Herd-level prevalence of bovine leukemia virus, Salmonella Dublin and Neospora caninum in Alberta, Canada, dairy herds using ELISA on bulk tank milk samples.

Endemic infectious diseases remain a major challenge for dairy producers worldwide. For effective disease control programs, up-to-date prevalence estimates are of utmost importance. The objective of this study was to estimate the herd-level prevalence of bovine leukemia virus (BLV), Salmonella Dublin, and Neospora caninum in dairy herds in Alberta, Canada using a serial cross-sectional study design. Bulk tank milk samples from all Alberta dairy farms were collected 4 times, in December 2021 (n = 489), April 2022 (n = 487), July 2022 (n = 487), and October 2022 (n = 480), and tested for antibodies against BLV, S. Dublin, and N. caninum using ELISAs. Herd-level apparent prevalence was calculated as positive samples divided by total tested samples at each time point. A mixed effect modified Poisson regression model was employed to assess the association of prevalence with region, herd size, herd type, and type of milking system. Apparent prevalence of BLV was 89.4, 88.7, 86.9 and 86.9% in December, April, July, and October, respectively, whereas for S. Dublin apparent prevalence was 11.2, 6.6, 8.6, and 8.5%, and for N. caninum apparent prevalence was 18.2, 7.4, 7.8, and 15.0%. For BLV, S. Dublin and N. caninum, a total of 91.7, 15.6, and 28.1% of herds, respectively, were positive at least once, whereas 82.5, 3.6, and 3.0% of herds were ELISA-positive at all 4 times. Compared with the north region, central Alberta had a high prevalence (prevalence ratio (PR) = 1.13) of BLV-antibody positive herds, whereas south Alberta had a high prevalence (PR = 2.56) of herds positive for S. Dublin antibodies. Furthermore, central (PR = 0.52) and south regions (PR = 0.46) had low prevalence of N. caninum-positive herds compared with the north. Hutterite colony herds were more frequently BLV-positive (PR = 1.13) but less frequently N. caninum-positive (PR = 0.47). Large herds (>7,200 L/day milk delivered ∼ > 250 cows) were 1.1 times more often BLV-positive, whereas small herds (≤3,600 L/day milk delivered ∼ ≤ 125 cows) were 3.2 times more often N. caninum-positive. For S. Dublin, Hutterite-colony herds were less frequently (PR = 0.07) positive than non-colony herds only in medium and large stratum but not in small stratum. Moreover, larger herds were more frequently (PR = 2.20) S. Dublin-positive than smaller herds only in non-colony stratum but not in colony stratum. Moreover, N. caninum prevalence was 1.6 times higher on farms with conventional milking systems compared with farms with an automated milking system. These results provide up-to-date information of the prevalence of these infections that will inform investigations of within-herd prevalence of these infections and help in devising evidence-based disease control strategies.

Global losses due to dairy cattle diseases: A comorbidity-adjusted economic analysis.

An economic simulation was carried out over 183 milk-producing countries to estimate the global economic impacts of 12 dairy cattle diseases and health conditions: mastitis (subclinical and clinical), lameness, paratuberculosis (Johne's disease), displaced abomasum, dystocia, metritis, milk fever, ovarian cysts, retained placenta, and ketosis (subclinical and clinical). Estimates of disease impacts on milk yield, fertility, and culling were collected from the literature, standardized, meta-analyzed using a variety of methods ranging from simple averaging to random-effects models, and adjusted for comorbidities to prevent overestimation. These comorbidity-adjusted disease impacts were then combined with a set of country-level lactational incidence and/or prevalence estimates, herd characteristics, and price estimates within a series of Monte Carlo simulations that estimated and valued the economic losses due to these diseases. It was estimated that total annual global losses are USD 65 billion (B). Subclinical ketosis, clinical mastitis, and subclinical mastitis were the costliest diseases modeled, resulting in mean annual global losses of approximately USD 18B, USD 13B, and USD 9B, respectively. Estimated global annual losses due to clinical ketosis, displaced abomasum, dystocia, lameness, metritis, milk fever, ovarian cysts, paratuberculosis, and retained placenta were estimated to be USD 0.2B, 0.6B, 0.6B, 6B, 5B, 0.6B, 4B, 4B, and 3B, respectively. Without adjustment for comorbidities, when statistical associations between diseases were disregarded, mean aggregate global losses would have been overestimated by 45%. Although annual losses were greatest in India (USD 12B), the USA (USD 8B), and China (USD 5B), depending on the measure of losses used (losses as a percent of GDP, losses per capita, losses as a percent of gross milk revenue), the relative economic burden of these dairy cattle diseases across countries varied markedly.

