• High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel; Fallico, Vincenzo; O'Sullivan, Orla; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Giblin, Linda; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 2012205 (Biomed Central, 17/11/2015)
      Background The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.
    • High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel; Fallico, Vincenzo; O'Sullivan, Orla; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Giblin, Linda; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 2012205 (Biomed Central, 17/11/2015)
      Background: The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results: Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions: In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.
    • Horn bud size of dairy-bred and suckler-bred calves at time of disbudding

      Marquette, Gabriela A.; McGee, Mark; Fisher, Andrew D.; Stanger, Kelly; Argüello, Anastasio; Earley, Bernadette; Teagasc Walsh Scholarship Programme (Biomed Central, 2021-06-16)
      Background Hot-iron disbudding is a common management procedure to prevent horn growth in calves. The study objective was to examine effect of age, breed and sex on horn bud size of dairy-bred and suckler-bred calves at time of disbudding. Results The left and right horn bud size (diameter and height in mm) of 279 calves, including dairy-bred Holstein-Friesian (Male (M) = 88) and 191 suckler-bred (86 Charolais, CH; (M = 39, Female (F) = 47), 67 Limousin, LM; (M = 32, F = 35) and 38 Simmental, SI; (M = 22, F = 16) sired)) was measured using a digital calliper at time of disbudding. Calves were retrospectively assigned to two age categories at time of disbudding: 1), 14 to 28 days (d) old and 2), 29 to 60 d old. Holstein-Friesian M calves had a greater horn bud diameter (16.97 v.14.45 mm) and height (7.79 v. 5.00 mm) compared to suckler-bred M calves (P < 0.01), with no difference (P > 0.05) among the suckler-bred calves. Suckler-bred M calves had a greater horn bud diameter (14.46 vs 13.29 mm) and height (5.01 vs 3.88 mm) compared to suckler-bred F calves (P < 0.05). The slopes of the lines of best fit show that horn bud diameter and height increased with age (P < 0.05) for HF, SI male and CH female calves while there was no relationship with age (P > 0.05) for CH and LM male calves, or for SI and LM female calves. Linear regression of age with diameter and with height for each breed and sex showed high variability in the data as indicated by R-squared values ranging from 0.003–0.41 indicating that in the case of the diameter and the height, the weight of the fitting effect was poor. Conclusions Calf age is not a good predictor of horn bud size and recommendations for the disbudding of calves should be based on horn bud size and not on age. The implications of these findings are that calves should be disbudded while horn development is still at the bud stage and when the bud is large enough to be easily palpable/visible, but not so large that disbudding could lead to severe tissue trauma.
    • A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover

      Nagy, Istvan; Barth, Susanne; Mehenni-Ciz, Jeanne; Abberton, Michael T; Milbourne, Dan; Department of Agriculture, Food and the Marine, Ireland; RSF 07–566 (Biomed Central, 13/02/2013)
      Background: White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. Results: We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. Conclusions: In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a “core set” of ancestrally derived homeolog specific variants in white clover.
    • Identifying challenges to manage body weight variation in pig farms implementing all-in-all-out management practices and their possible implications for animal health: a case study

