Browsing by Author "Khafipour, Ehsan"

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    Association of bovine major histocompatibility complex (BoLA) gene polymorphism with colostrum and milk microbiota of dairy cows during the first week of lactation
    (2018-11-12) Derakhshani, Hooman; Plaizier, Jan C; De Buck, Jeroen; Barkema, Herman W; Khafipour, Ehsan
    Abstract Background 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. Results 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. Conclusion 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.
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    Associations between digital dermatitis lesion grades in dairy cattle and the quantities of four Treponema species
    (2018-10-29) Beninger, Caroline; Naqvi, Syed A; Naushad, Sohail; Orsel, Karin; Luby, Chris; Derakhshani, Hooman; Khafipour, Ehsan; De Buck, Jeroen
    Abstract 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.
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    Biological observations in microbiota analysis are robust to the choice of 16S rRNA gene sequencing processing algorithm: case study on human milk microbiota
    (2020-09-18) Moossavi, Shirin; Atakora, Faisal; Fehr, Kelsey; Khafipour, Ehsan
    Abstract Background In recent years, the microbiome field has undergone a shift from clustering-based methods of operational taxonomic unit (OTU) designation based on sequence similarity to denoising algorithms that identify exact amplicon sequence variants (ASVs), and methods to identify contaminating bacterial DNA sequences from low biomass samples have been developed. Although these methods improve accuracy when analyzing mock communities, their impact on real samples and downstream analysis of biological associations is less clear. Results Here, we re-processed our recently published milk microbiota data using Qiime1 to identify OTUs, and Qiime2 to identify ASVs, with or without contaminant removal using decontam. Qiime2 resolved the mock community more accurately, primarily because Qiime1 failed to detect Lactobacillus. Qiime2 also considerably reduced the average number of ASVs detected in human milk samples (364 ± 145 OTUs vs. 170 ± 73 ASVs, p < 0.001). Compared to the richness, the estimated diversity measures had a similar range using both methods albeit statistically different (inverse Simpson index: 14.3 ± 8.5 vs. 15.6 ± 8.7, p = 0.031) and there was strong consistency and agreement for the relative abundances of the most abundant bacterial taxa, including Staphylococcaceae and Streptococcaceae. One notable exception was Oxalobacteriaceae, which was overrepresented using Qiime1 regardless of contaminant removal. Downstream statistical analyses were not impacted by the choice of algorithm in terms of the direction, strength, and significance of associations of host factors with bacterial diversity and overall community composition. Conclusion Overall, the biological observations and conclusions were robust to the choice of the sequencing processing methods and contaminant removal.
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    Composition and co-occurrence patterns of the microbiota of different niches of the bovine mammary gland: potential associations with mastitis susceptibility, udder inflammation, and teat-end hyperkeratosis
    (2020-04-14) Derakhshani, Hooman; Plaizier, Jan C; De Buck, Jeroen; Barkema, Herman W; Khafipour, Ehsan
    Abstract Background 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. Results 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. Conclusions 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.
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    Repeatability and reproducibility assessment in a large-scale population-based microbiota study: case study on human milk microbiota
    (2021-02-10) Moossavi, Shirin; Fehr, Kelsey; Khafipour, Ehsan; Azad, Meghan B
    Abstract Background Quality control including assessment of batch variabilities and confirmation of repeatability and reproducibility are integral component of high throughput omics studies including microbiome research. Batch effects can mask true biological results and/or result in irreproducible conclusions and interpretations. Low biomass samples in microbiome research are prone to reagent contamination; yet, quality control procedures for low biomass samples in large-scale microbiome studies are not well established. Results In this study, we have proposed a framework for an in-depth step-by-step approach to address this gap. The framework consists of three independent stages: (1) verification of sequencing accuracy by assessing technical repeatability and reproducibility of the results using mock communities and biological controls; (2) contaminant removal and batch variability correction by applying a two-tier strategy using statistical algorithms (e.g. decontam) followed by comparison of the data structure between batches; and (3) corroborating the repeatability and reproducibility of microbiome composition and downstream statistical analysis. Using this approach on the milk microbiota data from the CHILD Cohort generated in two batches (extracted and sequenced in 2016 and 2019), we were able to identify potential reagent contaminants that were missed with standard algorithms and substantially reduce contaminant-induced batch variability. Additionally, we confirmed the repeatability and reproducibility of our results in each batch before merging them for downstream analysis. Conclusion This study provides important insight to advance quality control efforts in low biomass microbiome research. Within-study quality control that takes advantage of the data structure (i.e. differential prevalence of contaminants between batches) would enhance the overall reliability and reproducibility of research in this field. Video abstract