Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA.

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2% (11/5483), 8.82% (485/5483), 1.22% (67/5483), and 4.94% (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39%, 0.11%, 0.01%, and 0.03%, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.

Establishment and Application of a Quadruplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, Porcine Circovirus Type 3, and Streptococcus suis.

Respiratory illnesses present a significant threat to porcine health, with co-infections involving Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Streptococcus suis (SS), Porcine Circovirus Type 2 (PCV2), and Porcine Circovirus Type 3 (PCV3) acting as the primary causative agents. As a result, the precise diagnosis of PRRSV, PCV2, PCV3 and SS is of paramount importance in the prevention and control of respiratory diseases in swine. Therefore, we conducted a molecular bioinformatical analysis to concurrently detect and differentiate PRRSV, PCV2, PCV3 and SS. We selected the ORF6 gene of PRRSV, the ORF2 gene of PCV2 and PCV3, and the glutamate dehydrogenase (GDH) gene of SS as targets. Specific primers and probes were designed for each pathogen, and following meticulous optimization of reaction conditions, we established a multiple TaqMan fluorescence quantitative PCR detection method. Subsequently, we subjected this method to a comprehensive assessment, evaluating its specificity, sensitivity, and repeatability. The research results demonstrated that the established multiple TaqMan fluorescence quantitative PCR detection method displays displayed exemplary specificity, with no instances of cross-reactivity with other pathogens. The method's minimum detection concentrations for PRRSV, PCV2, PCV3, and SS were 2.80 × 101 copies/µL, 1.96 × 102 copies/µL, 2.30 × 102 copies/µL, and 1.75 × 103 copies/µL, respectively. When applied to the analysis of 30 clinical samples, the results closely mirrored those obtained through Chinese standard uniplex real-time qPCR detection method for PRRSV, as well as the general PCR methods for SS, PCV2, and PCV3. This study underscores the robust specificity, high sensitivity, and consistent stability of the multiple TaqMan fluorescence quantitative PCR detection method that we have developed. It is ideally suited to the clinical monitoring of PRRSV, PCV2, PCV3, and SS, and it carries significant importance in ongoing efforts to prevent and manage respiratory diseases in porcine populations.

Fraxetin ameliorates symptoms of dextran sulphate sodium-induced colitis in mice.

Ulcerative colitis (UC) is one of the primary inflammatory bowel diseases (IBDs) and causes a serious threat to human public health around the world. Currently, there are no proven safe and effective treatment options to treat UC. Fraxetin (Fxt) is a widely recognized antioxidant and anti-inflammatory legume derived from ash bark. In the present study, we investigated the protective effect and mechanism of Fxt on UC. Our results showed that Fxt significantly attenuated the body weight, colon length reduction, tissue damage, and disease activity index induced by dextran sodium sulphate (DSS). Moreover, the DSS-induced activation of the NF-κB pathway and NLRP3 inflammasomes was inhibited, and the inflammatory response was reduced. Fxt restored gut barrier function by increasing the number of goblet cells and the levels of tight junction proteins (ZO-1 and occludin). In addition, Fxt can alter the intestinal microbiota by enhancing the diversity of the microbiota, increasing the relative abundance of beneficial bacteria and inhibiting the growth of harmful bacteria. These results revealed that Fxt alleviates DSS-induced colitis by modulating the inflammatory response, enhancing epithelial barrier integrity and regulating the gut microbiota. This study may provide a scientific basis for the potential therapeutic effect of Fxt in the prevention of colitis and other related diseases.

Microglia play an important role in PRV infection-induced immune responses of the central nervous system.

Pseudorabies virus (PRV) can infect multiple hosts and lead to fatal encephalitis. There is a significant increase in the number of microglia in the brain of animals infected with PRV. However, whether and how microglia contribute to central nervous system damage in PRV infection remain unknown. In the present study, we elucidated that PRV infection can cause more severe inflammatory cell infiltration, thicker and more numerous vessel sleeve walls, and more severe inflammatory responses in the brains of natural hosts (pigs) than in those of nonnatural hosts (mice). In a mice infection model, activated microglia restricted viral replication in the early stage of infection. Acute neuroinflammation caused by microglia hyperactivation at late-stage of infection. Furthermore, in vitro experiments revealed that microglia restricted viral replication and decreased viral infectivity. This may be associated with the phagocytic ability of microglia because we observed a significant increase in the expression of the membrane receptor TREM2 in microglia, which is closely related to phagocytosis, we observed that depletion of microglia exacerbated neurological symptoms, blood-brain barrier breakdown, and peripheral lymphocyte infiltration. Taken together, we revealed the dual role of microglia in protecting the host and neurons from PRV infection.

