Isolation, identification and characteristics of Bibersteinia trehalosi from goat.

A conditionally pathogenic bacterium called Bibersteinia trehalosi inhabits the upper respiratory tract of ruminants and is becoming a significant cause of pneumonia, especially in goats. In this study, we identified a gram-negative bacteria strain isolated from dead goat's lungs, which was named M01. By integrating the outcomes of its morphological and biochemical characterization with the investigation of the 16S rRNA gene sequence analysis, the isolate was identified as B. trehalosi. Based on antibiotic susceptibility tests, the isolate was shown to be resistant to ß-lactams, tetracyclines, and amphenicols. Its genome was discovered to comprise 2115 encoded genes and a circular chromosome measuring 2,345,568 bp using whole genome sequencing. Annotation of the VFBD database revealed that isolate M01 had four virulence genes encoding three virulence factors. The CARD database revealed that its genome has two antibiotic-resistance genes. Based on pathogenicity testing, isolate M01 was highly pathogenic to mice, primarily causing pneumonia, with an LD50 of 1.31 × 107 CFU/ml. Moreover, histopathology showed loss of alveolar structure and infiltration of lung inflammatory cells. Hence, the current study could provide sufficient information for prevention and control strategies for future epidemics of B. trehalosi in goat species.

Porcine circovirus type 2 ORF5 induces an inflammatory response by up-regulating miR-21 levels through targeting nuclear ssc-miR-30d.

Porcine circovirus type 2 (PCV2) infection leads to multi-system inflammation in pigs, and this effect can be achieved by upregulating host miR-21. The underlying mechanism of miR-21 regulates PCV2-induced inflammation is already known, however, how PCV2 regulates miR-21 levels and function using both autonomic and host factors remains to be further revealed. Here we present the first evidence that PCV2 ORF5 induces an inflammatory response by up-regulating miR-21 level through targeting nuclear miR-30d. In this study, we found that overexpression of ORF5 significantly increased miR-21 level and promoted the expression of inflammatory cytokines and activation of the NF-κB pathway, while ORF5 mutation had the opposite effect. Moreover, the differential expression of miR-21 could significantly change the pro-inflammatory effect of ORF5, indicating that ORF5 promotes inflammatory response by up-regulating miR-21. Bioinformatics analysis and clinical detection found that nuclear miR-30d was significantly down-regulated after ORF5 overexpression and PCV2 infection, and targeted pri-miR-21 and PCV2 ORF5. Functionally, we found that miR-30d inhibited the levels of miR-21 and inflammatory cytokines in cells. Mechanistically, we demonstrated that ORF5 inhibits miR-30d expression levels through direct binding but not via the circRNA pathway, and miR-30d inhibits miR-21 levels by targeting pri-miR-21. In summary, the present study revealed the molecular mechanism of ORF5 upregulation of miR-21, further refined the molecular chain of PCV2-induced inflammatory response and elucidated the role of miRNAs in it.

Elucidating the biological characteristics and pathogenicity of the highly virulent G2a porcine epidemic diarrhea virus.

Since the large-scale outbreak of porcine epidemic diarrhoea (PED) in 2010, caused by the genotype 2 (G2) variant of the porcine epidemic diarrhoea virus (PEDV), pig farms in China, even those vaccinated with the G2b vaccine, have experienced infections from the G2a variant, leading to significant economic losses. This study successfully isolated the G2a strain DY2020 from positive small intestine contents (SICs) by blind passage on Vero cells for four generations. The SICs were taken from Daye, Hubei Province, China. The biological characteristics were identified by indirect immunofluorescence assay (IFA) and transmission electron microscopy (TEM). The growth kinetics of the strain on Vero cells were detected by TCID50, and the virus titre could reach 107.35 TCID50 ml-1 (SD: 5.07×106). The pathogenicity towards colostrum-deprived piglets was conducted by assessing faecal viral shedding, morphometric analysis of intestinal lesions, and immunohistochemical staining. The results showed that DY2020 was highly virulent to colostrum-deprived piglets, with severe watery diarrhoea and other clinical symptoms appeared at 6 h post-infection (h p.i.), and all died within 30 h. Pathological tissue examination results showed that the lesions mainly occurred in the intestines of piglets, causing pathological changes such as shortening of intestinal villi. In summary, the discovery of the G2a strain DY2020 in this study is of great significance for understanding Hubei PEDV and provides an important theoretical basis for the development of new efficient PEDV vaccines.

