The gut microbiota contributes to the infection of bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and causes significant economic losses. Gut microbiota is a large microbial community and has a variety of biological functions. However, whether there is a correlation between gut microbiota and BVDV infection and what kind of relation between them have not been reported. Here, we found that gut microbiota composition changed in normal mice after infecting with BVDV, but mainly the low abundance microbe was affected. Interestingly, BVDV infection significantly reduced the diversity of gut microbiota and changed its composition in gut microbiota-dysbiosis mice. Furthermore, compared with normal mice of BVDV infection, there were more viral loads in the duodenum, jejunum, spleen, and liver of the gut microbiota-dysbiosis mice. However, feces microbiota transplantation (FMT) reversed these effects. The data above indicated that the dysbiosis of gut microbiota was a key factor in the high infection rate of BVDV. It is found that the IFN-I signal was involved by investigating the underlying mechanisms. The inhibition of the proliferation and increase in the apoptosis of peripheral blood lymphocytes (PBL) were also observed. However, FMT treatment reversed these changes by regulating PI3K/Akt, ERK, and Caspase-9/Caspase-3 pathways. Furthermore, the involvement of butyrate in the pathogenesis of BVDV was also further confirmed. Our results showed for the first time that gut microbiota acts as a key endogenous defense mechanism against BVDV infection; moreover, targeting regulation of gut microbiota structure and abundance may serve as a new strategy to prevent and control the disease.IMPORTANCEWhether the high infection rate of BVDV is related to gut microbiota has not been reported. In addition, most studies on BVDV focus on in vitro experiments, which limits the study of its prevention and control strategy and its pathogenic mechanism. In this study, we successfully confirmed the causal relationship between gut microbiota and BVDV infection as well as the potential molecular mechanism based on a mouse model of BVDV infection and a mouse model of gut microbiota dysbiosis. Meanwhile, a mouse model which is more susceptible to BVDV provided in this study lays an important foundation for further research on prevention and control strategy of BVDV and its pathogenesis. In addition, the antiviral effect of butyrate, the metabolites of butyrate-producing bacteria, has been further revealed. Overall, our findings provide a promising prevention and control strategy to treat this infectious disease which is distributed worldwide.

Study on the susceptibility of bovine coronavirus to BALB/c mice.

There are no other bovine coronavirus (BCoV) infection models except calves, which makes efficacy evaluation of vaccines and pathogenic mechanism research of BCoV inconvenient owing to their high value and inconvenient operation. This study aimed to establish a mouse model of BCoV infection. BCoV was used to infect 4-week-old male BALB/c mice and the optimal infection conditions were screened, including the following infection routes: gavage, intraperitoneal injection, and tail vein injection at doses of 1 × 108 TCID50, 2 × 108 TCID50 and 4 × 108 TCID50. Using the optimal infection conditions, BALB/c mice were infected with BCoV, and their body weight, blood routine, inflammatory factors, autopsy, virus distribution, and viral load were measured at 1, 3, 5, and 7 days after infection. The results showed that the optimal conditions for infecting BALB/c mice with BCoV HLJ-325 strain were continuous oral gavage for 3 days with a dose of 4 × 108 TCID50. On the 7th day after infection, there was significant extensive consolidation of the lungs and thinning of the colon wall. Significant inflammation was observed in various organs, especially in the colon and alveoli, where a large number of inflammatory cells infiltrate. Both BCoV Ag and nucleic acid are positive in visceral organs. The viral load in the colon and lungs was significantly higher than that in the other organs (p < 0.001). BCoV-infected mice showed a decreasing trend in body weight starting from day 5, and there was a significant difference compared to the control group on days 6 and 7 (p < 0.001). The total number of white blood cells and lymphocytes began to decrease and was significantly lower than that in the control group 24 h after infection (p < 0.001), and gradually returned to the control level. The cytokine TNF-α, IL-1ß, and IL-6 showed an increasing trend, significantly higher than the control group on day 5 and 7 (p < 0.001). These results indicate that the BCoV HLJ-325 strain can infect BALB/c mice and cause inflammatory reactions and tissue lesions. The most significant effect was observed on the seventh day after infection with a dose of 4 × 108 TCID50 and three consecutive gavages. This study established, for the first time, a BALB/c mouse model of BCoV infection, providing a technical means for evaluating the immune efficacy of BCoV vaccines and studying their pathogenic mechanisms.

