spectinomycin selection, with only ~5% of colony-forming units retaining the resistant phenotype after 24 h of growth without selection. These results demonstrate that G. vaginalis can be transformed by electroporation and that pKO403-lacZ′-Sp can be maintained and its genes expressed in this host, offering a starting point for the development of genetic tools for mechanistic studies of this important member of the vaginal microbiome.IMPORTANCEThe healthy human vaginal microbiome is mainly dominated by Lactobacillus spp. An imbalance or shift in this population can lead to a gynecological disorder known as bacterial vaginosis (BV). In BV, there is a reduction in Lactobacillus spp. and an overgrowth of mixed anaerobes and facultative bacteria including Gardnerella spp. The reason for this increase in the Gardnerella population and associated changes in the vaginal microbiota composition is yet not understood, and a lack of genetic tools is one of the major barriers to performing mechanistic research to study the biology of these clinically significant organisms. The first step in developing genetic tools is introducing foreign DNA. In this study, we have developed a protocol for transformation and identified a plasmid that can be maintained in G. vaginalis.

AbstractGardnerellaspp. significantly influence female reproductive health, and are indicators of bacterial vaginosis, a common gynecological disorder. Lack of genetic tools forGardnerellaspp. is a hindrance in fully understanding their role in the vaginal microbiome and no naturally occurring plasmids have yet been identified in these organisms. The aim of this study was to transformGardnerella vaginalisand characterize transformants carryingBifidobacterium-E. colishuttle vector pKO403-lacZ′-Sp.G. vaginalisATCC 49145 was selected for protocol development based on its high growth rate, lack of restriction activity and susceptibility to spectinomycin. Low efficiency (∼102cfu/µg of plasmid DNA) but reproducible transformation was achieved. The expression of the spectinomycin resistance gene and the β-galactosidase gene of pKO403-lacZ′-Sp inG. vaginalisATCC 49145 resulted in an increase in spectinomycin tolerance from 2 µg/ml (MIC) to >512 µg/ml, and an appreciable increase in β-galactosidase activity compared to the wild type. Plasmid copy number was determined to be ∼3 per genome copy. Plasmid was lost rapidly in the absence of spectinomycin selection, with only ∼13% of colony forming units retaining the resistant phenotype after 24 h of growth without selection. These results demonstrate thatG. vaginaliscan be transformed by electroporation and that pKO403-lacZ′-Sp can be maintained and its genes expressed in this host, offering a starting point for the development of genetic tools for mechanistic studies of this important member of the vaginal microbiome.ImportanceThe healthy human vaginal microbiome is mainly dominated byLactobacillusspp. An imbalance or shift in this population can lead to a gynecological disorder known as bacterial vaginosis (BV). In BV, there is a reduction inLactobacillusspp. and overgrowth of mixed anaerobes and facultative bacteria includingGardnerellaspp. The reason for this increase in theGardnerellapopulation and associated changes in the vaginal microbiota composition is yet not understood, and a lack of genetic tools is one of the major barriers to performing mechanistic research to study the biology of these clinically significant organisms. A first step in developing genetic tools is introducing foreign DNA. In this study, we have developed a protocol for transformation and identified a plasmid that can be maintained inG. vaginalis.

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.

Abstract This protocol describes an approach for the purification of high quality RNA from Gardnerella species based on a commercially available kit (PureLink RNA Mini Kit, Invitrogen). Initial RNA extraction following the manufacturer’s protocol resulted in poor RNA yield and quality from Gardnerella spp. based on A260/A280 measurements and RNA integrity number (RIN). Modifications made to the manufacturer’s recommended protocol including enzymatic lysis with lysozyme and proteinase K resulted in significantly higher RNA yield (≥25 ng/mL) and RNA quality (A260/A280 ~2.0, RIN ⪆ 8.0). RNA extracts resulting from this protocol are appropriate for use in gene expression studies based on real-time quantitative PCR or transcriptomics.

