Development and application of an indirect ELISA for the serological detection of bovine viral diarrhea virus infection based on the protein E2 antigen.

BACKGROUND: Bovine viral diarrhea virus (BVDV) causes continuous economic losses to the livestock industry. Monitoring antibodies with enzyme-linked immunosorbent assay (ELISA) is a valuable tool to ensure the purification of BVDV in cattle. However, currently available ELISA kits based on the whole BVDV virion are both costly and time-consuming. The E2 protein has good immunogenicity, induces the secretion of neutralizing antibodies and is an essential immunogen for serological detection. METHODS AND RESULTS: We developed a novel recombinant E2 protein-based indirect ELISA (rE2-iELISA) and conducted a serological survey for BVDV antibodies in 2021-2022 in Beijing, China. The results showed that E2 protein was successfully expressed with high immunogenicity and the optimal rE2-iELISA displayed high sensitivity, reproducibility and specificity. Clinical testing of 566 serum specimens indicated that 318 BVDV positive samples and 194 BVDV negative samples were tested by rE2-iELISA and the IDEXX BVDV ELISA-Ab kit, with a positive coincidence rate of 93.3%, a negative coincidence rate of 86.3%, and an overall coincidence rate of 90.5%. CONCLUSION: This study established an rE2-iELISA method, which is a highly sensitive, specific and robust ELISA-test validated to detect anti-BVDV antibodies. These findings indicate that the newly developed rE2-iELISA method has the potential to be used as a rapid, reliable and cost-effective screening tool for BVDV infection and provides technical support for the evaluation of vaccine efficacy in cattle herds in the future.

Lactobacillusjohnsonii L531 Protects against Salmonella Infantis-Induced Intestinal Damage by Regulating the NOD Activation, Endoplasmic Reticulum Stress, and Autophagy.

Salmonella enterica serovar Infantis (S. Infantis) is an intracellular bacterial pathogen. It is prevalent but resistant to antibiotics. Therefore, the therapeutic effect of antibiotics on Salmonella infection is limited. In this study, we used the piglet diarrhea model and the Caco2 cell model to explore the mechanism of probiotic Lactobacillus johnsonii L531 (L. johnsonii L531) against S. Infantis infection. L. johnsonii L531 attenuated S. Infantis-induced intestinal structural and cellular ultrastructural damage. The expression of NOD pathway-related proteins (NOD1/2, RIP2), autophagy-related key proteins (ATG16L1, IRGM), and endoplasmic reticulum (ER) stress markers (GRP78, IRE1) were increased after S. Infantis infection. Notably, L. johnsonii L531 pretreatment not only inhibited the activation of the above signaling pathways but also played an anti-S. Infantis infection role in accelerating autophagic degradation. However, RIP2 knockdown did not interfere with ER stress and the activation of autophagy induced by S. Infantis in Caco2 cells. Our data suggest that L. johnsonii L531 pretreatment alleviates the intestinal damage caused by S. Infantis by inhibiting NOD activation and regulating ER stress, as well as promoting autophagic degradation.

Anti-inflammatory effects of Lactobacillus johnsonii L531 in a pig model of Salmonella Infantis infection involves modulation of CCR6+ T cell responses and ER stress.

Probiotic pretreatment is an effective non-antibiotic strategy for preventing or controlling Salmonella infections. We found that Lactobacillus johnsonii L531, isolated from the colon of a clinically healthy weaned piglet, effectively prevented infection with Salmonella enterica serovar Infantis in a pig model. Newly weaned piglets were intragastrically administered Lactobacillus johnsonii L531 at 1.0 × 1010 CFU/day for 1 week before S. Infantis challenge. Pretreatment with L. johnsonii L531 lessened the severity of diarrhea and ileal inflammation in S. Infantis-infected piglets. Lactobacilli were more abundant in the ileum than jejunum after L. johnsonii L531 pretreatment. Treatment with L. johnsonii L531 reduced the abundance of total bacteria in the ileal mucosa and the production of lipocalin 2 in the jejunum of piglets challenged with Salmonella. Both intestinal morphology and transmission electron microscopy results indicated that L. johnsonii L531 alleviated intestinal tissue damage following S. Infantis challenge, especially in the villus and endoplasmic reticulum (ER). ER stress induced by S. Infantis was attenuated by L. johnsonii L531 treatment. The number of CD4- CCR6+ T cells decreased following S. Infantis challenge, but the percentage of CCR6- IFNγ+ T cells in peripheral blood increased. In intestinal mesenteric lymph nodes, S. Infantis increased the proportion of CCR6+ IFNγ+ T cells, whereas L. johnsonii L531 induced an increase in the proportion of CD4+ CCR6+ T cells in response to S. Infantis infection. Our data thus suggest that L. johnsonii L531 contributes to the maintenance of intestinal homeostasis by modulating T-cell responses and ER stress.

