Browsing by Author "De Buck, Jeroen"
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- ItemOpen AccessAge and Dose Dependent Susceptibility to Mycobacterium avium subspecies paratuberculosis Infection in Dairy Calves(2014-08-15) Mortier, Rienske Alice Rosa; De Buck, Jeroen; Barkema, Herman W.Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease (JD), a chronic enteritis in ruminants. Control programs focus on prevention of infection of susceptible individuals: calves < 6 months of age. However, this age-cut-off for susceptibility is not well supported scientifically. Additionally, control programs struggle with low sensitivity of diagnostic tests in the early stages of JD. The main objective of this thesis was to determine age-dependent susceptibility in dairy calves. Additionally, the course of immune responses as well as fecal shedding was assessed. Furthermore, gross lesions, histology and MAP-culture from tissues were used to confirm infection status of each calf, and to investigate age-dependent susceptibility. Fifty calves were inoculated per os on 2 consecutive days at 2 weeks and 3, 6, 9, or 12 months. Within each age group calves received either a high (5 x 109 CFU) or low dose (5 x 107 CFU) of MAP. Six calves served as a negative control group. Serum, whole blood and fecal samples were collected regularly until necropsy at 17 months of age. Macroscopic and histological lesions were assessed and bacterial culture was performed on tissue samples. Calves were successfully infected with MAP up to 1 year of age even with a low dose of MAP. Calves inoculated at 2 weeks, 3, or 6 months of age with a high dose of MAP had more severe necropsy lesions, were shedding MAP in feces more frequently, and had a stronger humoral and cellular immune response, than calves inoculated with a low dose. Shedding and humoral immune responses differed between individual calves and were detected in about half of the calves, which was more than anticipated. A dose-dependent cellular immune response was detected in each inoculated calf soon after inoculation using an interferon-gamma release assay and is therefore a good candidate test for early diagnosis. To conclude, calves are susceptible to MAP infection up to 1 year of age and could be infectious to other calves. Keeping the infection pressure low on-farm could reduce the severity of JD. Early diagnosis of MAP-infection is possible and this could improve the potential to control JD on-farm.
- ItemOpen AccessAssociation 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, EhsanAbstract 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.
- ItemOpen AccessAssociations 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, JeroenAbstract 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.
- ItemOpen AccessBacteriocins of bovine non-aureus staphylococci(2017) Carson, Domonique; Barkema, Herman W.; De Buck, Jeroen; Orsel, Karsina; Cobo, EduardoThe non-aureus staphylococci (NAS) species are among the most prevalent isolated from bovine milk and have been reported to inhibit major mastitis pathogens, likely by producing bacteriocins. This thesis is comprised of two sections, focusing on in vitro inhibition assays and in silico identification of bacteriocin gene clusters and bacteriocin resistance genes in NAS and Staphylococcus aureus, using isolates obtained from the Canadian Bovine Mastitis and Milk Quality Research Network. The first part determined the inhibitory capability of 441 bovine NAS isolates (comprising 25 species) against bovine S. aureus and human methicillin-resistant S. aureus (MRSA) and determined the presence of bacteriocin biosynthetic gene clusters in NAS whole genomes. Overall, 40 isolates from 9 species (S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus) inhibited growth of S. aureus in vitro; of which, 23 isolates (from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus) also inhibited MRSA. 105 putative bacteriocin gene clusters encompassing 6 different subclasses (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. The second part of the thesis determined the susceptibility of 139 bovine S. aureus isolates to a bacteriocin producing S. chromogenes isolate and identified and described the distribution of genes potentially associated with susceptibility and resistance in S. aureus whole genomes. Overall, 90 S. aureus isolates (65%) were resistant to inhibition by the S. chromogenes isolate. We identified 77 genes that were associated with an isolate being resistant. We also identified 76 genes that were associated with an isolate being susceptible to the S. chromogenes. Bacteriocin susceptibility and resistance seems to be linked to a large number of genes, the majority of which are annotated as hypothetical proteins and will need further assessment to determine their role in S. aureus susceptibility. Overall, bacteriocins may be a potential source of novel antimicrobials and this thesis represents the foundation to explore novel NAS bacteriocins.
