猪支原体肺炎发生的品种敏感差异及分子基础

Difference in Susceptibility to Mycoplasma Pneumonia Among Various Pig Breeds and Its Molecular Genetic Basis

【目的】检测并验证猪支原体肺炎发生的品种敏感差异,筛选相关差异表达基因,探究猪支原体肺炎发生的分子遗传基础,为猪该病发生的分子机制研究提供依据。【方法】以对肺炎支原体易感的梅山猪、耐受的长白猪及二者杂交选育的苏钟猪为材料,设立试验组(15头/品种)和对照组(5头/品种),试验组猪接种肺炎支原体Js强毒株,对照组猪注射等量生理盐水,所有猪隔离、无抗生素饲养,监测临床表现;处理后18、28 d测定日增重、抗体水平及肺部病变,28d试验结束时集中屠宰,评定肺部损伤,检测病原,确定肺炎支原体感染情况;选择肺炎支原体感染的梅山、长白、苏钟猪各2头,未感染猪各2头,构成芯片杂交试验猪群,应用Agilent猪表达谱基因芯片及品种内、品种间双重比较法,筛选差异表达基因,结合Gene Ontology(http://www.geneontology.org)及KEGG(http://www.genome.jp/kegg/)分析差异基因涉及的信号通路及调控网络。【结果】肺炎支原体处理后1—18d,试验组猪平均日增重显著低于对照猪(0.01<P<0.05),尤以梅山猪较为典型;19—28d,体重出现负增长,平均日增重极显著低于对照猪(P<0.01)。同时,病原处理后18d,长白猪和苏钟猪抗体水平变化不大,仍表现为阴性(s/p<0.3),而梅山猪抗体水平则迅速上升至阳性水平(s/p≥0.4),并显著高于长白、苏钟猪(均为0.01≤P<0.05);处理后28d,梅山猪抗体水平高达(0.97±0.26),极显著高于长白(0.15±0.10)、苏钟猪(0.46±0.20)(均为P<0.01)。而试验组猪在病原接种后的18d,梅山猪有11头表现出明显的支原体肺炎临床症状,长白猪仅2头出现轻微咳嗽,苏钟猪则有5头出现咳嗽、精神萎靡等初期病状;处理后23d,试验组1头梅山猪死于肺炎支原体感染;处理后28d,试验组梅山猪全部表现出支原体肺炎症状,长白猪7头出现初期感染症状;苏钟猪5头症状典型,6头稍有咳嗽,其余无明显异常。猪肺炎支原体检测与上述结果大体一致。试验猪的肺部X-ray透射及病理剖检结果则表明:处理后18d,试验组15头梅山猪肺部均见云絮状阴影,其中6头为典型的肺炎感染影像特征;长白猪仅1头出现肺炎影像特征,4头现少量云絮状阴影;苏钟猪病变数量和程度介于长白、梅山猪之间。处理后28d,梅山猪全部表现为典型的肺炎感染影像特征,长白猪和苏钟猪则分别有7头、13头出现肺炎影像特征。肺部病理剖检评分显示,梅山猪极显著高于长白猪(P<0.01),显著高于苏钟猪(0.01≤P<0.05)。表达谱芯片筛选结果表明,支原体肺炎患猪较健康对照猪表达上调基因49个,下调基因70个,涉及免疫反应、补体凝集、类固醇合成及代谢等18条信号调控通路。【结论】猪支原体肺炎的发生确实存在明显的品种间敏感差异,先天性免疫缺陷调控通路、Toll样受体信号通路及类固醇代谢通路在猪支原体肺炎感染炎症反应调控过程中发挥重要作用。

