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.

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.

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.

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.

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.

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.

Characterization of the Testis-Specific Angiotensin Converting Enzyme (tACE)-Interactome during Bovine Sperm Capacitation.

A comprehensive understanding of molecular and biochemical changes during sperm capacitation is critical to the success of assisted reproductive technologies. We reported involvement of the testis-specific isoform of Angiotensin Converting Enzyme (tACE) in bovine sperm capacitation. The objective of this study was to characterize the tACE interactome in fresh and heparin-capacitated bovine sperm through immunoprecipitation coupled with mass spectrometry. These interactions were validated by co-localization of tACE with beta-tubulin as an identified interactome constituent. Although interactions between tACE and several proteins remained unchanged in fresh and capacitated sperm, mitochondrial aldehyde dehydrogenase 2 (ALDH2), inactive serine/threonine protein-kinase 3 (VRK3), tubulin-beta-4B chain (TUBB4B), and tubulin-alpha-8 chain (TUBA8) were recruited during capacitation, with implications for cytoskeletal and membrane reorganization, vesicle-mediated transport, GTP-binding, and redox regulation. A proposed tACE interactional network with identified interactome constituents was generated. Despite tACE function being integral to capacitation, the relevance of interactions with its binding partners during capacitation and subsequent events leading to fertilization remains to be elucidated.

Invited review: Selective use of antimicrobials in dairy cattle at drying-off.

Administering intramammary antimicrobials to all mammary quarters of dairy cows at drying-off [i.e., blanket dry cow therapy (BDCT)] has been a mainstay of mastitis prevention and control. However, as udder health has considerably improved over recent decades with reductions in intramammary infection prevalence at drying-off and the introduction of teat sealants, BDCT may no longer be necessary on all dairy farms, thereby supporting antimicrobial stewardship efforts. This narrative review summarizes available literature regarding current dry cow therapy practices and associated impacts of selective dry cow therapy (SDCT) on udder health, milk production, economics, antimicrobial use, and antimicrobial resistance. Various methods to identify infections at drying-off that could benefit from antimicrobial treatment are described for selecting cows or mammary quarters for treatment, including utilizing somatic cell count thresholds, pathogen identification, previous clinical mastitis history, or a combination of criteria. Selection methods may be enacted at the herd, cow, or quarter levels. Producers' and veterinarians' motivations for antimicrobial use are discussed. Based on review findings, SDCT can be adopted without negative consequences for udder health and milk production, and concurrent teat sealant use is recommended, especially in udder quarters receiving no intramammary antimicrobials. Furthermore, herd selection should be considered for SDCT implementation in addition to cow or quarter selection, as BDCT may still be temporarily necessary in some herds for optimal mastitis control. Costs and benefits of SDCT vary among herds, whereas impacts on antimicrobial resistance remain unclear. In summary, SDCT is a viable management option for maintaining udder health and milk production while improving antimicrobial stewardship in the dairy industry.

Testis-Specific Isoform of Na+-K+ ATPase and Regulation of Bull Fertility.

An advanced understanding of sperm function is relevant for evidence-based male fertility prediction and addressing male infertility. A standard breeding soundness evaluation (BSE) merely identifies gross abnormalities in bulls, whereas selection based on single nucleotide polymorphisms and genomic estimated breeding values overlooks sub-microscopic differences in sperm. Molecular tools are important for validating genomic selection and advancing knowledge on the regulation of male fertility at an interdisciplinary level. Therefore, research in this field is now focused on developing a combination of in vitro sperm function tests and identifying biomarkers such as sperm proteins with critical roles in fertility. The Na+-K+ ATPase is a ubiquitous transmembrane protein and its α4 isoform (ATP1A4) is exclusively expressed in germ cells and sperm. Furthermore, ATP1A4 is essential for male fertility, as it interacts with signaling molecules in both raft and non-raft fractions of the sperm plasma membrane to regulate capacitation-associated signaling, hyperactivation, sperm-oocyte interactions, and activation. Interestingly, ATP1A4 activity and expression increase during capacitation, challenging the widely accepted dogma of sperm translational quiescence. This review discusses the literature on the role of ATP1A4 during capacitation and fertilization events and its prospective use in improving male fertility prediction.