Epidemiology, linkage to care and natural history of women of childbearing age with chronic hepatitis B: A population-based study.

Pregnant women with chronic hepatitis B (CHB) are a priority population for hepatitis B care. Identification of HBV status prior to pregnancy would facilitate timely maternal interventions and perinatal care. In our study, we aimed to study the epidemiology of CHB among women of childbearing age (WoCBA, 18-49 years) in Alberta, Canada. We retrospectively analysed Alberta Analytics databases to study CHB epidemiology, natural history and care linkage among WoCBA in Alberta, between April 2012 and March 2021. A Poisson regression was conducted to estimate incidence of newly identified CHB cases and prevalence trends, whereas predictors of care linkage were determined using logistic regression. Age/sex-adjusted incidence of newly identified CHB among WoCBA between 2015 and 2020 was 36.2/100,000 person/years, highest among individuals aged 30-39 years. Incidence of newly identified CHB decreased from 52.6 to 18.2/100,000 between 2015 and 2020, but prevalence increased from 131.7 to 248.6/100,000 in the same period. Newly identified CHB incident cases (n = 2124) had lower survival rates than age/sex-matched Canadians, with a standardized mortality ratio of 5.7 (95% CI 2.6-11.0). Increasing age (years) at diagnosis (HR, 1.2; 95% CI 1.1-1.3) was independently associated with mortality. Comorbid hepatocellular carcinoma, anti-HBV treatment and year of diagnosis were not significantly associated with mortality. Of the 1927 women with 2436 hepatitis B surface antigen-positive pregnancies from 2012 to 2020, only 27.6% had recommended HBV assessment during pregnancy. Of those women meeting criteria for antiviral therapy to prevent mother-to-child transmission (MTCT), only 66.4% received treatment. Suboptimal management during pregnancy and overall lower survival rates highlight the need to address care linkage barriers in women of childbearing age living with CHB.

Heme oxygenase activates calcium release from the endoplasmic reticulum of bovine mammary epithelial cells to promote TFEB entry into the nucleus to reduce the intracellular load of Mycoplasma bovis.

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.

Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis.

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis's in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Effects of bovine leukemia virus seropositivity and proviral load on milk, fat, and protein production of dairy cows.

The objective was to evaluate the effects of bovine leukemia virus (BLV) infection, as determined by BLV seropositivity and proviral load, on 305-d milk, fat, and protein production of dairy cows. A cross-sectional study was conducted among 1,712 cows from 9 dairy herds in Alberta, Canada. The BLV status was assessed using an antibody ELISA, whereas BLV proviral load in BLV-seropositive cattle was determined with quantitative PCR. Dairy Herd Improvement 305-d milk, fat, and protein production data were obtained for all enrolled cattle. Differences in these milk end points were assessed in 2 ways: first, by categorizing cows based on BLV serostatus (i.e., BLV positive or negative), and second, by categorizing based on BLV proviral load (i.e., BLV negative, low proviral load [LPL] BLV positive, and high proviral load [HPL] BLV positive). A mixed-effect multivariable linear regression model was used to assess differences in milk parameters. We found that BLV positivity, adjusted for parity and natural log-transformed somatic cell count (SCC), was not associated with reduction in 305-d milk, fat, or protein production. However, significant reductions in 305-d milk, fat, and protein yield occurred in HPL cows, but not in LPL cows, compared with BLV-negative cows, when adjusted for parity number and natural log-transformed SCC. In summary, BLV proviral load may predict effects of BLV infection on milk, fat, and protein production.