      Rodrigues da Costa, Maria; García Manzanilla, Edgar; Diana, Alessia; van Staaveren, Nienke; Torres-Pitarch, Alberto; Boyle, Laura A; Calderón Díaz, Julia A; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 14/S/832; et al. (Biomed Central, 2021-01-11)
      Background Managing body weight (BW) variation is a challenge in farrow-to-finish farms implementing all-in/all-out (AIAO) production systems due to the lack of “off-site” facilities to segregate slow growing pigs (SGP). This case study investigated different approaches to managing BW variation in a farrow-to-finish commercial pig farm with a self-declared AIAO management and the possible implications for animal health. Case presentation A total of 1096 pigs (1047 pigs born within 1 week plus 49 pigs born 1 week later) were tracked until slaughter as they moved through the production stages. Piglets were individually tagged at birth and their location on the farm was recorded on a weekly basis. In total, 10.3% of pigs died during lactation. Four main cohorts of pigs were created at weaning and retrospectively identified: cohort 1 = pigs weaned at 21 days (4.5%); cohort 2 = pigs weaned at 28 days (81.0%), which was sub-divided at the end of the first nursery stage into sub-cohort 2a = pigs split at 3 weeks post-weaning (29.7%); sub-cohort 2b = pigs split at 3 weeks post-weaning from cohort 2a and split again 5 weeks post-weaning (35.5%) and sub-cohort 2c = remaining smaller size pigs from cohort 2b (10.9%); cohort 3 = pigs weaned at 35 days (2.7%) and cohort 4 = pigs weaned at 49 days (1.5%) that were later mixed with SPG, delayed pigs from other cohorts and sick/injured pigs that recovered. Four strategies to manage BW variation were identified: i) earlier weaning (cohort 1); ii) delayed weaning of SGP (cohort 3 and 4); iii) re-grading pens by BW (sub-cohorts 2a, 2b and 2c) and, iv) delayed movement of SGP to the next production stage (several pigs from all cohorts). A higher percentage of delayed pigs presented pericarditis, pleurisy and enzootic pneumonia like lesions at slaughter compared with pigs under other strategies. Conclusion A variety of management practices were implemented to minimise BW variation during the production cycle. However, several cohorts of pigs were created disrupting AIAO management. Earlier weaning should only be practiced under specific circumstances where optimal animal health and welfare are guaranteed. Delayed weaning of SGP and delaying pigs to move to the next production stage could negatively affect animal health and should be avoided.
    • Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue

      Johnston, Dayle; Earley, Bernadette; Cormican, Paul; Murray, Gerard; Kenny, David A.; Waters, Sinead M.; McGee, Mark; Kelly, Alan K; McCabe, Matthew; Department of Agriculture, Food and the Marine; et al. (Biomed Central, 2017-05-02)
      Background Bovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences. Results Leptotrichiaceae, Mycoplasma, Pasteurellaceae, and Fusobacterium were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD. Leptotrichiaceae, Fusobacterium, Mycoplasma, Trueperella and Bacteroides had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Leptotrichiaceae, Mycoplasma and Pasteurellaceae showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two Leptotrichiaceae sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences. Conclusions The microbiomes of the cranial lung lobe and mediastinal lymph node from calves which died from BRD and from clinically healthy H-F calves have been characterised. Contigs corresponding to the abundant Leptotrichiaceae OTU were sequenced and found not to be identical to any known bacterial genus. This suggests that we have identified a novel bacterial species associated with BRD.
    • Immunoglobulin G from bovine milk primes intestinal epithelial cells for increased colonization of bifidobacteria

      Morrin, Sinead T; McCarthy, Geoffrey; Kennedy, Deirdre; Marotta, Mariarosaria; Irwin, Jane A; Hickey, Rita M.; Teagasc Walsh Fellowship Programme; 2014058 (SpringerOpen, 2020-06-18)
      Abstract A bovine colostrum fraction (BCF) was recently shown to enhance the adherence of several commensal organisms to intestinal epithelial cells through modulating the epithelial cell surface. In this study, the main components of the BCF were examined to investigate the active component/s responsible for driving the changes in the intestinal cells. The adherence of various bifidobacteria to HT-29 cells was increased when the intestinal cells were pre-incubated with immunoglobulin G (IgG). Modulation of the intestinal cells by IgG was concentration dependent with 16 mg/mL IgG resulting in a 43-fold increase in the adhesion of Bifidobacterium longum NCIMB 8809 to HT-29 cells. Periodate treatment of colostral IgG prior to performing the colonization studies resulted in a reduction in the adhesion of the strain to the intestinal cells demonstrating that the glycans of IgG may be important in modulating the intestinal cells for enhanced commensal adhesion. IgG isolated from mature milk also resulted in significant increases in adhesion of the Bifidobacterium strains tested albeit at reduced levels (3.9-fold). The impact of IgG on the HT-29 cells was also visualised via scanning electron microscopy. This study builds a strong case for the inclusion of IgG ingredients sourced from cow’s milk in functional foods aimed at increasing numbers of health promoting bacteria in the human gut.
    • The impact of cattle dung pats on earthworm distribution in grazed pastures

