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This collection contains articles published in the Open Access Biomed Central journals by Teagasc authors.

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• #### Blood and faecal biomarkers to assess dietary energy, protein and amino acid efficiency of utilization by growing and finishing pigs

Background Diet evaluation and optimization is a slow and expensive process and it is not possible to do it at a farm level. This study aimed to use the blood serum metabolite (BSM) and faecal volatile fatty acid (VFA) profiles as potential biomarkers to identify changes in protein, amino acid and energy dietary content in growing and finishing pig diets at farm level. Results Two studies were conducted. The first study (S1) included 20 pens of 11 pigs (87.0 ± 4.10 kg; 18 weeks old) assigned to 5 diets: control (C1), high or low crude protein (HP1 and LP1, respectively), and high or low net energy (HE1 and LE1, respectively). The second study (S2) included 28 pens of 11 pigs (41.3 ± 2.60 kg; 12 weeks old) assigned to 7 diets: control (C2), high or low crude protein (HP2 and LP2, respectively), high or low amino acid (HA2 and LA2, respectively), and high or low net energy (HE2 and LE2, respectively). Pigs were followed for 10 (S1) and 20 (S2) days, and blood and faecal samples were collected at 20 (S1) and 14 (S2) weeks of age. Data were analysed using general linear models and receiver operating characteristic curve analysis. Urea nitrogen showed the best results as a biomarker. Urea nitrogen was higher in pigs fed high protein diets, HP1 (13.6 ± 0.95 mg/dL) and HP2 (11.6 ± 0.61), compared to those fed low protein diets, LP1 (6.0 ± 0.95) and LP2 (5.2 ± 0.61; P < 0.001), showing good discrimination ability (Area under the curve (AUC) = 98.4 and 100%, respectively). These differences were not observed between diets LA2 (6.5 ± 0.61) and HA2 (8.7 ± 0.61; P > 0.05; AUC = 71.9%), which were formulated based on the ideal protein profile but with no excess of protein. Creatinine, triglycerides, branched-chain fatty acids, albumin, propionic acid, and cholesterol showed differences between at least 2 diets but only in one of the studies. Conclusions Urea nitrogen showed high accuracy to detect excess of crude protein in growing and finishing pig diets. Other biomarkers like BCFA showed promising results and need to be further studied.
• #### Modelling transmission of Mycobacterium avium subspecies paratuberculosis between Irish dairy cattle herds

Bovine paratuberculosis is an endemic disease caused by Mycobacterium avium subspecies paratuberculosis (Map). Map is mainly transmitted between herds through movement of infected but undetected animals. Our objective was to investigate the effect of observed herd characteristics on Map spread on a national scale in Ireland. Herd characteristics included herd size, number of breeding bulls introduced, number of animals purchased and sold, and number of herds the focal herd purchases from and sells to. We used these characteristics to classify herds in accordance with their probability of becoming infected and of spreading infection to other herds. A stochastic individual-based model was used to represent herd demography and Map infection dynamics of each dairy cattle herd in Ireland. Data on herd size and composition, as well as birth, death, and culling events were used to characterize herd demography. Herds were connected with each other through observed animal trade movements. Data consisted of 13 353 herds, with 4 494 768 dairy female animals, and 72 991 breeding bulls. We showed that the probability of an infected animal being introduced into the herd increases both with an increasing number of animals that enter a herd via trade and number of herds from which animals are sourced. Herds that both buy and sell a lot of animals pose the highest infection risk to other herds and could therefore play an important role in Map spread between herds.
• #### Lameness prevalence and management practices on Irish pasture-based dairy farms