Patient compliance with the implementation of a decolonization strategy for Staphylococcus aureus in hip and knee replacements.

BACKGROUND: A preoperative, in-community antimicrobial decolonization protocol combining chlorohexidine gluconate (CHG) sponges and mupirocin ointment to reduce surgical site infections amongst hip and knee replacement patients has been adopted in Alberta, Canada. Patient compliance with the protocol is essential for effectiveness. It is, therefore, important to understand patterns, and reasons why, patients do, and do not, comply. METHODS: A descriptive survey of patients having elective total hip or knee replacement at seven clinics in Alberta was conducted to determine patient compliance and reasons for noncompliance. Descriptive statistics and multivariate logistic regression were computed. RESULTS: Patient compliance was assessed in 3,427 patients. There were no differences in compliance based on the baseline protocols and enhanced protocols, but there was a difference based on clinic location. The odds of compliance with three CHG sponges were 4.47 times higher in rural versus urban clinics (P < .001). The most common reason for noncompliance for patients instructed to use 3 CHG sponges was "patient forgot". CONCLUSIONS: Compliance did not change when enhanced protocols were introduced; however, compliance differed by clinic location. Reasons for noncompliance included "sponges not provided", "patient forgot", and "surgery date moved". Results may inform clinics on areas where improvements could be made to increase patient compliance.

MicroRNA miR-223 modulates NLRP3 and Keap1, mitigating lipopolysaccharide-induced inflammation and oxidative stress in bovine mammary epithelial cells and murine mammary glands.

Bovine mastitis, the most prevalent and costly disease in dairy cows worldwide, decreases milk quality and quantity, and increases cow culling. However, involvement of microRNAs (miRNAs) in mastitis is not well characterized. The objective was to determine the role of microRNA-223 (miR-223) in regulation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and kelch like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) oxidative stress pathway in mastitis models induced by lipopolysaccharide (LPS) treatment of immortalized bovine mammary epithelial cells (bMECs) and murine mammary glands. In bMECs cultured in vitro, LPS-induced inflammation downregulated bta-miR-223; the latter interacted directly with the 3' untranslated region (3' UTR) of NLRP3 and Keap1. Overexpression of bta-miR-223 in bMECs decreased LPS and Adenosine 5'-triphosphate (ATP)-induced NLRP3 and its mediation of caspase 1 and IL-1ß, and inhibited LPS-induced Keap1 and Nrf2 mediated oxidative stress, whereas inhibition of bta-miR-223 had opposite effects. In an in vivo murine model of LPS-induced mastitis, increased miR-223 mitigated pathology in the murine mammary gland, whereas decreased miR-223 increased inflammatory changes and oxidative stress. In conclusion, bta-miR-223 mitigated inflammation and oxidative injury by downregulating the NLRP3 inflammasome and Keap1/Nrf2 signaling pathway. This study implicated bta-miR-223 in regulation of inflammatory responses, with potential as a novel target for treating bovine mastitis and other diseases.

Construction of a circRNA- lincRNA-lncRNA-miRNA-mRNA ceRNA regulatory network identifies genes and pathways linked to goat fertility.