      Bacher, M. G; Fenton, Owen; Bondi, G.; Creamer, Rachel E.; Karmarkar, M.; Schmidt, Olaf; Department of Agriculture, Food and the Marine; 13/S/468 (Biomed Central, 2018-12-19)
      Background Grazed grassland management regimes can have various effects on soil fauna. For example, effects on earthworms can be negative through compaction induced by grazing animals, or positive mediated by increases in sward productivity and cattle dung pats providing a food source. Knowledge gaps exist in relation to the behaviour of different earthworm species i.e. their movement towards and aggregation under dung pats, the legacy effects of pats and the spatial area of recruitment. The present study addressed these knowledge gaps in field experiments, over 2 years, using natural and simulated dung pats on two permanent, intensively grazed pastures in Ireland. Results Dung pats strongly affected spatial earthworm distribution, with up to four times more earthworms aggregating beneath pats, than in the control locations away from pats. In these earthworm communities comprising 11 species, temporally different aggregation and dispersal patterns were observed, including absence of individual species from control locations, but no clear successional responses. Epigeic species in general, but also certain species of the anecic and endogeic groups were aggregating under dung. Sampling after complete dung pat disappearance (27 weeks after application) suggested an absence of a dung pat legacy effect on earthworm communities. Based on species distributions, the maximum size of the recruitment area from which earthworms moved to pats was estimated to be 3.8 m2 per dung pat. Since actual grazing over 6 weeks would result in the deposition of about 300 dung pats per ha, it is estimated that a surface area of 1140 m2 or about 11% of the total grazing area can be influenced by dung pats in a given grazing period. Conclusions This study showed that the presence of dung pats in pastures creates temporary hot spots in spatial earthworm species distribution, which changes over time. The findings highlight the importance of considering dung pats, temporally and spatially, when sampling earthworms in grazed pastures. Published comparisons of grazed and cut grasslands probably reached incorrect conclusions by ignoring or deliberately avoiding dung pats. Furthermore, the observed intense aggregation of earthworms beneath dung pats suggests that earthworm functions need to be assessed separately at these hot spots.
    • Improved detection of biomarkers in cervico-vaginal mucus (CVM) from postpartum cattle

      Adnane, Mounir; Kelly, Paul M; Chapwanya, Aspinas; Meade, Kieran G; O'Farrelly, Cliona; The Algerian Ministry for High Education and Scientific Research; University of Tiaret, Algeria; Department of Agriculture, Food and the Marine, Ireland; ofarrecl-HRB-HRA_POR/2012/37; 13/S/472 (Biomed Central, 2018-09-29)
      Background In the postpartum cow, early diagnosis of uterine disease is currently problematic due to the lack of reliable, non-invasive diagnostic methods. Cervico-vaginal mucus (CVM) is an easy to collect potentially informative source of biomarkers for the diagnosis and prognosis of uterine disease in cows. Here, we report an improved method for processing CVM from postpartum dairy cows for the measurement of immune biomarkers. CVM samples were collected from the vagina using gloved hand during the first two weeks postpartum and processed with buffer alone or buffer containing different concentrations of the reducing agents recommended in standard protocols: Dithiothriotol (DTT) or N-Acetyl-L-Cysteine (NAC). Total protein was measured using the bicinchoninic acid (BCA) assay; interleukin 6 (IL-6), IL-8 and α1-acid glycoprotein (AGP) were measured by ELISA. Results We found that use of reducing agents to liquefy CVM affects protein yield and the accuracy of biomarker detection. Our improved protocol results in lower protein yields but improved detection of cytokines and chemokines. Using our modified method to measure AGP in CVM we found raised levels of AGP at seven days postpartum in CVM from cows that went on to develop endometritis. Conclusion We conclude that processing CVM without reducing agents improves detection of biomarkers that reflect uterine health in cattle. We propose that measurement of AGP in CVM during the first week postpartum may identify cows at risk of developing clinical endometritis.
    • In silico analysis highlights the frequency and diversity of type 1 lantibiotic gene clusters in genome sequenced bacteria