Pathological observation and transcriptomic analysis of thymus injury in PRRSV-infected piglets.

The thymus, the central immune organ in mammals, plays an important role in immune defense. Porcine reproductive and respiratory syndrome virus (PRRSV) infection in piglets can cause thymus injury and immunosuppression. However, the mechanisms of thymus injury remain unknown. This study was aimed at investigating the specific manifestations of thymus injury through the construction of a PRRSV-infected piglet model and histopathological observation. In this study, fourteen 40-day-old PRRSV-free piglets were randomly divided into two groups, eleven of which were intramuscularly injected with 3 mL of PRRSV WUH3 virus suspension (106 PFU /mL) in the infection group, and three of which were sham-inoculated with 3 mL of RPMI-1640 medium in the control group. Clinical necropsy and samples collection were performed on day 8 after artificial infection. With the Illumina platform, the transcriptomes of piglet thymus tissues from infected and control piglets were sequenced to explore the relationships of differentially expressed genes (DEGs) and signaling pathways with thymus injury. The immune organs of PRRSV-infected piglets were severely damaged. The histopathological findings in the thymus indicated that PRRSV infection was associated with a large decrease in lymphocytes, cell necrosis and cell apoptosis; an increase in blood vessels and macrophages; thymic corpuscle hyperplasia; and interstitial widening of the thymic lobules. The transcriptomic analysis results revealed that the Gene Ontology functions of DEGs were enriched primarily in biological processes such as angiogenesis, regulation of angiogenesis and positive regulation of cell migration. Moreover, greater numbers of blood vessels and macrophages were observed in the thymus in PRRSV-infected than control piglets. KEGG pathway enrichment analysis revealed that the DEGs were significantly enriched in the Toll-like receptor signaling pathway, chemokine signaling pathway, IL-17 signaling pathway and TNF signaling pathway. The expression of TLR8, IRF5, the chemokines CCL2, CCL3L1 and CCL5; and their receptors CCR1, CCR2 and CCR5 was significantly up-regulated in PRRSV infection, thus suggesting that these cytokines were associated with the pathological processes of thymus injury.

Japanese encephalitis virus induces apoptosis by activating the RIG-1 signaling pathway.

Japanese encephalitis virus (JEV) infection can cause brain tissue lesions characterized by neuronal death, and apoptosis is involved in JEV-induced neuronopathy. In the present study, mouse microglia were infected with JEV, and pyknosis with dark-staining nuclei of infected cells was detected using Hoechst 33342 staining. TUNEL staining showed that JEV infection promoted the apoptosis of BV2 cells, and the apoptosis rate was significantly increased at 24-60 hours postinfection (hpi) (P < 0.01) and was the highest at 36 h (P < 0.0001). Western blot results showed that the expression of the Bcl-2 protein in JEV-infected cells was downregulated significantly at 60 hpi (P < 0.001), whereas that of the Bax protein was observably upregulated at 60 hpi (P < 0.001). At the same time, the level of cytochrome c (Cyt c) was significantly increased (P < 0.001), and the expression levels of two apoptosis-related proteins, namely, cleaved caspase-3 (P < 0.01) and caspase-9 (P < 0.001), were elevated significantly. Immunofluorescence staining showed that the amount of Cyt c increased with time after infection. After BV2 cells were infected with JEV, the expression of RIG-1 increased significantly from 24 hpi to 60 h (P < 0.001). The expression of MAVS increased significantly at 24 h (P < 0.001) and decreased gradually from 24 h to 60 hpi. The expression of TBK1 and NF-κB (p65) was not significantly changed. The expression of p-TBK1 and p-NF-κB (p-p65) increased significantly within 24 h (P < 0.001) and decreased from 24 to 60 hpi. The expression levels of IRF3 and p-IRF3 peaked at 24 hpi (P < 0.001) and decreased gradually from 24 to 60 hpi. However, the expression levels of JEV proteins showed no significant change at 24 and 36 hpi but were markedly elevated at 48 and 60 hpi. Interference with the expression of the RIG-1 protein in BV2 cells resulted in a dramatic increase in the expression of the anti-apoptotic protein Bcl-2 (P < 0.05), whereas the pro-apoptotic protein Bax, cleaved caspase-9, and especially cleaved caspase-3 were downregulated (P < 0.05), and viral protein expression was notably reduced (P < 0.05). These results indicate that JEV induces apoptosis through mitochondrial-dependent apoptosis pathways, interfering with the expression of RIG-1 in BV2 cells can inhibit viral replication and inhibit apoptosis.