Prevalence of antimicrobial resistance genes and its association with restricted antimicrobial use in food-producing animals: a systematic review and meta-analysis.

BACKGROUND: There is ongoing debate regarding potential associations between restrictions of antimicrobial use and prevalence of antimicrobial resistance (AMR) in bacteria. OBJECTIVES: To summarize the effects of interventions reducing antimicrobial use in food-producing animals on the prevalence of AMR genes (ARGs) in bacteria from animals and humans. METHODS: We published a full systematic review of restrictions of antimicrobials in food-producing animals and their associations with AMR in bacteria. Herein, we focus on studies reporting on the association between restricted antimicrobial use and prevalence of ARGs. We used multilevel mixed-effects models and a semi-quantitative approach based on forest plots to summarize findings from studies. RESULTS: A positive effect of intervention [reduction in prevalence or number of ARGs in group(s) with restricted antimicrobial use] was reported from 29 studies for at least one ARG. We detected significant associations between a ban on avoparcin and diminished presence of the vanA gene in samples from animals and humans, whereas for the mecA gene, studies agreed on a positive effect of intervention in samples only from animals. Comparisons involving mcr-1, blaCTX-M, aadA2, vat(E), sul2, dfrA5, dfrA13, tet(E) and tet(P) indicated a reduced prevalence of genes in intervention groups. Conversely, no effects were detected for ß-lactamases other than blaCTX-M and the remaining tet genes. CONCLUSIONS: The available body of scientific evidence supported that restricted use of antimicrobials in food animals was associated with an either lower or equal presence of ARGs in bacteria, with effects dependent on ARG, host species and restricted drug.

Ouabain-induced activation of phospholipase C zeta and its contributions to bovine sperm capacitation.

The sperm-derived oocyte activating factor, phospholipase C zeta (PLC ζ), is the only PLC isoform reported in cattle. The objectives were to (1) localize PLC ζ in fresh and capacitated bovine sperm and (2) investigate the activation of PLC ζ during bull sperm capacitation and contributions of PLC activity to this process. We confirmed interaction of testis-specific isoform of Na/K-ATPase (ATP1A4) with PLC ζ (immunolocalization and immunoprecipitation) and tyrosine phosphorylation (immunoprecipitation) of PLC ζ (a post-translational protein modification commonly involved in activation of PLC in somatic cells) during capacitation. Furthermore, incubation of sperm under capacitating conditions upregulated PLC-mediated hyperactivated motility, tyrosine phosphoprotein content, acrosome reaction, and F-actin formation (flow cytometry), implying that PLC activity is enhanced during capacitation and contributing to these capacitation processes. In conclusion, we inferred that PLC ζ is activated during capacitation by tyrosine phosphorylation through a mechanism involving ATP1A4, contributing to capacitation-associated biochemical events.

Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro.

Mycoplasma species are the smallest prokaryotes capable of self-replication. To investigate Mycoplasma induced autophagy in mammalian cells, Mycoplasma bovis (M. bovis) and bovine mammary epithelial cells (bMEC) were used in an in vitro infection model. Initially, intracellular M. bovis was enclosed within a membrane-like structure in bMEC, as viewed with transmission electron microscopy. In infected bMEC, increased LC3II was verified by Western blotting, RT-PCR and laser confocal microscopy, confirming autophagy at 1, 3 and 6 h post-infection (hpi), with a peak at 6 hpi. However, the M. bovis-induced autophagy flux was subsequently blocked. P62 degradation in infected bMEC was inhibited at 3, 6, 12 and 24 hpi, based on Western blotting and RT-PCR. Beclin1 expression decreased at 12 and 24 hpi. Furthermore, autophagosome maturation was subverted by M. bovis. Autophagosome acidification was inhibited by M. bovis infection, based on detection of mCherry-GFP-LC3 labeled autophagosomes; the decreases in protein levels of Lamp-2a indicate that the lysosomes were impaired by infection. In contrast, activation of autophagy (with rapamycin or HBSS) overcame the M. bovis-induced blockade in phagosome maturation by increasing delivery of M. bovis to the lysosome, with a concurrent decrease in intracellular M. bovis replication. In conclusion, although M. bovis infection induced autophagy in bMEC, the autophagy flux was subsequently impaired by inhibiting autophagosome maturation. Therefore, we conclude that M. bovis subverted autophagy to promote its intracellular replication in bMEC. These findings are the impetus for future studies to further characterize interactions between M. bovis and mammalian host cells.