Oral Administration of a Select Mixture of Bacillus Probiotics Affects the Gut Microbiota and Goblet Cell Function following Escherichia coli Challenge in Newly Weaned Pigs of Genotype MUC4 That Are Supposed To Be Enterotoxigenic E. coli F4ab/ac Receptor Negative.

Structural disruption of the gut microbiota and impaired goblet cell function are collateral etiologic factors in enteric diseases. Low, moderate, or high doses of a Bacillus licheniformis-B. subtilis mixture (BLS mix) were orally administered to piglets of genotype MUC4 that are supposed to be F4-expressing enterotoxigenic Escherichia coli strain (F4+ ETEC) F4ab/ac receptor negative (i.e., MUC4-resistant piglets) for 1 week before F4+ ETEC challenge. The luminal contents were collected from the mucosa of the colon on day 8 after F4+ ETEC challenge. The BLS mix attenuated E. coli-induced expansion of Bacteroides uniformis, Eubacterium eligens, Acetanaerobacterium, and Sporobacter populations. Clostridium and Turicibacter populations increased following F4+ ETEC challenge in pigs pretreated with low-dose BLS mix. Lactobacillus gasseri and Lactobacillus salivarius populations increased after administration of BLS mix during E. coli infection. The beneficial effects of BLS mix were due in part to the expansion of certain Clostridium, Lactobacillus, and Turicibacter populations, with a corresponding increase in the number of goblet cells in the ileum via upregulated Atoh1 expression, in turn increasing MUC2 production and thus preserving the mucus barrier and enhancing host defenses against enteropathogenic bacteria. However, excessive BLS mix consumption may increase the risk for enteritis, partly through disruption of colonic microbial ecology, characterized by expansion of Proteobacteria and impaired goblet cell function in the ileum. Our findings suggest that oral administration of BLS mix reprograms the gut microbiota and enhances goblet cell function to ameliorate enteritis. IMPORTANCE: The present study is important for improving our understanding of the protective role of probiotics against Escherichia coli infection in piglets. Structural disruption of the gut microbiota and impaired goblet cell function are collateral etiologic factors in enteric diseases. In this study, low, moderate, or high doses of a Bacillus licheniformis-B. subtilis mixture (BLS mix) were orally administered to MUC4-resistant piglets for 1 week before the F4-expressing ETEC strain (F4+ ETEC) challenge. Our findings suggest that oral administration of BLS mix reprograms the gut microbiota and enhances goblet cell function to ameliorate enteritis.

Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation.

Escherichia coli is a major environmental pathogen causing bovine mastitis, which leads to mammary tissue damage and cell death. We explored the effects of the probiotic Lactobacillus rhamnosus GR-1 on ameliorating E. coli-induced inflammation and cell damage in primary bovine mammary epithelial cells (BMECs). Increased Toll-like receptor 4 (TLR4), NOD1, and NOD2 mRNA expression was observed following E. coli challenge, but this increase was attenuated by L. rhamnosus GR-1 pretreatment. Immunofluorescence and Western blot analyses revealed that L. rhamnosus GR-1 pretreatment decreased the E. coli-induced increases in the expression of the NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) and the serine protease caspase 1. However, expression of the adaptor protein apoptosis-associated speck-like protein (ASC, encoded by the Pycard gene) was decreased during E. coli infection, even with L. rhamnosus GR-1 pretreatment. Pretreatment with L. rhamnosus GR-1 counteracted the E. coli-induced increases in interleukin-1ß (IL-1ß), -6, -8, and -18 and tumor necrosis factor alpha mRNA expression but upregulated IL-10 mRNA expression. Our data indicate that L. rhamnosus GR-1 reduces the adhesion of E. coli to BMECs, subsequently ameliorating E. coli-induced disruption of cellular morphology and ultrastructure and limiting detrimental inflammatory responses, partly via promoting TLR2 and NOD1 synergism and attenuating ASC-independent NLRP3 inflammasome activation. Although the residual pathogenic activity of L. rhamnosus, the dosage regimen, and the means of probiotic supplementation in cattle remain undefined, our data enhance our understanding of the mechanism of action of this candidate probiotic, allowing for development of specific probiotic-based therapies and strategies for preventing pathogenic infection of the bovine mammary gland.

Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge.

Efficient strategies for treating enteritis caused by F4(+) enterotoxigenic Escherichia coli (ETEC)/verocytotoxigenic Escherichia coli (VTEC)/enteropathogenic E. coli (EPEC) in mucin 4 resistant (MUC4 RR; supposed to be F4ab/ac receptor-negative [F4ab/acR(-)]) pigs remain elusive. A low (3.9 × 10(8) CFU/day) or high (7.8 × 10(8) CFU/day) dose of Bacillus licheniformis and Bacillus subtilis spore mixture (BLS-mix) was orally administered to MUC4 RR piglets for 1 week before F4(+) ETEC/VTEC/EPEC challenge. Orally fed BLS-mix upregulated the expression of TLR4, NOD2, iNOS, IL-8, and IL-22 mRNAs in the small intestine of pigs challenged with E. coli. Expression of chemokine CCL28 and its receptor CCR10 mRNAs was upregulated in the jejunum of pigs pretreated with high-dose BLS-mix. Low-dose BLS-mix pretreatment induced an increase in the proportion of peripheral blood CD4(-)CD8(-) T-cell subpopulations and high-dose BLS-mix induced the expansion of CD4(-)CD8(-) T cells in the inflamed intestine. Immunostaining revealed that considerable IL-7Rα-expressing cells accumulated at the lamina propria of the inflamed intestines after E. coli challenge, even in pigs pretreated with either low- or high-dose BLS-mix, although Western blot analysis of IL-7Rα expression in the intestinal mucosa did not show any change. Our data indicate that oral administration of the probiotic BLS-mix partially ameliorates E. coli-induced enteritis through facilitating upregulation of intestinal IL-22 and IκBα expression, and preventing loss of intestinal epithelial barrier integrity via elevating ZO-1 expression. However, IL-22 also elicits an inflammatory response in inflamed intestines as a result of infection with enteropathogenic bacteria.

Oral administration of a select mixture of Bacillus probiotics generates Tr1 cells in weaned F4ab/acR- pigs challenged with an F4+ ETEC/VTEC/EPEC strain.

Although breeding of F4 receptor - negative (F4R(-)) pigs may prevent post-weaning diarrhea, the underlying immunity is poorly understood. Here, various doses of a Bacillus licheniformis and Bacillus subtilis mixture (BLS-mix) were orally administered to F4ab/acR(-) pigs for 1 week before F4 (K88) - positive ETEC/VTEC/EPEC challenge. Administration of BLS-mix increased the percentage of Foxp3(-)IL-10(+) T cells but not of Foxp3(+)IL-10(+) regulatory T (Treg) cells among peripheral blood CD4(+) T cells. A low dose of BLS-mix feeding resulted in increased the expression of IL-6, TNF-α, IL-10, and the transcription factors Foxp3 and T-bet mRNAs in the jejunum. Administration of either a low or high dose BLS-mix also led to an increase in the percentage of CD4(+)Foxp3(+) Treg cells among intraepithelial lymphocytes and CD4(+)IL-10(+) T cells in the small intestinal Peyer's patches and the lamina propria of F4ab/acR(-) pigs following F4(+) ETEC/VTEC/EPEC challenge. The increased number of IL-10-producing CD4(+) T cells was attributed to an increase in the proportion of Foxp3(-)IL-10(+) Treg cells rather than Foxp3(+)IL-10(+) Treg cells. Our data indicate that oral administration of BLS-mix to newly weaned F4ab/acR(-) pigs ameliorates enteritis in an F4(+) ETEC/VTEC/EPEC model; however, induction of IL-10-producing Foxp3(-) Treg cells by BLS-mix administration cannot account for the protection of newly weaned F4ab/acR(-) pigs from F4(+) ETEC/VTEC/EPEC infection, and that excessive generation of CD4(+)IL-10(+) T cells following consumption of BLS-mix during episodes of intestinal inflammation that is caused by enteric pathogens might prohibit clearance of the pathogen. Select probiotic mixtures may allow for tailoring strategies to prevent infectious diseases.

Dose-dependent effects of Lactobacillus rhamnosus on serum interleukin-17 production and intestinal T-cell responses in pigs challenged with Escherichia coli.

