Various body sites of vertebrates provide stable and nutrient-rich ecosystems for a diverse range of commensal, opportunistic, and pathogenic microorganisms to thrive. The collective genomes of these ...microbial symbionts (the microbiome) provide host animals with several advantages, including metabolism of indigestible carbohydrates, biosynthesis of vitamins, and modulation of innate and adaptive immune systems. In the context of the bovine udder, however, the relationship between cow and microbes has been traditionally viewed strictly from the perspective of host-pathogen interactions, with intramammary infections by mastitis pathogens triggering inflammatory responses (i.e., mastitis) that are often detrimental to mammary tissues and cow physiology. This traditional view has been challenged by recent metagenomic studies indicating that mammary secretions of clinically healthy quarters can harbor genomic markers of diverse bacterial groups, the vast majority of which have not been associated with mastitis. These observations have given rise to the concept of “commensal mammary microbiota,” the ecological properties of which can have important implications for understanding the pathogenesis of mastitis and offer opportunities for development of novel prophylactic or therapeutic products (or both) as alternatives to antimicrobials. Studies conducted to date have suggested that an optimum diversity of mammary microbiota is associated with immune homeostasis, whereas the microbiota of mastitic quarters, or those with a history of mastitis, are considerably less diverse. Whether disruption of the diversity of udder microbiota (dysbiosis) has a role in determining mastitis susceptibility remains unknown. Moreover, little is known about contributions of various biotic and abiotic factors in shaping overall diversity of udder microbiota. This review summarizes current understanding of the microbiota within various niches of the udder and highlights the need to view the microbiota of the teat apex, teat canal, and mammary secretions as interconnected niches of a highly dynamic microbial ecosystem. In addition, host-associated factors, including physiological and anatomical parameters, as well as genetic traits that may affect the udder microbiota are briefly discussed. Finally, current understanding of the effect of antimicrobials on the composition of intramammary microbiota is discussed, highlighting the resilience of udder microbiota to exogenous perturbants.
One mechanism through which S. cerevisiae may improve the performance of pigs is by altering the composition of the gut microbiota, a response that may be enhanced by early postnatal supplementation ...of probiotics. To test this hypothesis, newborn piglets (16 piglets/group) were treated with either S. cerevisiae yeast (5 x 109 cfu/pig: Low) or (2.5 x 1010 cfu/piglet: High) or equivalent volume of sterile water (Control) by oral gavage every other day starting from day 1 of age until weaning (28±1 days of age). Piglet body weight was recorded on days 1, 3, 7, 10, 17, 24 and 28 and average daily gain (ADG) calculated for the total period. At weaning, piglets were euthanized to collect cecum content for microbial profiling by sequencing of the 16S rRNA gene. ADG was higher in both Low and High yeast groups than in Control group (P<0.05). Alpha diversity analyses indicated a more diverse microbiota in the Control group compared with Low yeast group; the High yeast being intermediate (P < 0.01). Similarly, Beta diversity analyses indicated differences among treatments (P = 0.03), mainly between Low yeast and Control groups (P = 0.02). The sparse Partial Least Squares Discriminant Analysis (sPLS-DA) indicated that Control group was discriminated by a higher abundance of Veillonella, Dorea, Oscillospira and Clostridium; Low yeast treated pigs by higher Blautia, Collinsella and Eubacterium; and High yeast treated pigs by higher Eubacterium, Anaerostipes, Parabacteroides, Mogibacterium and Phascolarctobacterium. Partial Least Squares (PLS) analysis showed that piglet ADG was positively correlated with genus Prevotella in High yeast group. Yeast supplementation significantly affected microbial diversity in cecal contents of suckling piglets associated with an improvement of short chain fatty acid producing bacteria in a dose-dependent manner. In conclusion, yeast treatment improved piglet performance and shaped the piglet cecum microbiota composition in a dose dependent way.