Klebsiella pneumoniae infection causes mitochondrial damage and dysfunction in bovine mammary epithelial cells.

Klebsiella pneumoniae, an important cause of bovine mastitis worldwide, is strongly pathogenic to bovine mammary epithelial cells (bMECs). Our objective was to determine the role of mitochondrial damage in the pathogenicity of K. pneumoniae on bMECs, by assessing several classical indicators of mitochondrial dysfunction, as well as differentially expressed genes (DEGs). Two K. pneumoniae strains (HLJ-D2 and HB-AF5), isolated from cows with clinical mastitis (CM), were used to infect bMECs (MAC-T line) cultured in vitro. In whole-transcriptome analysis of bMECs at 6 h post-infection (hpi), there were 3453 up-regulated and 3470 down-regulated genes for HLJ-D2, whereas for HB-AF5, there were 2891 up-regulated and 3278 down-regulated genes (P < 0.05). Based on GO term enrichment of differentially expressed genes (DEGs), relative to the controls, the primary categories altered in K. pneumoniae-infected bMECs included cellular macromolecule metabolism, metabolic process, binding, molecular function, etc. Infections increased (P < 0.05) malondialdehyde concentrations and formation of reactive oxygen species in bMECs. Additionally, both bacterial strains decreased (P < 0.05) total antioxidant capacity in bMECs at 6 and 12 hpi. Furthermore, infections decreased (P < 0.05) mitochondrial membrane potential and increased (P < 0.01) mitochondrial calcium concentrations. Finally, severe mitochondrial swelling and vacuolation, as well as mitochondrial rupture and cristae degeneration, were detected in infected bMECs. In conclusion, K. pneumoniae infections induced profound mitochondrial damage and dysfunction in bMECs; we inferred that this caused cellular damage and contributes to the pathogenesis of K. pneumoniae-induced CM in dairy cows.

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.

Bacteriophages isolated from dairy farm mitigated Klebsiella pneumoniae-induced inflammation in bovine mammary epithelial cells cultured in vitro.

BACKGROUND: Klebsiella pneumoniae, an environmental pathogen causing mastitis in dairy cattle, is often resistant to antibiotics. K. pneumoniae was used as the host bacteria to support bacteriophage replication; 2 bacteriophages, CM8-1 and SJT-2 were isolated and considered to have therapeutic potential. In the present study, we determined the ability of these 2 bacteriophages to mitigate cytotoxicity, pathomorphological changes, inflammatory responses and apoptosis induced by K. pneumoniae (bacteriophage to K. pneumoniae MOI 1:10) in bovine mammary epithelial cells (bMECs) cultured in vitro. RESULTS: Bacteriophages reduced bacterial adhesion and invasion and cytotoxicity (lactate dehydrogenase release). Morphological changes in bMECs, including swelling, shrinkage, necrosis and hematoxylin and eosin staining of cytoplasm, were apparent 4 to 8 h after infection with K. pneumoniae, but each bacteriophage significantly suppressed damage and decreased TNF-α and IL-1ß concentrations. K. pneumoniae enhanced mRNA expression of TLR4, NF-κB, TNF-α, IL-1ß, IL-6, IL-8, caspase-3, caspase-9 and cyt-c in bMECs and increased apoptosis of bMECs, although these effects were mitigated by treatment with either bacteriophage for 8 h. CONCLUSIONS: Bacteriophages CM8-1 and SJT-2 mitigated K. pneumoniae-induced inflammation in bMECs cultured in vitro. Therefore, the potential of these bacteriophages for treating mastitis in cows should be determined in clinical trials.

