الفهرس 
Materials and methodsOnly 14 pages are availabe for public view
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Abstract
Allover the world, specially in Egypt and Arabian countries, there are great interest with Equine industry, which plays an important role in national incomes. EHV-1 disease is an important disease affecting equine industry. So, our attention directed to control this disease through vaccination process.
The present study was planned to prepare and evaluate an inactivated EHV-1 freeze dried vaccine, reconstituted in DEAE-Dextran solution as adjuvant. So, the study comprises the following investigation points:
1. Preparation of inactivated EHV-1 freeze dried vaccine:
1.1. Preparation of vaccine virus fluid:
The virus fluid was prepared from the freeze dried local isolate of EHV-1 which propagated in ECE (11-13 days old) for 12-13 passage.
1.2. Titration of vaccine virus fluid:
Titre of EHV-1 vaccine virus fluid ECE passage 12 in ECE 11-13 days old was 9 logio ElDso•
1.3. Inactivation of EHV-1:
EHV-1 was completely inactivated with BEI at concentration of 0.008 M for 24 hours at 37°C but residual infective virus was noted after 24 hours with 0.004 and 0.006 M BEI concentration at the same tifee=aml. x temperature. 1.4. Preparation of freeze dried vaccine:
The inactivated virus fluid was freeze dried according to the method described by Hillemon et al. (1951) and Ozawa et al. (1966).
1.5. Addition of DEAE-Dextran solution:
The locally prepared inactivated EHV-1 freeze dried vaccine was rconstituted in DEAE-Dextran solution (100 mg/ml tris HC1 buffer) at the time of inoculation. DEAE-Dextran acts as vaccine diluent and adjuvant.
2. Evaluation of the prepared vaccine: 2.1. Sterility test:
The final product of the vaccine was tested for its sterility using different bacterial media and they were free from any bacterial and fungal contaminants.
2.2. Safety test:
2.2.1. Residual infectivity test:
Detection of absence of residual infective virus in the inactivated virus fluids was tested in both ECE and mice. The inactivated virus fluids were free from any residual infective EHV-1.
2.2.2. Safety in horse and in mice:
Six horses and 10 mice received two doses from the final prepared inactivated EHV-1 freeze dried vaccine, reconstituted in DEAE-Dextran with 1 month interval for horses (3 ml/dose for horse) and 1 week interval for mice (0.2 ml/dose for mouse). They did not show any post vaccinal reaction (shock, toxicity, fever and/or hypersensitivity) or there is no isolated virus from the liver and lung tissue emulsion of inoculated mice when inoculated on CAM of ECE 11-13 days old.
2.3. Immunogenic potency of the locally prepared inactivated
EHV-1 freeze dried vaccine in mice:
Blood serum samples takes from the group of mice after 10 days from the 2nd dose of the prepared vaccine, gave mean ELISA titre 1946.2 and mean PHA titre 44.8 and positive AGPT. While, the blood serum samples collected from the control unvaccinated mice give negative result by all serological test. After challenge of vaccinated and control groups of mice, found that the EHV-1 was more rapidly cleared from vaccinated mice in comparison to unvaccinated control mice.
2.4. Keeping quality of the prepared vaccine:
Keeping quality of the locally prepared inactivated EHV-1 freze dried vaccine after keeping at different intervals (zero, 3, 6, 9 months) and (1 and 1.5 years) at different temperatures 4°C, -20°C and room temperature (25-37°C). Also, the keeping quality of reconstituted vaccine in sterile PBS and in DEAE-Dextran kept at 4°C was evaluated in mice. The locally prepared freeze dried vaccine could be kept at 4°C for one year and in room temperature for 6 months while at -20°C could be kept for more than 1.5 years. The reconstituted vaccine in sterile PBS could be kept for 3 months only while the vaccine reconstituted in DEAE-Dextran not stable for more than 2 hours.
2.5. Immunogenicity of locally prepared inactivated EHV-1 freeze dried vaccine in horses:
The immune response of four mares experimentally inoculated with the locally prepared inactivated EHV-1 freeze dried vaccine, reconstituted in DEAE-Dextran solution (100 mg/ml tris HC1 buffer) as adjuvant was traced for 13 months post vaccination using the following tests:
a.Agar gel precipitation test (AGPT).
b.Passive haemagglutination test (PHAT).
c.Enzyme linked immunosorbent assay (ELISA).
And d. Serum neutralization test (SNT).
The precipitating antibody started to appear after 2 weeks post vaccination and persist for 6 months only. The highest titre of PHA antibody and neutralizing antibody were at 3 MPV, while the peak of ELISA was at 4 MPV.
The obtained data clarify that:
1.The best concentration of BEI as an inactivator for EHV-1 was 0.008 M at 37°C for 24 hours.
2.The locally prepared inactivated EHV-1 freeze dried vaccine can be kept in -20°C for 1.5 years and in 4°C for layer and at room temperature not more than 6 months.
3.We advise to reconstitute the locally prepared inactivated freeze dried vaccine in DEAE-Dextran (100 mg/m1 tris HC1 buffer) just at the time
of animal inoculation.
4.Horses must be received two doses with one month interval and
revaccinated every 6 months.