【Objective】The objective of study is to detect susceptibility differences to M. hyopneumoniae among Chinese native pigs, western pigs and their crossbreed pigs, screen differentially expressed genes and explore the molecular genetic basis of M. hyopneumoniae infection in swine. It would The results of the study will provide a basis and possibility for researchers to investigate the molecular mechanisms of M. hyopneumoniae infection in swine. 【Method】 In this study, Meishan pigs（sensitive to M. hyopneumonia, n=20）, Landrace（excellent tolerance to M. hyopneumonia, n=20） and Suzhong pigs（cultivated breed derived from crossbred progeny of Meishan and Landrace pigs, n=20） were selected for artificial infection with M. hyopneumoniae. And the pigs were allocated randomly into the treatment group（15 pigs of each breed） or control group（5 pigs of each breed）. The pigs of treatment group were administered with M. hyopneumoniae strain JS. Meanwhile, the control pigs were injected with physiological saline solution. The treatment and control groups were isolated and fed an antibiotic-free diet throughout the experimental period. And then, clinical symptoms were observed daily, and average daily weight gain, antibodies level, X-ray transmission and M. hyopneumoniae pathogen were detected at 18 and 28 days post M. hyopneumoniae challenge, respectively. Twenty-eight days post challenge, the pigs were sacrificed and inspected for hepatization of the lung lobes as an indicator of Mycoplasma pneumonia. Then, 2 infected individuals and 2 non-infected individuals of each breed pigs by M. hyopneumoniae were selected to perform Agilent Pig 4x44 K Gene Expression Microarray analysis, and the double comparison method of inter- and intra-breed was used to investigate differential expression genes. Furthermore, Gene Ontology（http：//www. geneont ology. org） and KEGG pathway（http：//www. genome.jp/kegg/） were applied to analyze the roles of these differentially expressed genes involved in signaling pathway and regulatory network to discuss the molecular genetic basis in the process of M. hyopneumoniae infection in swine.【Result】 The results showed that, from day 1 to day 18 of experiment period, the average daily weight gain of the treated pigs was significantly lower than that of the control pigs（0.01≤P〈0.05）, especially for Meishan pigs. From day 19 to day 28, negative growth（an average daily weight gain of less than 0.00 kg） and a drop in average daily weight gain were recorded for pigs in the treatment group. And the average daily weight gain of treated pigs was significantly lower than that of the control pigs（P〈0.01）. Eighteen days post M. hyopneumoniae challenge, the antibody level of Landrace pigs and Suzhong pigs had little change and were low with the negative s/p ratio. But the s/p ratios of Meishan pigs were rapidly up to positive level, and were significantly higher than that of Landrace and Suzhong pigs（0.01≤P〈0.05）, respectively. Twenty-eight days after treatment, the antibody level of Meishan pigs was up to 0.97±0.26, which was significantly higher than those of Landrace（0.15±0.10） and Suzhong pigs（0.46±0.20）（P〈0.01, respectively）. At 18 days after treatment, clinical symptoms of mycoplasma pneumonia appeared in 11 Meishan pigs in the treatment group. Only two Landrace pigs showed signs of a slight cough. Five Suzhong pigs had early symptoms of mycoplasma pneumonia, such as coughing and lethargy. Twenty-three days into the experiment, one Meishan pig from the treatment group died after infection of M. hyopneumoniae. At the end of study, day 28, all Meishan pigs in the treatment group showed typical clinical symptoms and signs of mycoplasma pneumonia. Seven Landrace pigs from the treatment group had intermittent cough, lethargy and decreased appetite. Five Suzhong pigs had typical clinical symptoms of mycoplasma pneumonia, and further more six pigs had a slight cough. The results of M. hyopneumoniae pathogen detection were generally in accordance with description above. Moreover, on day 18 after treatment administration, the results of X-ray transmission detection revealed that 15 Meishan pigs in the treatment group had cloudy flocculent shadow, and amongst them, six animals had typical clinical symptoms and signs of mycoplasma pneumonia. While, only one of the Landrace pigs had a cloudy flocculent shadow and showed increased lung markings in the lung field, and four pigs had a small cloudy flocculent shadow. The number and degree of M. hyopneumoniae infection of Suzhong pigs appeared to be between that of Meishan and Landrace pigs. On day 28, all Meishan pigs in the treatment group had typical clinical symptoms and signs of mycoplasma pneumonia. Seven Landrace pigs and thirteen Suzhong pigs had clinical pathological evidence of mycoplasma pneumonia. At the end of experiment period, pathological examination of infected pigs indicated that the lung lesion scores of Meishan pigs（mean 23.20±2.54） were significantly higher than that of Landrace pigs（mean 10.27±8.18）（P〈0.01） and Suzhong pigs（mean 18.71±4.78）（0.01≤P 〈 0.05）. Profile microarray results showed that 119 differential genes were identified in Mycoplasma pneumonia pigs, including 49 up-regulated and 70 down-regulated genes, compared with the control group. These genes were involved in 18 signal pathways, such as B cell receptor signaling pathway, complement and coagulation cascades, steroid hormone biosynthesis and so on.【Conclusion】The obvious sensitivity differences do exist in the occurrence of swine mycoplasma pneumonia among various pig breeds. The primary immunodeficiency, Toll-like receptor signaling, and steroid metabolism pathways play important roles in the regulation of inflammatory response to M. hyopneumoniae infection in swine.