| Porcine Reproductive and Respiratory Syndrome(PRRS)is one of the most economically damaging diseases that seriously threaten pig production worldwide.The main feature is reproductive failure in sows and respiratory symptoms and high mortality in offspring.It has also immunological features such as persistent infection,immunosuppression and antibody-dependent enhancement(ADE).The disease is caused by porcine reproductive and respiratory syndrome virus(PRRSV).PRRSV has a very narrow cell tropism and only exhibits tropism for monocytes/macrophage cells.The virus primarily infects lungs,lymphoid tissues and certain differentiated macrophages in the placenta and replicates itselves,particularly porcine alveolar macrophages(PAM).Therefore,the detection of anti-PRRSV neutralizing antibodies based on PAM cells in this study is more in line with clinical features.Infection of the host with PRRSV first induces an innate immune response.The innate immune response consists mainly of the body's barrier structure,phagocytic cells,normal tissues and anti-bacterial and inflammatory factors in body fluids.Mononuclear cells and macrophages in infected pigs are activated to secrete cytokines such as IFN-?,TNF-?,and IL-1? and so on.These cytokines have bactericidal,inhibit viral replication and participate inflammatory response.Therefore,the dynamic changes of cytokines in serum after PRRSV infection at different time points can provide some basis for PRRSV induction of immune mechanism.For the above study,the PRRSV VR2332 strain was used to infect piglets by intranasal and injection routes to detect the viral proliferative patterns,antibody production patterns and IFN-?,TNF-? and IL-1? changes in the serum.In this study,nine healthy 20-day-old white piglets that were found to be negative PRRS,CSF,PCV2 and PR rabies antigens and antibody were used.Nine piglets were randomly divided into three groups.The first group was the intranasal group,the second group was the injection group and the third group was a control group.The first and second groups were inoculated with 5 m L(antigen volume 5×105 TCID50/m L).The third group was injected with 5 m L of healthy Mac-145 cell culture.On days 0th,3rd,7th,11 th,15th,19 th,23rd,27 th,31st,35 th,39th and 43 rd after infection,piglets of three groups were collected for coagulation and anticoagulation in the anterior vena cava.Coagulation serum was separated for PRRSV copy number,ELISA antibody and neutralizing antibody titer detection.Anticoagulant blood was collected for peripheral blood lymphocytes.And IFN-?,TNF-? and IL-1? were detected by established the real-time fluorescent quantitative PCR method.First,after PRRSV infects pigs via intranasal and injection,the PRRSV nucleic acid was detected by the separation of serum to compare the differences in the viremia.Serum samples were separated at different time points and RNA was extracted.RT-PCR method and the real-time fluorescent quantitative PCR method were used to detect PRRSV nucleic acid.And the results of positive detection of viral nucleic acid were consistent.PRRSV was detected on the 3rd days in the serum of two of the three piglets after the infection and the other one was detected on the 7th day in the intranasal infection group.The virus was greater than 103copies/?L on the 15 th day to the 19 th day.The virus the highest than 104copies/?L.On the 27 th day,the virus nucleic acid was negative and viremia disappeared.In the infected group,the virus was detected in the serum of the three piglets on the 3rd day and viral blood had appeared.On the 15 th to the 23 rd day,the virus copies had peaked.On the 19 th day,the viral could reach more than 105copies/?L.One piglet viremia disappeared on the 31 st day and the other two piglets on the 35 th day.Comparing the results of the two groups,the proliferation of the viral copy number of the injection group was consistently higher 101-102 times than that of the intranasal group and the viraemia duration was longer.After the end of the trial,PRRSV nucleic acid was detected in spleen,submandibular,inguinal,mesenteric lymph nodes and aseptically collected PAM cells from both groups.The results showed that PRRSV was detected in the submandibular,inguinal,and mesenteric lymph nodes,and aseptically collected PAM cells in the two experimental groups.And the number of copies in the injection group was higher than that in the intranasal group.This indicates that PRRSV was present in infected pigs.Second,after PRRSV infects pigs via intranasal and injections,ELISA antibodies and neutralizing antibodies was detected by the separation of serum.The differences in the rule of infection-induced antibody production in two different ways were compared.Serum samples separated at different time points were tested for ELISA antibodies.The anti-PRRSV antibody in the serum was detected to be positive on the 27 th day and then the antibody level was gradually increasing until the end of the test in two experimental groups.In the intranasal group,the ELISA antibody remained at the same level from the 27 th to the 43 rd day.The antibody level of the injection group gradually increased after the 27 th day and the ELISA antibody value was higher than the intranasal group from the 35 th to the 43 rd day.The results of the two groups of tests showed that the antibodies in the serum of the injection group were produced earlier than the