Escherichia coli infection induces ferroptosis in bovine mammary epithelial cells by activating the Wnt/ß-catenin pathway-mediated mitophagy.

Iron overload causes mitochondrial damage, and then activates mitophagy, which may directly trigger and amplify ferroptosis. Our objective was to investigate whether Escherichia coli (E. coli) isolated from clinical bovine mastitis induces ferroptosis in bovine mammary epithelial cells (bMECs) and if so, the underlying regulatory mechanism. E. coli infection caused mitochondrial damage, mitophagy, and ferroptosis. Rapamycin and chloroquine increased and suppressed ferroptosis, respectively, in E. coli-treated bMECs. Moreover, E. coli infection activated the Wnt/ß-catenin pathway, but foscenvivint alleviated it. In conclusion, E. coli infection induced ferroptosis through activation of the Wnt/ß-catenin pathway-promoted mitophagy, and it also suppressed GPX4 expression.

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.

Biological and genomic characterization of 4 novel bacteriophages isolated from sewage or the environment using non-aureus Staphylococci strains.

Non-aureus staphylococci (NAS) are an essential group of bacteria causing antimicrobial resistant intramammary infections in livestock, particularly dairy cows. Therefore, bacteriophages emerge as a potent bactericidal agent for NAS mastitis. This study aimed to obtain NAS-specific bacteriophages using bacterial strains isolated from cows with mastitis, subsequently evaluating their morphological, genomic, and lytic characteristics. Four distinct NAS bacteriophages were recovered from sewage or the environment of Chinese dairy farms; PT1-1, PT94, and PT1-9 were isolated using Staphylococcus chromogenes and PT1-4 using Staphylococcus gallinarum. Both PT1-1 (24/54, 44 %) and PT94 (28/54, 52 %) had broader lysis than PT1-4 (3/54, 6 %) and PT1-9 (10/54, 19 %), but PT1-4 and PT1-9 achieved cross-species lysis. All bacteriophages had a short latency period and good environmental tolerance, including surviving at pH=4-10 and at 30-60℃. Except for PT1-9, all bacteriophages had excellent bactericidal efficacy within 5 h of co-culture with host bacteria in vitro at various multiplicity of infection (MOIs). Based on whole genome sequencing, average nucleotide identity (ANI) analysis of PT1-1 and PT94 can be classified as the same species, consistent with whole-genome synteny analysis. Although motifs shared by the 4 bacteriophages differed little from those of other bacteriophages, a phylogenetic tree based on functional proteins indicated their novelty. Moreover, based on whole genome comparisons, we inferred that cross-species lysis of bacteriophage may be related to the presence of "phage tail fiber." In conclusion 4 novel NAS bacteriophages were isolated; they had good biological properties and unique genomes, with potential for NAS mastitis therapy.

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.

Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China.

Klebsiella pneumoniae can cause severe clinical mastitis in dairy cows, with K. pneumoniae type K57 (K57-KP) being the most common capsular serotype. To identify virulence factors and antimicrobial-resistance (AMR) genes of K57-KP with varying virulence, Galleria mellonella (greater wax moth) larvae were infected as a screening model to characterize virulence of 90 K57-KP strains, with 10 and 11 strains defined as virulent or attenuated, respectively, based on larval survival rates. Next, virulence of these 21 isolates was subsequently confirmed in adhesion and lactate dehydrogenase release assays, using bovine mammary epithelial cells cultured in vitro. Finally, genes associated with virulence and AMR were characterize with whole-genome sequencing. These 21 K57-KP strains were designated into 16 sequence types based on multi-locus sequence typing and allocated in phylogenetic analysis based on single nucleotide polymorphisms. We found great genetic diversity among isolates. In addition, adhesion-associated genes (e.g., fimA, sfaA, and focA) aminoglycoside-resistance genes (aph(6)-Id, strAB) were associated with virulence. This study provided new knowledge regarding virulence of K57-KP associated with bovine mastitis, which may inform development of novel diagnostic tools and prevention strategies for bovine mastitis.