The mechanism underlying the dose effect of probiotics on ameliorating diarrhea has not been fully elucidated. Here, low (1 × 10(9) CFU/ml) or high (1 × 10(11) CFU/ml) doses of Lactobacillus rhamnosus ATCC 7469 were administered orally to piglets for 1 week before F4 (K88)-positive enterotoxigenic Escherichia coli (F4(+) ETEC) challenge. Administration of a low, but not a high, dose of L. rhamnosus decreased the percentage of CD3(+) CD4(+) CD8(-) T cells in the peripheral blood. Notably, transiently increased serum concentrations of interleukin-17A (IL-17A) were observed after F4(+) ETEC challenge in pigs pretreated with a high dose of L. rhamnosus. Administration of L. rhamnosus increased the percentage of the small intestinal lamina propria CD3(+) CD4(+) CD8(-) cells and Peyer's patch CD3(+) CD4(-) CD8(-) and CD3(-) CD4(-) CD8(+) cells. The percentage of ileal intraepithelial CD3(+) CD4(-) CD8(+) cells increased only in the high-dose piglets. Administration of L. rhamnosus downregulated expression of ileal IL-17A after F4(+) ETEC challenge but had no effect on expression of gamma interferon (IFN-γ), IL-12, IL-4, and FOXP3 mRNA in the small intestine. Expression of jejunal IL-2, ileal transforming growth factor ß1 (TGF-ß1), and ileal IL-10 was upregulated in the low-dose piglets after F4(+) ETEC challenge. Our findings suggest that amelioration of infectious diarrhea in piglets by L. rhamnosus is associated with the generation of lamina propria CD3(+) CD4(+) CD8(-) T cells, the expansion of Peyer's patch CD3(+) CD4(-) CD8(-) and CD3(-) CD4(-) CD8(+) cells, and the attenuation of F4(+) ETEC-induced increase in CD3(+) CD4(+) CD8(+) T cells in the small intestine. However, consumption of high doses of L. rhamnosus may increase levels of serum IL-17A after F4(+) ETEC challenge, thus eliciting a strong proinflammatory response.

Effects of Spatial Expression of Activating Transcription Factor 4 on the Pathogenicity of Two Phenotypes of Bovine Viral Diarrhea Virus by Regulating the Endoplasmic Reticulum-Mediated Autophagy Process.

The endoplasmic reticulum (ER) stress response is a highly conserved stress-defense mechanism and activates the adaptive unfolded protein response (UPR) to mitigate imbalance. The ER stress-activated signaling pathways can also trigger autophagy to facilitate cellular repair. Bovine viral diarrhea virus (BVDV) utilizes the host cellular ER as the primary site of the life cycle. However, the interplay between cellular ER stress and BVDV replication remains unclear. This report reveals that cytopathic (cp) and noncytopathic (ncp) BVDV have distinct strategies to regulate UPR mechanisms and ER stress-mediated autophagy for their own benefit. Immunoblot analysis revealed that cp and ncp BVDV differentially regulated the abundance of ER chaperone GRP78 for viral replication, while the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α)-activating transcription factor 4 (ATF4) pathway of the UPR was switched on at different stages of infection. Pretreatment with ER stress inducer promoted virion replication, but RNA interference (RNAi) knockdown of ATF4 in BVDV-infected cells significantly attenuated BVDV infectivity titers. More importantly, the effector ATF4 activated by cp BVDV infection translocated into the nucleus to mediate autophagy, but ATF4 was retained in the cytoplasm during ncp BVDV infection. In addition, we found that cp BVDV core protein was localized in the ER to induce ER stress-mediated autophagy. Overall, the potential therapeutic target ATF4 may contribute to the global eradication campaign of BVDV. IMPORTANCE The ER-tropic viruses hijack the host cellular ER as the replication platform of the life cycle, which can lead to strong ER stress. The UPR and related transcriptional cascades triggered by ER stress play a crucial role in viral replication and pathogenesis, but little is known about these underlying mechanisms. Here, we report that cytopathic and noncytopathic BVDV use different strategies to reprogram the cellular UPR and ER stress-mediated autophagy for their own advantage. The cytopathic BVDV unconventionally downregulated the expression level of GRP78, creating perfect conditions for self-replication via the UPR, and the noncytopathic BVDV retained ATF4 in the cytoplasm to provide an advantage for its persistent infection. Our findings provide new insights into exploring how BVDV and other ER-tropic viruses reprogram the UPR signaling pathway in the host cells for replication and reveal the attractive host target ATF4 for new antiviral agents.