Bacteriophage has beneficial effects in a murine model of Klebsiella pneumoniae mastitis.

Bovine mastitis caused by Klebsiella pneumoniae is usually treated with antibiotics, thereby potentially increasing antimicrobial resistance. The objective of this study was to evaluate efficacy of a bacteriophage, isolated from dairy farm wastewater, as a treatment for a murine model of K. pneumoniae mastitis. A lytic bacteriophage CM8-1 was isolated, morphological and biological characteristics were assessed with transmission electron microscopy and double-layer plate, and its genome was sequenced and analyzed. Furthermore, effectiveness of this bacteriophage for treatment of a murine model of K. pneumoniae mastitis was evaluated based on the following mammary gland characteristics: morphological changes; number of K. pneumoniae; and mRNA and protein expression of pro-inflammatory factors TNF-α, IL-1ß, IL-6, and IL-8. Bacteriophage CM8-1 had an incubation period of 30 min and a burst time of 20 min. Its viability and adsorption were stable at 30 to 50°C, but decreased significantly at >60°C, with no significant change in viability or infectivity at pH 6 to 10. In a murine model of K. pneumoniae mastitis, injecting bacteriophage CM8-1 into the mammary gland 2 h after inoculation with K. pneumoniae resulted in reductions in bacterial counts in the murine mammary gland, improvements in mammary gland tissue morphology, and reductions in mRNA and protein expression of pro-inflammatory factors. Bacteriophage CM8-1 had stable biological characteristics and suppressed K. pneumoniae mastitis when injected into the mammary gland 2 h latera in mice bacterial inoculation.

Selenomethionine activates selenoprotein S, suppresses Fas/FasL and the mitochondrial pathway, and reduces Escherichia coli-induced apoptosis of bovine mammary epithelial cells.

Escherichia coli is a major environmental pathogen causing bovine mastitis, characterized by cell death and mammary tissue damage. Apoptosis, a form of cell death, has an important role in the pathogenesis of mastitis. Selenium, an essential trace element, protects against mastitis by acting through several biochemical pathways, potentially including prevention of apoptosis. Our objective was to investigate whether selenomethionine (SeMet) attenuated E. coli-induced apoptosis in bovine mammary epithelial cells (bMEC). These cells were cultured in vitro and treated with 0, 5, 10, 20, and 40 µM SeMet for 12 h, with or without E. coli (multiplicity of infection of 5) for 8 h. Treatment with SeMet/Z-IE(OMe)TD(OMe)-FMK (ZIK)/Z-LE(OMe)HD(OMe)-FMK (ZLK, specific inhibitors of caspase-8 and -9, respectively) significantly counteracted effects of E. coli on bMEC. Specifically, SeMet upregulated selenoprotein S (SeS) and increased mitochondrial membrane potential and the ratio of Bcl-2 and Bax. Furthermore, it decreased protein expressions of Fas, FasL, FADD, cleaved caspase-8, cytochrome c, cleaved caspase-9, and cleaved caspase-3, namely, decreasing protein expression of the Fas/FasL and mitochondrial pathways. Furthermore, it downregulated total apoptosis indexes in E. coli-infected bMEC. Although ZIK and ZLK (specific inhibitors of caspases 8 and 9, respectively) significantly inhibited Fas/FasL and the mitochondrial apoptotic pathway and apoptosis indexes, respectively, substantial apoptosis still occurred. In conclusion, SeMet attenuated E. coli-induced apoptosis in bMEC by activating SeS, associated with Fas/FasL and mitochondrial pathways.