Background: There is growing interest in the genetic improvement of fertility traits in female goats. With high-throughput genotyping, single-cell RNA sequencing (scRNA-seq) is a powerful tool for measuring gene expression profiles. The primary objective was to investigate comparative transcriptome profiling of granulosa cells (GCs) of high- and low-fertility goats, using scRNA-seq. Methods: Thirty samples from Ji'ning Gray goats (n = 15 for high fertility and n = 15 for low fertility) were retrieved from publicly available scRNA-seq data. Functional enrichment analysis and a literature mining approach were applied to explore modules and hub genes related to fertility. Then, interactions between types of RNAs identified were predicted, and the ceRNA regulatory network was constructed by integrating these interactions with other gene regulatory networks (GRNs). Results and discussion: Comparative transcriptomics-related analyses identified 150 differentially expressed genes (DEGs) between high- and low-fertility groups, based on the fold change (≥5 and ≤-5) and false discovery rate (FDR <0.05). Among these genes, 80 were upregulated and 70 were downregulated. In addition, 81 mRNAs, 58 circRNAs, 8 lincRNAs, 19 lncRNAs, and 55 miRNAs were identified by literature mining. Furthermore, we identified 18 hub genes (SMAD1, SMAD2, SMAD3, SMAD4, TIMP1, ERBB2, BMP15, TGFB1, MAPK3, CTNNB1, BMPR2, AMHR2, TGFBR2, BMP4, ESR1, BMPR1B, AR, and TGFB2) involved in goat fertility. Identified biological networks and modules were mainly associated with ovary signature pathways. In addition, KEGG enrichment analysis identified regulating pluripotency of stem cells, cytokine-cytokine receptor interactions, ovarian steroidogenesis, oocyte meiosis, progesterone-mediated oocyte maturation, parathyroid and growth hormone synthesis, cortisol synthesis and secretion, and signaling pathways for prolactin, TGF-beta, Hippo, MAPK, PI3K-Akt, and FoxO. Functional annotation of identified DEGs implicated important biological pathways. These findings provided insights into the genetic basis of fertility in female goats and are an impetus to elucidate molecular ceRNA regulatory networks and functions of DEGs underlying ovarian follicular development.

Broad-spectrum infrared thermography for detection of M2 digital dermatitis lesions on hind feet of standing dairy cattle.

Low-effort, reliable diagnostics of digital dermatitis (DD) are needed, especially for lesions warranting treatment, regardless of milking system or hygienic condition of the feet. The primary aim of this study was to test the association of infrared thermography (IRT) from unwashed hind feet with painful M2 lesions under farm conditions, with lesion detection as ultimate goal. Secondary objectives were to determine the association between IRT from washed feet and M2 lesions, and between IRT from unwashed and washed feet and the presence of any DD lesion. A total of 641 hind feet were given an M-score and IRT images of the plantar pastern were captured. Multivariable logistic regression analyses were done with DD status as dependent variable and maximum infrared temperature (IRTmax), lower leg cleanliness score and locomotion score as independent variables, and farm as fixed effect. To further our understanding of IRTmax within DD status, we divided IRTmax into two groups over the median value of IRTmax in the datasets of unwashed and washed feet, respectively, and repeated the multivariable logistic regression analyses. Higher IRTmax from unwashed hind feet were associated with M2 lesions or DD lesions, in comparison with feet without an M2 lesion or without DD, adjusted odds ratio 1.6 (95% CI 1.2-2.2) and 1.1 (95% CI 1.1-1.2), respectively. Washing of the feet resulted in similar associations. Dichotomization of IRTmax substantially enlarged the 95% CI for the association with feet with M2 lesions indicating that the association becomes less reliable. This makes it unlikely that IRTmax alone can be used for automated detection of feet with an M2 lesion. However, IRTmax can have a role in identifying feet at-risk for compromised foot health that need further examination, and could therefore function as a tool aiding in the automated monitoring of foot health on dairy herds.

Underlying genetic architecture of resistance to mastitis in dairy cattle: A systematic review and gene prioritization analysis of genome-wide association studies.

