Transcription Analysis On Response Of Porcine Alveolar Macrophages To Haemophilus Parasuis And Development Of Loop-Mediated Isothermal Amplification For Detection Of Haemophilus Parasuis

Haemophilus parasuis is the etiological agent of porcine polyserositis and arthritis (Glasser's disease) characterized by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. To date,15serovars of H.parasuis have been identified. H.parasuis infection can be acute or chronic, depending on the immunological status of the herd. The infection by H.parasuis has become an increasing threat to early-weaned pigs and in pig herds of high health status.The H.parasuis infection can be controlled by vaccination and antibiotic treatment. However, a key element for controlling the disease is to obtain a correct diagnosis of the causative agent. Isolation and microbiological culture of H.parasuis can be ineffective due to the fastidious growth of the bacteria, which can be aggravated by previous antibiotic treatment of affected animals. Many DNA based and immunological methods for Haemophilus parasuis detection have been developed, among these diagnostic tools, PCR-based methods are the most rapid and able to detect a small amount of bacteria chromosomes. Despite the simplicity and accuracy of PCR detection, the requirement of a high-precision thermal cycler makes it difficult for widespread use. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. In this study, we developed a novel LAMP method based on the sequence in16S rRNA gene for rapid detection of Haemophilus parasuis.The innate immune response in vertebrates is the first defense line against invading microorganisms. The main players in innate immunity are phagocytes such as neutrophils, dendritic cells and macrophages. As a major component of the host innate immunity, macrophages have essential roles in host defense to infection, because they often mediate the killing of microbes as well as initiate, maintain and resolve host inflammatory responses by releasing cytokines and chemokines. Bacterial pathogens that overcome host defenses ensure their ability to survive and propagate. The diversity of bacteria and the differences in their pathogenesis may lead to pathogen-specific responses of macrophages. A greater understanding of the complex interactions, which occur between the macrophages and pathogen, could lead to the identification of the host defense strategies and the complementary pathogen evasion strategies. The interactions between H.parasuis with porcine alveolar macrophages have been studied, but the detailed mechanisms of how porcine alveolar macrophages response to H.parasuis infection are not well elucidated. The high throughput cDNA microarray represents a powerful tool for analyzing the molecular events in bacteria-host cell interactions. This technology has been useful in identifying changes in gene expression both in cultured cells and in whole organisms infected with pathogens. In this study, we applied this high throughput cDNA microarray assay to improve our understanding of the innate immune response of macrophages to H.parasuis infection.1) Transcription analysis on response of porcine alveolar macrophages to Haemophilus parasuisA total of428differentially expressed (DE) genes were identified in the porcine alveolar macrophages (PAMs)6days after H.parasuis infection. These genes were principally related to inflammatory response, immune response, microtubule polymerization, regulation of transcript and signal transduction. Through the pathway analysis, the significant pathways mainly concerned with cell adhesion molecules(p=3.74E-12), cytokine-cytokine receptor interaction(p=2.18E-10), toll-like receptor signaling pathway(p=1.93E-05), MAPK signaling pathway(p=7.63E-04), suggesting that the host took different strategies to activate immune and inflammatory response upon H.parasuis infection. Partucularly, some DE genes related to phagocytosis, formation of phagolysosome,chemokine production and nitrix oxide production could contribute to explain the complicated mechanisms by which PAM played its functions. The global interactions network and six subnetworks of the proteins encoded by DE genes were analyzed by using STRING and IPA. Coronin1a,s100a4and s100a6were highlighted in the KEGG or STRING and were found to play important roles in the immune response. These observations suggest that the three genes may be novel candidates for disease-related DE genes. We cloned and sequenced the coronin la, s100a4and s100a6gene. coronin la:[GenBank:JN092377]; s100a4:[GenBank:JN122624.1]; s100a6:ID:[GenBank:DQ372082.1]. These three sequences were the first reporte of coronin1a, s100a4and s100a6in pigs. Phylogenetic analysis of the three genes indicated that poCORONIN1A and poS100A6belonged to the group containing the Bos taurus sequence and Equus caballus sequence respectively. Structural analysis indicated that the poCORONIN1A contained putative domains of Trp-Asp (WD) repeats signature, Trp-Asp (WD) repeats profile and Trp-Asp (WD) repeats circular profile at the N-terminus, poS100A4and poS100A6contained putative S-100calcium binding protein signature, EF-hand calcium-binding domain profile. Tissue distribution analysis indicated that coronin1a, s100a4and s100a6was highly expressed in pancreas, epencephalon and pancreas respectively. Further immunostimulation analysis indicated that mRNA levels of S100calcium-binding protein A4(S100A4) and S100calcium-binding protein A6(S100A6) in porcine PK-15cells increased within48h and were sustained after administration of lipopolysaccharide (LPS) and Poly (I:C) respectively. The s100a4and s100a6genes were found to be up-regulated significantly in lungs, spleen and lymph nodes in H.parasuis infected pigs.2) Development of loop-mediated isothermal amplification for detection of Haemophilus parasuisWe developed a LAMP assay targeting the16S rRNA gene for rapid detection of Haemophilus parasuis. The assay was carried out at61℃and finished within60min. The results obtained from testing31H.parasuis strains and other twenty-eight bacterial species strains showed that the LAMP was as specific as and more sensitive than nested PCR. Fifty-five lung samples were collected from55healthy pigs. All the samples were negative for H.parasuis by bacterial isolation, nested PCR and LAMP, respectively. Meanwhile,122lung samples were also collected from122pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR by picking up16additional cases (P=0.02, χ2test). The LAMP assay gave a same result comparing with the nested PCR when the two assays were respectively used to detect H.parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP was a highly sensitive and reliable method for detection of H.parasuis infection.