- ItemOpen AccessCathelicidin Mitigates Staphylococcus Aureus Mouse Mastitis and Prevents Bacterial Invasion in Mammary Epithelial Cells(2019-08-23) Cavalcante, Paloma; Cobo, Eduardo; Barkema, Herman; De Buck, Jeroen; Jenne, Craig; Gedamu, LashitewStaphylococcus aureus is an important cause of mastitis, increasingly problematic due to the presence of bacterial strains resistant to conventional antibiotics. The ability of S. aureus to invade host cells is key to its ability to escape immune defense and antibiotics. This study focused on functions of cathelicidin, a small cationic peptide secreted by epithelial cells and leukocytes, in the pathogenesis of S. aureus mastitis. We determined that endogenous murine cathelicidin (CRAMP; Camp) was key in controlling S. aureus infection, as cathelicidin knockout mice (Camp-/-) inframammary challenged with S. aureus had higher bacteria burden and more severe mastitis compared to wild-type mice. Both human and murine cathelicidins, LL37 and CRAMP, respectively, reduced invasion of S. aureus in mammary epithelia. This function was independent of TLR2 and CD36 cell surface expression. LL-37 internalized into mammary epithelial cells and impaired S. aureus growth in vitro. We conclude that cathelicidins are promising anti-infectious therapeutic targets in mastitis; endogenous or exogenous cathelicidins protected against S. aureus infection by preventing internalization and potentially by directly killing this pathogen.
- ItemOpen AccessCell wall proteome analysis of Mycobacterium smegmatis strain MC2 155(BioMed Central, 2010-04-22) He, Zhiguo; De Buck, Jeroen
- ItemOpen AccessCharacterization, expression and immunological analysis of the pe and ppe proteins of mycobacterium avium subspecies paratuberculosis(2011) Mackenzie, Nicholas Alexander; De Buck, JeroenJohne's Disease is a chronic, granulomatous enteritis of ruminant animals caused by Mycobacterium avium subspecies paratuberculosis. Unfortunately, available diagnostic tests cannot reliably detect animals in early stages of disease, hampering current control efforts which rely heavily on testing and culling of infected animals. In this work, two families of polymorphic proteins, the PE and PPE proteins, were tested for their potential as immunodiagnostic targets. These proteins are cell-surface exposed and hypothesized to be involved in virulence and antigenic variation. Using a bioinformatic approach, a number of highly specific sequences of potential diagnostic utility were identified within these two families. Representatives of the families were expressed and studied using in vitro transcription/translation, as well as recombinant expression and coexpression in E. coli. Both humoral and cell-mediated immune assays were then conducted on the expressed products using whole blood and sera from naturally-infected cattle and several potential diagnostic targets were identified.
- ItemOpen AccessCharacterizing the microbiota of bovine digital dermatitis(2021-03-24) Caddey, Benjamin; De Buck, Jeroen; Orsel, Karin; Morck, DouglasBovine digital dermatitis (DD) is a skin disease affecting cattle worldwide. As a significant contributor to infectious lameness, DD is increasingly an economic and animal welfare concern for dairy and beef producers. DD is a polymicrobial disease, with many different types of anaerobic bacteria strongly associated with lesions. No causative agents or etiopathogenesis mechanisms of DD are yet accepted due to the multiple different species present and microbiota variation among individual lesions. The involvement of Treponema in lesion development is generally accepted, whereas what combination of other anaerobic bacteria are involved is currently debated. Insufficient and incomplete identification and characterization of these additional species are a limiting factor in DD pathogenesis research. This thesis aimed to fill the gaps in knowledge of these additional anaerobes throughout DD lesions, while providing the first comprehensive description of DD microbiota in feedlot beef cattle. Through high throughput sequencing and culturing of DD tissue biopsies, we identified Treponema, Mycoplasma, Fusobacterium spp., Porphyromas levii, and Bacteroides pyogenes as potential DD pathogens, and used a multiplex qPCR for absolute quantification and reproducible characterization of species population dynamics. We identified T. medium, P. levii, and T. phagedenis as a group strongly associated with early lesion stages, thus potentially being involved in lesion formation. Through a meta-analysis of all publicly available DD metagenomic studies, we identify Treponema, Mycoplasma, Fusobacterium, and Porphyromonas as the primary DD-associated microbiota. We recommend focusing future DD research efforts on culturing and characterizing species of these groups in an effort to establish etiopathogenesis mechanisms of DD.