Mastitis, the most frequent disease in dairy cattle. Resistance to mastitis is a complex, polygenic trait controlled by several genes, each with small effects. Genome-wide association studies have been widely used to identify genomic variants associated with complex traits, including resistance to mastitis, to elucidate the underlying genetic architecture of the trait. However, no systematic review and gene prioritization analysis have been conducted to date on GWAS results for resistance to mastitis in dairy cattle. Hence, the objective was to perform a systematic review and gene prioritization analysis of GWAS studies to identify potential functional candidate genes associated with resistance to mastitis-related traits in dairy cattle. Four electronic databases were searched from inception to December 2020, supplemented with multiple sources of gray literature, to identify eligible articles. Annotation for genes and quantitative trait loci (QTL), and QTL enrichment analysis were conducted using GALLO. Gene prioritization analysis was performed by a guilty-by-association approach using GUILDify and ToppGene. From 52 articles included within this systematic review, 30 articles were used for further functional analyses. Gene and QTL annotation resulted in 9,125 and 43,646 unique genes and QTL, respectively, from 39 studies. In general, overlapping of genes across studies was very low (mean ± SD = 0.02% ± 0.07%). Most annotated genes were associated with somatic cell count-related traits and the Holstein breed. Within all annotated genes, 74 genes were shared among Holstein, Jersey, and Ayrshire breeds. Approximately 7.5% of annotated QTL were related to QTL class "health." Within the health QTL class, 2.6 and 2.2% of QTL were associated with clinical mastitis and somatic cell count-related traits. Enrichment analysis of QTL demonstrated that many enriched QTL were associated with somatic cell score located in Bos taurus autosomes 5, 6, 16, and 20. The prioritization analysis resulted in 427 significant genes after multiple test correction (false discovery rate of 5%) from 26 studies. Most prioritized genes were located in Bos taurus autosomes 19 and 7, and most top-ranked genes were from the cytokine superfamily (e.g., chemokines, interleukins, transforming growth factors, and tumor necrosis factor genes). Although most prioritized genes (397) were associated with somatic cell count-related traits, only 54 genes were associated with clinical mastitis-related traits. Twenty-four genes (ABCC9, ACHE, ADCYAP1, ARC, BCL2L1, CDKN1A, EPO, GABBR2, GDNF, GNRHR, IKBKE, JAG1, KCNJ8, KCNQ1, LIFR, MC3R, MYOZ3, NFKB1, OSMR, PPP3CA, PRLR, SHARPIN, SLC1A3, and TNFRSF25) were reported for both somatic cell count and clinical mastitis-related traits. Prioritized genes were mainly associated with immune response, regulation of secretion, locomotion, cell proliferation, and development. In conclusion, this study provided a fine-mapping of previously identified genomic regions associated with resistance to mastitis and identified key functional candidate genes for resistance to mastitis, which can be used to develop enhanced genomic strategies to combat mastitis by increasing mastitis resistance through genetic selection.

Prototheca bovis induces autophagy in bovine mammary epithelial cells via the HIF-1α and AMPKα/ULK1 pathway.

Prototheca bovis, a highly contagious pathogen, causes bovine mastitis, resulting in premature culling of affected cows and severe economic losses. Infection with P. bovis caused oxidative stress and apoptosis in bovine mammary epithelial cells (bMECs); however, mechanisms underlying P. bovis-induced autophagy remain unclear. Therefore, the autophagy flux induced by P. bovis in bMECs was analyzed by Western blot and laser scanning confocal microscopy. Expression levels of proteins in the HIF-1α and AMPKα/ULK1 pathway, including HIF-1α, AMPKα, p-AMPKα, ULK1, p-ULK1, mTOR, and p-mTOR, plus expression of autophagy-related genes including SQSTM1/p62, Atg5, Beclin1, and LC3II/LC3I, were quantified with Western blot. Infection with P. bovis induced autophagosomes and LC3 puncta in bMECs that were detected using transmission electron microscopy and laser scanning confocal microscopy, respectively. In addition, lysosome-associated proteins Rab7 and LAMP2a, and lysosomal activity were measured with Western blot and laser scanning confocal microscopy. Infection with P. bovis induced an unobstructed autophagic flux, increased protein expression of LC3II/LC3I, and decreased SQSTM1/p62 protein expression at 6 hpi. Furthermore, P. bovis upregulated protein expression in the HIF-1α and AMPKα/ULK1 pathway and increased the ratio of LC3II/LC3I, implying autophagy was activated in bMECs. However, deletion of AMPKα or ULK1 decreased LC3II/LC3I expression levels and LC3 puncta numbers, suggesting that autophagy was inhibited in bMECs. Additionally, deficiency of HIF-1α decreased protein expression of AMPKα and ULK1 as well as LC3 puncta numbers, and autophagy induced by P. bovis was also inhibited in bMECs. At 6 hpi, lysosome-associated protein Rab7 was decreased and LAMP2a was increased, indicating normal autophagy. In contrast, at 12 hpi, expression of Rab7 and LAMP2a proteins indicated that autophagy was inhibited in bMECs at that time. Therefore, we confirmed that P. bovis infection induced autophagy in bMECs via the HIF-1α and AMPKα/ULK1 pathway, with involvement of lysosome-associated protein Rab7 and LAMP2a.