Quercetin effectively improves LPS-induced intestinal inflammation, pyroptosis, and disruption of the barrier function through the TLR4/NF-κB/NLRP3 signaling pathway in vivo and in vitro.

Background: Inflammatory bowel diseases are characterized by the alterations of the mucosa and gastrointestinal physiology, and the core of these alterations is endothelial cells. Quercetin is a flavonoid presents in some traditional Chinese medicine, plants, and fruits. Its protective effects in several gastrointestinal tumors have been demonstrated, but its effects on bacterial enteritis and pyroptosis-related diseases have rarely been studied. Objective: This study aimed to evaluate the effect of quercetin on bacterial enteritis and pyroptosis. Design: In vitro experiments were performed using rat intestinal microvascular endothelial cells divided into seven groups: control group (no treatment), model group (10 µg/mL lipopolysaccharide (LPS)+1 mM adenosine triphosphate [ATP]), LPS group (10 µg/mL LPS), ATP group (1 mM ATP), and treatment groups (10 µg/mL LPS+1 mM ATP and 5, 10, and 20 µM quercetin). The expression of pyroptosis-associated proteins, inflammatory factors, tight junction proteins, and the percentage of late apoptotic and necrotic cells were measured. In vivo analysis was performed using specific pathogen-free Kunming mice pretreated with quercetin and the water extract of Cacumen Platycladi for 2 weeks followed by 6 mg/kg LPS on day 15. Inflammation in the blood and intestinal pathological changes were evaluated. Results: Quercetin used in vitro significantly reduced the expression of Toll-like receptor 4 (TLR4), NOD-like receptor 3 (NLRP3), caspase-1, gasdermin D, interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor-α. It also inhibited phosphorylation of nuclear factor-kappa B (NF-κB) p65 and increased cell migration and the expression of zonula occludens 1 and claudins, while reduced the number of late apoptotic cells. The in vivo results showed that Cacumen Platycladi and quercetin significantly reduced inflammation, protected the structure of the colon and cecum, and prevent fecal occult blood induced by LPS. Conclusions: These findings suggested the ability of quercetin to reduce inflammation induced by LPS and pyroptosis through TLR4/NF-κB/NLRP3 pathway.

Melatonin as Immune Potentiator for Enhancing Subunit Vaccine Efficacy against Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) is a pathogen associated with substantial economic losses in the dairy cattle industry. Currently, there are no effective vaccines against BVDV. Melatonin (MT) has been shown to have anti-inflammatory and anti-viral properties, and the use of MF59 in vaccines significantly enhances vaccine efficiency. Here, MT and MF59 were added into the Erns-LTB vaccine. Subsequently, their inhibitory activity on the NF-κB signaling pathway in Mardin-Darby Bovine Kidney cells and the hippocampus was assessed using western blot and quantitative reverse transcription PCR. The findings revealed that MT in the Erns-LTB vaccine decreases the phosphorylation of p65 proteins caused by BVDV infection. In addition, MT decreased the mRNA levels of IL-1ß and IL-6 in vitro, but increased the production of IFN-α, IFN-ß, Mx1 in vitro, brain-derived neurotrophic factor, cyclic amp response element-binding protein, and the stem cell factor in vivo. Furthermore, treatment with Erns-LTB + MF59 + MT stimulated the production of T lymphocytes, alleviated pathological damage, decreased expressions of BVDV antigen, and tight junction proteins in mice. These findings imply that MT has potential for use in the Erns-LTB vaccine to inhibit BVDV infection and regulate the immune responses of T-cells by inhibiting the NF-κB signaling pathway.

Salmonella Infantis Delays the Death of Infected Epithelial Cells to Aggravate Bacterial Load by Intermittent Phosphorylation of Akt With SopB.

