Research On Bivalent Engineering Vaccine Of Toxoplasma Gondii And Pesudorabies Virus

Toxoplasmosis, caused by Toxoplasma gondii, is widely distributed in warm-blooded animals and has significant zoonotic importance. Cats are the definitive host of T. gondii, may produce infectious oocysts. Oocysts is considered the main way of T. gondii infection, people could be infected T. gondii by ingestion of environmentally resistant oocytes. However, there are more and more research focused on the risk of human Toxoplasma-infection by consumption of meat or meat products contains T. gondii tissue cysts. Immune prevention is the main route for contral of toxoplasmosis. As the life cycle of T. gondii have a wide variety of complex factors that affect on Toxoplasma-immunity, the immune prevention of T. gondii present little progress; and, although there are many methods for detection of Toxoplasma gondii, but lack of a simple and rapid pathogen-diagnostic method in clinical. In the present study, we explored a diagnostic method of LAMP for detecting T. gondii DNA and a bivalent genetic engineering vaccines against T.gondii and pseudorabies virus.1. Development and application of LAMP assay for detecting T.gondii DNALoop-mediated isothermal amplification (LAMP) is invented by Notomi in 2000, as a new type method for nucleic acid amplification in vitro, mainly using 4 different primers identify the target genes of 6 a specific area, in isothermal conditions for efficient amplification. Gene amplification and product detection can be finished at one step, at present, the technology has been widely applied to viruses, bacteria, protozoa and other animal disease detection.In the present study describes the development of loop-mediated isothermal amplification (LAMP) specific to the single-copy gene MIC3 as a diagnostic tool of toxoplasmosis. A set of primers, composed of outer primers, inner primers and loop primers was designed from a published sequence data (GeneBank No. EU572718). The results confirmed that this LAMP method has good sensitivity and specificity, which can detect the limit to 0.4 tachyzoites. This LAMP and B1-based Nested-PCR were used to detect the 320 clinical samples of pigs at the same time, the positive rates were 11.6% and 9.69% respectively. Then, the LAMP method has been used to detect Toxoplasma gondii infection in pork,9 samples(9/45) from a slaughterhouse were positive,3 samples(3/68) from five market were detected positive, these results showed that the risk of Toxoplasma infection in pork is the fact. All the results indicated that LAMP can be widely used not only for the diagnosis of toxoplasmosis in pig, but also in daily monitoring of the risk of meat infected with T. gondii.2. Construction of recombinant pseudorabies viruses expressing SAG1, MIC3 and GRA7 gene of T.gondiiPseudorabies virus(PRV) can cause a variety of animals with fever, itching, and encephalitis as the main symptoms. Pseudorabies caused by the pseudorabies virus is one of the major infectious diseases threatening the global pig industry. Currently, pseudorabies virus(virulence genes knock-out) vaccine is one of most successful vaccines against animal disesases and a good vector, dozens of foreign genes were expressed in PRV.The DNA fragment containing the T.gondii SAG1(orMIC3, orGRA7) expression cassette [pCMV-SAG1(orMIC3,orGRA7)-BGH poly(A)] was released from pcDNA-SAG1(or pcDNA-MIC3, or pcDNA-GRA7) plasmid, and inserted into pgG-Uni, resulting in the recombinant transfer plasmids pgG-SAG1, pgG-MIC3 and pgG-GRA7. And then this plasmid was co-infected into IBRS-2 cells with genome of PRV TK-/gG-/EGFP+. When cytopathic effect was occurred in cells, recombinant virus TK-/gG-/SAG1(orMIC3, orGRA7)+was obtained from the cytopathic cells by plaque purification. The Three recombinant viruses expression of SAG1(orMIC3, orGRA7) protein was detected with Western blotting respectively. Results of TCID50 showed the insertion of ORF2 or ORF1-ORF2 gene had no influence on the propagation of recombinant viruses in IBRS-2 cells. And, the data also showed that the recombinant viruses are good genetic stability and safety.3. Immune effect and protective efficacy of recombinant pseudorabies virusesThree recombinant viruses were used to vaccinate mice with single or combined. All the mice inoculated with the recombinant virus produced specific antibodies to T. gondii lysis antigen (TLA), accompanied by a strong lymphocyte proliferation and the amounts of both IL-2 and IFN-y significantly increased. These results suggested that recombinant pseudorabies virus is capable of inducing strong humoral and Thl-type cellular immunity. Moreover, the mice immunization recombinant pseudorabies virus can produce protective immunity to resist lethal T. gondii RH strain attack, and can induce high levels of PRV neutralizing antibodies.Those with two or even three rPRV mixed immunized mice to produce higher levels of T. gondii-specific IgG antibody, lymphocyte proliferation stronger and more effective attacks against T. gondii. These results indicated that expression of protective antigen of T. gondii recombinant pseudorabies virus have developed the potential to become a new type of vaccine used to prevent and control both animals pseudorabies and toxoplasmosis.