      Marsh, Alan J.; O'Sullivan, Orla; Ross, R Paul; Cotter, Paul D.; Hill, Colin; Science Foundation Ireland (Biomed Central, 30/11/2010)
      Background: Lantibiotics are lanthionine-containing, post-translationally modified antimicrobial peptides. These peptides have significant, but largely untapped, potential as preservatives and chemotherapeutic agents. Type 1 lantibiotics are those in which lanthionine residues are introduced into the structural peptide (LanA) through the activity of separate lanthionine dehydratase (LanB) and lanthionine synthetase (LanC) enzymes. Here we take advantage of the conserved nature of LanC enzymes to devise an in silico approach to identify potential lantibiotic-encoding gene clusters in genome sequenced bacteria. Results: In total 49 novel type 1 lantibiotic clusters were identified which unexpectedly were associated with species, genera and even phyla of bacteria which have not previously been associated with lantibiotic production. Conclusions: Multiple type 1 lantibiotic gene clusters were identified at a frequency that suggests that these antimicrobials are much more widespread than previously thought. These clusters represent a rich repository which can yield a large number of valuable novel antimicrobials and biosynthetic enzymes.
    • In silico identification of bacteriocin gene clusters in the gastrointestinal tract, based on the Human Microbiome Project’s reference genome database

      Walsh, Calum J.; Guinane, Caitriona M.; Hill, Colin; Ross, R Paul; O'Toole, Paul W.; Cotter, Paul D.; Science Foundation Ireland; SFI/11/PI/1137 (Biomed Central, 16/09/2015)
      Background The human gut microbiota comprises approximately 100 trillion microbial cells which significantly impact many aspects of human physiology - including metabolism, nutrient absorption and immune function. Disturbances in this population have been implicated in many conditions and diseases, including obesity, type-2 diabetes and inflammatory bowel disease. This suggests that targeted manipulation or shaping of the gut microbiota, by bacteriocins and other antimicrobials, has potential as a therapeutic tool for the prevention or treatment of these conditions. With this in mind, several studies have used traditional culture-dependent approaches to successfully identify bacteriocin-producers from the mammalian gut. In silico-based approaches to identify novel gene clusters are now also being utilised to take advantage of the vast amount of data currently being generated by next generation sequencing technologies. In this study, we employed an in silico screening approach to mine potential bacteriocin clusters in genome-sequenced isolates from the gastrointestinal tract (GIT). More specifically, the bacteriocin genome-mining tool BAGEL3 was used to identify potential bacteriocin producers in the genomes of the GIT subset of the Human Microbiome Project’s reference genome database. Each of the identified gene clusters were manually annotated and potential bacteriocin-associated genes were evaluated. Results We identified 74 clusters of note from 59 unique members of the Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria and Synergistetes. The most commonly identified class of bacteriocin was the >10 kDa class, formerly known as bacteriolysins, followed by lantibiotics and sactipeptides. Conclusions Multiple bacteriocin gene clusters were identified in a dataset representative of the human gut microbiota. Interestingly, many of these were associated with species and genera which are not typically associated with bacteriocin production.
    • In vitro screening of different Pseudomonas fluorescens isolates to study lytic enzyme production and growth inhibition during antagonism of Fusarium oxysporum f. sp. cumini, wilt causing pathogen of cumin

      Rathore, Ridhdhi; Vakharia, Dinesh N; Rathore, Dheeraj S; Department of Biochemistry, College of Agriculture, Junagadh Agricultural University (Springer, 2020-05-12)
      Land plants exist in close association with bacterial and fungal microbes, where some associations can be pathogenic and others can be mutualistic/beneficial. One such relation exists between host plant, Cuminum cyminum L. (Cumin) and Fusarium oxysporum f. sp. cumini (Foc), the causal pathogen of cumin wilt and Pseudomonas fluorescens (Pf), where Pf acts as a bio-agent for inhibiting Foc and promoting plant growth of cumin. In this study, antagonism by 10 different Pf isolates against Foc was studied under laboratory conditions through percent growth inhibition and biochemical mechanisms. Among these Pf isolates, Pf-5 exhibited the highest in vitro growth inhibition (82.51%). A positive correlation was observed between percent growth inhibition and specific activities of hydrolytic enzymes, chitinase, β-1, 3 glucanase, and protease, where a negative correlation was observed with cell wall degrading enzymes, cellulase and polygalacturonase. To conclude, isolate Pf-5 could be a potential biocontrol agent for Fusarium wilt disease of cumin.
    • In vivo activity of Nisin A and Nisin V against Listeria monocytogenes in mice