Virulent Phage vB_EfaS_WH1 Removes Enterococcus faecalis Biofilm and Inhibits Its Growth on the Surface of Chicken Meat.

Enterococcus faecalis is a potential animal and human pathogen. Improper use of antibiotics encourages resistance. Bacteriophages and their derivatives are promising for treating drug-resistant bacterial infections. In this study, phylogenetic and electron microscopy analyses of phage vB_EfaS_WH1 (WH1) isolated from chicken feces revealed it to be a novel phage in the family Siphoviridae. WH1 showed good pH stability (4-11), temperature tolerance (4-60 °C), and broad E. faecalis host range (60% of isolates). Genome sequencing revealed a 56,357 bp double-stranded DNA genome with a G+C content of 39.21%. WH1 effectively destroyed E. faecalis EF01 biofilms, even at low concentrations. When WH1 was applied at 1 × 105 to 1 × 109 PFU/g to chicken breast samples stored at 4 °C, surface growing E. faecalis were appreciably eradicated after 24 h. The phage WH1 showed good antibacterial activity, which could be used as a potential biocontrol agent to reduce the formation of E. faecalis biofilm, and could also be used as an alternative for the control of E. faecalis in chicken products.

Identification of Embryonic Chicken Proteases Activating Newcastle Disease Virus and Their Roles in the Pathogenicity of Virus Used as In Ovo Vaccine.

In ovo vaccination is an attractive immunization approach for chickens. However, most live Newcastle disease virus (NDV) vaccine strains used safely after hatching are unsafe as in ovo vaccines due to their high pathogenicity for chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. Our previous studies reported that NDV strain TS09-C was a safe in ovo vaccine, and the F protein cleavage site (FCS) containing three basic amino acids (3B-FCS) was the crucial determinant of the attenuation of TS09-C in chicken embryos. Here, five trypsin-like proteases that activated NDV in chicken embryos were identified. The F protein with 3B-FCS was sensitive to the proteases Tmprss4, Tmprss9, and F7, was present in fewer tissue cells of chicken embryos, which limited the viral tropism, and was responsible for the attenuation of NDV with 3B-FCS, while the F protein with FCS containing two basic amino acids could be cleaved not only by Tmprss4, Tmprss9, and F7 but also by Prss23 and Cfd, was present in most tissue cells, and thereby was responsible for broad tissue tropism and high pathogenicity of virus in chicken embryos. Furthermore, when mixed with the protease inhibitors aprotinin and camostat, NDV with 2B-FCS exhibited greatly weakened pathogenicity in chicken embryos. Thus, our results extend the understanding of the molecular mechanism of NDV pathogenicity in chicken embryos and provide a novel molecular target for the rational design of in ovo vaccines, ensuring uniform and effective vaccine delivery and earlier induction of immune protection by the time of hatching. IMPORTANCE As an attractive immunization approach for chickens, in ovo vaccination can induce a considerable degree of protection by the time of hatching, provide support in closing the window in which birds are susceptible to infection, facilitate fast and uniform vaccine delivery, and reduce labor costs by the use of mechanized injectors. The commercial live Newcastle disease virus (NDV) vaccine strains are not safe for in ovo vaccination and cause the death of chicken embryos. The mechanism for viral pathogenicity in chicken embryos is poorly understood. In the present study, we identified five trypsin-like proteases that activate NDV in chicken embryos and elucidated their roles in the tissue tropism and pathogenicity of NDV used as in ovo vaccine. Finally, we revealed the molecular basis for the pathogenicity of NDV in chicken embryos and provided a novel strategy for the rational design of in ovo ND vaccines.

Fusion Protein Cleavage Site Containing Three Basic Amino Acids Attenuates Newcastle Disease Virus in Chicken Embryos: Use as an in ovo Vaccine.