Background Lameness is a painful disease, which negatively impacts dairy cow production and welfare. The aim of this observational study was to determine herd lameness prevalence, describe current lameness management practices and identify the presence of established risk factors for lameness on Irish pasture-based dairy farms. Farms were visited once during grazing (99 farms) and again during housing (85 farms). Lameness scoring was carried out at each visit (AHDB 0–3 scale); cows were classified as lame if they scored two or three. Farm management practices and infrastructure characteristics were evaluated via farmer questionnaires and direct measurements of farm infrastructure. Results Median herd-level lameness prevalence was 7.9% (interquartile range = 5.6 – 13.0) during grazing and 9.1% (interquartile range = 4.9 – 12.0) during housing; 10.9% of cows were lame at a single visit and 3.5% were lame at both visits (chronically lame or had a repeat episode of lameness). Fifty-seven percent of farmers were not familiar with lameness scoring and only one farm carried out lameness scoring. Only 22% of farmers kept records of lame cows detected, and 15% had a lameness herd health plan. Twenty-eight percent of farmers waited more than 48 h to treat a lame cow, and 21% waited for more than one cow to be identified as lame before treating. Six percent of farmers carried out routine trimming and 31% regularly footbathed (> 12 times per year). Twelve percent put severely lame cows in a closer paddock and 8% stated that they used pain relief to treat severely lame cows. Over 50% of farms had at least one cow track measurement that was classified as rough or very rough, and cow tracks were commonly narrow for the herd size. On 6% of farms, all cubicle beds were bare concrete (no matting or bedding) and on a further 6% of farms, there was a combination of cubicles with and without matting or bedding. On 56% of farms, all pens contained less than 1.1 cubicles per cow and on 28% of farms, a proportion of pens contained less than 1.1 cubicles per cow. Conclusions Overall, this study identified infrastructure and management practices which could be improved upon. The comparatively low lameness prevalence demonstrated, compared to fully housed systems, also highlights the benefits of a pasture-based system for animal welfare; however, there remains scope for improvement.
• #### Factors affecting retention of veterinary practitioners in Ireland: a cross-sectional study with a focus on clinical practice

Background Retention of veterinary practitioners has arisen as a significant problem in recent years in Ireland. No prior Irish peer-reviewed publications have addressed this problem. An online questionnaire was available through social media and via email to Irish vets from January to November 2019. The aim of this survey was to ascertain the factors contributing to the problem of vet retention in Ireland. Results A total of 370 eligible responses were received. The median age of respondents was 31 and the gender balance was 250 females (68%) to 118 males (32%). The majority of respondents worked in clinical practice 322 (89%), with 138 (42.8%) in mixed practice, 115 (35.7%) in small animal practice, 49 (15.2%) solely with farm animals and 20 (6.2%) in equine practice. Fifty-four percent of respondents described themselves as likely to be leaving their current job within two years and 32.8% as being likely to leave the profession. In total, 44 variables were assessed by univariate analysis and 27 variables were significantly (P < 0.05) associated with the likelihood of a respondent leaving their current job within 2 years (LCJ2), as a proxy measure of the problem of retention. All variables significant on univariate analysis at P < 0.2 were included in a multivariable logistic regression model. Factors associated with LCJ2 included satisfaction with work-life balance (Odds Ratio (OR) 0.33); satisfaction with working hours (OR 0.2); number of years qualified (OR 0.91); position as a practice owner/partner/director (OR 0.15); and log10salary (OR 0.03). Four variables were retained in a separate multivariable linear regression model as significant (P < 0.05) predictors of log10salary. Log10salary increased with years qualified. Males had an increased salary compared to females irrespective of years qualified. Part-time employees, vets on maternity leave or postgraduate vets had a lower log10salary. Compared to veterinary employees, self-employed or locum vets had a higher log10salary. Conclusions Veterinary employers should consider salary, working hours and the facilitation of a good work-life balance in order to successfully retain veterinary employees. The significant difference in salaries currently offered to male and female vets, and the high percentage of respondents considering leaving the profession, are important findings and warrant further investigation.
• #### Mycobacterium avium subspecies paratuberculosis infection in cattle – a review in the context of seasonal pasture-based dairy herds

Johne’s disease is an infectious disease affecting cattle, other ruminants and non-ruminant wildlife worldwide, caused by Mycobacterium avium subspecies paratuberculosis (MAP). This review provides an up-to-date concise overview of the pathogenesis of MAP, the significance of Johne’s disease in cattle and the use of diagnostic testing at both animal and herd level in the context of seasonal pasture-based herds. While MAP can only replicate intracellularly, the bacterium is sufficiently robust to survive for months in the environment. Transmission of MAP is mostly via the faecal-oral route, however in-utero transmission in also possible. The bacteria evade the immune system by persisting in macrophages in the small intestine submucosa, with this latent stage of infection lasting, in most cases, for at least two years before bacterial shedding and clinical signs begin. The slowly progressive nature of MAP infection, poor performance of diagnostic tests and management systems that expose susceptible calves to infection make control of Johne’s disease challenging, particularly in seasonal calving herds. Testing of individual animals provides little assurance for farmers and vets due to the poor sensitivity and, in the case of ELISA, imperfect specificity of the available tests. Repeated herd-level testing is utilised by the IJCP to detect infected herds, identify high risk animals, and provide increasing confidence that test-negative herds are free of infection. The IJCP aims to control the spread of Johne’s disease in cattle in Ireland, in order to protect non-infected herds, limit the economic and animal health impact of the disease, improve calf health and reassure markets of Johne’s disease control in Ireland.
• #### Antimicrobial resistance in Escherichia coli isolated from on-farm and conventional hatching broiler farms in Ireland