A Recombinant Chimera Vaccine Composed of LTB and Mycoplasma hyopneumoniae Antigens P97R1, mhp390 and P46 Elicits Cellular Immunologic Response in Mice.

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), leading to a mild and chronic pneumonia in swine. Relative control has been attained through active vaccination programs, but porcine enzootic pneumonia remains a significant economic challenge in the swine industry. Cellular immunity plays a key role in the prevention and control of porcine enzootic pneumonia. Therefore, the development of a more efficient vaccine that confers a strong immunity against M. hyopneumoniae is necessary. In this study, a multi-antigen chimera (L9m6) was constructed by combining the heat-labile enterotoxin B subunit (LTB) with three antigens of M. hyopneumoniae (P97R1, mhp390, and P46), and its immunogenic and antigenic properties were assessed in a murine model. In addition, we compared the effect of individual administration and multiple-fusion of these antigens. The chimeric multi-fusion vaccine induced significant cellular immune responses and high production of IgG and IgM antibodies against M. hyopneumoniae. Collectively, our data suggested that rL9m6 chimera exhibits potential as a viable vaccine candidate for the prevention and control of porcine enzootic pneumonia.

Tea Polyphenols Protects Tracheal Epithelial Tight Junctions in Lung during Actinobacillus pleuropneumoniae Infection via Suppressing TLR-4/MAPK/PKC-MLCK Signaling.

Actinobacillus pleuropneumoniae (APP) is the causative pathogen of porcine pleuropneumonia, a highly contagious respiratory disease in the pig industry. The increasingly severe antimicrobial resistance in APP urgently requires novel antibacterial alternatives for the treatment of APP infection. In this study, we investigated the effect of tea polyphenols (TP) against APP. MIC and MBC of TP showed significant inhibitory effects on bacteria growth and caused cellular damage to APP. Furthermore, TP decreased adherent activity of APP to the newborn pig tracheal epithelial cells (NPTr) and the destruction of the tight adherence junction proteins ß-catenin and occludin. Moreover, TP improved the survival rate of APP infected mice but also attenuated the release of the inflammation-related cytokines IL-6, IL-8, and TNF-α. TP inhibited activation of the TLR/MAPK/PKC-MLCK signaling for down-regulated TLR-2, TLR4, p-JNK, p-p38, p-PKC-α, and MLCK in cells triggered by APP. Collectively, our data suggest that TP represents a promising therapeutic agent in the treatment of APP infection.

The adcA and lmb Genes Play an Important Role in Drug Resistance and Full Virulence of Streptococcus suis.

Streptococcus suis is an recognized zoonotic pathogen of swine and severely threatens human health. Zinc is the second most abundant transition metal in biological systems. Here, we investigated the contribution of zinc to the drug resistance and pathogenesis of S. suis. We knocked out the genes of AdcACB and Lmb, two Zn-binding lipoproteins. Compared to the wild-type strain, we found that the survival rate of this double-mutant strain (ΔadcAΔlmb) was reduced in Zinc-limited medium, but not in Zinc-supplemented medium. Additionally, phenotypic experiments showed that the ΔadcAΔlmb strain displayed impaired adhesion to and invasion of cells, biofilm formation, and tolerance of cell envelope-targeting antibiotics. In a murine infection model, deletion of the adcA and lmb genes in S. suis resulted in a significant decrease in strain virulence, including survival rate, tissue bacterial load, inflammatory cytokine levels, and histopathological damage. These findings show that AdcA and Lmb are important for biofilm formation, drug resistance, and virulence in S. suis. IMPORTANCE Transition metals are important micronutrients for bacterial growth. Zn is necessary for the catalytic activity and structural integrity of various metalloproteins involved in bacterial pathogenic processes. However, how these invaders adapt to host-imposed metal starvation and overcome nutritional immunity remains unknown. Thus, pathogenic bacteria must acquire Zn during infection in order to successfully survive and multiply. The host uses nutritional immunity to limit the uptake of Zn by the invading bacteria. The bacterium uses a set of high-affinity Zn uptake systems to overcome this host metal restriction. Here, we identified two Zn uptake transporters in S. suis, AdcA and Lmb, by bioinformatics analysis and found that an adcA and lmb double-mutant strain could not grow in Zn-deficient medium and was more sensitive to cell envelope-targeting antibiotics. It is worth noting that the Zn uptake system is essential for biofilm formation, drug resistance, and virulence in S. suis. The Zn uptake system is expected to be a target for the development of novel antimicrobial therapies.