In ovo vaccination is an attractive immunization strategy for the poultry industry. However, although most live Newcastle disease virus (NDV) vaccine strains, such as LaSota and V4, can be used after hatching, they are pathogenic to chicken embryos when administered in ovo. We have previously reported that NDV strain TS09-C is a safe in ovo vaccine in specific-pathogen-free and commercial chicken embryos because it is attenuated in chicken embryos. However, the molecular basis of its attenuation is poorly understood. In this study, we firstly evaluated the safety of chimeric NDV strains after exchanging genes between strains TS09-C and LaSota as in ovo vaccines, and demonstrated that the attenuation of NDV in chicken embryos was dependent upon the origin of the fusion (F) protein. Next, by comparing the F protein sequences of TS09-C strain with those of LaSota and V4 strain, the R115 in cleavage site and F379 were found to be unique to TS09-C strain. The mutant viruses were generated by substituting one or two amino acids at position 115 and 379 in the F protein, and their safety as in ovo vaccine was evaluated. Mutation in residue 379 did not affect the viral embryonic pathogenicity. While the mutant virus rTS-2B (R115G mutation based on the backbone of TS09-C strain) with two basic amino acids in F cleavage site, was pathogenic to chicken embryos and similar with rLaSota in its tissue tropism, differing markedly from rTS09-C with three basic amino acids in F cleavage site. Together, these findings indicate that the F protein cleavage site containing three basic amino acids is the crucial determinant of the attenuation of TS09-C in chicken embryos. This study extends our understanding of the pathogenicity of NDV in chicken embryos and should expedite the development of in ovo vaccines against NDV.

Autophagy inhibitors alleviate Japanese encephalitis virus-induced cerebral inflammation in mice.

Japanese encephalitis (JE) is a zoonotic epidemic disease caused by Japanese encephalitis virus (JEV), and currently, no medicines are available to treat this disease. Autophagy modulators play an important role in the treatment of tumors, heart disease, and some viral diseases. The aim of this study was to investigate the effects of autophagy modulators on JEV infection and the host response in mice. The experimental mice were grouped as follows: DMEM (control), JEV, JEV+rapamycin (JEV+Rapa), JEV+wortmannin (JEV+Wort), JEV+chloroquine (JEV+CQ), Rapa, Wort, and CQ. The control group was treated with DMEM. The mice in other groups were infected with 105 PFU of JEV, and Rapa, Wort, and CQ were administered 2 h prior to JEV challenge and then administered daily for 10 consecutive days. All mice were monitored for neurological signs and survival. The damage of subcellular structures in the mouse brain was evaluated by transmission electron microscopy. The distribution of virus in the mouse brain was determined by RNAScope staining and immunohistochemical staining. The neuroinflammatory responses in the brain were examined via quantitative real-time PCR, and the signal pathways involved in neuroinflammation were identified by Western blot. The mice in the JEV+Wort and JEV+CQ groups showed milder neurological symptoms, less damage to the mitochondria in the brain tissue, and a higher survival rate than those in the JEV+Rapa and JEV groups. Compared with the JEV+Rapa and JEV groups, the distribution of JEV in the brain of mice in the JEV+Wort and JEV+CQ groups was lower, and the inflammatory response was weaker. No significant difference was observed in the expression of the PI3K/AKT/NF-κB pathway in mouse brain among the different groups. Our study suggests that the autophagy inhibitors Wort and CQ reduce JEV infection and weaken the inflammatory response, which does not depend on the PI3K/AKT/NF-κB pathway in mouse brain.

Precise localization and dynamic distribution of Japanese encephalitis virus in the rain nuclei of infected mice.

Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne zoonotic diseases, thereby posing a serious threat to human health. Although JEV is potentially neurotropic, its pathogenesis and distribution in the host have not been fully elucidated. In this study, an infected mouse model was established using a highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, combined with anatomical imaging of the mouse brain, were used to dynamically localize the virus and construct three-dimensional (3D) images. Consequently, onset of mild clinical signs occurred in some mice at 3.5 d post JEV infection, while most mice displayed typical neurological signs at 6 d post-infection (dpi). Moreover, brain pathology revealed typical changes associated with non-suppurative encephalitis, which lasted up to 8 d. The earliest detection of viral antigen was achieved at 3 dpi in the thalamus and medulla oblongata. At 6 dpi, the positive viral antigen signals were mainly distributed in the cerebral cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 8 dpi, the antigen signals gradually decreased, and the localization of JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was detected in the brain at 21 dpi. In this model, the viral antigen was first expressed in the reticular thalamic nucleus (Rt), and the virus content is relatively stable. The expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images showed that viral signals were mostly concentrated in the parietal cortex, occipital lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in mice, a large number of viral antigens were detected in denatured and necrotic neurons, suggesting that JEV directly causes neuronal damage. From the time of its entry, JEV is widely distributed in the central nervous system thereby causing extensive damage.