Genetic diversity and molecular epidemiology of outbreaks of Klebsiella pneumoniae mastitis on two large Chinese dairy farms.

Klebsiella pneumoniae is an opportunistic and environmental mastitis-causing pathogen, with potential for contagious transmission. Repetitive element sequence-based PCR was used to determine genetic diversity and explore potential transmission and reservoirs for mastitis caused by K. pneumoniae on 2 large Chinese dairy farms. A total of 1,354 samples was collected from the 2 dairy farms, including milk samples from cows with subclinical and clinical mastitis, bedding, feces, feed, teat skin, and milking liners. Environmental samples were collected from all barns and milking parlors and extramammary samples from randomly selected dairy cows on both farms. In total, 272 and 93 K. pneumoniae isolates were obtained from Farms A and B, respectively (with ~8K and 2K lactating cows, respectively). Isolation rates from clinical mastitis (CM), subclinical mastitis (SCM), and environmental or extramammary samples were 34, 23 and 37%, respectively for Farm A and 42, 3, and 34% for Farm B. The K. pneumoniae isolated from CM milk and extramammary or environmental sources had high genetic diversity (index of diversity >90%) on the 2 farms and from SCM on Farm A. However, on Farm B, 9 SCM isolates were classified as 2 genotypes, resulting in a relatively low index of diversity (Simpson's index of diversity = 0.39; 95% CI = 0.08-0.70). Genotypes of K. pneumoniae causing mastitis were commonly detected in feces, bedding, and milking liners (Farm A), or from teat skin, sawdust bedding, and feed (Farm B). Based on its high level of genetic diversity, we inferred K. pneumoniae was an opportunistic and environmental pathogen causing outbreaks of CM on these 2 large Chinese dairy farms. Nevertheless, that only a few genotypes caused SCM implied some strains had increased udder adaptability and a contagious nature or a common extramammary source. Finally, control of intramammary infections caused by K. pneumoniae on large Chinese dairy farms must consider farm-level predictors, as the 2 outbreaks had distinct potential environmental sources of infection.

Characterization of Streptococcus lutetiensis isolated from clinical mastitis of dairy cows.

Streptococcus lutetiensis, previously termed Streptococcus bovis type II/1, has rarely been associated with bovine mastitis. The objectives of this work were to characterize the molecular diversity, antimicrobial resistance profiles, virulence genes of Strep. lutetiensis (n = 37) isolated from bovine clinical mastitis, as well as its pathogenic effects in a murine mastitis model. Genetic relationships of isolates were determined by random amplified polymorphic DNA (RAPD)-PCR, virulence genes were detected by PCR. Antimicrobial susceptibility testing was carried out by broth microdilution technique. The pathogenic effects of Strep. lutetiensis were studied with 2 infection models: bovine mammary epithelial cells cultured in vitro and murine mammary infection in vivo. Streptococcus lutetiensis isolates were clustered into 5 RAPD-types (A-E), with a dominant type A representing 84% of isolates. Eighteen (49%), 16 (43%), and 9 (24%) isolates were resistant to ceftiofur, tetracycline, and erythromycin, respectively. Prevalence of multidrug resistance (resistant to ≥3 classes of antimicrobials) was 24% (9/37). The most prevalent virulence genes were bca (100%), speG (100%), hly (97%), scpB (95%), and ssa (95%). There was no difference between isolates from mild and moderate cases of bovine mastitis in prevalence of virulence genes. Streptococcus lutetiensis rapidly adhered to and subsequently invaded (1 and 3 h after infection, respectively) bovine mammary epithelial cells, resulting in elevated lactate dehydrogenase release (4 h after infection). Edema and hyperemia were observed in challenged mammary glands and bacteria were consistently isolated at 12, 24, and 48 h after infection. In addition, numerous neutrophils migrated into gland alveoli and interstitium of infected mammary tissue. We concluded that Strep. lutetiensis had potential to spread within a dairy herd and good adaptive ability in bovine mammary cells or tissue, which are generally characteristics of a contagious mastitis pathogen.