Background On-farm hatching (OH) systems are becoming more common in broiler production. Hatching conditions differ from conventional farms as OH chicks avoid exposure to handling, transport, post-hatch water and feed deprivation. In contrast, chicks in conventional hatching conditions (CH) are exposed to standard hatchery procedures and transported post hatching. The objectives of this pilot study were to investigate the prevalence and frequency of Escherichia coli resistant to antimicrobials, including presumptive ESBL/AmpC-producing E. coli, isolated from environmental and faecal samples from OH versus CH hatching systems, and to investigate the presence of ESBL/AmpC-producing encoding genes. Results Environmental samples were collected from one flock in 10 poultry farms (5 OH farms, 5 CH farms) on day 0 post disinfection of the facilities to assess hygiene standards. On D10 and D21 post egg/chick arrival onto the farm, samples of faeces, boot swabs and water drinker lines were collected. E. coli were isolated on MacConkey agar (MC) and MacConkey supplemented with cefotaxime (MC+). Few E. coli were detected on D0. However, on D10 and D21 E. coli isolates were recovered from faeces and boot swabs. Water samples had minimal contamination. In this study, 100% of cefotaxime resistant E. coli isolates (n=33) detected on selective media and 44% of E. coli isolates (84/192) detected on nonselective media were multidrug resistant (MDR). The antimicrobial resistance (AMR) genotype for the 15 ESBL/AmpC producing isolates was determined using multiplex PCR. Six of these were selected for Sanger sequencing of which two were positive for blaCMY-2, two for blaTEM-1 and two were positive for both genes. Conclusions There was no difference in E. coli isolation rates or prevalence of AMR found between the OH versus CH systems, suggesting that the OH system may not be an additional risk of resistant E. coli dissemination to broilers compared to the CH systems. The frequency of β-lactam resistant E. coli in boot swab and faeces samples across both OH (24/33 (73%)) and CH (9/33 (27%)) systems may indicate that hatcheries could be a reservoir and major contributor to the transmission of AMR bacteria to flocks after entry to the rearing farms.
• #### Sperm DNA methylation patterns at discrete CpGs and genes involved in embryonic development are related to bull fertility

Background Despite a multifactorial approach being taken for the evaluation of bull semen quality in many animal breeding centres worldwide, reliable prediction of bull fertility is still a challenge. Recently, attention has turned to molecular mechanisms, which could uncover potential biomarkers of fertility. One of these mechanisms is DNA methylation, which together with other epigenetic mechanisms is essential for the fertilising sperm to drive normal embryo development and establish a viable pregnancy. In this study, we hypothesised that bull sperm DNA methylation patterns are related to bull fertility. We therefore investigated DNA methylation patterns from bulls used in artificial insemination with contrasting fertility scores. Results The DNA methylation patterns were obtained by reduced representative bisulphite sequencing from 10 high-fertility bulls and 10 low-fertility bulls, having average fertility scores of − 6.6 and + 6.5%, respectively (mean of the population was zero). Hierarchical clustering analysis did not distinguish bulls based on fertility but did highlight individual differences. Despite this, using stringent criteria (DNA methylation difference ≥ 35% and a q-value < 0.001), we identified 661 differently methylated cytosines (DMCs). DMCs were preferentially located in intergenic regions, introns, gene downstream regions, repetitive elements, open sea, shores and shelves of CpG islands. We also identified 10 differently methylated regions, covered by 7 unique genes (SFRP1, STXBP4, BCR, PSMG4, ARSG, ATP11A, RXRA), which are involved in spermatogenesis and early embryonic development. Conclusion This study demonstrated that at specific CpG sites, sperm DNA methylation status is related to bull fertility, and identified seven differently methylated genes in sperm of subfertile bulls that may lead to altered gene expression and potentially influence embryo development.
• #### Ewe breed differences in the cervical transcriptome at the follicular phase of a synchronised oestrous cycle