Preparation of Escherichia coli ghost of anchoring bovine Pasteurella multocida OmpH and its immunoprotective effect.

Pasteurella multocida is a pathogen that can infect humans and animals. A ghost is an empty bacterial body devoid of cytoplasm and nucleic acids that can be efficiently presented by antigen-presenting cells. To study a novel ghost vector vaccine with cross-immune protection, we used bacteriophage PhiX174 RF1 and Pasteurella multocida standard strain CVCC393 as templates to amplify the split genes E and OmpH to construct a bidirectional expression vector E'-OmpH-pET28a-ci857-E. This is proposed to prepare a ghost Escherichia coli (engineered bacteria) capable of attaching and producing Pasteurella multocida OmpH on the inner membrane of Escherichia coli (BL21). The aim is to assess the antibody levels and the effectiveness of immune protection by conducting a mouse immunoprotective test. The bidirectional expression vector E'-OmpH-pET28a-ci857-E was successfully constructed. After induction by IPTG, identification by SDS-PAGE, western blot, ghost culture and transmission electron microscope detection, it was proven that the Escherichia coli ghost anchored to Pasteurella multocida OmpH was successfully prepared. The immunoprotective test in mice showed that the antibody levels of Pasteurella multocida inactivated vaccine, OmpH, ghost (aluminum glue adjuvant) and ghost (Freund's adjuvant) on day 9 after immunization were significantly different from those of the PBS control group (P < 0.01). The immune protection rates were 100%, 80%, 75%, and 65%, respectively, and the PBS negative control was 0%, which proved that they all had specific immune protection effects. Therefore, this study lays the foundation for the further study of ghosts as carriers of novel vaccine-presenting proteins.

Anti-BVDV Activity of Traditional Chinese Medicine Monomers Targeting NS5B (RNA-Dependent RNA Polymerase) In Vitro and In Vivo.

Natural products have emerged as "rising stars" for treating viral diseases and useful chemical scaffolds for developing effective therapeutic agents. The nonstructural protein NS5B (RNA-dependent RNA polymerase) of NADL strain BVDV was used as the action target based on a molecular docking technique to screen herbal monomers for anti-BVDV viral activity. The in vivo and in vitro anti-BVDV virus activity studies screened the Chinese herbal monomers with significant anti-BVDV virus effects, and their antiviral mechanisms were initially explored. The molecular docking screening showed that daidzein, curcumin, artemisinine, and apigenin could interact with BVDV-NADL-NS5B with the best binding energy fraction. In vitro and in vivo tests demonstrated that none of the four herbal monomers significantly affected MDBK cell activity. Daidzein and apigenin affected BVDV virus replication mainly in the attachment and internalization phases, artemisinine mainly in the replication phase, and curcumin was active in the attachment, internalization, replication, and release phases. In vivo tests demonstrated that daidzein was the most effective in preventing and protecting BALB/C mice from BVDV infection, and artemisinine was the most effective in treating BVDV infection. This study lays the foundation for developing targeted Chinese pharmaceutical formulations against the BVDV virus.

Short-term treatment with zingerone ameliorates dextran sulfate sodium-induced mouse experimental colitis.

