Browsing by Author "Liu, Gang"

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    Bacteriophages isolated from dairy farm mitigated Klebsiella pneumoniae-induced inflammation in bovine mammary epithelial cells cultured in vitro
    (2021-01-19) Shi, Yuxiang; Zhao, Wenpeng; Liu, Gang; Ali, Tariq; Chen, Peng; Liu, Yongxia; Kastelic, John P; Han, Bo; Gao, Jian
    Abstract 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-1β concentrations. K. pneumoniae enhanced mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, 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.
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    Characteristics of Escherichia coli Isolated from Bovine Mastitis Exposed to Subminimum Inhibitory Concentrations of Cefalotin or Ceftazidime
    (2018-11-01) Liu, Gang; Ding, Laidi; Han, Bo; Piepers, Sofie; Naqvi, S. Ali; Barkema, Herman W.; Ali, Tariq; De Vliegher, Sarne; Xu, Siyu; Gao, Jian
    Escherichia coli is a major udder pathogen causing clinical mastitis in dairy cattle and its heat stable endotoxin in powdered infant formula milk is a potential risk factor in neonatal infections. Cephalosporins are frequently used for treatment of mastitis caused by mastitis; however, use of these antimicrobials may induce antimicrobial resistance in E. coli. The objective of this study was to explore the in vitro effect of subminimum inhibitory concentrations (sub-MIC) of cefalotin (CF) and ceftazidime (CAZ) on the morphology, antimicrobial resistance, and endotoxin releasing characteristics of 3 E. coli isolates recovered from bovine clinical mastitis. The parent E. coli isolates, which were susceptible to CF and CAZ, were exposed to CF or CAZ separately at sub-MIC levels to produce 9 generations of induced isolates. Colonies of the CAZ-induced isolates from all 3 parent E. coli were smaller on blood agar and the bacteria became filamentous, whereas the CF-induced isolates did not demonstrate prominent morphological changes. After induction by CF or CAZ, many induced isolates showed resistance to cefoxitin, CAZ, CF, kanamycin, ampicillin, and amoxicillin/clavulanic acid while their parent isolates were susceptible to these antimicrobials. Notably, 5 CAZ-induced isolates from the same parent isolate were found to produce extended-spectrum beta-lactamase (ESBL) though none of the tested ESBL related genes could be detected. All CAZ-induced isolates released more endotoxin with a higher release rate, whereas endotoxin release of CF-induced E. coli isolates was not different from parent isolates. The exposure of cephalosporins at sub-MIC levels induced resistant Escherichia coli. We inferred that cephalosporins, especially CAZ, should be used prudently for treatment of clinical E. coli mastitis.
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    Klebsiella pneumoniae infection causes mitochondrial damage and dysfunction in bovine mammary epithelial cells
    (2021-02-10) Cheng, Jia; Zhang, Jv; Yang, Jingyue; Yi, Bing; Liu, Gang; Zhou, Man; Kastelic, John P; Han, Bo; Gao, Jian
    Abstract 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.
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    MicroRNA miR-223 modulates NLRP3 and Keap1, mitigating lipopolysaccharide-induced inflammation and oxidative stress in bovine mammary epithelial cells and murine mammary glands
    (2023-09-14) Zhou, Man; Barkema, Herman W.; Gao, Jian; Yang, Jingyue; Wang, Yue; Kastelic, John P.; Khan, Sohrab; Liu, Gang; Han, Bo
    Abstract 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-1β, 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.
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    Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro
    (2021-10-14) Liu, Yang; Deng, Zhaoju; Xu, Siyu; Liu, Gang; Lin, Yushan; Khan, Sohrab; Gao, Jian; Qu, Weijie; Kastelic, John P.; Han, Bo
    Abstract 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.
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    Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-κB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures
    (2021-12-11) Zhao, Wenpeng; He, Fumeng; Barkema, Herman W.; Xu, Siyu; Gao, Jian; Liu, Gang; Deng, Zhaoju; Shahid, Muhammad; Shi, Yuxiang; Kastelic, John P.; Han, Bo
    Abstract 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-1β 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-1β, and IL-1β 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.
