| Porcine reproductive and respiratory syndrome(PRRS)has been circulated globally and brought huge economic loss to the pig industry, especially the HP-PRRS caused by the American-type mutant strain which has caused serious loss and threat to the pig industry in our country. Therefore, it's extremely urgent to develop effective and practical diagnostic and preventive technologies.In the present research, a specific and sensitive RT-PCR diagnostic method which can differentiate high pathogenic and classical PRRSV was established, and the representative pathomorphological features caused by PRRSV were summarized based on a series of pathomorphological observations on PRRS cases of different disease stages. Finally, Comprehensive diagnostic method of PRRS which combines pathology and PCR was established and the investegation work involved in epidemic characteristic and host range of PRRSV in Liaoning were subsequently carried out for the purpose of isolation of Liaoning PRRSV strain and ascertainment of its gene type and hereditary variation. Also, nucleic vaccine of Liaoning strain of PRRSV based on ORF5 gene was expected to be built, which and immunopotentiator were applied together to assess the levels of humoral immunity and cellular immunity of the built nucleic vaccine through animal-immunizing experiment.Firstly, a RT-PCR differential detection method for HP-PRRSV strain and classical strain was established based on the Nsp2-specific primers design. Results show that the method is of strong specificity, high sensitivity and good repeatability. Morbid anatomic, histopathological and immunohistochemical method were applied to carry on the pathological observation for different infective stages of PRRS cases. The result shows that pathological dissection change and histopathological traits occur during the prodromal period and interstitial pneumonitis, degenerative phlegmasia of immune organs and nonsuppurative encephalitis occur during the period of obvious onset; pathological diagnosis of HP-PRRS as the additional basis of RT-PCR diagnostic method can demonstrate the infective stage of the disease. Application of pathological and PCR methods together makes the diagnosis of PRRS more accurate and objective.ELISA and PCR methods were applied to detect the PRRSV serum antibodies and pathogen genes in Liaoning. The results show that PRRSV antibody positive rate in serum of the total pigs in Liaoning is 55.1% and pathogen positive rate 19.5%. The pathogen distribution index is 100% and pathogen contamination index is 59.75% (unit: city region), showing that immunoprotection rate of PRRS is low and that pathogen distribution is wide and that contamination level is high in Liaoning. PRRS antibodies and pathogen were detected from different types of pig farms and different growth stages of pig herds in Liaoning, in which the antibody positive rate of farms immunized by attenuated live vaccine is the highest (79.8%); PRRSV pathogen positive rate of infected farms is the highest (81.6%). Antibody and pathogen positive rates are both high, 76.7% and 31.3% respectively. The highest proportion of screening out PRRSV mutant is incidence and piglets, for 93.4% and 86.7%; Mixed infection rate of PRRS and other diseases is serious, accounting for 80%. The mixed infection pathogens are streptococcus, pasteurella, salmonella, PCV2, HCV, and pseudorabies virus. In Liaoning, there may be many non-pig hosts, including animals of positive PRRSV serum antibodies such as chickens, ducks, pigeons, sparrows and sheep.The positive rate of sheep PRRSV antibodies is as high as 27.5%. It shows that there may be lots of non-pig hosts, which must be paid much attention to PRRS during the preventive and controlling course.One strain of American-type PRRSV was isolated by Marc-145 in Liaoning province, which is named PRRSV LN/0901 temporarily; Amino acid comparison, homology and phylogenetic tree analysis of Nsp2 and ORF5 gene sequence of the strain indicated that the genetic relationship of PRRSV LN/0901 is close to most epidemic mutant strains ever since 2006 and it's an independent branch, showing that the pathogen causing PRRS in Liaoning has mutated. The prediction and analysis of amino acid hydrophobicity and encoding protein transmembrane domain on ORF5 of PRRSV LN/0901 showed that there are 3 domains of high hydrophobicity in the amino acid domains of 8-28,63-78,110-128 and there are 3 transmembrane helix domains in amino acid domains of 15-32,66-88,103-125 at N-end, which indiacted that the strain ORF5 has lots of antigenic epitopes. There are common epitopes and different epitopes compared to the same kind of strains in our country and it can be important protein gene for studying and producing the genetic engineering vaccine in Liaoning.Based on this, the recombinant nucleic plasmid containing pCI-PRRSV LN/0901-ORF5 was built and successfully transfected to Marc-145 cells which was authenticated through RT-PCR, IFA and Western blotting methods. Mice were inoculated with the built nucleic vaccine and the immunopotentiator. ELISA,MTT and flow cytometry were used to detect the anti-PRRSV antibodies level, the proliferation of lymphocyte and its subclass, which showed that the newly built nucleic vaccine can stimulate mice to produce anti-PRRSV antibodies and also stimulate the proliferation of T-cells and its subclass; the immunogenicity of the built nucleic vaccine is stronger than inactivated vaccine. Transference factor and lentinan can significantly strengthen immune effect of the nucleic vaccine. The application of immunoadjuvant can strengthen immune effect more significantly.The nucleic vaccine built with local strain, PRRSV attenuated live vaccine, killed vaccine and transfer factors for pigs, microecologics, lentinan etc. were used to carry out pig immune comparison experiment. Through detecting the PRRSV-specific antibodies, lymphocyte and its subclass proliferation level in pigs, humoral and cellular immunity levels after the application of different kinds of vaccine and immunopotentiator were tested. The results show that the built nucleic vaccine can make pigs produce anti-PRRSV specific antibodies and neutral antibodies and obviously enhance the number of T cells and their subclass. The results also show that the humoral and cellular immune level is lower than attenuated live vaccine, nosignificantly than PRRSV killed vaccine. Application of transfer factors for pigs, microecologics, lentinan can significantly enhance the humoral and cellular immune level of PRRSV nucleic vaccine. The effect of mixed use of immunopotentiators is more significant than single use of one kind. Conclusionly, the research laid the foundation for developing comprehensive preventive technologies of PRRS in Liaoning province. |
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