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.

A soy lecithin nanoparticles-based extender effectively cryopreserves Holstein bull sperm.

Plant-based semen extenders, typically derived from soybean lecithin, are easier to modulate more and consistent in their composition than animal-based extenders. As large lecithin particles can, however, reduce effectiveness and solubility in bull semen extenders, sonication was used to create nano-lecithin (NL) particles of soybean lecithin. The objective was to determine the effects of lecithin type and concentration on the quality of frozen-thawed bovine sperm. We hypothesized that reducing the size of lecithin improves its interactions with the sperm and enhances the parameters that favor its motility, viability and fertility. Semen was collected from six mature Holstein bulls and ejaculates meeting minimum standards were pooled. Eight Tris-based extenders that contained 1, 2, 3, or 4 % of either conventional lecithin (L1-L4) or NL (NL1-NL4), plus two control extenders (one animal-based extender containing 20 % egg yolk [EY] and a commercial lecithin-based extender [BioXcell®]) were compared. Among soybean lecithin-based extenders, NL3 had the highest total and progressive sperm motility, and average path, straight-line and curvilinear sperm velocity, and was comparable to EY. Additionally, sperm mitochondrial activity was the highest in NL3, whereas sperm viability was highest in EY, NL3, and L4. Following in vitro fertilization of in vitro-matured bovine oocyes, NL3 had cleavage and hatching rates comparable to BioXcell®, but a lower blastocyst rate than EY. Overall, NL3 performed better than the other extenders for most end points, with efficiency comparable to EY. We, therefore, concluded that reducing lecithin particle size to a nano level improves sperm cryopreservation with optimal performance with 3 % NL.

Integrated Comparative Transcriptome and circRNA-lncRNA-miRNA-mRNA ceRNA Regulatory Network Analyses Identify Molecular Mechanisms Associated with Intramuscular Fat Content in Beef Cattle.

Intramuscular fat content (IMF), one of the most important carcass traits in beef cattle, is controlled by complex regulatory factors. At present, molecular mechanisms involved in regulating IMF and fat metabolism in beef cattle are not well understood. Our objective was to integrate comparative transcriptomic and competing endogenous RNA (ceRNA) network analyses to identify candidate messenger RNAs (mRNAs) and regulatory RNAs involved in molecular regulation of longissimus dorsi muscle (LDM) tissue for IMF and fat metabolism of 5 beef cattle breeds (Angus, Chinese Simmental, Luxi, Nanyang, and Shandong Black). In total, 34 circRNAs, 57 lncRNAs, 15 miRNAs, and 374 mRNAs were identified by integrating gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Furthermore, 7 key subnets with 16 circRNAs, 43 lncRNAs, 7 miRNAs, and 237 mRNAs were detected through clustering analyses, whereas GO enrichment analysis of identified RNAs revealed 48, 13, and 28 significantly enriched GO terms related to IMF in biological process, molecular function, and cellular component categories, respectively. The main metabolic-signaling pathways associated with IMF and fat metabolism that were enriched included metabolic, calcium, cGMP-PKG, thyroid hormone, and oxytocin signaling pathways. Moreover, MCU, CYB5R1, and BAG3 genes were common among the 10 comparative groups defined as important candidate marker genes for fat metabolism in beef cattle. Contributions of transcriptome profiles from various beef breeds and a competing endogenous RNA (ceRNA) regulatory network underlying phenotypic differences in IMF provided novel insights into molecular mechanisms associated with meat quality.

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.

Morphologic, Steroidogenic, and Transcriptomic Assessment of the Corpus Luteum in Holstein Cows after Spontaneous or Hormone-Induced Ovulation.