- ItemOpen AccessComposition 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, EhsanAbstract 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.
- ItemOpen AccessDetecting total immunoglobulins in diverse animal species with a novel split enzymatic assay(2019-10-28) Drikic, Marija; Olsen, Steven; De Buck, JeroenAbstract Background Total immunolobulin G concentration is a useful, albeit underutilized, diagnostic parameter for health assessments of non-domestic animal species, due to a lack of functional diagnostic tools. Traditional assays, including enzyme-linked immunosorbent assay or radial immunodiffusion, require development of specific reagents (e.g., polyclonal antisera and appropriate protocols) for each animal species, precluding wide and easy adoption in wildlife welfare. As an alternative, bacterial virulence factors able to bind IgGs in antigen-independent manner can be used. To further simplify the diagnostic procedure and increase the number of species recognized by an assay, in this study a recently developed Split Trehalase immunoglobulin assay (STIGA) with bIBPs as a sensing elements was used to detect antibodies in 29 species from 9 orders. Three bacterial immunoglobulin binding proteins (protein G, protein A and protein L) were incorporated into STIGA reagents to increase the number of species recognized. Results IgG concentrations were detected through glucose production and produced signals were categorized in 4 categories, from not active to strong signal. Activation was detected in almost all tested animal species, apart from birds. Incorporation of Protein G, Protein A and Protein L allowed detection of IgGs in 62, 15.5 and 6.9% of species with a strong signal, respectively. Assays combining 2 bacterial immunoglobulin binding proteins as sensing element generally gave poorer performance than assays with the same bacterial immunoglobulin binding proteins fused to both trehalase fragments. Conclusions STIGA assays have potential to be further developed into an easily adoptable diagnostic test for total amount of IgGs in almost any serum sample, independent of species.
- ItemEmbargoDetection of Johne’s Disease on dairy farms using different qPCR target genes for Mycobacterium avium subsp. paratuberculosis in young stock(2023-08) Martins, Larissa; Barkema, Herman W.; Orsel, Karin; De Buck, Jeroen; Pearson, JenniferYoung stock can shed Mycobacterium avium subspecies paratuberculosis (MAP) in feces, present antibody titers and transmit MAP to other young stock. However, most Johne’s disease (JD) control programs do not include young stock in MAP testing strategies, which might be one of the reasons why only a few JD control programs were able to eradicate MAP. This study aimed to include young stock in a JD testing strategy and improve diagnostic tests. A literature review conducted reported that young stock can shed MAP as early as 4 mo of age. However, due to the chronic characteristic of the disease, it was considered important to improve current diagnostic tests and develop new tests, such as phage-based and metabolomics tests. A tentative inclusion of young stock in the MAP testing strategy was evaluated based on direct fecal qPCR and ELISA every 2 mo from animals between 2-12 mo of age. A sudden rise in MAP prevalence was detected at the second sampling, 2 mo after the start of the study. Although the high MAP prevalence was explained in part by the presence of MAP infections in the herd, it was not possible to explain the specificity of the ISMAP02 gene, which raises doubts about different Mycobacterium species being detected by the same assay. Furthermore, an in depth evaluation of the main MAP target genes for qPCR assays was proposed across different sample types and MAP concentrations. Overall, all MAP target genes were able to detect samples with high MAP concentration. IS900 and ISMAP02 consistently identified MAP in all sample types. However, the genes mbtA, hspX and F57 presented issues to detect samples with mid to low MAP concentrations.