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    Rapid and reliable detection of Leishmania antibodies in canine serum with double-antigen sandwich homogeneous chemical luminescence
    (2024-07-30) Zhao, Xiangjun; Ma, Licai; Jin, Yipeng; Barkema, Herman W.; Kastelic, John P.; Wang, Lu; Wen, Kai; Liu, Gang
    Abstract Background Leishmaniasis, caused by Leishmania spp. parasites, is an important zoonotic disease globally, posing severe threats to humans and animals. In the absence of effective vaccines, reliable serological diagnostic methods are critical for disease control. However, the enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay have limitations due to complexity, time required and/or sensitivity. Therefore, our objective was to develop an accurate, rapid and user-friendly detection method of canine leishmania antibody based on double-antigen sandwich homogeneous chemical luminescence. Methods Homogeneous chemiluminescent technology was employed, and expressed recombinant fusion proteins containing full-length K9, K39 and K26 repeat sequences were used as diagnostic antigens. To establish a dual-antigen sandwich serological assay capable of detecting various antibody types, a factorial design was used to optimize concentrations of diagnostic antigen-receptor microspheres and of biotinylated diagnostic antigens, as well as of reaction solution composition and reaction duration. To evaluate and validate this newly developed method, we collected 41 Leishmania-positive serum samples, 30 Leishmania-negative control serum samples and 78 clinical serum samples for which no diagnostic information was available. Comparative analyses were performed using parasitological testing and an indirect ELISA as reference methods, focusing on diagnostic sensitivity and specificity. Results Sodium dodecyl sulfate–polyacrylamide gel electrophoresis confirmed the purification of the diagnostic antigens, which exhibited clear bands without impurities. Based on results from the 41 Leishmania-positive samples and 30 Leishmania-negative samples, there was sufficient sensitivity to detect samples diluted up to 256-fold, with analytical specificity of 100%. Overall diagnostic sensitivity was 100% and diagnostic specificity was 93.3%. Diagnostic performance was highly consistent between the newly developed method and the indirect ELISA (Kappa = 0.82, P < 0.01). Testing could be completed within 35 min with the new method Conclusions We have developed a novel double-antigen sandwich homogeneous chemical luminescence method to detect canine Leishmania antibodies, with high sensitively and specificity, a short incubation interval and a simple protocol. This streamlined approach not only offers a sensitive and efficient method for clinical diagnosis but also has great potential for use in automated testing. Graphical Abstract
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    Re-emergence of canine Leishmania infantum infection in mountain areas of Beijing
    (2023-03-30) Liu, Gang; Wu, Yuanheng; Wang, Lei; Liu, Yang; Huang, Wei; Li, Yifan; Gao, Mengbo; Kastelic, John; Barkema, Herman W.; Xia, Zhaofei; Jin, Yipeng
    Abstract Canine Leishmaniasis (CanL) is an endemic infectious disease in China, causing visceral Leishmaniasis (VL) and resulting in important public health problem. However, in the last 3 y, endemic trends have changed considerably and spatial–temporal aggregation areas have shifted from northwestern to central China. Although Beijing was an endemic area for CanL in the last century, this disease has not been reported in Beijing since control programs were implemented in the 1950s. In the present study, PCR and immunochromatographic (ICT) were used to estimate prevalence of Leishmania infection in domestic dogs living in Beijing, a VL re -emergencearea. In total, 4420 canine blood samples were collected at vet clinics in 14 districts of Beijing. Overall prevalence (percentage of dogs seropositive and/or PCR positive) of CanL infection in Beijing was 1.22% (54/4420). However, prevalence of CanL in the western mountain areas was 4.68% (45/961), significantly higher than that (0.26%, 9/3459) of the plains. In addition, multilocus sequence typing (MLST) of seven enzyme-coding genes was used to examine phylogenetic relationships of CanL strains. Forty-one Leishmania infantum isolates were well separated from the other strains and divided into five major clades (A to E) by MLST analysis. All clades were closely related to strains from Sichuan Province and Gansu Province. A phylogenetic tree, based on the MLST, revealed that L. infantum in Beijing was genetically related to strains from western endemic of Mountain type VL in China. In conclusion, CanL has re-emerged in Beijing, and almost 5% of dogs living in Beijing’s mountain areas were infected with L. infantum. The phylogenetic tree based on MLST effectively distinguished species of Leishmania and reflected geographical origins. Because dogs are considered a natural reservoir, comprehensive control measures including surveillance, phylogenetic analyses and management should be implemented to mitigate or eliminate Leishmaniasis.
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    Virulence gene profiles: alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China
    (2018-03-02) Zhang, Limei; Gao, Jian; Barkema, Herman W; Ali, Tariq; Liu, Gang; Deng, Youtian; Naushad, Sohail; Kastelic, John P; Han, Bo
    Abstract Background Staphylococcus aureus, a common cause of bovine mastitis, is known for its ability to acquire to antimicrobial resistance and to secrete numerous virulence factors that can exacerbate inflammation. In addition, alpha-hemolysin has an important role in S. aureus infections, diversity of the hla gene (that produces alpha-hmolysin) in S. aureus isolated from bovine mastitis has not been well characterized. The objective was, therefore, to determine diversity of virulence genes, hla gene sequences, and clonal profiles of S. aureus from bovine mastitis in Chinese dairy herds, and to evaluate inter-relationships. Results The antimicrobials resistance varies from as low as 1.9% (2/103) for CTX to as high as 76.7% (79/103) for penicilin in the 103 isolates and 46 (44.7%) S. aureus were determined as multi-resistant isolates with diverse resistance patterns. Thirty-eight virulence gene patterns (with variable frequencies) were identified in the 103 isolates and correlated with MLST types, indicating a great diversity. Although the hla gene also had great diversity (14 genotypes), Hla peptides were relatively more conserved. With 7 clonal complexes identified from 24 spa types and 7 MLST types. Regarding the letter, ST 97 was the dominant type in S. aureus from bovine mastitis in China. Furthermore, based on phylogenetic analysis, there was a distinct evolutionary relationship between the hla gene and MLST. Conclusion Multi-resistant S. aureus occurred in bovine mastitis with diverse resistance patterns. The diversity of virulence gene profiles, especially the hla gene and, their relationship with molecular types were reported for the first time in S. aureus from bovine mastitis, which will be useful for future studies on immunogenicity and vaccine development. In addition, based on the distinct evolutionary relationship between the hla gene and MLST types, we inferred that the hla gene has potential role for molecular typing of S. aureus.