lncRNA-miRNA-mRNA ceRNA Network Involved in Sheep Prolificacy: An Integrated Approach.

Understanding the molecular pattern of fertility is considered as an important step in breeding of different species, and despite the high importance of the fertility, little success has been achieved in dissecting the interactome basis of sheep fertility. However, the complex mechanisms associated with prolificacy in sheep have not been fully understood. Therefore, this study aimed to use competitive endogenous RNA (ceRNA) networks to evaluate this trait to better understand the molecular mechanisms responsible for fertility. A competitive endogenous RNA (ceRNA) network of the corpus luteum was constructed between Romanov and Baluchi sheep breeds with either good or poor genetic merit for prolificacy using whole-transcriptome analysis. First, the main list of lncRNAs, miRNAs, and mRNA related to the corpus luteum that alter with the breed were extracted, then miRNA−mRNA and lncRNA−mRNA interactions were predicted, and the ceRNA network was constructed by integrating these interactions with the other gene regulatory networks and the protein−protein interaction (PPI). A total of 264 mRNAs, 14 lncRNAs, and 34 miRNAs were identified by combining the GO and KEGG enrichment analyses. In total, 44, 7, 7, and 6 mRNAs, lncRNAs, miRNAs, and crucial modules, respectively, were disclosed through clustering for the corpus luteum ceRNA network. All these RNAs involved in biological processes, namely proteolysis, actin cytoskeleton organization, immune system process, cell adhesion, cell differentiation, and lipid metabolic process, have an overexpression pattern (Padj < 0.01). This study increases our understanding of the contribution of different breed transcriptomes to phenotypic fertility differences and constructed a ceRNA network in sheep (Ovis aries) to provide insights into further research on the molecular mechanism and identify new biomarkers for genetic improvement.

Nrf2 and NF-κB/NLRP3 inflammasome pathways are involved in Prototheca bovis infections of mouse mammary gland tissue and mammary epithelial cells.

Prototheca bovis is a serious pathogen for animals, but pathogenesis of P. bovis mastitis is unclear. The objective was to characterize how P. bovis induces inflammatory responses in mouse mammary gland tissue and mammary epithelial cells (mMECs). Prototheca bovis damaged mammary gland tissue and mitochondrial structure, and induced oxidative stress, as evident by significant increases in mtROS and MDA concentrations and significant decreases in T-SOD activity in both mammary gland tissue and mMECs. Expression of Nrf2, HO-1 and Keap1 proteins was significantly changed in mammary gland tissue and mMECs after P. bovis infection. Additionally, cytokines (IL-1ß, IL-6 and IL-18) and protein expressions in NF-κB and in the NLRP3 inflammasome pathway were significantly increased in mammary gland tissue and mMECs. In the P. bovis group, treatment with N-acetyl-l-cysteine (NAC) significantly decreased protein expression in NF-κB and the NLRP3 inflammasome pathway, as well as IL-1ß, IL-6 and IL-18, whereas protein expression in the Nrf2 pathway was significantly changed. Inhibition of NF-κB or NLRP3 significantly decreased expression of IL-1ß and IL-18 proteins in mMECs infected with P. bovis. Additionally, activating Nrf2 inhibited expression of NLRP3 and IL-1ß. In conclusion, P. bovis induced an inflammatory response via the NF-κB/NLRP3 inflammasome pathway; however, scavenging ROS or activating Nrf2 mitigated the inflammatory response in infected mMECs.