- ItemOpen AccessDeveloping a Non-aureus Staphylococcus Intramammary Probiotic as a Preventative Measure for Bovine Mastitis(2023-05-11) Vu, Dennis; De Buck, Jeroen; Storey, Douglas; Barkema, HermanBovine mastitis is the most common and economically important disease affecting the dairy industry. Intramammary infection (IMI) with Staphylococcus aureus is the leading cause of contagious mastitis. Interestingly, non-aureus staphylococci (NAS) are frequently found in cows with subclinical mastitis, but with a severity less than with S. aureus. Antibiotics are the main method for preventing and treating mastitis. Misuse and overuse of antibiotics have resulted in the emergence of antimicrobial-resistant bacteria, and thus, alternative treatments are required. Bacteriocins, antimicrobial peptides produced by bacteria, are a promising alternative. We hypothesized that by creating a NAS probiotic through genetically engineering a bacteriocin gene cluster into its genome, it will be able to inhibit S. aureus and prevent mastitis. To achieve this, we needed to find a persistent and non-inflammatory NAS strain that can colonize cow mammary glands by using an experimental mammary infusion model. After finding a persistent and non-inflammatory NAS, we will perform a bacteriocin gene cluster knock-in using allelic replacement. Finally, the probiotic will then be characterized through gene expression and killing assays. This thesis aimed to create an alternative treatment to prevent the growth of mastitis pathogens during the dry period. Ultimately, this will lower the usage of antibiotics and give us another preventative tool against mastitis. We identified S. warneri 2993 as the most persistent and non-inflammatory NAS but unfortunately, we were not able to perform our bacteriocin gene knock-in. Instead, we recommend future studies to re-attempt this gene knock-in but with a different bacteriocin gene cluster for an increased likelihood of success. The probiotic will then need testing in ¬in vivo¬ IMI experiments in mice and then in cows following the protocols we have designed.
- ItemOpen AccessDevelopment of a Gram-type Specific Method for Detection of Bovine Mastitis Pathogens by Combining Loop-mediated Isothermal Amplification (LAMP) and Split Trehalase Technologies(2022-07-20) Miao, Zhuohan; De Buck, Jeroen; Finney, Constance; Niu, DongyanBovine mastitis, which results mainly from intramammary infections (IMI) caused by Gram-positive or Gram-negative bacteria, is a huge burden on the global dairy industry. Oftentimes the presence of an infection can be deduced by measuring the somatic cell count (SCC) or released enzymes, or directly detected by the presence of mastitis pathogens. While many laboratory diagnostic methods (e.g., SCC determination, bacteria culturing, qPCR) exist, few have been modified and adopted for on-farm use due to the requirement of sterility, dedicated equipment and well-trained personnel for implementation, time restraints, and accuracy. As such, the development of new methods that can sensitively and specifically detect mastitis-causing pathogens on-farm would be very valuable. We aimed to detect and distinguish Gram-positive and Gram-negative mastitis bacteria to inform on the proper and prompter treatment/antibiotic to prescribe through two steps: (i) develop PCR and LAMP assay for a point-of-care (POC) amplification of Gram-positive and Gram-negative bacteria and (ii) use a modified amplicon binding split trehalase assay (ABSTA) to detect this amplification by a specific protein-DNA binding and the complementation of split trehalase. To do this, split trehalase fusion proteins HisTreA-C-SpoIIID and HisTreA-N-SpoIIID were purified, and the binding with modified oligonucleotides incorporated with SpoIII-specific recognition sequences was tested to verify and optimize the complementation conditions. Next, recognition sequences were introduced in Gram-type specific PCR primers, allowing for specific detection of PCR amplicons by ABSTA. We also integrated the recognition sequences in three Gram-type specific LAMP primer sets designed based on the pathogens’ 16S rRNA genes, allowing for specific detection of Staphylococcus, Streptococcus and Gram-negative bacteria LAMP products by ABSTA. The salt concentration, protein reagents versus DNA substrate ratio, direction and linker length of tandem recognition sites required for the complementation were optimized. Binding with purified PCR products demonstrated Gram-type detection specificity with the protein reagents, whereas the complementation appeared to be inhibited by agents in unpurified PCR products. The sensitivity of detection of bacterial genomic DNA of Escherichia coli, Staphylococcus devriesii and Streptococcus uberis ranged from 2 to 24. The sensitivity of detection of E. coli in spiked milk samples was 11 CFU/ml, but 4.9 × 104 CFU/ml and 2.0 × 105 CFU/ml for S. devriesei and S. uberis, respectively. The analytical specificity of the newly designed LAMP primer sets was evaluated with ten mastitis isolates at two genomic DNA copy number levels. In conclusion, the combination of bacterial genetic target amplification by LAMP and ABSTA demonstrated high sensitivity and specificity towards genomic DNA of Gram-positive and Gram-negative bacteria, enhancing the potential of developing a timesaving, user-friendly and cost-effective on-farm diagnostic method for educated treatment decisions of bovine mastitis causative pathogens.