BACKGROUND: Ulcerative colitis (UC) is a relapsing and chronic inflammatory disease of the gastrointestinal tract, which seriously threatens human health. Zingerone (ZO) has been proven to be effective for many diseases. The purpose of this study is to investigate the protective effects and potential mechanisms of ZO extracted from ginger on dextran sulfate sodium (DSS)-induced mouse ulcerative colitis (UC). RESULTS: The results showed that ZO alleviated the weight loss of UC model mice, reduced the disease activity index scores, and inhibited the shortening of colon length. ZO also improved DSS-induced pathological changes in colon tissue and inhibited the secretion of pro-inflammatory cytokines in colon and mesenteric lymph nodes. Further mechanism analysis found that ZO inhibited DSS-induced nuclear factor-κB pathway activation, and regulated peroxisome proliferator-activated receptor γ (PPARγ) expression. To further explore whether PPARγ was involved in the anti-UC effect of ZO, PPARγ inhibitor GW9662 was used. Although ZO also showed a protective effect on GW9662-treated colitis mice, the protective role was significantly weakened. Importantly, the administration of GW9662 significantly aggravated UC compared with the ZO + DSS group. In addition, we preliminarily found that ZO had the effects of inhibiting DSS-induced oxidative stress, maintaining intestinal barrier, and inhibiting the content of LPS and the population of Escherichia coli. CONCLUSIONS: These results indicated that supplementation with ZO might be a new dietary strategy for the treatment of UC. © 2022 Society of Chemical Industry.

Phloretin is protective in a murine salmonella enterica serovar typhimurium infection model.

Salmonella, an important zoonotic pathogen, causes significant morbidity and mortality in both humans and animals. Phloretin mainly isolated from strawberries and apples has the effects of treating inflammation and pathogenic bacteria, but its protective efficacy and mechanism of action against Salmonella spp. are less clear. In this study, we found that phloretin alleviated body weight loss, colon length shortening, and colonic pathological damage caused by S. Typhimurium. Phloretin also decreased S. Typhimurium translocation to the mesenteric lymph nodes (MLN) and spleen. Further mechanism studies showed that phloretin significantly inhibited inflammation and oxidative stress levels in the colonic tissue. Phloretin also prevented S. Typhimurium-mediated impairment in the colon epithelium barrier by the regulation ZO-1 and occludin levels. Interestingly, phloretin did not inhibit S. typhimurium growth in vitro, but reduced the internalization of S. Typhimurium into Caco-2 cells. Taken together, these findings indicated that phloretin may be a new dietary strategy to combat the disease.

Mechanism of blood-brain barrier disruption by an Escherichia coli from lambs with severe diarrhea and meningoencephalitis.

Escherichia coli (E. coli) is a common conditional pathogen that is associated with a variety of infections in humans and animals. Although there are increasing reports regarding the infection of E. coli to domestic animals and poultry, the infection of E. coli in lambs is relatively less reported, especially on meningoencephalitis. Here, we reported the isolation of an E. coli strain designated as NMGCF-19 from lambs characterized with severe diarrhea and neurological disorder, and demonstrated that NMGCF-19 as the causative agent has the ability to disrupt the blood-brain barrier (BBB) to cause the meningoencephalitis using a mouse model. Investigation on the mechanism regarding the NMGCF-19-related meningoencephalitis revealed a significant decreased expression of ZO-1 and occludin in mouse brain tissue in comparison with the control mice. Moreover, infection of NMGCF-19 increased the expression of proinflammatory cytokines TNF-α, IL-6, IL-1ß and IL-18, up-regulated HMGB1 level, and activated TLR2/TLR4/MyD88 and NLRP3 inflammasome pathways. These findings indicated that NMGCF-19 likely invades the brain tissue by disrupting the tight junction (TJ) architecture and causes the meningoencephalitis via increasing inflammatory response and activating TLR2/TLR4/MyD88 and NLRP3 inflammasome pathways.

Discovery of a virus of the species Enterovirus F in goats.