There is evidence that replacing the gonadotropin-releasing hormone (GnRH) with porcine luteinizing hormone (pLH) to synchronize ovulation prior to artificial insemination (AI) increased pregnancy per AI in dairy cows without affecting blood progesterone (P4) concentrations. Whether morphologic, steroidogenic, and transcriptomic differences exist among corpora lutea (CL) formed after ovulation induced by GnRH and pLH is unclear. Our main objective, therefore, was to compare CL characteristics between GnRH- and pLH-induced CL. In 24 non-lactating Holstein cows, ovulations were spontaneous (Spont-Ov) or induced with 100 µg GnRH, 25 mg pLH, or 1 mg estradiol benzoate (EB), with CL excised 12 d after ovulation. In pLH- versus GnRH-treated cows, the duration of elevated LH (above baseline) was prolonged (10 versus 6 h, respectively, p < 0.01), but CL dimensions, pixel intensity of CL images, proportions of steroidogenic and non-steroidogenic luteal cells, and mean plasma LH did not significantly differ. Post-ovulation mean plasma P4 (ng/mL) did not differ among Spont-Ov (3.0) pLH (3.1) or GnRH (3.0) cows but were lower in EB cows (2.0). In vitro P4 concentration was greater in luteal explants of pLH-treated cows than in all other groups (combined means, 16.0 vs. 12.3 µg/mL, p < 0.02). Relative abundance of mRNA for oxytocin receptor (OXTR) was 2-fold higher (p < 0.01) in CL of pLH vs. GnRH cows and highest in Spont-Ov CL. In summary, pLH-treated cows had a longer LH peak, and greatest luteal tissue concentrations and in vitro production of P4. We inferred that increased P4 concentrations at the ovarian-uterine level in pLH-treated cows could have promoted embryo development and increased pregnancy per AI.

Alternatives to antibiotics for treatment of mastitis in dairy cows.

Mastitis is considered the costliest disease on dairy farms and also adversely affects animal welfare. As treatment (and to a lesser extent prevention) of mastitis rely heavily on antibiotics, there are increasing concerns in veterinary and human medicine regarding development of antimicrobial resistance. Furthermore, with genes conferring resistance being capable of transfer to heterologous strains, reducing resistance in strains of animal origin should have positive impacts on humans. This article briefly reviews potential roles of non-steroidal anti-inflammatory drugs (NSAIDs), herbal medicines, antimicrobial peptides (AMPs), bacteriophages and their lytic enzymes, vaccination and other emerging therapies for prevention and treatment of mastitis in dairy cows. Although many of these approaches currently lack proven therapeutic efficacy, at least some may gradually replace antibiotics, especially as drug-resistant bacteria are proliferating globally.

Factors associated with antimicrobial resistant enterococci in Canadian beef cattle: A scoping review.

Introduction: Antimicrobial resistance (AMR) is a global health concern, occurring when bacteria evolve to render antimicrobials no longer effective. Antimicrobials have important roles in beef production; however, the potential to introduce AMR to people through beef products is a concern. This scoping review identifies factors associated with changes in the prevalence of antimicrobial-resistant Enterococcus spp. applicable to the Canadian farm-to-fork beef continuum. Methods: Five databases (MEDLINE, BIOSIS, Web of Science, Embase, and CAB Abstracts) were searched for articles published from January 1984 to March 2022, using a priori inclusion criteria. Peer-reviewed articles were included if they met all the following criteria: written in English, applicable to the Canadian beef production context, primary research, in vivo research, describing an intervention or exposure, and specific to Enterococcus spp. Results: Out of 804 screened articles, 26 were selected for inclusion. The included articles discussed 37 factors potentially associated with AMR in enterococci, with multiple articles discussing at least two of the same factors. Factors discussed included antimicrobial administration (n = 16), raised without antimicrobials (n = 6), metal supplementation (n = 4), probiotics supplementation (n = 3), pen environment (n = 2), essential oil supplementation (n = 1), grass feeding (n = 1), therapeutic versus subtherapeutic antimicrobial use (n = 1), feeding wet distiller grains with solubles (n = 1), nutritional supplementation (n = 1) and processing plant type (n = 1). Results were included irrespective of their quality of evidence. Discussion: Comparability issues arising throughout the review process were related to data aggregation, hierarchical structures, study design, and inconsistent data reporting. Findings from articles were often temporally specific in that resistance was associated with AMR outcomes at sampling times closer to exposure compared to studies that sampled at longer intervals after exposure. Resistance was often nuanced to unique gene and phenotypic resistance patterns that varied with species of enterococci. Intrinsic resistance and interpretation of minimum inhibitory concentration varied greatly among enterococcal species, highlighting the importance of caution when comparing articles and generalizing findings. Systematic Review Registration: [http://hdl.handle.net/1880/113592].