Digital dermatitis (DD) presents as painful, ulcerative or proliferative lesions that lead to bovine lameness affecting economic efficiency and animal welfare. Although DD etiological agent(s) have ...not been established, it is widely accepted that DD is a polymicrobial disease significantly associated with species of Treponema and the non-linear disease progression may be attributed to interactions among infecting bacteria. We postulated the morphological changes associated with DD lesion grades are related to interactions among infecting species of Treponema. We developed a novel species-specific qPCR that can identify the absolute abundance of the four of the most common species of Treponema in DD, T. phagedenis, T. medium, T. pedis and T. denticola, in a single reaction. We found species abundance and the number of distinct Treponema species present is higher in active, ulcerative lesions than in healing lesions, chronic lesions, and DD-free skin. Treponema spp. were present in both DD-free skin and M3 lesions following treatment with oxytetracycline. We have also found positive correlations among T. phagedenis, T. medium and T. pedis indicating they are significantly more likely to be found together than apart and their absolute quantities tend to increase together, a relationship which is not present with T. denticola. Further, we found Treponema, particularly viable T. denticola, in lesions 5 days post treatment with oxytetracycline (M3). Our findings suggest that pathogenicity may be closely associated with Treponema abundance, particularly T. phagedenis, T. medium and T. pedis, and interactions among them, independent of T. denticola. Our results provide a novel, consistent method to identify species of Treponema within DD lesions and associate Treponema spp. and abundance with morphological changes related to host pathogenicity.
Illumina technology currently dominates bacterial genomics due to its high read accuracy and low sequencing cost. However, the incompleteness of draft genomes generated by Illumina reads limits their ...application in comprehensive genomics analyses. Alternatively, hybrid assembly using both Illumina short reads and long reads generated by single molecule sequencing technologies can enable assembly of complete bacterial genomes, yet the high per-genome cost of long-read sequencing limits the widespread use of this approach in bacterial genomics. Here we developed a protocol for hybrid assembly of complete bacterial genomes using miniaturized multiplexed Illumina sequencing and non-barcoded PacBio sequencing of a synthetic genomic pool (SGP), thus significantly decreasing the overall per-genome cost of sequencing.
We evaluated the performance of SGP hybrid assembly on the genomes of 20 bacterial isolates with different genome sizes, a wide range of GC contents, and varying levels of phylogenetic relatedness. By improving the contiguity of Illumina assemblies, SGP hybrid assembly generated 17 complete and 3 nearly complete bacterial genomes. Increased contiguity of SGP hybrid assemblies resulted in considerable improvement in gene prediction and annotation. In addition, SGP hybrid assembly was able to resolve repeat elements and identify intragenomic heterogeneities, e.g. different copies of 16S rRNA genes, that would otherwise go undetected by short-read-only assembly. Comprehensive comparison of SGP hybrid assemblies with those generated using multiplexed PacBio long reads (long-read-only assembly) also revealed the relative advantage of SGP hybrid assembly in terms of assembly quality. In particular, we observed that SGP hybrid assemblies were completely devoid of both small (i.e. single base substitutions) and large assembly errors. Finally, we show the ability of SGP hybrid assembly to differentiate genomes of closely related bacterial isolates, suggesting its potential application in comparative genomics and pangenome analysis.
Our results indicate the superiority of SGP hybrid assembly over both short-read and long-read assemblies with respect to completeness, contiguity, accuracy, and recovery of small replicons. By lowering the per-genome cost of sequencing, our parallel sequencing and hybrid assembly pipeline could serve as a cost effective and high throughput approach for completing high-quality bacterial genomes.