Coinfection of porcine deltacoronavirus and porcine epidemic diarrhea virus altered viral tropism in gastrointestinal tract in a piglet model.

Coinfection of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) is one of common findings in diarrheal piglets that cause massive economic losses to the pig industry globally. However, the mechanism of the co-infection is unclear. In this study, neonatal non-colostrum-fed piglets were exposed orally with a single infection of PDCoV or PEDV, or coinfection of PDCoV and PEDV. Clinically all viral infected piglets developed watery diarrhea and dehydration in 24 h post-exposure (hpe) and were succumbed to viral diarrhea disease and euthanized at 72 hpe. Histopathologically, acute gastroenteritis is evident in all viral infected piglet. Immunohistochemistry, RNAscope and RT-qPCR demonstrated that PEDV tropism changes from epithelial cells of small intestine to gastric epithelial cells and macrophages in Peyer's patches in the ileum. These findings suggest that coinfection of PDCoV and PEDV can alter PEDV tropism that may affect the outcome of viral disease in piglets. This animal model can be used for the pathogenesis and vaccination of viral coinfection in piglet in the future.

Differential expression and correlation analysis of miRNA-mRNA profiles in swine testicular cells infected with porcine epidemic diarrhea virus.

The variant virulent porcine epidemic diarrhea virus (PEDV) strain (YN15) can cause severe porcine epidemic diarrhea (PED); however, the attenuated vaccine-like PEDV strain (YN144) can induce immunity in piglets. To investigate the differences in pathogenesis and epigenetic mechanisms between the two strains, differential expression and correlation analyses of the microRNA (miRNA) and mRNA in swine testicular (ST) cells infected with YN15, YN144, and mock were performed on three comparison groups (YN15 vs Control, YN144 vs Control, and YN15 vs YN144). The mRNA and miRNA expression profiles were obtained using next-generation sequencing (NGS), and the differentially expressed (DE) (p-value < 0.05) mRNA and miRNA were obtained using DESeq R package. mRNAs targeted by DE miRNAs were predicted using the miRanda algortithm. 8039, 8631 and 3310 DE mRNAs, and 36, 36, and 22 DE miRNAs were identified in the three comparison groups, respectively. 14,140, 15,367 and 3771 DE miRNA-mRNA (targeted by DE miRNAs) interaction pairs with negatively correlated expression patterns were identified, and interaction networks were constructed using Cytoscape. Six DE miRNAs and six DE mRNAs were randomly selected to verify the sequencing data by real-time relative quantitative reverse transcription polymerase chain reaction (qRT-PCR). Based on bioinformatics analysis, we discovered the differences were mostly involved in host immune responses and viral pathogenicity, including NF-κB signaling pathway and bacterial invasion of epithelial cells, etc. This is the first comprehensive comparison of DE miRNA-mRNA pairs in YN15 and YN144 infection in vitro, which could provide novel strategies for the prevention and control of PED.

Receptor-interacting serine/threonine kinase 1- and 3-dependent inflammation induced in lungs of chicken infected with Pasteurella multocida.

Fowl cholera is a serious, highly contagious disease caused by the bacterium Pasteurella multocida (P. multocida) in a range of avian species and is characterized by an acute form of septicaemia. The pathogenic mechanism of chicken lung injury caused by the bacterium is unclear. Therefore, P. multocida Q (a reference standard strain isolated from chicken) and 1G1 (a clinic isolated strain from duck) were selected to infect chickens, establishing fowl cholera-induced laying hen models. Several important proteins involved in the process of lung injury were identified and quantified using immunohistochemistry and WB. The results showed that chicken lungs infected with bacteria for 24 h showed congestion and edema. The inflammatory factors HMGB1 and IL-6, intercellular matrix MMP, the cell apoptosis-associated caspase-3 and necrotic apoptosis signal molecules RIPK1 and RIPK3 were widely expressed in the lungs of group Q and were significantly different compared with those of 1G1 group and uninfected group (P < 0.05). The results indicated that RIPK1 and RIPK3 are involved in the injury process of chicken lungs after infection with P. multocida, and the mechanisms of lung injury induced by different strains are different.

