| Viruses of avian leukosis(AL) belong to the genus Alpharetrovirus of the Retroviridae family, causing a variety of benign and malignant tumors that spread in chickens. The target cells of ALV are immune cells. Infection of ALVs induce immunosuppression, which is accompany by second infection of other pathogens. Subgroup J virus was first isolated from meat-type chickens in the late 1980 s in the United Kingdom and and has been reported in many areas of the world. There were scattered reports about myelocyte-like tumors and hemangioma caused by ALV-J in many areas of China, causing a tremendous economic loss to the chicken industry in our country. A group of suspected new subgroup of ALVs have been isolated from indigenous chicken breeds in China recently, but the pathogenesis of these newly isolates remains to be determined. Avian coccidiosis is caused by obligate intracellular protozoan parasites of the genus Eimeria. The Eimeria enters the host by penetration of intestinal epithelial cells causing serious damage to the physical integrity and mucosal immunity of the gut. Eimeria infected chickens are often co-infected with many other intestinal parasites, bacteria or some viruses. E. tenella is one of the most pathogenic one,which mainly infects the epithelial cells. Eimeria species are ubiquitous in environment. Up to 90 % of chicken flocks can be exposed, with a high prevalence of diseased chickens.Uncontrolled outbreaks cause high mortality. Co-infections with different pathogens(bacteria,viruses, yeasts, or parasites) is an observed common feature, and that is more frequent in diseased chickens,resulting most of the time in higher pathogenesis. This is a great challenge for disease diagnose and control. ALVs and chicken coccidia are two important avian pathogens prevalent in chickens in many countries. Both ones cause damage to immune system and often co-infected with other pathogens. The present study carried out an epidemiological survey and pathogenesis studies on co-infections of ALVs and chicken coccidia, and tried to understand the pathogenetic mechanism of co-infections by high throughput technologies.The gut microbiome is extremely complex and comprises a large variety of commensal microbes. Gut microbial genome is known as the host "second genome". Role of the intestinal flora ranging from harvesting energy from the diet, stimulating the proliferation of the intestinal epithelium to developing the immune system in the host. Various physiological and psychological health problems are also related to the intestinal flora disturbance.Therefore, the function of intestinal flora have received increasing attention from researchers in recent years. In poultry, the microbiota of the ceca is very diverse and 1 g(wet weight) of cecal content may contain 1011 bacteria, including various conditioned pathogens. These conditioned pathogens provide a wealth of resources for secondary infections when the host immune alters. High throughput sequencing and bioinformatics analysis of the macro genome is an effective method to analyze gut microbiome, which can directly analyze the distribution of bacteria in the intestine compared with the traditional method. With high throughput technology, it is convenient to detect the intestinal flora changes after pathogen infection,which may help us to clarify the pathogenic mechanism.In the post genomic era, the protein, as the direct executor of biological function, has become a research focus in the field of life science. Proteomics is an effective method for high-throughput screening and analysis of protein expression, protein modification, protein interaction and function of the body organs, tissues or cells. Compared with the traditional studies on a single protein, it reflects the overall state of the body more accurately. As a new method on systemic biology researches, proteomeic technology provides a powerful method to screen the key protein in the pathogenic infection, and to further reveal the pathogenic mechanisms.1. Epidemiological survey on co-infections of ALVs and coccidia in chicken flocksThe present study carried out an epidemiological survey on co-infections of ALVs and coccidia in chicken flocks by pathogen detection combined with molecular biology technology. The results showed that coccidia was detected in most of chicken flocks in our investigation(Jining, Feicheng, Xintai), with the detection rate up to 90 % in some flocks.ALV was detected only in some flocks from Feicheng of Taian, with the detection rate up to33.3 %. The gp85 nucleotide sequence was amplified and sequenced. The results showed that these flocks were infected with ALV-J. The statistical analysis found that co-infection rate ofALVs and coccidia was up to 13.33 % in Feicheng of Taian, which demonstrated the co-infections of ALVs and coccidia in chicken flocks for the first time.2. Genomic sequence analysis and biological characteristics of rescued clone of ALV strain JS11C1We determined the complete genome sequence, constructed an infectious