Development And Application Of Direct Immunofluorescence Assay For Detection Of Porcine Reproductive And Respiratory Syndrome

Porcine reproductive and respiratory syndrome(PRRS)is induced by Porcine reproductive and respiratory syndrome virus(PRRSV)infection. It is characterized by severe reproductive failure in sows, including abortion, stillbirths and weak piglets, dyspnea and high mortality in piglets and respiratory disease in fattening pigs. Several affected pigs have a clinical manifestation of cyanosis in the ears, so it's also called"the blue ear disease". Since June 2006, a pig disease characterized by persisting high fever, dyspnea, skin cyanosis, reproductive failure in sows and high morbidity and mortality broke out in partial regions of Jiangxi, Hunan, Hubei and Anhui provinces in China, and it has caused immense economic losses in the pig industry. The research results of etiology showed that a highly pathogenic PRRSV variation strain was the main pathogen of"high fever syndrome". RT-PCR has been used for etiologic diagnosis of this strain, but it requires good experimental facilities and the detection expense of RT-PCR is high, which restrict its extended application on a large scale in the clinic.In this study, healthy piglets were inoculated with a highly pathogenic PRRSV-HBR variation strain which was isolated from clinical case of"high fever syndrome"for preparating antiserum. The immunoglobulin (IgG) was extracted from the antiserum by ammonium sulfate precipitation method, and then it was labeled with fluorescein isothiocyanate (FITC). After optimizing the reaction conditions, a direct immunofluorescence assay (DIFA) was developed for the detection of antigens in the virus-infected cell culture monolayer and tissue sections of pigs. The virus propagation dynamics in MARC-145 cells was detected by the DIFA. The results showed that the antigen positive cells appeared at 16h PI and reached the propagation peak at 60h to 72h PI, then these infected cells began to disrupt at 96h PI. The antigen distribution in the tissues of PRRSV-inoculated pigs was also detected. The high antigen positive rate was found in inguinal lymph nodes, jejunum, testis, spleen, mandibular lymph nodes, mesenteric lymph nodes and tonsil, respectively. The mean positive detection rate of PRRSV in 38 clinical samples from 10 farms of Shandong, Neimenggu, Jilin and Heilongjiang was 63.2%(24/38). The DIFA had a good sensitivity, specificity and repeatability, and the agreement rate was 96.3% when the presence of PRRSV was confirmed by RT-PCR. There was no antigen across reaction with several other viruses of pig diseases and the FITC-conjugated antibody was stable for 6 months at 4℃. This study provided a rapid, sensitive, specific and effective test facility for clinical diagnosis of porcine reproductive and respiratory syndrome(PRRS).