Porcine CXCR1/2 antagonist CXCL8(3-72)G31P inhibits lung inflammation in LPS-challenged mice.

Swine pneumonia is a great threat for pig industry around the world, which is usually accompanied with neutrophils infiltration in the airway. Although interleukin-8 (CXCL8) and its receptors, CXC chemokine receptor 1 and 2 (CXCR1/2) in human have been well documented, the expression and function of CXCR1/2 is still unknown in swine. To explore the feasibility to develop new veterinary anti-inflammatory drugs targeting porcine CXCR1/2, we detected CXCR1/2 expression in swine pneumonia through Real-Time PCR and immunohistochemistry for the first time. Two porcine CXCR1/2 antagonists, CXCL8(3-72)N11R/G31P (pN11R) and CXCL8(3-72)G31P (pG31P) were prepared and their anti-inflammatory effects were evaluated using cell chemotaxis assays and animal experiments. Our data showed that CXCR1/2 expression, which was closely related to neutrophil infiltration in the lung, was significantly up-regulated in swine pneumonia. The pN11R and pG31P could effectively inhibit the directional migration of neutrophils in vitro. In vivo data also indicated that both pN11R and pG31P significantly relieved LPS-induced pneumonia in mice through decreasing the expression of TNF-α, CXCL8, and IL-1ß, and inhibiting neutrophil influx into the lung. pG31P was more efficient. Our study suggested that it is possible to develop new veterinary anti-inflammatory drugs targeting porcine CXCR1/2, and pG31P is a promising candidate.

Necroptosis of Splenic Macrophages Induced by Streptococcus gallolyticus subsp. pasteurianus.

A previous study demonstrated that a highly virulent strain of Streptococcus gallolyticus subsp. pasteurianus, designated as the AL101002 strain, induced high mortality in ducklings with splenic lesions. In this study, 42 ducklings were subcutaneously inoculated with the AL101002 strain to study changes in splenic lesions over time. The spleens from these ducklings were significantly enlarged by congestion and edema, and/or showed multiple marbled areas 14 days postinoculation (dpi). The AL101002 strain was reisolated from the spleens and blood and confirmed by immunohistochemistry (IHC) with the use of anti-AL101002 antibody. Histopathologically, the main lesion was macrophage necrosis in the spleens from 1 to 7 dpi. Terminal dUTP nick-end labeling assay, transmission electron microscopy, and IHC by anti-macrosialin antibody (CD68) demonstrated that macrophage necrosis was necroptosis, which was further confirmed by quantitative (real-time) reverse-transcriptase PCR analysis. Two major factors of apoptosis, caspase 3 and caspase 8, did not significantly change during the AL101002 infection, suggesting that apoptosis signals were not activated. However, the key factor mixed lineage kinase like was increased significantly (P < 0.05) from Day 1 to Day 14 dpi. Inflammatory cytokine interleukin-1ß and interleukin-6 had significantly (P < 0.01) upregulated expression in the spleens on Day 1 dpi. Tumor necrosis factor α was downregulated from Day 1 to Day 5 dpi, but increased from Day 7 to Day 14. Our results demonstrated that AL101002 strain mainly infects macrophages and resulted in macrophage necroptosis and suggested that macrophage necroptosis in spleens is involved in the pathogenesis of S. gallolyticus subsp. pasteurianus infection in ducklings.

The Role of Necroptosis, Apoptosis, and Inflammation in Fowl Cholera-Associated Liver Injury in a Chicken Model.