Crohn's disease therapeutic dietary intervention (CD-TDI): study protocol for a randomised controlled trial.

INTRODUCTION: Dietary patterns that might induce remission in patients with active Crohn's disease (CD) are of interest to patients, but studies are limited in the published literature. We aim to explore the efficacy of the CD therapeutic dietary intervention (CD-TDI), a novel dietary approach developed from best practices and current evidence, to induce clinical and biomarker remission in adult patients with active CD. METHODS AND ANALYSIS: This study is a 13-week, multicentre, randomised controlled trial in patients with mild-to-moderate active CD at baseline. One hundred and two patients will be block randomised, by sex, 2:1 to the intervention (CD-TDI) or conventional management. Coprimary outcomes are clinical and biomarker remission, defined as a Harvey Bradshaw Index of <5 and a faecal calprotectin of <250 µg/g, respectively.Secondary outcomes include gut microbiota diversity and composition, faecal short-chain fatty acids, regulatory macrophage function, serum and faecal metabolomics, C reactive protein, peripheral blood mononuclear cell gene expression profiles, quality of life, sedentary time and physical activity at 7 and/or 13 weeks. Predictive models of clinical response to a CD-TDI will be investigated. ETHICS AND DISSEMINATION: The research protocol was approved by the Conjoint Health Research Ethics Board at the University of Calgary (REB19-0402) and the Health Research Ethics Board-Biomedical Panel at the University of Alberta (Pro00090772). Study findings will be presented at national and international conferences, submitted for publication in abstracts and manuscripts, shared on social media and disseminated through patient-education materials. TRIAL REGISTRATION NUMBER: NCT04596566.

Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-κB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures.

Emergence of bovine mastitis caused by Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1ß and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1ß, and IL-1ß proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.

Invited review: Bovine leukemia virus-Transmission, control, and eradication.

Bovine leukemia virus (BLV) infection, endemic in North American dairy herds, has production-limiting effects. A literature review of available papers published since 1995 concerning BLV transmission and its control was conducted. Although confirmed transmission routes were reviewed (blood, natural breeding, in utero, colostrum, and milk), there is still a lack of detailed information on other specific risks for transmission (e.g., contact transmission and hoof-trimming knives). Eradication of BLV has been achieved by combined management, segregation, and culling approaches. In contrast, although sole implementation of best management practices aimed at prevention of BLV transmission has decreased within-herd BLV prevalence, it has not eradicated BLV from a herd. Therefore, control and eradication of BLV by best management practices only should be further investigated. Additionally, the role of proviral load in infected cattle was investigated. Cattle with a high proviral load seem to be more likely to infect others, whereas those with a very low proviral load seem to have low risks of transmitting BLV. Information on proviral load could be taken into account when controlling BLV in high-prevalence herds. In conclusion, there is a need for detailed, large-scale studies investigating roles of specific transmission routes, knowing proviral load of infected individuals.

Effects of different culture media on growth of Treponema spp. isolated from digital dermatitis.