Background Cervical artificial insemination (AI) with frozen-thawed semen results in unacceptably low pregnancy rates internationally. The exception is in Norway, where vaginal deposition of frozen-thawed semen to a natural oestrous routinely yields pregnancy rates in excess of 70%. Previous studies by our group has demonstrated that this is due to differences in cervical sperm transport. However, a potentially important contributory factor is that ewes are inseminated to a natural oestrous in Norway but to a synchronised oestrous across most of the rest of the world. In this study, we interrogated the gene expression of the sheep cervix of four ewe breeds with known differences in pregnancy rates following cervical AI using frozen-thawed semen under the effect of exogenous hormones to synchronise the oestrous cycle. These four ewe breeds (n = 8 to 11 ewes per breed) are from two countries: Ireland (Belclare and Suffolk; medium and low fertility, respectively) and Norway (Norwegian White Sheep (NWS) and Fur; both with high fertility compared to the Irish ewe breeds). Results RNA extracted from cervical biopsies collected from these breeds was analysed by RNA-sequencing and differential gene expression analysis. Using the low-fertility Suffolk breed as a reference level; 27, 1827 and 2641 genes were differentially expressed in Belclare, Fur and NWS ewes, respectively (P <  0.05 and FC > 1.5). Gene ontology (GO) analysis revealed that Fur and NWS had an up-regulation of enriched pathways involved in muscle contraction and development compared to Suffolk. However, there was a down-regulation of the immune response pathway in NWS compared to Suffolk. In addition, GO analysis showed similar expression patterns involved in muscle contraction, extracellular matrix (ECM) development and cell-cell junction in both Norwegian ewe breeds, which differed to the Irish ewe breeds. Conclusions This novel study has identified a number of conserved and breed-specific biological processes under the effect of oestrous synchronisation that may impact cervical sperm transport during the follicular phase of the reproductive cycle.
• #### Engaging with selective dry cow therapy: understanding the barriers and facilitators perceived by Irish farmers

Background Selective dry cow therapy (SDCT) is widely promoted in dairy farming as a method to reduce antimicrobial usage. New legislation introduced by the European Union will restrict and regulate the prophylactic and metaphylactic use of antibiotics from January 2022. Blanket dry cow therapy continues to be a practice engaged in by many farmers in Ireland and for many of these farmers, moving towards SDCT would require a significant infrastructural, behavioural and/or cultural change on their farm. Existing research has reported the important need to understand farmers’ motivations to initiate any substantial behaviour change. However, it is currently unknown what farmers know, think and believe about SDCT in Ireland. The aim of this study was to use qualitative methods to explore what barriers and facilitators farmers perceived to exist with SDCT and explore if they had chosen to implement SDCT after voluntarily participating in a funded dry cow consult with a trained veterinarian, with the objective of maximising the dry period udder health performance and moving safely to SDCT. Results In this study, 19 farmers were contacted, and telephone interviews were conducted regarding farmers’ beliefs about the consequences of SDCT. Audio recordings were professionally transcribed verbatim and analysed qualitatively using an inductive thematic analysis. The analysis identified 6 barriers and 6 facilitators to implementing SDCT. A significant fear of increasing mastitis incidence was evident that caused reluctance towards SDCT and reliance on antibiotics. Mixed perceptions on SDCT, infrastructure limitations, a perceived lack of preventive advice as well as peer influence were presented as barriers to SDCT. Farmers can build confidence when a graded approach to SDCT is implemented, which could help overcome the fear of SDCT and reliance on antibiotics. Regulatory pressure, high standards of farm hygiene and use of targeted veterinary consults were found to facilitate SDCT. Education was suggested to motivate farmers in the future uptake of SDCT. Despite cited negative influences, peer influence can be utilised to encourage the farming community. Conclusions This study prioritises areas to facilitate the major behaviour change required as a dairy industry in order to move from blanket dry cow therapy to SDCT.
• #### Horn bud size of dairy-bred and suckler-bred calves at time of disbudding

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.
• #### Genetic and economic benefits of foreign sire contributions to a domestic sheep industry; including an Ireland-New Zealand case study

