Assessment Of The Pigs Susceptibility To PRRSV In Jiang Su Province And Anhui Province

Porcine reproductive and respiratory syndrome(PRRS),commonly known as"blue ear disease",is a disease that could cause reproductive disorders and respiratory syndrome by the porcine reproductive and respiratory syndrome virus(PRRSV).In 2006,The outbreak of pig ardent fever caused huge economic losses to pig industry in China;PRRSV has been confirmed to be the main causative virus.While the existing vaccines and bio-security measures cann't effectively limit the infection.The future key consideration will be the disease resistance in the genetic level.There are many local pig breeds in China,but their resistance to PRRSV had not been studied.In this study,we established a method to assess the resistance to PRRSV in vitro.Then,five porcine breeds in JiangSu province and AnHui province respectively were evaluated,and the association between PRRSV resistance and swine immune-related factors were assayed.This study aimed to lay a foundation to PRRS resistance breeding,and will promote the protection and utilization of the resources of local porcine breeds in China.The study includes four parts:1.PRRSV NJGC infected pigletsIn order to get qualified PPRSV strain for evaluating pigs' susceptibility to PRRSV,5 piglets were injected with PRRSV NJGC to confirm the virulence and occurrence regularity of PRRSV NJGC.The results showed that the body temperature of piglets gradually increased in the first day after inoculating,and the highest temperature appeared on the eighth day(40.9-42.3?).Piglets showed lethargic,dyspneic,loss of appetite,diarrheic,leptystic,with their ears showed blue-violet and other typical symptoms of PRRS.All piglets died in 11-13d after inoculation.When its were dissected,we found their lungs were dark purple-like lesions with some necrotic area;their livers were hyperemic with gray-white nodule and liver cell necrosis;the spleen and kidney were oedematous with a large number of needle-like bleeding spots;the lymph nodes,especially inguinal lymph node and hilar lymph node,showed enlarged,hemorrhagic and necrotic;and their hyperemia,hemorrhage and ulcers were seen in intestines.Moreever,PRRSV mRNA was expressed in several tissues(including heart,liver,spleen,lung,kidney,lymph nodes)of dead pigs examined by RT-PCR.The results showed that PRRSV NJGC was a virulent strain,and it's suitable to evaluate the susceptibility of pig to PRRSV.2.Establish a model for assessment on pigs susceptibility to PRRSV that based on replication differences of PRRSV in PAMTo establish a model for assessment on pigs susceptibility to PRRSV,we examined the proliferation of PRRSV NJGC.The purified PAMs were inoculated with PRRSV NJGC,and the expressions of PRRSV mRNA of PAMs were examined by Real-time quantitative PCR(qRT-PCR)after being infected PRRSV 4?8?12?18?24?32?36?48?60 and 72h.Meanwhile,the viral titer of supernatant of PAM was examined.The results showed that PRRSV began to proliferate in PAM after being infected 4h,increased rapidly from 8 to 12h,and reached the highest level in 12h,while decreased rapidly from 12 to 18h,and changed slowly from 24 to 72h.The viral titer of supernatant of PAM increased rapidly from 6 to 12h,and reached Plateau phase between 12h and 24h,then entered into decline phase.With the result of replication of PRRSV in PAM and the virus titer of supernatant of PAM,we chose 6,12,18,24 and 36h as a point to assess pigs' susceptibility to PRRSV.3.Assessment of the pigs susceptibility to PRRSV in JiangSu province and Anhui provinceTo assess pigs susceptibility to PRRSV in Jiangsu and Anhui province,PAMs were separated and purified from 5 breeds(Erhualian,Meishan,Suzhong pig,Jiangquhai,Dingyuan pig)and infected with PRRSV NJGC in vitro.The expression of PRRSV mRNA of PAM infected at different time were examined by qRT-PCR,and Reed-Muench method was used to determine the virus titer of supernatant of PAM.qRT-PCR revealed that the proliferation trend of PRRSV NJGC was similar in different pig breeds,and replication of PRRSV reached the highest level at 12h,and decreased quickly from 12h to 18h in each pig breed's PAM.The virus titer of supernatant of PAM also has similar trendy in different pigs' breeds.Virus titer increased from 6h to 12h,and reached plateau phase from 12h to 24h.The result of assessment on pig breeds' susceptibility to PRRSV in Jiangsu province and Anhui province showed that Jiangquhai pig has lower susceptibility than other four breeds,while Dingyuan pig has a higher susceptibility.The susceptibility to PRRSV of Jiangquhai pig was significantly lower other than Dingyuan pig and Suzhong pig(P<0.05).All these results laid a firm foundation of PRRS resistance breeding of pigs.4.The expression difference of cytokine,Sn and TLR3 among infected PAM of pig breedsTo study the association between pig susceptibility to PRRSV infection in PAM and the expression of cytokine or receptor genes,three immune markers and two receptor genes were examined with cDNA.The results showed that IL-8 and IL-10 increased,while TNF-? was reduced in infected PAM.IL-10 expression after infected with PRRSV in descending order:Dingyuan pigs,Jiangquhai pigs,Meishan pigs,Erhualian,Suzhong pigs;IL-8 expression from low to high:Dingyuan pigs,Jiangquhai pigs,Meishan pigs,Erhualian,Suzhong pigs;TNF-a expression from low to high:Dingyuan pigs,Jiangquhai pigs,Meishan pigs,Suzhong pigs,Erhualian.These results illustrated that these markers were significantly associated with PPRS virus-resistant.The reduced or delayed growth of PRRSV was associated with high levels of interleukin-10(IL-10)mRNA and interleukin-8(IL-8)mRNA accumulation and reduction of synthesis of tumor necrosis factor-?(TNF-?).IFN-? mRNA expression increased in infected PAM in vitro,and it's cDNA level was that the higher expression level of IFN-y,the weaker susceptibility to PRRSV in our researched Chinese pigs.This result suggested that PRRSV can induce the expression of IFN-?,and high level of IFN-y may inhibit the replication of PRRSV in PAM.The expression of Sn and TLR3 mRNA increased in infected PAM.Sn and TLR3 expressions of Jiangquhai pig were the lowest in the four pig breeds,and TLR3 cDNA reached a significant low level(P<0.01)at the Twelfth hour.These results suggested that PRRSV can induce the Sn and TLR3 expression,and the expression of Sn may associate with pigs susceptibility to PRRS V.