- ItemOpen AccessDistribution of Staphylococcus non-aureus isolated from bovine milk in Canadian herds(2016) Condas, Larissa; Barkema, Herman; De Buck, Jeroen; Liljebjelke, Karen; Kastelic, John; Middleton, John; De Vliegher, Sarne; Armstrong, GlenThe Staphylococci non-aureus (SNA) species are among the most prevalent isolated from bovine milk. However, the role of each species within the SNA group still needs to be fully understood. Knowing which SNA species are most common in bovine intramammary infections (IMI), as well as their epidemiology, is essential to the improvement of udder health on dairy farms worldwide. This thesis is comprised of two studies on the epidemiology of SNA species in bovine milk, and used molecular methods to identify of isolates obtained from the Canadian Bovine Mastitis and Milk Quality Research Network. The first study focused on the prevalence of SNA species on Canadian dairy farms and potential associations of SNA positive mammary quarters with bulk milk somatic cell count (BMSCC), barn type, parity, month of lactation and quarter location. Overall SNA represented 9% of the isolates from culture positive mammary quarters and the most common species were S. chromogenes, S. simulans, S. xylosus, S. haemolyticus, and S. epidermidis. Province and barn type were associated with SNA species distribution; Albertan bedded-packs were mostly affected by S. chromogenes, Maritimes free-stall herds by S. epidermidis, and Ontario and Quebec tie-stalls by S. xylosus. Staphylococcus arlettae, S. cohnii, and S. gallinarum were isolated from quarters of herds with high BMSCC. Fresh heifers and cows in later lactation were most frequently infected by S. chromogenes. The second study focused on the distribution of the same species in SNA positive-quarters according to udder inflammation status, classified according to low and high SCC and clinical mastitis. Average somatic cell count (SCC) for the SNA as a group was 70,000 cells/mL, driven mostly by S. chromogenes, S. haemolyticus, S. xylosus and S. epidermidis. Species-specific prevalence of SNA-positive quarters was higher in high (≥ 200,000 cells/mL) than in low SCC (< 200,000 cells/mL) samples for the 11 most frequently isolated SNA species. Staphylococcus sciuri was more frequently isolated from clinical mastitis samples. Considering SNA as a group will misrepresent the role of individual species on farms. Ultimately, adopting molecular identification of SNA species along with future research in species-specific risk factors are necessary to fully elucidate the importance of of the different SNA species on udder health and possible species-specific interventions.