Here, we report two novel enteroviruses, designated as SD-S67 and SD-S68, isolated from a goat farm. Their complete genome sequences were determined and found to be 7455 and 7465 nucleotides in length, respectively. Molecular characterization revealed that SD-S67 is closely related to bovine enterovirus strain 261 and that SD-S68 to caprine enterovirus strain CEV-JL14. Phylogenetic analysis showed that SD-S67 clustered with members of the species Enterovirus F, and that SD-S68 clustered with enteroviruses of goats and sheep. Recombination analysis showed that SD-S67 is likely to have undergone several recombination events in the process of its evolution. To the best of our knowledge, this is the first report of an enterovirus F isolate from a goat and of a coinfection with enteroviruses of different species in the same goat herd.

Leptospira interrogans induces uterine inflammatory responses and abnormal expression of extracellular matrix proteins in dogs.

Leptospira interrogans (L. interrogans), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans. In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans, to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans.

Efficacy of cefepime, ertapenem and norfloxacin against leptospirosis and for the clearance of pathogens in a hamster model.

Animals and humans with severe leptospirosis may require empirical treatment. Although many antibiotics are active against multiple leptospira serovars in vitro, their efficacy in vivo is limited. We evaluated the efficacy of cefepime (daily dose: 2, 5, 10, and 20 mg/kg), ertapenem (daily dose: 2.5, 5, and 10 mg/kg) and norfloxacin (daily dose: 20, 40, and 80 mg/kg) for the treatment of leptospirosis and the ability to clear leptospira in target organs (liver, kidney, lung, heart, and spleen) in a lethal hamster model using Leptospira interrogans serovar Autumnalis. The histopathology of infected kidney, lung and liver was also evaluated using hematoxylin and eosin stain (H&E stain). All untreated animals, serving as a negative control, died with leptospira existing in the target organs between the 5th and 7th day after infection. All of the treated groups displayed improved survival compared to the untreated group and demonstrated a dose-dependent decrease in the presence of leptospira in the target organs. Cefepime showed survival benefit comparable to the standard treatment, doxycycline. We conclude that all of the antibiotics tested in vivo produce a statistically significant survival advantage, alleviate tissue injury and decrease the abundance of leptospira in target organs.

Inhibitory effects of astragalin on lipopolysaccharide-induced inflammatory response in mouse mammary epithelial cells.

BACKGROUND: Tea brewed from the leaves of persimmon or Rosa agrestis have several medical functions including treating allergy, antiatopic dermatitis, and anti-inflammatory effects. The objective of this study was to investigate the molecular mechanisms of astragalin, a main flavonoid component isolated from these herbs, in modifying lipopolysaccharide (LPS)-induced signaling pathways in primary cultured mouse mammary epithelial cells (mMECs). MATERIALS AND METHODS: The mMECs were treated with LPS in the absence or presence of different concentrations of astragalin. The expression of proinflammatory cytokines tumor necrosis factor α, and interleukin 6, as well as nitric oxide production were determined by enzyme-linked immunosorbent assay and Griess reaction, respectively. Cyclooxygenase-2, inducible nitric oxide synthase, toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), inhibitor protein of NF-κB (IκBα), P38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase were measured by Western blot. RESULTS: The results showed that astragalin suppressed the expression of tumor necrosis factor α, interleukin 6, and nitric oxide in a dose-dependent manner in mMECs. Western blot results showed that the expression of inducible nitric oxide synthase and cyclooxygenase-2 was inhibited by astragalin. Besides, astragalin efficiently decreased LPS-induced TLR4 expression, NF-κB activation, IκBα degradation, and the phosphorylation of p38, extracellular signal-regulated kinase in BMECs. CONCLUSIONS: Our results indicated that astragalin exerts anti-inflammatory properties possibly via the inactivation of TLR4-mediated NF-κB and mitogen-activated protein kinases signaling pathways in LPS-stimulated mMECs. Thus, astragalin may be a potential therapeutic agent for bovine mastitis.