Theaflavin Ameliorates Streptococcus suis-Induced Infection In Vitro and In Vivo.

Streptococcus suis (S. suis) is one of the most important zoonotic pathogens that threaten the lives of pigs and humans. Even worse, the increasingly severe antimicrobial resistance in S. suis is becoming a global issue. Therefore, there is an urgent need to discover novel antibacterial alternatives for the treatment of S. suis infection. In this study, we investigated theaflavin (TF1), a benzoaphenone compound extracted from black tea, as a potential phytochemical compound against S. suis. TF1 at MIC showed significant inhibitory effects on S. suis growth, hemolytic activity, and biofilm formation, and caused damage to S. suis cells in vitro. TF1 had no cytotoxicity and decreased adherent activity of S. suis to the epithelial cell Nptr. Furthermore, TF1 not only improved the survival rate of S. suis-infected mice but also reduced the bacterial load and the production of IL-6 and TNF-α. A hemolysis test revealed the direct interaction between TF1 and Sly, while molecular docking showed TF1 had a good binding activity with the Glu198, Lys190, Asp111, and Ser374 of Sly. Moreover, virulence-related genes were downregulated in the TF1-treated group. Collectively, our findings suggested that TF1 can be used as a potential inhibitor for treating S. suis infection in view of its antibacterial and antihemolytic activity.

4-Phenyl-butyric Acid Inhibits Japanese Encephalitis Virus Replication via Inhibiting Endoplasmic Reticulum Stress Response.

Japanese encephalitis virus (JEV) infection causes host endoplasmic reticulum stress (ERS) reaction, and then induces cell apoptosis through the UPR pathway, invading the central nervous system and causing an inflammation storm. The endoplasmic reticulum stress inhibitor, 4-phenyl-butyric acid (4-PBA), has an inhibitory effect on the replication of flavivirus. Here, we studied the effect of 4-PBA on JEV infection both in vitro and vivo. The results showed that 4-PBA treatment could significantly decrease the titer of JEV, inhibit the expression of the JEV NS3 protein (in vitro, p < 0.01) and reduce the positive rate of the JEV E protein (in vivo, p < 0.001). Compared to the control group, 4-PBA treatment can restore the weight of JEV-infected mice, decrease the level of IL-1ß in serum and alleviate the abnormalities in brain tissue structure. Endoplasmic reticulum stress test found that the expression level of GRP78 was much lower and activation levels of PERK and IRE1 pathways were reduced in the 4-PBA treatment group. Furthermore, 4-PBA inhibited the UPR pathway activated by NS3, NS4b and NS5 RdRp. The above results indicated that 4-PBA could block JEV replication and inhibit ER stress caused by JEV. Interestingly, 4-PBA could reduce the expression of NS5 by inhibiting transcription (p < 0.001), but had no effect on the expression of NS3 and NS4b. This result may indicate that 4-PBA has antiviral activity independent of the UPR pathway. In summary, the effect of 4-PBA on JEV infection is related to the inhibition of ER stress, and it may be a promising drug for the treatment of Japanese encephalitis.

Mutations in troABCD against Copper Overload in a copA Mutant of Streptococcus suis.

