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Molecular Analysis of Intestinal Flora and Expression of IL-6, IL-2, SIgA, IFN-γ, CD3in Digestive, Respiratory, Immune System of Rhesus Monkey Infected with Streptococcus Pneumoniae

Author: LuoQiHui
Tutor: ChengAnChun
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Intestinal Flora Molecular Analysis IL-6 IL-2 sIgA IFN-γ CD3 Streptococcus Pneumoniae Rhesus monkey
CLC: S858.9
Type: PhD thesis
Year: 2012
Downloads: 82
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Abstract


Streptococcus pneumoniae is the main pathogen of Community acquired pneumonia. To investigate its pathogenesis, in this experiment we used rhesus monkeys as experiment animals to isolate and identify streptococcus pneumoniae. After obtaining the tissues, we detected histopathological changes in digestive system, respiratory apparatus and immune system by H.E staining. The intestinal flora plays a very important role in nutrition, metabolism and the host immune protection, and the structure of intestinal flora has very close relation with the development and changes in many diseases. In this assay, we chose two molecular ecology methods to detect structure of gut flora in healthy and infected with streptococcus pneumoniae rhesus monkeys. Expression changes of IL-6, IL-2, sIgA, IFN-y and CD3proteins were detected after rhesus monkey infected streptococcus pneumonia spontaneously by immunohistochemistry. The results in this study were as follows.1. Olive green hemolysis strains have been separated from blood and these organs including heart, liver, spleen and lung of infected rhesus monkeys. The results of bile solubility test and optochin-resistant test show that separated strain are streptococcus pneumoniae and resistant to optochin. The virulence test of mouse show the separated bacteria are virulent strains. Analysis of16S rRNA sequence indicates its homology is up to99%. All these tests show that those monkeys were infected with virulent streptococcus pneumoniae.2. Histopathological changes show that many macrophages and granulocytes are observed in lymph node; enlarged splenic sinusoid crevice and more lymphocytes; Lung and trachea with absolute bleeding, interalveolar septum thinckening and great inflammatory cell infiltrating; Hepatic tissue with catholic bleeding, a part of hepatocyte denaturation and mortification; Glandular cell in the stratum mucosum of gastrointestinal tract with catholic denaturization and mortification, accompanied by glandular organ atrophy and structure destroyed, lymphocytes of stratum mucosum increased greatly; Esophagus without obvious pathological changes. All the above changes indicate infected rhesus monkeys have typical histopathological changes caused by streptococcus pneumoniae.3. In this study, two methods were taken for DNA extract, and the result show extraction kit was better than normal methods in purity and production. Then16S rRNA V3region was amplified by common primers, and the PCR products were analyzed by DGGE electrophoresis. DGGE graph were analysed by Quantity one software(Bio-rad) and Cluster anlysis by UPGAMA. According to the data, we calculated Shannon-wiener diversity index, Pielou index, Margalef index and Berger-Parker index. These entire indexes were analysed by SPSS19.0for determinating structure morphology of bacteria community. The DGGE results indicate, healthy monkeys have stable enterobacteria, and the band number in cecum and colon are the most, next in rectum, ileum and jejunum, in duodenum the least. But in rhesus monkeys infected with streptococcus pneumoniae, the band number decrease significantly (P<0.01). Great difference in band number is observed among different monkeys and different intestines. Shannon-wiener diversity index, Pielou index, Margalef index and Berger-Parker index in guts of healthy monkey are higher than that of infected monkeys. This result consists with clinical and pathologic symptom which mean the diversity of gut flora decreased in infected rhesus monkeys,4. In this essay, SYBR Green Ⅰ FQ-PCR were established for detecting Bifidobacteria, Lactobacillus, Bacteroides, Escherichia coli, Enterococcus and Bacillus in alimentary tracts of rhesus monkeys. Results show the above six bacteria genus are the dominant flora in rhesus monkeys, the contents of Bifidobacteria and Lactobacillus among them are the most (logcopies were7.692±0.905and7.529±0.979, respectively). The contents of each genus in cecum, colon and rectum are higher than that in the former intestine. The dominant bacteria in infected monkeys change a lot. The logcopies of Lactobacillus, Bifidobacteria and Bacteroides decrease significantly (P<0.05), and logcopies of Bacillus and Enterococcus are lowered to some extent, but that of E.coli increase a little. These results revealed gut flora were disbalanced in rhesus monkeys infected with streptococcus pneumoniae, which is easy to form vicious cycle with inflammatory reaction provocated by infection. Also this research provides some new idea in the relation between gut flora and monkey’s health.5. Immunohistochemisty method was applied in this study for detecting protein expression, and all the results were as follows.(1) Results in Digestive system (including esophagus, stomach, liver, jejunum and cecum)(compared with normal group):The level of IL-6, IFN-y and CD3protein increased, IL-2and sIgA proteins decrease, proteins expression mainly localized in mucous layer. IL-6and IFN-y proteins positive cells areas are significantly higher than those in healthy monkeys (p<0.01), especially in jejunum, cecum and stomach. IL-2positive cells areas decrease significantly in infected group (p<0.01), but optical density value show that IL-2positive cells increase significantly in infected group (p<0.01). There are no obvious difference between infected and normal groups concerning CD3and sIgA proteins positive cells areas, only in jejunum, sIgA show decrease; in jejunum and liver, CD3positive cells increase significantly (p<0.05). Optical density value of positive cells express CD3proteins increase in liver and jejunum significantly (p<0.05).(2) Respiratory apparatus (trachea and lung) results:in the infected group, positive cells areas of IFN-y proteins increase significantly in lung, trachea and blood vessel (p<0.05); sIgA proteins also increase, but only obvious in trachea; IL-2proteins increase slightly, CD3and IL-6proteins show no significant difference. As for the optical density value of positive cells in infected group, IFN-y, IL-2proteins increase obviously in trachea (p<0.05), IgA anf IFN-y proteins increase obviously in blood vessel and lung (p<0.05), there are no significant difference in CD3and IL-6.(3) Immune system (spleen and lymph nodes) results. In infected group, positive cells areas of IFN-y, IL-2proteins increase significantly in spleen, and there are no significant difference in lymph nodes, except for CD3, the other cytokines all increase in infected group. All the five cytokines’ optical density value of positive cells areas increase in infected group, IL-6proteins show extreme significantly increase(p<0.01), IFN-y, IL-2proteins increase significantly (p<0.05).From above imunohistochemisty results, antibody expression point the influence of Streptococcus pneumoniae put on mucous immune system, which may involve in pathogenicity of Streptococcus pneumoniae. Compared with normal group, in infected group, the positive cells areas of IL-2proteins increased in respiratory apparatus and immune system and decrease in digestive system; IL-6proteins decrease in respiratory apparatus, and increase in Immune system and digestive system; IFN-y proteins increase in digestive system; CD3proteins decrease in Respiratory apparatus and Immune system and increase in digestive system; IgA proteins increased in respiratory apparatus and immune system and decrease in digestive system. All of the factors interact with each other and participate in regulating humoral immunity and cytoimmunity, and form a network to help host to resist disoperation resulting from Streptococcus pneumoniae.

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