The Gut Microbiota Contributes to the Development of LPS-Induced Orchitis by Disrupting the Blood-Testosterone Barrier in Mice.

Orchitis is a frequent inflammatory reproductive disease that causes male infertility and a decline in sperm quality. Gut microbiota can regulate systemic and local inflammation, spermatogenesis and blood-testosterone barrier (BTB). In this study, we investigated correlation between gut microbiota and orchitis by establishing a mouse gut microbiota imbalance model induced by antibiotics (ABX) treatment and orchitis model induced by lipopolysaccharide (LPS) infection. Based on these two models, 16s rRNA sequencing and feces microbiota transplantation (FMT) experiments were combined to examine the function and regulatory mechanisms of the gut microbiota in host defense against orchitis. Compared with control mice, gut microbiota imbalance resulted in increasing inflammatory responses, modulating oxidative stress related enzyme activity, testosterone levels and the permeability of blood testosterone barrier, which are restored after FMT. Subsequently, we tested the relationship between the gut microbiota imbalance and testicular inflammation severity in orchitis. It was found that the ABX and LPS co-treated mice had more severe inflammatory responses, lower testosterone levels and greater permeability of the BTB than the LPS-treated mice, but these changes could be partially recovered by gut microbiota transplantation. In conclusion, these above results proved for the first time that gut microbiota is involved in the pathogenesis of orchitis, which laid a good foundation for the subsequent development of anti-orchitis drugs and probiotic targeting intestinal flora.

The protective role of phlorizin against lipopolysaccharide-induced acute orchitis in mice associated with changes in gut microbiota composition.

Objective: Orchitis is a common reproductive disease of male animals, which has serious implications to human and animal reproduction. Additionally, phlorizin (PHN), a common polyphenol in apples and strawberries, has a variety of biological activities, including antioxidant, anti-inflammatory, anti-diabetic, and anti-aging activities. We aimed to determine the protective effects and potential mechanisms of PHN in lipopolysaccharide (LPS)-induced acute orchitis in mice. Method: After 21 days of PHN pretreatment, mice were injected with LPS to induce testicular inflammation, and then the changes of testicular tissue structure, expression of inflammatory factors, testosterone level, expression of testosterone-related genes, adhesion gene and protein expression were detected, and the structural changes in the intestinal flora after PHN treatment were further detected by 16SRNA. Result: Our results demonstrated that PHN treatment reduced LPS-induced testicular injury and body and testicular weight losses. The mRNA expression levels of pro-inflammatory cytokines-related genes and antioxidant enzyme activity were also decreased and elevated, respectively, by PHN administration; however, PHN treatment also reduced the LPS-induced decrease in testosterone levels in the testes. Additionally, further studies found that PHN increased the expression of marker proteins zonula occludens-1 (ZO-1) and occludin associated with the blood testosterone barrier compared with that in LPS treatment groups. To further examine the potential mechanisms of the protective effect of PHN on LPS-induced testicular injury, we compared the differences of gut microbiota compositions between the 100 mg/kg PHN treatment group and the control group using 16SRNA. Metagenomic analyses indicated that the abundances of Bacteroidetes, Muribaculaceae, Lactobacillaceae, uncultured bacterium f Muribaculaceae, and Lactobacillus in the PHN treatment group improved, while potential microbes that can induce intestinal diseases, including Verrucomicrobia, Epsilonbacteraeota, Akkermansiaceae, and Akkermansia decreased in the PHN treatment group. Conclusion: Our results indicate that PHN pretreatment might alleviate orchitis by altering the composition of gut microflora, which may provide a reference for reducing the occurrence of acute orchitis in male animals.