Bovine digital dermatitis (DD) is a skin disorder that is a significant cause of infectious lameness in cattle around the world. However, very little is known about the etiopathogenesis of the ...disease and the microbiota associated with DD in beef cattle. In this study, we provide a comprehensive characterization of DD and healthy skin microbiota of feedlot beef cattle. We also developed and validated a novel multiplex quantitative PCR (qPCR) assay to quantify the distribution of DD-associated bacterial species across DD lesion stages. We determined the DD-associated microbiota with deep amplicon sequencing of the V3-V4 hypervariable region of the 16S rRNA gene, followed by the application of novel and existing qPCR assays to quantify species distributions of Treponema,
,
, and
across lesion stages. Deep amplicon sequencing revealed that Treponema,
,
, and
were associated with DD lesions. Culturing of DD biopsy specimens identified Porphyromonas levii, Bacteroides pyogenes, and two
spp. within DD lesions. Using species-specific qPCR on DD lesion DNA, we identified P. levii in 100% of active lesion stages. Early-stage lesions were particularly associated with Treponema medium, T. phagedenis, and
. This study suggests a core DD microbial group consisting of species of Treponema,
,
, and
, which may be closely tied with the etiopathogenesis of DD. Further characterizations of these species and
spp. are necessary to understand the microbial factors involved in DD pathogenesis, which will help elucidate DD etiology and facilitate more targeted and effective mitigation and treatment strategies.
Previous work, primarily in dairy cattle, has identified various taxa associated with digital dermatitis (DD) lesions. However, there is a significant gap in our knowledge of DD microbiology in beef cattle. In addition, characterization of bacteria at the species level in DD lesions is limited. In this study, we provide a framework for the accurate and reproducible quantification of major DD-associated bacterial species from DNA samples. Our findings support DD as a polymicrobial infection, and we identified a variety of bacterial species spanning multiple genera that are consistently associated with DD lesions. The DD-associated microbiota identified in this study may be capable of inducing the formation and progression of DD lesions and thus should be primary targets in future DD pathogenesis studies.
The interplay between host genotype and commensal microbiota at different body sites can have important implications for health and disease. In dairy cows, polymorphism of bovine major ...histocompatibility complex (BoLA) gene has been associated with susceptibility to several infectious diseases, most importantly mastitis. However, mechanisms underlying this association are yet poorly understood. In the present study, we sought to explore the association of BoLA gene polymorphism with the dynamics of mammary microbiota during the first week of lactation.
Colostrum and milk samples were collected from multiparous Holstein dairy cows at the day of calving and days 1 and 6 after calving. Microbiota profiling was performed using high-throughput sequencing of the V1-V2 regions of the bacterial 16S rRNA genes and ITS2 region of the fungal ribosomal DNA. Polymorphism of BoLA genes was determined using PCR-RFLP of exon 2 of the BoLA-DRB3. In general, transition from colostrum to milk resulted in increased species richness and diversity of both bacterial and fungal communities. The most dominant members of intramammary microbiota included Staphylococcus, Ruminococcaceae, and Clostridiales within the bacterial community and Alternaria, Aspergillus, Candida, and Cryptococcus within the fungal community. Comparing the composition of intramammary microbiota between identified BoLA-DRB3.2 variants (n = 2) revealed distinct clustering pattern on day 0, whereas this effect was not significant on the microbiota of milk samples collected on subsequent days. On day 0, proportions of several non-aureus Staphylococcus (NAS) OTUs, including those aligned to Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus sciuri, and Staphylococcus haemolyticus, were enriched within the microbiota of one of the BoLA-DRB3.2 variants, whereas lactic acid bacteria (LAB) including Lactobacillus and Enterococcus were enriched within the colostrum microbiota of the other variant.
Our results suggest a potential role for BoLA-gene polymorphism in modulating the composition of colostrum microbiota in dairy cows. Determining whether BoLA-mediated shifts in the composition of colostrum microbiota are regulated directly by immune system or indirectly by microbiota-derived colonization resistant can have important implications for future development of preventive/therapeutic strategies for controlling mastitis.
