Correlation Of Cytokines Like TNF-? And The Formation Of Microthrombus In Pigs Lungs Infected By PRRSV

Porcine reproductive and respiratory syndrome(PRRS)caused by Porcine reproductive and respiratory syndrome virus(PRRSV)has been seriously affecting the economy of pig farms.People regard the disease resistance breeding as a key method of prevention and control due to the PRRS is difficult to control.As early as the 1950 s,the researchers had developed a swine erysipelas resistant variety through several generations of breeding.At present,a new breed called Etongliangtouwu(ETLTW)has been bred by Professor Liu Bang of Huazhong Agricultural University,which has Tongcheng pig as the female parent and big white pig as the male parent.It not only has the advantages of good meat quality and taste,but also maintains the characteristics of PRRSV resistance of Tongcheng pig.The researches on PRRSV has been hot all the time,but most of them are about the pathogenesis and variety differences of PRRSV induced inflammation.However,the causes of PRRSV leading to tissue microthrombosis were rarely studied.In this thesis,ETLTW is taken as the research object,the histopathology and electron microscopy is used to observe the pathological changes of lung tissue in ETLTW which is infected by PRRSV.The distribution and change of PRRSV,TNF-?,eNOS and TF are observed by immunohistochemistry,q PCR and Western blot.Therefore,the pathological characteristics and pathogenesis of PRRSV infection in ETLTW are described,which not only accumulate pathological research data for PRRSV disease resistance breeding,but also enrich the research content of PRRSV pathogenesis.This research mainly includes the following contents: 1 Clinical symptoms and general pathological changes of lung tissue in ETLTW which infected with PRRSVAfter three days of infection,the symptoms with high fever in the ETLTW appeared,the body temperature rose to above 40 ?,the mental state depresses,the appetite lesseded obviously.The pigs like to lie down,the reaction is slow,and the breathing is difficult.Some pigs developed skin cyanosis and neurological symptoms after six days infection,including convulsions,muscle tremors,and slight paralysis of the hind limbs.The normal control group had good mental state,normal appetite and normal body temperature of 38.5?39.5 ?.It was found that the lungs are dark red due to serious congestion in the infected pigs after autopsy.The apical,heart and septal lobes of the lungs were usually solid in texture,dark red in color and increased in weight.Some infected pigs suffered from edema in their lungs,and the enlargement of it.The capsule was tense and glossy,and severe pigs had obvious rib indentation in the back of the lungs.Most of infected pigs suffered different degrees of interstitial widening.There were no abnormality found in the lungs from the normal control group.The results show that the serious damage to the lung caused by PRRSV,with the respiratory function of the lung is reduced,and the lack of oxygen supply at the ends of the body due to breathing difficulties in the infected pigs.2 Histopathological changes of lung in ETLTW which infected with PRRSVCompared with the normal control group,the alveolar septa of infected pigs were widened in varying degrees;the capillary of alveolar wall was seriously congested and bleeding,with a large number of exfoliated epithelial cells and macrophages in some trachea and bronchia.Few infected pigs' lungs were filled with exudate and macrophages,there were lots of exfoliated alveolar epithelial cells,lymphocytes and megaphages in the alveolar cavity cells,and numbers of lymphocytes around parts of the bronchi,with a large number of microthrombus around the alveolar wall.Alveolar collapse in few infected pigs was found,and a large number of necrotic cells in the alveolar cavity.The results show that the varying degrees of interstitial pneumonia in infected pigs were caused by PRRSV,and varying degrees of microthrombus was found in alveolar capillary in this thesis,which indicates that the blood vessels are damaged by PRRSV,and lead to formation of micro thrombus.3 The relationship between microthrombotic changes of lung tissue and virus and mortality in ETLTW which infected with PRRSVThe pathological changes of 46 infected pigs in pulmonary microthrombus were observed.According to the criteria of microthrombus,22 infected pigs were mild microthrombus,14 infected pigs were moderate microthrombus,and 10 infected pigs were severe microthrombus.By analyzing the degree of microthrombotic lesions and mortality,we found that the mortality of severe microthrombotic group was higher than the other two groups,indicating that a large number of microthrombotic pulmonary tissues may be one of the causes of high mortality of PRRS.4 Distribution and expression of PRRSV,TNF-?,eNOS and TF in lung tissue of ETLTW which infected with PRRSVMaking use of immunohistochemistry,we found that PRRSV did not expressed in normal lung tissue,and the lung tissue infected with PRRSV mainly distributed in alveolar macrophages and few lymphocytes.The expressions of PRRSV in mild,moderate and severe microthrombotic lung tissues were analyzed,which were found are increased step by step,and the expressions of PRRSV in severe microthrombotic groups were significantly higher than the other two groups.The results show that the PRRSV is involved in the process of microthrombotic formation,results in the rise of mortality of infected pigs.The expression and distribution of TNF-? m RNA and protein was detected by immunohistochemistry,fluorescence q PCR and Western blot.Compared with the control pigs,the number and staining of TNF-? positive signal in the lung tissues of infected pigs increased.The positive signal of TNF-? antigen is mainly distributed in alveolar macrophages and lymphocytes.The expressions of TNF-? in mild,moderate and severe microthrombotic lung tissues were analyzed,and the rising severity of the microthrombotic lesions according to the increase of TNF-? expression has been found.The expression of TNF-? in severe microthrombotic group is significantly higher than that in mild and moderate microthrombotic groups.The results show that the expression of TNF-? in infected pig lung tissue is up-regulated to promote the formation of microthrombosis.In this study,immunohistochemistry,q PCR and Western blot are used to detect the expression and distribution of eNOS m RNA and protein.It was found that eNOS mainly distributed in alveolar type II cells,alveolar macrophages,vascular endothelial cells and bronchial smooth muscle cells.Compared with the normal lung tissue,the number of positive signal staining in the infected group is significantly lower than that in the control group.The results show that the expression of eNOS in the lung tissue of infected pigs is down-regulated to be involved in the formation of microthrombus in the lung tissue of infected pigs.The results of TF immunohistochemistry show that it is mainly distributed in alveolar macrophages,lymphocytes and vascular endothelial cells and plasma,it is brownish yellow.Compared with the normal control group,the positive signal staining and quantity in the infection group are significantly higher than that in the normal control group.The results of positive rate analysis show that the slight,the moderate and the severe microthrombus groups presented a gradient upward trend.It is shown that the external coagulation system is started by TF and then participate the process of microthrombosis.The above results show that the pathological changes of lung tissue in ETLTW which infected with PRRSV are mainly interstitial pneumonia.In addition,there were a lot of microthrombosis in pulmonary capillaries.The expressions of PRRSV,TNF-?,eNOS and TF are closely related to the severity of pulmonary microthrombosis.The mortality of PRRSV infected pigs are increased by microthrombosis in the lungs.