The activation of liver X receptors in Madin-Darby bovine kidney cells and mice restricts infection by bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and is an important pathogen that represents a serious threat to the development of the cattle industry by causing significant economic losses. Liver X receptors (LXRs) are members of the nuclear receptor superfamily and have become attractive therapeutic targets for cardiovascular disease. In the present study, we found that LXRs in both Madin-Darby bovine kidney (MDBK) cells and mice were associated with BVDV infection. GW3965, an agonist for LXRs, significantly inhibited BVDV RNA and protein levels in MDBK cells. In vivo studies in a mouse model also confirmed the inhibitory role of GW3965 in BVDV replication and the ameliorating effect of GW3965 on pathological injury to the duodenum. In vitro investigations of the potential mechanisms involved showed that GW3965 significantly inhibited BVDV-induced increases in cholesterol levels and viral internalization. Furthermore, the antiviral activity of GW3965 was significantly reduced following cholesterol replenishment, thus demonstrating that cholesterol was involved in the resistance of GW3965 to BVDV replication. Further studies indicated the role of ATP-binding cassette transporter A1 (ABCA1) and cholesterol-25-hydroxylase (CH25H) in the antiviral activity of GW3965. We also demonstrated the significant antiviral effect of 25hydroxycholesterol (25HC), a product of the catalysis of cholesterol by CH25H. In addition, the anti-BVDV effects of demethoxycurcumin (DMC), cyanidin-3-O-glucoside (C3G), and saikosaponin-A (SSA), three natural agonizts of LXRs, were also confirmed in both MDBK cells and mice. However, the antiviral activities of these agents were weakened by SR9243, a synthetic inhibitor of LXRs. For the first time, our research demonstrated that the activation of LXRs can exert significant anti-BVDV effects in MDBK cells and mice.

Phlorizin Limits Bovine Viral Diarrhea Virus Infection in Mice via Regulating Gut Microbiota Composition.

Phlorizin (PHZ) is one of the main pharmacologically active ingredients in Lithocarpus polystachyus. We have previously shown that PHZ inhibits the replication of bovine viral diarrhea virus (BVDV), but the exact antiviral mechanism, especially in vivo, is still unknown. Here, we further confirm that PHZ has good protective effects in BVDV-infected mice. We analyzed BVDV-induced CD3+, CD4+, and CD8+ T cells among peripheral blood lymphocytes and found that PHZ significantly restored their percentage. Metagenomic analyses revealed that PHZ markedly improved the richness and diversity of intestinal microbiota and increased the abundance of potentially health-related microbes (families Lachnosipiraceae, Ruminococcaceae, and Oscillospiraceae). Specifically, the relative abundance of short chain fatty acid (SCFA)-producing bacteria, including Lachnospiraceae_UCG-006, unclassified_f_Ruminococcaceae, Oscillibacter, Intestinimonas, Blautia, and Lachnoclostridium increased significantly after PHZ treatment. Interestingly, BVDV-infected mice that received fecal microbiota from PHZ-treated mice (PHZ-FMT) had a significantly lower viral load in the duodenum and jejunum than untreated mice. Pathological lesions of duodenum and jejunum were also greatly reduced in the PHZ-FMT group, confirming a significant antiviral effect. These findings show that gut microbiota play an important role in PHZ's antiviral activity and suggest that their targeted intervention might be a promising endogenous strategy to prevent and control BVDV.

Blockade of BTLA alone or in combination with PD-1 restores the activation and proliferation of CD8+ T cells during in vitro infection with NCP BVDV.