Salmonella Infantis has emerged as a major clinical pathogen causing gastroenteritis worldwide in recent years. As an intracellular pathogen, Salmonella has evolved to manipulate and benefit from the cell death signaling pathway. In this study, we discovered that S. Infantis inhibited apoptosis of infected Caco-2 cells by phosphorylating Akt. Notably, Akt phosphorylation was observed in a discontinuous manner: immediately 0.5 h after the invasion, then before peak cytosolic replication. Single-cell analysis revealed that the second phase was only induced by cytosolic hyper-replicating bacteria at 3-4 hpi. Next, Akt-mediated apoptosis inhibition was found to be initiated by Salmonella SopB. Furthermore, Akt phosphorylation increased mitochondrial localization of Bcl-2 to prevent Bax oligomerization on the mitochondrial membrane, maintaining the mitochondrial network homeostasis to resist apoptosis. In addition, S. Infantis induced pyroptosis, as evidenced by increased caspase-1 (p10) and GSDMS-N levels. In contrast, cells infected with the ΔSopB strain displayed faster but less severe pyroptosis and had less bacterial load. The results indicated that S. Infantis SopB-mediated Akt phosphorylation delayed pyroptosis, but aggravated its severity. The wild-type strain also caused more severe diarrhea and intestinal inflammatory damage than the ΔSopB strain in mice. These findings revealed that S. Infantis delayed the cells' death by intermittent activation of Akt, allowing sufficient time for replication, thereby causing more severe inflammation.

Lactobacillus johnsonii L531 Ameliorates Escherichia coli-Induced Cell Damage via Inhibiting NLRP3 Inflammasome Activity and Promoting ATG5/ATG16L1-Mediated Autophagy in Porcine Mammary Epithelial Cells.

Escherichia coli (E. coli), a main mastitis-causing pathogen in sows, leads to mammary tissue damage. Here, we explored the effects of Lactobacillus johnsonii L531 on attenuating E. coli-induced inflammatory damage in porcine mammary epithelial cells (PMECs). L. johnsonii L531 pretreatment reduced E. coli adhesion to PMECs by competitive exclusion and the production of inhibitory factors and decreased E. coli-induced destruction of cellular morphology and ultrastructure. E. coli induced activation of NLRP3 inflammasome associated with increased expression of NLRP3, ASC, and cleaved caspase-1, however, L. johnsonii L531 inhibited E. coli-induced activation of NLRP3 inflammasome. Up-regulation of interleukin (Il)-1ß, Il-6, Il-8, Il-18, tumor necrosis factor alpha, and chemokine Cxcl2 expression after E. coli infection was attenuated by L. johnsonii L531. E. coli infection inhibited autophagy, whereas L. johnsonii L531 reversed the inhibitory effect of E. coli on autophagy by decreasing the expression of autophagic receptor SQSTM1/p62 and increasing the expression of autophagy-related proteins ATG5, ATG16L1, and light chain 3 protein by Western blotting analysis. Our findings suggest that L. johnsonii L531 pretreatment restricts NLRP3 inflammasome activity and induces autophagy through promoting ATG5/ATG16L1-mediated autophagy, thereby protecting against E. coli-induced inflammation and cell damage in PMECs.

Oral Administration of a Select Mixture of Lactobacillus and Bacillus Alleviates Inflammation and Maintains Mucosal Barrier Integrity in the Ileum of Pigs Challenged with Salmonella Infantis.

Salmonella is important as both a cause of clinical disease in swine and as a source of food-borne transmission of disease to humans. Lactobacillus and Bacillus are often used as antibiotic substitutes to prevent Salmonella infection. In this study, we evaluated the effects of a select mixture of Lactobacillus johnsonii L531, Bacillus licheniformis BL1721 and Bacillus subtilis BS1715 (LBB-mix) in prevention of Salmonella enterica serovar Infantis infection in a pig model. LBB-mix was orally administered to newly weaned piglets for seven days before S. Infantis challenge. LBB-mix pretreatment ameliorated S. Infantis-induced fever, leukocytosis, growth performance loss, and ileal inflammation. Pre-administration of LBB-mix reduced the number of Salmonella in the feces but increased the number of goblet cells in the ileum. S. Infantis infection resulted in an increase in cell death in the ileum, this increase was attenuated by LBB-mix consumption. Claudin 1 and cleaved caspase-1 expression was decreased in the ileum of pigs challenged with S. Infantis, but not in pigs pretreated with LBB-mix. In conclusion, our data indicate that a select LBB-mix has positive effects on controlling S. Infantis infection via alleviating inflammation and maintaining the intestinal mucosal barrier integrity in pigs.

Frequency of Diarrheagenic Virulence Genes and Characteristics in Escherichia coli Isolates from Pigs with Diarrhea in China.

