Application of Four Genotyping Methods to Mycoplasma bovis Isolates Derived from Western Canadian Feedlot Cattle.

Mycoplasma bovis is a significant pathogen of feedlot cattle, responsible for chronic pneumonia and polyarthritis syndrome (CPPS). M. bovis isolates (n = 129) were used to compare four methods of phylogenetic analysis and to determine if the isolates' genotypes were associated with phenotypes. Metadata included the health status of the animal from which an isolate was derived (healthy, diseased, or dead), anatomical location (nasopharynx, lung, or joint), feedlot, and production year (2006 to 2018). Four in silico phylogenetic typing methods were used: multilocus sequence typing (MLST), core genome MLST (cgMLST), core genome single nucleotide variant (cgSNV) analysis, and whole-genome SNV (wgSNV) analysis. Using Simpson's diversity index (D) as a proxy for resolution, MLST had the lowest resolution (D = 0.932); cgSNV (D = 0.984) and cgMLST (D = 0.987) generated comparable results; and wgSNV (D = 1.000) provided the highest resolution. Visual inspection of the minimum spanning trees found that the memberships of the clonal complexes and clades had similar structural appearances. Although MLST had the lowest resolution, this methodology was intuitive and easy to apply, and the PubMLST database facilitates the comparison of sequence types across studies. The cg methods had higher resolution than MLST, and the graphical interface software was user-friendly for nonbioinformaticians, but the proprietary software is relatively expensive. The wgSNV approach was the most robust for processing poor-quality sequence data while offering the highest resolution; however, application of its software requires specialized training. None of the four methods could associate genotypes with phenotypes.

Comparison of Two Multilocus Sequence Typing Schemes for Mycoplasma bovis and Revision of the PubMLST Reference Method.

Mycoplasma bovis causes pneumonia, pharyngitis, otitis, arthritis, mastitis, and reproductive disorders in cattle and bison. Two multilocus sequence typing (MLST) schemes have been developed for M. bovis, with one serving as the PubMLST reference method, but no comparison of the schemes has been undertaken. Although the PubMLST scheme has proven to be highly discriminatory and informative, the recent discovery of isolates missing one of the typing loci, adh-1, raises concern about its suitability for continued use. The goal of our study was to compare the performance of the two MLST schemes and identify a new reference scheme capable of fully typing all isolates. We evaluated 448 isolates from diverse geographic and anatomic sites that collectively represent cattle, bison, deer, and a goat. The discrimination indexes (DIs) for the PubMLST and the alternative scheme are 0.909 (91 sequence types [STs]) and 0.842 (77 STs), respectively. Although the PubMLST scheme outperformed the alternative scheme, the adh-1 locus must be retired from the PubMLST scheme if it is to be retained as a reference method. The DI obtained using the six remaining PubMLST loci (0.897, 79 STs) fails to reach the benchmark recommended for a reference method (0.900), mandating the addition of a seventh locus. Comparative analysis of genome sequences from the isolates used here identified the dnaA locus from the alternative scheme as the optimal replacement for adh-1 This revised scheme, which will be implemented as the new PubMLST reference method, has a DI of 0.914 and distinguishes 88 STs from the 448 isolates evaluated.

Transcriptome Analysis Identifies Plasmodiophora brassicae Secondary Infection Effector Candidates.

Plasmodiophora brassicae (Wor.) is an obligate intracellular plant pathogen affecting Brassicas worldwide. Identification of effector proteins is key to understanding the interaction between P. brassicae and its susceptible host plants. To date, there is very little information available on putative effector proteins secreted by P. brassicae during a secondary infection of susceptible host plants, resulting in root gall production. A bioinformatics pipeline approach to RNA-Seq data from Arabidopsis thaliana (L.) Heynh. root tissues at 17, 20, and 24 d postinoculation (dpi) identified 32 small secreted P. brassicae proteins (SSPbPs) that were highly expressed over this secondary infection time frame. Functional signal peptides were confirmed for 31 of the SSPbPs, supporting the accuracy of the pipeline designed to identify secreted proteins. Expression profiles at 0, 2, 5, 7, 14, 21, and 28 dpi verified the involvement of some of the SSPbPs in secondary infection. For seven of the SSPbPs, a functional domain was identified using Blast2GO and 3D structure analysis and domain functionality was confirmed for SSPbP22, a kinase localized to the cytoplasm and nucleus.