      Campion, Alicia; Casey, Patrick G.; Field, Des; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Programme for Research in Third-Level Institutions; Irish Research Council for Science, Engineering and Technology; Enterprise Ireland; Science Foundation Ireland (Biomed Central, 01/02/2013)
      Background: Lantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo. Results: Here we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model. More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 105 cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen. Conclusion: This analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.
    • Inhibitory activity of Lactobacillus plantarum LMG P-26358 against Listeria innocua when used as an adjunct starter in the manufacture of cheese

      Mills, Susan; Serrano, L Mariela; Griffin, Carmel; O'Connor, Paula M.; Schaad, Gwenda; Bruining, Chris; Hill, Colin; Ross, R Paul; Meijer, Wilco C (Biomed Central, 30/08/2011)
      Lactobacillus plantarum LMG P-26358 isolated from a soft French artisanal cheese produces a potent class IIa bacteriocin with 100% homology to plantaricin 423 and bacteriocidal activity against Listeria innocua and Listeria monocytogenes. The bacteriocin was found to be highly stable at temperatures as high as 100°C and pH ranges from 1-10. While this relatively narrow spectrum bacteriocin also exhibited antimicrobial activity against species of enterococci, it did not inhibit dairy starters including lactococci and lactobacilli when tested by well diffusion assay (WDA). In order to test the suitability of Lb. plantarum LMG P-26358 as an anti-listerial adjunct with nisin-producing lactococci, laboratory-scale cheeses were manufactured. Results indicated that combining Lb. plantarum LMG P-26358 (at 108 colony forming units (cfu)/ml) with a nisin producer is an effective strategy to eliminate the biological indicator strain, L. innocua. Moreover, industrial-scale cheeses also demonstrated that Lb. plantarum LMG P-26358 was much more effective than the nisin producer alone for protection against the indicator. MALDI-TOF mass spectrometry confirmed the presence of plantaricin 423 and nisin in the appropriate cheeses over an 18 week ripening period. A spray-dried fermentate of Lb. plantarum LMG P-26358 also demonstrated potent anti-listerial activity in vitro using L. innocua. Overall, the results suggest that Lb. plantarum LMG P-26358 is a suitable adjunct for use with nisin-producing cultures to improve the safety and quality of dairy products.
    • Integrated analysis of the local and systemic changes preceding the development of post-partum cytological endometritis

      Foley, Cathriona; Chapwanya, Aspinas; Callanan, John J; Whiston, Ronan; Miranda-CasoLuengo, Raúl; Lu, Junnan; Meijer, Wim G; Lynn, David J; O'Farrelly, Cliona; Meade, Kieran G (Biomed Central, 2015-10-19)
      Background The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility postpartum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the postpartum dairy cow. Methods Endometrial biopsy RNA was extracted from 15 cows at 7 and 21 days postpartum (DPP), using the Qiagen RNeasy® Plus Mini kit and quality determined using an Agilent 2100 bioanalyser. Disease status was determined by histpathology based on inflammatory cell infiltrate. RNA-seq of both mRNA and miRNA libraries were performed on an Illumina® HiSeq™ 2000. Paired reads were aligned to the bovine genome with Bowtie2 and differentially expressed genes were identified using EdgeR. Significantly over-represented Gene Ontology terms were identified using GO-seq, and pathway analysis was performed using KEGG. Quanititative real-time PCR was also performed for validation (ABI 7500 fast). Haematology was assessed using an automated ADVIA 2120 analyser. Serum proteins were evaluated by ELISA and metabolite analysis was performed using a Beckman Coulter AU 400 clinical analyser. Terminal-restriction fragment length polymorphism (T-RFLP) was used to obtain fingerprints of the microbial communities present. Results Next-generation sequencing from endometrial biopsies taken at 7 DPP identified significant induction of inflammatory gene expression in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor and NFκB pathways, 73 genes and 31 miRNAs were significantly differentially expressed between healthy cows (HC, n = 9) and cows which subsequently developed CE at 7 DPP (n = 6, FDR < 0.1). While significant differential expression of 4197 genes in the transcriptome of healthy cows between 7 and 21 DPP showed the transition from a proinflammatory to tissue profliferation and repair, only 31 genes were differentially expressed in cows with CE (FDR < 0.1), indicating the arrest of such a transition. A link betwene the dysregulated inflammatory response and the composition of the uterine microbial communities was suggested by the presence of significant differences in uterine bacterial tRFLP profiles between HC and CE groups. Furthermore, inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) expression levels were detected in plasma at 7 DPP in cows that developed CE. Conclusion Our data suggests that the IL1 and IL17 inflammatory cascade activated early postpartum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common early inflammatory profile, elevated and differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE postpartum.
    • Integrated analysis of the local and systemic changes preceding the development of post-partum cytological endometritis