clone of ALV strain JS11C1, and performed comparison analysis using the whole genome sequence or elements with that of other ALSVs all available in GenBank. The results showed that the full-length sequence of the JS11C1 DNA provirus genome was 7707 bp, which is consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The rescued infectious clone of JS11C1 showed similar growth rate and biological characteristics to its original virus. All the comparison analysis based on whole genomes support the opinion that JS11C1 and other similar isolates are relatively far related to any known subgroups of ALVs and might be classified as a new subgroup K. It may be the first infectious clone of these ALVs constructed. Therefore, it established a good technical platform to further study biological characteristics in vitro, genome structure and function,and molecular mechanism of its pathogenesis.3. Co-infections of different subgroups of ALVs and E. tenella on the pathogenesis effects in different chicken linesCo-infections of different subgroups of ALVs(J: r NX0101 and K: rJS11C1) and E.tenella on the pathogenesis effects in different chicken lines(SPF, Hy-line Brown and AA broiler chickens) were studied. The results showed that co-infections of ALVs and E. tenella induced a higher mortality rate and a lower body weight in all chicken lines compared to a single-pathogen infection. The ALV infection speeded up the disease symptoms induced by E.tenella and the E. tenella infection extended the ALV-J viremia in co-infected chickens.Thymus atrophy, decrease in the humoral response levels to pathogen and NDV vaccine,modification in the blood lymphocyte sub-populations and transcriptional cytokine disorders were found in co-infected chickens compared to chickens infected with one pathogen alone and to controls. In conclusion we underline a synergy between ALVs and E. tenella that results in increasing pathogenesis in different chicken lines.4. The effects of co-infections of ALV and E. tenella on chicken cecal microbiomeThe effects of co-infections of ALV and E. tenella on chicken cecal microbiome were studied to elucidate the possible mechanism of pathogensis. ALVs infection increase the richness and diversity of cecal microflora. There is a clear association with loss of important bacterial populations in concert with an enrichment of potentially pathogenic populations and ALVs infection. Specific members of opportunistic pathogens were enriched by infection of different ALV subgroups. The most obvious character of cecal microflora from E. tenella infected chickens was the changes of the structure of intestinal flora to a large degree. The richness and diversity of cecal microflora were decreased sharply. Similar to ALV infection,loss of important bacterial populations and an enrichment of potentially pathogenic populations were also observed. Cecal microbiota of chickens co-infected with r-NX0101 and E. tenella showed diversely changes degrees, ranging from the status of r-NX0101 infection alone to E. tenella infection alone. Proliferation of some bacteria in the individual was observed. Whereas, synergy of r-JS11C1 and E. tenella to increase many conditioned pathogens was observed in co-infected chickens. Thus there is a higher possibility of secondary infections for co-infected chickens, which may be related to the increased pathogenesis.5. The effects of co-infections of ALV and E. tenella on proteome of chicken spleenProteome of spleen from healthy chickens and chickens single or co-infected with different subgroups of ALVs and E. tenella were studied by iTRAQ technology. A total of1737 proteins were identified in chicken spleen. Functional annotation on the identified proteins showed that most of the proteins in spleen take part in the basic biological metabolic processes. Many proteins are also involved in immune related biological processes and pathways related to different pathogens and various disease. r-NX0101 infection mainly affects the transcription, translation and post-translation of the host, and the expression of tumor formation and migration related matrix adhesion proteins, which are closely related to the tumor formation characteristics of subgroup J of ALV. Infection of r-JS11C1 mainly enriched the expression of host proteins related to post-modifications. Infection of E. tenella has a great influence on the structure of host cell and tissue as a result of the invasion of the parasite. Co-infections of r-NX0101 and E. tenella significantly enriched cytolysis related proteins as well as many differential proteins that take part in immunological pathways.Co-infections of r-JS11C1 and E. tenella are mainly involved in expression of proteins related to stress and nervous system diseases. This provides new ideas and theoretical basis to elucidate the pathogenic mechanism of co- infections, which will promote to better understanding of the pathogenic mechanism and the interaction between virus and host. |
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