Transcriptome analysis of response to Plasmodiophora brassicae infection in the Arabidopsis shoot and root.

BACKGROUND: Clubroot is an important disease caused by the obligate parasite Plasmodiophora brassicae that infects the Brassicaceae. As a soil-borne pathogen, P. brassicae induces the generation of abnormal tissue in the root, resulting in the formation of galls. Root infection negatively affects the uptake of water and nutrients in host plants, severely reducing their growth and productivity. Many studies have emphasized the molecular and physiological effects of the clubroot disease on root tissues. The aim of the present study is to better understand the effect of P. brassicae on the transcriptome of both shoot and root tissues of Arabidopsis thaliana. RESULTS: Transcriptome profiling using RNA-seq was performed on both shoot and root tissues at 17, 20 and 24 days post inoculation (dpi) of A. thaliana, a model plant host for P. brassicae. The number of differentially expressed genes (DEGs) between infected and uninfected samples was larger in shoot than in root. In both shoot and root, more genes were differentially regulated at 24 dpi than the two earlier time points. Genes that were highly regulated in response to infection in both shoot and root primarily were involved in the metabolism of cell wall compounds, lipids, and shikimate pathway metabolites. Among hormone-related pathways, several jasmonic acid biosynthesis genes were upregulated in both shoot and root tissue. Genes encoding enzymes involved in cell wall modification, biosynthesis of sucrose and starch, and several classes of transcription factors were generally differently regulated in shoot and root. CONCLUSIONS: These results highlight the similarities and differences in the transcriptomic response of above- and below-ground tissues of the model host Arabidopsis following P. brassicae infection. The main transcriptomic changes in root metabolism during clubroot disease progression were identified. An overview of DEGs in the shoot underlined the physiological changes in above-ground tissues following pathogen establishment and disease progression. This study provides insights into host tissue-specific molecular responses to clubroot development and may have applications in the development of clubroot markers for more effective breeding strategies.

The respiratory and fecal microbiota of beef calves from birth to weaning.

The development and growth of animals coincide with the establishment and maturation of their microbiotas. To evaluate the respiratory and fecal microbiotas of beef calves from birth to weaning, a total of 30 pregnant cows, and their calves at birth, were enrolled in this study. Deep nasal swabs and feces were collected from calves longitudinally, starting on the day of birth and ending on the day of weaning. Nasopharyngeal, vaginal, and fecal samples were also collected from cows, and the microbiotas of all samples were analyzed. The fecal microbiota of calves was enriched with Lactobacillus during the first 8 weeks of life, before being displaced by genera associated with fiber digestion, and then increasing in diversity across time. In contrast, the diversity of calf respiratory microbiota generally decreased with age. At birth, the calf and cow nasal microbiotas were highly similar, indicating colonization from dam contact. This was supported by microbial source-tracking analysis. The structure of the calf nasal microbiota remained similar to that of the cows, until weaning, when it diverged. The changes were driven by a decrease in Lactobacillus and an increase in genera typically associated with bovine respiratory disease, including Mannheimia, Pasteurella, and Mycoplasma. These three genera colonized calves early in life, though Mannheimia was initially transferred from the cow reproductive tract. Path analysis was used to model the interrelationships of calf respiratory and fecal microbiotas. It was observed that respiratory Lactobacillus and fecal Oscillospiraceae UCG-005 negatively affected the abundance of Mannheimia or Pasteurella.IMPORTANCEIn beef cattle production, bovine respiratory disease (BRD) accounts for most of the feedlot morbidities and mortalities. Metaphylaxis is a common management tool to mitigate BRD, however its use has led to increased antimicrobial resistance. Novel methods to mitigate BRD are needed, including microbiota-based strategies. However, information on the respiratory bacteria of beef calves prior to weaning was limited. In this study, it was shown that the microbiota of cows influenced the initial composition of both respiratory and fecal microbiotas in calves. While colonization of the respiratory tract of calves by BRD-associated genera occurred early in life, their relative abundances increased at weaning, and were negatively correlated with respiratory and gut bacteria. Thus, microbiotas of both the respiratory and gastrointestinal tracts have important roles in antagonism of respiratory pathogens and are potential targets for enhancing calf respiratory health. Modulation may be most beneficial, if done prior to weaning, before opportunistic pathogens establish colonization.