intranasal group.And the antibody titer was also higher than that of the intranasal group.Based on the PAM cell microneutralization assay,the neutralization antibody level after immunization with the PRRSV VR2332 attenuated strain was tested in this study.The detection result was determined based on the 70% virus inhibition standard.In the intranasal group,neutralization tests were performed on the 3rd,11 th,19th,27 th,35th and 43 th days after immunization to measure the neutralizing antibody levels.The results showed that the neutralizing antibody titer was less than 1:2 on the 35 th day before.The neutralizing antibody titer at day 43 was 1:2.This result indicates that the neutralizing antibody level is very low in the intranasal group.In the injection group,the neutralization titers were less than 1:2 on the 3rd,11 th and 19 th days after immunization.And the neutralization titers on the 27 th,35th and 43 th days were 1:4,1:8 and 1:4 respectively.The results of this experiment showed that the neutralizing antibodies produced in the serum of the injection group were in a gradually increasing trend and dropped to a titer on the 43 th day.The results of the two groups compared that the injection group with neutralizing antibody titers were earlier and higher than in the nasal group.Third,after infection of pigs with PRRSV by intranasal and injection,m RNA transcriptional level of IFN-?,TNF-? and IL-1? was detected by isolate peripheral blood lymphocytes.The different routes of infection were compared.For the detection of cytokines after infection with PRRSV,the real-time fluorescent quantitative PCR assay was established in this study.The results of IFN-? m RNA transcriptional level showed that compared with the control group,the transcription level of IFN-? m RNA in the intranasal group decreased rapidly on the 3rd day after infection.It was basically the same as that in the control group on the 7th day.And then rose to the 15 th day.The amount reached the highest.Followed by the control group from the 19 th day to the 23 rd day and gradually increased after the 27 th day and the transcriptional level with the control group tended to be the same level.Compared with the control group,the injection group decreased rapidly on the 7th day.Afterwards,it showed an upward trend until the 15 th day which was higher than that of the control group.The m RNA transcriptional level reached the highest on the 19 th day.And decreased with the control group and basically leveled with the control group after the 35 th day.Comparing the results of the two groups,the time for the decrease of IFN-? transcription in the intranasal group was earlier than that of the injection group and the m RNA transcriptional level in the intranasal group on the 27 th day was basically at the same level as the control group,while the injection group was in the 35 th.The results of TNF-? m RNA transcriptional level showed that the intranasal group rose rapidly on the 3rd day after infection was significantly higher than the control group and reached the highest on the 7th day.Although it showed an upward trend,the transcriptional level was higher until the 19 th day.The injection group also showed an upward trend after infection,which was significantly higher than that of the control group.The rising trend was significant in the 7-23 th day and reached the highest on the 15 th day.The m RNA transcriptional level in both groups was significantly higher than that in the control group.Compared with the two groups of experimental results,the TNF-? m RNA transcriptional level in the intranasal group was higher than that in the injection group and the highest value appeared earlier.However,the amount of TNF-? in the intranasal group was higher than that in the injection group before the 15 th day.After 15 days,the injection group was significantly higher than the intranasal group until the end of the trial.The results of IL-1? m RNA transcriptional level showed that the IL-1? m RNA transcriptional level in the intranasal group increased sharply on the 3rd day after infection,which was significantly higher than that of the control group.It reached the highest level on the 7th day and then it tended to decrease until the 19 th day.The m RNA transcriptional level of the 19 th day and control group was basically at the same level.The trend of the test result after infection in the injection group was basically the same as that of the intranasal group.Comparing the results of the two groups,the IL-1? m RNA transcriptional level in the intranasal group was higher on the third day than in the injection group.The injection group reached the highest on the 7th day and was higher than the intranasal group.Both groups were compared with the control group on the 19 th day at the same level.Afterwards,we used the Pearson regression analysis of SPSS 18.0 software to analyze the correlation between copies of PRRSV at each time point and the m RNA transcriptional level of the corresponding cytokines in the two experimental groups.Compared with the correlation between the PRRSV copies and m RNA transcriptional level of cytokines was higher than the injection group.The results of indicated that the change of PRRSV proliferation after intranasal infection may have more complicated relationships with the production of IFN-?,TNF-? and IL-1?. |
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