Intestinal pathogenic Escherichia coli (InPEC) is a leading cause of postweaning diarrhea (PWD) in pigs. Here, a total of 455 E. coli strains were isolated from small intestinal content or feces from pigs with PWD in 56 large-scale (>500 sows; 10,000 animals per year) swine farms between 2014 and 2016. The frequency of occurrence of selected virulence factors for InPEC pathotypes was detected in 455 isolates by real-time PCR. Sequence types (STs), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility profiles of 171 E. coli isolates from 56 swine farms were further determined. The heat-labile enterotoxin (LT) was the most common (61.76%), followed by heat-stable enterotoxin (STb) (33.19%), stx2e (21.54%), STa (15.00%), eae (8.98%), cnf2 (5.71%), stx2 (5.71%), F18 (3.25%), and F4 (2.25%) with rates varying by geographic area and year of isolation. Notably, hybrids of E. coli isolates were potentially more virulent, as some InPEC hybrids (virotype F18:LT:eae:stx2e) can rapidly cause cell death in vitro. Genotypic analysis revealed that the most prominent genotype was ST10 (12.87%). The PFGE patterns were heterogeneous but were not ST or virotype related. A total of 94.15% of isolates were multidrug-resistant, with average resistance rates ranging from 90.05% for nalidixic acid to 2.34% for meropenem. Our investigation contributes to establishing the etiology of diarrhea and developing intervention strategies against E. coli-associated diarrheal disease in the future.

Lactobacillus johnsonii L531 reduces pathogen load and helps maintain short-chain fatty acid levels in the intestines of pigs challenged with Salmonella enterica Infantis.

In the current study, we screened Lactobacillus strains isolated from the colon of clinically healthy weaned piglets for potential probiotic properties and isolated Lactobacillus. johnsonii L531, which produced high levels of beneficial metabolites (butyric, acetic, and lactic acid) in vitro. We also evaluated the efficacy of this metabolites-producing probiotic in treating Salmonella. Infantis infection. Oral administration of L. johnsonii L531 to newly weaned piglets significantly decreased levels of Salmonella colonization in colonic and jejunal contents, accelerated the clearance of Salmonella in feces after infection, and reduced S. Infantis translocation to the spleen. Pretreatment with SCFAs-promoting probiotic L. johnsonii L531 significantly ameliorated the depletion of SCFAs induced by S. Infantis infection and led to significantly greater weight gain and better feed conversion ratios compared to piglets challenged only with S. Infantis. These data provide further evidence that SCFAs-promoting probiotic L. johnsonii L531 treatment could be a suitable nonantibiotic alternative for controlling Salmonella infection and maintaining metabolic homeostasis, thereby enhancing the gut health of piglets during the critical weaning period.

Uterine Microbiota of Dairy Cows With Clinical and Subclinical Endometritis.

The objective of this study was to characterize the uterine microbiota of dairy cows with clinical and subclinical endometritis and to identify the potential bacterial genera as well as their interactions associated with uterine disease. Uterine flush samples (n = 27) were collected from 13 healthy, 5 subclinical endometritic (SE), and 9 clinical endometritic (CE) cows at 30 days postpartum. Microbial DNA from uterine flush samples was subjected to sequencing of the 16S rRNA gene on the Illumina MiSeq platform. The uterine microbiota of healthy, SE, and CE cows had similarly complex microbial diversity, and shared 293 of 445 operational taxonomic units. However, endometritic and healthy cows could be discriminated by the relative abundance of bacterial genera. In CE cows, the uterine microbiota was characterized by increased abundance of Fusobacterium and unique presence of Trueperella and Peptoniphilus. For SE cows, known intrauterine pathogens were almost absent and the uterine microbiota was characterized by enrichment of Lactobacillus and Acinetobacter. Analysis of correlations between bacterial genera showed that the uterine microbiota exhibited two co-occurrence groups (i.e., the Lactococcus and the Fusobacterium COGs), indicating that the synergistic effect by co-occurred bacteria may be an important aspect of pathogenesis. Our findings support that common uterine pathogens are not associated with subclinical endometritis at 30 days postpartum and indicate the need of investigating the role of commensal bacteria such as Lactobacillus, and Acinetobacter in the inflammatory process of uterine endometrium.

Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Activation of NLRP3 and NLRC4 Inflammasomes With Differential Requirement for ASC.