Background Importation of foreign genetics is a widely used genetic improvement strategy. However, even if the foreign genetic merit is currently greater than the domestic genetic merit, differences in foreign and domestic trends mean that the long-term competitiveness of an importation strategy cannot be guaranteed. Gene flow models are used to quantify the impact that a specific subpopulation, such as foreign genetics, can have over time on the genetic or economic benefit of a domestic industry. Methods We used a deterministic recursive gene flow model to predict the commercial performance of lambs born across various subpopulations. Numerous breeding strategies were evaluated by varying market share, proportions of rams selected for mating, genetic trend, superiority of foreign genetics over domestic genetics and frequency of importation. Specifically, an Ireland-New Zealand case study was simulated to quantify the potential gain that could be made by using foreign sire contributions (New Zealand) in a domestic sheep industry (Ireland). Results Genetic and economic gains were generated from alternative breeding strategies. The ‘base scenario’ (i.e. representing the current industry) predicted an average genetic merit value of €2.51 for lambs born and an annualised cumulative benefit of €45 million (m) after 20 years. Maximum genetic (€9.45 for lambs born) and economic (annualised cumulative benefit of €180 m after 20 years) benefits were achieved by implementing the ‘PRO-intense-market scenario’ which involved shifting market share away from conservative domestic breeders and reducing the proportion of rams that were selected for mating by progressive domestic breeders from the top 40% to the top 20%, without the use of any foreign genetics. The ‘PROFOR scenario’, which considered the use of foreign and progressive domestic genetics, predicted an average genetic merit value of €7.37 for lambs born and an annualised cumulative benefit of €144 m, after 20 years. Conclusions Our results demonstrate that there is opportunity for a domestic industry to increase industry benefits without the use of foreign genetics but through an attempt to shift the market share away from conservative domestic breeders towards progressive domestic breeders. However, the importation and use of progressive foreign genetics may be an effective method to trigger a change in behaviour of conservative domestic breeders towards the use of progressive genetics.
• #### MAP, Johne’s disease and the microbiome; current knowledge and future considerations

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne’s disease in ruminants. As an infectious disease that causes reduced milk yields, effects fertility and, eventually, the loss of the animal, it is a huge financial burden for associated industries. Efforts to control MAP infection and Johne’s disease are complicated due to difficulties of diagnosis in the early stages of infection and challenges relating to the specificity and sensitivity of current testing methods. The methods that are available contribute to widely used test and cull strategies, vaccination programmes also in place in some countries. Next generation sequencing technologies have opened up new avenues for the discovery of novel biomarkers for disease prediction within MAP genomes and within ruminant microbiomes. Controlling Johne’s disease in herds can lead to improved animal health and welfare, in turn leading to increased productivity. With current climate change bills, such as the European Green Deal, targeting livestock production systems for more sustainable practices, managing animal health is now more important than ever before. This review provides an overview of the current knowledge on genomics and detection of MAP as it pertains to Johne’s disease.
• #### Why do Irish pig farmers use medications? Barriers for effective reduction of antimicrobials in Irish pig production

Abstract Background In addressing the threat of antimicrobial resistance, it is critical to understand the barriers to the uptake of strategies for the reduction of antimicrobial use (AMU) in the pig industry. In several EU countries, factors such as education level, habits and social pressures are recognised as affecting farmers’ decision-making process in relation to AMU. However, there is a lack of information on the Irish scenario. The aim of this study was to investigate pig farmers’ perspectives and their behaviour towards AMU to identify potential barriers to effectively reduce AMU in Irish pig production. We conducted face-to-face semi-structured interviews with 30 pig farmers, 5 pig veterinarians and 4 focus groups of pig farm personnel. We employed qualitative analyses to explore the objective of the study. Results Qualitative analysis revealed six convergent themes as potential barriers: perceptions about the need for AMU on farm, concept of animal welfare and associated management practices, legislation, culture, economics and standards of communication/type of advice-network. Overall, pig farmers believed that there is poor communication between stakeholders (i.e. farmers, vets and advisors) and a lack of reliable people to approach for advice. They considered themselves as operating responsibly in terms of AMU compared to their national and international colleagues and expressed the importance of a so-called ‘Irish solution’ to the problem of AMU because it was associated with what ‘has always been done’ and was therefore considered reliable and safe. Conclusions Barriers and challenges were in line with those identified in other EU countries highlighting similarities in behavioural and attitudinal patterns among pig farmers. Overall, farmers appeared to be more likely to rely on previous experiences or to wait for an imposed change (e.g. legislation) instead of taking personal action. Thus, considerable behavioural and attitudinal changes are needed to adopt a more responsible AMU in Irish pig production and to develop effective intervention strategies.
• #### Molecular-genetic characterization of human parvovirus B19 prevalent in Kerala State, India