Streptococcus suis is a major swine pathogen that is increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Metal homeostasis plays a critical role during the process of bacterial infection. In this study, RNA sequencing was used to identify potential candidate genes involved in the maintenance of intracellular copper homeostasis. CopA was identified as the primary copper exporter in S. suis. The copA deletion mutant strain was found to be more sensitive to copper and accumulated more intracellular copper than the wild-type (WT) parent strain. In addition, adding manganese increased the ability of S. suis to resist copper, and the manganese transporter, TroABCD, was involved in tolerance to copper. The copA deletion mutant strain accumulated less copper when supplemented with manganese. Furthermore, when cultured with copper, the double deletion mutant (ΔcopAΔtroA) exhibited improved growth compared to the copA deletion mutant strain. In addition, the double deletion mutant (ΔcopAΔtroA) accumulated less copper than the copA deletion mutant strain. These data were consistent with a model wherein defective TroABCD resulted in decreased cellular copper accumulation and protected the strain against copper poisoning. IMPORTANCE Metal homeostasis plays a critical role during the process of bacterial infection. We identified three important potential candidate genes involved in maintenance of intracellular copper homeostasis. CopA was demonstrated to be the main copper exporter in Streptococcus suis, and manganese increased the tolerance of S. suis to copper. The double deletion mutant (ΔcopAΔtroA) improved growth ability over the copA deletion mutant strain in the presence of high concentrations of copper and accumulated less copper. These findings are consistent with a model wherein defective TroABCD resulted in decreased cellular accumulation of copper and protected the strain against copper poisoning.

Signaling Lymphocytic Activation Molecule Family Member 1 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease in domestic swine. Signaling lymphocytic activation molecule family member 1 (SLAMF1) is a costimulatory factor that is involved in innate immunity, inflammation, and infection. Here, we demonstrate that overexpression of the SLAMF1 gene inhibited PRRSV replication significantly and reduced the levels of key signaling pathways, including MyD88, RIG-I, TLR2, TRIF, and inflammatory factors IL-6, IL-1ß, IL-8, TNF-ß, TNF-α, and IFN-α in vitro. However, the knockdown of the SLAMF1 gene could enhance replication of the PRRSV and the levels of key signaling pathways and inflammatory factors. Overall, our results identify a new, to our knowledge, antagonist of the PRRSV, as well as a novel antagonistic mechanism evolved by inhibiting innate immunity and inflammation, providing a new reference and direction for PRRSV disease resistance breeding.

Metabolomics and proteomics analyses revealed mechanistic insights on the antimicrobial activity of epigallocatechin gallate against Streptococcus suis.

Streptococcus suis (S. suis) is a highly virulent zoonotic pathogen and causes severe economic losses to the swine industry worldwide. Public health security is also threatened by the rapidly growing antimicrobial resistance in S. suis. Therefore, there is an urgent need to develop new and safe antibacterial alternatives against S. suis. The green tea polyphenol epigallocatechin gallate (EGCG) with a number of potential health benefits is known for its antibacterial effect; however, the mechanism of its bactericidal action remains unclear. In the present, EGCG at minimal inhibitory concentration (MIC) showed significant inhibitory effects on S. suis growth, hemolytic activity, and biofilm formation, and caused damage to S. suis cells in vitro. EGCG also reduced S. suis pathogenicity in Galleria mellonella larvae in vivo. Metabolomics and proteomics analyses were performed to investigate the underlying mechanism of antibacterial activity of EGCG at MIC. Many differentially expressed proteins involved in DNA replication, synthesis of cell wall, and cell membrane, and virulence were down-regulated after the treatment of S. suis with EGCG. EGCG not only significantly reduced the hemolytic activity of S. suis but also down-regulated the expression of suilysin (Sly). The top three shared KEGG pathways between metabolomics and proteomics analysis were ABC transporters, glycolysis/gluconeogenesis, and aminoacyl-tRNA biosynthesis. Taken together, these data suggest that EGCG could be a potential phytochemical compound for treating S. suis infection.

Epidemic and Evolutionary Characteristics of Swine Enteric Viruses in South-Central China from 2018 to 2021.