Escherichia coli is a common cause of mastitis in dairy cows. The adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) synergizes with caspase-1 to regulate inflammasome activation during pathogen infection. Here, the ASC gene was knocked out in bovine mammary epithelial (MAC-T) cells using clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated (Cas)-9 technology. MAC-T cells were pre-incubated with and without Lactobacillus rhamnosus GR-1 and then exposed to E. coli. Western blot analysis demonstrated increased expression of NLRP3 and NLRC4 following E. coli infection, but this increase was attenuated by pre-incubation with L. rhamnosus GR-1, regardless of ASC knockout. Western blot and immunofluorescence analyses revealed that pre-incubation with L. rhamnosus GR-1 decreased E. coli-induced caspase-1 activation at 6 h after E. coli infection, as also observed in ASC-knockout MAC-T cells. The E. coli-induced increase in caspase-4 mRNA expression was inhibited by pre-incubation with L. rhamnosus GR-1. ASC knockout diminished, but did not completely prevent, increased production of IL-1ß and IL-18 and cell pyroptosis associated with E. coli infection, whereas pre-incubation with L. rhamnosus GR-1 inhibited this increase. Our data indicate that L. rhamnosus GR-1 suppresses activation of ASC-dependent NLRP3 and NLRC4 inflammasomes and production of downstream IL-lß and IL-18 during E. coli infection. L. rhamnosus GR-1 also inhibited E. coli-induced cell pyroptosis, in part through attenuation of NLRC4 and non-canonical caspase-4 activation independently of ASC.

Distribution and Antimicrobial Resistance of Salmonella Isolated from Pigs with Diarrhea in China.

Salmonella can cause enteric diseases in humans and a wide range of animals, and even outbreaks of foodborne illness. The aim of this study was to investigate the frequency and distribution of serovars, and antimicrobial resistance in Salmonella isolates from pigs with diarrhea in 26 provinces in China from 2014 to 2016. A total of 104 Salmonella isolates were identified and the dominant serovar was S. 4,[5],12:i:- (53.9%). All Salmonella isolates were resistant to trimethoprim-sulfamethoxazole, and many were resistant to ampicillin (80.8%) and tetracycline (76.9%). Among 104 Salmonella isolates, aac(6')-Ib-cr was the dominant plasmid-mediated quinolone resistance gene (80.8%), followed by qnrS (47.1%). The pulsed-field gel electrophoresis results suggest that the Salmonella isolates from different regions were genetically diverse, and ST34 was the most prevalent. S. 4,[5],12:i:- isolates is the widespread presence of heavy metal tolerance genes. The fact that the same sequence types were found in different regions and the high similarity coefficient of S. 4,[5],12:i:- isolates from different regions indicate the clonal expansion of the isolates, and the isolates carried various antimicrobial resistance genes. The multidrug resistant Salmonella can be widely detected in pigs, which will present a challenge for farm husbandry.

Anti-inflammatory capacity of Lactobacillus rhamnosus GG in monophasic variant Salmonella infected piglets is correlated with impeding NLRP6-mediated host inflammatory responses.

In this study, we investigated the effect of Lactobacillus rhamnosus GG strain (LGG) in ameliorating enteritis in newly weaned pigs following challenge with a monophasic variant of Salmonella enterica serovar Typhimurium (serotyped as 4,[5],12:i:-), which has been linked to disease in humans and livestocks over the past 10 years. In weaned pigs, S. enterica serovar 4,[5],12:i:- induced the mRNA expression of toll like receptor (TLR) 5 and TLR4, while increasing interleukin (IL)-8 and IL-6 mRNA expression in the jejunum. The monophasic variant Salmonella stimulated the expression of nucleotide-binding oligomerization domain-containing protein 1/2 (NOD1/2) mRNA in the ileum, which was accompanied by phosphorylation of IκB-α, an inhibitor of NF-κB, activating the NF-κB pathway and promoting the release of inflammatory cytokines. Oral administration of LGG attenuated the Salmonella-induced increases in the expression of NOD1 mRNA of jejunal and ileal tissues. LGG promoted the secretion of immunoglobulin A in different intestinal segments but did not induce expression of polymeric immunoglobulin receptor. LGG also impeded the activation of the Nod-like receptor protein (NLRP) 6/apoptosis-associated speck-like protein/caspase-1 inflammasome and decreased the production of IL-18 in the ileum during Salmonella infection. In contrast, activation of the NLRP3 inflammasome was not altered. Our data indicate that LGG accelerated the clearance of Salmonella in the early phase of infection and prevented the excessive inflammatory responses in S. enterica serovar 4,[5],12:i:- model. LGG ameliorates inflammation induced by infection with the monophasic variant Salmonella via inhibition of the canonical NF-κB pathway and attenuation of the NLRP6-mediated inflammasome in the intestine.