- ItemOpen AccessEvaluation of age-dependent susceptibility in calves infected with two doses of Mycobacterium avium subspecies paratuberculosis using pathology and tissue culture(BioMed Central, 2013-10-07) Mortier, Rienske A. R.; Barkema, Herman W.; Bystrom, Janet M.; Illanes, Oscar; Orsel, Karin; Wolf, Robert; Atkins, Gordon; De Buck, Jeroen; Medicine
- ItemOpen AccessExploring the ‘Nemabiome’: Deep Amplicon Sequencing to Investigate Community Structure and Drug Resistance in Parasitic Gastrointestinal Nematodes of Livestock(2018-01-12) Avramenko, Russell; Gilleard, John; Colwell, Douglas; De Buck, Jeroen; Wasmuth, JamesParasitic gastrointestinal nematodes are one of the most important pathogen groups impacting livestock health, welfare and production worldwide. Control in both animals and humans is heavily dependent on the prophylactic administration of anthelmintic drugs. However, there are widespread and increasing reports of anthelmintic drug resistance in many parasitic nematode species making current approaches to control unsustainable. Consequently, there is an urgent need for better tools for diagnosis, surveillance and research to support the development of sustainable approaches to parasite control. Although co-infection with multiple nematode species within a single host is common, there are few tools with which to study the composition of these complex parasite communities. Nematode species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. This thesis introduces the concept of the ‘nemabiome’ as the community of mixed nematode species inhabiting the gastrointestinal tract of the host. Chapter 2 presents the development of a deep-amplicon (metabarcoding) sequencing approach, targeting the ITS-2 rDNA locus, to define the species composition of the nemabiome. This approach is analogous to deep-amplicon sequencing methodologies used to study the bacterial ‘microbiome’. Chapter 3 uses this methodology to assess the species composition of nematode communities in cattle from Canada, the United States and Brazil. It also describes changes in the nemabiome before and after anthelmintic treatment in Canadian cattle. Chapter 4 illustrates the ability of this approach to describe the nemabiome of commercial and conservation bison herds in western Canada, which previously had little information regarding species prevalence. Chapter 5 introduces another deep-amplicon sequencing application; screening for DNA sequence polymorphisms associated with benzimidazole drug resistance in mixed nematode infections. The isotype-1 β-tubulin locus is screened for three polymorphisms associated with benzimidazole resistance. Chapter 6 describes the application of this approach to screen for benzimidazole resistance associated polymorphisms in the cattle and bison parasite populations characterised in Chapters 3 and 4. These approaches represent vital tools needed to conduct epidemiological studies, surveillance of parasite species, improve the sensitivity of drug efficacy testing and assess anthelmintic resistance on a large scale.
- ItemOpen AccessFecal shedding and tissue infections demonstrate transmission of Mycobacterium avium subsp. paratuberculosis in group-housed dairy calves(2017-04-28) Corbett, Caroline S; De Buck, Jeroen; Orsel, Karin; Barkema, Herman WAbstract Current Johne’s disease control programs primarily focus on decreasing transmission of Mycobacterium avium subsp. paratuberculosis (MAP) from infectious adult cows to susceptible calves. However, potential transmission between calves is largely overlooked. The objective was to determine the extent of MAP infection in calves contact-exposed to infectious penmates. Thirty-two newborn Holstein–Friesian calves were grouped into 7 experimental groups of 4, consisting of 2 inoculated (IN) calves, and 2 contact-exposed (CE) calves, and 1 control pen with 4 non-exposed calves. Calves were group housed for 3 months, with fecal samples were collected 3 times per week, blood and environmental samples weekly, and tissue samples at the end of the trial. The IN calves exited the trial after 3 months of group housing, whereas CE calves were individually housed for an additional 3 months before euthanasia. Control calves were group-housed for the entire trial. All CE and IN calves had MAP-positive fecal samples during the period of group housing; however, fecal shedding had ceased at time of individual housing. All IN calves had MAP-positive tissue samples at necropsy, and 7 (50%) of the CE had positive tissue samples. None of the calves had a humoral immune response, whereas INF-γ responses were detected in all IN calves and 5 (36%) CE calves. In conclusion, new MAP infections occurred due to exposure of infectious penmates to contact calves. Therefore, calf-to-calf transmission is a potential route of uncontrolled transmission on cattle farms.