Comparative Genomic Analyses of Lactococcus garvieae Isolated from Bovine Mastitis in China.

Lactococcus garvieae is an emerging zoonotic pathogen, but there are few reports regarding bovine mastitis. The prevalence of L. garvieae represents an increasing disease threat and global public health risk. Thirty-nine L. garvieae isolates were obtained from 2,899 bovine clinical mastitis milk samples in 6 provinces of China from 2017 to 2021. Five clonal complexes were determined from 32 multilocus sequence types (MLSTs) of L. garvieae: sequence type 46 (ST46) was the predominant sequence type, and 13 novel MLSTs were identified. All isolates were resistant to chloramphenicol and clindamycin, but susceptible to penicillin, ampicillin, amoxicillin-clavulanic acid, imipenem, ceftiofur, enrofloxacin, and marbofloxacin. Based on genomic analyses, L. garvieae had 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase. Most isolates had lsaD and mdtA antimicrobial resistance (AMR) genes. Based on COG (Clusters of Orthologous Genes database) results, the functions of defense, transcription and replication, and recombination and repair were enhanced in unique genes, whereas functions of translation, ribosomal structure, and biogenesis were enhanced in core genes. The KEGG functional categories enriched in unique genes included human disease and membrane transport, whereas COG functional categories enriched in core genes included energy metabolism, nucleotide metabolism, and translation. No gene was significantly associated with host specificity. In addition, analysis of core genome single nucleotide polymorphisms (SNPs) implied potential host adaptation of some isolates in several sequence types. In conclusion, this study characterized L. garvieae isolated from mastitis and detected potential adaptations of L. garvieae to various hosts. IMPORTANCE This study provides important genomic insights into a bovine mastitis pathogen, Lactococcus garvieae. Comprehensive genomic analyses of L. garvieae from dairy farms have not been reported. This study is a detailed and comprehensive report of novel features of isolates of L. garvieae, an important but poorly characterized bacterium, recovered in the past 5 years in 6 Chinese provinces. We documented diverse genetic features, including predominant sequence type ST46 and 13 novel MLSTs. Lactococcus garvieae had 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase and resistance to chloramphenicol and clindamycin. Most isolates had lsaD and mdtA antimicrobial resistance genes. However, no gene was significantly associated with host specificity. This is the first report that characterized L. garvieae isolates from bovine mastitis and revealed potential host adaptations of L. garvieae to various hosts.

Invited review: Selective treatment of clinical mastitis in dairy cattle.

Treatment of clinical mastitis (CM) and use of antimicrobials for dry cow therapy are responsible for the majority of animal-defined daily doses of antimicrobial use (AMU) on dairy farms. However, advancements made in the last decade have enabled excluding nonsevere CM cases from antimicrobial treatment that have a high probability of cure without antimicrobials (no bacterial causes or gram-negative, excluding Klebsiella spp.) and cases with a low bacteriological cure rate (chronic cases). These advancements include availability of rapid diagnostic tests and improved udder health management practices, which reduced the incidence and infection pressure of contagious CM pathogens. This review informed an evidence-based protocol for selective CM treatment decisions based on a combination of rapid diagnostic test results, review of somatic cell count and CM records, and elucidated consequences in terms of udder health, AMU, and farm economics. Relatively fast identification of the causative agent is the most important factor in selective CM treatment protocols. Many reported studies did not indicate detrimental udder health consequences (e.g., reduced clinical or bacteriological cures, increased somatic cell count, increased culling rate, or increased recurrence of CM later in lactation) after initiating selective CM treatment protocols using on-farm testing. The magnitude of AMU reduction following a selective CM treatment protocol implementation depended on the causal pathogen distribution and protocol characteristics. Uptake of selective treatment of nonsevere CM cases differs across regions and is dependent on management systems and adoption of udder health programs. No economic losses or animal welfare issues are expected when adopting a selective versus blanket CM treatment protocol. Therefore, selective CM treatment of nonsevere cases can be a practical tool to aid AMU reduction on dairy farms.