AbstractBovine reproductive failure, which includes infertility, abortion, and stillbirth in cattle, leads to significant economic losses for beef and milk producers. Diagnosing the infectious causes of bovine reproductive failure is challenging as there are multiple pathogens associated with it. The traditional stepwise approach to diagnostic testing is time-consuming and can cause significant delays. In this study, we have developed a syndromic next-generation sequencing panel (BovReproSeq), for the simultaneous detection of 17 pathogens (bacteria, virus and protozoa) associated with bovine reproductive failure. This targeted approach involves amplifying multiple pathogen-specific targets using ultra-multiplex PCR, followed by sequencing with the Oxford Nanopore platform and subsequent analysis of the data using a custom bioinformatic pipeline to determine the presence or absence of pathogens. We tested 116 clinical samples and found that BovReproSeq results matched with current diagnostic methods for 93% of the samples, and most of the disagreements occurring in samples with very low pathogen loads (Ct > 35). At the optimal read-count threshold of 10 reads (minimum number of reads to classify the sample as positive), the clinical sensitivity of the assay was approximately 82%, while clinical specificity was 100%. The overall accuracy of the assay was 98.8%. Matthew’s Correlation Coefficient (correlation coefficient of binary classification) was approximately 0.90 and F1 score (harmonic mean of Precision and Recall) was 0.90, indicating excellent overall performance. Our study presents a significant advancement in detecting the infectious agents associated with bovine reproductive failure and the BovReproSeq panel’s ability to detect 17 pathogens makes it a promising tool for veterinary diagnostics.ImportanceBovine reproductive failure causes substantial economic losses to beef and milk producers, and infectious disease contributes significantly to this syndrome. Etiologic diagnosis is complicated since multiple pathogens can be involved and infections with some pathogens are asymptomatic or cause similar clinical signs. A stepwise approach to diagnostic testing is time-consuming and increases the risk of missing the correct diagnosis. BovReproSeq is a next-generation sequencing based diagnostic panel that allows detection of 17 reproductive failure pathogens simultaneously.

ObjectiveBartonella 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. ResultsDNA 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.

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.

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.

Gardnerella spp. express and export enzymes for the breakdown of glycogen into glucose, maltose, and malto-oligosaccharides for consumption by the vaginal microbiota but how the expression of these “public goods” is affected by substrate and product levels in the environment is not known. Accurate measurement of relative gene expression using real-time quantitative PCR relies on the identification of appropriate reference genes whose expression levels remain constant under the conditions of the study. Currently, no reference genes have been identified for gene expression analysis of Gardnerella spp. The objectives of this study were to identify reference genes and apply them in determining the relative gene expression levels of genes encoding α-amylase and α-amylase-pullulanase in media supplemented with substrate (glycogen) or a preferred product (maltotriose). Ten candidate reference genes were evaluated and analysis of Cq values from qPCR using multiple algorithms identified uppS (encoding polyprenyl diphosphate synthase) as the top comprehensively ranked reference gene followed by gatA (encoding Asp-tRNA/Glu-tRNA amidotransferase subunit gatA). Interpretation of the Cq values for α-amylase and α-amylase-pullulanase was performed by applying these two reference genes in the calculation of relative gene expression levels. α-amylase-pullulanase gene expression was upregulated in media supplemented with 1% glycogen in comparison to media supplemented with 1% maltotriose suggesting a regulatory mechanism in G. swidsinskii that responds to nutrient availability. No significant difference in gene expression of α-amylase was observed suggesting expression is not influenced by substrate availability. The RNA purification protocol and reference genes validated in this study will be useful in future studies of gene expression in Gardnerella .Importance Knowledge of the factors affecting growth of vaginal microbiota is critical to understanding how vaginal dysbiosis is initiated and maintained. Overgrowth of Gardnerella species including G. swidsinskii is a hallmark of bacterial vaginosis. These organisms break down vaginal glycogen and the products become available for uptake by Gardnerella and other microbiota. Measuring how expression of genes encoding glycogen degrading enzymes relates to relative abundance of substrate and products in the environment requires development of protocols for RNA purification and identification of reference genes for RT-qPCR.### Competing Interest StatementThe authors have declared no competing interest.