Auction market placement and a rest stop during transportation affect the respiratory bacterial microbiota of beef cattle.

Background: Bovine respiratory disease (BRD) is a significant health problem in beef cattle production, resulting in considerable economic losses due to mortalities, cost of treatment, and reduced feed efficiency. The onset of BRD is multifactorial, with numerous stressors being implicated, including transportation from farms to feedlots. In relation to animal welfare, regulations or practices may require mandatory rest times during transportation. Despite this, there is limited information on how transportation and rest stops affect the respiratory microbiota. Results: This study evaluated the effect of cattle source (ranch-direct or auction market-derived) and rest stop duration (0 or 8 h of rest) on the upper respiratory tract microbiota and its relationship to stress response indicators (blood cortisol and haptoglobin) of recently weaned cattle transported for 36 h. The community structure of bacteria was altered by feedlot placement. When cattle were off-loaded for a rest, several key bacterial genera associated with BRD (Mannheimia, Histophilus, Pasteurella) were increased for most sampling times after feedlot placement for the ranch-direct cattle group, compared to animals given no rest stop. Similarly, more sampling time points had elevated levels of BRD-associated genera when auction market cattle were compared to ranch-direct. When evaluated across time and treatments several genera including Mannheimia, Moraxella, Streptococcus and Corynebacterium were positively correlated with blood cortisol concentrations. Conclusion: This is the first study to assess the effect of rest during transportation and cattle source on the respiratory microbiota in weaned beef calves. The results suggest that rest stops and auction market placement may be risk factors for BRD, based solely on increased abundance of BRD-associated genera in the upper respiratory tract. However, it was not possible to link these microbiota to disease outcome, due to low incidence of BRD in the study populations. Larger scale studies are needed to further define how transportation variables impact cattle health.

The Isolation and Characterization of Bacteriophages Infecting Avian Pathogenic Escherichia coli O1, O2 and O78 Strains.

Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78, are important serogroups relating to chicken health, being responsible for colibacillosis. In this study, we isolated and characterized bacteriophages (phages) from hen feces and human sewage in Alberta with the potential for controlling colibacillosis in laying hens. The lytic profile, host range, pH tolerance and morphology of seven APEC-infecting phages (ASO1A, ASO1B, ASO2A, ASO78A, ASO2B, AVIO78A and ASO78B) were assessed using a microplate phage virulence assay and transmission electron microscopy (TEM). The potential safety of phages at the genome level was predicted using AMRFinderPlus and the Virulence Factor Database. Finally, phage genera and genetic relatedness with other known phages from the NCBI GenBank database were inferred using the virus intergenomic distance calculator and single gene-based phylogenetic trees. The seven APEC-infecting phages preferentially lysed APEC strains in this study, with ECL21443 (O2) being the most susceptible to phages (n = 5). ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5-9.0 after 4 h of incubation. Based on TEM, phages were classed as myovirus, siphovirus and podovirus. No genes associated with virulence, antimicrobial resistance or lysogeny were detected in phage genomes. Comparative genomic analysis placed six of the seven phages in five genera: Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (<70%), phage ASO78B was not assigned a genus in the siphovirus and could represent a new genus in class Caudoviricetes. The tail fiber protein phylogeny revealed variations within APEC-infecting phages and closely related phages. Diverse APEC-infecting phages harbored in the environment demonstrate the potential to control colibacillosis in poultry.

Genome-Wide Association Study of Nucleotide Variants Associated with Resistance to Nine Antimicrobials in Mycoplasma bovis.

Antimicrobial resistance (AMR) studies of Mycoplasma bovis have generally focused on specific loci versus using a genome-wide association study (GWAS) approach. A GWAS approach, using two different models, was applied to 194 Mycoplasma bovis genomes. Both a fixed effects linear model (FEM) and a linear mixed model (LMM) identified associations between nucleotide variants (NVs) and antimicrobial susceptibility testing (AST) phenotypes. The AMR phenotypes represented fluoroquinolones, tetracyclines, phenicols, and macrolides. Both models identified known and novel NVs associated (Bonferroni adjusted p < 0.05) with AMR. Fluoroquinolone resistance was associated with multiple NVs, including previously identified mutations in gyrA and parC. NVs in the 30S ribosomal protein 16S were associated with tetracycline resistance, whereas NVs in 5S rRNA, 23S rRNA, and 50S ribosomal proteins were associated with phenicol and macrolide resistance. For all antimicrobial classes, resistance was associated with NVs in genes coding for ABC transporters and other membrane proteins, tRNA-ligases, peptidases, and transposases, suggesting a NV-based multifactorial model of AMR in M. bovis. This study was the largest collection of North American M. bovis isolates used with a GWAS for the sole purpose of identifying novel and non-antimicrobial-target NVs associated with AMR.