Contextualized understandings of dairy farmers' perspectives on antimicrobial use and regulation in Alberta, Canada.

Antimicrobial resistance (AMR) has been largely attributed to antimicrobial use (AMU). To achieve judicious AMU, much research and many policies focus on knowledge translation and behavioral change mechanisms. To address knowledge gaps in contextual drivers of decisions made by dairy farmers concerning AMU, we conducted ethnographic fieldwork to investigate one community's understanding of AMU, AMR, and associated regulations in the dairy industry in Alberta, Canada. This included participation in on-farm activities and observations of relevant interactions on dairy farms in central Alberta for 4 mo. Interviews were conducted with 25 dairy farmers. The interviews were analyzed using thematic analysis and yielded several key findings. Many dairy farmers in this sample: (1) value their autonomy and hope to maintain agency regarding AMU; (2) have shared cultural and immigrant identities which may inform their perspectives of future AMU regulation as it relates to their farming autonomy; (3) feel that certain AMU policies implemented in other contexts would be impractical in Alberta and would constrain their freedom to make what they perceive to be the best animal welfare decisions; (4) believe that their knowledge and experience are undervalued by consumers and policy makers; (5) are concerned that the public does not have a complex understanding of dairy farming and, consequently, worry that AMU policy will be based on misguided consumer concerns; and (6) are variably skeptical of a link between AMU in dairy cattle and AMR in humans due to their strict adherence to milk safety protocols that is driven by their genuine care for the integrity of the product. A better understanding of the sociocultural and political-economic infrastructure that supports such perceptions is warranted and should inform efforts to improve AMU stewardship and future policies regarding AMU.

Selective treatment of nonsevere clinical mastitis does not adversely affect cure, somatic cell count, milk yield, recurrence, or culling: A systematic review and meta-analysis.

Treatment of clinical mastitis (CM) contributes to antimicrobial use on dairy farms. Selective treatment of CM based on bacterial diagnosis can reduce antimicrobial use, as not all cases of CM will benefit from antimicrobial treatment, e.g., mild and moderate gram-negative infections. However, impacts of selective CM treatment on udder health and culling are not fully understood. A systematic search identified 13 studies that compared selective versus blanket CM treatment protocols. Reported outcomes were synthesized with random-effects models and presented as risk ratios or mean differences. Selective CM treatment protocol was not inferior to blanket CM treatment protocol for the outcome bacteriological cure. Noninferiority margins could not be established for the outcomes clinical cure, new intramammary infection, somatic cell count, milk yield, recurrence, or culling. However, no differences were detected between selective and blanket CM treatment protocols using traditional analyses, apart from a not clinically relevant increase in interval from treatment to clinical cure (0.4 d) in the selective group and higher proportion of clinical cure at 14 d in the selective group. The latter occurred in studies co-administering nonsteroidal anti-inflammatories only in the selective group. Bias could not be ruled out in most studies due to suboptimal randomization, although this would likely only affect subjective outcomes such as clinical cure. Hence, findings were supported by a high or moderate certainty of evidence for all outcome measures except clinical cure. In conclusion, this review supported the assertion that a selective CM treatment protocol can be adopted without adversely influencing bacteriological and clinical cure, somatic cell count, milk yield, and incidence of recurrence or culling.

Melatonin improves post-thaw sperm quality after mild testicular heat stress in rams.