      Foley, Cathriona; Chapwanya, Aspinas; Callanan, John J; Whiston, Ronan; Miranda-CasoLuengo, Raúl; Lu, Junnan; Meijer, Wim G; Lynn, David J; O'Farrelly, Cliona; Meade, Kieran G (Biomed Central, 2015-10-19)
      Background The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility postpartum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the postpartum dairy cow. Methods Endometrial biopsy RNA was extracted from 15 cows at 7 and 21 days postpartum (DPP), using the Qiagen RNeasy® Plus Mini kit and quality determined using an Agilent 2100 bioanalyser. Disease status was determined by histpathology based on inflammatory cell infiltrate. RNA-seq of both mRNA and miRNA libraries were performed on an Illumina® HiSeq™ 2000. Paired reads were aligned to the bovine genome with Bowtie2 and differentially expressed genes were identified using EdgeR. Significantly over-represented Gene Ontology terms were identified using GO-seq, and pathway analysis was performed using KEGG. Quanititative real-time PCR was also performed for validation (ABI 7500 fast). Haematology was assessed using an automated ADVIA 2120 analyser. Serum proteins were evaluated by ELISA and metabolite analysis was performed using a Beckman Coulter AU 400 clinical analyser. Terminal-restriction fragment length polymorphism (T-RFLP) was used to obtain fingerprints of the microbial communities present. Results Next-generation sequencing from endometrial biopsies taken at 7 DPP identified significant induction of inflammatory gene expression in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor and NFκB pathways, 73 genes and 31 miRNAs were significantly differentially expressed between healthy cows (HC, n = 9) and cows which subsequently developed CE at 7 DPP (n = 6, FDR < 0.1). While significant differential expression of 4197 genes in the transcriptome of healthy cows between 7 and 21 DPP showed the transition from a proinflammatory to tissue profliferation and repair, only 31 genes were differentially expressed in cows with CE (FDR < 0.1), indicating the arrest of such a transition. A link betwene the dysregulated inflammatory response and the composition of the uterine microbial communities was suggested by the presence of significant differences in uterine bacterial tRFLP profiles between HC and CE groups. Furthermore, inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) expression levels were detected in plasma at 7 DPP in cows that developed CE. Conclusion Our data suggests that the IL1 and IL17 inflammatory cascade activated early postpartum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common early inflammatory profile, elevated and differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE postpartum.
    • Integrated analysis of the local and systemic changes preceding the development of post-partum cytological endometritis