Background Human parvovirus B19V is a DNA virus, and a member of the family Parvoviridae, that causes various clinical manifestations, from asymptomatic to persistent infection that is associated with different autoimmune diseases. The parvovirus B19 evolves with a very high mutation rate that is closer to those of existing RNA viruses. Globally circulating B19V is currently classified into three genotypes, but their distribution is not spatially and temporally correlated. Except for a few recent reports on B19V entry into the human host and its genetic diversity, there is a lack of sufficient studies on this virus from distinct geographical locations and no clear understanding of its evolution has been documented. Methods To better understand the evolution of the Human parvo B19V virus from India's southern part, a geographically distinct location with no reports of B19V genomes, we have screened for B19V in 456 suspected cases using VP1/2 surface marker genes, and its characteristics were studied in detail. Amongst 456 clinically suspected B19V samples, 7.2% (33/456) were found positive by nested PCR (nPCR) were subsequently validated by real-time PCR, Sanger sequencing, and metagenome analysis. Results Human parvovirus B19 infection was shown among 33 of 456 patients when tested by nPCR; 30 among these were also positive by qPCR and were subsequently confirmed by sequencing 75% nPCR positive samples and 76% qPCR positive samples were from patients with age. ≥ 50 years respectively (Additional file 1: Table S1). The complete VP1/2 gene assembly from the South Indian strain showed three novel mutations (T122A, V128I, I283V), which might significantly impact the stability and virulence of the B19V virus circulating in this part of the world. These mutations might be crucial for its adaptive evolutionary strategies facilitating the spread and infectivity potential of the virus. In maximum likelihood phylogeny of VP1/2 sequences, the South Indian B19V strain forms a separate clade closer to the existing genotype two strains circulating worldwide. Conclusion Our study contributes to a better understanding of the human parvovirus's genetic and evolutionary characteristics in South India. Also, it highlights the possibility that a positive selection pressure acting on VP1/2 could increase the survival and replication capabilities of the viruses.
• #### The effect of disinfectant ingredients on teat skin bacteria associated with mastitis in Irish dairy herds

Background Teat disinfection is an important step in the control of mastitis within a dairy herd. The objective of this study was to evaluate the effectiveness of 96 commercially available teat disinfectant products in Ireland against bacterial isolates on teat skin. Teat disinfection products were applied to the teats of seventeen Holstein–Friesian cows. A split-udder model was used where one cow received two different teat disinfection products on each day. A composite swab sample was taken of the left teats and the right teats before and after teat disinfectant application. Swab samples were plated onto 3 different selective agars to enumerate bacterial counts of streptococcal, staphylococcal and coliform isolates. Results Streptococcal isolates were the most prominent bacterial group recovered on teat swabs taken before the application of a teat disinfection product (55.0%), followed by staphylococcal isolates (41.3%) and coliform isolates (3.7%). Products were reclassified by active ingredients (n = 9) for analysis. These ingredient groups included; chlorhexidine, chlorine dioxide, diamine, iodine, iodine and lactic acid, lactic acid, lactic acid and chlorhexidine, lactic acid and hydrogen peroxide, and lactic acid and salicylic acid. The ingredient group, chlorine dioxide, resulted in comparable reductions to the iodine group for streptococcal isolates. The ingredient group, iodine combined with lactic acid, resulted in the greatest reduction of staphylococcal isolates. When observing products individually, a product containing 1.6% w/w lactic acid combined with hydrogen peroxide was the most effective at reducing streptococcal isolates on the teat skin, whereas a product containing lactic acid combined with 0.6% w/w chlorhexidine was the most effective against staphylococcal isolates. Minor differences were observed regarding the relationship between effectiveness and active ingredient concentration between products. Conclusions This study suggests that some teat disinfectant products achieve a higher reduction in bacterial levels against different specific bacterial groups on teat skin than other products. Therefore, when choosing a teat disinfectant product, the bacteria in the dairy herds’ environment should be considered. Further studies are necessary to evaluate products efficacy against new IMIs and any possible effects on teat skin condition.
• #### Effect of a single, oral administration of selenitetriglycerides, at two dose rates, on blood selenium status and haematological and biochemical parameters in Holstein-Friesian calves

