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Studies On Gene Immunization Of Chickens Against Infectious Laryngotracheitis

Posted on:2001-02-06Degree:DoctorType:Dissertation
Country:ChinaCandidate:S S MengFull Text:PDF
GTID:1103360002452452Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Infectious Laryngotracheitis (ILT) is an acute respiratory infection of chickens that can result in severe production losses due to mortality and decressed egg production. The disease occurs worldwide and is one of the major health and economic problems in the poultry industry. The causative agent of ILT is Infectious Laryngotracheitis Virus (ILTV), a member of the Alphaherpesvirinae subfamily of the Herpesviridae. To date gB is known to be a main immunogen of ILTV and gC, gD may also play a important role in inducing protective immunity. At present ILT is controlled by the use of modified live vaccines, all ILTV live vaccines remain certain virulence, and are able to establish latent infection for life time, the vaccine viruses may increase virulence during in vivo passaging. Therefore, there is an urgent demand for safer vaccines which could be used to control and possibly eradicate ILTV. Gene vaccination against ILTV was carried out in this research with recombinant eukaryotic expression plasmids containing the immunogen genes of ILTV strain Wanggang. This research may open a new approach to prevent and control ILT. gB and gD genes of ILTV were amplified from ILTV strain WG(isolated from Wanggang, Harbin, China)by polymerase chain reaction (PCR) based on the published sequences of ILTV strain SA2. The PCR products were cloned into pCR3-Uni vector and sequenced. Sequence analysis indicated that the gene coding for gB of ILTV WG with 2619bp in length is identical to those of ILTV strains Throne 882 and 632, and displayed 99.8% nucleotide identities with ILTV SA2 . The gene coding for gD of ILTV WG with 11 34bp in length displayed 97% nucleotide identities with ILTV SA2. A fragment containing gC gene of ILTV WG was previously cloned in pBluecript 5K by Dr. Xin Guo, and subcloned in the study into pCR3-uni vector. Sequence analysis indicated that the gC gene of ILTV WG with 1245bp in length displayed 99% nucleotide identities with ILTV SA2. The obtained recombinant plasmids were designated as PgB, PgC and PgD, respectively. To test immune efficiency of PgB, PgC, and PgD, SPF chickens were divided into seven groups (20 chickens for each): (A) normal saline; (B) pCR3-Uni, (C) commercial ILTV live vaccine; (D) PgB; (E) PgB+PgC+PgD, (F) PgB+CpG, (G) PgB+PgC+PgD+CpG. 3-week-old chickens in group B, D and E were immunized by four sites intramuscular injection, the dose of each injected plasmid for a chicken is lOOj.tg. Plasmids with the same dose (1 OOj.tg) for each in group E were mixed before injection. Two DNA inoculations were given, one at time 0 and the second two weeks later. In group C, 5-week-old chickens were inoculated by eye drop with commercial ILTV vaccine. The proliferative responses of lymphocytes from thymus and bursa of chickens were measured by MTT assay, antibody responses were assayed using indirect ELISA. Challenges with Lethal dose of ILTV WG were administered at four weeks after the second DNA inoculation, the protective immunity was observed. The results showed that recombinant plasmid DNA induced strong proliferative responses of lymphocytes, seroconversion was observed by two weeks after first immunization, antibody responses peaked after second immunization while the control groups showed no changes. After challenge, 79% birds in group D or E, 100% birds in group C, and 14% birds in group A or B were survived, respectively. The results indicated that strong protective efficiency against ILTV was induced by gene vaccination. To test immune enhancement of CpG to gene vaccination, chickens were immunized with a mixture of bacterial...
Keywords/Search Tags:Infectious Laryngotracheitis Virus, gB, gC, gD, DNA vaccine, CpG DNA
PDF Full Text Request
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