      Foley, Cathriona; Chapwanya, Aspinas; Callanan, John J; Whiston, Ronan; Miranda-CasoLuengo, Raúl; Lu, Junnan; Meijer, Wim G; Lynn, David J; O'Farrelly, Cliona; Meade, Kieran G (Biomed Central, 2015-10-19)
      Background The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility postpartum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the postpartum dairy cow. Methods Endometrial biopsy RNA was extracted from 15 cows at 7 and 21 days postpartum (DPP), using the Qiagen RNeasy® Plus Mini kit and quality determined using an Agilent 2100 bioanalyser. Disease status was determined by histpathology based on inflammatory cell infiltrate. RNA-seq of both mRNA and miRNA libraries were performed on an Illumina® HiSeq™ 2000. Paired reads were aligned to the bovine genome with Bowtie2 and differentially expressed genes were identified using EdgeR. Significantly over-represented Gene Ontology terms were identified using GO-seq, and pathway analysis was performed using KEGG. Quanititative real-time PCR was also performed for validation (ABI 7500 fast). Haematology was assessed using an automated ADVIA 2120 analyser. Serum proteins were evaluated by ELISA and metabolite analysis was performed using a Beckman Coulter AU 400 clinical analyser. Terminal-restriction fragment length polymorphism (T-RFLP) was used to obtain fingerprints of the microbial communities present. Results Next-generation sequencing from endometrial biopsies taken at 7 DPP identified significant induction of inflammatory gene expression in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor and NFκB pathways, 73 genes and 31 miRNAs were significantly differentially expressed between healthy cows (HC, n = 9) and cows which subsequently developed CE at 7 DPP (n = 6, FDR < 0.1). While significant differential expression of 4197 genes in the transcriptome of healthy cows between 7 and 21 DPP showed the transition from a proinflammatory to tissue profliferation and repair, only 31 genes were differentially expressed in cows with CE (FDR < 0.1), indicating the arrest of such a transition. A link betwene the dysregulated inflammatory response and the composition of the uterine microbial communities was suggested by the presence of significant differences in uterine bacterial tRFLP profiles between HC and CE groups. Furthermore, inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) expression levels were detected in plasma at 7 DPP in cows that developed CE. Conclusion Our data suggests that the IL1 and IL17 inflammatory cascade activated early postpartum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common early inflammatory profile, elevated and differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE postpartum.
    • Inter- and intra-reproducibility of genotypes from sheep technical replicates on Illumina and Affymetrix platforms

      Berry, Donagh; O'Brien, Aine; Wall, E.; McDermott, Kevin; Randles, Shane; Flynn, Paul; Park, Stephen D. E.; Grose, Jenny; Weld, Rebecca; McHugh, Noirin; et al. (Biomed Central, 2016-11-10)
      Background Accurate genomic analyses are predicated upon access to accurate genotype input data. The objective of this study was to quantify the reproducibility of genotype data that are generated from the same genotype platform and from different genotyping platforms. Methods Genotypes based on 51,121 single nucleotide polymorphisms (SNPs) for 84 animals that were each genotyped on Illumina and Affymetrix platforms and for another 25 animals that were each genotyped twice on the same Illumina platform were compared. Genotypes based on 11,323 SNPs for an additional 21 animals that were genotyped on two different Illumina platforms by two different service providers were also compared. Reproducibility of the results was measured as the correlation between allele counts and as genotype and allele concordance rates. Results A mean within-animal correlation of 0.9996 was found between allele counts in the 25 duplicate samples that were genotyped on the same Illumina platform and varied from 0.9963 to 1.0000 per animal. The mean (minimum, maximum) genotype and allele concordance rates per animal between the 25 duplicate samples were equal to 0.9996 (0.9968, 1.0000) and 0.9993 (0.9937, 1.0000), respectively. The concordance rate between the two different Illumina platforms was also near 1. A mean within-animal correlation of 0.9738 was found between genotypes that were generated on the Illumina and Affymetrix platforms and varied from 0.9505 to 0.9812 per animal. The mean (minimum, maximum) within-animal genotype and allele concordance rates between the Illumina and Affymetrix platforms were equal to 0.9711 (0.9418, 0.9798) and 0.9845 (0.9695, 0.9889), respectively. The genotype concordance rate across all genotypes increased from 0.9711 to 0.9949 when the SNPs used were restricted to those with three high-resolution genotype clusters which represented 75.2% of the called genotypes. Conclusions and implications Our results suggest that, regardless of the genotype platform or service provider, high genotype concordance rates are achieved especially if they are restricted to high-quality extracted DNA and SNPs that result in high-quality genotypes.
    • Investigation of bovine abortion and stillbirth/perinatal mortality - similar diagnostic challenges, different approaches

