Virulence assessment of four Glaesserella parasuis strains isolated in Liaoning province of China.

Glaesserella parasuis (G. parasuis) is a part of the normal upper respiratory microbiota of healthy swine. In many studies, the serovars 1, 4, 5, and 12 of G. parasuis are considered to be highly virulent and its serovars 3, 6, 7, 9, and 11 are considered to be non-virulent. Until now, researchers have found that non-virulent strains of G. parasuis cause an increasing number of diseases. However, little is known concerning why non-virulent strains cause disease with the virulence changes. In present study, four G. parasuis strains were evaluated for their cytotoxicity property, which aims to compare their virulence. The results showed that highly virulent strains XX0306 and CY1201, as well as, non-virulent strains HLD0115 and YK1603 caused a series of pathological changes, increased lactate dehydrogenase (LDH) release, and decreased cell activity. In addition, compared to the control group, both highly and non-virulent strains showed similar trends, demonstrating that the method of classifying the virulence of G. parasuis based on its serovar is worth further deliberation. Hence, we investigated the adhesion capacity and invasion rate of G. parasuis, the results indicated that XX0306 and HLD0115 had the strongest adhesion and invasion ability, which contradicts the classification of the virulence of G. parasuis based on its serovar. The apoptosis degree induced by highly virulent strains was more intensive than non-virulent strains, as measured by annexin V and propidium iodide (PI) double staining. Through testing the expression of apoptosis-related genes Bcl-2 and Bax, we found highly virulent strains induced apoptosis by inhibiting the expression of Bcl-2.

Transcriptomics analysis reveals key lncRNAs and genes related to the infection of porcine lung macrophages by Glaesserella parasuis.

Glaesserella parasuis (G. parasuis) is the pathogen of Glässer's disease in pig herds, which can cause severe inflammatory responses. However, at present, the pathogenic mechanism of G. parasuis is not very clear. LncRNAs can regulate the expression of mRNA in a variety of ways, thereby causing host cells to produce a variety of functional changes in response to bacterial infection. Here, we detected the changes in lncRNAs and mRNAs of 3D4/21 cells after G. parasuis CY1201 strain (serotype 13) infection. A total of 876 lncRNAs and 2166 mRNAs were differentially expression in 3D4/21 cells after G. parasuis infection. GO and KEGG enrichment analysis showed that the differentially up-regulated lncRNA target genes were mainly involved in the response to extracellular stimuli, cell receptor signaling pathways and chemokine signaling pathways. The differentially down-regulated lncRNA target genes were mainly involved in ERK1/ERK2 cascade reaction and adhesion junctions. 44 lncRNAs were screened that might be related in inflammation. CeRNA regulatory network of the top five difference inflammation-related lncRNAs showed that the up-regulated lncRNA group involved 5 lncRNAs, 50 miRNAs and 49 mRNAs. Meanwhile, there were 26 miRNAs and 36 mRNAs in the top five down-regulated lncRNA group. Our results contribute to understand the basic role of lncRNAs in 3D4/21 cells during G. parasuis infection, and lay the foundation for following research.

Application of mouse model for evaluation of recombinant LpxC and GmhA as novel antigenic vaccine candidates of Glaesserella parasuis serotype 13.

Glaesserella parasuis (G. parasuis) has been one of the bacteria affecting the large-scale swine industry. Lack of an effective vaccine has limited control of the disease, which has an effect on prevalence. In order to improve the cross-protection of vaccines, development on subunit vaccines has become a hot spot. In this study, we firstly cloned the lpxC and gmhA genes from G. parasuis serotype 13 isolates, and expressed and purified their proteins. The results showed that LpxC and GmhA can stimulate mice to produce IgG antibodies. Through testing the cytokine levels of interleukin 4 (IL-4), IL-10 and interferon-γ (IFN-γ), it is found that recombinant GmhA, the mixed LpxC and GmhA can stimulate the body to produce Th1 and Th2 immune responses, while recombinant LpxC and inactivated bacteria can only produce Th2 immune responses. On the protection rate for mice, recombinant LpxC, GmhA and the mixture of LpxC and GmhA can provide 50%, 50% and 60% protection for lethal dose of G. parasuis infection, respectively. The partial protection achieved by the recombinant LpxC and GmhA supports their potential as novel vaccine candidate antigens against G. parasuis.