Digital dermatitis (DD) lesions in cattle are characterized by the presence of multiple Treponema species. Current culture media for isolating treponemes generally uses serum supplementation from different animals to target particular Treponema sp.; however, their suitability for DD Treponema isolation has not been fully determined. We studied the effect of culture media (OTEB, NOS and TYGV) and serum supplementation on mixed Treponema spp. dynamics. Bacterial growth was evaluated by direct microscopic count, optical density, wet weight and a species-specific qPCR and the correlations between these independent methods were calculated. Wet weight, optical density and bacterial count correlated best with each other. Different Treponema species performed differently under the tested culture media. T. phagedenis growth was enhanced in OTEB media supplemented with bovine fetal serum (BFS) or horse serum (HS). T. medium had lower generation time when culture media were supplemented with rabbit serum (RS). Lowest generation time for T. pedis and T. denticola were obtained in NOS media supplemented with HS and OTEB media supplemented with BFS, respectively. Detection of cystic forms observed after 5 days of culture did not differ among the culture media. Correlation between different Treponema spp. growth quantification techniques indicated that alternative quantification methods such as qPCR and wet weight could be used depending on the purpose. We conclude that effects of culture media and serum supplementation on mixed Treponema spp. communities should be taken into account when isolating a specific Treponema species.

Dietary patterns, food groups and nutrients in Crohn's disease: associations with gut and systemic inflammation.

This study examined associations between dietary intake and gut and systemic inflammation assessed by fecal calprotectin ≤ or > 100 µg/mg (FCP), C-reactive protein ≤ or > 5 mg/L (CRP) and serum cytokine profiles in Crohn's disease (CD) patients in clinical remission. A 3-month observational study was conducted at the University of Calgary in Calgary, Alberta, Canada between 2016 and 2018 in 66 outpatients with CD in clinical remission. FCP was obtained from stool samples at baseline and 3-months and serum CRP and serum cytokines were assessed at 3-months only (n = 41). Dietary intakes were collected using 3-day food records at baseline and 3-months and categorized as: PREDIMED Mediterranean diet scores (pMDS) total and individual components, the dietary inflammatory index (DII), food groups, and common micro- and macro-nutrients. Statistical models were developed to identify relationships between dietary factors and FCP, CRP and cytokine levels. Daily intake of leafy green vegetables was associated with FCP ≤ 100 µg/mg (p < 0.05). Increasing omega 6:3 ratio was associated with CRP ≤ 5 mg/L (p = 0.02). Different cytokines were significantly associated with various dietary variables. Future studies in patients with greater disease activity should be undertaken to explore these relationships.

In vitro immune responses of bovine mammary epithelial cells induced by Escherichia coli, with multidrug resistant extended-spectrum ß-lactamase, isolated from mastitic milk.

Bovine mastitis is an inflammatory condition of mammary glands causing huge economic losses for dairy industries. Infection with extended-spectrum ß-lactamase (ESBL)-producing sequence types (ST) 410-Escherichia coli (ESBL-ST410 E. coli) is considered a leading cause of bovine mastitis in China. However, pathogenic effects of these strains in an in vitro model, e.g. bovine mammary epithelial cells (bMECs), are unknown. Therefore, our objectives were to explore pathogenesis (adhesion and invasion, inflammation, oxidative stress and apoptosis) of ESBL-E. coli (highly prevalent in bovine mastitis) in bMECs. Non-pathogenic E. coli DH5α and a prototypical E. coli P4 were included as negative and positive controls, respectively. The bMECs were infected with our isolated ST410 strains, plus DH5α and P4, with assessment of the following end points: adhesive and invasive capabilities; lactate dehydrogenase (LDH) activities; inflammatory responses, including concentrations of interleukin-1ß (IL-1ß), IL-6, IL-10 and tumor necrosis factor-α; oxidative stress including intracellular reactive oxygen species production, malondialdehyde concentrations, activities of glutathione peroxidase and superoxide dismutase; and apoptosis. All ST410 strains had greater adhesive and invasive capabilities and increased LDH release, with varying degrees of inflammatory responses, oxidative stress and apoptosis compared to blank and DH5α groups, similar to P4-infected bMECs. In particular, ST410(4) was more likely than the other 3 isolates to adhere to and invade bMECs and increase LDH activities, cytokine release, oxidative stress and apoptosis. Thus, ST410 isolates had pathogenic manifestations of adhesive and invasive capabilities; furthermore, they induced inflammation, oxidative stress and apoptosis in bMECs. Finally, ST410(4) was the most pathogenic strain.