الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Foot and mouth disease (FMD) is a highly contagious viral disease among cloven hooved domestic and wild animals including cattle, buffaloes, sheep, goats and pigs. It has seven serotypes (A, O, SAT1, SAT2, SAT3, Asia 1 and C) causing huge economic losses due to reduced milk yields, abortions, delayed conception, perinatal mortality and premature culling. Controlling of FMD in susceptible animals such as cattle, sheep and goats attained by vaccination. Animal Health organizations resorted to the use of Honey bee venom (HBV) as an alternative apitherapy for FMD infection. This antiviral compound has pharmacologically active components including mainly melittin and other peptides, enzymes and biogenic amines that have antibacterial, anti- parasitic and antiviral properties.
The present study aimed to find a tool to limit the severity of FMD virus infection and its spread during outbreaks through the use of antiviral agents especially those of natural origin in addition to enhance the immunogenicity of the inactivated locally produced FMD vaccine. Such agent of choice was honey bee venom (HBV). So, the study included the following experimental steps:
1- Determination of the in vitro cell toxicity of HBV on Baby Hamster Kidney (BHK) cell culture, by applying 2-fold serial dilutions, which revealed that it is safe at concentrations of 62.5, 31.25, 15.63, 7.81, 3.9 and 1.95 µg/ml according to dilutions of 1/16, 1/32, 1/64, 1/128, 1/256 and 1/512. These safe concentrations showed normal cell shape with normal growth behaviour. This result was confirmed by in vivo intraperitoneal inoculation of HBV in 55 G. pigs with 0.4 ml and 55 of 2-3 days old baby Swiss mice with 0.2 ml.
2- In vitro study, HBV was used as antiviral agent against FMDV serotypes (O, A, SAT2) using all of the safe concentrations revealed that there is 100% reduction in the virus titer of the three FMD virus serotypes except with the dilution 1/512 (1.95 µg/ml), which showed antiviral effect with 81.25, 85.7, and 89.8% against serotype O, A and SAT2 respectively.
3- The invivo experiment was confirmed by inoculation of G. pigs and mice which withstood the experimental infection at percentage of 100 up to a concentration of 3.9 µg/ml while 1.95 µg/ml showed less protection against the three serotypes. Untreated infected G. pigs showed characteristics vesicle on planter surface of limbs, tongue and mouth while untreated mice showed paralysis of the hind limbs.
4- The safe concentration of HBV (0.4ml of concentration 3.9ug/ml) was used to treat G. pigs as a model of infected animals with FMDV serotypes. The temperature, clinical sign and titration of viruses serotypes were recorded after treatment with the safe concentration. On the 7th day post infection was observed rise in rectal temperature (38.5±0.5b - 38.9±0.6b ℃), began to decrease recording its lower degree (35.9±0.4b,c - 36.2±0.2b ℃) on the 7th day post treatment. In addition to these symptoms, positive reactions on the tongue and feet with eye redness were observed on the 1st day post treatment and on the tongue and feet on the 3rd day post treatment of G. pigs infected with O and SAT2 viruses in addition to eye redness in those infected with serotype A. But there were no clinical signs recorded on the 7th day post treatment. By the 7th day post infection in the infected untreated animals, the virus titers were increased by time to reach 8.5; 8 and 7.0 log10 TCID50/ml for serotype O, A and SAT2 respectively. Whereas in infected treated animals, these titers were decreased gradually by the 3rd day post treatment to reach zero level by the 9th day post treatment. Also, the same result
appeared by treatment of sheep (2 ml of concentration 3.9 µg/ml) infected with different FMD serotypes. The mean rectal temperature of infected sheep was raised showing higher degree (41±0.1b – 41.5±0.1b,c ℃) by the 7th DPI and slightly decrease by the 3rd DPT (40±0.1c – 40.2±0.3b ℃) returning to its normal temperature (39.5±0.2a – 39.6±0.1a ℃) by the 7th DPT. Also, in the infected untreated animals, the virus titers didn’t change up to the 7th day post infection (8.5±0.13a, 8±0.25a and 7±0.41a log10 TCID50/ml for serotype O, A and SAT2 respectively, whereas these titers were decreased gradually by the 3rd day post treatment (6.5±0.14b, 6±0.54b and 5.5±0.23b,c log10 TCID50/ml for serotype O, A and SAT2 respectively) by the 3rd DPT to reach zero level by the 9th day post treatment in the infected treated sheep.
5- HBV was simultaneously inoculated with the commercial FMD vaccine in sheep to evaluate its effect on the cellular immunity determined by Lymphocyte blastogenesis assay, IL-6 and IL-12 and the humeral immunity determined by SNT and ELISA. The results revealed that HBV induced high immune response from strength and duration rather than using the vaccine alone. The optical density of lymphocyte blastogenesis carried out on blood samples collected from vaccinated sheep with FMD alone reached its peak value by the 14th DPV (0.95±0.13b), but in sheep received HBV with FMD vaccine reached its peak value by the 7th DPV (1.15±0.25b). At the 35th DPV, the lowest values (0.78±0.21c) and (0.76±0.12bc) were recorded for FMD vaccine and FMD vaccine with HBV respectively.
6- IL-6 and IL-12 were estimated in the sera of vaccinated sheep and showed higher levels in sheep vaccinated by FMD vaccine with HBV than in sheep vaccinated with FMD vaccine alone by the 14th DPV (3.97±0.32b and 3.78±0.16b for IL-6 and 6.8±0.32b and 6.2±0.19b for IL-12 respectively. By the 35th DPV, the levels of IL-6 and IL-12 were decreased to 3.33±0.51c and
3.12±0.15b,c for IL-6 and 5.3±0.51c and 4.8±0.19b,c for IL-12 in sheep received HBV with FMD vaccine and in sheep received the vaccine alone.
7- Monitoring of the humeral immunity, it was found that HBV enhances the immune response of vaccinated sheep to the trivalent FMD vaccine resulted in earlier induction of specific FMD serotypes antibodies where reaching their peak on earlier time with prolonged duration of immunity. It was found that FMD serotypes antibodies were detected by application of SNT and solid phase indirect ELISA which showed that vaccinated sheep exhibited FMD serotype protective antibody titers (1.55±0.09 log10 and 1.8±0.1 log10 by SNT and ELISA respectively) by the second week post vaccination with FMD vaccine alone and (1.75±0.09 log10 by SNT and 1.95. ±0.13 log10 by ELISA) with administration of HBV by the first week post vaccination. These titers recorded their peaks (3.05±0.03 log10 by SNT and 3.13±0.03 log10 by ELISA) on the 12th WPV and 3.25±0.09 log10 by SNT and 3.45±0.04 log10 by ELISA on the 10th WPV using the vaccine alone and with HBV respectively. Such antibodies were gradually decreased to reach its lowest protective levels (1.60±0.04 log10 by SNT and 1.82±0.03 log10 by ELISA) on the 32nd WPV with FMD vaccine alone while in the case of administration of HBV these values were (1.65 ±0.15 log10 by SNT and 1.8±0.03 log10 by ELISA) on the 38th WPV. Non protective FMD serotype antibody titers (less than 1.5 and 1.8 log10 by SNT and ELISA respectively) were recorded by the 36th and 40th WPV with FMD vaccine alone and FMD vaccine with HBV respectively.
We can conclude that HBV used as antiviral against FMDV serotypes during outbreaks and also as an immune stimulant simultaneously with FMD vaccine to enhance the humeral and cellular immunity.