      Mee, John F (Biomed Central, 2020-09-04)
      Abstract This pracademic paper reviews current bovine foetopathy (abortion and stillbirth) case definitions, reporting and triage, and causes and time-of-death and proposes veterinary practitioner-focused investigative standard operating procedures (SOPs). Issues of under- and over-triage and intra-institutional SOP harmonisation are also discussed. It is proposed that an ‘observable abortion’ (120–260 days of gestation) is a more practitioner-friendly definition of abortion for reporting and benchmarking purposes and that the term ‘peristillbirth’ can replace stillbirth and perinatal mortality. Diagnosis of bovine foetopathy involves an investigative triad of the farmer, veterinary practitioner and the veterinary diagnostic laboratory. However, the poor sensitivity of abortion reporting undermines the value of currently adopted scanning/passive surveillance; parallel active surveillance/sentinel herd models should also be employed. The approach to abortion investigation differs from that of peristillbirth. The former should include collecting a herd and case history, examination and sampling of dam and cohorts and sampling of the foetus and placenta. A sample selection decision tree is provided to assist test selection. In peristillbirths, non-infectious and periparturient causes-of-death are more important hence the anamnesis must focus on peristillbirth risk factors and calving management. The foetopsy, while including the sampling menu appropriate to aborted foetuses, must also include a detailed internal and external examination of the carcass for lesions indicative of periparturient causes-of-death. In addition, for aborted foetuses the time-of-death is not important as the foetus is generally not viable; however, for the peristillbirth the time-of-death is critical as it provides useful information for the farmer to address modifiable risk factors and to alter their perinatal management. Reporting of the ultimate cause-of-death is more useful to prevent future abortions and peristillbirths though the proximate cause-of-death is often reported in the absence of a complete clinical anamnesis. Finally, the common reasons for diagnosis not reached (DNR) and the limitations of current investigative approaches are discussed.
    • Investigation of molecular mechanisms underlying tetracycline resistance in thermophilic Campylobacter spp. suggests that previous reports of tet(A)-mediated resistance in these bacteria are premature

      Lynch, Caoimhe; Hawkins, Kayleigh; Lynch, Helen; Egan, John; Bolton, Declan J.; Coffey, Aidan; Lucey, Brigid; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 15/F/641; et al. (Biomed Central, 2019-11-09)
      The true prevalence of tet(A), which codes for a tetracycline efflux pump, in thermophilic Camplyobacter spp. requires clarification after reports emerged in Iran (2014) and Kenya (2016) of the novel detection of tet(A) in Campylobacter. During our investigation of antibiotic resistance mechanisms in a sample of Irish thermophilic Campylobacter broiler isolates, it was determined that 100% of tetracycline-resistant isolates (n = 119) harboured tet(O). Accessory tetracycline-resistance mechanisms were considered as tetracycline minimum inhibitory concentrations ranged from 4 to ≥ 64 mg/L. Primers previously reported for the detection of tet(A) in Campylobacter failed to produce an amplicon using a positive control strain (Escherichia coli K12 SK1592 containing the pBR322 plasmid) and a selection of Campylobacter isolates. Accordingly, we designed new tet(A)-targeting primers on SnapGene2.3.2 that successfully generated a 407 bp product from the positive control strain only. Further in silico analysis using BLASTn and SnapGene2.3.2 revealed that previously reported Campylobacter tet(A) sequences deposited on GenBank shared 100% homology with Campylobacter tet(O). We postulate that this gave rise to the erroneous report of a high tet(A) prevalence among a pool of Kenyan broiler Campylobacter isolates that were tested using primers designed based on these apparent tet(A) sequences. In conclusion, further work would be required to determine whether the homology between tet(A) potentially present in Campylobacter and known tet(A) genes would be sufficient to allow amplification using the primers designed in our study. Finally, the existence of tet(A) in thermophilic Campylobacter spp. remains to be demonstrated.