Abstract Background Fungi constitute an important yet frequently neglected component of the human microbiota with a possible role in health and disease. Fungi and bacteria colonise the infant ...gastrointestinal tract in parallel, yet most infant microbiome studies have ignored fungi. Milk is a source of diverse and viable bacteria, but few studies have assessed the diversity of fungi in human milk. Results Here we profiled mycobiota in milk from 271 mothers in the CHILD birth cohort and detected fungi in 58 (21.4%). Samples containing detectable fungi were dominated by Candida , Alternaria , and Rhodotorula , and had lower concentrations of two human milk oligosaccharides (disialyllacto-N-tetraose and lacto-N-hexaose). The presence of milk fungi was associated with multiple outdoor environmental features (city, population density, and season), maternal atopy, and early-life antibiotic exposure. In addition, despite a strong positive correlation between bacterial and fungal richness, there was a co-exclusion pattern between the most abundant fungus ( Candida ) and most of the core bacterial genera. Conclusion We profiled human milk mycobiota in a well-characterised cohort of mother-infant dyads and provide evidence of possible host-environment interactions in fungal inoculation. Further research is required to establish the role of breastfeeding in delivering fungi to the developing infant, and to assess the health impact of the milk microbiota in its entirety, including both bacterial and fungal components.
The aim of this study was to assess the synergistic effects of co-fermentation of glucose, starch, and cellulose using anaerobic digester sludge (ADS) on the biohydrogen (H sub(2)) production and the ...associated microbial communities. Yields of H sub(2) were 1.22, 1.00, and 0.15 mol H sub(2)/mol hexose-added in fermentation reactions containing glucose, starch, and cellulose as mono-substrates. The H sub(2) yields were greater by an average of 27 plus or minus 4% than expected in all the different co-substrate conditions, which affirmed that co-fermentation of different substrates improved the H sub(2) potential. Glucose addition to starch and/or cellulose favored acetate synthesis, while cellulose degradation was associated with the propionate synthesis pathway. Illumina sequencing technology and bioinformatics analyses revealed operational taxonomic units (OTUs) in the Phyla Bacteroides, Chloroflexi, Firmicutes, Proteobacteria, Spirochaetes, Synergistes, and Thermotogae were common in mono- and co-substrate batches. However, OTUs in the Phyla Acidobacteria, Actinobacteria, and Bacteroidetesee were unique to only the co-substrate conditions.
Within complex microbial ecosystems, microbe-microbe interrelationships play crucial roles in determining functional properties such as metabolic potential, stability and colonization resistance. In ...dairy cows, microbes inhabiting different ecological niches of the udder may have the potential to interact with mastitis pathogens and therefore modulate susceptibility to intramammary infection. In the present study, we investigated the co-occurrence patterns of bacterial communities within and between different niches of the bovine mammary gland (teat canal vs. milk) in order to identify key bacterial taxa and evaluate their associations with udder health parameters and mastitis susceptibility.
Overall, teat canal microbiota was more diverse, phylogenetically less dispersed, and compositionally distinct from milk microbiota. This, coupled with identification of a large number of bacterial taxa that were exclusive to the teat canal microbiota suggested that the intramammary ecosystem, represented by the milk microbiota, acts as a selective medium that disfavors the growth of certain environmental bacterial lineages. We further observed that the diversity of milk microbiota was negatively correlated with udder inflammation. By performing correlation network analysis, we identified two groups of phylogenetically distinct hub species that were either positively (unclassified Bacteroidaceae and Phascolarctobacterium) or negatively (Sphingobacterium) correlated with biodiversity metrics of the mammary gland (MG). The latter group of bacteria also showed positive associations with the future incidence of clinical mastitis.
Our results provide novel insights into the composition and structure of bacterial communities inhabiting different niches of the bovine MG. In particular, we identified hub species and candidate foundation taxa that were associated with the inflammatory status of the MG and/or future incidences of clinical mastitis. Further in vitro and in vivo interrogations of MG microbiota can shed light on different mechanisms by which commensal microbiota interact with mastitis pathogens and modulate udder homeostasis.
Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under ...monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/β) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.
PDF