Background Selenitetriglycerides are biologically active, organic forms of selenium formed as a result of the modification of selenic acid and sunflower oil. Studies in rats have shown that they are well absorbed and of low toxicity. There are no published studies on selenitetriglycerides supplementation in calves. Results In this study, selenitetriglycerides were administered once orally on the 2nd day of life at a dose of 0.5 or 1 mg Se/kg body weight to each of six Holstein-Friesian calves while six control calves were not supplemented. Blood for determination of selenium concentration, glutathione peroxidase activity, haematological parameters, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase activities and glucose, total protein, albumin, triglycerides, cholesterol, urea, and creatinine concentration was collected before supplementation (day 0) and 1, 2, 5, 10 and 14 days after supplementation. Selenitetriglycerides administration increased (P < 0.01) serum selenium concentration in supplemented calves as early as day1, from a mean of 63.4 to 184.22 µg/l in calves receiving selenium at a dose of 0.5 mg/kg BW, and from 63.17 to 200.33 µg/l in calves receiving 1 mg/kg. Serum selenium concentrations remained significantly higher compared to the control group throughout the experiment. Glutathione peroxidase activity was higher in supplemented than control calves, significantly so in animals receiving the 1 mg/kg dose of Se on the 10th and 14th days (P < 0.05). There were no significant differences in the haematological and biochemical parameters between the groups. Conclusions This experiment showed that supplementation with selenitetriglycerides could significantly improve blood selenium status in calves without adverse effects on haematological or biochemical parameters. These findings are essential prerequisites for future studies on selenitetriglycerides supplementation to manage clinical selenium deficiency in calves.
• #### Isolation and characterisation of ΦcrAss002, a crAss-like phage from the human gut that infects Bacteroides xylanisolvens

Background The gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual’s core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible. Results Faeces from a healthy donor with high levels of crAss-like phage was used to initiate a faecal fermentation in a chemostat, with selected antibiotics chosen to inhibit rapidly growing bacteria and selectively enrich for Gram-negative Bacteroidales. This had the objective of promoting the simultaneous expansion of crAss-like phages on their native hosts. The levels of seven different crAss-like phages expanded during the fermentation, indicating that their hosts were also present in the fermenter. The enriched supernatant was then tested against individual Bacteroidales strains isolated from the same faecal sample. This resulted in the isolation of a previously uncharacterised crAss-like phage of candidate genus IV of the proposed Alphacrassvirinae sub-family, ΦcrAss002, that infects the gut commensal Bacteroides xylanisolvens. ΦcrAss002 does not form plaques or spots on lawns of sensitive cells, nor does it lyse liquid cultures, even at high titres. In keeping with the co-abundance of phage and host in the human gut, ΦcrAss002 and Bacteroides xylanisolvens can also co-exist at high levels when co-cultured in laboratory media. Conclusions We report the isolation and characterisation of ΦcrAss002, the first representative of the proposed Alphacrassvirinae sub-family of crAss-like phages. ΦcrAss002 cannot form plaques or spots on bacterial lawns but can co-exist with its host, Bacteroides xylanisolvens, at very high levels in liquid culture without impacting on bacterial numbers. Video abstract
• #### Bovine milk somatic cell transcriptomic response to Staphylococcus aureus is dependent on strain genotype

Background Mastitis is an economically important disease of dairy cows with Staphylococcus aureus a major cause worldwide. Challenge of Holstein-Friesian cows demonstrated that S. aureus strain MOK124, which belongs to Clonal Complex (CC)151, caused clinical mastitis, while strain MOK023, belonging to CC97, caused mild or subclinical mastitis. The aim of this study was to elucidate the molecular mechanisms of the host immune response utilising a transcriptomic approach. Milk somatic cells were collected from cows infected with either S. aureus MOK023 or MOK124 at 0, 24, 48, 72 and 168 h post-infection (hpi) and analysed for differentially expressed (DE) genes in response to each strain. Results In response to MOK023, 1278, 2278, 1986 and 1750 DE genes were found at 24, 48, 72 and 168 hpi, respectively, while 2293, 1979, 1428 and 1544 DE genes were found in response to MOK124 at those time points. Genes involved in milk production (CSN1, CSN10, CSN1S2, CSN2, a-LACTA and PRLR) were downregulated in response to both strains, with a more pronounced decrease in the MOK124 group. Immune response pathways such as NF-κB and TNF signalling were overrepresented in response to both strains at 24 hpi. These immune pathways continued to be overrepresented in the MOK023 group at 48 and 72 hpi, while the Hippo signalling, extracellular matrix interaction (ECM) and tight junction pathways were overrepresented in the MOK124 group between 48 and 168 hpi. Cellular composition analysis demonstrated that a neutrophil response was predominant in response to MOK124, while M1 macrophages were the main milk cell type post-infection in the MOK023 group. Conclusions A switch from immune response pathways to pathways involved in maintaining the integrity of the epithelial cell layer was observed in the MOK124 group from 48 hpi, which coincided with the occurrence of clinical signs in the infected animals. The higher proportion of M1 macrophages in the MOK023 group and lack of substantial neutrophil recruitment in response to MOK023 may indicate immune evasion by this strain. The results of this study highlight that the somatic cell transcriptomic response to S. aureus is dependent on the genotype of the infecting strain.
• #### A pilot study on the prevalence of lice in Irish beef cattle and the first Irish report of deltamethrin tolerance in Bovicola bovis