Activation of the Anaphase Promoting Complex Reverses Multiple Drug Resistant Cancer in a Canine Model of Multiple Drug Resistant Lymphoma.

Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that went into remission showed that APC substrate mRNAs were decreased, indicating that the APC was activated during remission. In vitro validation using canine lymphoma cells selected for resistance to chemotherapeutic drugs confirmed that APC activation restored MDR chemosensitivity, and that APC activity was reduced in MDR cells. This supports the idea that rapidly pushing MDR cells that harbor high loads of chromosome instability through mitosis, by activating the APC, contributes to improved survival and disease-free duration.

Investigation of Macrolide Resistance Genotypes in Mycoplasma bovis Isolates from Canadian Feedlot Cattle.

Mycoplasma bovis is associated with bovine respiratory disease (BRD) and chronic pneumonia and polyarthritis syndrome (CPPS) in feedlot cattle. No efficacious vaccines for M. bovis exist; hence, macrolides are commonly used to control mycoplasmosis. Whole genome sequences of 126 M. bovis isolates, derived from 96 feedlot cattle over 12 production years, were determined. Antimicrobial susceptibility testing (AST) of five macrolides (gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin) was conducted using a microbroth dilution method. The AST phenotypes were compared to the genotypes generated for 23S rRNA and the L4 and L22 ribosomal proteins. Mutations in domains II (nucleotide 748; E. coli numbering) and V (nucleotide 2059 and 2060) of the 23S rRNA (rrl) gene alleles were associated with resistance. All isolates with a single mutation at Δ748 were susceptible to tulathromycin, but resistant to tilmicosin and tildipirosin. Isolates with mutations in both domain II and V (Δ748Δ2059 or Δ748Δ2060) were resistant to all five macrolides. However, >99% of isolates were resistant to tildipirosin and tilmicosin, regardless of the number and positions of the mutations. Isolates with a Δ748 mutation in the 23S rRNA gene and mutations in L4 and L22 were resistant to all macrolides except for tulathromycin.

Comparison of multilocus sequence types found among North American isolates of Mycoplasma bovis from cattle, bison, and deer, 2007-2017.

A prior multilocus sequence typing (MLST) study reported that Mycoplasma bovis isolates from North American bison possess sequence types (STs) different from those found among cattle. The 42 bison isolates evaluated were obtained in 2007 or later, whereas only 19 of 94 (~20%) of the available cattle isolates, with only 1 from North America, were from that same time. We compared STs of additional, contemporary, North American cattle isolates with those from bison, as well as isolates from 2 North American deer, all originating during the same timeframe, to more definitively assess potential strain-related host specificity and expand our understanding of the genetic diversity of M. bovis. From 307 isolates obtained between 2007 and 2017 (209 from cattle, 96 from bison, 2 from deer), we identified 49 STs, with 39 found exclusively in cattle and 5 exclusively in bison. Four STs were shared between bison and cattle isolates; one ST was found in cattle and in a deer. There was no clear association between ST and the health status of the animal of origin. An MLST-based phylogeny including 41 novel STs identified in our study reveals that STs found in bison fall within several divergent lineages that include STs found exclusively in cattle.

Identification of Plasmodiophora brassicae effectors - A challenging goal.

Clubroot is an economically important disease affecting Brassica plants worldwide. Plasmodiophora brassicae is the protist pathogen associated with the disease, and its soil-borne obligate parasitic nature has impeded studies related to its biology and the mechanisms involved in its infection of the plant host. The identification of effector proteins is key to understanding how the pathogen manipulates the plant's immune response and the genes involved in resistance. After more than 140 years studying clubroot and P. brassicae, very little is known about the effectors playing key roles in the infection process and subsequent disease progression. Here we analyze the information available for identified effectors and suggest several features of effector genes that can be used in the search for others. Based on the information presented in this review, we propose a comprehensive bioinformatics pipeline for effector identification and provide a list of the bioinformatics tools available for such.