- ItemOpen AccessGene Expression Study Of Mycobacterium avium subspecies paratuberculosis Infected Cows – “A road to identify transcripts that could serve as biomarkers for early diagnosis of Johne’s disease”(2014-02-05) David, Joel; De Buck, Jeroen; Barkema, Herman; Ghosh, Subrata; Guan, LeLuoCurrent diagnostic tools lack sensitivity to detect JD early after infection. Hence, this study aimed to find biomarkers for early diagnosis of JD. An infection trial was set up with a high dose (n=5) and low dose (n=5) oral challenge with Mycobacterium avium subsp. paratuberculosis at 2 weeks of age and 5 non-infected control animals. Whole blood was collected at a 3-month interval (3, 6, 9, 12 and 15 months) for gene expression analysis using Affymetrix® GeneChip® Bovine Genome Array on samples obtained at 3, 6 and 9 months after infection. Microarray findings were confirmed using qPCR and longitudinal assays profiling transcripts over 15-month period was carried out. This study found differential gene expression at each sampled time point and found transcripts for potential biomarker use. CD46 was upregulated and BOLA and BNBD9-like genes were downregulated over the entire 15-months. Overall immune response was inhibited in MAP-challenged animals.
- ItemOpen AccessIdentifying genes in Mycobacterium avium subspecies paratuberculosis that are essential for survival in dairy calves(2017) Mirto, Amanda; De Buck, Jeroen; Barkema, Herman; Behr, Marcel; Dong, TaoMycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic wasting disease common in dairy cattle. Currently, there are no effective vaccines to prevent infection and fecal bacterial shedding. To determine potential targets for a live- attenuated vaccine strain 12 Holstein-Friesian dairy calves were inoculated with 5 x 10^10 CFUs on two consecutive days with a mutant library from MAP strain A1-157. MAP was cultured 2 and 4 mo post inoculation from intestinal tissues and lymph nodes. Quantitative PCR was used to assess changes in proportions of select gene mutants between intestinal tissues and the inoculum; this method failed to confirm previously described essential genes. After applying filtering parameters, targeted next generation sequencing discovered 42 genes absent from all tissues which are involved in DNA transcription, pathogenesis, metabolism and biosynthesis, and oxidation-reduction reactions. These results provide many potential genes for attenuation of MAP for the creation of candidate vaccine strains.
- ItemOpen AccessInvestigating the virulence of Treponema phagedenis strains isolated from digital dermatitis lesions in a murine abscess model(2023-07) Scott, Colton; De Buck, Jeroen; Chaconas, George; Harrison, JoeDigital Dermatitis (DD) is an ulcerative foot lesion in the heel bulbs of dairy cattle. DD is a polymicrobial disease with no precise etiology, although key pathogenic genera have been consistently found disproportional and abundant in diseased tissue as opposed to healthy skin. One such genera is Treponema, a member genus of the phylum Spirochaetes. Within Treponema, many different phylotypes are found in DD, however the species Treponema phagedenis is uniformly found in copious quantities and deep within the skin layers in the active, ulcerative stages of disease. The pathogenic mechanisms these bacteria use to persist in the skin and the role they play in the larger pathology of DD is widely unknown. To explore the pathogenesis and virulence of Treponema phagedenis, isolates of this species were investigated in a subcutaneous murine abscess model. For this purpose, mice were subcutaneously inoculated with T. phagedenis in two infection trials. In the first trial, a dosage study was conducted to elucidate the pathogenicity of strains across three different spirochete per inoculum (SPI) doses, based on abscess volumes. In the second trial, isolates were inoculated with the dose of 5 x 109 SPI selected based on trial 1 results, to determine the expression level of 11 putative virulence genes and gain insight into the virulence of strains. To do this, abscesses were harvested for RNA extraction followed by RT-qPCR. From the RT-qPCR assay, the relative fold change of these genes was surmised by comparing the expression levels of in vitro culture samples against in vivo murine samples. During this analysis, it was determined that genes encoding for two metal-ion import lipoproteins, were found highly upregulated during infection. In addition, two genes involved in the adherence of treponemes to the host were found moderately expressed versus the in vitro samples. Conversely, two genes involved in motility and chemotaxis were found to not be significantly upregulated or utilized during infection. This gene expression analysis highlights the preference in strategy for T. phagedenis to persist and adhere in the host rather than engage motility and disseminate.