Swine enteric viruses are a major cause of piglet diarrhea, causing a devastating impact on the pork industry. To further understand the molecular epidemiology and evolutionary diversity of swine enteric viruses, we carried out a molecular epidemiological investigation of swine enteric viruses (PEDV, PDCoV, PoRVA, and TGEV) on 7107 samples collected from pig farms in south-central China. The results demonstrated that PEDV is the predominant pathogen causing piglet diarrhea, and its infection occurs mainly in relatively cold winter and spring in Hunan and Hubei provinces. The positive rate of PEDV showed an abnormal increase from 2020 to 2021, and that of PoRVA and PDCoV exhibited gradual increases from 2018 to 2021. PEDV-PoRVA and PEDV-PDCoV were the dominant co-infection modes. A genetic evolution analysis based on the PEDV S1 gene and ORF3 gene revealed that the PEDV GII-a is currently epidemic genotype, and the ORF3 gene of DY2020 belongs to a different clade relative to other GII-a strains isolated in this study. Overall, our results indicated that the variant PEDV GII-a is the main pathogen of piglet diarrhea with a trend of outbreak. G9 is the dominant PoRVA genotype and has the possibility of outbreak as well. It is therefore critical to strengthen the surveillance of PEDV and PoRVA, and to provide technical reserves for the prevention and control of piglet diarrhea.

Mycoplasma hyopneumoniae Infection Activates the NOD1 Signaling Pathway to Modulate Inflammation.

Mycoplasma hyopneumoniae is a highly contagious pathogen causing porcine enzootic pneumonia, which elicits prolonged inflammatory response modulated by pattern recognition receptors (PRRs). Although significant advances have been achieved in understanding the Toll-Like receptors that recognize M. hyopneumoniae, the role of nucleotide-binding oligomerization domain 1 (NOD1) in M. hyopneumoniae infected cells remains poorly understood. This study revealed that M. hyopneumoniae activates the NOD1-RIP2 pathway and is co-localized with host NOD1 during infection. siRNA knockdown of NOD1 significantly impaired the TRIF and MYD88 pathway and blocked the activation of TNF-α. In contrast, NOD1 overexpression significantly suppressed M. hyopneumoniae proliferation. Furthermore, we for the first time investigated the interaction between M. hyopneumoniae mhp390 and NOD1 receptor, and the results suggested that mhp390 and NOD1 are possibly involved in the recognition of M. hyopneumoniae. These findings may improve our understanding of the interaction between PRRs and M. hyopneumoniae and the function of NOD1 in host defense against M. hyopneumoniae infection.

PerR-Regulated Manganese Import Contributes to Oxidative Stress Defense in Streptococcus suis.

Streptococcus suis has been increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Metal homeostasis plays a critical role in the antioxidative capability of bacteria, thus facilitating the escape of pathogenic species from the innate immunity systems of hosts. Here, we revealed that manganese increased the ability of S. suis to resist oxidative stress. RNA sequencing was used to identify potential candidate genes involved in the maintenance of intracellular manganese homeostasis. Four genes, termed troABCD, were identified by NCBI BLASTp analysis. The troA, troB, troC, and troD deletion mutant strains exhibited decreased intracellular manganese content and tolerance to H2O2 compared to the wild-type strain. Thus, troABCD were determined to be involved in manganese uptake and played an important role in H2O2 tolerance in S. suis. Furthermore, the inactivation of perR increased the survival of H2O2-pulsed S. suis 2.18-fold and elevated the intracellular manganese content. H2O2-pulsed S. suis and perR deletion mutants upregulated troABCD. This finding suggested that H2O2 released the suppression of troABCD by perR. In addition, an electrophoretic mobility shift assay (EMSA) showed that PerR at 500 ng binds to the troABCD promoter, indicating that troABCD were directly regulated by PerR. In conclusion, this study revealed that manganese increases tolerance to H2O2 by upregulating the expression of troABCD. Moreover, PerR-regulated Mn import in S. suis and increased the tolerance of S. suis to oxidative stress by regulating troABCD. IMPORTANCE During infection, it is extremely important for bacteria to defend against oxidative stress. While manganese plays an important role in this process, its role is unclear in S. suis. Here, we demonstrated that manganese increased S. suis tolerance to oxidative stress. Four manganese ABC transporter genes, troABCD, were identified. Oxidative stress increased the content of manganese in the cell. Furthermore, PerR increased the tolerance to oxidative stress of S. suis by regulating troABCD. Manganese played an important role in bacterial defense against oxidative stress. These findings provide novel insight into the mechanism by which S. suis resists oxidative stress and approaches to inhibit bacterial infection by limiting manganese intake.