LncRNA-MEG3 Regulates the Inflammatory Responses and Apoptosis in Porcine Alveolar Macrophages Infected with Haemophilus parasuis Through Modulating the miR-210/TLR4 Axis.

Haemophilus parasuis (H. parasuis, HPS) can elicit serious inflammatory responses and cause enormous economic loss to swine industry worldwide. However, the factors responsible for systemic infection and inflammatory responses of HPS have not yet been fully clarified. In this study, we found that lncRNA-MEG3 was significantly up-regulated in porcine alveolar macrophages (PAMs) infected with HPS. The gain- and loss-of-function analysis confirmed that lncRNA-MEG3 participated in the inflammatory responses and apoptosis in HPS-infected PAMs, which was assessed via several inflammatory cytokine genes (TNF-α, IL-1ß, and IL-6) and apoptotic factors (Bcl-2, Bax, and C-caspase-3). Based on biotin-labeled RNA pull-down assay, we found that lncRNA-MEG3 bound with miR-210 in HPS-infected PAMs. Based on both overexpression and knockdown analysis of lncRNA-MEG3, our results indicated that lncRNA-MEG3 promoted the expression of TLR4 in HPS-infected PAMs. Using dual-luciferase reporter assays, we showed that lncRNA-MEG3 positively regulated the expression of TLR4 gene in HPS-infected PAMs through miR-210 pathway. Taken together, our results indicated that lncRNA-MEG3 participated in the inflammatory responses and apoptosis in HPS-infected PAMs through modulating the miR-210/TLR4 axis. The results from this investigation provided significant information for a novel target to control HPS infection in swine.

Comparative Transcriptomic Analyses of Haemophilus parasuis Reveal Differently Expressed Genes among Strains with Different Virulence Degrees.

Haemophilus parasuis is commonly found in the upper respiratory tract of the pigs. Some isolates of H. parasuis can lead to both pneumonia and Glässer's disease of pigs with severe clinical symptoms. The virulence-associated genes for the various degrees of virulence observed in H. parasuis remains poorly understood. In the present study, we identified the differentially expressed genes between YK1603 (non-virulent strain) and XM1602 (moderately virulent strain) or CY1201 (highly virulent strain) of H. parasuis using Illumina sequencing technique. In comparison to YK1603, a total of 195 genes were significantly changed in CY1201, of which 71 genes were up-regulated and 124 genes were down-regulated, whereas 705 genes were significantly changed in XM1602, of which 415 genes were up-regulated and 290 genes were down-regulated. The enriched analysis of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways on the differentially expressed genes showed that both enriched main GO terms and KEGG pathways appear to be different between the two kinds of comparision: CY1201 versus YK1603, and XM1602 versus YK1603. Based on real-time PCR technique, on the whole, it was confirmed that the expression of ten genes: lpxL, tbpB, kdtA, waaQ, oapA, napA, ptsH, mmsA, lpxM, and lpxB were agreement with the findings in Illumina sequencing analysis. These identified genes might participate in the regulation of a wide range of biological process involved in virulence of H. parasuis, such as phosphotransferase system and ABC transporters. Our results from this study provide a new way to gain insight into the virulent mechanisms of H. parasuis.

Effect of exposure routes on the relationships of lethal toxicity to rats from oral, intravenous, intraperitoneal and intramuscular routes.

The lethal toxicity values (log 1/LD(50)) of 527 aliphatic and aromatic compounds in oral, intravenous, intramuscular and intraperitoneal routes were used to investigate the relationships of log 1/LD(50) from different exposure routes. Regression analysis shows that the log 1/LD(50) values are well correlated between intravenous and intraperitoneal or intramuscular injections. However, the correlations between oral and intravenous or intraperitoneal routes are relatively poor. Comparison of the average residuals indicates that intravenous injection is the most sensitive exposure route and oral administration is the least sensitive exposure route. This is attributed to the difference in kinetic process of toxicity testing. The toxic effect of a chemical can be similar or significantly different between exposure routes, depending on the absorption rates of chemicals into blood. Inclusion of hydrophobic parameter and fractions of ionic forms can improve the correlations between intravenous and intraperitoneal or oral routes, but not between intraperitoneal and oral routes. This is due to the differences of absorption rate in different exposure environments from different routes. Several factors, such as experimental uncertainty, metabolism and toxic kinetics, can affect the correlations between intravenous and intraperitoneal or oral routes.