Bovine viral diarrhea virus (BVDV) infection can result in typical peripheral blood lymphopenia and immune dysfunction. However, the molecular mechanism underlying the onset of lymphopenia remains unclear. B and T lymphocyte attenuator (BTLA) is a novel immune checkpoint molecule that primarily inhibits activation and proliferation of T cells. Blockade of BTLA with antibodies can boost the proliferation and anti-viral immune functions of T cells. Nonetheless, the immunomodulatory effects of BTLA in CD8+ T cells during BVDV infection remain unknown. Therefore, BTLA expression was measured in bovine peripheral blood CD8+ T cells infected with BVDV in vitro. Furthermore, the effects of BTLA or PD-1 blockade on CD8+ T cell activation, proliferation, and anti-viral immunological activities were investigated, as well as expression of signaling molecules downstream of BTLA, both alone and in combination. The results demonstrated that BTLA and PD-1 mRNA and protein levels were considerably increased in CD8+ T cells infected with cytopathic and non-cytopathic (NCP) BVDV. Surprisingly, as compared to blockade of either BTLA or PD-1, blockade of both dramatically increased proliferation and expression of CD25 and p-EKR of CD8+ T cells infected with NCP BVDV. Furthermore, blockade of BTLA, but not PD-1, had no effect on BVDV replication or IFN-γ expression. These findings confirmed the immunomodulatory roles of BTLA during BVDV infection, as well as the synergistic role of BTLA and PD-1 in NCP BVDV infection, thereby providing new insights to promote activation and the anti-viral immunological activities of CD8+ T cells.

PI3K/AKT mediated De novo fatty acid synthesis regulates RIG-1/MDA-5-dependent type I IFN responses in BVDV-infected CD8+T cells.

Bovine viral diarrhea virus (BVDV) has caused massive economic losses in the cattle business worldwide. Fatty acid synthase (FASN), a key enzyme of the fatty acid synthesis (FAS) pathway, has been shown to support virus replication. To investigate the role of fatty acids (FAs) in BVDV infection, we infected CD8+T lymphocytes obtained from healthy cattle with BVDV in vitro. During early cytopathic (CP) and noncytopathic (NCP) BVDV infection in CD8+ T cells, there is an increase in de novo lipid biosynthesis, resulting in elevated levels of free fatty acids (FFAs) and triglycerides (TG). BVDV infection promotes de novo lipid biosynthesis in a dose-dependent manner. Treatment with the FASN inhibitor C75 significantly reduces the phosphorylation of PI3K and AKT in BVDV-infected CD8+ T cells, while inhibition of PI3K with LY294002 decreases FASN expression. Both CP and NCP BVDV strains promote de novo fatty acid synthesis by activating the PI3K/AKT pathway. Further investigation shows that pharmacological inhibitors targeting FASN and PI3K concurrently reduce FFAs, TG levels, and ATP production, effectively inhibiting BVDV replication. Conversely, the in vitro supplementation of oleic acid (OA) to replace fatty acids successfully restored BVDV replication, underscoring the impact of abnormal de novo fatty acid metabolism on BVDV replication. Intriguingly, during BVDV infection of CD8+T cells, the use of FASN inhibitors prompted the production of IFN-α and IFN-ß, as well as the expression of interferon-stimulated genes (ISGs). Moreover, FASN inhibitors induce TBK-1 phosphorylation through the activation of RIG-1 and MDA-5, subsequently activating IRF-3 and ultimately enhancing the IFN-1 response. In conclusion, our study demonstrates that BVDV infection activates the PI3K/AKT pathway to boost de novo fatty acid synthesis, and inhibition of FASN suppresses BVDV replication by activating the RIG-1/MDA-5-dependent IFN response.

Survey of mycotic mastitis in dairy cows from Heilongjiang Province, China.