In this study, we aimed to compare uterine microbial profiles in postpartum dairy cows, determined by bacteriological culture and next-generation sequencing, using three uterine sampling techniques (swab, cytobrush, and lavage) and induced phases of the estrous cycle (estrus and diestrus). Fifteen healthy postpartum dairy cows at 53 +/- 5 days postpartum were enrolled in the study. Uterine samples were collected during a fixed-time artificial insemination protocol. Viable bacteria were aerobically cultured from part of each sample, and bacterial isolates were identified through Sanger sequencing of the 16S rRNA gene. Total genomic DNA was extracted from the remainder of undiluted samples to quantify bacterial load using 16S rRNA qPCR and characterize the microbiome by metagenomic sequencing of the V1-V3 region of the 16S rRNA gene. Microbial profiles and composition were analyzed using the Shannon-Weaver diversity index and principal component analysis, respectively. Out of 87 samples, 88 % (77/87) were culture positive. The proportion of culture-positive uterine samples did not differ between sampling techniques (P = 0.39) or estrous cycle phases (P = 0.99). However, swab, cytobrush, and lavage techniques yielded 1.5, 9 and 9 times greater bacterial loads (P < 0.01), respectively, during diestrus than estrus phase. Moreover, during diestrus phase, the cytobrush method yielded 3 and 6 times more bacteria (P < 0.01) than both the lavage and swab methods. The most abundant bacterial genera identified from both bacteriological culture and metagenomic sequencing were Bacillus and Enterococcus, regardless of sampling technique or phases of the estrous cycle. Bacterial genera in moderate to low abundance through metagenomic sequencing included Streptococcus, Oscillospiraceae, and Lachnospiraceae. Notably, the uterine microbial profiles and composition, determined by metagenomic sequencing, did not differ by sampling techniques (P = 0.55 and P = 0.60, respectively) or estrous cycle phases (P = 0.34 and P = 0.17, respectively). In conclusion, our results suggest that any of the sampling techniques can be reliably used to study the uterine microbiome of healthy cows at random phases of the estrous cycle. However, it is important to consider potential differences in bacterial yield as a confounding factor.

MOTIVATION:Sanger sequencing of taxonomic marker genes (e.g. 16S/18S/ITS/rpoB/cpn60) represents the leading method for identifying a wide range of microorganisms including bacteria, archaea, and fungi. However, the manual processing of sequence data and limitations associated with conventional BLAST searches impede the efficient generation of strain libraries essential for cataloging microbial diversity and discovering novel species. RESULTS:isolateR addresses these challenges by implementing a standardized and scalable three-step pipeline that includes: (1) automated batch processing of Sanger sequence files, (2) taxonomic classification via global alignment to type strain databases in accordance with the latest international nomenclature standards, and (3) straightforward creation of strain libraries and handling of clonal isolates, with the ability to set customizable sequence dereplication thresholds and combine data from multiple sequencing runs into a single library. The tool's user-friendly design also features interactive HTML outputs that simplify data exploration and analysis. Additionally, in silico benchmarking done on two comprehensive human gut genome catalogues (IMGG and Hadza hunter-gather populations) showcase the proficiency of isolateR in uncovering and cataloging the nuanced spectrum of microbial diversity, advocating for a more targeted and granular exploration within individual hosts to achieve the highest strain-level resolution possible when generating culture collections. AVAILABILITY AND IMPLEMENTATION:isolateR is available at: https://github.com/bdaisley/isolateR.

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. Keywords bats , chiroptera , , , gammaherpesvirus , , , pulmonary intravascular macrophage

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.