Green tea polyphenols inhibit growth, pathogenicity and metabolomics profiles of Streptococcus suis.

Streptococcus suis (SS) is an important pathogen in pigs and can also cause severe infection in humans. Currently, more and more drug resistance is reported, resulting in the search for new drugs being needed urgently. Green tea polyphenols (GTP) was reported to inhibit many bacteria. However, SS response to GTP has not been studied before. In this report, the effect of GTP on growth, cell integrity, pathogenicity and metabolic pathway of SS was examined. The GTP inhibited growth, led to cellular damage, and attenuated pathogenicity of SS. Finally, GTP affected many important metabolic pathways of SS, such as ABC transporters, pyrimidine metabolism, protein digestion and absorption. The results provide new insight into the prevention and control of SS infection.

Expression profile of long non-coding RNAs in porcine lymphnode response to porcine circovirus type 2 infection.

Porcine circovirus type 2 (PCV2) can cause various clinical diseases in pigs, resulting in huge losses for the pig farms all over the world. In order to develop a new strategy to control PCV2, it is essential to understand its mechanisms firstly, especially PCV2 interferes with the host's innate immunity. In the present study, lncRNA and mRNA expression profiles in porcine lymphnode response to PCV2 infection were deeply sequenced and analyzed. 3271 novel lncRNAs were identified in all. 1898 mRNAs and 282 lncRNAs showed differential expression between control and PCV2-infected groups. The bioinformatics analysis including lncRNA-mRNA co-expression network construction, as well as GO and KEGG pathway analysis focused on the DEGs was carried out. The results indicated that lncRNAs might participate in PCV2 infection-induced the pathogenesis of immunosuppression through regulating the host's immune responses, biological regulation, response to stimulus, cellular component organization or biogenesis and metabolism. And these differentially expressed lncRNAs might play important roles in response to PCV2 infection in the host's innate immune system. These findings provided a large-scale survey of dysregulated lncRNAs after PCV2 infection, especially the lncRNAs responded to host's innate immune within the lymphnode. This study will provide a novel insight into the lncRNAs' functions and the possible immunosuppressive mechanism induced by PCV2 infection. However, further research will be required to verify the characteristic function of the dysregulated lncRNAs.

Three novel immunogenic proteins determined through 2-Dimensional electrophoresis and mass spectrometry with immune serum confer protection against challenge with porcine Pasteurella multocida in mouse models.

Pasteurella multocida is an important zoonotic pathogen that causes multiple diseases in both animals and humans. Test of good immunogenic proteins is beneficial for vaccine development and disease control. In the present study, we determined four novel immunogenic proteins of P. multocida by using 2-DE MALDI-TOF MS with immune serum. These four proteins included a trimethylamine-N-oxide reductase TorA, a translation elongation factor Ts, a phosphoglyceromutase PGAM, and a peroxiredoxin PrX. Among these proteins, TorA, Prx, and PGAM were successfully expressed by using E. coli. Western-blotting assays showed that recombinant TorA, Prx, and/or PGAM displayed good reactions with infectious sera of P. multocida serogroups A, B, D and F. Immunization of either rTorA, rPrx, and/or rPGAM induced significantly high levels of antibodies as well as IFN-γ, IL-4 and IL-10 in mice (P < 0.01). Protective efficacy tests revealed that vaccination of either rTorA, rPrx, and/or rPGAM protected 60% ~ 80% of the tested mice against the challenge with P. multocida field isolate. Our results obtained from the present study suggest that these three proteins could be tested as good vaccine candidates against P. multocida infections.