A survey of the prevalence rate, pathogenic subspecies, and risk factors of mycotic mastitis in dairy cows from Heilongjiang Province, China, was conducted. Milk samples from 412 cows with chronic mastitis were collected and cultured on 8 % sheep blood agar, MacConkey agar, and Sabouraud agar with chloramphenicol. Counting of the morphologically distinct colonies was performed, as well as the isolation and identification of organisms through phenotypical and physiological criteria. Four hundred seventy-eight aerobic microorganisms were isolated. Yeasts and yeast-like fungi 35.6 % (170/478) and bacteria 64.4 % (308/478) were isolated. The fungal isolates were identified as Candida (79.4 %), Trichosporon (5.9 %), Aspergillus (7.1 %), Cryptococcus (2.4 %), and Rhodotorula (4.1 %). More than ten species of yeast were isolated including Candida krusei 50/135 (37 %), Candida rugosa 16/135 (11.9 %), and Candida lusitaniae 15/135 (11.1 %). A higher positivity (18.5 and 56.3 %) (P ≤0.05) was observed in cows from environmental temperatures of 0-15 and 15-35 °C than those at <0 °C and in cows affected by the disease for >45 and 30-45 days compared with cows suffering 10-30 days. Meanwhile, a statistically significant difference (44.9 vs. 31.4 %) (P ≤0.05) was observed under extensive raising systems vs. intensive raising systems. It appears that Candida is a major pathogen of mycotic mastitis of dairy cows. Extensive raising system, high environmental temperature (15-35 °C), and the duration of the disease (>30 days) were important risk factors of the incidence of mycotic mastitis. Here, we provide a theoretical foundation for research into preventing and treating mycotic mastitis of dairy cows in China.

Total flavones from Sonchus arvensis L. ameliorate colitis by adjusting the gut microbiota.

OBJECTIVE: Sonchus arvensis L. is traditional Chinese food and medicine. We investigated protective effects of flavones from Sonchus arvensis L. (SAF) on colitis induced by dextran sulfate sodium (DSS) in mice by regulating gut microbiota (GM). METHOD: C57BL/6 mice were divided randomly: control group (CL); DSS group (ML); positive control + DSS group (AN); SAF + DSS (FE) group. The protective effects of SAF on ulcerative colitis (UC) were estimated by food intake, water intake, bodyweight loss, diarrhea, blood in stools, colon length, histology, disease activity index (DAI) score, and blood parameters. The sequencing of 16S rRNA gene was detected to investigate effect of SAF on GM. RESULTS: SAF attenuate bodyweight loss significantly. The DAI score was lower in FE group than that in ML group. Colon length was improved significantly in ML group. Pathologic changes could be ameliorated after SAF was administered to UC mice. SAF improved blood parameters of model mice. 16S rRNA sequencing revealed that it was very important to ameliorate colitis with bacteria of the phylum Verrucomicrobiota, class Verrucomicrobiae, order Verrucomicrobiales, family Akkermansiaceae, and genus Akkermansia. CONCLUSION: The SAF protective effect against colitis induced by DSS in mice may have a connection with GM diversity.

In Vivo and In Vitro Antiviral Activity of Phlorizin Against Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) is one of the most serious pathogens affecting the cattle industry worldwide. Phlorizin, a kind of flavonoids extracted from apple tree roots, leaves, and fruits, has a variety of biological functions and has been widely used as a herbal supplement and food additive. Here, BALB/c mouse and Madin-Darby bovine kidney (MDBK) cells were used to explore the effect and mechanism of phlorizin against BVDV infection. The results showed that phlorizin significantly inhibited CP BVDV replication and improved the histopathological changes of duodenum and spleen in mice. In vitro studies also confirmed the activity of phlorizin against CP BVDV. Exploration on its potential mechanism suggested that phlorizin inhibited CP BVDV-induced beclin-1 level and the conversion rate of LC3B-I to LC3B-II. Interestingly, although phlorizin also showed a protective effect on MDBK cells, which were treated with 3-methyladenine A (3-MA), the effect was significantly weakened. Furthermore, phlorizin suppressed the stage of BVDV replication but showed no effect on stages of attachment and internalization. Our data further indicated that phlorizin promoted IFN-α and IFN-ß levels, decreased IL-1ß and IL-6 expression, and regulated RIG-I, MDA5, TLR3, and NLRP3 levels. Similar to CP BVDV results, in vivo and in vitro, phlorizin inhibited NCP BVDV (NY-1 and YNJG2020 strains) infection. These results were the first to be discovered that phlorizin might be used as a new dietary strategy for controlling BVDV infection.