Fowl cholera resulting from infection with Pasteurella multocida causes huge economic losses in the poultry industry. Necrotic hepatitis is reported to be a significant lesion associated with fowl cholera in chickens. Clarifying the underlying molecular mechanism of hepatic injury caused by P. multocida infection is needed to develop new strategies to control fowl cholera. Pasteurella multocida Q (the standard reference strain) and P. multocida 1G1 (a clinical strain) were used to infect healthy laying hens. Clinical signs were observed and gross lesions in livers were observed postmortem. Histologic lesions and the localization and expression of protein molecules associated with necroptosis, apoptosis, and inflammation in hepatic tissues were examined by hematoxylin and eosin staining and immunohistochemistry. Western blot analysis was used to determine the expression of liver injury-related genes. Necroptotic molecules such as RIPK1 (receptor interaction protein kinases 1), RIPK3 (receptor interaction protein kinases 3), and MLKL (mixed lineage kinase domain-like protein) were observed by immunostaining primarily in the cytoplasm of hepatocytes within or around necrotic foci, and inflammatory mediators HMGB1 (high-mobility group box 1) and IL-6 (interleukin-6) were found in the cytoplasm of heterophils, monocytes/macrophages, and hepatic sinusoids. In addition, MMP9 (matrix metalloproteinase 9) and TIMP1 (tissue inhibitor of metalloproteinase 1) were observed in hepatic parenchymal cells, inflammatory cells, and interstitial spaces, whereas the apoptotic effector molecule caspase-3 (cysteine-containing aspartic proteolytic enzymes 3) was mainly found in hepatocytes. The expression of RIPK1, RIPK3, and MLKL was significantly higher in the infected chickens than in the controls. HMGB1 and IL-6 protein levels were also increased in infected chickens relative to those in controls. Both MMP9 and TIMP1 were highly expressed in infected chickens. In addition, caspase-3 protein levels were significantly elevated in infected chickens. Necroptosis, apoptosis, and inflammation played a significant role in hepatic injury caused by P. multocida.

Genome Sequence of a Virulent Genotype III Newcastle Disease Virus Isolated from Laying Ducks in China.

Here, we report the complete genome sequence of a virulent Newcastle disease virus (NDV) strain HN1007, isolated from diseased duck flocks in Henan, China, in 2010. The isolate has a genome length of 15,186 nucleotides, and was classified as a member of genotype III of class II.

Inducible Expression of both ermB and ermT Conferred High Macrolide Resistance in Streptococcus gallolyticus subsp. pasteurianus Isolates in China.

Streptococcus gallolyticus subsp. pasteurianus is an under-recognized pathogen and zoonotic agent causing opportunistic infections in humans. Despite increasing recognition of this subspecies as a cause for human infectious diseases, limited information is known about its antibiotic resistance mechanism. In this study, we aim to identify the molecular mechanism underlying the high macrolide resistance of six S. gallolyticus subsp. pasteurianus isolates from dead ducklings collected in several natural outbreaks in China during 2010-2013. All isolates exhibited multi-drug resistance including high macrolide resistance (MIC ≥ 1024 mg/L for erythromycin, and 512 mg/L for clarithromycin). Efflux-encoding mefA and mefE genes were not detectable in these isolates. The presence of 23S rRNA mutations in specific isolates did not significantly change macrolide MICs. No nucleotide substitutions were found in genes encoding ribosomal proteins L4 or L22. The ermB and ermT genes were found in the genomes of all isolates. These two genes were acquired independently in one highly virulent isolate AL101002, and clustered with Tn916 and IS1216, respectively. The expression of both ermB and ermT in all isolates was erythromycin inducible and yielded comparable macrolide MICs in all six isolates. Taken together, inducible expression of both ermB and ermT conferred high macrolide resistance in these S. gallolyticus subsp. pasterianus isolates. Our findings reveal new macrolide resistance features in S. gallolyticus subsp. pasteurianus by both ermB and ermT.

Development of a novel thermostable Newcastle disease virus vaccine vector for expression of a heterologous gene.

Thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease for village poultry flocks, due to their independence of cold chains for delivery and storage. To explore the potential use of thermostable NDV as a vaccine vector, an infectious clone of thermostable avirulent NDV strain TS09-C was developed using reverse genetics technology. The GFP gene, along with the self-cleaving 2A gene of foot-and-mouth disease virus and ubiquitin monomer (2AUbi), were inserted immediately upstream of the NP (nucleocapsid protein), M (matrix protein) or L (large polymerase protein) gene translation start codon in the TS09-C infectious clone. Detection of GFP expression in the recombinant virus-infected cells showed that the recombinant virus, rTS-GFP/M, with the GFP gene inserted into the M gene expressed the highest level of GFP. The rTS-GFP/M virus retained the same thermostability, growth dynamics and pathogenicity as its parental rTS09-C virus. Vaccination of specific-pathogen-free chickens with the rTS-GFP/M virus conferred complete protection against virulent NDV challenge. Taken together, the data suggested that the rTS09-C virus could be used as a vaccine vector to develop bivalent thermostable vaccines against Newcastle disease and the target avian diseases for village chickens, especially in the developing and least-developed countries.