Background Pediculosis in cattle causes significant itching, irritation and stress to the animal, often resulting in skin damage and poor coat condition. The control of bovine pediculosis in Ireland is based predominantly on commercial insecticides belonging to one of two chemical classes, the synthetic pyrethroids and the macrocyclic lactones. In recent years, pyrethroid tolerance has been reported in a number of species of livestock lice in the United Kingdom and Australia. Results In this pilot survey, lice were detected in 16 (94%) out of 17 herds visited. Two species of lice, Bovicola bovis and Linognathus vituli were identified. In vitro contact bioassays showed evidence of deltamethrin tolerance in Bovicola bovis collected from 4 farms. This was confirmed by repeatedly assessing louse infestations on treated animals on one farm. Conclusions To our knowledge this is the first record of insecticide tolerant populations of lice in Irish cattle. The results also provide new data on the species of lice infesting beef cattle in Ireland and the prevalence and control of louse infestations in Irish beef cattle herds.
• #### Characterization and adaptation of Caldicellulosiruptor strains to higher sugar concentrations, targeting enhanced hydrogen production from lignocellulosic hydrolysates

Abstract Background The members of the genus Caldicellulosiruptor have the potential for future integration into a biorefinery system due to their capacity to generate hydrogen close to the theoretical limit of 4 mol H2/mol hexose, use a wide range of sugars and can grow on numerous lignocellulose hydrolysates. However, members of this genus are unable to survive in high sugar concentrations, limiting their ability to grow on more concentrated hydrolysates, thus impeding their industrial applicability. In this study five members of this genus, C. owensensis, C. kronotskyensis, C. bescii, C. acetigenus and C. kristjanssonii, were developed to tolerate higher sugar concentrations through an adaptive laboratory evolution (ALE) process. The developed mixed population C. owensensis CO80 was further studied and accompanied by the development of a kinetic model based on Monod kinetics to quantitatively compare it with the parental strain. Results Mixed populations of Caldicellulosiruptor tolerant to higher glucose concentrations were obtained with C. owensensis adapted to grow up to 80 g/L glucose; other strains in particular C. kristjanssonii demonstrated a greater restriction to adaptation. The C. owensensis CO80 mixed population was further studied and demonstrated the ability to grow in glucose concentrations up to 80 g/L glucose, but with reduced volumetric hydrogen productivities ( $$Q_{{{\text{H}}_{2} }}$$ Q H 2 ) and incomplete sugar conversion at elevated glucose concentrations. In addition, the carbon yield decreased with elevated concentrations of glucose. The ability of the mixed population C. owensensis CO80 to grow in high glucose concentrations was further described with a kinetic growth model, which revealed that the critical sugar concentration of the cells increased fourfold when cultivated at higher concentrations. When co-cultured with the adapted C. saccharolyticus G5 mixed culture at a hydraulic retention time (HRT) of 20 h, C. owensensis constituted only 0.09–1.58% of the population in suspension. Conclusions The adaptation of members of the Caldicellulosiruptor genus to higher sugar concentrations established that the ability to develop improved strains via ALE is species dependent, with C. owensensis adapted to grow on 80 g/L, whereas C. kristjanssonii could only be adapted to 30 g/L glucose. Although C. owensensis CO80 was adapted to a higher sugar concentration, this mixed population demonstrated reduced $$Q_{{{\text{H}}_{2} }}$$ Q H 2 with elevated glucose concentrations. This would indicate that while ALE permits adaptation to elevated sugar concentrations, this approach does not result in improved fermentation performances at these higher sugar concentrations. Moreover, the observation that planktonic mixed culture of CO80 was outcompeted by an adapted C. saccharolyticus, when co-cultivated in continuous mode, indicates that the robustness of CO80 mixed culture should be improved for industrial application.