The objective was to determine effects of slow-release melatonin on post-thaw sperm quality in rams exposed to mild testicular heat stress (HS; scrotal neck insulation). Twelve yearling Dorset rams were randomly and equally allocated to receive either 36 mg melatonin in 1 ml corn oil or 1 ml corn oil injected subcutaneously (SQ); 15 day later, all rams had HS for 96 h (start of HS = start of Week 0). Semen was collected before HS and once weekly from Weeks 1 to 7, extended in Steridyl CSS One Step, held at 5°C for ~3 h, loaded into 0.5 ml straws, held 5 cm above liquid nitrogen for 10 min and then plunged. Computer assisted semen analysis (CASA) was conducted on frozen-thawed sperm. There were group and week effects for total and progressive motility (p < .001), plus group and week effects and group*week interactions (p < .001) for post-thaw total abnormalities, acrosome integrity, post-thaw sperm DNA fragmentation index (DFI) and high mitochondrial membrane potential (HMMP). Post-thaw sperm total and progressive motility, acrosome integrity and HMMP were higher (p < .05) in melatonin versus control groups from Weeks 1 to 7, and the melatonin group reached baseline level (pre-heat stress) at Week 7 (75.79 ± 0.96, 65.48 ± 1.51, 75.00 ± 0.89 and 67.00 ± 1.06, respectively; mean ± SEM). Conversely, post-thaw sperm total abnormalities and DFI were lower (p < .05) in melatonin versus control, and both reached baseline at Week 7 in the melatonin group (26.00 ± 0.57 and 5.66 ± 0.17, respectively). Coiled tails, distal midpiece reflexes, distal cytoplasmic droplets, ruffled acrosomes, bowed midpieces, pyriform heads and knobbed acrosomes were the most common abnormalities in both groups, with lower percentages in melatonin-treated rams. Results supported our hypothesis that HS reduces post-thaw sperm quality, and that melatonin lessens those reductions, manifested by significantly better total and progressive motility, acrosome integrity and HMMP, and fewer sperm total abnormalities and DFI.

Biological and genomic characteristics of two bacteriophages isolated from sewage, using one multidrug-resistant and one non-multidrug-resistant strain of Klebsiella pneumoniae.

Bovine mastitis caused by multi-drug resistant (MDR) Klebsiella pneumoniae is difficult to treat with antibiotics, whereas bacteriophages may be a viable alternative. Our objective was to use 2 K. pneumoniae strains, 1 MDR and the other non-MDR, to isolate phages from sewage samples and compare their biological and genomic characteristics. Additionally, phage infected mouse mammary gland was also analyzed by H&E staining and ELISA kits to compare morphology and inflammatory factors, respectively. Based on assessments with double agar plates and transmission electron microscopy, phage CM_Kpn_HB132952 had clear plaques surrounded by translucent halos on the bacterial lawn of K. pneumoniae KPHB132952 and belonged to Siphoviridae, whereas phage CM_Kpn_HB143742 formed a clear plaque on the bacterial lawn of K. pneumoniae KPHB143742 and belonged to Podoviridae. In 1-step growth curves, CM_Kpn_HB132952 and CM_Kpn_HB143742 had burst sizes of 0.34 and 0.73 log10 PFU/mL, respectively. The former had a latent period of 50 min and an optimal multiplicity of infection (MOI) of 0.01, whereas for the latter, the latent period was 30 min (MOI = 1). Phage CM_Kpn_HB132952 had better thermal and acid-base stability than phage CM_Kpn_HB143742. Additionally, both phages had the same host range rate but different host ranges. Based on Illumina NovaSeq, phages CM_Kpn_HB132952 and CM_Kpn_HB143742 had 140 and 145 predicted genes, respectively. Genomic sequencing and phylogenetic tree analysis indicated that both phages were novel phages belonging to the Klebsiella family. Additionally, the histopathological structure and inflammatory factors TNF-α and IL-1ß were not significantly different among phage groups and the control group. In conclusion, using 1 MDR and 1 non-MDR strain of K. pneumoniae, we successfully isolated two phages from the same sewage sample, and demonstrated that they had distinct biological and genomic characteristics.

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.