Rapid and simultaneous detection of Japanese encephalitis virus by real-time nucleic acid sequence-based amplification.

Japaneses encephalitis (JE) is most common zoonoses caused by Japanese encephalitis virus (JEV) with a high mortality and disability rate. To take timely preventive and control measures, early and rapid detection of JE RNA is necessary. But due to characteristic brief and low viraemia, JE RNA detection remains challenging. In this study, a real-time nucleic acid sequence-based amplification (RT-NASBA) was developed for rapid and simultaneous detection of JEV. Four pairs of primer were designed using a multiple genome alignment of all JEV strains from GenBank. NASBA assay established and optimal reaction conditions were confirmed by using primers and probe on ns1 gene of JEV. The specificity and sensitivity of the assay were compared with RT-PCR by using serial RNA and virus cultivation dilutions. The results showed that JEV RT-NASBA assay was established, and robust signals could be observed in 10 min with high specificity. The limit of dectetion of RT-NASBA was 6 copies per reaction. The assay was thus 100 to 1, 000 times more sensitive than RT-PCR. The cross-reaction was performed with other porcine pathogens, and negative amplification results indicated the high specificity of this method. The novel JEV RT-NASBA assay could be used as an efficient molecular biology tool to diagnose JEV, which would facilitate the surveillance of reproductive failure disease in swine and would be beneficial for public health security.

Tea polyphenols inhibit the growth and virulence of ETEC K88.

Diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) causes high levels of morbidity and mortality in neonatal piglets. Owing to the abuse of antibiotics and emergence of drug resistance, antibiotics are no longer considered only beneficial, but also potentially harmful drugs. Supplements that can inhibit the growth of bacteria are expected to replace antibiotics. Tea polyphenols have numerous important biological functions, including antibacterial, antiviral, antioxidative, anti-inflammatory, and antihypertensive effects. We investigated the role of tea polyphenols in ETEC K88 infection using a mouse model. Pretreating with tea polyphenols attenuated the symptoms induced by ETEC K88. Furthermore, in a cell adherence assay, tea polyphenols inhibited ETEC K88 adherence to IPEC-J2 cells. When cells were infected with ETEC K88, mRNA and protein levels of claudin-1 were significantly decreased compared with those of control cells. However, when cells were pretreated with tea polyphenols, claudin-1 mRNA and protein levels were higher than those in cells without pretreatment upon cell infection with ETEC K88. TLR2 mRNA levels were also higher following cell infection with ETEC K88 when cells were pretreated with tea polyphenols. These data revealed that tea polyphenols could increase the barrier integrity of IPEC-J2 cells by upregulating expression of claudin-1 through activation of TLR2. Tea polyphenols had beneficial effects on epithelial barrier function. Therefore, tea polyphenols could be used as a novel strategy to control and treat pig infections caused by ETEC K88.

Isolation and evolutionary analyses of porcine epidemic diarrhea virus in Asia.

Porcine epidemic diarrhea virus (PEDV) is a leading cause of diarrhea in pigs worldwide. Virus isolation and genetic evolutionary analysis allow investigations into the prevalence of epidemic strains and provide data for the clinical diagnosis and vaccine development. In this study, we investigated the genetic characteristics of PEDV circulation in Asia through virus isolation and comparative genomics analysis. APEDV strain designated HB2018 was isolated from a pig in a farm experiencing a diarrhea outbreak. The complete genome sequence of HB2018 was 28,138 bp in length. Phylogenetic analysis of HB2018 and 207 PEDVs in Asia showed that most PEDV strains circulating in Asia after 2010 belong to genotype GII, particularly GII-a. The PEDV vaccine strain CV777 belonged to GI, and thus, unmatched genotypes between CV777 and GII-a variants might partially explain incomplete protection by the CV777-derived vaccine against PEDV variants in China. In addition, we found the S protein of variant strains contained numerous mutations compared to the S protein of CV777, and these mutations occurred in the N-terminal domain of the S protein. These mutations may influence the antigenicity, pathogenicity, and neutralization properties of the variant strains.